Booster vaccination is required to elicit and maintain COVID-19 vaccine-induced immunity in SIV-infected macaques.

ABSTRACTProlonged infection and possible evolution of SARS-CoV-2 in patients living with uncontrolled HIV-1 infection highlight the importance of an effective vaccination regimen, yet the immunogenicity of COVID-19 vaccines and predictive immune biomarkers have not been well investigated. Herein, we report that the magnitude and persistence of antibody and cell-mediated immunity (CMI) elicited by an Ad5-vectored COVID-19 vaccine are impaired in SIV-infected macaques with high viral loads (> 105 genome copies per ml plasma, SIVhi) but not in macaques with low viral loads (< 105, SIVlow). After a second vaccination, the immune responses are robustly enhanced in all uninfected and SIVlow macaques. These responses also show a moderate increase in 70% SIVhi macaques but decline sharply soon after. Further analysis reveals that decreased antibody and CMI responses are associated with reduced circulating follicular helper T cell (TFH) counts and aberrant CD4/CD8 ratios, respectively, indicating that dysregulation of CD4+ T cells by SIV infection impairs the COVID-19 vaccine-induced immunity. Ad5-vectored COVID-19 vaccine shows no impact on SIV loads or SIV-specific CMI responses. Our study underscores the necessity of frequent booster vaccinations in HIV-infected patients and provides indicative biomarkers for predicting vaccination effectiveness in these patients.

Recombinant Simian Varicella Virus-Simian Immunodeficiency Virus Vaccine Induces T and B Cell Functions and Provides Partial Protection against Repeated Mucosal SIV Challenges in Rhesus Macaques.

HIV vaccine mediated efficacy, using an expanded live attenuated recombinant varicella virus-vectored SIV rSVV-SIVgag/env vaccine prime with adjuvanted SIV-Env and SIV-Gag protein boosts, was evaluated in a female rhesus macaques (RM) model against repeated intravaginal SIV challenges. Vaccination induced anti-SIV IgG responses and neutralizing antibodies were found in all vaccinated RMs. Three of the eight vaccinated RM remained uninfected (vaccinated and protected, VP) after 13 repeated challenges with the pathogenic SIVmac251-CX-1. The remaining five vaccinated and infected (VI) macaques had significantly reduced plasma viral loads compared with the infected controls (IC). A significant increase in systemic central memory CD4+ T cells and mucosal CD8+ effector memory T-cell responses was detected in vaccinated RMs compared to controls. Variability in lymph node SIV-Gag and Env specific CD4+ and CD8+ T cell cytokine responses were detected in the VI RMs while all three VP RMs had more durable cytokine responses following vaccination and prior to challenge. VI RMs demonstrated predominately SIV-specific monofunctional cytokine responses while the VP RMs generated polyfunctional cytokine responses. This study demonstrates that varicella virus-vectored SIV vaccination with protein boosts induces a 37.5% efficacy rate against pathogenic SIV challenge by generating mucosal memory, virus specific neutralizing antibodies, binding antibodies, and polyfunctional T-cell responses.

Development of a nonhuman primate challenge model to evaluate CD8+ T cell responses to an adenovirus-based vaccine expressing SIV proteins upon repeat-dose treatment with checkpoint inhibitors.

Targeting immune checkpoint receptors expressed in the T cell synapse induces active and long-lasting antitumor immunity in preclinical tumor models and oncology patients. However, traditional nonhuman primate (NHP) studies in healthy animals have thus far demonstrated little to no pharmacological activity or toxicity for checkpoint inhibitors (CPIs), likely due to a quiescent immune system. We developed a NHP vaccine challenge model in Mauritius cynomolgus monkey (MCMs) that elicits a strong CD8+ T cell response to assess both pharmacology and safety within the same animal. MHC I-genotyped MCMs were immunized with three replication incompetent adenovirus serotype 5 (Adv5) encoding Gag, Nef and Pol simian immunodeficiency virus (SIV) proteins administered 4 weeks apart. Immunized animals received the anti-PD-L1 atezolizumab or an immune checkpoint-targeting bispecific antibody (mAbX) in early development. After a single immunization, Adv5-SIVs induced T-cell activation as assessed by the expression of several co-stimulatory and co-inhibitory molecules, proliferation, and antigen-specific T-cell response as measured by a Nef-dependent interferon-γ ELIspot and tetramer analysis. Administration of atezolizumab increased the number of Ki67+ CD8+ T cells, CD8+ T cells co-expressing TIM3 and LAG3 and the number of CD4+ T cells co-expressing 4-1BB, BTLA, and TIM3 two weeks after vaccination. Both atezolizumab and mAbX extended the cytolytic activity of the SIV antigen-specific CD8+ T cell up to 8 weeks. Taken together, this vaccine challenge model allowed the combined study of pharmacology and safety parameters for a new immunomodulatory protein-based therapeutic targeting CD8+ T cells in an NHP model.

