Prototype Pathogens for Vaccine and Monoclonal Antibody Countermeasure Development: NIAID Workshop Process and Outcomes for Viral Families of Pandemic Potential.

Given the increased risk of pandemics driven by emerging and reemerging infectious diseases, it is imperative that the United States and global scientific community be better prepared for future threats by prioritizing and launching key research programs and strategies. In December 2021, the National Institute of Allergy and Infectious Diseases (NIAID) published its pandemic preparedness plan, which focuses on the prototype pathogen approach for medical countermeasure development. The plan was introduced before its release at a NIAID-hosted workshop in November 2021 that featured scientific experts from the extramural community, government, and the private sector and focused on selection of prototype pathogens from 10 viral families with pandemic risk and moderate resources. This article will serve as an introduction to this special issue and will briefly define the prototype pathogen approach, describe the workshop goals and process for outcomes, and provide an outline of the viral working group articles to follow and future directions for NIAID.

Prototype Pathogen Approach for Vaccine and Monoclonal Antibody Development: A Critical Component of the NIAID Plan for Pandemic Preparedness.

Severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) emerged 20 years ago, presaging a series of subsequent infectious disease epidemics of international concern. The recent emergence of SARS-CoV-2 has underscored the importance of targeted preparedness research to enable rapid countermeasure development during a crisis. In December 2021 the National Institute of Allergy and Infectious Diseases (NIAID), building upon the successful strategies developed during the SARS-CoV-2 response and to prepare for future pandemics, published a pandemic preparedness plan that outlined a research strategy focused on priority pathogens, technology platforms, and prototype pathogens. To accelerate the discovery, development, and evaluation of medical countermeasures against new or previously unknown pathogens of pandemic potential, we present here a strategy of research directed at select prototype pathogens. In this manner, leveraging a prototype pathogen approach may serve as a powerful cornerstone in biomedical research preparedness to protect public health from newly emerging and reemerging infectious diseases.

Humoral immunity induced by mucosal and/or systemic SIV-specific vaccine platforms suggests novel combinatorial approaches for enhancing responses.

Combinatorial HIV/SIV vaccine approaches targeting multiple arms of the immune system might improve protective efficacy. We compared SIV-specific humoral immunity induced in rhesus macaques by five vaccine regimens. Systemic regimens included ALVAC-SIVenv priming and Env boosting (ALVAC/Env); DNA immunization; and DNA plus Env co-immunization (DNA&Env). RepAd/Env combined mucosal replication-competent Ad-env priming with systemic Env boosting. A Peptide/Env regimen, given solely intrarectally, included HIV/SIV peptides followed by MVA-env and Env boosts. Serum antibodies mediating neutralizing, phagocytic and ADCC activities were induced by ALVAC/Env, RepAd/Env and DNA&Env vaccines. Memory B cells and plasma cells were maintained in the bone marrow. RepAd/Env vaccination induced early SIV-specific IgA in rectal secretions before Env boosting, although mucosal IgA and IgG responses were readily detected at necropsy in ALVAC/Env, RepAd/Env, DNA&Env and DNA vaccinated animals. Our results suggest that combined RepAd priming with ALVAC/Env or DNA&Env regimen boosting might induce potent, functional, long-lasting systemic and mucosal SIV-specific antibodies.

Antibodies to gp120 and PD-1 expression on virus-specific CD8+ T cells in protection from simian AIDS.

