Vaccine Adjuvants: Methods and Protocols (Methods in Molecular Biology, Vol 626)

ME T H O D S

IN

MO L E C U L A R BI O L O G Y

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For other titles published in this series, go to www.springer.com/series/7651

TM

Vaccine Adjuvants Methods and Protocols

Edited by

Gwyn Davies St. George’s University of London, London, UK

Editor Gwyn Davies Department of Cardiac & Vascular Sciences St. George’s University of London Cranmer Terrace London Tooting United Kingdom SW17 0RE [email protected]

ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-60761-584-2 e-ISBN 978-1-60761-585-9 DOI 10.1007/978-1-60761-585-9 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2009943283 © Springer Science+Business Media, LLC 2010 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Cover illustration: Detection of antigen presention in vivo by immunofluorescence. 100 μg of EαGFP protein plus 1 μg LPS was injected into the neck scruff and 30 min or 24 hours later the skin injection site and draining brachial lymph nodes (BLNs) were collected, processed and stained. The cover illustration shows intrinsic EαGFP fluorescence (green) 30 min post injection at skin injection site. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)

Preface Over the last few years the field of research and development of vaccine adjuvants has become a very exciting and stimulating area to work in. Its scope is huge, from discoveries in fundamental immunology and mechanisms by which adjuvants are able to influence immune responses to antigens, to the safe application of products that modify immune responses, to optimization of adjuvant formulation with antigens, and last but not least, to the industrial and commercial considerations that are essential if an adjuvant is to be developed in a vaccine product. Its potential is also huge, as we understand more and more about how adjuvants may steer the immune system toward the responses required by unmet vaccination needs. Indeed, one of the complexities in putting a book like this together has been the decisions involving what to leave out rather than what to include. Where are the boundaries with pure immunology, and how do you avoid simply producing another vaccine protocol book? Also, much has recently been published about vaccine adjuvants, but relatively little specifically addressing the techniques and methods that are applied to understanding how adjuvants produce their effects in vaccines. This volume, then, aims to provide some guidance on how to go about assessing the activity of adjuvant products. Of course, the fact that there are very different mechanisms by which an adjuvant effect can be generated, reviewed in Chapter1, and different routes of administration of vaccines, means that many different aspects have to be addressed. The general philosophy of the volume has been not to describe individual adjuvants in development or in use in vaccines, although some types are described, but rather to provide information on measuring the responses produced by adjuvants. No preconception is made as the types of vaccine that could benefit from formulation with an adjuvant. Two chapters describe what might be considered as reference adjuvants. First, methods for the use of aluminium salts, that until recently were the only adjuvant products in licensed human vaccines, and then Freund’s adjuvants, are discussed. Use of the latter, particularly the complete Freund’s, is now restricted, but it has served as a reference for much adjuvant research, and so it is included here. There have inevitably been some overlaps with vaccine studies, although we have tried to keep the focus on the adjuvant throughout. There is also a small amount of overlap between some chapters, but it seemed preferable to maintain the integrity of each. Unfortunately, it has not been possible to include all relevant topics; commercial and other considerations have prevented some from being presented. Some topics are covered by reviews. The AS04 adjuvant has been described previously, but the approval of vaccines containing this adjuvant has opened up possibilities for novel adjuvants, and we considered a description of the methods used in its development as highly informative for this volume. The importance of adjuvant safety cannot be overestimated and a review has been dedicated to this. Also, there is a review of the use of cytokines as adjuvants. I sincerely hope that the methods presented here will have an enabling effect for those interested in working in vaccine adjuvant research and development. Also, I hope they

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will help to stimulate discussion on how we can best standardize adjuvant testing so that meaningful comparisons can be made, and above all, so that useful predictions can be made on which new products will most effectively and safely help to solve the current challenges in vaccination. Gwyn Davies

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v ix

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Immunology of Vaccine Adjuvants . . . . . . . . . . . . . . . . . . . . . . . . Carla M.S. Ribeiro and Virgil E.J.C. Schijns

1

2.

Preclinical Development of AS04 . . . . . . . . . . . . . . . . . . . . . . . . . Nathalie Garçon

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Nonclinical Safety Assessment of Vaccines and Adjuvants . . . . . . . . . . . . . Jayanthi J. Wolf, Catherine V. Kaplanski, and Jose A. Lebron

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Aluminum Adjuvants: Preparation, Application, Dosage, and Formulation with Antigen . . . . . . . . . . . . . . . . . . . . . . . . . . Erik B. Lindblad and Niels E. Schønberg

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Freund’s Complete and Incomplete Adjuvants, Preparation, and Quality Control Standards for Experimental Laboratory Animals Use . . . . . . . . . . . Duncan E.S. Stewart-Tull

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5.

6.

Liposomal Adjuvants: Preparation and Formulation with Antigens . . . . . . . . Jean Haensler

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Micro/Nanoparticle Adjuvants: Preparation and Formulation with Antigens . . . Padma Malyala and Manmohan Singh

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Adjuvant Activity on Human Cells In Vitro . . . . . . . . . . . . . . . . . . . . 103 Dominique De Wit and Michel Goldman

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Adjuvant Activity on Murine and Human Macrophages . . . . . . . . . . . . . 117 Valerie Quesniaux, Francois Erard, and Bernhard Ryffel

. . . . . . . . . . . . . . . . . . . . . 131

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In Vitro Effects of Adjuvants on B Cells Jörg Vollmer and Hanna Bellert

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NKT Cell Responses to Glycolipid Activation . . . . . . . . . . . . . . . . . . . 149 Josianne Nitcheu Tefit, Gwyn Davies, and Vincent Serra

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Tracking Dendritic Cells In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . 169 Catherine M. Rush and James M. Brewer

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Adjuvant Effects on Antibody Titre . . . . . . . . . . . . . . . . . . . . . . . . 187 Barry Walker and Ian Feavers

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Functional Antibody Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Ian Feavers and Barry Walker

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Determining Adjuvant Activity on T-Cell Function In Vivo: Th Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Thomas Lindenstrøm, Peter Andersen, and Else Marie Agger

16.

Quantitative Multiparameter Assays to Measure the Effect of Adjuvants on Human Antigen-Specific CD8 T-Cell Responses . . . . . . . . . . . . . . . 231 Laurent Derré, Camilla Jandus, Petra Baumgaertner, Vilmos Posevitz, Estelle Devêvre, Pedro Romero, and Daniel E. Speiser

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Large-Animal Model for Establishing E/T Ratio of Adjuvants . . . . . . . . . . 251 Luuk A. Th. Hilgers

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Determining the Activity of Mucosal Adjuvants . . . . . . . . . . . . . . . . . . 261 Barbara C. Baudner and Giuseppe Del Giudice

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Adjuvant Activity of Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Michael G. Tovey and Christophe Lallemand

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

Contributors ELSE MARIE AGGER • Adjuvant Research, Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark PETER ANDERSEN • Adjuvant Research, Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark BARBARA C. BAUDNER • Novartis Vaccines and Diagnostics, Siena, Italy PETRA BAUMGAERTNER • Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne, Switzerland HANNA BELLERT • Coley Pharmaceutical GmbH, A Pfizer Company, Düsseldorf, Germany JAMES M. BREWER • Centre for Biophotonics, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, Scotland, UK GWYN DAVIES • European Adjuvant Advisory Committee, Cardiac and Vascular Sciences, St. George’s University of London, London, UK DOMINIQUE DE WIT • Institut d’Immunologie Médicale, Université Libre de Bruxelles, Charleroi, Belgium GIUSEPPE DEL GIUDICE • Novartis Vaccines and Diagnostics, Siena, Italy LAURENT DERRÉ • Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne, Switzerland ESTELLE DEVÊVRE • Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne, Switzerland FRANCOIS ERARD • Molecular Immunology and Embryology, University and CNRS, Orleans, France; IIDMM, University of Cape Town, Cape Town, South Africa IAN FEAVERS • National Institute for Biological Standards and Controls, Potters Bar, Hertfordshire, UK NATHALIE GARÇON • Global Adjuvant Center for Vaccine, GlaxoSmithKline Biologicals, Wavre, Belgium MICHEL GOLDMAN • Institut d’Immunologie Médicale, Université Libre de Bruxelles, Charleroi, Belgium JEAN HAENSLER • Sanofi Pasteur, Marcy l Etoile, France LUUK A. TH. HILGERS • Nobilon International BV, Boxmeer, the Netherlands CAMILLA JANDUS • Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne, Switzerland CATHERINE V. KAPLANSKI • Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA CHRISTOPHE LALLEMAND • Laboratory of Viral Oncology, FRE2937 CNRS, Institut André Lwoff, Villejuif, France JOSE A. LEBRON • Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA

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ERIK B. LINDBLAD • Adjuvant Department, Brenntag Biosector, Frederikssund, Denmark THOMAS LINDENSTRØM • Adjuvant Research, Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark PADMA MALYALA • Novartis Vaccines and Diagnostics, Cambridge, MA, USA VILMOS POSEVITZ • Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne, Switzerland VALERIE QUESNIAUX • Molecular Immunology and Embryology, University and CNRS, Orleans, France; IIDMM, University of Cape Town, Cape Town, South Africa CARLA M.S. RIBEIRO • Department of Cell Biology & Immunology, Wageningen University, Wageningen, The Netherlands PEDRO ROMERO • Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne, Switzerland CATHERINE M. RUSH • Vascular Biology Unit, School of Medicine, James Cook University, Townsville, Queensland, Australia BERNHARD R YFFEL • Molecular Immunology and Embryology, University and CNRS, Orleans, France; IIDMM, University of Cape Town, Cape Town, South Africa VIRGIL E.J.C. SCHIJNS • Department of Cell Biology & Immunology, Wageningen University, Wageningen, The Netherlands NIELS E. SCHØNBERG • Adjuvant Department, Brenntag Biosector, Frederikssund, Denmark VINCENT SERRA • Wittycell SAS, Evry, France MANMOHAN SINGH • Novartis Vaccines and Diagnostics, Cambridge, MA, USA DANIEL E. SPEISER • Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne, Switzerland DUNCAN E.S. STEWART-TULL • Division of Infection and Immunity, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, Scotland, UK JOSIANNE NITCHEU TEFIT • Wittycell SAS, Evry, France MICHAEL G. TOVEY • Laboratory of Viral Oncology, FRE2937 CNRS, Institut André Lwoff, Villejuif, France JÖRG VOLLMER • Coley Pharmaceutical GmbH, A Pfizer Company, Düsseldorf, Germany BARRY WALKER • National Institute for Biological Standards and Controls, Potters Bar, Hertfordshire, UK JAYANTHI J. WOLF • Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA

Chapter 1 Immunology of Vaccine Adjuvants Carla M.S. Ribeiro and Virgil E.J.C. Schijns Abstract In recent times vaccine adjuvants, or immunopotentiators, received abundant attention in the media as critical ingredients of current and future vaccines. Indeed, vaccine adjuvants are recognized to make the difference between competing vaccines based on identical antigens. Moreover, it is recognized that vaccines designed for certain indications require a matching combination of selected antigen(s) together with a critical immunopotentiator that selectively drives the required immune pathway with minimal adverse reactions. Recently, the mechanistic actions of some immunopotentiators have become clearer as a result of research focused on innate immunity receptors. These insights enable more rational adjuvant and vaccine design, which, ideally, is based on predictable immunophenotypes following vaccination. This chapter addresses immunopotentiators, classed according to their (presumed) mechanisms of action. They are categorized functionally in two major groups as facilitators of signal 1 and/or signal 2. The mode(s) of action of some well-known adjuvant prototypes is discussed in the context of this classification. Key words: Adjuvant, signal 1, signal 2, stranger, danger.

1. Introduction Vaccines have become one of most successful life-saving instruments in modern medicine due to their extremely efficient and cost-effective prevention of infectious disease (1). Traditionally, vaccines comprise either live-attenuated, replicating pathogens or non-replicating, inactivated pathogens or their subunits (2). Live vaccines, still used to immunize against measles or rubella, are safe for the majority of recipients. Although cost-effective and rather easy to manufacture, live vaccines may cause disease when given to a recipient with an unrecognized immunodeficiency (3). Inactivated vaccines consist of killed pathogens or G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_1, © Springer Science+Business Media, LLC 2010

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isolated non-replicating subunits. They are safe for immunocompromised individuals, but they often show limited immunogenicity. The vast majority of current vaccines act by inducing antibodies. However, many new vaccine targets require the induction of specific cell-mediated responses, in addition to antibodies (4). Special adjuvants, or immunopotentiators, are therefore required to elicit adequate immunity, for example, enhancement of T-cell responses, by targeting certain innate immune cells in most cases, with the additional benefits that less antigen doses and fewer administrations are necessary (5). Vaccine adjuvants come in many forms and are more or less effective at inducing the onset, magnitude, duration, and quality of an immune response against a co-formulated antigen. Hence, adjuvants (from Latin adjuvare meaning “to help”) can be defined as a group of structurally heterogeneous compounds able to enhance or modulate the intrinsic immunogenicity of an antigen (6). They can be classed according to their chemical nature or physical properties, yet related compounds frequently have divergent immunomodulating capacities. For example, saponin variants may differ in their capacity to stimulate Th1- or Th2-type immunity (7). Alternatively, adjuvants have been clustered according to the immunological events they induce (2, 8), although for many the exact mechanism of action is unknown. In 1989 Charles Janeway Jr. aptly termed vaccine adjuvants “the immunologist’s dirty little secret” (9). It reflected the general ignorance on the mechanisms of action of most known adjuvants at the time. Yet rational vaccine design involves a logical choice of immunopotentiator based on its mode of action and its expected effect on efficacy and safety of the vaccine (3). Despite the tremendous impact of the adjuvant choice, even today the key processes critical for immune induction in general, and those evoked by distinct adjuvants, in particular, are unknown and subject of debate among immunologists and vaccinologists (10).

2. Signal 1 and Signal 2 Facilitators

At present two major functional classes of vaccine adjuvants have been defined (8). The first category includes so-called facilitators of signal 1, influencing the fate of the vaccine antigen in time, place, and concentration, ultimately improving immunoavailability of the antigen. The second major group constitutes facilitators of signal 2, providing the correct co-stimulation signals for the antigen-specific adaptive immune cells during antigen recognition. Both classes of adjuvant are not mutually exclusive (Table 1.1). For a schematic illustration see Fig. 1.1.

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Table 1.1 Classification of adjuvants according to their stimulatory action Adjuvant category

Concept

Critical feature

Example

Signal 1

Improving immunoavailability

Time, place, dose of antigen

Alum-containing adjuvants, oil-based emulsions

Stranger (PAMP)

Lipopolysaccharide

Signal 2

Improving co-stimulation

Danger (DAMP)

Heat-shock proteins

Recombinant co-stimulus

Recombinant interferon

Release of natural immune system brakes

CTLA-4 inhibitory antibody

According to the two-signal model (11, 12) both the presentation of antigen (signal 1) and the additional secondary signals (signal 2) are required for activation of specific T and B lymphocytes, which form the adaptive arm of the immune system.

a

b

Fig. 1.1. The two-signal model. Recent advances in immunology have shown that the magnitude and specificity of the signals perceived by the innate immune cells following infection (and vaccination) can shape subsequent adaptive immune responses. Activation of (depicted) (a) T helper (Th) cells requires at least two different signals (b) from the antigen-presenting cell (APC) including signal 1 (antigen presentation) and signal 2 (co-stimulation).

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Secondary signals are delivered by co-stimulatory or co-inhibitory signals, and their overall balance and constellation determines the magnitude and quality of the ensuing adaptive immune reaction (Fig. 1.1). Stimulatory second signals (in most cases for adjuvants) inform the T cells that the presented antigens are proper subjects to initiate an immune response. Charles Janeway predicted that microbial pathogens are evolutionarily distant from their hosts and contain conserved molecular patterns, so-called pathogen-associated microbial patterns (PAMPs), which can be recognized by so-called pathogen recognition receptors (PRRs) (9). Today we know that PRRs exist. They include Toll-like receptors (TLRs), NOD-like receptors, dectin-1 or RIG-like helicases which are predominantly found on cells of the innate immune system. The rather recent discovery of PRRs and their capacity to detect molecular structures unique to pathogens (also referred to as signal 0) provided a mechanistic concept of the key upstream events leading to co-stimulation. This insight formed a breakthrough for rational vaccine design based on PRR ligands, comprising various PAMPs and later also endogenous TLR ligands, such as heat-shock proteins (Hsp) (5). Nowadays the innate immune system is studied extensively after the general appreciation by the scientific community that this system plays a critical role in PAMP sensing and instruction of adaptive immune reactions. It is considered critical in signal 2 induction and downstream activation of distinct T helper cell subsets. The category of signal 2 facilitating adjuvants has since been mechanistically defined at the molecular level (please see Section 4).

3. Signal 1 Facilitators In contrast to molecular events involved in the generation of signal 2, very little is known about the mechanisms governing the adjuvant effect of signal 1 facilitators. When soluble antigen is injected subcutaneously it is presented in two waves in the secondary draining lymph nodes (LN). Within 30 min free antigen enters the LN by afferent lymph vessels and is presented by resident dendritic cells (DC), while tissueresident DCs that acquire antigen at the injection site migrate to LN within 12–24 h and present Ag in MHC class II complexes. They sustain the activation of Ag-specific CD4+ cells initially activated by resident DC (13). For most vaccine adjuvants it is not known whether their activity is required at the site of injection or in the local

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draining lymph node. Naive T and B cells normally do not enter non-lymphoid areas, such as most injection sites, of the body efficiently. They rather circulate between secondary lymphoid organs, including the spleen and lymph nodes, via the blood and efferent lymphatics. Only memory and effector lymphocytes are able to penetrate non-lymphoid tissues during an inflammation (14, 15). Hence, the antigen has to reach the secondary lymphoid tissues in order to be recognized by adaptive immune cells. The geographical concept of immune reactivity (16) proposed that time, place, and dose of the antigen are critical factors for induction of adaptive T- and B-cell responses. It is supported by experimental observations of reduced immune responses early after surgical removal of the injection site (17) and in lymph node-deficient hosts (18). No immune response develops when the antigen is not able to reach the lymph node due to interruption of the afferent lymphatics (19–22). Also the fact that augmented immune responses were observed after repeated injection of minute amounts of poorly immunogenic antigens support this concept (23, 24). Obviously, motility and migration have a key role in many biological processes. However, despite the paramount importance of this class of vaccine adjuvants and their extensive application in existing vaccines, the mechanisms underlying the activity of presumed signal 1 facilitators remain a mystery. For prototype examples such as oil-based emulsions and aluminum-based adjuvants some aspects of its alchemy are described in the next section. 3.1. Oil-Based Emulsions

Already in 1968 Herbert attempted to mimic slow release of antigen by daily injections of tiny doses of ovalbumin in mice and noted an antibody production profile similar to a single full dose of ovalbumin formulated in a W/O emulsion (24). Interestingly, the antibody level in the circulation dropped soon after the daily injections were stopped. Freund studied the role of the depot function in rabbits by measuring antibody formation after surgical removal of the vaccine from the site of injection. Excisions performed between 30 min and 4 h after injection resulted in a decrease of the immune response when compared to the response in animals in which the injected areas were not removed. Remarkably, excisions performed after one or more days did not seem to affect the antibody response (17, 25). Hence, as yet there is no conclusive evidence for slow (i.e., more than 1 day) release as explanation for immune stimulating features of Freund’s adjuvants. Despite such investigations, little is known about the mechanisms responsible for adjuvant activity of the W/O emulsion in general. The structural requirements as well as the cellular and molecular immunological mechanisms within the host remain poorly

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understood. Indeed, this extremely powerful immune activating formulation has been shrouded in obscurity and is often referred as an example of “the immunologist’s dirty little secret.” As local reactions and residues represent major safety concerns for vaccines based on W/O emulsions, there is a clear demand for alternatives. Understanding the mechanism underlying W/O emulsion activity may help to further improve their formulation, thereby diminishing or eliminating potential hazards. Alternatively, such insights will be highly useful in developing novel vehicles in general. Dupuis and coworkers (26) showed that DCs internalize vaccine antigen and labeled adjuvant MF-59, an oil-in-water emulsion, after intramuscular injection. How DCs know where to go is likely based on chemokine-based attraction, preceded by recruitment of granulocytes and monocytes/macrophages as shown recently (27). In general, these cells are rapidly recruited into sites of tissue injury in response to inoculation with live or inactivated microorganisms, probably as a result of locally produced chemotactic factors. The transient influx of neutrophils (PMNs) or other innate immune cells (27) likely affects DC function. Upon arrival in the lymph nodes DC’s antigen capture and processing capacity declines, while their immunostimulatory function is up-regulated. 3.2. Aluminum-Based Adjuvants

