Objective: The rectal mucosa is a critical site of HIV vulnerability. We sought to identify transcriptomic features of rectal mucosal tissue prior to exposure associated with support or restriction of HIV replication. Design: Rectal tissue from HIV-negative cisgender men (n=57) underwent concurrent i) RNAseq transcriptomic analyses (2 biopsies/participant) and ii) challenge with HIV in the ex vivo explant model of infection (3 biopsies challenged/participant) as part of a larger cohort study to understand the rectal mucosal immune environment among men who have sex with men. Methods: P24 was quantified in the explant supernatants over a culture period of 18 days via ELISA. Participant median p24 log Area Under the Curve was correlated with bulk transcriptomic data (Illumina HiSeq3000) to identify associations between gene expression and p24 production. Significant differentially expressed genes (DEG) were identified via DESeq2 analysis, and analyzed with Reactome to identify pathways of interest. Results: 183 DEG (181 upregulated, 2 downregulated) were associated with higher p24 accumulation in the ex vivo challenge model, including T cell activation, B cell function, and chemokine DEG. Reactome analysis of the upregulated genes identified ‘Adaptive Immune System’, ‘Cytokine Signaling in Immune System’, and ‘Innate Immune System’ as significant upregulated pathways. Conclusions: For the first time, we identified rectal tissue transcriptomic signatures associated with increased p24 production utilizing an ex vivo model. Our findings are highly relevant to HIV transmission and the early establishment of HIV reservoirs in humans, and future studies should examine the identified pathways as targets for new or improved biomedical prevention or treatment interventions.

The RV144 human immunodeficiency virus type 1 (HIV-1) vaccine trial showed a strong association between anti-gp70 V1V2 scaffold (V1V2) and anti-V2 hot spot peptide (V2 HS) antibody responses and reduced risk of HIV infection. Accordingly, a primary goal for HIV vaccines is to enhance the magnitude and breadth of V1V2 and V2 HS antibody responses in addition to neutralizing antibodies. Here, we tested the immunogenicity and efficacy of HIV-1 C.1086 gp140 boosts administered sequentially after priming with CD40L-adjuvanted DNA/simian-human immunodeficiency virus (SHIV) and boosting with modified vaccinia virus Ankara (MVA)-SHIV vaccines in rhesus macaques. The DNA/MVA vaccination induced robust vaccine-specific CD4 and CD8 T cell responses with a polyfunctional profile. Two gp140 booster immunizations induced very high levels (~2 mg/ml) of gp140 binding antibodies in serum, with strong reactivity directed against the homologous (C.1086) V1V2, V2 HS, V3, and gp41 immunodominant (ID) proteins. However, the vaccine-induced antibody showed 10-fold (peak) and 32-fold (prechallenge) weaker binding to the challenge virus (SHIV1157ipd3N4) V1V2 and failed to bind to the challenge virus V2 HS due to a single amino acid change. Point mutations in the immunogen V2 HS to match the V2 HS in the challenge virus significantly diminished the binding of vaccine-elicited antibodies to membrane-anchored gp160. Both vaccines failed to protect from infection following repeated SHIV1157ipd3N4 intrarectal challenges. However, only the protein-boosted animals showed enhanced viral control. These results demonstrate that C.1086 gp140 protein immunizations administered following DNA/ MVA vaccination do not significantly boost heterologous V1V2 and V2 HS responses and fail to enhance protection against heterologous SHIV challenge. IMPORTANCE HIV, the virus that causes AIDS, is responsible for millions of infections and deaths annually. Despite intense research for the past 25 years, there remains no safe and effective vaccine available. The significance of this work is in identifying the pros and cons of adding a protein boost to an already wellestablished DNA/MVA HIV vaccine that is currently being tested in the clinic. Characterizing the effects of the protein boost can allow researchers going forward to design vaccines that generate responses that will be more effective against HIV. Our results in rhesus macaques show that boosting with a specific HIV envelope protein does not significantly boost antibody responses that were identified as immune cor- relates of protection in a moderately successful RV144 HIV vaccine trial in humans and highlight the need for the development of improved HIV envelope immunogens.

Heterologous prime boost with DNA and recombinant modified vaccinia virus Ankara (rMVA) vaccines is considered as a promising vaccination approach against human immunodeficiency virus (HIV-1). To further enhance the efficacy of DNA-rMVA vaccination, we investigated humoral and cellular immune responses in mice after three sequential immunizations with DNA, a combination of DNA and virus-like particles (VLP), and rMVA expressing HIV-1 89.6 gp120 envelope proteins (Env). DNA prime and boost with a combination of VLP and DNA vaccines followed by an rMVA boost induced over a 100-fold increase in Env-specific IgG antibody titers compared to three sequential immunizations with DNA and rMVA. Cellular immune responses were induced by VLP-DNA and rMVA vaccinations at high levels in CD8 T cells, CD4 T cells, and peripheral blood mononuclear cells secreting interferon (IFN)-γ, and spleen cells producing interleukin (IL)-2, 4, 5 cytokines. This study suggests that a DNA and VLP combination vaccine with MVA is a promising strategy in enhancing the efficacy of DNA-rMVA vaccination against HIV-1.