Recombinant Herpesvirus Vectors: Durable Immune Responses and Durable Protection against Simian Immunodeficiency Virus SIVmac239 Acquisition.

A prophylactic vaccine that confers durable protection against human immunodeficiency virus (HIV) would provide a valuable tool to prevent new HIV/AIDS cases. As herpesviruses establish lifelong infections that remain largely subclinical, the use of persistent herpesvirus vectors to deliver HIV antigens may facilitate the induction of long-term anti-HIV immunity. We previously developed recombinant (r) forms of the gamma-herpesvirus rhesus monkey rhadinovirus (rRRV) expressing a replication-incompetent, near-full-length simian immunodeficiency virus (SIVnfl) genome. We recently showed that 8/16 rhesus macaques (RMs) vaccinated with a rDNA/rRRV-SIVnfl regimen were significantly protected against intrarectal (i.r.) challenge with SIVmac239. Here we investigated the longevity of this vaccine-mediated protection. Despite receiving no additional booster immunizations, the protected rDNA/rRRV-SIVnfl vaccinees maintained detectable cellular and humoral anti-SIV immune responses for more than 1.5 years after the rRRV boost. To assess if these responses were still protective, the rDNA/rRRV-SIVnfl vaccinees were subjected to a second round of marginal-dose i.r. SIVmac239 challenges, with eight SIV-naive RMs serving as concurrent controls. After three SIV exposures, 8/8 control animals became infected, compared to 3/8 vaccinees. This difference in SIV acquisition was statistically significant (P = 0.0035). The three vaccinated monkeys that became infected exhibited significantly lower viral loads than those in unvaccinated controls. Collectively, these data illustrate the ability of rDNA/rRRV-SIVnfl vaccination to provide long-term immunity against stringent mucosal challenges with SIVmac239. Future work is needed to identify the critical components of this vaccine-mediated protection and the extent to which it can tolerate sequence mismatches in the challenge virus. IMPORTANCE We report on the long-term follow-up of a group of rhesus macaques (RMs) that received an AIDS vaccine regimen and were subsequently protected against rectal acquisition of simian immunodeficiency virus (SIV) infection. The vaccination regimen employed included a live recombinant herpesvirus vector that establishes persistent infection in RMs. Consistent with the recurrent SIV antigen expression afforded by this herpesvirus vector, vaccinees maintained detectable SIV-specific immune responses for more than 1.5 years after the last vaccination. Importantly, these vaccinated RMs were significantly protected against a second round of rectal SIV exposures performed 1 year after the first SIV challenge phase. These results are relevant for HIV vaccine development because they show the potential of herpesvirus-based vectors to maintain functional antiretroviral immunity without the need for repeated boosting.

Cervico-Vaginal Inflammatory Cytokine and Chemokine Responses to Two Different SIV Immunogens.

Studies have shown that vaccine vectors and route of immunization can differentially activate different arms of the immune system. However, the effects of different HIV vaccine immunogens on mucosal inflammation have not yet been studied. Because mucosal sites are the primary route of HIV infection, we evaluated the cervico-vaginal inflammatory cytokine and chemokine levels of Mauritian cynomolgus macaques following immunization and boost using two different SIV vaccine immunogens. The PCS vaccine delivers 12 20-amino acid peptides overlapping the 12 protease cleavage sites, and the Gag/Env vaccine delivers the full Gag and full Env proteins of simian immunodeficiency virus. We showed that the PCS vaccine prime and boosts induced short-lived, lower level increases of a few pro-inflammatory/chemotactic cytokines. In the PCS-vaccine group only the levels of MCP-1 were significantly increased above the baseline (P = 0.0078, Week 6; P = 0.0078, Week 17; P = 0.0234; Week 51) following multiple boosts. In contrast, immunizations with the Gag/Env vaccine persistently increased the levels of multiple cytokines/chemokines. In the Gag/Env group, higher than baseline levels were consistently observed for IL-8 (P = 0.0078, Week 16; P = 0.0078, Week 17; P = 0.0156, Week 52), IL-1ß (P = 0.0234, Week 16; P = 0.0156, Week 17; P = 0.0156, Week 52), and MIP-1α (P = 0.0313, Week 16; P = 0.0156, Week 17; P = 0.0313, Week 52). Over time, repeated boosts altered the relative levels of these cytokines between the Gag/Env and PCS vaccine group. 18 weeks after final boost with a higher dosage, IP-10 levels (P = 0.0313) in the Gag/Env group remained higher than baseline. Thus, the influence of vaccine immunogens on mucosal inflammation needs to be considered when developing and evaluating candidate HIV vaccines.

