Nina Malkevitch

Protection against mucosal simian immunodeficiency virus SIV (mac251) challenge by using replicating adenovirus-SIV multigene vaccine priming and subunit boosting

ABSTRACT Whereas several recent AIDS vaccine strategies have protected rhesus macaques against a pathogenic simian/human immunodeficiency virus (SHIV)89.6P challenge, similar approaches have provided only modest, transient reductions in viral burden after challenge with virulent, pathogenic SIV, which is more representative of HIV infection of people. We show here that priming with replicating adenovirus recombinants encoding SIV env/rev, gag, and/or nef genes, followed by boosting with SIV gp120 or an SIV polypeptide mimicking the CD4 binding region of the envelope, protects rhesus macaques from intrarectal infection with the highly pathogenic SIVmac251. Using trend analysis, significant reductions in acute-phase and set point viremia were correlated with anti-gp120 antibody and cellular immune responses, respectively. Within immunization groups exhibiting significant protection, a subset (39%) of macaques have exhibited either no viremia, cleared viremia, or controlled viremia at the threshold of detection, now more than 40 weeks postchallenge. This combination prime-boost strategy, utilizing replication competent adenovirus, is a promising alternative for HIV vaccine development.

Potent, persistent induction and modulation of cellular immune responses in rhesus macaques primed with Ad5hr-simian immunodeficiency virus (SIV) env/rev, gag, and/or nef vaccines and boosted with SIV gp120.

ABSTRACT Immunity elicited by multicomponent vaccines delivered by replication-competent Ad5hr-simian immunodeficiency virus (SIV) recombinants was systematically investigated. Rhesus macaques were immunized mucosally at weeks 0 and 12 with Ad5hr-SIVsmH4env/rev, with or without Ad5hr-SIVmac239gag or Ad5hr-SIVmac239nef, or with all three recombinants. The total Ad5hr dosage was comparably adjusted among all animals with empty Ad5hr-ΔE3 vector. The macaques were boosted with SIV gp120 in monophosphoryl A-stable emulsion adjuvant at 24 and 36 weeks. Controls received Ad5hr-ΔE3 vector or adjuvant only. By ELISPOT analysis, all four SIV gene products elicited potent cellular immune responses that persisted 42 weeks post-initial immunization. Unexpectedly, modulation of this cellular immune response was observed among macaques receiving one, two, or three Ad5hr-SIV recombinants. Env responses were significantly enhanced throughout the immunization period in macaques immunized with Ad5hr-SIV env/rev plus Ad5hr-SIV gag and tended to be higher in macaques that also received Ad5hr-SIV nef. Macaques primed with all three recombinants displayed significant down-modulation in numbers of gamma interferon (IFN-γ)-secreting cells specific for SIV Nef, and the Env- and Gag-specific responses were also diminished. Modulation of antibody responses was not observed. Down-modulation was seen only during the period of Ad5hr-recombinant priming, not during subunit boosting, although SIV-specific IFN-γ-secreting cells persisted. The effect was not attributable to Ad5hr replication differences among immunization groups. Vaccine delivery via replication-competent live vectors, which can persistently infect new cells and continuously present low-level antigen, may be advantageous in overcoming competition among complex immunogens for immune recognition. Effects of current multicomponent vaccines on individual immune responses should be evaluated with regard to future vaccine design.

A call for replicating vector prime-protein boost strategies in HIV vaccine design.

A key challenge to HIV vaccine development is the integration of HIV proviral DNA into the host genome upon infection. Therefore, an optimal vaccine should block infection within hours of viral exposure, providing ‘sterilizing immunity’ at mucosal sites and in blood via potent, broadly reactive antibody to the HIV envelope glycoprotein. This is difficult due to the envelope’s conformational complexity and sequence diversity. Antibodies that do not completely prevent infection nevertheless could reduce the viral infectious burden, allowing strong cellular immunity to control viremia, delay disease progression and prevent viral transmission, while also providing help for T- and B-cell responses. Rapidly responsive, potent, persistent immunity might best be achieved using prime–boost strategies incorporating a replicating vector and an optimally designed envelope subunit.

