Development of an oral prime–boost strategy to elicit broadly neutralizing antibodies against HIV-1

Vaccine 20 (2002) 1968–1974

Note

Development of an oral prime–boost strategy to elicit broadly neutralizing antibodies against HIV-1 Anthony L. Devico a , Timothy R. Fouts a , Mohamed T. Shata a , Roberta Kamin-Lewis a,b , George K. Lewis a,∗ , David M. Hone a a

b

Division of Vaccine Research, Institute of Human Virology, University of Maryland Biotechnology Institute, 725 W. Lombard Street, Baltimore, MD 21201, USA Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA Received 18 June 2001; accepted 28 November 2001

Abstract Given the increasing incidence of HIV-1 infection world-wide, an affordable, effective vaccine is probably the only way that this virus will be contained. Accordingly, our group is developing an oral prime–boost strategy with the primary goal of eliciting broadly neutralizing antibodies against HIV-1 to provide sterilizing immunity for this virus. Our secondary goal is to elicit broadly cross-reactive anti-viral CD8+ T cells by this strategy to blunt any breakthrough infections that occur after vaccination of individuals who fail to develop sterilizing immunity. This article describes our progress in the use of the live attenuated intracellular bacteria, Salmonella and Shigella, as oral delivery vehicles for DNA vaccines and the development of conformationally constrained HIV-1 Env immunogens that elicit broadly neutralizing antibodies. © 2002 Published by Elsevier Science Ltd. Keywords: Prime–boost strategy; Neutralizing antibodies; HIV-1

1. Introduction The primary endpoint of HIV-1 vaccine development is to identify an immunogen that can be formulated in a way such that the vaccine is effective and affordable for world-wide use. Our strategy to achieve this goal is shown in Fig. 1. There are three major hurdles that must be cleared for this strategy to be successful. First, the immunogen must elicit broad immunity that is effective against genetically disparate variants of HIV-1. The last 2 years have witnessed a focusing of efforts on two non-exclusive strategies by new studies on the mechanisms of protective immunity in macaque models. There is now little doubt that protective immunity can be afforded by either passive transfer of neutralizing antibodies [1–3] or by the induction of strong virus-specific CD8+ T cell responses by vaccination [4,5]. Most important, these two sets of studies reveal distinct patterns of protective immunity. The passive transfer of neutralizing antibodies affords what appears to be sterilizing immunity [1–3]. By contrast, ∗

Corresponding author. Tel.: +1-410-706-4688. E-mail address: [email protected] (G.K. Lewis).

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strong CD8+ responses allow infection but result in the control of viremia and disease in short-term analyses [4,5]. Clearly, these experiments are exciting and demonstrate conclusively that it is possible to elicit protective immunity against primate immunodeficiency viruses. As will be detailed below, our approach is predicated on the idea that sterilizing immunity, mediated by broadly neutralizing antibodies, is not only preferable but attainable. Second, an effective vaccine against HIV-1 must elicit protective immunity at each portal of HIV-1 entry. Thus, a successful vaccine must protect against mucosal and parenteral exposures to HIV-1. This poses a dilemma for the use of any vaccine candidate that is designed to be delivered parenterally as it is known that parenteral immunization with non-replicating vaccines usually induces little or no mucosal immunity [6]. It is also known that recall responses can be elicited in both the mucosal and systemic compartments after boosting in either compartment; however, this requires priming in the mucosal compartment [7]. Priming in the systemic compartment only allows recall responses in the systemic compartment and these only occur when the boost is given parenterally [7]. Taken together, these studies show that mucosal immunity requires mucosal priming and

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Fig. 1. Criteria for our oral prime–boost strategy to elicit broadly neutralizing antibodies against HIV-1.

