Namal P.M. Liyanage

Adjuvant-dependent innate and adaptive immune signatures of risk of SIVmac251 acquisition.

A recombinant vaccine containing Aventis Pasteurs canarypox vector (ALVAC)–HIV and gp120 alum decreased the risk of HIV acquisition in the RV144 vaccine trial. The substitution of alum with the more immunogenic MF59 adjuvant is under consideration for the next efficacy human trial. We found here that an ALVAC–simian immunodeficiency virus (SIV) and gp120 alum (ALVAC–SIV + gp120) equivalent vaccine, but not an ALVAC–SIV + gp120 MF59 vaccine, was efficacious in delaying the onset of SIVmac251 in rhesus macaques, despite the higher immunogenicity of the latter adjuvant. Vaccine efficacy was associated with alum-induced, but not with MF59-induced, envelope (Env)-dependent mucosal innate lymphoid cells (ILCs) that produce interleukin (IL)-17, as well as with mucosal IgG to the gp120 variable region 2 (V2) and the expression of 12 genes, ten of which are part of the RAS pathway. The association between RAS activation and vaccine efficacy was also observed in an independent efficacious SIV-vaccine approach. Whether RAS activation, mucosal ILCs and antibodies to V2 are also important hallmarks of HIV-vaccine efficacy in humans will require further studies.

Antibody to the gp120 V1/V2 Loops and CD4+ and CD8+ T Cell Responses in Protection from SIVmac251 Vaginal Acquisition and Persistent Viremia

The human papillomavirus pseudovirions (HPV-PsVs) approach is an effective gene-delivery system that can prime or boost an immune response in the vaginal tract of nonhuman primates and mice. Intravaginal vaccination with HPV-PsVs expressing SIV genes, combined with an i.m. gp120 protein injection, induced humoral and cellular SIV-specific responses in macaques. Priming systemic immune responses with i.m. immunization with ALVAC-SIV vaccines, followed by intravaginal HPV-PsV–SIV/gp120 boosting, expanded and/or recruited T cells in the female genital tract. Using a stringent repeated low-dose intravaginal challenge with the highly pathogenic SIVmac251, we show that although these regimens did not demonstrate significant protection from virus acquisition, they provided control of viremia in a number of animals. High-avidity Ab responses to the envelope gp120 V1/V2 region correlated with delayed SIVmac251 acquisition, whereas virus levels in mucosal tissues were inversely correlated with antienvelope CD4+ T cell responses. CD8+ T cell depletion in animals with controlled viremia caused an increase in tissue virus load in some animals, suggesting a role for CD8+ T cells in virus control. This study highlights the importance of CD8+ cells and antienvelope CD4+ T cells in curtailing virus replication and antienvelope V1/V2 Abs in preventing SIVmac251 acquisition.

Comparative analysis of SIV-specific cellular immune responses induced by different vaccine platforms in rhesus macaques

To identify the most promising vaccine candidates for combinatorial strategies, we compared five SIV vaccine platforms including recombinant canary pox virus ALVAC, replication-competent adenovirus type 5 host range mutant RepAd, DNA, modified vaccinia Ankara (MVA), peptides and protein in distinct combinations. Three regimens used viral vectors (prime or boost) and two regimens used plasmid DNA. Analysis at necropsy showed that the DNA-based vaccine regimens elicited significantly higher cellular responses against Gag and Env than any of the other vaccine platforms. The T cell responses induced by most vaccine regimens disseminated systemically into secondary lymphoid tissues (lymph nodes, spleen) and effector anatomical sites (including liver, vaginal tissue), indicative of their role in viral containment at the portal of entry. The cellular and reported humoral immune response data suggest that combination of DNA and viral vectors elicits a balanced immunity with strong and durable responses able to disseminate into relevant mucosal sites.

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.