Vaccine targeting SIVmac251 protease cleavage sites protects macaques against vaginal infection.

After over 3 decades of research, an effective anti-HIV vaccine remains elusive. The recently halted HVTN702 clinical trial not only further stresses the challenge to develop an effective HIV vaccine but also emphasizes that unconventional and novel vaccine strategies are urgently needed. Here, we report that a vaccine focusing the immune response on the sequences surrounding the 12 viral protease cleavage sites (PCSs) provided greater than 80% protection to Mauritian cynomolgus macaques against repeated intravaginal SIVmac251 challenges. The PCS-specific T cell responses correlated with vaccine efficacy. The PCS vaccine did not induce immune activation or inflammation known to be associated with increased susceptibility to HIV infection. Machine learning analyses revealed that the immune microenvironment generated by the PCS vaccine was predictive of vaccine efficacy. Our study demonstrates, for the first time to our knowledge, that a vaccine which targets only viral maturation, but lacks full-length Env and Gag immunogens, can prevent intravaginal infection in a stringent macaque/SIV challenge model. Targeting HIV maturation thus offers a potentially novel approach to developing an effective HIV vaccine.

A single lentivector DNA based immunization contains a late heterologous SIVmac251 mucosal challenge infection.

Variety of conventional vaccine strategies tested against HIV-1 have failed to induce protection against HIV acquisition or durable control of viremia. Therefore, innovative strategies that can induce long lasting protective immunity against HIV chronic infection are needed. Recently, we developed an integration-defective HIV lentiDNA vaccine that undergoes a single cycle of replication in target cells in which most viral antigens are produced. A single immunization with such lentiDNA induced long-lasting T-cell and modest antibody responses in cynomolgus macaques. Here eighteen months after this single immunization, all animals were subjected to repeated low dose intra-rectal challenges with a heterologous pathogenic SIVmac251 isolate. Although the viral set point in SIVmac-infected cynomolgus is commonly lower than that seen in Indian rhesus macaques, the vaccinated group of macaques displayed a two log reduction of peak of viremia followed by a progressive and sustained control of virus replication relative to control animals. This antiviral control correlated with antigen-specific CD4+ and CD8+ T cells with high capacity of recall responses comprising effector and central memory T cells but also memory T cell precursors. This is the first description of SIV control in NHP model infected at 18 months following a single immunization with a non-integrative single cycle lentiDNA HIV vaccine. While not delivering sterilizing immunity, our single immunization strategy with a single-cycle lentivector DNA vaccine appears to provide an interesting and safe vaccine platform that warrants further exploration.

Immunotherapy with DNA vaccine and live attenuated rubella/SIV gag vectors plus early ART can prevent SIVmac251 viral rebound in acutely infected rhesus macaques.

Anti-retroviral therapy (ART) has been highly successful in controlling HIV replication, reducing viral burden, and preventing both progression to AIDS and viral transmission. Yet, ART alone cannot cure the infection. Even after years of successful therapy, ART withdrawal leads inevitably to viral rebound within a few weeks or months. Our hypothesis: effective therapy must control both the replicating virus pool and the reactivatable latent viral reservoir. To do this, we have combined ART and immunotherapy to attack both viral pools simultaneously. The vaccine regimen consisted of DNA vaccine expressing SIV Gag, followed by a boost with live attenuated rubella/gag vectors. The vectors grow well in rhesus macaques, and they are potent immunogens when used in a prime and boost strategy. We infected rhesus macaques by high dose mucosal challenge with virulent SIVmac251 and waited three days to allow viral dissemination and establishment of a reactivatable viral reservoir before starting ART. While on ART, the control group received control DNA and empty rubella vaccine, while the immunotherapy group received DNA/gag prime, followed by boosts with rubella vectors expressing SIV gag over 27 weeks. Both groups had a vaccine "take" to rubella, and the vaccine group developed antibodies and T cells specific for Gag. Five weeks after the last immunization, we stopped ART and monitored virus rebound. All four control animals eventually had a viral rebound, and two were euthanized for AIDS. One control macaque did not rebound until 2 years after ART release. In contrast, there was only one viral rebound in the vaccine group. Three out of four vaccinees had no viral rebound, even after CD8 depletion, and they remain in drug-free viral remission more than 2.5 years later. The strategy of early ART combined with immunotherapy can produce a sustained SIV remission in macaques and may be relevant for immunotherapy of HIV in humans.