Aluminum-containing adjuvants such as aluminum hydroxide and aluminum phosphate adjuvants (often referred to as alum) are generally assumed to adsorb the Ag on the alum particle which forms an Ag depot at the injection site. This is believed to prolong availability of Ag (signal 1) for Ag-presenting cells (APCs). However, this concept has been challenged in recent times. Various mechanisms have been proposed to be responsible for its adjuvant effect. Alum has been shown to fix complement (28), to cause granulomas (29), to recruit eosinophils and neutrophils (30), and induce the appearance in the spleen of IL-4secreting cells (31). Yet, how alum achieves these effects remains unknown. Interestingly, alum does not promote classical direct dendritic cell (DC) maturation (32), which questions whether its activity is mediated by pattern recognition receptors. Kool and coworkers showed that intraperitoneally injected alum triggers local inflammatory CD11b+ Ly6G– Ly6C+ F4/80int monocyte-type cells to differentiate into inflammatory dendritic cells, which is associated with the secretion of uric acid (33). Treatment with uricase reduced recruitment of antigen (OVA)-labeled monocytes in alum-immunized mice. However, effects on alum-driven antigenspecific antibody production were not measured. This concept is

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strongly challenged by earlier data showing that uric acid crystals augment CTL responses to co-injected antigen, which, by contrast, was not observed for aluminum hydroxide in the same animal experiments (34). Hence, uric acid polarizes the immune response toward a type-1 response-associated CTL reaction, while a Th2-type response is the hallmark of alum adjuvanted vaccines. The lack of proper CTL priming by alum represents its major deficiency as vaccine adjuvant. Both (35) and very recently (30) showed that alum is able to activate the NALP3 inflammasome in human peripheral blood mononuclear cells (PBMCs), and primary peritoneal macrophages of mice, respectively. However, such activation is dependent on priming of the cells with lipopolysaccharide (LPS), questioning whether inflammasome activation is involved during vaccination in the absence of a co-stimulatory LPS signal. By contrast, Sokoloska and coworkers showed in 2007 that alum-containing adjuvants directly stimulate the release of IL-1β and IL-18 via caspase-1 activation from mouse dendritic cells. Remarkably, in this study responses were noted without priming by LPS (36). In NALP3-deficient mice a partial (3- to 5fold) reduction of alum-induced antigen-specific antibody formation (IgE and IgG1 isotypes) was observed using OVA or human serum albumin (HAS) as antigens (30, 35). On the contrary, Franchi and Nunez showed that NALP3-deficient mice showed normal antibody responses when immunized in the context of alum adjuvant (37). However, of particular importance is a recent study (38) demonstrating that mice lacking MyD88, and therefore unable to respond to TLR signals as well as IL-1β and IL-18 (39), are still capable to generate normal antibody responses when augmented by repository vaccine adjuvants including aluminum hydroxide, Freund’s incomplete and complete adjuvant. Of importance is also the study of Pollock et al., who showed that endogenous IL-18 facilitates alum-induced IL-4 production, but effects on humoral immune responses such as OVA-specific immunoglobulin G1 (IgG1) and IgE production, the hallmark of alum’s adjuvant effects, remained unaffected in IL-18-deficient mice (40). Moreover, in an allergic asthma model, IL-1R1-deficient mice sensitized by OVA formulated in alum developed typical pulmonary Th2 responses, eosinophilic inflammation, antibody responses and CD4(+) T-cell priming in lymph nodes similar to normal mice (41). Together, these data do not support the recently proposed NALP3 pathway (30), nor the uric acid concept (33) for alum adjuvant activity. In addition, there is no evidence for IL-18 or IL-1β dependence.

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4. Signal 2 Facilitators 4.1. Signal Zero, PAMPs, and Stranger Motifs

The innate immune system of vertebrates uses germlineencoded pattern recognition receptors (PRRs) to sense invading pathogens. These PRRs are able to recognize so-called pathogenassociated molecular patterns (PAMPs), for example, peptidoglycan or unmethylated CpG DNA. PAMPs are unique and conserved molecular structures of a given microbial class (bacteria, viruses, fungi, and protozoa). Depending on their location these PRRs can be secreted receptors (e.g., pentraxins) found in blood and lymph associated with complement or opsonization, membrane receptors (e.g., C-type-like receptors, Toll-like receptors) on APC associated with endocytosis or induction of nuclear factorkB (NF-kB)- and mitogen-activated protein kinases (MAPKs)dependent signaling pathways or cytosolic receptors on APCs associated with induction of NF-KB and MAPK signaling pathways (42) (Fig. 1.2).

Fig. 1.2. Overview of major classes of pattern recognition receptors. MBL, mannose binding lectin; CRP, C-reactive protein; SR, scavenger receptor; CR, complement receptor; MR, mannose receptor; TREM, triggering receptor expressed on myeloid cells; TLR, Toll-like receptor; NOD, nucleotide oligomerization domain; NALP, Nacht, LRR and PYRIN domaincontaining proteins; RIG, retinoic acid-inducible gene; LDL, low-density protein, LPS, lipopolysaccharide, LTA, lipoteichoic acid, MDP, muramyl dipeptide, ss, single stranded. NBS- Nucleotide binding site, CARD - Caspax recruitment domain

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Among the PRRs, Toll-like receptors (TLRs) are the most well-studied and best-described class of receptors. They have been shown to play an essential role in the initial response of the innate immune system to infection. TLRs are type I transmembrane proteins with an extracellular domain of interspersed leucine-rich repeat (LRR) motifs that are involved in recognition of PAMPs. The cytoplasmic domain is characterized by a Toll/IL-1 receptor (TIR) motif which is involved in signal transduction (43). TLRs are either expressed on the plasma or endosomal membrane of APCs and they respond to specific bacterial, viral, fungal, and protozoan PAMPs. Recognition of PAMPs by TLRs results in recruitment of a set of TIR domain-containing adaptor proteins such as MyD88. These interactions trigger intracellular downstream cascades eventually leading to activation of nuclear factor-kB (NF-kB) and mitogen-activated protein kinases (MAPKs), which results in induction of inflammatory cytokines (e.g., interleukin-1β, tumor necrosis factor-α) and co-stimulatory ligands (B7-1 and B7-2). Importantly, TLRs not only trigger signal 2. They may also play a role in antigen presentation (signal 1). TLRs may control the generation of T-cell receptor (TCR) ligands from the phagosome guaranteeing the presentation of both microbial components and antigen to activated APCs (44). TLR engagement can initiate and also shape the adaptive immune response, for instance, by skewing the immune system toward a Th1 or Th2 response (45) or by activating or inhibiting Treg cells (46). Moreover, vaccine adjuvants that contain TLR ligands can induce higher avidity T-cell responses (47). This suggest the need for antigen and TLR agonist to be co-delivered, in order to target the same phagosome cargo of one APC and thereby induce optimal antigen presentation and subsequent stimulation of Ag-specific T-cell responses (6). Indeed, TLRs ability to link innate and adaptive immunity represents a promising mechanism to be explored in the design of new vaccines (5). However, antigen targeting and engulfment can also be mediated by other receptors present in the surface of APC, such as C-type lectin receptors (CLRs) and triggering receptors expressed on myeloid cells (TREMs). CLRs, including mannose receptor and DC-SIGN, recognize sugar moieties (e.g., N-acetylglucosamine, mannose) at the surface of the pathogens enabling binding to an array of bacteria, virus, and fungi. Intracellular cytosolic receptors such as NODs (nucleotide-binding oligomerization domain proteins), NOD-like receptors (NLRs), and retinoic acid-inducible gene I-like helicases (RLHs) recognize structures from intracellular bacteria or viruses, but possibly also aluminum-containing adjuvants (30) as mentioned earlier. These receptors may form new targets for vaccine adjuvant development.

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4.2. Danger Signals, Alarm Signals, or Damage-Associated Molecular Patterns (DAMPs)

According to the so-called danger theory, originally proposed by (48), the immune system evolved to focus primarily on danger rather than on microbial non-self signals described above (48–50). Accordingly, antigens can be divided into two major groups: those associated with danger and antigens positioned in a harmless situation. Danger signals can be released by stressed or damaged tissue, as organelles of cells undergoing necrotic (but not apoptotic) death. Although such organelles or molecules from dead cells are not well defined at the molecular level, likely candidates responsible for observed immune activation include mitochondrial and nuclear fractions of necrotic cells as well as heat-shock proteins (HSPs), cellular DNA, or uric acid (30). These may be referred to as danger-associated molecular patterns (DAMPs) which lead directly or indirectly to up-regulation of co-stimulatory signals for APCs. Together with antigens released from dying cells these danger signals are recognized by APCs which may trigger an immune response if the host is not tolerant for these antigens. Also endogenous cytokines, such as type I IFN produced by infected cells, can be considered a DAMP. Indeed, several adjuvants are known to cause local damage at the injection site and may act by induction of danger signals. Examples include aluminum hydroxide (30), saponins and possibly also oil-based emulsions (51). Theoretically, the danger model may explain, in part, some of the mystery of signal 1 facilitating adjuvants described earlier.

4.3. Recombinant Cytokines or Co-stimulatory Molecules Mimicking Endogenous Immune Amplifiers

Protection conferred by some vaccines, such as influenza subunit vaccine, is mainly achieved by induction of a humoral response. A humoral response is mediated by antibodies and their effector functions, which include neutralization and opsonization of pathogen and activation of the complement cascade. Nevertheless, to ensure that the pathogen is effectively eradicated, it is not only important to attain an adequate amount of antibody but also to generate a specific immunoglobulin isotype. Antibody isotypes differ in their ability to activate the complement cascade or the binding to receptors on phagocytes. Therefore, it is of great importance to use adjuvants which promote the synthesis of specific antibody isotypes which confer more protective activity (52). Synthesis of particular antibody isotypes is strongly influenced by the combination of locally produced cytokines. For example, interleukin-5 (IL-5) or transforming growth factor β can augment IgA antibody formation, while interleukin-4 (IL-4) and interleukin-13 (IL-13) are able to induce switching toward the IgE isotype, and interferon-γ (IFN-γ) increases the synthesis of IgG2a antibody (53). A number of cytokines represent the hallmarks of a polarized immune reaction. For example, IFN-γ and IL-12 are associated with Th1-type immune reactions, while

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IL-4, IL-5, and IL-13 are related to Th2-type immune pathways. Use of such molecules in recombinant form may skew responses in the desired direction. Therefore, recombinant cytokines constitute serious adjuvant candidates that are especially suitable for subunit vaccines, which are poorly immunogenic when compared to whole killed or live-attenuated pathogens (54). In addition, ligands and receptors of co-stimulatory pathways represent an attractive target for the development of adjuvants. For instance, type I IFNs promote DC maturation by increasing expression of co-stimulatory molecules including CD40, CD80, and CD86 and major histocompatibility complex (MHC) antigen. Recombinant IFNs co-delivered with influenza vaccine have been shown to enhance protection against virus challenge (55). Also CD40 stimulation by agonistic antibodies exerts adjuvant effects (56). As a result, various co-stimulatory agonists are currently considered as an important new class of adjuvants (57). 4.4. Release of the Brakes

As mentioned earlier TLR agonists can constitute potent adjuvants for infectious diseases. However, it has been demonstrated that certain TLR agonists were able to promote the induction of IL-10-secreting Treg cells (58). A recent study revealed that TLR agonists can induce IL-12 and IL-10 production concomitantly and therefore promote the development of Th1 and importantly also Treg cells (59). So during a physiological immune response stimulating signals are accompanied by natural inhibitory signals. It is now recognized that CTLA-4 blockade (60), or depletion of Treg cell development, enhances the efficacy of therapeutic vaccination against tumors (61). Also, selective inhibitors of MAPKp38 in dendritic cell vaccines suppressed IL-10 and enhanced IL-12 production, thereby augmenting Th1 response and suppressing Treg cells (59). Hence, attenuation of regulatory Tcell induction by proper TLR agonist, or inhibition of natural immune response attenuating signals, may improve the efficacy of some vaccines and represents a variant of signal 2 facilitation (59).

5. Outlook Despite accumulated knowledge on the adjuvant mechanisms of signal 2 facilitators described above, we know very little about the classical adjuvants, which are presumed to facilitate signal 1. In particular we lack knowledge on the most upstream events, the earliest interactions between adjuvant and the tissue at the injection site. Future studies will certainly unravel these events and contribute to rational vaccine design.

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Chapter 2 Preclinical Development of AS04 Nathalie Garçon Abstract Recent knowledge on vaccine-induced immunity led to the development of vaccine Adjuvant Systems specially designed and adapted to vaccine needs. AS04 is such a tailored Adjuvant System developed by GlaxoSmithKline Biologicals. This chapter focuses on the methods that were used during the preclinical evaluation of AS04. AS04 consists of the combination of aluminum salts and 3 -O-deacylated monophosphoryl lipid A (MPL), a detoxified lipid A derivative with retained immunostimulatory capacity. MPL also induces considerably less pro-inflammatory cytokines, as compared to the parent LPS molecule. Preclinical evaluation of AS04 allowed the determination of the optimal size of MPL particles. The added value of MPL in AS04-based formulations was evidenced by higher vaccine-elicited antibody responses, as well as the induction of higher levels of memory B cells, as compared to aluminum alone formulations. Preclinical evaluation demonstrated the relevance of using AS04 in situations where high and long-lasting antibody levels are needed. This represents the basis for the successful application of AS04 in vaccines against hepatitis B virus and human papillomavirus. Key words: AS04, Adjuvant System, MPL, formulation, immune response, TLR-4, vaccine.

1. Introduction Over the last century, vaccines have demonstrated their potential in reducing mortality and morbidity due to infectious diseases, being at the same time one of the safest and most cost-saving products developed for health care. The subtle molecular mechanisms that are involved in the initiation of the body’s response to a vaccine are now better understood. Indeed, in the last 20 years, progress has been huge in understanding the immune response mechanisms, particularly the intricate relationship between innate and adaptive immunity. At G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_2, © Springer Science+Business Media, LLC 2010

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the center of the immune system, the antigen-presenting cells (APCs) represent the cell population that is able to recognize the type of pathogen and to distribute the work appropriately to the different immune effectors according to the pathogen involved. APCs stimulate the other immune factors after being activate through innate receptors on their membrane that specifically recognize pathogen molecules called pathogen-associated molecular patterns (PAMPs). The most studied of these innate receptors is the family of Toll-like receptors (TLRs) in which up to 10 members have been identified so far (1, 2). The impact of this growing knowledge on vaccinology is remarkable. Indeed, mostly to increase the safety profile, the last generation of vaccines is composed of protein subunits and recombinant antigens instead of whole pathogens. This evolution toward less toxicity, unfortunately, brought a decrease in immunogenicity for some antigens, as the stimulatory signals provided by several PAMPs present in the whole pathogen had been lost, and with them the ability to adequately stimulate APCs. To compensate such loss, it has become evident that an appropriate adjuvantation was required (3). The use of adjuvants is not new. They were first recognized in the 1920–1930s, when it was observed that increased antibody responses were induced by adding aluminum salts to a given antigen (4, 5). Such aluminum salts have been for 70 years the only adjuvant approved for human use, but their action might be too restricted for the new challenges that have appeared with the subunit vaccines and to respond to the still unmet medical needs. The recently acquired knowledge in immunology makes possible the use of TLRdependent or TLR-independent molecule combinations that are able to act at the APC level to specifically activate the desired arms of the immune system. With such a versatile approach, it is feasible to adapt the adjuvantation to the specific immunological needs required for a given pathogen and/or a given target population. GlaxoSmithKline Biologicals (GSK Bio) has been developing such tailor-made Adjuvant Systems (AS), destined to be combined with the right antigen(s) and intended to promote a fast, strong, and sustained immune response, supported by cellmediated immunity. One of these proprietary Adjuvant Systems, AS04, is now present in two registered vaccines, FENDrixTM , the hepatitis B vaccine destined to pre- and hemodialysis patients, and CervarixTM , the human papillomavirus (HPV-16/18) cervical cancer vaccine, the first vaccine with a new adjuvant to be registered in the US, and approved in 100 additional countries including Japan. The chapter below discusses the methods that were used during the preclinical evaluation of AS04, which allowed successful development of the adjuvant in vaccines.

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2. The AS04 Adjuvant System AS04 originates from the need to ensure better vaccination outcomes in subjects with challenging immunological conditions or challenging pathogens. AS04 is composed of an aluminum salt (aluminum hydroxide for CervarixTM or aluminum phosphate for FENDrixTM ) combined with a TLR-4-dependent immunostimulant, namely the 3 -O-deacylated monophosphoryl lipid A (MPL). The combination of both molecules results in an AS able to promote a strong and sustained protection through the induction of high and persistent antibody titers, concomitant with effective B- and T-cell memory responses (6, 7). 2.1. Aluminum Salts

As already mentioned, aluminum salts are the oldest and also most widely used vaccine adjuvants. They are principally recognized to enhance antibody responses and their mechanism of action, though not fully understood, has been partially unveiled (for review, see (3)). It had been long accepted that adsorption of the antigen onto aluminum and its slow release at the site of injection (depot effect) were key to the adjuvant effect until it was demonstrated that adsorption, and thus the depot effect, might not be the crucial mechanism for the induction of the immune response. Results in mice rather suggested that aluminum salts impact on the APC function, inducing a Th2-biased immune response characterized by the promotion of antibody production via IL-4 signaling.

2.2. Monophosphoryl Lipid A (MPL)

MPL is a purified, detoxified derivative of the lipopolysaccharide (LPS) that is found on the outer membrane of the R595 strain of Salmonella minnesota. Detoxification is carried out through successive acid and base treatments. After purification through a succession of chromatography steps, it yields a molecule with the same immunomodulatory properties but with considerably less toxicity than the parent LPS molecule (8, 9). LPS is a group of structurally related complex molecules composed of three covalently linked regions (1): the innermost region is lipid A, containing glucosamine disaccharide units that carry long chains of fatty acids (2), the central region is the core oligosaccharide, and (3) the outer region is represented by the Ospecific polysaccharide chains, containing repeated oligosaccharide units. MPL displays the same basic structure as the lipid A region of LPS. It is noteworthy that there is biosynthetic variability in the assembly of the lipid A moiety and loss of fatty acids from the lipid A backbone during the hydrolytic steps of the manufacturing process. The resulting MPL is a mixture of

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HO

HO

O

O

P HO

O

O O

O NH

A

OH

HO HO NH

B C Number of Fatty Acids 6 5

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A C14(OC14) C14(OC14) C14OH ΔC14 C14OH Δ-C 14 H H

Position B C14 (OC12) C14 (OC12) C14 (OC12) C14 (OC12) C14 (OC12) C14 (OC12) C14 (OC12) C14 (OC12)

C C14(OC16) C14OH C14(OC16) C14(OC16) C14OH C14OH C14(OC16) C14OH

C14(OC16) = 3-(R)-hexadecanoyloxytetradecanoyl C14(OC14) = 3-(R)-tetradecanoyloxytetradecanoyl C14(OC12) = 3-(R)-dodecanoyloxytetradecanoyl C14OH = 3-(R)-hydroxytetradecanoyl Δ-C14 = tetradecanoyl

Fig. 2.1. Major 3-O-deacyl monophosphoryl lipid A congeners in MPL. Congener species all contain the same backbone consisting of a β -1 ,6-linked disaccharide of 2-deoxy-2aminoglucose, phosphorylated at the 4 position, but contain variable numbers and types of fatty acyl groups at the 2, 2 , and 3 positions. The 1, 3, 4, and 6 positions of the backbone are unsubstituted in all monophosphoryl lipid A species present in MPL. The 2, 2 , and 3 positions may be substituted with tetradecanoic, 3-(R)-hydroxytetradecanoic, or 3-(R)-acyloxytetradecanoic acids, depending on the position, so that the total number of fatty acyl groups varies from three to six (previously published in (6)).

closely related 3 -O-deacylated monophosphoryl lipid A species, called congeners (Fig. 2.1), and is the one referred to in the GSK Bio Adjuvant Systems. The composition of the mixture is always the same in reproducible manufacturing conditions and may vary when a different process is used. The resulting monophosphoryl lipid A then may display different properties. The Gram-negative bacterial LPS are PAMPs, specifically recognized by TLR-4 (10, 11), a receptor at the surface of the tissueresident macrophages and of immature APCs. Activation by LPS elicits a cascade of signals ultimately resulting in an immune response that is appropriate to combat the infection. MPL also acts through TLR-4 binding, leading to an activation pattern which is similar to that induced by LPS. 2.3. Formulation

Due to its hydrophobic nature, MPL is present in aqueous solution as a particulate structure and can be characterized by its particle size. It is available as a lyophilized triethylamine salt (TEA MPL) and the lyophilized powder is resuspended in water before it is processed to reach a particle size that allows sterile filtration,

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as measured by photon correlation light scattering. MPL bulk preparations are stable over time in terms of both chemical composition and physical properties. For vaccine preparation, MPL is adsorbed on preformed aluminum salt. MPL can be characterized by chromatographic analysis when present in aqueous solution or after desorption from the aluminum salts. If desorption is not possible because of the high binding affinity, a destructive method, such as gas chromatography–mass spectrometry, must be used to quantify MPL. As for any substance to be injected in humans, sterility and pyrogenicity need to be tested. Due to its nature, pyrogenicity of MPL cannot be evaluated with the classical limulus amebocyte lysate (LAL) test (12, 13) but only with the rabbit pyrogenicity assay (14).