that strong mucosal priming most often leads not only to mucosal immunity but to systemic immunity as well. Note that in the studies cited above mucosal challenge was prevented by the intravenous transfer of neutralizing IgG antibodies suggesting that parenteral immunization might confer mucosal protection. On the other hand, there is evidence for the presence of IgA neutralizing antibodies in mucosal fluids of exposed-uninfected commercial sex workers [8] and that these antibodies can block the transcytosis of HIV-1 across epithelial barriers in tissue culture [9]. These data suggest that mucosal antibodies contribute to protective immunity, particularly in situations of low-level virus exposure, such as those likely to be at play in mucosal transmission consequent to sexual intercourse. In addition, there is also evidence that transient mucosal exposure to SIV can results in protective immunity to a subsequent SIV challenge [10,11], making it highly likely that mucosal immunity will prove beneficial, if not necessary, to protect against mucosal exposure to HIV-1. For these reasons, our strategy employs mucosal priming. In addition to the apparent benefits of mucosal immunity elicited by mucosal priming, there are practical reasons for pursuing this route of immunization. Third, because of the rapid emergence of HIV-1 in developing nations, affordability of a vaccine is of paramount importance. Many of the current HIV-1 vaccines in clinical trials include a soluble envelope subunit protein immunogen in conjunction with one or another viral vector encoding this and other proteins of HIV-1. The costs of producing quantities of highly purified recombinant proteins are high and likely to make such vaccines less affordable in developing nations. To overcome this problem, we are exploiting live attenuated intracellular bacteria, including Salmonella and Shigella, as oral delivery vehicles for DNA vaccines encoding immunogens that elicit both strong CD8+ T cell responses in addition to broadly neutralizing antibody responses. Oral vaccines have the additional advantage in that

they obviate the need for needles, which is a major cause of iatrogenic infections in developing nations. Based on these considerations, we are developing an oral prime–boost strategy in which the first dose of the vaccine will be an oral inoculation of attenuated Salmonella or Shigella carrying a DNA vaccine encoding an immunogen that can elicit broadly neutralizing antibodies. In addition, this DNA vaccine can include an immunogen, such as an optimized CTL vaccine that is under development at Oxford University [12–14]. The second dose of the vaccine will be a parenteral inoculation of a soluble protein (in the case of the immunogen that elicits broadly neutralizing antibodies) or a viral vector encoding the relevant immunogens. In the sections to follow, we will briefly describe our progress in the development of the two essential components of this strategy: an oral delivery system for DNA vaccines and an immunogen that elicits broadly neutralizing antibodies against HIV-1.

2. Live attenuated intracellular bacteria as oral delivery vehicles for HIV-1 DNA vaccines Our group has pursued the development of live attenuated intracellular bacteria as oral delivery vehicles for HIV-1 vaccines for the last decade. We were attracted to this idea because of the increasing availability of well-attenuated strains of both Salmonella typhi [15–25] and Shigella flexneri [19,26–30]. Our progress and that of others in the development of this strategy has been reviewed recently [31]. It can be summarized as follows. Using attenuated Salmonella typhimurium in mice as the model the following observations have been made. First, when the immunogen is expressed cytoplasmically by the bacterium, it was necessary to drive the expression off of a strong promoter using a mulitcopy, balanced lethal in order to see T cell proliferative responses after multiple inoculations [32,33]. No CTL

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Fig. 2. Induction of CD8+ T cell responses using an attenuated S. typhimurium secreting an HIV-1 gp120MN immunogen and an attenuated S. flexneri carrying a DNA vaccine encoding a codon-optimized gp120MN that is expressed only in eukaroytic cells. Panel (a) shows the prokaryotic and eukaryotic expression cassettes. The attenuated S. typhimurium expresses gp120MN fused with the components of the E. coli ␣-hemolysin, which is a type I secretion system. Panel (b) shows a component of a time-course experiment in which Balbc mice were primed intranasally with S. flexneria carrying the codon-optimized gp120MN DNA vaccine followed by orogastric boosting with S. typhimurium secreting gp120MN. IFN-␥ ELISPOTS were measured after stimulation of spleen cells with the immundominant P18 epitope of the gp120MN V3 region. Depletion studies confirmed that the ELISPOTS are CD8+ (data not shown).

responses specific for the HIV-1 immunogen were obtained in those experiments. Stronger responses obtained after a single oral inoculation required secretion of the immunogen into the periplasm by a type II secretion system [34]. In that study, both T cell proliferative and local IgA antibody responses were obtained. However, CTL responses were highly sporadic suggesting that those responses were on the cusp of detection. Most recently, we have exploited a type I secretion system, the Eschericia coli ␣-hemolysin (see [35] for review and [36] for structure), to develop a strain that can reproducibly prime and boost CTL responses. The

composition of one such a vector using S. typhimurium is illustrated in Fig. 2a and its use in a prime–boost experiment with a Shigella-DNA vaccine carrying the same immunogen is shown in Fig. 2b. The use of live attenuated intracellular bacteria as oral delivery vehicles for protein immunogens suffers one major drawback. They are not useful as delivery vehicles when the protein expressed by the bacterium is requires post-translational modifications unique to eukaryotic cells for the proper folding of the protein. In this regard, the HIV-1 Env protein is a prime example.

