Elsevier

Vaccine

Volume 29, Issue 47, 3 November 2011, Pages 8530-8541
Vaccine

A heat shock protein based polyvalent vaccine targeting HSV-2: CD4+ and CD8+ cellular immunity and protective efficacy

https://doi.org/10.1016/j.vaccine.2011.07.011Get rights and content

Abstract

Efforts to develop a subunit vaccine against genital herpes have been hampered by lack of knowledge of the protective antigens of HSV-2, the causative agent of the disease. Vaccines based either on selected antigens or attenuated live virus approaches have not demonstrated meaningful clinical activity. We present here results of a therapeutic vaccine candidate, HerpV (formerly called AG-707), consisting of 32 HSV-2 peptides derived from 22 HSV-2 proteins, complexed non-covalently to the HSP70 chaperone and formulated with QS-21 saponin adjuvant. HerpV is observed to be immunogenic, generating CD4+ and CD8+ T cell responses in three mouse strains including HLA-A2 transgenic mice. Optimal T cell stimulation was dependent on the synergistic adjuvant properties of QS-21 with hsp70. The vaccine provided significant protection from viral challenge in a mouse prophylaxis model and showed signals of activity in a guinea pig therapeutic model of existing infection. Peripheral blood mononuclear cells from human HSV-2+ subjects also showed reactivity in vitro to a subset of individual peptides and to the pool of all 32 peptides. Recombinant human Hsc70 complexed with the 32 peptides also stimulated the expansion of CD8+ T cells from HSV-2+ subjects in vitro. These studies demonstrate that HerpV is a promising immunotherapy candidate for genital herpes, and provide a foundation for evaluating HerpV in human HSV-2+ subjects with the intent of eliciting CD4+ and CD8+ T cell responses to a broad array of viral antigens.

Introduction

Genital herpes, caused primarily by infection with HSV-2, is a persistent condition in humans which has eluded all prophylactic and therapeutic vaccine development efforts. Most recently, an 8000 subject placebo controlled prophylactic vaccine study in HSV-2 seronegative women failed to show clinical activity [1]. While it is possible that the narrow antigen composition of this vaccine which consisted only of glycoprotein D was a key factor contributing to its lack of efficacy, a placebo controlled study of a single gene deletion HSV-2 replication defective virus vaccine also lacked efficacy in the therapeutic setting [2]. These two studies are representative bookends of the approaches to vaccine development in the herpes field. Reasons for their failure can be speculated: the widely tested glycoproteins D (and B) may simply not be protective antigens in humans given the wealth of evidence of their eliciting neutralizing antibodies in humans [3]. Additionally, genetically attenuated live virus vaccines which theoretically could present a vast number of antigens to the immune system may lack sufficient immunogenicity due to the built-in replication blocks which minimize virulence. Recent evidence collected in HSV-2 seropositive as well as in HSV-2 immune seronegative subjects has demonstrated the considerable breadth of viral antigens recognized by T cells [4], [5], [6], [7]. The data point to the distinct possibility that a herpes vaccine based on a single or small number of antigens may be unlikely to be protective in the genetically diverse human population regardless of how effectively it primes antibody and/or T cell responses. Here, we report the results of our attempt to address the twin challenges of finding the protective antigens in HSV-2 and delivering them in a manner which activates the immune system robustly with particular focus on cellular immunity believed to be crucial for viral control [8], [9], [10].