Antiretroviral therapy partly reverses the systemic and mucosal distribution of NK cell subsets that is altered by SIVmac251 infection of macaques

We characterized three subsets of NK cells in blood, and two subsets in mucosal tissues. SIVmac251 infection increased total and CD16(+) NK cells in the blood. In the rectum, we observed a significant increase in total and NKG2A(+) NK cells during SIV infection. In contrast, the NKp44(+) subset significantly depleted in acute infection and continued to decline in frequency during chronic phase. During SIV infection, blood CD16 and mucosal NKG2A(+) subsets had increased cytotoxic potential. Intriguingly, the NKp44(+) NK cell subtype that likely mediates mucosal homeostasis via the production of cytokines, acquired cytotoxicity. Antiretroviral therapy significantly increased the frequency of mucosal NKG2A(+) NK cells and peripheral CD16(+) NK cells. However, it failed to restore the normal frequency of NKp44(+) NK cells in the rectum. Thus, SIVmac251 infection causes changes in the distribution and function of NK cells and antiretroviral therapy during chronic infection only partially restores NK homeostasis and function.

Boosting of ALVAC-SIV Vaccine-Primed Macaques with the CD4-SIVgp120 Fusion Protein Elicits Antibodies to V2 Associated with a Decreased Risk of SIVmac251 Acquisition

The recombinant ALVAC vaccine coupled with the monomeric gp120/alum protein have decreased the risk of HIV and SIV acquisition. Ab responses to the V1/V2 regions have correlated with a decreased risk of virus acquisition in both humans and macaques. We hypothesized that the breadth and functional profile of Abs induced by an ALVAC/envelope protein regimen could be improved by substituting the monomeric gp120 boost, with the full-length single-chain (FLSC) protein. FLSC is a CD4-gp120 fusion immunogen that exposes cryptic gp120 epitopes to the immune system. We compared the immunogenicity and relative efficiency of an ALVAC-SIV vaccine boosted either with bivalent FLSC proteins or with monomeric gp120 in alum. FLSC was superior to monomeric gp120 in directing Abs to the C3 α2 helix, the V5 loop, and the V3 region that contains the putative CCR5 binding site. In addition, FLSC boosting elicited significantly higher binding Abs to V2 and increased both the Ab-dependent cellular cytotoxicity activity and the breadth of neutralizing Abs. However, the FLSC vaccine regimen demonstrated only a trend in vaccine efficacy, whereas the monomeric gp120 regimen significantly decreased the risk of SIVmac251 acquisition. In both vaccine regimens, anti-V2 Abs correlated with a decreased risk of virus acquisition but differed with regard to systemic or mucosal origin. In the FLSC regimen, serum Abs to V2 correlated, whereas in the monomeric gp120 regimen, V2 Abs in rectal secretions, the site of viral challenge, were associated with efficacy.

HIV vaccine candidate activation of hypoxia and the inflammasome in CD14 + monocytes is associated with a decreased risk of SIV mac251 acquisition

Qualitative differences in the innate and adaptive responses elicited by different HIV vaccine candidates have not been thoroughly investigated. We tested the ability of the Aventis Pasteur live recombinant canarypox vector (ALVAC)–SIV, DNA–SIV and Ad26–SIV vaccine prime modalities together with two ALVAC–SIV + gp120 protein boosts to reduce the risk of SIVmac251 acquisition in rhesus macaques. We found that the DNA and ALVAC prime regimens were effective, but the Ad26 prime was not. The activation of hypoxia and the inflammasome in CD14+CD16− monocytes, gut-homing CCR5-negative CD4+ T helper 2 (TH2) cells and antibodies to variable region 2 correlated with a decreased risk of SIVmac251 acquisition. By contrast, signal transducer and activator of transcription 3 activation in CD16+ monocytes was associated with an increased risk of virus acquisition. The Ad26 prime regimen induced the accumulation of CX3CR1+CD163+ macrophages in lymph nodes and of long-lasting CD4+ TH17 cells in the gut and lungs. Our data indicate that the selective engagement of monocyte subsets following a vaccine prime influences long-term immunity, uncovering an unexpected association of CD14+ innate monocytes with a reduced risk of SIVmac251 acquisition.Distinct monocyte subsets associate with different outcomes of SIV vaccines.