3. The Preclinical Development of AS04 3.1. Immunological Evaluation of MPL

As previously mentioned, MPL exists as particles of different size in aqueous solutions. Therefore, besides studies on the optimal concentration, the impact of MPL particle size on the immune response was evaluated. Groups of Balb/c mice (n = 10) were immunized at days 0 and 15 by the intra-footpad route with 70 μL of the vaccine formulation (1/7 of the human dose), equivalent to 10 μL of aluminum hydroxide, 0.4 μg of hepatitis B surface antigen (HBsAg), and different amounts of MPL (Fig. 2.2). At day 7 after the second immunization, blood samples were taken and serum anti-HBsAg IgG2a antibody levels were measured by ELISA. These experiments in mice established that both parameters have an impact on the humoral response (Fig. 2.2). In the conditions of the experiment, small size MPL (100 nm) showed a typical dose–response curve, while larger MPL particles (500 nm) induced a bell-shaped humoral response curve. The results obtained with small MPL particles were confirmed in two open, randomized clinical trials in healthy adults that compared the immunogenicity of four different formulations. In these trials, hepatitis B vaccines, containing different concentrations of MPL (200 nm in size), 500 μg of aluminum salts, and 20 μg of HBsAg were administered to healthy adults (18–40 years), according to a 0 and 6 month immunization schedule. As negative control, the same formulation without MPL was used in a three-dose regimen (0, 1, and 6 months). One month after the last immunization, specific antibody levels were measured in serum by ELISA. The effect observed was dose related and a plateau was reached at 50 μg of MPL per human

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anti-HBsAg antibodies (EU/mL)

4000 100 nm MPL 500 nm MPL

3500 3000 2500 2000 1500 1000 500 0 0

0.8

3.1 12.5 MPL (μg)

50

Fig. 2.2. Effect of MPL particle size and concentration on mouse IgG2a antibody response to hepatitis B surface antigen (HBsAg). Mice (n = 10 per group) were vaccinated with combinations of HBsAg/aluminum with MPL particles of two different sizes (100 or 500 nm) at concentrations ranging from 0 to 50 μg per vaccine dose. At day 7 after the second injection, serum samples were assayed for anti-HBsAg IgG2a antibodies by ELISA. Results are expressed as ELISA Units/mL (previously published in (6)).

dose (Fig. 2.3). Based on these preclinical results, manufacturability, and dose ranging in humans, the AS04 formulation selected for vaccination of adults is 50 μg per vaccine dose of MPL particles with a size allowing sterile filtration. The beneficial effects of AS04 on the immune responses, and particularly the added value of MPL (in AS04) as compared with aluminum alone, were established in animal models. Groups of mice were immunized at days 0 and 21 with the combination of both HPV-16 and HPV-18 L1 virus-like particles adjuvanted with aluminum salt alone or with aluminum salt supplemented with MPL (AS04). At days 14 and 37 after the second immunization, blood samples were taken and anti-HPV-16 and anti-HPV18 antibody levels were determined by ELISA. These experiments showed that the presence of MPL in the formulation greatly enhances the antibody levels elicited by vaccination (Fig. 2.4). 3.2. Safety Studies

Safety assessment is an integrated part of adjuvant development. To date, guidelines have been issued by the European authorities (15) and WHO (16) to regulate testing of vaccine or new adjuvant safety. Different safety aspects have been evaluated in several animal species. Altogether, the various preclinical safety evaluations did not indicate any adverse effect or systemic toxicity of MPL and/or AS04. Only transient inflammatory markers were observed locally, coherent with the use of an immunomodulator.

Preclinical Development of AS04

21

20000

anti-HBsAg antibodies (mIU/mL)

18000 16000 14000 12000 10000 8000 6000 4000 2000 0 12.5

25

50 MPL (μg)

100

no MPL

Fig. 2.3. Effect of MPL concentration on human antibody response to hepatitis B surface antigen (HBsAg). Healthy human adults (18–40 years) were vaccinated with combinations of HBsAg/aluminum with MPL particles of 200 nm at concentrations ranging from 12.5 to 100 μg per vaccine dose. At day 30 after the second injection, serum samples were assayed for anti-HBsAg antibodies by ELISA. Results are expressed as ELISA Units/mL (previously published in (6)).

10

× 105

14

anti-VLP16

× 105

anti-VLP18

GMT antibody titers (EU/mL)

GMT antibody titers (EU/mL)

12 8

6

4

2

10 8 6 4 2

0

0 Al(OH)3

AS04

Al(OH)3 14d post II

AS04

37d post II

Fig. 2.4. Antibody response in mice after immunization with adjuvanted HPV-16/18 L1 VLP vaccine. Mice (n = 12 per group) were vaccinated with the combination of HPV-16 and HPV-18 L1 VLPs adjuvanted with MPL adsorbed to aluminum salt (AS04) or with aluminum salt alone. After two intra-muscular injections (0 and 21 days), serum samples collected 14and 37-day post II were assayed for anti-HPV-16 or HPV-18 L1 VLP antibody response by ELISA. Results are expressed as ELISA Units/mL (GMT ± 95% CI) (previously published in (7)).

Garçon

The mode of action of an adjuvant needs to be established to the best of the current knowledge. Together with preclinical safety evaluation, insights into the mode of action allow the design of clinical trials and the safety follow-up during subsequent vaccine development. Despite detoxification, MPL has been shown to retain the capacity to act as an immunostimulant, like the original LPS molecule. The reduced endotoxicity of MPL has been related to its lower aptitude to induce pro-inflammatory cytokines, such as TNF-α, and to its favorable effect on IL-10 synthesis. TNF-α expression in response to MPL was assessed on human CD14+ cell population (Fig. 2.5a). Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were cultured for 6 h in the presence of MPL (10 μg/mL) or LPS (0.1 μg/mL). The frequency of TNF-α-producing CD14+ cells was measured by the combination of intracellular staining and flow cytometry analysis. CD14 is a surface marker associated with TLR-4, essentially found on macrophages, and TNF-α is a pro-inflammatory cytokine. Fixed and permeabilized PBMCs were incubated with anti-CD14 and anti-TNF-α antibodies. This allowed the determination of the percentage of TNF-α-producing CD14+ cells, which is representative of the pro-inflammatory potency of the immunomodulator. Alternatively, TNF-α production was also measured in the U937 bioassay (Fig. 2.5b). For that, a human 50

16000

A

B

14000 40

cytokine production (pg/mL)

3.3. Functional Properties

cytokine producing cells (%)

22

30

20

10

12000 10000 8000 6000 4000 2000

0

control LPS

MPL

control LPS

MPL

Fig. 2.5. Comparative analysis of the production of the pro-inflammatory cytokine TNF-α upon stimulation by LPS or MPL. (A) PBMCs were purified from healthy human adults and cultured in the presence of LPS or MPL. Flow cytometry associated with intracellular staining was used to determine the frequency (%) of TNF-α-producing CD14+ cells (macrophages). (B) Human U937 cells, differentiated into macrophage-like cells, were cultured in the presence of LPS or MPL. TNF-α levels in culture supernatants were measured by ELISA and expressed in picograms per milliliter (previously published in (6)).

Preclinical Development of AS04

23

U937 monocytic cell line (ATCC, CRL-1597.2) was cultured in 10% FCS–RPMI medium (5 × 105 cells/mL) in 24-well culture plates and was differentiated into macrophage-like cells in the presence of phorbol myristate acetate (PMA, 30 ng/mL) for 72 h. After differentiation, the cells were washed with medium, resuspended in the presence of MPL (10 μg/mL) or LPS (0.1 μg/mL), and incubated for 4 h. Quantitation of TNF-α was carried out with ELISA. The intensity of the pro-inflammatory response was approximately 15 times lower with MPL than with the parent molecule. Since MPL is adsorbed onto aluminum salts in the adjuvant formulation, it was necessary to explore whether aluminumadsorbed MPL maintains its ability to act as a TLR-4 agonist, like free MPL. U937 bioassay was used, in conjunction with ELISA. U937 cells were cultured in 10% FCS–RPMI medium (5 × 105 cells /mL) in 24-well culture plates and were differentiated into macrophage-like cells in the presence of PMA (30 ng/mL) for 72 h. After differentiation, the cells were washed with medium and resuspended in the presence of various doses of aluminum hydroxide, MPL, or both (AS04) for 6 h (Fig. 2.6). After incubation, supernatants of the culture plate wells were collected and the concentration of TNF-α determined by ELISA. The results were expressed as relative potency, using an internal 6

Relative potency

5

4

3

2

1

0 10 _

30 _ 100 _

_

_

_

1

3

10

10 1

30 100 Al(OH)3 3 10 MPL

Fig. 2.6. Aluminum-coupled MPL displays the same potency for inducing TNF-α as free MPL. U937 cells were incubated and differentiated into macrophage-like cells in the presence of various doses of aluminum alone, MPL alone, or AS04 (MPL coupled onto aluminum). TNF-α in culture supernatants was measured by ELISA, and results were expressed as relative potency compared to an internal MPL reference (previously published in (6)).

24

Garçon

MPL reference (10 μg/mL) as comparator. It was observed that aluminum-adsorbed MPL displays the same potency for inducing TNF-α than free MPL, which indicates that adsorption of MPL onto aluminum does not impact on its immunological properties.

4. The Added Value of AS04 in the Clinic

The selection of AS04 as adjuvant for the HPV-16/18 cervical cancer vaccine was based on clinical studies in which a higher antibody response was consistently observed against the vaccine antigens formulated with AS04, compared with aluminum hydroxide or non-adjuvanted formulations. The induction of higher antibody levels by AS04 reflected the agonistic TLR-4 activity of MPL, in addition to the antibody-inducing effect of aluminum, highlighting the benefit of using combination of molecules in an Adjuvant System. In these clinical studies, volunteers received three intramuscular administration of HPV-16 and HPV-18 L1 VLP antigens formulated with either AS04 (aluminum hydroxide + MPL) or aluminum hydroxide alone. Blood samples were taken at different time points and the levels of anti-HPV-16 and antiHPV-18 antibodies were measured by ELISA. Briefly, microwell titer plates were coated with either purified recombinant L1 VLP-16 (210 ng/100 μL) or L1 VLP-18 (270 ng/100 μL) overnight at 4◦ C. After blocking of the active sites, serial dilutions of the human sera were added to the wells, followed, after washing, by peroxidase-conjugated goat anti-human IgG. Bound IgG were revealed by addition of tetramethylbenzidine, a peroxidase substrate, which causes a colorimetric reaction; this reaction was stopped with sulfuric acid and quantified by optical density measurements at 450 and 620 nm (Fig. 2.7). Vaccination must not only elicit immediate high titers of antibody but also afford protection over the long term. Persistent serum antibody levels after vaccination reflect the generation of antigen-specific memory B cells. In this respect, the added value of MPL in eliciting memory B cells was investigated on the same panel of volunteers as above. The quantification of antigenspecific memory B cells was performed with a memory B-cell ELISPOT, adapted from the assay developed by the Lanzavecchia laboratory (17). Briefly, PBMCs were purified from blood samples and plated into culture plates in the presence of CpG (3 μg/mL), which induces the pool of memory B cells to differentiate into antibody-producing plasma cells. Among the pool, HPV-16- and HPV-18-specific plasma cells were detected by plating the cultures onto HPV-16 or HPV-18 pre-coated 96-well filter plates (ELISPOT plates). In wells containing anti-HPV-16

Preclinical Development of AS04

GMT antibody titers (EU/ml)

10000

6000

anti-HPV16

*

anti-HPV18 *

5000

8000 Al(OH)3 AS04

6000

25

4000

Al(OH)3 AS04

*

3000 4000

*

*

2000 *

2000

*

0

8

*

*

*

*

*

0

*

1000 0

16

24

32

Time (months)

40

48

0

8

16

24

32

40

48

Time (months)

Fig. 2.7. AS04 Adjuvant System induces a higher and longer lasting antibody response to HPV-16/18 L1 VLP antigens in humans. In two separate clinical trials, human subjects were vaccinated with HPV-16 and HPV-18 L1 VLPs adjuvanted with AS04 or with aluminum salt alone. Antibody responses against HPV-16 or HPV-18 L1 VLPs were evaluated by ELISA at several time points and expressed as geometric mean titers (GMT) in ELISA Units/mL. Significant differences (p < 0.05) between the antibody titers of the AS04 and the aluminum salt group are indicated by asterisks (n = 9–19 subjects for the aluminum salt group, n = 21–37 subjects for the AS04 group). Arrows indicate vaccination time points (previously published in (7)).

or anti-HPV-18-producing plasma cells, the antibodies directly bound the coated antigens. After washing the cells, bound antibodies are locally detected with a biotinylated mouse anti-human IgG, followed by peroxidase-labeled avidin and the addition of a peroxidase substrate to obtain a colored spot. Quantification of the number of spots occurred in an automated ELISPOT plate reader. Results are expressed as the frequency or percentage of HPV-16- or HPV-18-specific antibody-producing plasma cells (reflecting the specific memory B cells) among the total IgGproducing plasma cell population in PBMC, as determined in parallel with a control ELISPOT assay for total IgG. The results (Fig. 2.8) show that AS04-adjuvanted antigens elicit much higher levels of memory B cells than when adjuvanted with aluminum hydroxide alone. This can be attributed to the direct effect of MPL on the APCs, which induces an adequate cascade stimulation to induce long-term immune responses.

5. Conclusions Over the last decades, GSK Bio has accumulated a unique experience in combining immunologically active molecules to develop the family of Adjuvant Systems, each member being specifically designed for a given antigen and a given population. AS04 is one of the Adjuvant Systems developed by GSK Bio. Preclinical investigations showed that the presence of AS04 in a vaccine greatly enhances the production of antibodies against the vaccine antigen, with no safety issue. AS04 also induces

Frequency of HPV16/18-specific memory B cells

26

Garçon

HPV16

16000

HPV18

3000 (13)*

(14) Q3

12000 2000 8000 (11)

1000 4000 (21) (6)

0

(8)

pre

Median

(22) (12)

(11)

(13)

(5)

Q1

(9)

0 day 60

day 210

pre

Al(OH)3

day 60

day 210

AS04

Fig. 2.8. Frequency of HPV-16- and HPV-18-specific memory B cells in humans. Subjects from two separate clinical trials were vaccinated with HPV-16 and HVP-18 L1 VLPs adjuvanted with AS04 or aluminum salt alone. Memory B-cell responses directed against HPV-16 or HPV-18 L1 VLPs were quantified by ELISPOT at two time points post vaccination. Results are represented as the frequency of HPV-16- or HPV-18-specific memory B cells per 106 PBMCs. The number of subjects is given in parenthesis. Asterisk represents significant difference between the aluminum and the AS04 group (p < 0.05) (previously published in (7)).

high levels of memory cells, which accounts for sustained production of specific antibodies. These characteristics are found particularly in CervarixTM , the GSK Bio HPV-16 and HPV18 AS04-adjuvanted cervical cancer vaccine, that has demonstrated long-term efficacy against HPV-16 and HPV-18 infection and associated cervical lesions (18, 19), but also in FENDrixTM , an AS04-adjuvanted hepatitis B vaccine destined to pre- and hemodialysis patients, able to induce high and persistent-specific antibody responses (20, 21). The use of AS04 in other candidate vaccines is currently under investigation.

Acknowledgments The author is thankful to Ulrike Krause and Pascal Cadot for their assistance in the preparation of the chapter. FENDrix and Cervarix are trade marks of the GlaxoSmithKline group of companies. References 1. Takeda, K., Akira, S. (2005) Toll-like receptors in innate immunity. Int Immunol 17(1), 1–14. 2. Beutler, B., Jiang, Z., Georgel, P., et al. (2006) Genetic analysis of host resistance:

Toll-like receptor signaling and immunity at large. Annu Rev Immunol 24, 353–389. 3. McKee, A. S., Munks, M. W., Marrack, P. (2007) How do adjuvants work? Impor-

Preclinical Development of AS04

4.

5. 6.

7.

8.

9.

10.

11.

12.

13.

tant considerations for new generation adjuvants. Immunity 27(5), 687–690. Glenny, A. T., Buttlem, G. A. H., Stevens, M. F. (1931) Rate of disappearance of diphtheria toxoid injected into rabbits and guinea pigs: toxoid precipitated with alum. J Pathol 34, 267–275. Ramon, G. (1926) Procédés pour accroître la production des antitoxines. Ann Inst Pasteur 40, 1–10. Garçon, N., Van Mechelen, M., Wettendorff, M. (2006) Development and evaluation of AS04, a novel and improved immunological adjuvant system containing MPL and aluminium salt, in (Schijns V., O’Hagan D., eds.) Immunopotentiators in Modern Vaccines. Elsevier Academic Press, London, pp 161–177. Giannini, S. L., Hanon, E., Moris, P., et al. (2006) Enhanced humoral and memory B cellular immunity using HPV16/18 L1 VLP vaccine formulated with the MPL/aluminium salt combination (AS04) compared to aluminium salt only. Vaccine 24(33–34), 5937–5949. Myers, K. R., Truchot, A. T., Word, J., Hudson, Y., Ulrich, J. T. (1990) A critical determinant of lipid A endotoxic activity, in (Nowotny A., Spitzer J. J., Ziegler E. J., eds.) Cellular and Molecular Aspects of Endotoxin Reactions. Elsevier Science Publishing Co., New York, pp 145–156. Johnson, D. A., Keegan, D. S., Sowell, C. G., et al. (1999) 3-O-Deacyl monophosphoryl lipid A derivatives: synthesis and immunostimulant activities. J Med Chem 42(22), 4640–4649. Hirschfeld, M., Ma, Y., Weis, J. H., Vogel, S. N., Weis, J. J. (2000) Cutting edge: repurification of lipopolysaccharide eliminates signaling through both human and murine Tolllike receptor 2. J Immunol 165(2), 618–622. Tapping, R. I., Akashi, S., Miyake, K., Godowski, P. J., Tobias, P. S. (2000) Toll-like receptor 4, but not Tolllike receptor 2, is a signaling receptor for Escherichia and Salmonella lipopolysaccharides. J Immunol 165(10), 5780–5787. Iwanaga, S., Morita, T., Harada, T., et al. (1978) Chromogenic substrates for horseshoe crab clotting enzyme. Its application for the assay of bacterial endotoxins. Haemostasis 7(2–3), 183–188. Nakamura, S., Morita, T., Iwanaga, S., Niwa, M., Takahashi, K. (1977) A sensi-

14.

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22.

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tive substrate for the clotting enzyme in horseshoe crab hemocytes. J Biochem 81(5), 1567–1569. European Pharmacopea. (1971). Test for pyrogens, Vol. 11. Published under the direction of the Council of Europe, Maisonneuve SA, Sainte-Ruffine, France, pp 58–60. The European Medicines Agency (EMEA). (2005) Committee for medicinal products for human use (CHMP). Guideline on adjuvants in vaccines for human use. (EMEA/CHMP/VEG/134716/2004) (http://www.emea.europa.eu/pdfs/human/ vwp/13471604en.pdf). World Health Organization. (2005) Guidelines on nonclinical evaluation of vaccines. WHO Technical Report Series No. 927, Annex 1, pp 31–63 (http://www.who.int/ biologicals/publications/trs/areas/vaccines/ nonclinical_evaluation/en/). Bernasconi, N. L., Traggiai, E., Lanzavecchia, A. (2002) Maintenance of serological memory by polyclonal activation of human memory B cells. Science 298(5601), 2199–2202. Harper, D. M., Franco, E. L., Wheeler, C., et al. (2004) Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial. Lancet 364(9447), 1757–1765. Harper, D., Gall, S., Naud, P., et al. (2008) Sustained immunogenicity and high efficacy against HPV-16/18 related cervical neoplasia: long-term follow up through 6.4 years in women vaccinated with CervarixTM (GSK’s HPV 16/18 AS04 candidate vaccine). Gynecol Oncol 109(1), 158–159. Kong, N. C. T., Beran, J., Kee, S. A., et al. (2008) A new adjuvant improves the immune response to hepatitis B vaccine in hemodialysis patients. Kidney Int 73(7), 856–862. Beran, J. (2008) Safety and immunogenicity of a new hepatitis B vaccine for the protection of patients with renal insufficiency including pre-haemodialysis and haemodialysis patients. Expert Opin Biol Ther 8(2), 235–247. Kool, M., Pétrilli, V., De Smedt, T., Rolaz, A., Hammad, H., van Nimwegen, M., Bergen, I. M., Castillo, R., Lambrecht, B. N., Tschopp, J. (2008) Cutting edge: Alum adjuvant stimulates inflammatory dendritic cells through activation of the NALP3 inflammasome. J Immunol 181(6), 3755–3759, Sep 15.