We have previously shown that macaques vaccinated with DNA vectors expressing SIVmac239 antigens developed potent immune responses able to reduce viremia upon high-dose SIVmac251 challenge. To further improve vaccine-induced immunity and protection, we combined the SIVmac239 DNA vaccine with protein immunization using inactivated SIVmac239 viral particles as protein source. Twenty-six weeks after the last vaccination, the animals were challenged intrarectally at weekly intervals with a titrated dose of the heterologous SIVsmE660. Two of DNA-protein coimmunized macaques did not become infected after 14 challenges, but all controls were infected by 11 challenges. Vaccinated macaques showed modest protection from SIVsmE660 acquisition compared with naive controls (P = 0.050; stratified for TRIM5α genotype). Vaccinees had significantly lower peak (1.6 log, P = 0.0048) and chronic phase viremia (P = 0.044), with 73% of the vaccinees suppressing viral replication to levels below assay detection during the 40-wk follow-up. Vaccine-induced immune responses associated significantly with virus control: binding antibody titers and the presence of rectal IgG to SIVsmE660 Env correlated with delayed SIVsmE660 acquisition; SIV-specific cytotoxic T cells, prechallenge CD4+ effector memory, and postchallenge CD8+ transitional memory cells correlated with control of viremia. Thus, SIVmac239 DNA and proteinbased vaccine protocolswere able to achieve high, persistent, broad, and effective cellular and humoral immune responses able to delay heterologous SIVsmE660 infection and to provide long-term control of viremia. These studies support a role of DNA and protein-based vaccines for development of an efficacious HIV/AIDS vaccine.

Generating durable humoral immunity through vaccination depends upon effective interactions of follicular helper T (Tfh) cells with germinal center (GC) B cells. Th1 polarization of Tfh cells is an important process shaping the success of Tfh-GC B cell interactions by influencing costimulatory and cytokinedependent Tfh help to B cells. However, the question remains as to whether adjuvant-dependent modulation of Tfh cells enhances HIV-1 vaccine-induced antienvelope (anti-Env) antibody responses. We investigated whether an HIV-1 vaccine platform designed to increase the number of Th1-polarized Tfh cells enhances the magnitude and quality of anti-Env antibodies. Utilizing a novel interferoninduced protein 10 (IP-10)-adjuvanted HIV-1 DNA prime followed by a monophosphoryl lipid A and QS-21 (MPLA+QS-21)-adjuvanted Env protein boost (DIP-10 PALFQ) in macaques, we observed higher anti-Env serum IgG titers with greater cross-clade reactivity, specificity for V1V2, and effector functions than in macaques primed with DNA lacking IP-10 and boosted with MPLA-plus-alum-adjuvanted Env protein (DPALFA) The DIP-10 PALFQ vaccine regimen elicited higher anti-Env IgG1 and lower IgG4 antibody levels in serum, showing for the first time that adjuvants can dramatically impact the IgG subclass profile in macaques. The DIP-10 PALFQ regimen also increased vaginal and rectal IgA antibodies to a greater extent. Within lymph nodes, we observed augmented GC B cell responses and the promotion of Th1 gene expression profiles in GC Tfh cells. The frequency of GC Tfh cells correlated with both the magnitude and avidity of anti-Env serum IgG. Together, these data suggest that adjuvant-induced stimulation of Th1-Tfh cells is an effective strategy for enhancing the magnitude and quality of anti-Env antibody responses. IMPORTANCE The results of the RV144 trial demonstrated that vaccination could prevent HIV transmission in humans and that longevity of anti-Env antibodies may be key to this protection. Efforts to improve upon the prime-boost vaccine regimen used in RV144 have indicated that booster immunizations can increase serum anti-Env antibody titers but only transiently. Poor antibody durability hampers efforts to develop an effective HIV-1 vaccine. This study was designed to identify the specific elements involved in the immunological mechanism necessary to produce robust HIV-1-specific antibodies in rhesus macaques. By clearly defining immune-mediated pathways that improve the magnitude and functionality of the anti-HIV-1 antibody response, we will have the foundation necessary for the rational development of an HIV-1 vaccine.