Reduction of peak viremia by an integration-defective SIV proviral DNA vaccine in rhesus macaques.

An integrase-defective SIV (idSIV) vaccine delivered by a DNA prime and viral particle boost approach can suppress viral loads (VLs) during the acute infection stage after intravenous SIVmac239 challenge. This study investigated how idSIV DNA and viral particle immunization alone contributed to the suppression of VLs in Chinese rhesus macaques after SIV challenge. Two macaques were immunized with idSIV DNA five times and two macaques were immunized with idSIV viral particles three times. Cellular and humoral immune responses were measured in the vaccinated macaques after immunization. The VLs and CD4+ T cell counts were monitored for 28 weeks after the intravenous SIVmac239 challenge. The SIV-specific T cell responses were only detected in the DNA-vaccinated macaques. However, binding and neutralizing antibodies against autologous and heterologous viruses were moderately better in macaques immunized with viral particles than in macaques immunized with DNA. After the challenge, the mean peak viremia in the DNA group was 2.3 logs lower than that in the control group, while they were similar between the viral particle immunization and control groups. Similar CD4+ T cell counts were observed among all groups. These results suggest that idSIV DNA immunization alone reduces VLs during acute infection after SIV challenge in macaques and may serve as a key component in combination with other immunogens as prophylactic vaccines.

A Recombinant Rhesus Monkey Rhadinovirus Deleted of Glycoprotein L Establishes Persistent Infection of Rhesus Macaques and Elicits Conventional T Cell Responses.

A replication-competent, recombinant strain of rhesus monkey rhadinovirus (RRV) expressing the Gag protein of SIVmac239 was constructed in the context of a glycoprotein L (gL) deletion mutation. Deletion of gL detargets the virus from Eph family receptors. The ability of this gL-minus Gag recombinant RRV to infect, persist, and elicit immune responses was evaluated after intravenous inoculation of two Mamu-A*01+ RRV-naive rhesus monkeys. Both monkeys responded with an anti-RRV antibody response, and quantitation of RRV DNA in peripheral blood mononuclear cells (PBMC) by real-time PCR revealed levels similar to those in monkeys infected with recombinant gL+ RRV. Comparison of RRV DNA levels in sorted CD3+ versus CD20+ versus CD14+ PBMC subpopulations indicated infection of the CD20+ subpopulation by the gL-minus RRV. This contrasts with results obtained with transformed B cell lines in vitro, in which deletion of gL resulted in markedly reduced infectivity. Over a period of 20 weeks, Gag-specific CD8+ T cell responses were documented by major histocompatibility complex class I (MHC-I) tetramer staining. Vaccine-induced CD8+ T cell responses, which were predominantly directed against the Mamu-A*01-restricted Gag181-189CM9 epitope, could be inhibited by blockade of MHC-I presentation. Our results indicate that gL and the interaction with Eph family receptors are dispensable for the colonization of the B cell compartment following high-dose infection by the intravenous route, which suggests the existence of alternative receptors. Further, gL-minus RRV elicits cellular immune responses that are predominantly canonical in nature.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with a substantial disease burden in sub-Saharan Africa, often in the context of human immunodeficiency virus (HIV) infection. The related rhesus monkey rhadinovirus (RRV) has shown potential as a vector to immunize monkeys with antigens from simian immunodeficiency virus (SIV), the macaque model for HIV. KSHV and RRV engage cellular receptors from the Eph family via the viral gH/gL glycoprotein complex. We have now generated a recombinant RRV that expresses the SIV Gag antigen and does not express gL. This recombinant RRV was infectious by the intravenous route, established persistent infection in the B cell compartment, and elicited strong immune responses to the SIV Gag antigen. These results argue against a role for gL and Eph family receptors in B cell infection by RRV in vivo and have implications for the development of a live-attenuated KSHV vaccine or vaccine vector.

Vaccination of Macaques with DNA Followed by Adenoviral Vectors Encoding Simian Immunodeficiency Virus (SIV) Gag Alone Delays Infection by Repeated Mucosal Challenge with SIV.