Corrigendum: Adjuvant-dependent innate and adaptive immune signatures of risk of SIVmac251 acquisition

Monica Vaccari, Shari N Gordon, Slim Fourati, Luca Schifanella, Namal P M Liyanage, Mark Cameron, Brandon F Keele, Xiaoying Shen, Georgia D Tomaras, Erik Billings, Mangala Rao, Amy W Chung, Karen G Dowell, Chris Bailey-Kellogg, Eric P Brown, Margaret E Ackerman, Diego A Vargas-Inchaustegui, Stephen Whitney, Melvin N Doster, Nicolo Binello, Poonam Pegu, David C Montefiori, Kathryn Foulds, David S Quinn, Mitzi Donaldson, Frank Liang, Karin Loré, Mario Roederer, Richard A Koup, Adrian McDermott, Zhong-Min Ma, Christopher J Miller, Tran B Phan, Donald N Forthal, Matthew Blackburn, Francesca Caccuri, Massimiliano Bissa, Guido Ferrari, Vaniambadi Kalyanaraman, Maria G Ferrari, DeVon Thompson, Marjorie Robert-Guroff, Silvia Ratto-Kim, Jerome H Kim, Nelson L Michael, Sanjay Phogat, Susan W Barnett, Jim Tartaglia, David Venzon, Donald M Stablein, Galit Alter, Rafick-Pierre Sekaly & Genoveffa Franchini Nat. Med.; doi:10.1038/nm.4105; corrected online 16 June 2016

Do CD16+NKG2A+NK Cells Recruited to the Gut Combined with Passively Administered SIV Specific Antibodies Prevent SIVmac251 Acquisition in Macaques?

OA04.04 Background: Growing evidence suggests that NK cells and antibodies that mediate ADCC can control HIV infection. ADCC was shown to have an inverse correlation with risk of HIV infection in the RV144 vaccine trial that showed 31% protection from HIV acquisition. We recently showed the recruitment of CD16+ NKG2A+ NK cells to the gut during ALVAC/SIV/gp120 vaccine regimen in macaques. Here we investigated whether NKG2A+ NK cells together with vaccine induced anti-envelop IgG at mucosa can protect from SIVmac251 acquisition. Methods: IgG was purified from sera following 8 additional immunizations with ALVAC-SIV/gp120/Alum of 8 macaques previously exposed to SIVmac251 remaining uninfected. To demonstrate that systemically given IgG can localize to the gut, CY5 labeled anti-human IgG was subcutaneously administered to macaques and anti-human antibodies were measured in serum and rectum by ELISA and immunohistochemistry (IHC) at 6h, 24h and weekly for 4 weeks. Two macaque cohorts (A and B) were vaccinated with ALVAC-SIV (SIV766 Gag-Pro gp120TM) at 0, 4, 12 and 24 weeks. ALUM was administered together with the vaccine at 12 and 24 weeks. Purified IgG will be given to group A (n=8) subcutaneously (24h) and intra-rectally (IR) (2h) prior to the repeated low dose challenge with SIVmac251 IR in July 2014. Group B (n=8) and unvaccinated group (n=12) will be challenged simultaneously. Results: Purified IgG from 8 protected animals showed 1.87% of gp120 specific activity by ELISA. Anti-human IgG was detected in serum and rectal biopsies by ELISA and IHC up to 3 weeks post antibody administration. Cytometric analysis of rectal biopsies showed recruitment of NKG2A+NK (p=0.03) cells to the gut of vaccines. Conclusions: Our current data shows the recruitment of NKG2A+NK cells to the gut and successful delivery of antibodies to the gut mucosa. Protection after SIV challenge in the end of July 2014, will confirm our hypotheses that the vaccine mediated recruitment of NK cell together with antibodies can prevent SIV acquisition.