Here, we report the results of a late boost and three additional series of simian immunodeficiency virus (SIV) challenges in seven DNA/modified vaccinia virus Ankara (MVA)-vaccinated rhesus macaques who resisted a first series of rectal challenges. During 29 additional challenges delivered over 2.3 years, all animals became infected. However, 13 blips of virus in six macaques and anamnestic Env-specific rectal IgA responses in three of the six suggested that local control of infections was occurring during the serial challenge.

A better understanding of the distribution and functional capacity of CD4 T helper (Th) and CD8 T cytotoxic (Tc) cell subsets in the rectal mucosa (RM), a major site for HIV acquisition and replication, in adults is needed. In this study, we compared the distribution of Th and Tc cell subsets between blood and RM compartments in 62 HIV negative men, focusing primarily on IL-17-producing CD4 and CD8 T cells due to their importance in establishing and maintaining mucosal defenses, and examined associations between the frequencies of Th17 and Tc17 cell subsets and the availability of highly HIV-susceptible target cells in the RM. The RM exhibited a distinct immune cell composition comprised of higher frequencies of Th2, Th17, and Tc17 cells compared to the peripheral blood. The majority of Tc17 cells in RM were quadruple-cytokine producers (IL-17A + , IFN-γ + , TNF-α + , and IL4 + ), whereas most Th17 cells in blood and RM were single IL-17A producers or dual-cytokine producers (IL-17A + TNF-α + ). In a separate cohort of 21 HIV positive men, we observed similar tissue distributions of Th and Tc cell subsets, although Tc17 cell frequencies in both blood and tissues were very low. Higher frequencies of multi-cytokine-producing Th17 and Tc17 cells in RM of HIV negative men positively correlated with increased mucosal HIV target cells, suggesting a need to further characterize the effector functions of these cells and their role in HIV acquisition and pathogenesis.

Mucosal surfaces are vulnerable to human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection and thus are key sites for eliciting vaccine-mediated protection. Vaccine protocols carried out at the Yerkes Primate Research Center utilized SIVmac239-based immunization strategies with intrarectal and intravaginal SIVsmE660 challenge of rhesus macaques. We investigated whether there were genetic signatures associated with SIVsmE660 intrarectal and intravaginal transmissions in vaccinated and unvaccinated monkeys. When transmitted/founder (T/F) envelope (Env) sequences from 49 vaccinated and 15 unvaccinated macaques were compared to each other, we were unable to identify any vaccine breakthrough signatures. In contrast,when the vaccinated and control T/F Envs were combined and compared to the challenge stock, residues at gp120 positions 23, 45, 47, and 70 (Ile-Ala-Lys-Asn [I-A-K-N]) emerged as signatures of mucosal transmission. However, T/F Envs derived from intrarectal and intravaginal infections were not different. Our data suggest that the vaginal and rectal mucosal environments both imposed a strong selection bias for SIVsmE660 variants carrying I-A-K-N that was not further enhanced by immunization.These findings, combined with the strong conservation of A-K-N in most HIV-2/SIVsmm isolates and the analogous residues in HIV-1/SIVcpz isolates, suggest that these residues confer increased transmission fitness to SIVsmE660.

Antibodies and cytotoxic T cells represent 2 arms of host defense against pathogens. We hypothesized that vaccines that induce both high-magnitude CD8+ T cell responses and antibody responses might confer enhanced protection against HIV. To test this hypothesis, we immunized 3 groups of nonhuman primates: (a) Group 1, which includes sequential immunization regimen involving heterologous viral vectors (HVVs) comprising vesicular stomatitis virus, vaccinia virus, and adenovirus serotype 5-expressing SIVmac239 Gag; (b) Group 2, which includes immunization with a clade C HIV-1 envelope (Env) gp140 protein adjuvanted with nanoparticles containing a TLR7/8 agonist (3M-052); and (c) Group 3, which includes a combination of both regimens. Immunization with HVVs induced very high-magnitude Gag-specific CD8+ T cell responses in blood and tissue-resident CD8+ memory T cells in vaginal mucosa. Immunization with 3M-052 adjuvanted Env protein induced robust and persistent antibody responses and long-lasting innate responses. Despite similar antibody titers in Groups 2 and 3, there was enhanced protection in the younger animals in Group 3, against intravaginal infection with a heterologous SHIV strain. This protection correlated with the magnitude of the serum and vaginal Env-specific antibody titers on the day of challenge. Thus, vaccination strategies that induce both CD8+ T cell and antibody responses can confer enhanced protection against infection.

Generating highly functional antibodies against HIV-1 is critical to prevent infection. Here we evaluated the ability of CD40L (a co-stimulatory molecule for B cells and dendritic cells) as an adjuvant to prevent mucosal infection from neutralization-susceptible (SIVE660) and neutralization-resistant (SIV251) SIVs.