Vaccines aimed at inducing T cell responses to protect against human immunodeficiency virus (HIV) infection have been under development for more than 15 years. Replication-defective adenovirus (rAd) vaccine vectors are at the forefront of this work and have been tested extensively in the simian immunodeficiency virus (SIV) challenge macaque model. Vaccination with rAd vectors coding for SIV Gag or other nonenvelope proteins induces T cell responses that control virus load but disappointingly is unsuccessful so far in preventing infection, and attention has turned to inducing antibodies to the envelope. However, here we report that Mauritian cynomolgus macaques (MCM), Macaca fascicularis, vaccinated with unmodified SIV gag alone in a DNA prime followed by an rAd boost exhibit increased protection from infection by repeated intrarectal challenge with low-dose SIVmac251. There was no evidence of infection followed by eradication. A significant correlation was observed between cytokine expression by CD4 T cells and delayed infection. Vaccination with gag fused to the ubiquitin gene or fragmented, designed to increase CD8 magnitude and breadth, did not confer resistance to challenge or enhance immunity. On infection, a significant reduction in peak virus load was observed in all vaccinated animals, including those vaccinated with modified gag These findings suggest that a nonpersistent viral vector vaccine coding for internal virus proteins may be able to protect against HIV type 1 (HIV-1) infection. The mechanisms are probably distinct from those of antibody-mediated virus neutralization or cytotoxic CD8 cell killing of virus-infected cells and may be mediated in part by CD4 T cells.IMPORTANCE The simian immunodeficiency virus (SIV) macaque model represents the best animal model for testing new human immunodeficiency virus type 1 (HIV-1) vaccines. Previous studies employing replication-defective adenovirus (rAd) vectors that transiently express SIV internal proteins induced T cell responses that controlled virus load but did not protect against virus challenge. However, we show for the first time that SIV gag delivered in a DNA prime followed by a boost with an rAd vector confers resistance to SIV intrarectal challenge. Other partially successful SIV/HIV-1 protective vaccines induce antibody to the envelope and neutralize the virus or mediate antibody-dependent cytotoxicity. Induction of CD8 T cells which do not prevent initial infection but eradicate infected cells before infection becomes established has also shown some success. In contrast, the vaccine described here mediates resistance by a different mechanism from that described above, which may reflect CD4 T cell activity. This could indicate an alternative approach for HIV-1 vaccine development.

Replicating Adenovirus-SIV Immunization of Rhesus Macaques Induces Mucosal Dendritic Cell Activation and Function Leading to Rectal Immune Responses.

Inducing strong mucosal immune responses by vaccination is important for providing protection against simian immunodeficiency virus (SIV). A replicating adenovirus type 5 host range mutant vector (Ad5hr) expressing SIV proteins induced mucosal immune responses in rectal tissue associated with delayed SIV acquisition in female rhesus macaques, but the initial mechanisms leading to the induced immunity have not been elucidated. As dendritic cells (DCs) are known to orchestrate both innate and adaptive effector immune cell responses, we investigated their role here. Rhesus macaques were immunized twice mucosally with a replicating Ad5hr expressing SIV Env, Gag, and Nef (Ad-SIV) or empty Ad5hr vector (Ad-Empty). DC subsets and their activation were examined in rectal tissue, blood, and LNs at 3 timepoints after each immunization. Plasmacytoid DCs, myeloid DCs, and Langerhans cells were significantly increased in the rectal mucosa, but only myeloid DCs were significantly increased in blood post-immunizations. All rectal DC subsets showed increased frequencies of cells expressing activation markers and cytokines post-immunization, blood DCs showed mixed results, and LN DCs showed few changes. Rectal DCs responded strongly to the vector rather than expressed SIV antigens, but rectal DC frequencies positively correlated with induced rectal antigen-specific memory T and B cells. These correlations were confirmed by in vitro co-cultures showing that rectal Ad-SIV DCs induced proliferation and antigen-specific cytokine production by autologous naïve T cells. Our results highlight the rapid response of DCs to Ad immunization and their role in mucosal immune activation and identify initial cellular mechanisms of the replicating Ad-SIV vaccine in the rhesus macaque model.

Analysis of Complement-Mediated Lysis of Simian Immunodeficiency Virus (SIV) and SIV-Infected Cells Reveals Sex Differences in Vaccine-Induced Immune Responses in Rhesus Macaques.