We compared the relative efficacies against simian immunodeficiency virus (SIV) challenge of three vaccine regimens that elicited similar frequencies of SIV-specific CD4(+) and CD8(+) T-cell responses but differed in the level of antibody responses to the gp120 envelope protein. All macaques were primed with DNA plasmids expressing SIV gag, pol, env, and Retanef genes and were boosted with recombinant modified vaccinia Ankara virus (MVA) expressing the same genes, either once (1 × MVA) or twice (2 × MVA), or were boosted once with MVA followed by a single boost with replication-competent adenovirus (Ad) type 5 host range mutant (Ad5 h) expressing SIV gag and nef genes but not Retanef or env (1 × MVA/Ad5). While two of the vaccine regimens (1 × MVA and 1 × MVA/Ad5) protected from high levels of SIV replication only during the acute phase of infection, the 2 × MVA regimen, with the highest anti-SIV gp120 titers, protected during the acute phase and transiently during the chronic phase of infection. Mamu-A*01 macaques of this third group exhibited persistent Gag CD8(+)CM9(+) effector memory T cells with low expression of surface Programmed death-1 (PD-1) receptor and high levels of expression of genes associated with major histocompatibility complex class I (MHC-I) and MHC-II antigen. The fact that control of SIV replication was associated with both high titers of antibodies to the SIV envelope protein and durable effector SIV-specific CD8(+) T cells suggests the hypothesis that the presence of antibodies at the time of challenge may increase innate immune recruiting activity by enhancing antigen uptake and may result in improvement of the quality and potency of secondary SIV-specific CD8(+) T-cell responses.

NK and CD4+ T cell cooperative immune responses correlate with control of disease in a macaque simian immunodeficiency virus infection model.

Control of infectious disease may be accomplished by successful vaccination or by complex immunologic and genetic factors favoring Ag-specific multicellular immune responses. Using a rhesus macaque model, we evaluated Ag-specific T cell-dependent NK cell immune responses in SIV-infected macaques, designated "controlling" or "noncontrolling" based on long-term chronic viremia levels, to determine whether NK cell effector functions contribute to control of SIV infection. We observed that Gag stimulation of macaque PBMCs induced subset-specific NK cell responses in SIV-controlling but not SIV-noncontrolling animals, as well as that circulatory NK cell responses were dependent on Ag-specific IL-2 production by CD4(+) central memory T cells. NK cell activation was blocked by anti-IL-2-neutralizing Ab and by CD4(+) T cell depletion, which abrogated the Gag-specific responses. Among tissue-resident cells, splenic and circulatory NK cells displayed similar activation profiles, whereas liver and mucosal NK cells displayed a decreased activation profile, similar in SIV-controlling and -noncontrolling macaques. Lack of T cell-dependent NK cell function was rescued in SIV-noncontrolling macaques through drug-mediated control of viremia. Our results indicate that control of disease progression in SIV-controlling macaques is associated with cooperation between Ag-specific CD4(+) T cells and NK cell effector function, which highlight the importance of such cell-to-cell cooperativity in adaptive immunity and suggest that this interaction should be further investigated in HIV vaccine development and other prophylactic vaccine approaches.

Replicating adenovirus-simian immunodeficiency virus (SIV) recombinant priming and envelope protein boosting elicits localized, mucosal IgA immunity in rhesus macaques correlated with delayed acquisition following a repeated low-dose rectal SIV(mac251) challenge.

We have shown that sequential replicating adenovirus type 5 host range mutant human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) recombinant priming delivered first intranasally (i.n.) plus orally and then intratracheally (i.t.), followed by envelope protein boosting, elicits broad cellular immunity and functional, envelope-specific serum and mucosal antibodies that correlate with protection from high-dose SIV and simian/human immunodeficiency virus (SHIV) challenges in rhesus macaques. Here we extended these studies to compare the standard i.n./i.t. regimen with additional mucosal administration routes, including sublingual, rectal, and vaginal routes. Similar systemic cellular and humoral immunity was elicited by all immunization routes. Central and effector memory T cell responses were also elicited by the four immunization routes in bronchoalveolar lavage fluid and jejunal, rectal, and vaginal tissue samples. Cellular responses in vaginal tissue were more compartmentalized, being induced primarily by intravaginal administration. In contrast, all immunization routes elicited secretory IgA (sIgA) responses at multiple mucosal sites. Following a repeated low-dose intrarectal (i.r.) challenge with SIV(mac251) at a dose transmitting one or two variants, protection against acquisition was not achieved except in one macaque in the i.r. immunized group. All immunized macaques exhibited reduced peak viremia compared to that of controls, correlated inversely with prechallenge serum antienvelope avidity, antibody-dependent cellular cytotoxicity (ADCC) titers, and percent antibody-dependent cell-mediated viral inhibition. Both antibody avidity and ADCC titers were correlated with the number of exposures required for infection. Notably, we show for the first time a significant correlation of vaccine-induced sIgA titers in rectal secretions with delayed acquisition. Further investigation of the characteristics and properties of the sIgA should elucidate the mechanism leading to this protective effect.