Chapter 3 Nonclinical Safety Assessment of Vaccines and Adjuvants Jayanthi J. Wolf, Catherine V. Kaplanski, and Jose A. Lebron Abstract To ensure the safe administration of vaccines to humans, vaccines (just like any new chemical entity) are evaluated in a series of nonclinical safety assessment studies that aim at identifying the potential toxicities associated with their administration. The nonclinical safety assessment of vaccines, however, is only part of a testing battery performed prior to human administration, which includes (1) the evaluation of the vaccine in efficacy and immunogenicity studies in animal models, (2) a quality control testing program, and (3) toxicology (nonclinical safety assessment) testing in relevant animal models. Although each of these evaluations plays a critical role in ensuring vaccine safety, the nonclinical safety assessment is the most relevant to the evaluation in human clinical trials, as it allows the identification of potential toxicities to be monitored in human trials, and in some cases, eliminates candidates that have unacceptable risks for human testing. This review summarizes the requirements for the nonclinical testing of vaccines and adjuvants needed in support of all phases of human clinical trials. Key words: Vaccine, safety, adjuvant, nonclinical safety assessment.

1. Introduction There is probably no other type of medicinal product that is responsible for saving lives to the same extent as vaccines, which have been credited for saving hundreds of millions of lives over the past 100 years. Despite all the therapeutic advances in medicine, vaccines remain an integral part of the diseasefighting arsenal by preventing and/or ameliorating the effects of a gamut of infectious diseases. Given the success of vaccines, many companies and government institutes are working on developing new vaccines. Unlike small molecule drugs, vaccines are one of the most diverse type of medicinal products including purified and recombinant proteins, polysaccharide preparations, G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_3, © Springer Science+Business Media, LLC 2010

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plasmid DNA, recombinant viruses, virus-like particles, living irradiated cells, synthetic peptides, and attenuated or live organisms—viruses, bacteria, or parasites. Vaccines are often formulated with adjuvants, which are substances that enhance the immune response induced by the vaccine antigens. Like vaccines, adjuvants also encompass a range of products including inorganic salts (e.g., aluminum-based adjuvants), oligonucleotides (e.g., CpG DNA sequences), oil emulsions (e.g., MF59), and R saponin-based mixtures (e.g., QS-21 and ISCOMATRIX ). More recently, vaccine administration is being evaluated using novel delivery devices, which include needleless injectors, electroporation, microneedles, and nanoparticles. However, regardless of the considerable variety in vaccine formulation and delivery methods, the nonclinical safety assessment of vaccines follows a common approach aimed at evaluating their potential for local and systemic toxicity. This review focuses on summarizing the regulatory expectations and types of toxicology studies available for the nonclinical safety assessment of vaccines and adjuvants, concluding with a discussion on how to interpret the toxicology data and determine its implications for human safety.

2. Regulatory Considerations Several regulatory guidelines have been published (Table 3.1) which provide information about the nonclinical safety assessment studies that need to be performed for new vaccines and adjuvants. Nonclinical safety assessment programs are usually designed on a case-by-case basis, since some vaccine types under development require special considerations. The addition of a novel adjuvant in vaccine formulations needs to be justified in preclinical studies in order to demonstrate that inclusion of the adjuvant will provide a benefit to immunogenicity and/or efficacy. Dose–response studies in animals are performed to select the vaccine antigen and adjuvant concentrations to be tested in subsequent nonclinical and clinical studies. Toxicology studies for vaccines with adjuvants are designed to evaluate the safety profile of the adjuvant and adjuvant/vaccine combination. The World Health Organization (WHO) (1) and European Medicines Agency (EMEA) (2) have published guidelines on the overall nonclinical safety evaluation of vaccines. The EMEA has published a comprehensive guideline for vaccine adjuvants which covers aspects of nonclinical and clinical testing for new adjuvants (3). In addition to these and other regulatory guidelines for vaccines (Table 3.1), nonclinical safety assessment studies need to be performed in compliance with Good Laboratory Practices (GLP) regulations as detailed in 21 CFR Part 58 (4).

Guideline on Adjuvants in Vaccines for Human Use (3) Note for Guidance on Pharmaceutical and Biological Aspects of Combined Vaccines (7) Note for Guidance on the Quality, Preclinical and Clinical Aspects of Gene Transfer Medicinal Products (8) Points to Consider on the Manufacture and Quality Control of Human Somatic Cell Therapy Medicinal Products (9)

Adjuvanted vaccines Combination vaccines Viral vector and DNA vaccines Cell-based vaccines

(continued)

Note for Guidance on Preclinical Pharmacological and Toxicological Testing of Vaccines (2)

All vaccines

European Medicines Agency (EMEA)

ICH S6: Preclinical Safety Evaluation of BiotechnologyDerived Pharmaceuticals (6)

Recombinant vaccines (although it is more relevant for other biologics)

Guidelines for Assuring the Quality and Nonclinical Safety Evaluation of DNA Vaccines (5)

DNA vaccines

International Conference on Harmonization (ICH)

WHO Guidelines on Nonclinical Evaluation of Vaccines (1)

All vaccines

World Health Organization (WHO)

Guideline

Vaccine class

Regulatory agency

Table 3.1 Guidelines for the nonclinical safety assessment of vaccines Nonclinical Safety Assessment of Vaccines and Adjuvants 31

Guidance for Industry. Considerations for Developmental Toxicity Studies for Preventative and Therapeutic Vaccines for Infectious Disease Indications (11) Guidance for Industry: Considerations for Plasmid DNA Vaccines for Infectious Disease Indications (12) Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy (13) Points to Consider in the Production and Testing of New Drugs and Biologicals Produced by Recombinant DNA Technology (14)

Vaccines for women of childbearing potential DNA vaccines Viral vector and cell-based vaccines Recombinant protein/peptide vaccines

United States Food and Drug Administration (FDA)

21 CFR Part 610: General Biological Product Standards (10)

All biological products

United States Code of Federal Regulations (US CFR)

Guideline

Vaccine class

Regulatory agency

Table 3.1 (continued)

32 Wolf, Kaplanski, and Lebron

Nonclinical Safety Assessment of Vaccines and Adjuvants

33

3. Toxicology Studies Performed for Vaccines 3.1. Selection of Animal Models

Unlike toxicology programs for new chemical entities which require two species, one rodent and one nonrodent, the toxicology program for vaccines typically use a single species which must be demonstrated to be a relevant animal model based on the immunogenicity or efficacy of the vaccine in the selected species. In general, vaccine toxicology programs use either rodents (rats or mice) or non-rodents (rabbits). Non-human primates are only used if there is no other option; for example, for a therapeutic vaccine, non-human primates might be the only species that has homology with the human antigen. The size of the treatment group in toxicology studies depends on the species that is chosen. Generally, 10 animals/gender/group/necropsy time points are used for studies with rodents, whereas fewer animals per group are used in studies with larger animals.

3.2. Immunogenicity Evaluation

The immunogenicity of the vaccine candidate is typically evaluated within the toxicology study, as recommended in the WHO guideline (1). The measure of the expected immune response allows a demonstration of the exposure to the vaccine and confirms the relevance of the animal model for evaluating the potential toxicity of the vaccine. In addition, this measure might help the toxicologist with data interpretation, particularly in correlating any observed toxic effects with the degree of the specific immune response to the vaccine. The evaluation of the immune response to the vaccine relies on immunoassays that are developed in order to measure the most relevant endpoint, i.e., antibody response or cellular immune response. For the measure of specific antibodies, standard ELISA formats are now frequently replaced by multiplex assays for multiple antigens vaccine candidates. The simultaneous evaluation of multiple antigen-specific antibody responses in a single serum R sample is achieved using technologies such as Luminex or elecR  trochemiluminescence detection (ECLA, e.g., MSD ) (15). When the candidate vaccine targets the cellular arm of the immune response, assays measuring cytokine-secreting antigenspecific T lymphocytes (such as γ-interferon ELIspot (16)) allow the evaluation of the most relevant endpoint. However, these assays are quite resource intensive, since peripheral blood mononuclear cells collected from animals administered the candidate vaccine need to be stimulated ex vivo with the corresponding peptide antigen(s) and the number of cytokine-secreting T lymphocytes is evaluated.

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3.3. Toxicology Studies

Vaccine toxicology studies evaluate the inherent toxicity of the vaccine formulation, which includes any adjuvants and excipients, in addition to evaluating any toxicity that might be due to the vaccine immune response. The types of toxicology studies that could be performed are listed below; however, since vaccine safety assessment programs are designed on a case-by-case basis not all of these studies will need to be performed. The vaccine formulation used in toxicology studies should be representative of the proposed clinical formulation; therefore, for adjuvanted vaccines the vaccine and adjuvant are tested together. The adjuvant alone can be included as a control group in these studies, but is usually not tested in separate studies unless it is a new synthetic adjuvant. In the latter case, the new adjuvant is regarded as a new chemical entity and requires single- and repeat-dose toxicity studies in two species (one rodent and one nonrodent), assessment of genotoxicity, and possibly other tests (3). More details on considerations for new synthetic adjuvants are provided in Section 3.4.

3.3.1. Single-Dose Toxicity Studies

The purpose of single-dose toxicity studies is to determine the acute effects of a vaccine by evaluating parameters such as mortality, clinical signs, body weight, and food consumption. Since single-dose evaluations can be included within a repeat-dose toxicity study, separate single-dose toxicity studies are typically not performed for vaccines intended for clinical use in a repeat-dose schedule.

3.3.2. Repeat-Dose Toxicity Studies

Repeat-dose toxicity studies evaluate the effects of repeated administrations of the vaccine in animals. The same route of administration as the clinical route is used in the animal study, and usually one more dose is administered in animals when compared with the number of doses in the clinical regimen (this is sometimes referred to as the “N+1” rule (17)). The full human dose of the vaccine needs to be tested, or when not possible due to formulation constraints, then the maximum feasible dose should be administered into the selected animal species. An adjuvant-alone control group or saline control group could be included in the study as a concurrent control. Antemortem parameters evaluated include mortality, physical signs, body weights, food consumption, ophthalmic examinations, urinalyses, hematology, serum biochemistry, coagulation, and immunogenicity assessments as described in Section 3.2. Necropsies are usually performed at two time points, a few days (e.g., 2–7 days) after the last vaccine dose (to determine the early effects after vaccine dosing) and 2–4 weeks after the last vaccine dose (to detect any delayed toxicity and determine whether any detected effects have resolved over time). Postmortem evaluations include gross examination of all major organs, organ weights for selected organs, and histopathology

Nonclinical Safety Assessment of Vaccines and Adjuvants

35

on a complete list of tissues (following the WHO vaccine guidance) (1). Histopathological examination is usually focused on the pivotal organs (brain, kidneys, liver, reproductive organs), immune organs (spleen, thymus, draining lymph nodes), and the site of vaccine administration (e.g., quadriceps at the injection site and skin over the quadriceps, if the vaccine is administered intramuscularly) (1). In cases where there are specific theoretical safety concerns, additional parameters may be included in the study. For example, potential pathogenic autoimmune responses against a particular tissue could be evaluated by more detailed immunohistochemistry. When using an oligonucleotide-based adjuvant, the development of anti-DNA or anti-RNA antibodies could be evaluated. Potential systemic inflammatory responses could be evaluated by examining inflammatory biomarkers such as serum IL-6. Treatment-related effects that are typically observed in vaccine repeat-dose toxicity studies include inflammation at the site of injection, enlargement and hyperplasia of lymph nodes draining the injection sites, increase in spleen weight, and hematological and serum biochemical changes (such as increases in white blood cells, increases in serum globulin, and decreases in serum albumin). These observations are regarded as the intended immunological and inflammatory responses to the vaccine and are not considered adverse effects unless unusually severe. For vaccines, in most cases the highest dose level tested (full human dose) is the No-Observed-Adverse-Effects-Level (NOAEL). However, with the addition of novel adjuvants that might cause severe reactogenicity, more than one vaccine/adjuvant dose levels could be tested in the toxicology study such that a NOAEL can be determined. 3.3.3. Local Tolerance Studies

The purpose of local tolerance studies is to evaluate potential irritation at the injection site both macroscopically and histopathologically. In the interest of reducing animal use, an assessment of local tolerance can be performed within the repeat-dose toxicity study.

3.3.4. Safety Pharmacology Studies

The purpose of safety pharmacology studies is to evaluate the potential for undesirable effects of a substance on physiological functions, particularly on the cardiovascular system, respiratory system, and central nervous system (18). Separate safety pharmacology studies are generally not performed for vaccines (17). Safety pharmacology evaluations, such as body temperature, electrocardiogram, and central nervous system evaluations, could be incorporated into the repeat-dose toxicity study (1), if needed.

3.3.5. Developmental and Reproductive Toxicity Studies

Developmental and reproductive toxicity studies are needed for vaccines that will be administered to women of childbearing potential, since these studies provide information on potential

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effects of the vaccine on fertility, fetal development, and postnatal development of the offspring (11). According to the US FDA guideline on vaccine developmental toxicity studies (11), for vaccines indicated for women of childbearing potential, subjects may be included in clinical trials without developmental toxicity studies, provided appropriate precautions are taken to avoid vaccination during pregnancy. Data from the developmental toxicity studies can then be supplied with the Biologics License Application submission. In the developmental toxicity study, female animals are immunized a few weeks before mating in order to ensure peak immune responses during the critical phases of pregnancy (e.g., organogenesis). Vaccine booster doses are then administered during gestation (embryo-fetal period) and lactation (postnatal period) to evaluate potential direct embryotoxic effects of the components of the vaccine formulation and to maintain an immune response throughout the remainder of gestation. If an adjuvant is included in the vaccine, an adjuvant-alone control group could also be included. 3.3.6. Genotoxicity Studies

According to the “WHO Guidelines on Nonclinical Evaluation of Vaccines” (1) and the EMEA “Note for Guidance on Preclinical Pharmacological and Toxicological Testing of Vaccines” (2), genotoxicity studies are generally not required for vaccines. For adjuvants of biological origin, genotoxicity studies might not be regarded as relevant (6). However, for synthetic adjuvants, which are considered to be new chemical entities, the standard tests that are used to assess the potential for gene mutation, chromosome aberrations, and primary DNA damage are needed (19).

3.3.7. Carcinogenicity Studies

According to the “WHO Guidelines on Nonclinical Evaluation of Vaccines” (1) and the EMEA “Note for Guidance on Preclinical Pharmacological and Toxicological Testing of Vaccines” (2), carcinogenicity studies are generally not required for vaccines. Carcinogenicity studies are also not needed for adjuvants. According to the EMEA “Guideline on Adjuvants in Vaccines for Human Use” (3), adjuvants are intended to be used only a few times with low dosages such that the risk of induction of tumors by these compounds is very small.

3.3.8. Other Toxicity Studies

Specialized toxicity studies are needed for certain types of vaccines. For example, virulence and neurovirulence studies may be needed for new live attenuated virus vaccines that have either a theoretical or an established potential for reversion of attenuation (20) or neurotropic activity (1). Note that the current vaccine strains of measles, mumps, and varicella viruses that have a good safety record should not require re-evaluation in the neurovirulence test(s) when there are minimal changes to seed lots or to manufacture (20). Biodistribution studies, and in some cases integration studies, are performed for nucleic acid and viral

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vector-based vaccines to determine the tissue distribution following administration and the potential for the vector to integrate into the host genome (5, 12). 3.4. Toxicology Studies Required for Adjuvants Alone

4. Evaluation of Toxicology Data and Risk Assessment

New chemically synthesized adjuvants are regarded as new chemical entities and require a separate toxicological testing program, in addition to their evaluation as part of the vaccine formulation in the tests described in Section 3.3. Separate tests required for novel chemical adjuvants include single- and/or repeat-dose toxicity studies in two species, one rodent and one nonrodent, unless a specific scientific rationale can be provided for testing in a single species (3). Since adjuvants may stimulate the immune system, tests for the induction of systemic hypersensitivity in appropriate models might be needed, although no test is really considered predictive. For synthetic chemical-based adjuvants, an assessment of genotoxicity is needed using the standard battery of tests (e.g., potential for gene mutation, chromosome aberrations, and primary DNA damage) (19). Pharmacokinetic studies (e.g., determining serum concentrations of antigens) are not required for vaccines (1, 2); however, these studies might help in further understanding the mechanism of action of the adjuvant. Additionally, an in vivo test for pyrogenicity might be needed, with the possibility of using alternative in vitro tests for fever-inducing substances if such tests are validated (3). The results from toxicology studies of the adjuvant alone could be included in a separate Drug Master File that is referenced in the Investigational New Drug (IND) application, which contains toxicology data for vaccine adjuvant–antigen combinations.

The goals of the nonclinical safety assessment program are to identify (1) a safe starting dose in the human clinical trials (2), potential toxicities and their reversibility, and (3) target organs of toxicity. The next step after the toxicological studies are completed is to interpret the results with these goals in mind and taking into consideration the intended indication, the target population (e.g., infants, adults), the margin-of-safety (MOS), and the limitations, if any, in extrapolating the results in animals to human safety. The intended indication plays a critical role in the risk/benefit evaluation and can be classified into two major categories— prophylactic and therapeutic. Prophylactic vaccines, for example, influenza, rotavirus, and hepatitis B vaccines, are designed to prevent disease. These types of vaccines are generally administered to healthy individuals, most of which are infants and

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children. In contrast, therapeutic vaccines, such as vaccines for cancer and Alzheimer’s disease, are designed to take advantage of the patient’s immune system to treat a disease already manifested in the patient. Thus, given that the targeted population of prophylactic and therapeutic vaccines are different (healthy vs. disease), and that tolerance for risk is directly proportional to the disease severity, the expectation is that prophylactic vaccines have minimal adverse effects and higher MOS, while some adverse effects might be tolerated with therapeutic vaccines, with smaller MOS. Generally, the safe starting human dose for vaccines corresponds to the NOAEL identified in the repeat-dose toxicity study. For vaccines, the MOS is usually expressed as the fold excess by body weight between humans and the animal species since vaccines are generally given to animals at a full human dose regardless of the animal body weight. For example, if a full human dose of a vaccine is administered into a 0.25 kg rat, and the vaccine is intended for adult individuals (assume an average body weight of 70 kg), then the MOS based on body weight (assuming the human dose evaluated in rats is the NOAEL) will be calculated as the ratio between the human weight and the rat weight since both will be receiving the full human dose; thus, 70 kg/0.25 kg = 280-fold MOS based on body weight. Another consideration when extrapolating toxicology results to potential human risk is the animal model used for the toxicology studies. For example, when vaccine adjuvants that are tolllike receptor 9 (TLR-9) agonists are evaluated in rodents, the adjuvant may cause toxicities resulting from an overstimulation of the immune system (exaggerated pharmacology). These toxicities may be more pronounced in rodents than in non-human primates or humans, which have a more limited distribution of TLR-9. Thus, the animal model used for the toxicology study needs to be taken into account when developing the overall vaccine risk assessment. Finally, note that the assignment of the risk/benefit ratio of the vaccine needs to be continuously refined as new data from the clinic and additional nonclinical studies becomes available.

5. Conclusion In this review we highlighted the regulatory expectations and types of toxicology studies available for the nonclinical safety assessment of vaccines and provided a discussion on the interpretation of the toxicology data and its implications for human safety. We also discussed how the nonclinical safety assessment strategy

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is impacted by the type of indication, and whether the vaccine uses novel adjuvants. Although use of these novel approaches promises to make available vaccines currently not available to prevent serious diseases such as HIV and malaria, increasing safety concerns on the use of these novel technologies have resulted in increased regulatory requirements for both the nonclinical and the clinical development aspects of vaccines. The ultimate goal of vaccine developers should be to develop safe and efficacious vaccines. Although the nonclinical safety assessment is an integral part toward achieving this goal, it is important to remember that ensuring the safety of vaccines is a multi-component approach that includes (1) the evaluation of vaccines in animal models of efficacy and immunogenicity, (2) a quality control testing program, (3) nonclinical safety assessment studies, and (4) clinical studies. This multi-component approach is what ultimately will support the marketing application and eventual licensure of the vaccine.