An effective human immunodeficiency virus (HIV) vaccine has yet to be developed, and defining immune correlates of protection against HIV infection is of paramount importance to inform future vaccine design. The complement system is a component of innate immunity that can directly lyse pathogens and shape adaptive immunity. To determine if complement lysis of simian immunodeficiency virus (SIV) and/or SIV-infected cells represents a protective immune correlate against SIV infection, sera from previously vaccinated and challenged rhesus macaques were analyzed for the induction of antibody-dependent complement-mediated lysis (ADCML). Importantly, the vaccine regimen, consisting of a replication-competent adenovirus type 5 host-range mutant SIV recombinant prime followed by a monomeric gp120 or oligomeric gp140 boost, resulted in overall delayed SIV acquisition only in females. Here, sera from all vaccinated animals induced ADCML of SIV and SIV-infected cells efficiently, regardless of sex. A modest correlation of SIV lysis with a reduced infection rate in males but not females, together with a reduced peak viremia in all animals boosted with gp140, suggested a potential for influencing protective efficacy. Gag-specific IgG and gp120-specific IgG and IgM correlated with SIV lysis in females, while Env-specific IgM correlated with SIV-infected cell lysis in males, indicating sex differences in vaccine-induced antibody characteristics and function. In fact, gp120/gp140-specific antibody functional correlates between antibody-dependent cellular cytotoxicity, antibody-dependent phagocytosis, and ADCML as well as the gp120-specific IgG glycan profiles and the corresponding ADCML correlations varied depending on the sex of the vaccinees. Overall, these data suggest that sex influences vaccine-induced antibody function, which should be considered in the design of globally effective HIV vaccines in the future.IMPORTANCE An HIV vaccine would thwart the spread of HIV infection and save millions of lives. Unfortunately, the immune responses conferring universal protection from HIV infection are poorly defined. The innate immune system, including the complement system, is an evolutionarily conserved, basic means of protection from infection. Complement can prevent infection by directly lysing incoming pathogens. We found that vaccination against SIV in rhesus macaques induces antibodies that are capable of directing complement lysis of SIV and SIV-infected cells in both sexes. We also found sex differences in vaccine-induced antibody species and their functions. Overall, our data suggest that sex affects vaccine-induced antibody characteristics and function and that males and females might require different immune responses to protect against HIV infection. This information could be used to generate highly effective HIV vaccines for both sexes in the future.

Gag and env conserved element CE DNA vaccines elicit broad cytotoxic T cell responses targeting subdominant epitopes of HIV and SIV Able to recognize virus-infected cells in macaques.

HIV sequence diversity and the propensity of eliciting immunodominant responses targeting inessential variable regions are hurdles in the development of an effective AIDS vaccine. We developed a DNA vaccine comprising conserved elements (CE) of SIV p27Gag and HIV-1 Env and found that priming vaccination with CE DNA is critical to efficiently overcome the dominance imposed by Gag and Env variable regions. Here, we show that DNA vaccinated macaques receiving the CE prime/CE+full-length DNA co-delivery booster vaccine regimens developed broad, potent and durable cytotoxic T cell responses targeting conserved protein segments of SIV Gag and HIV Env. Gag CE-specific T cells showed robust anamnestic responses upon infection with SIVmac239 which led to the identification of CE-specific cytotoxic lymphocytes able to recognize epitopes covering distinct CE on the surface of SIV infected cells in vivo. Though not controlling infection overall, we found an inverse correlation between Gag CE-specific CD8+ T cell responses and peak viremia. The T cell responses induced by the HIV Env CE immunogen were recalled in some animals upon SIV infection, leading to the identification of two cross-reactive epitopes between HIV and SIV Env based in sequence homology. These data demonstrate that a vaccine combining Gag and Env CE DNA subverted the normal immunodominance patterns, eliciting immune responses that included subdominant, highly conserved epitopes. These vaccine regimens augment cytotoxic T cell responses to highly conserved epitopes in the viral proteome and maximize response breadth. The vaccine-induced CE-specific T cells were expanded upon SIV infection, indicating that the predicted CE epitopes incorporated in the DNA vaccine are processed and exposed by infected cells in their natural context within the viral proteome.

Modified Newcastle Disease virus as an improved vaccine vector against Simian Immunodeficiency virus.

SIV infection in macaques is a relevant animal model for HIV pathogenesis and vaccine study in humans. To design a safe and effective vaccine against HIV, we evaluated the suitability of naturally-occurring avirulent Newcastle disease virus (NDV) strains and several modified versions of NDV as vectors for the expression and immunogenicity of SIV envelope protein gp160. All the NDV vectors expressed gp160 protein in infected cells. The gp160 expressed by these vectors formed oligomers and was incorporated into the NDV envelope. All the NDV vectors expressing gp160 were attenuated in chickens. Intranasal immunization of guinea pigs with modified NDV vectors such as rNDV-APMV-2CS/gp160 and rNDV-APMV-8CS/gp160 (NDV strain LaSota containing the cleavage site sequences of F protein of avian paramyxovirus (APMV) serotype 2 and 8, respectively), and rNDV-BC-F-HN/gp160 (NDV strain BC containing LaSota F cleavage site and LaSota F and HN genes) elicited improved SIV-specific humoral and mucosal immune responses compared to other NDV vectors. These modified vectors were also efficient in inducing neutralizing antibody responses to tier 1 A SIVmac251.6 and tier 1B SIVmac251/M766 strains. This study suggests that our novel modified NDV vectors are safe and immunogenic and can be used as vaccine vector to control HIV.