Genomic analysis reveals pre- and postchallenge differences in a rhesus macaque AIDS vaccine trial: insights into mechanisms of vaccine efficacy.

We have employed global transcriptional profiling of whole blood to identify biologically relevant changes in cellular gene expression in response to alternative AIDS vaccine strategies with subsequent viral challenge in a rhesus macaque vaccine model. Samples were taken at day 0 (prechallenge), day 14 (peak viremia), and week 12 (set point) from animals immunized with replicating adenovirus type 5 host range (Ad5hr) recombinant viruses expressing human immunodeficiency virus HIV(env)(89.6P), simian immunodeficiency virus SIV(gag)(239), or SIV(nef)(239) alone or in combination with two intramuscular boosts with HIV(89.6P)gp140ΔCFI protein (L. J. Patterson et al., Virology 374:322-337, 2008), and each treatment resulted in significant control of viremia following simian-human immunodeficiency virus SHIV(89.6P) challenge (six animals per group plus six controls). At day 0, 8 weeks after the last treatment, the microarray profiles revealed significant prechallenge differences between treatment groups; data from the best-protected animals led to identification of a network of genes related to B cell development and lymphocyte survival. At peak viremia, expression profiles of the immunized groups were extremely similar, and comparisons to control animals reflected immunological differences other than effector T cell functions. Suggested protective mechanisms for vaccinated animals included upregulation of interleukin-27, a cytokine known to inhibit lentivirus replication, and increased expression of complement components, which may synergize with vaccine-induced antibodies. Divergent expression profiles at set point for the immunized groups implied distinct immunological responses despite phenotypic similarities in viral load and CD4(+) T cell levels. Data for the gp140-boosted group provided evidence for antibody-dependent, cell-mediated viral control, whereas animals immunized with only the replicating Ad5hr recombinants exhibited a different evolution of the B cell compartment even at 3 months postchallenge. This study demonstrates the sensitivity and discrimination of gene expression profiling of whole blood as an analytical tool in AIDS vaccine trials, providing unique insights into in vivo mechanisms and potential correlates of protection.

Multiple vaccine-elicited nonneutralizing antienvelope antibody activities contribute to protective efficacy by reducing both acute and chronic viremia following simian/human immunodeficiency virus SHIV89.6P challenge in rhesus macaques.

We have shown that following priming with replicating adenovirus type 5 host range mutant (Ad5hr)-human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) recombinants, boosting with gp140 envelope protein enhances acute-phase protection against intravenous simian/human immunodeficiency virus (SHIV)(89.6P) challenge compared to results with priming and no boosting or boosting with an HIV polypeptide representing the CD4 binding site of gp120. We retrospectively analyzed antibodies in sera and rectal secretions from these same macaques, investigating the hypothesis that vaccine-elicited nonneutralizing antibodies contributed to the better protection. Compared to other immunized groups or controls, the gp140-boosted group exhibited significantly greater antibody activities mediating antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell-mediated viral inhibition (ADCVI) in sera and transcytosis inhibition in rectal secretions. ADCC and ADCVI activities were directly correlated with antibody avidity, suggesting the importance of antibody maturation for functionality. Both ADCVI and percent ADCC killing prechallenge were significantly correlated with reduced acute viremia. The latter, as well as postchallenge ADCVI and ADCC, was also significantly correlated with reduced chronic viremia. We have previously demonstrated induction by the prime/boost regimen of mucosal antibodies that inhibit transcytosis of SIV across an intact epithelial cell layer. Here, antibody in rectal secretions was significantly correlated with transcytosis inhibition. Importantly, the transcytosis specific activity (percent inhibition/total secretory IgA and IgG) was strongly correlated with reduced chronic viremia, suggesting that mucosal antibody may help control cell-to-cell viral spread during the course of infection. Overall, the replicating Ad5hr-HIV/SIV priming/gp140 protein boosting approach elicited strong systemic and mucosal antibodies with multiple functional activities associated with control of both acute and chronic viremia.