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It is well known that gp120, the outer envelope glycoprotein of HIV-1, is heavily glycosylated by N-linked sugars [37] and that bacteria cannot fold this protein to a functional form [32,38,39]. This makes it nearly impossible to elicit broadly neutralizing antibodies against a gp120 expressed by Salmonella or Shigella. For this reason, we sought other means of expressing the HIV-1 antigens in order to exploit Salmonella as a delivery system for a vaccine against HIV-1. This led to our use of Salmonella and Shigella as delivery vehicles for DNA vaccines. The ability of intracellular gram-negative bacteria to “transfect” infected cells with plasmids encoding eukaryotic expression systems was discovered independently by three groups [40–42] including ourselves [41]. Although it is still early in the game, it appears that this is a robust method for the delivery of DNA vaccines in addition to being able to transfect eukaryotic cells in vitro. The first published vaccine study reported antigen specific T cell proliferative responses and S. flexneri to deliver a DNA vaccine encoding LacZ in addition to the induction of anti-LacZ antibodies [43]. A more extensive study reported the induction of antibody responses and both class I and class II restricted T cell responses to LacZ after immunization with S. typhimurium carrying a eukaryotic expression plasmid encoding LacZ [44]. 1 These early studies presaged the appearance of a growing literature in which live attenuated intracellular bacteria have been used to deliver DNA vaccines including several comprehensive reviews [40–43,45–70]. Our studies are in press (Shata et al., in press) and will not be shown here save for the example in Fig. 1, where attenuated S. flexneri was used to elicit CD8+ T cell responses in mice using an HIV-1 MN DNA vaccine. We have made the following observations using attenuated S. typhimurium and S. flexneri. (1) Potent CD8+ T cell responses can be elicited using these systems after a single oral dose of the vaccine provide a “codon-optimized” HIV-1 DNA vaccine [71,72] is used. (2) The DNA vaccine delivered orally by Salmonella or Shigella is at least as immunogenic as naked DNA given intramuscularly and might be much more immunogenic when Shigella is used as the vector (Shata et al., in press). (3) The CD8+ T cell response elicited after a single oral inoculation with these vectors approximates those seen with the same HIV-1 immunogen delivered by vaccinia (Shata et al., in press). (4) HIV-1 Env DNA vaccines delivered by Salmonella or Shigella can prime for humoral responses elicited by soluble proteins. (5) By using an appropriate “molecular adjuvant” in the DNA vaccine, these antibody responses can be main1 Darji et al. show that invasive strains of Shigella flexneri and Escherichia coli, that undergo Iysis upon entry into mammalian cells because of impaired cell wall synthesis, can act as stable DNA delivery systems to their host. This direct gene transfer is efficient, of broad host cell range and the replicative or integrative vectors so delivered are stably inherited and expressed by the cell progeny. DNA delivery by abortive invasion of eukaryotic cells by bacteria is of potential interest for stimulation of mucosal immunity and for in vivo or ex vivo gene therapy of human diseases.

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tained at high levels long after immunization (Fouts et al., in preparation). Taken together, it is now clear that in animal models live attenuated intracellular bacteria can deliver DNA vaccines that are capable of inducing both humoral and CD8+ T cell responses in a variety of systems. The challenges at hand for their development as HIV-1 vaccines are to identify bacterial strains that will deliver the DNA vaccines in humans and to identify an immunogen that will elicit broadly neutralizing antibody responses in addition to strong CD8+ T cell responses.

3. Conformationally constrained Env immunogens elicit broadly neutralizing antibodies against HIV-1 Despite much effort, it has been difficult to elicit broadly neutralizing antibodies against genetically diverse primary isolates of HIV-1 using conventional Env immunogens, such as monomeric gp120 or oligomeric gp140 (reviewed in [73–76]). Typically, these immunogens elicit neutralizing antibodies specific for isolate specific epitopes and offer little in the way of cross-neutralization among diverse isolates, even for T cell line adapted viruses. This has led to interest in conformationally altered Env immunogens that expose otherwise cryptic epitopes that correspond to forms of Env that are involved in virus binding and entry, which are steps in the virus replicative cycle that all HIV-1 variants must negotiate. Three sets of studies carried out over the past decade (actually predating the discovery of coreceptors) demonstrate that such immunogens can indeed be produced and used to elicit broadly neutralizing responses using defined Env immunogens. The first published experiments were carried out approximately 8 years ago by Celada et al. [77] who used non-covalent complexes of CD4 and gp120 to raise neutralizing monoclonal antibodies specific for laboratory isolates of HIV-1. These antibodies were not characterized in detail but they appear to be specific for conformationally altered CD4. Approximately 5 years ago, Kang et al. [78,79] also used non-covalent gp120–CD4 complexes as immunogens to enhance the immunogenicity of neutralizing epitopes exposed on gp120. Our original studies used covalently cross-linked complexes of gp120 and CD4 and were the first to produce complex specific antibodies and polyclonal responses that neutralize primary isolates of HIV-1 across multiple clades using mice and goats as the animal models [80,81]. More recently, our group in collaboration with Ranajit Pal (ABL, Kensington, MD) has confirmed the existence of broadly neutralizing antibodies against a wide array of primary HIV-1 isolates using sera from Rhesus macaques immunized with chemically cross-linked gp120–CD4 complexes similar to those described in [80,81]. These antibodies can be purified from immune sera and appear to be specific for previously unknown epitopes that are resident only on the gp120–CD4 complex (Fouts Pal et al., in preparation). Taken together these studies strongly