Control of Heterologous Simian Immunodeficiency Virus SIVsmE660 Infection by DNA and Protein Coimmunization Regimens Combined with Different Toll-Like-Receptor-4-Based Adjuvants in Macaques.

We developed a method of simultaneous vaccination with DNA and protein resulting in robust and durable cellular and humoral immune responses with efficient dissemination to mucosal sites and protection against simian immunodeficiency virus (SIV) infection. To further optimize the DNA-protein coimmunization regimen, we tested a SIVmac251-based vaccine formulated with either of two Toll-like receptor 4 (TLR4) ligand-based liposomal adjuvant formulations (TLR4 plus TLR7 [TLR4+7] or TLR4 plus QS21 [TLR4+QS21]) in macaques. Although both vaccines induced humoral responses of similar magnitudes, they differed in their functional quality, including broader neutralizing activity and effector functions in the TLR4+7 group. Upon repeated heterologous SIVsmE660 challenge, a trend of delayed viral acquisition was found in vaccinees compared to controls, which reached statistical significance in animals with the TRIM-5α-resistant (TRIM-5α R) allele. Vaccinees were preferentially infected by an SIVsmE660 transmitted/founder virus carrying neutralization-resistant A/K mutations at residues 45 and 47 in Env, demonstrating a strong vaccine-induced sieve effect. In addition, the delay in virus acquisition directly correlated with SIVsmE660-specific neutralizing antibodies. The presence of mucosal V1V2 IgG binding antibodies correlated with a significantly decreased risk of virus acquisition in both TRIM-5α R and TRIM-5α-moderate/sensitive (TRIM-5α M/S) animals, although this vaccine effect was more prominent in animals with the TRIM-5α R allele. These data support the combined contribution of immune responses and genetic background to vaccine efficacy. Humoral responses targeting V2 and SIV-specific T cell responses correlated with viremia control. In conclusion, the combination of DNA and gp120 Env protein vaccine regimens using two different adjuvants induced durable and potent cellular and humoral responses contributing to a lower risk of infection by heterologous SIV challenge.IMPORTANCE An effective AIDS vaccine continues to be of paramount importance for the control of the pandemic, and it has been proven to be an elusive target. Vaccine efficacy trials and macaque challenge studies indicate that protection may be the result of combinations of many parameters. We show that a combination of DNA and protein vaccinations applied at the same time provides rapid and robust cellular and humoral immune responses and evidence for a reduced risk of infection. Vaccine-induced neutralizing antibodies and Env V2-specific antibodies at mucosal sites contribute to the delay of SIVsmE660 acquisition, and genetic makeup (TRIM-5α) affects the effectiveness of the vaccine. These data are important for the design of better vaccines and may also affect other vaccine platforms.

Vaccine-modified NF-kB and GR signaling in cervicovaginal epithelium correlates with protection.

Cervicovaginal epithelium plays a critical role in determining the outcome of virus transmission in the female reproductive tract (FRT) by initiating or suppressing transmission-facilitating mucosal immune responses in naïve and SIVmac239Δnef-vaccinated animals, respectively. In this study, we examined the very early responses of cervical epithelium within 24 h after vaginal exposure to SIV in naive and SIVmac239Δnef-vaccinated rhesus macaques. Using both ex vivo and in vivo experimental systems, we found that vaginal exposure to SIV rapidly induces a broad spectrum of pro-inflammatory responses in the epithelium associated with a reciprocal regulation of NF-kB and glucocorticoid receptor (GR) signaling pathways. Conversely, maintenance of high-level GR expression and suppression of NF-kB expression in the epithelium were associated with an immunologically quiescent state in the FRT mucosa and protection against vaginal challenge in SIVmac239Δnef-vaccinated animals. We show that the immunologically quiescent state is induced by FCGR2B-immune complexes interactions that modify the reciprocal regulation of NF-kB and GR signaling pathways. Our results suggest that targeting the balance of NF-kB and GR signaling in early cervicovaginal epithelium responses could moderate mucosal inflammation and target cell availability after vaginal infection, thereby providing a complementary approach to current prevention strategies.

DNA prime/protein boost vaccination elicits robust humoral response in rhesus macaques using oligomeric simian immunodeficiency virus envelope and Advax delta inulin adjuvant.