Sequential priming with simian immunodeficiency virus (SIV) DNA vaccines, with or without encoded cytokines, and a replicating adenovirus-SIV recombinant followed by protein boosting does not control a pathogenic SIVmac251 mucosal challenge.

Previously, combination DNA/nonreplicating adenovirus (Ad)- or poxvirus-vectored vaccines have strongly protected against SHIV(89.6P), DNAs expressing cytokines have modulated immunity elicited by DNA vaccines, and replication-competent Ad-recombinant priming and protein boosting has strongly protected against simian immunodeficiency virus (SIV) challenge. Here we evaluated a vaccine strategy composed of these promising components. Seven rhesus macaques per group were primed twice with multigenic SIV plasmid DNA with or without interleukin-12 (IL-12) DNA or IL-15 DNA. After a multigenic replicating Ad-SIV immunization, all groups received two booster immunizations with SIV gp140 and SIV Nef protein. Four control macaques received control DNA plasmids, empty Ad vector, and adjuvant. All vaccine components were immunogenic, but the cytokine DNAs had little effect. Macaques that received IL-15-DNA exhibited higher peak anti-Nef titers, a more rapid anti-Nef anamnestic response postchallenge, and expanded CD8(CM) T cells 2 weeks postchallenge compared to the DNA-only group. Other immune responses were indistinguishable between groups. Overall, no protection against intrarectal challenge with SIV(mac251) was observed, although immunized non-Mamu-A*01 macaques as a group exhibited a statistically significant 1-log decline in acute viremia compared to non-Mamu-A*01 controls. Possible factors contributing to the poor outcome include administration of cytokine DNAs to sites different from the Ad recombinants (intramuscular and intratracheal, respectively), too few DNA priming immunizations, a suboptimal DNA delivery method, failure to ensure delivery of SIV and cytokine plasmids to the same cell, and instability and short half-life of the IL-15 component. Future experiments should address these issues to determine if this combination approach is able to control a virulent SIV challenge.

A simplified method for the rapid fluorometric assessment of antibody-dependent cell-mediated cytotoxicity.

We demonstrate that the FATAL cytolysis assay can be adapted into a rapid and fluorometric antibody-dependent cellular cytotoxicity assay (RFADCC). The RFADCC relies on double-staining target cells with a membrane dye (PKH-26) and a viability dye (CFSE) prior to the addition of antibody and effector cells. We used the RFADCC to assess dose-dependent and envelope-specific anti-human immunodeficiency virus (HIV) ADCC responses mediated by monoclonal antibody-2G12 and human sera. Using the assay, we also detected early anti-simian immunodeficiency virus (SIV) ADCC responses in rhesus macaques infected with pathogenic SIV(mac251). Importantly, the RFADCC was further useful in monitoring anti-HIV and anti-SIV ADCC responses elicited by immunizing chimpanzees and rhesus macaques with replicating adenovirus-based AIDS vaccine candidates. In comparison to the standard chromium release assay, the RFADCC provides a higher cell killing readout and is advantageous in allowing use of viably frozen as well as fresh effector cells, thus facilitating assay standardization. The RFADCC is therefore a simple, reliable, and highly sensitive method that can be applied to assess the ADCC activity of monoclonal antibodies as well as ADCC responses elicited by HIV or SIV infection or by AIDS vaccine candidates.

Enhanced immunity and protective efficacy against SIVmac251 intrarectal challenge following ad-SIV priming by multiple mucosal routes and gp120 boosting in MPL-SE.