The HSV-2 vaccine described here is the rodent equivalent of a heat shock protein based genital herpes vaccine candidate, also named HerpV, which recently completed Phase 1 clinical testing (A. Wald, see submitted companion manuscript). HSP–peptide complexes isolated from infected or malignant cells represent a natural antigenic fingerprint of those cells [11]. Such complexes can also be reconstituted in vitro which offers the possibility of selecting and synthesizing specific antigen sequences of interest which may be delivered with recombinant HSP [12]. Upon injection into a host, HSP–peptide complexes bind to HSP receptors on antigen presenting cells leading to internalization of the complexes and presentation of the chaperoned peptides by MHC class I and II molecules [13], [14], [15], [16]. In this manner immunization with HSP–peptide complexes primes/boosts T cell responses specific for the chaperoned peptides [11]. Using the approach of reconstituting HSP–peptide complexes in vitro, Rouse and co-workers demonstrated the immunogenicity and protective activity of mouse HSP70 complexed with defined MHC class I and II binding peptides of gD, gB and ICP-27 in the acute and memory phase in mice [17], [18]. Similarly, Navaratnam et al. used gp96, another stress protein, to form in vitro complexes with three mouse cytotoxic T cell (CTL) epitopes of the bovine herpes virus (BHV) serine/threonine protein kinase and glycoprotein H [19]. Such complexes elicited CTLs which killed peptide pulsed as well as BHV infected target cells. HSP vaccines have been widely tested in animal models of other infectious diseases as well, including tuberculosis, listeria and LCMV [20], [21]. Immunization of cancer patients with autologous cancer-derived HSP–peptide complexes has also consistently shown immunological and clinical activity [22], [23], [24], [25], [26], [27]. The saponin QS-21, a component of the HerpV vaccine approach, has also demonstrated potent adjuvant activity in multiple indications over the past 20 years in animal models and human clinical trials [28], [29].

Our results show that vaccination with the multi-valent HerpV candidate vaccine induces an immune response to multiple epitopes and protection from HSV-2 in several animal models. A companion article describes the results of a Phase 1 safety and immunogenicity study of HerpV in HSV-2+ subjects (A. Wald, see submitted companion manuscript).

Section snippets

Selection of HSV-2 peptides in HerpV

HerpV used in the rodent studies consists of 32 synthetic HSV-2 derived peptides (each 35 amino acids long) non-covalently complexed with HSP70 purified from normal tissue (described below). The peptide sequences are derived from 10 tegument, 8 envelope and 4 other HSV-2 proteins, representative of proteins expressed during all phases of viral replication [30] (Table 1). The rationale by which the ∼85 known HSV-2 proteins were winnowed to 22 proteins from which the 32 peptides in HerpV are

Discussion

This study is the first demonstration of immunogenicity and protective efficacy of a polyvalent herpes vaccine comprising a large array of long synthetic viral peptides, which do not contain any previously defined protective T cell epitopes. The finding that HerpV stimulates not only CD4+ but also CD8+ T cells fulfills what are likely to be key requirements for a successful herpes vaccine. Biological activity of the peptides was shown to be dependent on HSP70, to which the peptides were

HSP70 and peptides

Mouse and guinea pig HSP70 was purified from mouse and guinea pig liver and kidney tissue lysate by ATP agarose affinity and DEAE Sepharose anion exchange chromatography columns, concentrated and subjected to 0.22 μM filtration. Human Hsc70 cDNA was sourced directly from ATCC (IMAGE Consortium Code 612844), sequenced to confirm identity and expressed in BL21 (DE3) E. coli using the pET-24a(+) expression vector (Novagen). rh-Hsc70 protein was purified from lysed E. coli cell paste by Q Sepharose

Acknowledgements

For technical expertise and insightful discussions: Nicholas Messinese, Aaron Wilson, Erik Devereaux, Hao Tang and Andrei Varnavski.

Conflict of interest statement: AM, CM, HC, JP, KL, AL, RMC, AT, SM and DLL each had/have direct ownership of stock of Agenus, Inc. PKS was a consultant to Agenus, Inc. and has direct ownership of Agenus, Inc. stock.

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    1

    Current address: National Institute of Allergy and Infectious Diseases.

    2

    Current address: Pfizer Vaccine Research.

    3

    Current address: Novartis.

    4

    Current address: GlaxoSmithKline.

    5

    Current address: Sanofi Pasteur.

    6

    Current address: NKT Therapeutics Inc.

    7

    These authors contributed equally to this work.

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