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Fig. 3. Depiction of the location of the flexible linker on a molecular model of the FLSC described in [82]. The structure was generated from the coordinates deposited in the Protein Data Base 1CG1 from [84].

suggest that gp120–CD4 complexes be considered as HIV-1 vaccine candidates. For this reason, we have developed a second generation gp120–CD4 immunogen that can be formulated as a DNA vaccine, as a soluble subunit protein vaccine, and even as a viral vector vaccine [82]. This construct is a chimeric protein made by linking the C-terminus of HIV-1 Ba-L gp120 with the N-terminus of the D1D2 domains of human CD4 by a flexible peptide spacer resulting in a monomeric molecule denoted as full-length single chain (FLSC) [82]. Physical-chemical studies showed that this FLSC is primarily a monomer in which the gp120 and CD4 moieties predominantly form an intramolecular complex that is apparently favored by the geometry of the peptide spacer (Fig. 3) [82]. Most important, this molecule constituitively binds to CCR5 indicating that it is in a conformationally-stable form that is distinct from free gp120 that exists in solution as a relatively disordered structure [83]. This picture is confirmed by immunochemical analyses in which conformational epitopes associated with CD4 binding are constitutively exposed [82]. Preliminary studies suggest that FLSC harbors epitopes recognized by broadly neutralizing antibodies and that it is immunogenic in laboratory animals. For this reason, we are developing FLSC as the major immunogen, both as a DNA vaccine and as a soluble subunit protein, in our oral prime–boost strategy.

4. Summary In summary, we have developed the principal components of an oral prime–boost strategy to elicit both strong CD8+ T cell responses and broadly neutralizing antibodies against HIV-1. We are at the juncture where this strategy can be tested in human volunteers, which is a major focus of our current HIV-1 vaccine effort.

Acknowledgements Supported by NIH Grants: AI041914 (DH), AI043756 (DH), AI44736 (RKL), HL59796 (ALD), HL63647 (ALD), AI47066 (ALD), AI47490 (GKL), AI43046 (GKL), AI3892 (GKL), and IAVI (GKL, DH). References [1] Mascola JR, Lewis MG, Stiegler G, et al. Protection of macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies. J Virol 1999;73(5): 4009–18. [2] Mascola JR, Stiegler G, VanCott TC, et al. Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat Med 2000;6(2):207–10. [3] Baba TW, Liska V, Hofmann-Lehmann R, et al. Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian human immunodeficiency virus infection. Nat Med 2000;6(2):200–6. [4] Schmitz JE, Kuroda MJ, Santra S, et al. Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes. Science 1999;283(5403):857–60. [5] Amara RR, Villinger F, Altman JD, et al. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science 2001;292(5514):69–74. [6] de Aizpurua HJ, Russell-Jones GJ. Oral vaccination: identification of classes of proteins that provoke an immune response upon oral feeding. J Exp Med 1988;167(2):440–51. [7] Herremans TM, Reimerink JH, Buisman AM, Kimman TG, Koopmans MP. Induction of mucosal immunity by inactivated poliovirus vaccine is dependent on previous mucosal contact with live virus. J Immunol 1999;162(8):5011–8. [8] Kaul R, Plummer F, Clerici M, Bomsel M, Lopalco L, Broliden K. Mucosal IgA in exposed, uninfected subjects: evidence for a role in protection against HIV infection. AIDS 2001;15(3):431–2. [9] Devito C, Broliden K, Kaul R, et al. Mucosal and plasma IgA from HIV-1 exposed-uninfected individuals inhibit HIV-1 transcytosis across human epithelial cells. J Immunol 2000;165(9):5170–6.

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