Acknowledgments The authors would like to thank Dr. Brian Ledwith and Dr. Tom Monticello for reviewing this chapter. References 1. WHO Guidelines on Nonclinical Evaluation of Vaccines. (2003) WHO/BS/03.1969. WHO, Geneva, Switzerland. 2. Note for Guidance on Preclinical Pharmacological and Toxicological Testing of Vaccines. (1997) EMEA. CPMP/SWP/465/95. 3. Guideline on Adjuvants in Vaccines for Human Use. (2005) EMEA. EMEA/CHMP/VEG/134716/2004. 4. Good Laboratory Practice Regulations. (2009) Code of Federal Regulations, Title 21, Part 58 (21 CFR 58). 5. Guidelines for Assuring the Quality and Nonclinical Safety Evaluation of DNA Vaccines (2005) WHO, Geneva, Switzerland. 6. ICH S6: Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals. (1997) International Conference on Harmonization. 7. Note for Guidance on Pharmaceutical and Biological Aspects of Combined Vaccines. (1998) EMEA. CPMP/BWP/477/98. 8. Note for Guidance on the Quality, Preclinical and Clinical Aspects of Gene Trans-

9.

10. 11.

12. 13. 14.

fer Medicinal Products. (2001) EMEA. CPMP/BWP/3088/99. Points to Consider on the Manufacture and Quality Control of Human Somatic Cell Therapy Medicinal Products. (2001) EMEA. CPMP/BWP/41450/98. General Biological Products Standards. (2009) Code of Federal Regulations, Title 21, Part 610 (21 CFR 610). Guidance for Industry. Considerations for Developmental Toxicity Studies for Preventative and Therapeutic Vaccines for Infectious Disease Indications. (2006) CBER, FDA. Guidance for Industry: Considerations for Plasmid DNA Vaccines for Infectious Disease Indications. (2007) CBER, FDA. Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy. (1998) CBER, FDA. Points to Consider in the Production and Testing of New Drugs and Biologicals Produced by Recombinant DNA Technology. (1985) CBER, FDA.

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15. Opalka, D., Lachman, C. E., MacMullen, S. A., et al. (2003) Simultaneous quantitation of antibodies to neutralizing epitopes on virus-like particles for human papillomavirus types 6, 11, 16, and 18 by a multiplexed luminex assay. Clin Diagn Lab Immunol 10, 108–115. 16. Casimiro, D. R., Tang, A., Perry, H. C., et al. (2002) Vaccine-induced immune responses in rodents and nonhuman primates by use of a humanized human immunodeficiency virus type 1 pol gene. J Virol 76, 185–194. 17. Gruber, M. F. (2003) Non-clinical safety assessment of vaccines, in CBER Counter Terrorism Workshop, Bethesda, MD.

18. ICH. (2000) Safety Pharmacology Studies for Human Pharmaceuticals. Topic S7A, Step 5, ICH Harmonized Tripartite Guideline. International Conference on Harmonization, Geneva, Switzerland. 19. ICH. (1997) S2B Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. International Conference on Harmonization, Geneva, Switzerland. 20. The Organisers, IABs. (2006) IABs scientific workshop on neurovirulence tests for live virus vaccines. Biologicals 34, 233–236.

Chapter 4 Aluminum Adjuvants: Preparation, Application, Dosage, and Formulation with Antigen Erik B. Lindblad and Niels E. Schønberg Abstract Important new knowledge about the effect of aluminum adjuvants on the immune response in terms of their impact on cytokine profiles, uptake by antigen-presenting cells (APC), and surface marker expression has been published in recent years. However, although the knowledge about these adjuvants is thus more comprehensive now than ever before, the user is often still confined to a more empirical approach when confronted with practical issues when it comes to the handling and use of these adjuvants. In this chapter we have given focus to the user’s perspective, discussing practicalities like dosage, temperature stability, relevant monographs, and preparation with antigen. Key words: Aluminum hydroxide gel, aluminum phosphate gel, monographs, dosage, autoclaving, freezing, adsorption.

1. Introduction Aluminum compounds have the longest history and by far the most comprehensive record of use as adjuvants in practical vaccination of both animals and humans. Both aluminum hydroxide (AlhydrogelTM ) and aluminum phosphate (Adju-PhosTM ) adjuvants are generally regarded as safe to use in human vaccines when used in accordance with current vaccination schedules (1–3). Vaccine preparations based on adsorption of the antigen onto a preformed aluminum hydroxide or aluminum phosphate adjuvant are referred to as aluminum-adsorbed vaccines (see Note 1). In practical vaccination the adsorption of antigen onto preformed aluminum hydroxide and aluminum phosphate gels has G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_4, © Springer Science+Business Media, LLC 2010

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facilitated standardization of vaccine production and has now almost completely substituted the original old technique where potassium alum was made to co-precipitate with the antigen by exposing the potassium alum + antigen mixture to alkaline conditions (4). The historical and more detailed theoretical background of the aluminum adjuvants have been left out, primarily as they are beyond the scope of this chapter which is meant to provide a more practical approach, but also since they have been reviewed extensively elsewhere (5–9). 1.1. Preparation of Aluminum Adjuvants

Aluminum hydroxide and aluminum phosphate adjuvants are generally prepared by exposing aqueous solutions of aluminum ions to alkaline conditions in a well-defined and controlled chemical environment. Various soluble aluminum salts can be used for the production of aluminum hydroxide, typically aluminum sulfate or chloride is used. However, the experimental conditions— temperature, concentration, and even the rate of addition of reagents—strongly influence the results (10, 11). Anions present at time of preparation may co-precipitate and change the characteristics away from those of “pure” aluminum hydroxide. One very important example of an anion having such influence is phosphate. Aluminum phosphate gel can be seen as an example of such a preparation where the soluble aluminum salts are exposed to alkaline conditions in the presence of sufficient amounts of phosphate ions.

1.2. Application of Aluminum Adjuvants

Aluminum adjuvants have been used in both experimental immunology (12, 13) and to raise murine monoclonals of the IgG1 isotype as well as polyclonal antisera for analytical purposes, immunoprecipitation assays, etc. In veterinary medicine aluminum adjuvants have been used in a large number of vaccine formulations against viral (14–18) and bacterial (19–22) diseases, as well as in attempts to make antiparasite vaccines (23–26). In human vaccination aluminum adjuvants have been primarily used in tetanus, diphtheria, pertussis, and poliomyelitis vaccines as part of standard child vaccination programs for more than 50 years in many countries, including combination vaccines. Later aluminum adjuvants were also introduced in hepatitis A, hepatitis B virus vaccines, and in the new vaccines against human papillomavirus (HPV)-induced cervical cancer. Other aluminumadsorbed vaccines, against, e.g., anthrax and botulinus toxin, are available for special risk groups. Aluminum phosphate has a potential of being applied also in DNA vaccines with promising results, whereas aluminum hydroxide has been shown to inhibit the transcription of the nucleotide in DNA vaccines. The content of phosphate in the DNA molecule

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apparently gives a high binding affinity of the nucleotide to the aluminum hydroxide, which in turn prevents the host RNA from getting access to and translating the nucleotides into protein (27). One obvious limitation for the application of aluminum adjuvants lies in the clear Th2 profile of these adjuvants with adsorbed protein vaccines. A Th2-biased immune response is not likely to protect against diseases for which Th1 immunity and MHC class I-restricted CTLs are essential for protection, such as with, e.g., intracellular parasites or tuberculosis (28). Another limitation lies in the fact that traditional aluminum-adsorbed vaccines are frost sensitive and therefore not lyophilizable. 1.3. Dosing Aluminum Adjuvants

Although there are no generally accepted limits for the dosages of aluminum adjuvants to be used in experimental immunology, many countries have established local animal ethics committees that have to approve the individual experimental protocol before commencing the experiments. For the use of aluminum adjuvants (and other adjuvants as well) to raise polyclonal antibodies for, e.g., analytical and diagnostic purposes ECVAM (a generally recognized European body engaged in animal ethics) established a set of guidelines in 1999 (29). In the preclinical phase of vaccine development the optimum dose of adjuvant is normally determined empirically in a pilot trial, but helpful guidelines are available in the literature. In veterinary vaccines there is no defined maximum limit for the allowed content of aluminum adjuvants. Here the dose is normally set from a balance between efficacy and local reactogenicity. In vaccines for humans the allowed amount of aluminum in adsorbed vaccines is subject to limitations. These limits are 1.25 mg aluminum per dose in Europe (30) and in USA 0.85 mg aluminum per dose if determined by assay, 1.14 mg if determined by calculation, and 1.25 mg if safety and efficacy data justifies it (31). These limit values refer to aluminum calculated as metallic aluminum and not as the salt.

1.4. Temperature Stability of Aluminum Adjuvants

In practical terms there are two major considerations to be observed here. The first is that aluminum adjuvants are sensitive to freezing. Aluminum hydroxide and phosphate adjuvants are suspensions of hydrated colloid particles (aluminum oxyhydroxide and aluminum hydroxyphosphate, respectively) with a slow sedimentation in water. The slow sedimentation is partly due to the oriented water molecules that are an intrinsic constituent of the adjuvants (giving buoyancy), and partly because the adjuvant particles have a charge that gives electrical repulsion among the particles in the suspension and to some extent prevents packing. If the adjuvant is subjected to freezing then the free supernatant water as well as the oriented water molecules will eventually

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freeze, and upon thawing, the water molecules formerly oriented in the gel structure itself will not take up their former position and as a result the gel structure is irreversibly damaged (Fig. 4.1). Aluminum adjuvants thus damaged by freezing lose their protein adsorption capacity (Fig. 4.2). At the other end of the temperature scale autoclaving is commonly applied to achieve sterility of the adjuvant preparations. It is a well-established observation that autoclaving leads to a slight

Fig. 4.1. Two vials of aluminum hydroxide gel adjuvant. The vial to the left has not been exposed to freezing. The vial to the right has been frozen and subsequently thawed. The gel structure in this vial is collapsed and the colloid water released into the supernatant.

30,00 25,00

mg HSA/mL

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20,00 15,00 10,00 5,00 0,00 0% destruction

25% destruction

75% 50% destruction destruction

100% destruction

Fig. 4.2. Reduction of protein adsorption capacity (HSA onto aluminum hydroxide gel) after controlled partial and complete freezing of the adjuvant prior to incubation with antigen.

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reduction of protein adsorption as well as a minor reduction of pH in non-buffered adjuvant preparations. Burrell and coworkers investigated the effects of autoclaving on aluminum hydroxide and aluminum phosphate. They found that autoclaving aluminum hydroxide increased the degree of crystallinity as measured by the width at half-length of the major band in the X-ray diffractogram. It was confirmed that the pH decreased during autoclaving, suggesting that deprotonation and dehydration reactions resulted in a reduced surface area as both the protein adsorption and the viscosity decreased following autoclaving. With aluminum phosphate adjuvant the amorphous structure remained after autoclaving; however, also in this case deprotonation and dehydration reactions occurred as evidenced by a decrease in pH. In addition the protein adsorption capacity, rate of acid neutralization at pH 2.5, and PZC also decreased, indicating that the reactions resulted in a decreased surface area (32). 1.5. Relevant Monographs

A number of pharmacopeias and handbooks contain monographs on aluminum hydroxide and aluminum phosphate. However, these may apply to preparations intended for oral intake as antacids, and not for parenteral use as adjuvants. Examples of such monographs are the USP monograph on aluminum hydroxide gel and the Ph. Eur. monographs on hydrated aluminum phosphate no. 1598 and 2166. Such monographs have focus on acid neutralizing capacity and may encompass the option of adding flavors like peppermint oil or eucalyptus to make the taste better. In addition, there is little focus on the sterility of the preparation. Handbook of Pharmaceutical Excipients contain monographs on both aluminum hydroxide and aluminum phosphate for use in vaccines (33, 34). A monograph on aluminum hydroxide specifically for use in adsorbed vaccines was launched by the European Pharmacopoeia in July 2004 (35).

1.6. Formulation with Antigen

As a general guideline for many protein antigens adsorption is best accomplished in the pH interval between the isoelectric point (IEP) of the protein antigen and the point of zero charge (PZC) of the adjuvant, which is the equivalence of the IEP, but for the adjuvant (Fig. 4.3). This applies for both aluminum hydroxide and aluminum phosphate adjuvants. In this interval the adjuvant and the antigen will have opposite electrical charges, facilitating electrostatic attraction and adsorption (36). In the formulation with antigen a pH close to the physiological pH is preferred, i.e., pH 6–8, to reduce vaccination discomfort. Applying this principle aluminum hydroxide is the preferred adjuvant for adsorption of antigens with an acidic IEP and aluminum phosphate is the preferred adjuvant for adsorption of antigens with an alkaline IEP (see Note 2).

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Fig. 4.3. In the pH range between the isoelectric point (IEP) of the antigen and the point of zero charge (PZC) of the mineral adjuvant there is a basis for electrostatic attraction, due to opposite charge. The alkaline PZC for Al(OH)3 makes it suitable for adsorption of acidic IEP proteins as exemplified by albumin, whereas the acidic PZC of AlPO4 makes it suitable for adsorption of alkaline IEP proteins, as exemplified by hen egg lysozyme.

Antigen preparations that contain fragments of phospholipid membranes or phosphorylated proteins may in addition adsorb to aluminum hydroxide in particular by an alternative mechanism, known as ligand exchange (37). However, the same mechanism may reduce adsorption by electrostatic attraction if anions with affinity for ligand exchange with hydroxyl on the surface of aluminum hydroxide, in particular phosphate ions, are used in the formulation (e.g., in buffers) (38). In general incubation for 1–2 h at room temperature or over night at 4◦ C for both aluminum hydroxide and aluminum phosphate adjuvant is recommended to allow adsorption to take place.

2. Materials 2.1. Production of Aluminum Adjuvants 2.1.1. Aluminum Hydroxide ad modus Gupta and Rost

1. 5× aluminum chloride/sodium acetate solution: 0.257 M aluminum chloride and 0.05 M sodium acetate (62.05 g aluminum chloride and 6.8 g of sodium acetate per liter of distilled water). Sterilize by autoclaving or by filtration (0.2 μm) (see Note 3). The pH of the 5× concentrate is between 3 and 4. Store at room temperature (20–30◦ C) (39). 2. 5× sodium hydroxide solution: 0.257 M sodium hydroxide (10.25 g of sodium hydroxide per liter of distilled water). Sterilize by autoclaving or filter sterilization (see Note 3).

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The pH of the 5× concentrate is between 12 and 14. Store at room temperature (20–30◦ C). 3. Acetic acid (sterilized). 2.1.2. Method for Preparation of Aluminum Phosphate Adjuvant ad modus WHO (40)

1. Sterile distilled water. 2. K1 filter sheets. 3. Aluminum chloride solution: dissolve 3 kg of pure aluminum chloride (AlCl3 ·6H2 O) in distilled water to a final volume of 30 L. The solution is then filtered through a soft (K1) filter sheet. 4. Trisodium phosphate: dissolve 4.73 kg of pure trisodium phosphate dodecahydrate (Na3 PO4 ·12H2 O) in distilled water to a final volume of 30 L. The solution is then filtered through K1 sheets. 5. 0.36% sodium chloride (NaCl) in water (100 L).

2.2. Testing of Aluminum Adjuvants

1. Bunsen burner.

2.2.1. Determination of Aluminum

3. pH meter with combined pH electrode.

2. Precision balance, d = 1 mg. 4. Titriplex solution, pH 6.0: 37.224 g Titriplex III (ethylenediaminetetraacetic acid, analysis quality GR) in H2 O ad 1,000 mL (0.100 M final). Adjust to pH 6.0 using sodium acetate powder. 5. Xylenol orange potassium nitrate mixture: grind together 1 g xylenol orange tetrasodium salt and 99 g KNO3 in a mortar until a fine powder with a homogenous color is obtained. (KNO3 is added to improve the stability.) 7. Zinc sulfate solution (0.100 M): 28.754 g zinc sulfate heptahydrate (ZnSO4 ·7H2 O) analysis quality GR in H2 O ad 1 L. 8. Saturated sodium acetate solution: sodium acetate trihydrate (CH3 COONa·3H2 O) analysis quality GR in water. 9. 32% w/v sodium hydroxide (NaOH) solution: 320 g NaOH in Aqua purificata ad 1,000 mL. 10. Concentrated hydrochloric acid (HCl 37%). 11. Aluminum titrisol standard solution: dilute the contents of one vial of standard liquid [aluminum chloride (AlCl3 ) in H2 O, 1.000 g Al ± 0.002 g (Merck) which contains 1.000 g Al] to 100 mL with Aqua purificata. This solution then contains 10 mg Al per milliliter and has a density of 1.039 g/mL.

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2.2.2. Determination of the Protein Adsorption Capacity

1. Test-tubes: Immuno Tube MiniSorp 12 mL (Biotech Line) 2. Orbit rotator 3. Desiccator with silica gel 4. Bench centrifuge 5. Double beam UV–VIS spectrophotometer 6. Imidazole buffer, pH 6.5: 3.4 g imidazole, 2.2 g NaCl, and 18 mL 1 M HCl in water ad 1,000 mL. Filter through a 0.45 μm filter (Sartorius) and store in a refrigerator 7. HSA standard solution: 200 mg HSA (ICN or Sigma) in 100 mL imidazole buffer 8. BCA Protein Assay Reagent A, 250 mL (Pierce) 9. BCA Protein Assay Reagent B, 25 mL (Pierce) 10. HSA stock solution: 2.0 g HSA (ICN or Sigma) in 200 mL imidazole buffer

3. Methods 3.1. Production of Aluminum Adjuvants

3.1.1. Aluminum Hydroxide ad modus Gupta and Rost

Whereas the production details of the commercially available aluminum adjuvants are subject to confidentiality, a recipe for the production of aluminum hydroxide adjuvant in laboratory scale has been published by Gupta and Rost (39). A method for preparing aluminum phosphate adjuvant has previously been published by WHO (40). 1. Stir the contents continuously during the procedure at 40–60 rpm (39). 2. First add 0.257 M NaOH solution (20% of final volume) to a mixing vessel. 3. Add sterile distilled water (50–55% of final volume). 4. Add 0.257 M aluminum chloride/sodium acetate (AlCl3 /CH3 COONa) solution (20% of final volume) to the mixture at a rate of 1–2 L/min. During the addition of this solution, monitor pH and maintain it between 5.5 and 6.5 (optimal pH for tetanus and diphtheria toxoids) or any other range suitable for a particular antigen. 5. Adjust to the final volume with sterile distilled water. Mix the suspension for 2 h. 6. Adjust the final pH to 5.9–6.1 (for tetanus and diphtheria toxoids) or to the optimal pH with 5 N NaOH or 5 N acetic acid.

Aluminum Adjuvants

3.1.2. Method for Preparation of Aluminum Phosphate Adjuvant ad modus WHO (40)

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1. Filter 150 L of distilled water through K1 sheets into a container of 300 L capacity. Slowly add the 30 L of AlCl3 solution and mix well. 2. Add the Na3 PO4 solution with good stirring until a pH of 5.0 is reached (about 27–29 L are necessary). 3. Finally, add 30 L of distilled water, mix the suspension well, and leave standing for 7 days. 4. After 7 days siphon off the clear supernatant and add the same volume of sterile distilled water. Mix the suspension well and again leave standing for 7 days. 5. After 7 days again siphon off the clear supernatant, add distilled water to bring the volume to 150 L, and then add 100 L of 0.36% NaCl. The total volume is now 250 L. 6. Distribute the resulting suspension of AlPO4 with continuous mixing into 6 L volumes in 10 L bottles steamed for 30 min and autoclaved for 40 min at 121◦ C. 7. After 2 or 3 days test the sterility of the phosphate suspension. 8. This procedure yields bottles each containing 6 L of AlPO4 suspension with 6 mg of aluminum phosphate per milliliter or 1.32 mg of aluminum per milliliter (see Note 4).