Previously, 39% of rhesus macaques primed orally, intranasally, and intratracheally with adenovirus (Ad)-simian immunodeficiency virus (SIV) recombinants and boosted with gp120 in monophosphoryl lipid A-stable emulsion (MPL-SE) remained aviremic or cleared or controlled viremia at the threshold of detection following SIV(mac251) intrarectal challenge (Study B). In contrast, no macaques primed orally and intranasally with Ad-SIV recombinants and boosted with gp120 in Quillaja Saponaria-21 exhibited undetectable viremia post-challenge (Study A). We conducted a detailed comparison of the studies to elucidate the effect of different vaccine regimens on induced immunity associated with the different challenge outcomes. Quantitative viral load comparisons were statistically analyzed. All immune responses were assessed at identical timepoints post-immunization, and cellular immunity was re-evaluated on cryopreserved cells from Study B macaques to match Study A data acquired with frozen cells. Study B exhibited greater protective efficacy, increased levels of p11C and p54m tetramer positive cells and a trend toward enhanced interferon-gamma secreting cells in response to Env and Gag peptides, modestly enhanced serum neutralizing antibodies, and greater positivity in anti-gp120 rectal IgA and IgG antibodies. Study A macaques exhibited greater positivity in salivary IgA anti-gp120 antibodies. Thus, the vaccine regimen using oral-intranasal-intratracheal priming and protein boosting in MPL-SE was superior, eliciting greater protective efficacy against pathogenic SIV(mac251) and enhanced SIV-specific immunity, systemically and at rectal sites. The mechanism(s) by which binding antibodies, lacking neutralizing activity against the primary challenge virus, may contribute to protection requires further study.

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.

Replicating adenovirus-simian immunodeficiency virus (SIV) vectors efficiently prime SIV-specific systemic and mucosal immune responses by targeting myeloid dendritic cells and persisting in rectal macrophages, regardless of immunization route.

Although priming with replicating adenovirus type 5 host range mutant (Ad5hr)-human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) recombinants, followed by HIV/SIV envelope boosting, has proven highly immunogenic, resulting in protection from SIV/simian-human immunodeficiency virus (SHIV) challenges, Ad5hr recombinant distribution, replication, and persistence have not been examined comprehensively in nonhuman primates. We utilized Ad5hr-green fluorescent protein and Ad5hr-SIV recombinants to track biodistribution and immunogenicity following mucosal priming of rhesus macaques by the intranasal/intratracheal, sublingual, vaginal, or rectal route. Ad recombinants administered by all routes initially targeted macrophages in bronchoalveolar lavage (BAL) fluid and rectal tissue, later extending to myeloid dendritic cells in BAL fluid with persistent expression in rectal mucosa 25 weeks after the last Ad immunization. Comparable SIV-specific immunity, including cellular responses, serum binding antibody, and mucosal secretory IgA, was elicited among all groups. The ability of the vector to replicate in multiple mucosal sites irrespective of delivery route, together with the targeting of macrophages and professional antigen-presenting cells, which provide potent immunogenicity at localized sites of virus entry, warrants continued use of replicating Ad vectors.

Strong viremia control in vaccinated macaques does not prevent gradual Th17 cell loss from central memory.

It has been proposed that microbial translocation might play a role in chronic immune activation during HIV/SIV infection. Key roles in fighting bacterial and fungal infections have been attributed to Th17 and Tc17 cells. Th17 cells can be infected with HIV/SIV, however whether effective vaccination leads to their maintenance following viral challenge has not been addressed. Here we retrospectively investigated if a vaccine regimen that potently reduced viremia post-challenge preserved Th17 and Tc17 cells, thus adding benefit in the absence of sterilizing protection. Rhesus macaques were previously vaccinated with replication-competent Adenovirus recombinants expressing HIVtat and HIVenv followed by Tat and gp140 protein boosting. Upon SHIV(89.6P) challenge, the vaccines exhibited a significant 4 log reduction in chronic viremia compared to sham vaccinated controls which rapidly progressed to AIDS [39]. Plasma and cryopreserved PBMC samples were examined pre-challenge and during acute and chronic infection. Control macaques exhibited a rapid loss of CD4(+) cells, including Th17 cells. Tc17 cells tended to decline over the course of infection although significance was not reached. Immune activation, assessed by Ki-67 expression, was associated with elevated chronic viremia of the controls. Significantly increased plasma IFN-γ levels were also observed. No increase in plasma LPS levels were observed suggesting a lack of microbial translocation. In contrast, vaccinated macaques had no evidence of immune activation within the chronic phase and preserved both CD4(+) T-cells and Tc17 cells in PBMC. Nevertheless, they exhibited a gradual, significant loss of Th17 cells which concomitantly displayed significantly higher CCR6 expression over time. The gradual Th17 cell decline may reflect mucosal homing to inflammatory sites and/or slow depletion due to ongoing low levels of SHIV replication. Our results suggest that potent viremia reduction during chronic SHIV infection will delay but not prevent the loss of Th17 cells.