A Replication Competent Adenovirus 5 Host Range Mutant-Simian Immunodeficiency Virus (SIV) Recombinant Priming/Subunit Protein Boosting Vaccine Regimen Induces Broad, Persistent SIV-Specific Cellular Immunity to Dominant and Subdominant Epitopes in Mamu-A*01 Rhesus Macaques

CTL are important in controlling HIV and SIV infection. To quantify cellular immune responses induced by immunization, CD8+ T cells specific for the subdominant Env p15m and p54m epitopes and/or the dominant Gag p11C epitope were evaluated by tetramer staining in nine macaques immunized with an adenovirus (Ad) 5 host range mutant (Ad5hr)-SIVenv/rev recombinant and in four of nine which also received an Ad5hr-SIVgag recombinant. Two Ad5hr-SIV recombinant priming immunizations were followed by two boosts with gp120 protein or an envelope polypeptide representing the CD4 binding domain. Two mock-immunized macaques served as controls. IFN-γ-secreting cells were also assessed by ELISPOT assay using p11C, p15m, and p54m peptide stimuli and overlapping pooled Gag and Env peptides. As shown by tetramer staining, Ad-recombinant priming elicited a high frequency of persistent CD8+ T cells able to recognize p11C, p15m, and p54m epitopes. The presence of memory cells 38 wk postinitial immunization was confirmed by expansion of tetramer-positive CD8+ T cells following in vitro stimulation. The SIV-specific CD8+ T cells elicited were functional and secreted IFN-γ in response to SIV peptide stimuli. Although the level and frequency of response of peripheral blood CD8+ T cells to the subdominant Env epitopes were not as great as those to the dominant p11C epitope, elevated responses were observed when lymph node CD8+ T cells were evaluated. Our data confirm the potency and persistence of functional cellular immune responses elicited by replication competent Ad-recombinant priming. The cellular immunity elicited is broad and extends to subdominant epitopes.

Human Immunodeficiency Virus Type 1 Induces Apoptosis in CD4+ but Not in CD8+ T Cells in Ex Vivo-Infected Human Lymphoid Tissue

ABSTRACT Progression of human immunodeficiency virus (HIV) disease is associated with massive death of CD4+ T cells along with death and/or dysfunction of CD8+ T cells. In vivo, both HIV infection per se and host factors may contribute to the death and/or dysfunction of CD4+ and CD8+ T cells. Progression of HIV disease is often characterized by a switch from R5 to X4 HIV type 1 (HIV-1) variants. In human lymphoid tissues ex vivo, it was shown that HIV infection is sufficient for CD4+T-cell depletion. Here we address the question of whether infection of human lymphoid tissue ex vivo with prototypic R5 or X4 HIV variants also depletes or impairs CD8+ T cells. We report that whereas productive infection of lymphoid tissue ex vivo with R5 and X4 HIV-1 isolates induced apoptosis in CD4+ T cells, neither viral isolate induced apoptosis in CD8+ T cells. Moreover, in both infected and control tissues we found similar numbers of CD8+ T cells and similar production of cytokines by these cells in response to phorbol myristate acetate or anti-CD3–anti-CD28 stimulation. Thus, whereas HIV-1 infection per se in human lymphoid tissue is sufficient to trigger apoptosis in CD4+ T cells, the death of CD8+ T cells apparently requires additional factors.

Boosting of SIV-specific immune responses in rhesus macaques by repeated administration of Ad5hr-SIVenv/rev and Ad5hr-SIVgag recombinants.

Previous non-human primate studies have shown replication competent adenovirus (Ad) HIVenv/rev and SIVenv/rev recombinants to be promising vaccine candidates. To broaden induced immunity in rhesus macaques, an Ad type 5 host range (Ad5hr) mutant vector with an inserted SIV gag gene was added to the vaccine regimen. Immunity to the encoded SIV Env, Rev, and Gag gene products was evaluated following two immunizations with the same recombinants. The vaccines were administered intranasally plus orally via stomach tube at weeks 0 and 12. The recombinants replicated well in the upper respiratory tract but poorly in the gut, suggesting enteric-coated capsules might improve oral delivery to the intestine. SIV-specific cellular immunity was induced in all 16 immunized macaques. Fourteen exhibited positive interferon-gamma (IFN-gamma) ELISPOT responses, and nine, including two lacking IFN-gamma responses, exhibited SIV-specific T-cell proliferative activity. IFN-gamma secreting peripheral blood mononuclear cells (PBMCs) in response to SIV Gag, Env, and Rev peptides were induced in 73, 53, and 27% of macaques, respectively, and were boosted two- to four-fold by the second immunization. A persistent response to Gag was evident at least 10 weeks thereafter. p11C tetramer staining confirmed elicitation of SIV Gag-specific CD8+ T-cells in Mamu-A*01 macaques. Proliferative responses were more frequent after the second immunization, and binding antibody titers to SIV gp120 were significantly boosted by the immunization regimen. We conclude that a second administration of recombinants based in the same Ad5hr vector can effectively boost immunity to inserted gene products, obviating development of several recombinants in different Ad serotypes for multiple immunizations.