3.2. Testing of Aluminum Adjuvants

This section presents two of the most important tests that are recommended prior to using preformed aluminum adjuvants in vaccine production. Commercial producers of aluminum adjuvants supply a Certificate of Analysis (CoA) with each batch supplied. It is highly recommended that the end user verifies the analytical data of the CoA in their own incoming material control. In addition, the end user should undertake testing for additional critical parameters that may apply to their specific, intended use of the adjuvant, i.e., vaccine-specific requirements (see Note 5). Below we describe the necessary tests for (1) determination of the aluminum concentration in the product (important for the dosing into the vaccine) and (2) a method for quantitative determination of the protein adsorption capacity (in the context of standardization it may be preferable over the pass–not pass limit test of the Ph. Eur. monograph 1664). In the example presented, the test is based on the adsorption of human serum albumin onto aluminum hydroxide, but the principle can be utilized with other proteins as well, including purified toxoids.

3.2.1. Determination of Aluminum

The aluminum content in aluminum hydroxide gel can be determined by titration of Titriplex–EDTA. The test is based on the principles given in USP and European Pharmacopoeia test 2.5.11 (complexometric titrations). The test is carried out in duplicates.

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The result of the test will be given as the mean of the two duplicates. 1. Determination of volume B by measuring precisely 50 mL 0.100 M Titriplex solution, pH 6.0, and adding a small amount of xylenol orange potassium nitrate mixture to produce a clear yellowish brown color. Titrate with 0.100 M ZnSO4 until the yellowish brown changes to pink. The amount (mL) of 0.100 M ZnSO4 used is B. 2. Prepare quantity A by shaking the product bottle well and take a sample of 6 g of the product if you analyze aluminum hydroxide adjuvant (Alhydrogel) or 10 g of the product if you analyze aluminum phosphate adjuvant (Adju-Phos). The exact amount is noted. This amount is hereafter called A. 3. Determine quantity b by transferring the 6 g if analyzing Alhydrogel or 10 g if analyzing Adju-Phos (quantity A) of the product from step 2 above into a 250 mL beaker. As a reference use 6 g of the Titrisol standard solution. Add 50 mL H2 O and 5 mL 32% w/v NaOH solution. In case there should be any Al-containing product on the sides of the beaker, rinse with as little water as necessary. The Adju-Phos should now be a clear solution. Heat Alhydrogel using a Bunsen burner until the solution is clear. Allow the solution to cool down to room temperature and then neutralize the solution by drop-wise addition of concentrated HCl. During this neutralization a precipitate of aluminum oxide forms. Re-dissolve this precipitate by further dropwise addition of concentrated HCl until pH = 1.0. Now add precisely 50 mL of 0.100 M Titriplex solution, pH 6.0, heat until boiling, and then keep warm using a pilot light for approximately 10 min. Allow the solution to cool down to room temperature and adjust the pH to 6.0 using saturated sodium acetate solution. Add a little amount of xylenol orange-KNO3 (to produce a clear yellowish brown color, very little is required). Titrate with 0.100 M ZnSO4 until the yellowish brown color changes to pink. The amount (mL) of 0.1 M ZnSO4 used is b. 4. Calculate mg/mL Al = “C” using the following formula: C = (B − b) × d × M × 27 A where B and b are defined above in the text d = density = approx. 1.03 g/mL for Alhydrogel 2.0%, approx. 1.01 g/mL for Adju-Phos 1.039 g/mL for the aluminum standard solution. M = the molarity of ZnSO4

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27 = the molecular weight of Al A is defined above in the text. 3.2.2. Determination of the Protein Adsorption Capacity (see Notes 6–8)

Determination of the protein adsorption capacity of the adjuvant is highly recommended and can be measured by a variety of analytical methods (see Notes 7 and 8). It is normally done by comparing the protein content in the aqueous phase of the antigen solution before and after adsorption onto the adjuvant. We use a test based on a modified BCA (bicinchoninic acid) assay. The BCA method was developed in 1985 by Smith et al. in USA (41) and seems in some respect to be superior to other methods for protein determination with respect to sensitivity as well as user friendliness. It is based on the principle that copper ions will, through interaction with peptide bonds, pass from the Cu++ state to the Cu+ state and that Cu+ has a high binding affinity to BCA. The complex will then have a light absorption maximum at 562 nm, i.e., this complex may be used for a photometric protein determination. This test principle has been utilized in the present method, adapted by Lindblad for determining the protein adsorption capacity of Alhydrogel. When the test is carried out, the protein solution should be at room temperature, just as all other applied reagents. All reagents have to be used within 2 days. Prepare a standard curve for every lot of HSA. 1. Plot a standard curve for HSA by preparing the samples of different protein concentration as shown in Table 4.1. For each HSA concentration 0.2 mL is transferred into a test tube. Prepare two tubes with “blanks,” referred to as Sample 0.

Table 4.1 Preparation of the standard row of HSA in imidazol buffer Name

Concentration (mg/mL)

HSA standard solution (mL)

Imidazole buffer (mL)

0 (blank)

0.0

0

10

1

0.2

1

9

2

0.4

2

8

3

0.6

3

7

4

0.8

4

6

5

1.0

5

5

6

1.2

6

4

7

1.4

7

3

8

1.6

8

2

9

1.8

9

1

10

2.0

10

0

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Lindblad and Schønberg

2. Prepare a working solution (W.S.) by mixing 50 mL of Reagent A with 1 mL of Reagent B. Mix thoroughly. Add 4.0 mL W.S. to each tube and mix thoroughly. Incubate samples for 2 h at room temperature. 3. Measure the absorbance at 562 nm for the standards against a blank sample and plot absorbance against HSA concentration. 4. Using the HSA stock solution (2.0 g HSA in 200 mL imidazole buffer) prepare nine samples in 10 mL test tubes [0 (i.e., blank), 1, 2, . . . 8] as shown in Table 4.2.

Table 4.2 For Alhydrogel 2% Preparation of Alhydrogel with HSA prior to adsorption HSA stock solution (mL)

Imidazole buffer (mL)

0

0.00

7.00

1

26

2.60

4.40

2

30

3.00

4.00

3

34

3.40

3.60

4

38

3.80

3.20

5

42

4.20

2.80

6

46

4.60

2.40

7

50

5.00

2.00

8

54

5.40

1.60

Name 0 (blank)

Amount of HSA added (mg)

5. Shake the Alhydrogel flask thoroughly and add 2.0 mL of the Alhydrogel to each of the test tubes and attach them to the wheel of the blood rotator. Let the Alhydrogel adsorb the HSA for at least 1 h by rotating at room temperature. 6. After adsorption, transfer the tubes to the bench centrifuge and spin down by centrifugation for 10 min at 2,550 × g. For each sample 0.2 mL is transferred to a test tube. Avoid transferring any sediment (i.e., fine fractions of Alhydrogel), as this may influence the readings (see Note 9). 7. Prepare a working solution (W.S.) by mixing thoroughly four parts of reagent B with 200 parts of reagent A. For the testing of each batch of Alhydrogel use approximately 40 mL of W.S. 8. To each sample tube, add 4.0 mL W.S. and mix thoroughly. Incubate samples for 2 h at room temperature. 9. Measure the absorbance of the samples at 562 nm. As reference use the imidazole buffer.

Aluminum Adjuvants

53

Fig. 4.4. Determining the protein adsorption capacity of aluminum adjuvant (exemplified by adsorption of HSA onto aluminum hydroxide gel). The crossing point of the graph through the measurements with free HSA in the supernatant with the X-axis gives the adsorption capacity for the 2 mL sample taken into analysis. The result is divided by 2 to state mg HSA adsorbed per milliliter Alhydrogel.

10. Calculate the binding capacity of that particular batch (mg HSA adsorbed per milliliter Alhydrogel). The following is an example calculation of the case where the standard curve shows that a sample after adsorption, centrifuging, and filtering has a HSA concentration of 0.225 mg HSA per milliliter. The total volume was 9 mL as the tube from which the sample was taken contained 7 mL sample and 2 mL Alhydrogel. The total amount of free protein after adsorption was thus 0.225 mg × 9 = 2.03 mg. The total amount of protein before adsorption in the same test tube was 38.00 mg. The difference, equivalent to the amount

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adsorbed, is thus (38.00 – 2.03) mg = 36 mg protein, and since it was adsorbed on 2.0 mL Alhydrogel, it corresponds in this single test to an adsorption capacity of 36 divided by 2 (equivalent to 18 mg HSA per milliliter Alhydrogel). This calculation should be made for each of the adsorption samples (see Note 10). The calculated values for free HSA in the supernatant (Y-axis) is plotted against the amount of HSA added to the sample (X-axis) (Fig. 4.4). The adsorption capacity for the 2 mL Alhydrogel added is given by the intercept between a line through the calculated values for free HSA in the supernatant and the X-axis.

4. Notes 1. In the literature the word “alum” is sometimes used to describe both aluminum hydroxide and aluminum phosphate gels, but that is incorrect use of terminology. Potassium alum, KAl(SO4 )2 ·12H2 O, is in accordance with the chemical definition of an alum, whereas neither aluminum hydroxide nor aluminum phosphate is. Although both aluminum hydroxide and aluminum phosphate adjuvants are chemically referred to as Al(OH)3 and AlPO4 , respectively, neither of these formulas should be seen as stoichiometrically true. As a consequence any calculation that takes basis in the formulas mentioned will only be an approximation. 2. Antigens with a distinct polarity in terms of one part of the molecule having a clearly acidic IEP and a distant part of the same molecule having a clearly alkaline IEP may bind well to both, e.g., Al(OH)3 and AlPO4 . But in such cases there may be a difference in the orientation of the adsorbed molecule (42). 3. If filtration is used, test the compatibility of filter membranes with the solution. 4. Cleaning of equipment: aluminum adjuvants may leave a white deposit on stainless steel and aluminum surfaces in tanks and holding vessels upon prolonged exposure or drying. Such deposits are not easy to rinse away with water alone, but can be removed exposing the surfaces to 4–5% NaOH at 80◦ C. 5. The Ph. Eur. monograph 1664 contains a requirement for a Limulus Amoebocyte Lysate (LAL) testing of the aluminum hydroxide adjuvant. The LAL test can be performed either as a clot assay or as a chromogenic assay.

Aluminum Adjuvants

55

Although these assays have a long history of use for the purpose neither of the two procedures are particularly well suited for the testing of aluminum hydroxide, due to some intrinsic characteristics of the aluminum adjuvant. The binding affinity of LPS to aluminum hydroxide is well established and was much higher than to aluminum phosphate (43). With the clot assay, the problem is that aluminum hydroxide acts as an adsorbant, meaning that the clotting of reagents, which, in applications where there is no adsorbant present, is specific for the LAL–endotoxin interaction, cannot be deemed as a specific reaction in this particular application. With the chromogenic assay, problems also arise due to the adsorption of endotoxin by the aluminum hydroxide. This adsorption has been measured to be in the magnitude of 283 μg/mL Al (43). Lipid A is typically composed of a glucosamine (GlcN) disaccharide backbone [β-D-glucosaminyl-(1-6)-α-D-glucosamine disaccharide] which carries two ester-bound phosphate groups at positions 1 and 4 , and amide or ester-linked long fatty acids at positions 2, 2 , 3, and 3 (44, 45). The adsorption of endotoxin by aluminum hydroxide gel is due to two mechanisms, which are a direct consequence of the structure of the endotoxin molecule: At physiological pH aluminum hydroxide adjuvant is positively charged, whereas endotoxin typically is negatively charged at pH conditions higher than 2. As a result there is attraction between the positively charged aluminum hydroxide and the negatively charged endotoxin. As described above, the LPS contains phosphate residue that can displace surface hydroxyl from the aluminum hydroxide gel. This process is known as ligand exchange, and the affinity is very high indeed. The impact is profound when, for example, preparing a standard curve by making a dilution series of LPS from, e.g., E. coli. If aluminum hydroxide is present LPS will be taken up by the adjuvant and as a consequence will not be freely accessible in the supernatant, i.e., before the adjuvant is saturated. If aluminum hydroxide is excluded from the LPS dilutions used for the standard curve, good standard curves can be prepared, but cannot be correlated with aluminum hydroxide-containing samples. LPS cannot be determined by “spiking” a sample of aluminum hydroxide with a controlled amount of LPS and then correlating it to the standard curve from the LAL assay. 6. It has been observed that acute toxicity is reduced in adsorbed vaccines as compared to the non-adsorbed antigen preparation. It is conceivable that the acute toxicity is reduced simply by a delayed release of toxic vaccine

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constituents, like pertussis toxin, peptidoglycans from Gram-negative cell walls, or LPS (lipopolysaccharides) from the injection site. 7. ELISA methods have been designed (46) in which aluminum-adsorbed antigens could be used directly as antigens in ELISA assays. ELISA methods were also applied for in vitro assessment of various viral antigens, i.e., pseudorabies, porcine parvovirus, and infectious bovine rhinotracheitis vaccines adsorbed onto aluminum hydroxide adjuvant (47). 8. In a complex mixture of antigens some individual components may adsorb to the adjuvant to a larger extent than others, meaning that what is actually adsorbed is not reflecting the composition of the original complex solution quantitatively. The reason for this could be either the presence of phosphorylated amino acids or differences in the isoelectric point of some of the components. An HPLC chromatogram can be run on the complex antigen mixture prior to adsorption and compared with the bands of a HPLC run on the supernatant of the same mixture after adsorption. Unadsorbed components will retain their peak pattern, whereas missing peaks or reduced height of peaks are indicative of complete or partial protein adsorption. Similarly, if an antiserum raised against the complex antigen mixture is available, a crossed, two-dimensional immunoelectrophoresis before and after adsorption may reveal if single components from the complex solution of proteins remain unadsorbed. In this case it is based on the precipitation band pattern from the electrophoresis (48). 9. The fine fraction of particles of aluminum adjuvants may give lead to light scatter that interferes with photometrical readings. For that reason it is recommended to pass the supernatants through a 0.22 μm nitrocellulose sterility filter after harvest and before further processing to retain such particles. 10. This way of calculating gives an approximate value. The reason for this is that Alhydrogel is a suspension of hydrated particles in water. After centrifugation the 2 mL of Alhydrogel is packed at the bottom of the test tube together with the adsorbed protein. Hence, it is only an approximation to state that concentration of HSA measured in the free supernatant relates to the 9 mL.

Aluminum Adjuvants

57

References 1. Immunological Adjuvants. (1976) Technical Report Series 595 World Health Organization, Geneva. 2. Edelman, R. (1980) Vaccine adjuvants. Rev Infect Dis 2(3), 370–383. 3. Goldenthal, K. L., Cavagnaro, J. A., Alving, C., Vogel, F. R. (1993) NCVDG working groups: safety evaluation of vaccine adjuvants. AIDS Res Hum Retrovirus 9(suppl. 1), s47–s51. 4. Volk, V. K., Bunney, W. E. (1942) Diphtheria immunization with fluid toxoid and alumprecipitated toxoid. Am J Pub Health 32, 690–699. 5. Hem, S. L., White, J. L. (1984) Characterization of aluminum hydroxide for use as an adjuvant in parenteral vaccines. J Parent Sci Tech 38(1), 2–11. 6. Hem, S. L., HogenEsch, H. (2007) Aluminum-containing adjuvants: properties, formulation and use, in (Singh, M., ed.), Vaccine Adjuvants and Delivery Systems. John Wiley & Sons, Hoboken, NJ, pp 81–114. 7. Lindblad, E. B. (2004) Aluminum compounds for use in vaccines. Immunol Cell Biol 82, 497–505. 8. Lindblad, E. B. (2004) Aluminum adjuvants – in retrospect and prospect. Vaccine 22, 3658–3668. 9. Lindblad, E. B. (2006) Mineral adjuvants, in (Schijns, V. and O’Hagan, D. eds.), Immunopotentiators in Modern Vaccines. Elsevier Science Publishers, Burlington, MA, pp 217–233. 10. Willstätter, R., Kraut, H. (1923) Über ein Tonerde-Gel von der Formel Al(OH)3 II: mitteilung über Hydrate und Hydrogele. Ber Dtsch Chem Ges 56, 1117–1121. 11. Willstätter, R., Kraut, H. (1924) Über die Hydroxide und ihre Hydrate in den verschiedenen Tonerde-Gelen. V: mitteilung über Hydrate und Hydrogele. Ber Dtsch Chem Ges 57, 1082–1091. 12. Uede, T., Huff, T. F., Ishizaka, K. (1982) Formation of IgE binding factors by rat T lymphocytes. V. Effect of adjuvant for the priming immunization on the nature of IgE binding factors formed by antigenic stimulation. J Immunol 129(4), 1384–1390. 13. Uede, T., Ishizaka, K. (1982) Formation of IgE binding factors by rat T lymphocytes. VI. Cellular mechanisms for the formation of IgE-potentiating factor and IgEsuppressive factor by antigenic stimulation of antigen primed spleen cells. J Immunol 129(4), 1391–1397.

14. McDougall, J. S. (1969) Avian infectious bronchitis: the protection afforded by an inactivated virus vaccine. Vet Rec 85, 378–380. 15. Wilson, J. H. G., Hermann-Dekkers, W. M., Leemans-Dessy, S., de Meijer, J. W. (1977) Experiments with an inactivated hepatitis leptospirosis vaccine in vaccination programmes for dogs. Vet Rec 100, 552–554. 16. Sellers, R. F., Herniman, K. A. J. (1974) Early protection of pigs against foot-andmouth disease. Br Vet J 130, 440–445. 17. Hyslop, N. S. G., Morrow, A. W. (1969) The influence of aluminium hydroxide content, dose volume and the inclusion of saponin on the efficacy of inactivated foot-and-mouth disease vaccines. Res Vet Sci 10(2), 109–120. 18. Pini, A., Danskin, D., Coackley, W. (1965) Comparative evaluation of the potency of beta-propiolactone inactivated newcastle disease vaccines prepared from a lentogenic and a velogenic strain. Vet Rec 77(5), 127–129. 19. Thorley, C. M., Egerton, J. R. (1981) Comparison of alum-adsorbed or non-alumadsorbed oil emulsion vaccines containing, either pilate or non-pilate bacteroides nodosus cells in inducing and maintaining resistance of sheep to experimental foot rot. Res Vet Sci 30, 32–37. 20. McCandlish, I. A. P., Thompson, H., Wright, N. G. (1978) Vaccination against canine bordetellosis using an aluminium hydroxide adjuvant vaccine. Res Vet Sci 25, 51–57. 21. Nagy, L. K., Penn, C. W. (1974) Protective antigens in bovine pasteurellosis. Dev Biol Stand 26, 65–76. 22. Ris, D. R., Hamel, K. L. (1979) Leptospira interrogans serovar pomona vaccines with different adjuvants in cattle. NZ Vet J 27, 169–171. 23. Leland, S. E., Sofield, W. L., Minocha, H. C. (1988) Immunogenic effects of culturederived exoantigens of Cooperia punctata on calves before and after challenge exposure with infective larvae. Am J Vet Res 49(3), 366–379. 24. Monroy, F. G., Adams, J. H., Dobson, C., Bast, I. J. (1989) Nematospiroides dubius: influence of adjuvants on immunity in mice vaccinated with antigens isolated by affinity chromatography from adult worms. Exp Parasitol 68(1), 67–73. 25. Carlow, C. K. S., Bianco, A. E. (1987) Resistance of Onchocerca lienalis microfilariae in mice conferred by egg antigens of homologous and heterologous onchocerca species. Parasitology 94(3), 485–496.

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26. Gamble, H. R., Murrell, K. D., Marti, H. P. (1986) Inoculation of pigs against Trichinella spiralis using larval excretorysecretory antigens. Am J Vet Res 47(11), 2396–2399. 27. Kwissa, M., Lindblad, E. B., Schirmbeck, R., Reimann, J. (2003) Co-delivery of a DNA vaccine and a protein vaccine with aluminum phosphate stimulates a potent and multivalent immune response. J Mol Med 81(8), 502–510. 28. Lindblad, E. B., Elhay, M. J., Silva, R., Appelberg, R., Andersen, P. (1997) Adjuvant modulation of immune responses to tuberculosis subunit vaccines. Infect Immun 65(2), 623– 629. 29. Leenaars, P. P. A. M., Hendriksen, C. F. M., de Leeuw, W. A., Carat, F., Delahaut, P., Fischer, R., Halder, M., Hanley, W. C., Hartinger, J., Hau, J., Lindblad, E. B., Nicklas, W., Outschoorn, I. M., Stewart-Tull, D. E. S. (1999) The production of polyclonal antibodies in laboratory animals: the report and recommendations of ECVAM/FELASA Workshop 35. ATLA 27, 70–102. 30. Ph. Eur. 6.ed. (2009) Vaccines for human use pp. 3971. 31. Code of Federal Regulations. (2009) 21, vol. 7: sec. 610.15 (Constituent Materials), Revised April 1. 32. Burrell, L. S., Lindblad, E. B., White, J. L., Hem, S. L. (1999) Stability of aluminumcontaining adjuvants to autoclaving. Vaccine 17, 2599–2603. 33. Hem, S. L., Klepak, P., Lindblad, E. B. (2009) Monograph: aluminum hydroxide adjuvant, in Handbook of Pharmaceutical Excipients. Pharmaceutical Press Ltd., London. 34. Hem, S. L., Klepak, P., Lindblad, E. B. (2009) Monograph: aluminum phosphate adjuvant, in Handbook of Pharmaceutical Excipients. Pharmaceutical Press Ltd., London. 35. European Pharmacopeia. (2004) Monograph 1664: Aluminium hydroxide, hydrated, for adsorption (Aluminii hydroxicum hydricum ad adsorptionem). 36. Seeber, S. J., White, J. L., Hem, S. L. (1991) Predicting the adsorption of proteins by aluminium-containing adjuvants. Vaccine 9, 201–203. 37. Morefield, G. L., Jiang, D., RomeroMendez, I. Z., Geahlen, R. L., HogenEsch, H., Hem, S. L. (2005) Effect of phosphorylation of ovalbumin on adsorption by aluminum-containing adjuvants and elution upon exposure to interstitial fluid. Vaccine 23(12), 1502–1506.