Vaccine-elicited SIV and HIV envelope-specific IgA and IgG memory B cells in rhesus macaque peripheral blood correlate with functional antibody responses and reduced viremia.

An effective HIV vaccine requires strong systemic and mucosal, cellular and humoral immunity. Numerous non-human primate studies have investigated memory T cells, but not memory B cells. Humoral immunologic memory is mediated by long-lived antibody-secreting plasma cells and differentiation of memory B cells into short-lived plasma blasts following re-exposure to immunizing antigen. Here we studied memory B cells in vaccinated rhesus macaques. PBMC were stimulated polyclonally using CD40 Ligand, IL-21 and CpG to induce B cell proliferation and differentiation into antibody secreting cells (ASCs). Flow cytometry was used for phenotyping and evaluating proliferation by CFSE dilution. B cell responses were quantified by ELISPOT. Methodology was established using PBMC of vaccinated elite-controller macaques that exhibited strong, multi-functional antibody activities. Subsequently, memory B cells elicited by two replicating Ad-recombinant prime/envelope boost regimens were retrospectively evaluated pre- and post-SIV and SHIV challenges. The vaccine regimens induced SIV and HIV Env-specific IgG and IgA memory B cells. Prior to challenge, IgA memory B cells were more numerous than IgG memory B cells, reflecting the mucosal priming immunizations. Pre- and post-challenge memory B cells were correlated with functional antibody responses including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated viral inhibition (ADCVI) and transcytosis inhibition. Post-challenge, Env-specific IgG and IgA memory B cells were correlated with reduced chronic viremia. We conclude that functional antibody responses elicited by our prime/boost regimen were effectively incorporated into the memory B cell pool where they contributed to control of viremia following re-exposure to the immunizing antigen.

Rapid SIV Env-specific mucosal and serum antibody induction augments cellular immunity in protecting immunized, elite-controller macaques against high dose heterologous SIV challenge.

Three Indian rhesus macaques, Ad-SIV primed/protein boosted and exposed twice to high-dose mucosal SIV(mac251) challenges, exhibited elite control of viremia over 6.5 years. They were negative for host factors associated with control of SIV infection. After a third intrarectal challenge with SIV(smE660), all controlled viremia, with one (macaque #5) maintaining undetectable viremia in blood. Acquisition was not blocked, but virus was contained in the jejunum and draining lymph nodes. Polyfunctional memory T cell responses and high-titered neutralizing and non-neutralizing serum and mucosal antibodies were present before and maintained post-challenge. The level of protection seen for animal #5 was predicted from analyses of gene transcription in jejunum 2 weeks post-challenge. Macaques #7 and #9, exhibiting lower pre-challenge cellular and humoral immunity, partially controlled the SIV(smE660) challenge. Initial vaccine-induced control by macaque #5 extended to the SIV(smE660) challenge due to multiple immune mechanisms that were boosted and augmented by cryptic SIV exposure.

Replicating adenovirus vector prime/protein boost strategies for HIV vaccine development.

BACKGROUND: In recent years the HIV vaccine field introduced a number of promising vaccine candidates into human clinical trials. OBJECTIVE: To briefly discuss the advances made in vaccine development and HIV pathogenesis and give an overview of the body of work our lab has generated in multiple animal models on replication-competent Adenovirus recombinant vaccines. METHODS: Emphasis is placed on comparative examination of vaccine components, routes of immunization and challenge models using replicating Adenovirus vectors. RESULTS/CONCLUSION: The findings make the case that replicating Adenovirus vectors are superior in priming multiple arms of the immune system, and in conjunction with protein boosting, have resulted in dramatic protective efficacy leading to their advancement to Phase I trials. Implications of the recent halting of the Merck Ad5-HIV Phase IIb clinical trial of our vaccine approach and other vectored vaccines are discussed.

Replicating adenovirus HIV/SIV recombinant priming alone or in combination with a gp140 protein boost results in significant control of viremia following a SHIV89.6P challenge in Mamu-A*01 negative rhesus macaques.