Potent, persistent cellular immune responses elicited by sequential immunization of rhesus macaques with Ad5 host range mutant recombinants encoding SIV Rev and SIV Nef

Vaccines incorporating multiple HIV components should elicit broad immunity and protection against a spectrum of HIV strains. Early regulatory and accessory gene products are attractive candidates for such vaccines. Here, immunogenicity studies on SIV Rev and Nef expressed in replication competent Adenovirus type 5 host range mutant vectors (Ad5hr) are summarized. Interferon-gamma (IFN-gamma)-secreting cells in response to Env and Rev peptides were enumerated by ELISPOT after two sequential immunizations of 55 macaques with Ad5hr-SIVenv/rev. Responses to SIV Nef were assessed in 16 macaques also immunized with Ad5hr-SIVnef. Potent cellular immunity to both Rev and Nef was induced following the second Ad-recombinant immunization and persisted for at least 30 weeks. Persistent cellular immunity to SIV Env was also seen, with a mean of 700 IFN-gamma-secreting cells per million PBMC. Rev and Env responses were positively correlated. While greater responses to early gene products occur in natural infection, as immunogens Rev and Nef elicited the same number of IFN-gamma secreting cells as Env, after adjusting for differences in protein size. The same percentage of macaques also responded to Rev, Nef, and Env: 59, 63, and 64%, respectively. Overall, Ad5hrSIVenv/rev and -SIVnef were highly effective immunogens. Their contribution to protective efficacy will be addressed in future studies.

Evaluation of combination DNA/replication-competent Ad-SIV recombinant immunization regimens in rhesus macaques

Combination vaccine regimens in which priming with recombinant DNA is followed by boosting with recombinant viral vectors have been shown in previous studies to effectively enhance cellular immunity. However, no information exists concerning possible synergy of the cellular immune response when DNA immunization is followed by administration of a recombinant vector able to replicate. As our approach makes use of replication-competent Ad HIV and SIV recombinants, we performed a pilot experiment in six rhesus macaques in which we compared immunogenicity resulting from priming with one or two DNA recombinants encoding the SIVsmH4 env and rev genes with that elicited by a single replication-competent Ad5hr-SIV env/rev priming immunization. All macaques were subsequently administered an Ad5hr-SIV env/rev booster immunization followed by two immunizations with SIV gp120 protein. The choice of the env gene as target immunogen allowed comparison of induced cellular immune responses as well as binding and neutralizing antibodies elicited in serum and mucosal secretions. We report here that all immunized monkeys developed strong cellular immunity to the SIV envelope as shown by secretion of interferon-gamma, lysis of envelope-expressing target cells, and/or proliferation in response to gp120 or inactivated SIV. Similarly, all macaques developed anti-gp120 binding antibodies and neutralizing antibodies in serum and IgG and IgA binding antibodies in mucosal secretions. We did not observe consistently enhanced immune responses in any immunization group. We conclude that two sequential immunizations with the same replication-competent Ad5hr-SIV recombinant is as effective as priming with one or two recombinant DNA vaccines followed by a single Ad5hrSIV recombinant immunization.

Prime-boost AIDS Vaccine Strategies Based on Replication-Competent Adenovirus Recombinants

The potent, persistent immunity needed to prevent HIV infection might be best achieved by priming cellular immunity with replicating vectors and boosting antibodies with optimally designed envelopes. Replicating Ad vaccines infect epithelial cells on mucosal surfaces and thus also elicit mucosal immunity. In chimpanzees, compared to non-replicating Ad vaccines, at the same or lower dose replicating Ad vaccines were better at eliciting cellular immunity and priming antibody responses. Mismatched envelope boosts induced broad neutralizing activity to diverse R5 viruses and cross-clade ADCC activity. Multigenic Ad-SIV vaccines and SIV envelope subunit boosts elicited strong protection in 39% of rhesus macaques challenged mucosally with SIVmac251. Durability of protection against a second challenge was established in 73% of previously protected animals, associated with persistent cellular immunity. Induction of memory cells and broad, strong functional antibodies illustrates the promise of this prime-boost vaccine strategy. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005