38. Rinella, J. V., White, J. L., Hem, S. L. (1995) Effect of anions on model aluminumadjuvant-containing vaccines. J Colloid Interface Sci 172, 121–130. 39. Gupta, R. K., Rost, B. E. (2000) Aluminum compounds as vaccine adjuvants, in (O’Hagan D. T., ed.), Vaccine Adjuvants, Preparation Methods and Research Protocols. Humana Press, Totowa, NJ, pp 65–89. 40. WHO (1977). World Health Organization Manual for the production and control of vaccines: Diphtheria toxoid; Appendix D.21: Preparation of aluminium phosphate suspension. BLG/UNDP/77.1. Rev. 1, pp 90–91 41. Smith, P. K., Krohn, R. I., Hermanson, G. T., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150, 76–85. 42. Dagoussat, N., Robillard, V., Haeuw, J. F., Plotnicky-Gilquin, H., Power, U., Corvaia, N., Nguyen, T., Bonnefoy, J. Y., Beck, A. (2001) A novel bipolar mode of attachment to aluminium-containing adjuvants by BBG2Na, a recombinant subunit hRSV vaccine. Vaccine 19(30), 4143–4152. 43. Shi, Y., HogenEsch, H., Regnier, F. E., Hem, S. L. (2001) Detoxification of endotoxin by aluminium hydroxide adjuvant. Vaccine 19, 1747–1752. 44. Burton, A. J., Carter, H. E. (1964) Purification and characterization of lipid A component of the lipopolysaccharides from Escherichia coli. Biochemistry 3, 411–418. 45. Gmeiner, J., Lüderitz, O., Westphal, O. (1969) Biochemical studies on lipopolysaccharides of Salmonella R. mutans. Eur J Biochem 7, 370–379. 46. Thiele, G. M., Rogers, J., Collins, M., Yasuda, N., Smith, D., McDonald, T. L. (1990) An enzyme-linked immunosorbent assay for the detection of antitetanus toxoid antibody using aluminiumadsorbed coating antigen. J Clin Lab Anal 4, 126–129. 47. Katz, J. B., Hanson, S. K., Patterson, P. A., Stoll, I. R. (1989) In vitro assessment of viral antigen content in inactivated aluminium hydroxide adjuvanted vaccines. J Virol Methods 25, 101–108. 48. Weeke, B., Weeke, E., Løwenstein, H. (1975) The adsorption of serum proteins to aluminium hydroxide gel examined by means of quantitative immunoelectrophoresis, in (Axelsen, N. H., ed.), Quantitative Immunoelectrophoresis. New Developments and Applications. Universitetsforlaget, Denmark, pp 149–154.

Chapter 5 Freund’s Complete and Incomplete Adjuvants, Preparation, and Quality Control Standards for Experimental Laboratory Animals Use Duncan E.S. Stewart-Tull Abstract Quality control and quality assurance procedures are discussed for the agreed benchmark standard Freund’s complete adjuvant (FCA). In addition, the use of the incomplete adjuvant (FIA) in the preparation of antisera is discussed. A major problem is the use of a safe and suitable mineral oil in FCA and FIA; manufacturers should provide infra-red spectra and gas liquid chromatography analyses. A range of safety tests, toxicity, pyrogenicity and endotoxin assays and advice on practical procedures for the use of these adjuvants are described. Key words: Freund’s complete adjuvant (FCA), Freund’s incomplete adjuvant (FIA), immunopotentiator, Mycobacterium tuberculosis, Montanide, immunization procedures, haemolytic activity, creatine kinase assay, rabbit pyrogenicity test, mouse weight gain test.

1. Introduction It is now more than 80 years since Lewis and Loomis (1) found that tuberculous guinea-pigs inoculated with sheep erythrocytes produced higher levels of sheep haemolysins than those in healthy animals. They injected rabbits with 50 μg of Mycobacterium bovis cells and obtained similar results (2). Their studies were continued by Dienes who injected guinea-pigs with 1–2 mg Mycobacterium tuberculosis O5 to induce a tuberculous nodule which was then inoculated with ovalbumin (3, 4). In 1993, EU and USA scientists and administrators discussed the harmonization of regulatory procedures for veterinary products. One opinion was G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_5, © Springer Science+Business Media, LLC 2010

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that “adjuvants are too reactive for inclusion in vaccines” (5). In terms of human vaccines Robbins was of the opinion that “we are prepared to accept no toxicity, any toxicity that we accept is a compromise” (6). This ignored the previous experience in the 1950s when some 18,000 American Service personnel were injected with an oil-adjuvanted influenza vaccine (7–10). In addition, 23,917 human beings were injected with an oil-adjuvanted poliomyelitis vaccine but only 93 individuals had an adverse reaction and only 14 developed nodules at the site of injection (11, 12). 1.1. Freund’s Complete Adjuvant

Subsequently, Freund used a water-in-mineral oil emulsion containing M. tuberculosis cells—this was termed Freund’s complete adjuvant, FCA (13). Adverse reactions to FCA reported from earlier studies included the formation of an epithelioid granuloma at the injection site (14–16). This effect was particularly noticeable in the New Guinea vaccination trial of 1964 during which pregnant Maprick tribal women were vaccinated with an adjuvanted tetanus vaccine in an attempt to prevent neonatal deaths. It was their normal practice to cover the umbilical stump on the newborn with mud and this sometimes caused a fatal disease. Unfortunately, in this trial many women developed similar granulomatous lesions at the site of injection, some of which had to be surgically removed (17). This was a setback for the use of an adjuvanted vaccine, but little attention was directed to the quality and composition of the mineral oil or the emulsifying agent. A detailed assessment of FCA is provided in a number of review articles (18, 19). It is important to qualify the constituents used in the preparation of experimental FCAs as they may vary considerably in their composition. The inclusion of dead M. tuberculosis is deemed to be unacceptable for use in human vaccines because of sensitization to other proteins, e.g. tuberculin. However, in 1990 the late Prof. Jonas Salk did confirm to me “If I thought that the inclusion of FCA would provide an effective vaccine in the fight against AIDS I should have no hesitation in recommending its use.” It should also be recognised that several existing commercial vaccines in routine use do contain microgram levels of an immunomodulator as shown in Table 5.1 (18). Conversely, the use of Freund’s incomplete adjuvant—FIA, without the mycobacterial component, did not cause the formation of such acute granulomatous adverse reactions in many experimental studies. Consequently, it was assumed that the mycobacterial cell component alone was responsible for the adverse reactions at the site of injection of FCA. Such conclusions, however, ignored the effects that the emulsifiers or mineral oils, used in the preparation of FCA, might have had on the formation of adverse histological reactions at the site of injection. Prior to the 1970s, the early studies used oils produced by the

Freund’s Complete and Incomplete Adjuvants

61

Table 5.1 Immunomodulator content of some vaccines Approximate weight (μg) of immunopotentiator injected during normal immunization schedules Vaccine

Peptidoglycan

Lipopolysaccharide

BCG

3.0–5.0

–

Whooping cough

6.5–50.0

6.0–35.0

Cholera

0.3–12.0

0.3–7.0

acid treatment or oleum method in the petroleum industry. At the beginning of the 1970s white mineral oils were produced by the single or double hydrogenation procedure and superior grades of oil were obtained which lacked the more toxic contaminants responsible for the adverse reactions. Therefore, it was not surprising that at the North Atlantic Treaty Organization meeting in Sounion, Greece, in 1989 it was agreed that Freund’s complete adjuvant (produced by the Statens Seruminstitut, Copenhagen, and available from Brenntag Biosector, Frederikssund, Denmark) consisting of 85% Marcol 52, 15% Arlacel A (mannide monooleate) and 500 μg heat-killed, dried M. tuberculosis 2.0 mg/mL and aluminium hydroxide adjuvant (Brenntag, Denmark) should be accepted as the “gold standard adjuvants” for experimental use (20). The FCA preparation was chosen as the standard adjuvant preparation because of its extensive use in the development of experimental vaccines in animals, its strong adjuvant effect and the considerable literature describing its activity. The mixture is used in a 1:1 ratio with the antigen-containing aqueous phase. The combination of mineral oil (Marcol 52) and dead mycobacterial organisms in FCA produces a specific cellular reaction in experimental animals since the mycobacteria stimulate the formation of epithelioid macrophage cells and also the maturation of plasmablasts to plasma cells. This effect was greater when the oil and mycobacterial fraction were combined. Numerous studies have shown that the oil emulsion was responsible for the retention of the antigen at the site of inoculation. This depot provides a slow and prolonged antigenic stimulus to antibody-forming cells. In addition, FCA stimulates both humoral and cell-mediated immune responses (see Note 1). In order to measure efficacy with a new adjuvant preparation it is important that its activity should be compared to these gold standards. Efficacy is dependent upon the vigorous mixing of the FCA and the antigen until a stable emulsion has formed, see below.

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There are increasing concerns about the general use of FCA for the routine production of antisera because Freund’s incomplete adjuvant (FIA) will, in most cases, achieve an acceptable result. From an ethical viewpoint there are few reasons to use FCA for routine antisera production and it is reasonable to use FIA. Some suggestions have been made that FCA should be limited to essential studies on the cell-mediated immune responses or where it is to be used as the standard adjuvant when a comparison is made with new adjuvanted vaccines. It should be emphasised that FIA or less reactive FCA products are more likely to be used. A non-ulcerative, commercial preparation of Non-Ulcerative Freund-type complete adjuvant—NUFA—has been introduced by Morris of Guildhay Ltd. (Guildford, Surrey, UK); this can be administered by intramuscular, subcutaneous and intradermal routes in small doses at multiple sites (see Note 2). The intramuscular site creates a longer depot stimulus and fewer adverse reactions than the other routes. 1.2. Freund’s Incomplete Adjuvant (FIA)

The killed M. tuberculosis component of FCA is not included in FIA. The latter is routinely used for boosting immunizations subsequent to a primary injection of FCA. It can also be used for the initial immunization, particularly when a strong antigen is used or moderate antibody levels are sufficient. As with FCA, the FIA is available from The Statens Seruminstitut, Copenhagen, and is composed of 85% Marcol and 15% Arlacel A. The search for less-reactogenic hydrocarbons and emulsifiers has continued. The Montanide series of oils (SEPPIC, France) based on anhydromannitol ether octadecanoate (oleic acid is a naturally occurring carboxylic acid in fats and oils—CH3 (CH2 )7 CH: CH (CH2 )7 COOH) is non-toxic in the Berlin test in a 50:50 (v/v) emulsion. The LD50 value in mice is equivalent to 25.0 g/kg bodyweight. Montanide ISA 720 is a ready-touse preparation, containing a highly refined emulsifier, a natural, metabolizable oil with a pharmaceutical grade mineral oil; Drakeol 6VR has been the choice for experimental studies in Glasgow and no adverse reactions have been recorded (see Notes 3 and 4).

1.3. The Mineral Oil

Many early studies employed the use of ill-defined mineral oils obtained from the petroleum industry. Indeed for my own early studies in the 1960s a free supply of Bayol F was obtained from the Esso Petroleum Company. An engineer walked to a outlet point in the Thames-side refinery where he suggested that “This cut-off should provide a supply of oil with the correct carbon chain length”. There may be concomitant adverse side-effects because of the reactogenicity of some types of mineral oil, particularly the formation of the epithelioid macrophage granuloma at the site of

Freund’s Complete and Incomplete Adjuvants

63

injection and local ulceration when the injection is given subcutaneously. My research in Japan with Shozo Kotani’s group was done with “a specially formulated mineral oil for adjuvant research”, but this caused severe disruption of cell membrane bilayers with consequent gross tissue destruction. Subsequently, gas liquid chromatography studies in Glasgow revealed that this mineral oil preparation contained short-chain hydrocarbons, nC6 H14 to nC10 –H22, and these were highly reactogenic and immunosuppressive. The most suitable mineral oils for experimental vaccine inclusion were shown to be fully saturated hydrocarbons, nC16 H34 –nC19 H40 (22) (see Note 4). The type of emulsion may affect the level of antibody stimulation; the injection of ovalbumin in a water-in-oil emulsion produced a stronger immune response in mice than in an oil-in-water emulsion. The hydrophilic and lipophilic groups should be in equilibrium in the adjuvant emulsion for the effective immunological activity of Freund’s adjuvants.

2. Materials 2.1. Preparation of Freund’s Complete Adjuvanted Experimental Vaccines

1. Heat-killed M. tuberculosis cells (the FCA available from Brenntag, or the Guildhay NUFA non-ulcerative Freund’s adjuvant (see above) is recommended) 2. Bacillus Calmette Guérin (BCG vaccine BP, BNF [id] John Bell and Croydon, London, UK) or from health organizations or direct from Movianto (SSI brand) 3. Mineral oil: examples are Bayol F (e.g. Esso is one source, but if a local source is available QA-QC information should be forthcoming), Marcol 52 or Drakeol 6VR (local source plus QA-QC record) or equivalent 4. Emulsifier such as Arlacel A (mannide mono-oleate) 5. Montanide 720 (Seppic)

2.2. Preparation of Freund’s Incomplete Adjuvanted Experimental Vaccines

1. Mineral oil: e.g. Bayol F, Marcol 52 or Drakeol 6VR.

2.3. Immunization Procedures

1. Experimental vaccine prepared as in Section 2.1.

2.4. Comparative Tests to Measure the Safety and Efficacy of Adjuvants

1. Cyanmethaemoglobin standard (Merck Ltd: Cyanmethaemoglobin standard for photometric determinations of haemoglobin or Sigma haemoglobin standard)

2. Emulsifier such as Arlacel A (mannide mono-oleate). 3. Montanide 720 (Seppic).

2. Experimental animal.

2. Drabkins reagent

64

Stewart-Tull

3. Creatine kinase diagnostic kit (Sigma) 4. Rabbits (adult New Zealand White, obtained from a reputable breeder) 5. Swiss white mice 6. Guinea-pigs

3. Methods 3.1. Preparation of Freund’s Complete Adjuvanted Experimental Vaccines

1. Dissolve the antigen, 10.0 mg in 0.2 mL sterile physiological saline (see Note 5). 2. Suspend 1.0 mg heat-killed, M. tuberculosis cells evenly in 0.6 mL sterile mineral oil by mild ultrasonication (see Note 6). 3. Add 0.2 mL of an emulsifier (see Note 7) to this oil phase and mix thoroughly. Alternatively, a suitable Montanide, e.g. Montanide 720, combined oil and emulsifier can be used. 4. Add the aqueous phase to the oil phase and mix thoroughly until a creamy white emulsion is produced. This can be achieved by either drawing up into a 1.0-mL glass Luer syringe fitted with a medium bore needle, or passing the mixture from syringe to syringe through a sterile adapter or by mild ultrasonication. 5. The nature of the resulting emulsion should be tested to ensure that a water-in-mineral oil emulsion has been prepared. Expel a drop of the emulsion onto the surface of water in a shallow dish: An oil-in-water emulsion will immediately disperse over the surface, whereas a water-in-oil emulsion will retain the integrity of the drop. 6. Store the final mixture at 4◦ C or at room temperature, depending on the nature of the antigen, but do not freeze because this will cause the emulsion to breakdown.

3.2. Preparation of Freund’s Incomplete Adjuvanted Experimental Vaccines

1. The procedure is as described in the Section 3.1, but the mycobacterial component or its equivalent is omitted from the mixture (see Note 8).

3.3. Immunization Procedures

1. The experimental vaccine should be warmed to 37◦ C before injection (see Note 9). This avoids shock to the small animal and helps the vaccine to flow from the syringe. 2. The size of the injection dose will depend on the animal and the route of injection. For example, 0.2 mL of a waterin-oil emulsion containing 2.0 mg of antigen and 200 μg

Freund’s Complete and Incomplete Adjuvants

65

M. tuberculosis im into the left hindlimb of a guinea-pig is suitable. On the other hand, 200 μg M. tuberculosis in the vaccine dose will completely suppress the humoral response in a mouse; in this case 25–50 g is an optimal dose. Different regimens may be required for larger animals; those for rabbits and sheep have been described where it may be advisable to use multiple injection sites (23). 3.4. Comparative Tests to Measure the Safety and Efficacy of Adjuvants

In 1988 at the First NATO Advanced Studies Institute conference on “Immunological Adjuvants and Vaccines” a unified approach to the assessment of immunological adjuvants was discussed (20, 25). This followed our previous studies on acceptable procedures to allow QA and QC in the use of mineral oils for use in adjuvant emulsions (21). In view of the expansion of the European Community it is important that these issues are widely discussed by researchers and industrialists so that each member country can accept agreed procedures acceptable to the EU and also to other regulatory bodies. By 1990, at the Second NATO conference on “Vaccines—Recent trends and progress” some recommendations were made and accepted; as mentioned previously it was agreed that FCA and aluminium hydroxide should be classed as the standard adjuvants against which new products would be compared (20, 23, 24). In addition, a number of QC and QA assays were discussed as shown below (and see Notes 11 and 12).

3.4.1. Haemolytic Activity of Adjuvants: Spectrophotometric Determination of Haemoglobin Release

The spectrophotometric determination of haemoglobin released from fresh whole rabbit blood by the accepted method gave the most reproducible and stable results. 1. Prepare reference solutions of cyanmethaemoglobin from 0.055 g/L to 0.85 g/L and measure the A540 nm values. 2. Add the test sample of fresh blood (20 μL), collected in a heparinized tube, to 4.0 mL of Drabkins reagent, mix and leave for 5 min after which the A540 nm values are recorded. 3. Prepare a standard curve with the reference standards and use to measure rabbit test results for haemoglobin concentration. If the percentage haemolysis in the negative control is >5.0% or in the positive control is 20% the test is invalid (25).

3.4.2. Haemolytic Activity of Adjuvants: Creatine Kinase Assay

There is a slight advantage with this method as a commercial kit is available from Sigma Chemical Company (Creatine kinase diagnostic kit Cat 520 or 520-C). 1. Three days after the injection of the adjuvanted vaccine a sample of blood is taken and the creatine kinase (CK) activity is measured according to the manufacturer’s instructions (see Note 10).

66

Stewart-Tull

3.4.3. Rabbit Pyrogenicity Assay

If the result of Test 1 is negative whether the adjuvant is required to stimulate a humoral or cell-mediated response a pyrogenicity test should be done (see Note 11). 1. All glassware, diluents and solutions to be used should be pyrogen-free. 2. The rabbits are required to be sham-tested 7 days prior to use. 3. Before the actual test, the rectal temperature of the rabbit is taken for every 30 min up to 90 min prior to the injection into a marginal ear vein of sterile physiological saline. If the temperature varies by either >0.2◦ C or if the range of readings exceeds 0.4◦ C the rabbit would be excluded. 4. Subsequently, the test sample, the immunomodulator in sterile physiological saline, is injected and the process is repeated; animals with a temperature rise of >0.6◦ C fail the test (26, 27).

3.4.4. Endotoxin Assays: Mouse Weight Gain Test

If the experimental vaccine fails the test described in 3.3.1, 3.3.2 or 3.3.3 above, it is unnecessary to proceed to further tests as the result indicates that the adjuvant is too reactive. This is a standard and reliable method to assess the presence of endotoxin as the mouse shows a decrease in total weight during the 24 h after inoculation. 1. If the animal shows a steady rise in body weight for 7 days after inoculation the inoculum is acceptable. 2. Conversely, if the animal continues to lose weight or even dies the inoculum fails the toxicity test (29).