The partial success of the RV144 trial underscores the importance of envelope-specific antibody responses for an effective HIV-1 vaccine. Oligomeric HIV-1 envelope proteins delivered with a potent adjuvant are expected to elicit strong antibody responses with broad neutralization specificity. To test this hypothesis, two SIV envelope proteins were formulated with delta inulin-based adjuvant (Advax) and used to immunize nonhuman primates. Oligomeric gp140-gp145 from SIVmac251 and SIVsmE660 was purified to homogeneity. Oligomers showed high-affinity interaction with CD4 and were highly immunogenic in rabbits, inducing Tier 2 SIV-neutralizing antibodies. The immunogenicity of an oligomeric Env DNA prime and protein boost together with Advax was evaluated in Chinese rhesus macaques. DNA administration elicited antibodies to both envelopes, and titres were markedly enhanced following homologous protein boosts via intranasal and intramuscular routes. Strong antibody responses were detected against the V1 and V2 domains of gp120. During peak immune responses, a low to moderate level of neutralizing activity was detected against Tier 1A/1B SIV isolates, with a moderate level noted against a Tier 2 isolate. Increased serum antibody affinity to SIVmac251 gp140 and generation of Env-specific memory B cells were observed in the immunized macaques. Animals were subjected to low-dose intravaginal challenge with SIVmac251 one week after the last protein boost. One out of three immunized animals was protected from infection. Although performed with a small number of macaques, this study demonstrates the utility of oligomeric envelopes formulated with Advax in eliciting broad antibody responses with the potential to provide protection against SIV transmission.

Vaccine-induced immune responses against both Gag and Env improve control of simian immunodeficiency virus replication in rectally challenged rhesus macaques.

The ability to control lentivirus replication may be determined, in part, by the extent to which individual viral proteins are targeted by the immune system. Consequently, defining the antigens that elicit the most protective immune responses may facilitate the design of effective HIV-1 vaccines. Here we vaccinated four groups of rhesus macaques with a heterologous vector prime/boost/boost/boost (PBBB) regimen expressing the following simian immunodeficiency virus (SIV) genes: env, gag, vif, rev, tat, and nef (Group 1); env, vif, rev, tat, and nef (Group 2); gag, vif, rev, tat, and nef (Group 3); or vif, rev, tat, and nef (Group 4). Following repeated intrarectal challenges with a marginal dose of the neutralization-resistant SIVmac239 clone, vaccinees in Groups 1-3 became infected at similar rates compared to control animals. Unexpectedly, vaccinees in Group 4 became infected at a slower pace than the other animals, although this difference was not statistically significant. Group 1 exhibited the best post-acquisition virologic control of SIV infection, with significant reductions in both peak and chronic phase viremia. Indeed, 5/8 Group 1 vaccinees had viral loads of less than 2,000 vRNA copies/mL of plasma in the chronic phase. Vaccine regimens that did not contain gag (Group 2), env (Group 3), or both of these inserts (Group 4) were largely ineffective at decreasing viremia. Thus, vaccine-induced immune responses against both Gag and Env appeared to maximize control of immunodeficiency virus replication. Collectively, these findings are relevant for HIV-1 vaccine design as they provide additional insights into which of the lentiviral proteins might serve as the best vaccine immunogens.

Immunologic and Virologic Mechanisms for Partial Protection from Intravenous Challenge by an Integration-Defective SIV Vaccine.

The suppression of viral loads and identification of selection signatures in non-human primates after challenge are indicators for effective human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) vaccines. To mimic the protective immunity elicited by attenuated SIV vaccines, we developed an integration-defective SIV (idSIV) vaccine by inactivating integrase, mutating sequence motifs critical for integration, and inserting the cytomegalovirus (CMV) promoter for more efficient expression in the SIVmac239 genome. Chinese rhesus macaques were immunized with idSIV DNA and idSIV particles, and the cellular and humoral immune responses were measured. After the intravenous SIVmac239 challenge, viral loads were monitored and selection signatures in viral genomes from vaccinated monkeys were identified by single genome sequencing. T cell responses, heterologous neutralization against tier-1 viruses, and antibody-dependent cellular cytotoxicity (ADCC) were detected in idSIV-vaccinated macaques post immunization. After challenge, the median peak viral load in the vaccine group was significantly lower than that in the control group. However, this initial viral control did not last as viral set-points were similar between vaccinated and control animals. Selection signatures were identified in Nef, Gag, and Env proteins in vaccinated and control macaques, but these signatures were different, suggesting selection pressure on viruses from vaccine-induced immunity in the vaccinated animals. Our results showed that the idSIV vaccine exerted some pressure on the virus population early during the infection but future modifications are needed in order to induce more potent immune responses.