Previously, replicating adenovirus type 5 host range (Ad5hr)-HIV/SIV recombinant priming in combination with SIV envelope boosting, resulted in significant, durable protection in 39% of rhesus macaques after SIVmac251 challenge. Both Env-specific antibody mediating ADCC, and cellular immunity correlated with protection. Here we evaluate the relative immunogenicities of novel HIV proteins and their contribution to protection in a SHIV89.6P model. All groups were primed with Ad-HIVenv89.6P, SIVgag239, and SIVnef239 recombinants. One group was not boosted, one received HIV89.6Pgp140DeltaCFI protein, and one a novel HIV-1 poly-peptide "peptomer". The HIV89.6Pgp140DeltaCFI protein in adjuvant strongly boosted Env-specific antibody and memory T cell responses in blood and tissue, resulting in significant reductions in acute and set point viremia. Macaques not boosted, showed a significant reduction in set point viremia, a full 32 weeks after the last Ad priming immunization. The HIV peptomer-boosted group showed a trend toward chronic viremia reduction, but was not protected.

Comparative study of Tat vaccine regimens in Mauritian cynomolgus and Indian rhesus macaques: influence of Mauritian MHC haplotypes on susceptibility/resistance to SHIV(89.6P) infection.

Protection afforded by HIV Tat-based vaccines has differed in Indian rhesus and Mauritian cynomolgus macaques. We evaluated native Tat and Ad-HIVtat priming/Tat-boosting regimens in both species. Both vaccines were immunogenic. Only the Ad-tat regimen modestly reduced acute viremia in rhesus macaques after SHIV(89.6P) challenge. Confounding variables uncovered in Mauritian macaques included significant associations of susceptibility to infection with MHC class IB and class II H2 and H5 haplotypes, and resistance to infection with class IB haplotypes H3 and H6. Although protection here was limited, Tat-based vaccines incorporating other HIV components have shown greater efficacy. Combination strategies should be further explored.

A replication-competent adenovirus-human immunodeficiency virus (Ad-HIV) tat and Ad-HIV env priming/Tat and envelope protein boosting regimen elicits enhanced protective efficacy against simian/human immunodeficiency virus SHIV89.6P challenge in rhesus macaques.

We previously demonstrated that replication-competent adenovirus (Ad)-simian immunodeficiency virus (SIV) recombinant prime/protein boost regimens elicit potent immunogenicity and strong, durable protection of rhesus macaques against SIV(mac251). Additionally, native Tat vaccines have conferred strong protection against simian/human immunodeficiency virus SHIV(89.6P) challenge of cynomolgus monkeys, while native, inactivated, or vectored Tat vaccines have failed to elicit similar protective efficacy in rhesus macaques. Here we asked if priming rhesus macaques with replicating Ad-human immunodeficiency virus (HIV) tat and boosting with the Tat protein would elicit protection against SHIV(89.6P). We also evaluated a Tat/Env regimen, adding an Ad-HIV env recombinant and envelope protein boost to test whether envelope antibodies would augment acute-phase protection. Further, expecting cellular immunity to enhance chronic viremia control, we tested a multigenic group: Ad-HIV tat, -HIV env, -SIV gag, and -SIV nef recombinants and Tat, Env, and Nef proteins. All regimens were immunogenic. A hierarchy was observed in enzyme-linked immunospot responses (with the strongest response for Env, followed by Gag, followed by Nef, followed by Tat) and antibody titers (with the highest titer for Env, followed by Tat, followed by Nef, followed by Gag). Following intravenous SHIV(89.6P) challenge, all macaques became infected. Compared to controls, no protection was seen in the Tat-only group, confirming previous reports for rhesus macaques. However, the multigenic group blunted acute viremia by approximately 1 log (P = 0.017), and both the multigenic and Tat/Env groups reduced chronic viremia by 3 and 4 logs, respectively, compared to controls (multigenic, P = 0.0003; Tat/Env, P < 0.0001). The strikingly greater reduction in the Tat/Env group than in the multigenic group (P = 0.014) was correlated with Tat and Env binding antibodies. Since prechallenge anti-Env antibodies lacked SHIV(89.6P)-neutralizing activity, other functional anti-Env and anti-Tat activities are under investigation, as is a possible synergy between the Tat and Env immunogens.