3.4.5. Practical Procedures in the Preparation and Application of Adjuvant Mixtures

1. Standard control antigens: egg-white ovalbumin was originally chosen because it was a poor antigen but influenza haemagglutinin was also recommended (20). 2. The choice of test animals (see Note 13): It was pointed out that guinea-pigs and rabbits do not respond to influenza virus and outbred mice could introduce a considerable degree of laboratory to laboratory variation (20). Therefore, it was agreed to use guinea-pigs and mice. The mice should have (a) variable genetic background and similar H-2 haplotypes and (b) a constant genetic background and variable H-2. Dr Chella David (USA) recommended the following scheme: mouse strain and histocompatibility specificity 1, BALB/c H-2d ; 2, DRA/c H-2d ; 3, C57BL/10 H-2b ; 4, C3H H-2k ; 5 C3H.B10 H-2b . Tests should be done on either mouse groups 1, 2, 4 and 5 or with groups 3, 4 and 5. Other animals suggested for testing vaccines before field trials were goats and monkeys (see Note 14). It seems that an international agreement would be required in this matter.

Freund’s Complete and Incomplete Adjuvants

67

Table 5.2 Doses and sites of injection for various animals Injection sites

Species Mice or hamsters Guinea-pigs or rats

Maximum volume per injection site

Primary response

Secondary response

50 μL

Im

im

im into one hindlimb

oral im into one hindlimb

200 μL 200 μL 300 μL

3.4.6. Recommended Volumes of Injection Doses and Routes of Inoculation for Animals

oral

Rabbits

250 μL (if in multiple sites Tc ) distilled water or appropriate aqueous buffer. The total lipid concentration should be within the range of 0–400 mg/mL. 2. Place the flask in a temperature-controlled water bath and stir the mixture at >Tc until all the lipid material has transferred into a milky suspension. 3. Leave the suspension with stirring at >Tc for about 1 h, whereupon vesicles of diverse sizes, usually MLVs, are formed. Crude MLVs are usually not compatible with sterile filtration on a 0.2 μm membrane and need further processing for size reduction and homogenization. Two readily scalable processes for the size reduction of liposomes, extrusion and high-pressure homogenization (see Note 8), are described below but sonication methods (bath or probe sonication) can also be used for the size reduction of liposomes, at least at lab scale. 4. To reduce the size of MLVs by extrusion (19), transfer the MLV suspension into the prewarmed (>Tc ) chamber of a LipexTM extruder and extrude the liposomes under nitrogen pressure (100–700 psi) through two stacked polycarbonate filters of calibrated porosity (≤ 200 nm), according to the LipexTM extruder operating instructions. It may not be possible to extrude some preparations of MLVs straight through 0.2 or 0.1 μm filters. In this case, sequential extrusions through filters of decreasing porosity (0.8 μm– 0.4 μm–0.2 μm–0.1 μm) may be required. In all cases, several passes (up to seven depending on the lipid composition of the liposomes) through two stacked filters may

Liposomal Adjuvants: Preparation and Formulation with Antigens

79

be required to obtain a homogeneous suspension of mostly SUVs that is compatible with sterile filtration. 5. Alternatively, to reduce the size of MLVs by high-pressure homogenization (20), process the MLV suspension for a number of full cycles through the interaction chamber (at >Tc ) of the M-110Y MicrofluidizerTM or the EmulsiFlexTM C3 homogenizer. The number of cycles required to obtain liposomes that pass a sterile filter (≤ 0.2 μm) depends on the lipid composition of the liposomes. In some cases, a single cycle at moderate-high pressure (10,000 psi) may be sufficient to obtain sterile-filterable vesicles. 3.1.3. Preparation of SUVs Directly by Ethanol Injection

When the starting lipid or mixture of lipids is readily soluble in ethanol, it may be advantageous to use the method known as “ethanol injection technique” for the direct preparation of SUVs (21) (see Note 9). 1. Dissolve the desiccated lipid material in absolute ethanol to a concentrated stock solution. Heating (up to 60◦ C) may be necessary to increase solubility of the lipid(s) to concentrations close to the limit of saturation, as the injection step will dilute this ethanol solution by a factor of 20 (see below). 2. For the preparation of 2 mL of liposomes, place 1.9 mL of the aqueous phase (distilled water or appropriate buffer as described above) in a 5 mL jacketed glass vial and allow the temperature to equilibrate at >Tc by circulating water from a temperature-controlled water bath. 3. Place the vial on a magnetic stirrer and stir the aqueous phase vigorously at about 1,000 rpm by using a magnetic stirring bar. 4. Withdraw 100 μL of the concentrated lipid stock solution by using a standard gastight Hamilton syringe (volume Tc and collect the suspension for sterile filtration (see Note 11). 7. The lipid concentration in this liposome suspension will be 20 times less than in the ethanol stock solution and ethanol will be present in a final concentration of 5% v/v. At such low concentration, residual ethanol is compatible with parenteral pharmaceutical products (22) and should not interfere with liposome or antigen stability.

80

Haensler

3.1.4. Association of Antigens with Preformed Liposomes

Potent adjuvant effects can be obtained by simple mixing of antigen solutions with preformed liposomes (see Notes 12 and 13): 1. Simply mix the sterile-filtered antigen solution(s) with sterile-filtered liposomes by adding the appropriate volume of antigen to the appropriate volume of liposomes in aseptic conditions into a glass vial under a flow-hood. 2. Homogenize the mixture by gently stirring or shaking the vial.

3.1.5. Encapsulation of Soluble Antigens into Small Liposomes

Entrapment of antigens into small vesicles may be carried out by the “dehydration–rehydration” procedure in the presence of sucrose (23) (see Note 14). 1. Mix the solution of SUVs as prepared in Section 3.1.2 or 3.1.3 with the solution containing the antigen(s) and sucrose at a concentration of 3 mg per mg of lipid (see Note 15). 2. Sample the mixture into freeze-drying vials and freeze-dry overnight under vacuum (< 0.1 Torr). 3. Rehydrate the resulting powder at a temperature >Tc with as little volume of water as possible (ca. 5 μL/mg lipid) until a wet slurry is formed and allow the slurry to stand at >Tc for 60 min. 4. Dilute the slurry twice in water at >Tc and further incubate for 30 min. 5. Make up the resulting liposome suspension to its final concentration by dilution with the appropriate buffer and filter through a 0.2 μm filter for sterilization. If the reconstituted liposomes are too large for sterile filtration, they can be extruded in the presence of non-entrapped material to reduce their size to less than 200 nm in diameter, with much of the originally entrapped antigen remaining associated with the vesicles.

3.1.6. Reconstitution of Hydrophobic Antigens into Liposomes by the Detergent Removal Technique

Some antigens, especially membrane proteins, are solubilized by using detergents and chaotropic agents such as urea or arginine. Liposomes containing such antigens can be formed by the “detergent removal method” to produce a detergent-free formulation. The detergent should have a high critical micelle concentration (CMC), so that it can easily be removed by dialysis. N-octyl-β-Dglucopyrannoside (OG), with a CMC of 25 mM in water, is often used (24) (see Note 16). 1. Solubilize the SUVs as prepared in Section 3.1.2 or 3.1.3 to obtain a clear solution of mixed lipid/detergent micelles by stepwise addition under stirring of solid OG (or from a

Liposomal Adjuvants: Preparation and Formulation with Antigens

81

concentrated stock solution) up to a detergent/lipid molar ratio of 10:1 (see Note 17). 2. Add the desired amount of antigen solution to the mixed lipid/detergent micelles under gentle stirring at an appropriate temperature for antigen stability. The solution should remain clear; if not, more detergent should be added until a clear solution is obtained. 3. Place the clear solution into a Spectra/Por dialysis tubing (molecular weight cut off < molecular weight of antigen) and dialyze at 5◦ C against 100–500 volumes of the selected liposome buffer over 36 h with one change of the external buffer at 16 h (see Note 18). 4. After the dialysis, collect the detergent-free suspension of liposomes (generally SUVs with nearly 100% of the lipophilic antigens inserted into their membranes), make up to its final concentration by dilution with the liposome buffer, and filter through a 0.2 μm filter for sterilization. If the reconstituted liposomes are too large for sterile filtration, their size can be reduced by extrusion through two (stacked) polycarbonate filters of 10,000). Variance (% SD/GMT) decreased with increased GMT and was between 10 and 30% in groups with low antibody titres and less than 10% in groups with high titres. Relative to toxicity, no significant local reactions to phosphate-buffered saline or antigen without adjuvant were noted except in a few individuals, which displayed discoloration and loss of structure of < 1 cm3 1 week PT2, resulting in toxicity score of 20,000, respectively. Oily emulsions manifested necrosis, abscess, fibrosis, granulomatous and purulent inflammation, and vaccine residues with little difference between 1 week PT2 and 4 weeks PT1. The negative and positive controls indicated a large window for the analysis of both efficacy and toxicity of adjuvants. 13. A typical study included between 10 and 25 groups of five animals per group. As animals were marked individually, E/T ratio of individual animals and groups could be determined. References 1. Mestas, J., Hughes, C. W. (2004) Of mice and not men: differences between mouse and human immunology. J Immunol 172, 2731–2738. 2. Hulst, M. M., Westra, D. F., Wensvoort, G., Moormann, R. J. M. (1993) Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hog cholera. J Virol 67, 6479–6486. 3. Bokhout, B. A., Bianchi, A. T., van der Heijden, P. J., Scholten, J. W., Stok, W. (1986) The influence of a water-in-oil emulsion on humoral immunity. Comp Immunol Microbiol Infect Dis 9, 161–168. 4. Hilgers, L. A., Lejeune, G., Nicolas, I., Fochesato, M., Boon, B. (1999) Sulfolipocyclodextrin in squalane-in-water as a novel and safe vaccine adjuvant. Vaccine 17, 219–228. 5. Blom, A. G., Hilgers, L. A. T. (2004) Sucrose fatty acid sulphate esters as novel vaccine

adjuvants: effect of the chemical composition. Vaccine 23, 743–754. 6. Hilgers, L. A. T., Platenburg, P. L. I., Luitjens, A., Groenveld, B., Dazelle, T., FerrariLaloux, M., Weststrate, M. W. (1994) A novel non-mineral oil-based adjuvant. I. Efficacy of a synthetic sulfolipopolysaccharide in squalane-in-water emulsion in laboratory animals. Vaccine 12, 653–660. 7. Hilgers, L. A. T., Platenburg, P. L. I., Luitjens, A., Groenveld, B., Dazelle, T., Weststrate, M. W. (1994) A novel non-mineral oil-based adjuvant. II. Efficacy of a synthetic sulfolipopolysaccharide in squalane-in-water emulsion in pigs. Vaccine 12, 661–665. 8. Bouma, A., de Smit, A. J., de Kluiver, E. P., Terpstra, C., Moormann, R. J. M. (1999) Efficacy and stability of a subunit vaccine based on glycoprotein E2 of classical swine fever virus. Vet Microbiol 66, 101–114.

Large-Animal Model for Establishing E/T Ratio of Adjuvants 9. Terpstra, C., Bloemraad, M., Gielkens, A. L. J. (1984) The neutralizing peroxidase-linked assay for detection of antibody against swine fever. Vet Microbial 16, 123–128. 10. US Department of Health and Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research.

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(2009). Guidance for Industry: toxicity grading scale for healthy adult and adolescent volunteers enrolled in preventive vaccine clinical trials (Draft guidance 2005). Available at http://www.fda.gov/cber/gdlns/toxvac.htm [accessed January 2009].

Chapter 18 Determining the Activity of Mucosal Adjuvants Barbara C. Baudner and Giuseppe Del Giudice Abstract Mucosal vaccination offers the advantage of blocking pathogens at the portal of entry, improving patient’s compliance, facilitating vaccine delivery, and decreasing the risk of unwanted spread of infectious agents via contaminated syringes. Recent advances in vaccinology have created an array of vaccine constructs that can be delivered to mucosal surfaces of the respiratory, gastrointestinal, and genitourinary tracts using intranasal, oral, and vaginal routes. Due to the different characteristics of mucosal immune response, as compared with systemic response, mucosal immunization requires particular methods of antigen presentation. Welltolerated adjuvants that enhance the efficacy of such vaccines will play an important role in mucosal immunization. Among promising mucosal adjuvants, mutants of cholera toxin and the closely related heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli present powerful tools, augmenting the local and systemic serum antibody response to co-administered antigens. In this chapter, we describe the formulation and application of vaccines using the genetically modified LTK63 mutant as a prototype of the family of these mucosal adjuvants and the tools to determine its activity in the mouse model. Key words: Mucosal vaccination, mucosal adjuvants, LT mutants, ELISA, antibody response, T-cell response, ELISPOT.

1. Introduction One of the most notable benefits of mucosal immunization is the fact that both systemic and mucosal immunity are triggered, which is particularly advantageous in the case of vaccination against diseases caused by mucosal pathogens. The vast majority of infections occur, or start from, mucosal surfaces, which leads to the hypothesis that protective immunization against these pathogens needs a successful mucosal immune response. G. Davies (ed.), Vaccine Adjuvants, Methods in Molecular Biology 626, DOI 10.1007/978-1-60761-585-9_18, © Springer Science+Business Media, LLC 2010

261

262

Baudner and Giudice

Therefore, the mucosal route appears to be the most appropriate means for immunization. However, most clinically relevant vaccine candidates show weak immunogenicity when delivered mucosally and poor transport characteristics across biological barriers. This implies the need for adjuvants to potentiate their protective immune response or to improve their presentation and targeting. Mucosal adjuvants are components which are co-administered with a vaccine to enhance the immunogenicity of vaccine antigens (1). A better understanding of mucosal immunity, cellular immunology, and molecular biology during the past decades and novel technologies led to the discovery of new adjuvants (2, 3). Some of the more promising adjuvants include microspheres, immune-stimulating complexes (ISCOMS), liposomes, CpG DNA, cytokines, monophosphoryl lipid A, virus-like particles, and modified bacterial toxins (1). Among bacterial toxins, heat-labile enterotoxin (LT) from Escherichia coli and cholera toxin (CT) from Vibrio cholerae are known to be potent mucosal immunogens and have shown to serve as excellent adjuvants for co-administered antigens (4–6). LT and CT belong to the family of adenosine 5 -diphosphate (ADP)-ribosylating bacterial toxins and have an A–B structure (7, 8). The A subunit is enzymatically active and is responsible for increased intracellular accumulation of cyclic adenosine monophosphate (cAMP), thought to be responsible for the toxicity of both LT and CT. The pentameric B subunit of LT and CT binds to cell membrane surface receptors by its receptorbinding site, through interaction mainly with GM1 (9–11). This allows the transfer of the A subunit to the cytoplasm of the cell. Consequently, both LT and CT are toxic in their native state, and several mutants have been generated with reduced toxicity while maintaining the adjuvanticity of these molecules (12, 13) and are able to enhance local IgA, systemic IgG, and cellular immune responses to co-administered vaccine antigens after both oral and nasal immunization (13–19). The LTK63 and LTR72 mutants have been successfully used to immunize a variety of animal species with various vaccine antigens. These mucosal vaccine formulations have proven efficacious each time challenge with infectious pathogen or lethal toxin was feasible. Table 18.1 gives a summary of the available studies with relevant references (17, 19–36). More recently, LTK63 has been proven to exert its mucosal adjuvanticity in human volunteers after intranasal immunization with influenza vaccine (37). This chapter will focus on the use of LTK63 mutant as a prototype of this family of mucosal adjuvants. Its activity/adjuvanticity for the induction of systemic and mucosal antibodies, and increased T-cell and B-cell activity against co-administered vaccine antigens will be evaluated.

PO

PO

IN

IN

H. pylori urease

H. pylori NAP

Myc. Tub. Ags

Hib conjugates

IN

IN

SC

Measles antigens

HIV-1 gag

HIV-1 gag

IN

PO

H. pylori VacA

Pertussis antigens

PO

H. pylori CagA

IN

IN

H. pylori CagA

IN/SC

TC

Diphtheria toxoid

Pneumo conjugates

IN

Tetanus fragment C

MenC conjugates

Route

Antigen

Mouse

Mouse

Mouse

Mouse

Mouse

Mouse

Mouse

Guinea pig/mouse

Mouse

Mouse

Mouse

Mouse

Mouse

Mouse

Mouse

Species

+

ND

ND

+

+

+

+

+

+

+

+

+

+

+

+

Serum

ND

ND

ND

+

+

+

+

+

ND

ND

+

+

+

ND

+

Mucosal

Antibody response

ND

ND

ND

+

+

ND

ND

ND

ND

ND

ND

ND

+

ND

ND

CD4+

T-cell response

+

+

+

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

CD8+

ND

ND

ND

Yes

Yes

ND

ND

Yes

Yes

Yes

Yes

Yes

Yes

ND

Yes

Protection

Table 18.1 LTK63 induces strong, protective immune responses in various animal models following various mucosal routes

(continued)

(27)

(27)

(26)

(17)

(19, 25)

(24)

(21)

(23)

(22)

(22)

(22)

(22)

(22)

(21)

(20)

References

Determining the Activity of Mucosal Adjuvants 263

IN

PO

IM

IN

IN

IN

Flu HA

Flu HA

Flu HA

Toxoplasma gondii antigens

Malaria antigens

Ricin A

Mouse

Mouse

Mouse

Mouse

Mouse

Mouse

Goat

Moue

Mouse

Species

+

+

+

+

+

+

+

+

ND

Serum

+

+

+

-

+

+

+

+

ND

Mucosal

Antibody response

+

+

+

ND

ND

ND

ND

ND

ND

CD4+

T-cell response

Notes: IN, intranasal; PO, per os; IM, intramuscular; SC, subcutaneous; TC, transcutaneous; ND, not determined.

IN

Vaginal

Goat HSV-1

IN

RSV M2 peptide

HSV-2 gD2

Route

Antigen

Table 18.1 (continued)

ND

+

ND

ND

ND

ND

ND

ND

+

CD8+

Yes

Yes

Yes

ND

ND

Yes

Yes

ND

Yes

(36)

(35)

(34)

unpublished data

(33)

(32)

(31)

(30)

(28, 29)

Protection References

264 Baudner and Giudice

Determining the Activity of Mucosal Adjuvants

265

The mutant LTK63 is generated by site-directed mutagenesis of single-stranded DNA by classic techniques using defined oligonucleotides (38) and procedures have been extensively reported in laboratory textbooks (12, 39, 40); furthermore other mutants such as the LTR72 which contains some residual enzymatic activity have been generated (13). LTK63 is the result of a substitution of serine 63 in the A subunit with a lysine which dramatically reduces its enzymatic activity (14–17). LTK63 has been shown to be a potent mucosal adjuvant for inducing CTL with a co-administered peptide immunogen (14). The residual toxicity of LT mutants can be very easily assessed in vitro and in vivo. LT exhibits toxicity on Y1 adrenal cells, which undergo rounding when cultured as a monolayer in the presence of these toxins (41). Furthermore the residual ADPribosyltransferase activity can be addressed biochemically in vitro using appropriate substrates (42). The in vivo toxicity of these

Fig. 18.1. Schematic representation of determining the activity of mucosal adjuvants.

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Baudner and Giudice

molecules can be tested in ligated ileal loops in rabbits, where, if toxic, they induce accumulation of fluid that increases with the amount of toxin inoculated (43). For the in vivo evaluation of the mucosal adjuvanticity of LTK63 vaccine, antigens will be formulated with and without LTK63 and given by various mucosal routes (intranasal, intragastric, vaginal, or rectal) using a mouse model. Mice are immunized according to standard schedules and mucosal washes and tissues as well as serum samples and splenocytes are harvested and prepared for stimulation (Fig. 18.1). Methods for assaying adjuvant- and antigen-specific antibody titers by ELISA, cell-mediated immunity by T-cell proliferation, and the frequency of antibody-secreting cells by ELISPOT will be described (Fig. 18.1).

2. Materials 2.1. Preparation of Immunogen/ Adjuvant Vaccine Formulations

1. Immunogen, for example, an existing or an experimental vaccine 2. Adjuvant: LTK63 or other LT mutants (Novartis vaccines) 3. Phosphate-buffered saline (PBS) 4. Sodium bicarbonate

2.2. Immunization of Animals

1. Female, 6- to 8-week-old inbred BALB/c mice (Charles River) or other strain of mice as appropriate based on the vaccine model 2. Vaccine formulations (see Sections 2.1 and 3.1) 3. Gavage needles for intragastric immunizations (Luer-Lock stainless-steel, 50-mm × 1-mm gavage with a round tip) 4. Sodium bicarbonate 5. Sterile saline 6. Syringe (