Complete Rescue of HTLV-1p12KO Infectivity by Depletion of Monocytes Together with NK and CD8+ T Cells.

The transient depletion of monocytes alone prior to exposure of macaques to HTLV-1 enhances both HTLV-1WT (wild type) and HTLV-1p12KO (Orf-1 knockout) infectivity, but seroconversion to either virus is not sustained over time, suggesting a progressive decrease in virus expression. These results raise the hypotheses that either HTLV-1 persistence depends on a monocyte reservoir or monocyte depletion provides a transient immune evasion benefit. To test these hypotheses, we simultaneously depleted NK cells, CD8+ T cells, and monocytes (triple depletion) prior to exposure to HTLV-1WT or HTLV-1p12KO. Remarkably, triple depletion resulted in exacerbation of infection by both viruses and complete rescue of HTLV-1p12KO infectivity. Following triple depletion, we observed rapid and sustained seroconversion, high titers of antibodies against HTLV-1 p24Gag, and frequent detection of viral DNA in the blood and tissues of all animals when compared with depletion of only CD8+ and NK cells, or monocytes alone. The infection of macaques with HTLV-1WT or HTLV-1p12KO was associated with higher plasma levels of IL-10 after 21 weeks, while IL-6, IFN-γ, IL-18, and IL-1ß were only elevated in animals infected with HTLV-1WT. The repeat depletion of monocytes, NK, and CD8+ cells seven months following the first exposure to HTLV-1 did not further exacerbate viral replication. These results underscore the contribution of monocytes in orchestrating anti-viral immunity. Indeed, the absence of orf-1 expression was fully compensated by the simultaneous depletion of CD8+ T cells, NK cells, and monocytes, underlining the primary role of orf-1 in hijacking host immunity.

In Vivo Treatment with Insulin-like Growth Factor 1 Reduces CCR5 Expression on Vaccine-Induced Activated CD4+ T-Cells.

At the heart of the DNA/ALVAC/gp120/alum vaccine's efficacy in the absence of neutralizing antibodies is a delicate balance of pro- and anti-inflammatory immune responses that effectively decreases the risk of SIVmac251 acquisition in macaques. Vaccine efficacy is linked to antibodies recognizing the V2 helical conformation, DC-10 tolerogenic dendritic cells eliciting the clearance of apoptotic cells via efferocytosis, and CCR5 downregulation on vaccine-induced gut homing CD4+ cells. RAS activation is also linked to vaccine efficacy, which prompted the testing of IGF-1, a potent inducer of RAS activation with vaccination. We found that IGF-1 changed the hierarchy of V1/V2 epitope recognition and decreased both ADCC specific for helical V2 and efferocytosis. Remarkably, IGF-1 also reduced the expression of CCR5 on vaccine-induced CD4+ gut-homing T-cells, compensating for its negative effect on ADCC and efferocytosis and resulting in equivalent vaccine efficacy (71% with IGF-1 and 69% without).

HTLV-1 p12 modulates the levels of prion protein (PrPC) in CD4+ T cells.

Introduction: Infection with human T cell lymphotropic virus type 1 (HTLV-1) is endemic in Brazil and is linked with pro-inflammatory conditions including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic neuroinflammatory incapacitating disease that culminates in loss of motor functions. The mechanisms underlying the onset and progression of HAM/TSP are incompletely understood. Previous studies have demonstrated that inflammation and infectious agents can affect the expression of cellular prion protein (PrPC) in immune cells. Methods: Here, we investigated whether HTLV-1 infection affected PrPC content in cell lines and primary CD4+cells in vitro using flow cytometry and western blot assays. Results: We found that HTLV-1 infection decreased the expression levels of PrPC and HTLV-1 Orf I encoded p12, an endoplasmic reticulum resident protein also known to affect post-transcriptionally cellular proteins such as MHC-class I and the IL-2 receptor. In addition, we observed a reduced percentage of CD4+ T cells from infected individuals expressing PrPC, which was reflected by IFN type II but not IL-17 expression. Discussion: These results suggested that PrPC downregulation, linked to both HTLV-1 p12 and IFN-γ expression in CD4+ cells, may play a role in the neuropathogenesis of HTLV-1 infection.

Cholera toxin B scaffolded, focused SIV V2 epitope elicits antibodies that influence the risk of SIVmac251 acquisition in macaques.

Introduction: An efficacious HIV vaccine will need to elicit a complex package of innate, humoral, and cellular immune responses. This complex package of responses to vaccine candidates has been studied and yielded important results, yet it has been a recurring challenge to determine the magnitude and protective effect of specific in vivo immune responses in isolation. We therefore designed a single, viral-spike-apical, epitope-focused V2 loop immunogen to reveal individual vaccine-elicited immune factors that contribute to protection against HIV/SIV. Method: We generated a novel vaccine by incorporating the V2 loop B-cell epitope in the cholera toxin B (CTB) scaffold and compared two new immunization regimens to a historically protective 'standard' vaccine regimen (SVR) consisting of 2xDNA prime boosted with 2xALVAC-SIV and 1xΔV1gp120. We immunized a cohort of macaques with 5xCTB-V2c vaccine+alum intramuscularly simultaneously with topical intrarectal vaccination of CTB-V2c vaccine without alum (5xCTB-V2/alum). In a second group, we tested a modified version of the SVR consisting of 2xDNA prime and boosted with 1xALVAC-SIV and 2xALVAC-SIV+CTB-V2/alum, (DA/CTB-V2c/alum). Results: In the absence of any other anti-viral antibodies, V2c epitope was highly immunogenic when incorporated in the CTB scaffold and generated highly functional anti-V2c antibodies in the vaccinated animals. 5xCTB-V2c/alum vaccination mediated non-neutralizing ADCC activity and efferocytosis, but produced low avidity, trogocytosis, and no neutralization of tier 1 virus. Furthermore, DA/CTB-V2c/alum vaccination also generated lower total ADCC activity, avidity, and neutralization compared to the SVR. These data suggest that the ΔV1gp120 boost in the SVR yielded more favorable immune responses than its CTB-V2c counterpart. Vaccination with the SVR generates CCR5- α4ß7+CD4+ Th1, Th2, and Th17 cells, which are less likely to be infected by SIV/HIV and likely contributed to the protection afforded in this regimen. The 5xCTB-V2c/alum regimen likewise elicited higher circulating CCR5- α4ß7+ CD4+ T cells and mucosal α4ß7+ CD4+ T cells compared to the DA/CTB-V2c/alum regimen, whereas the first cell type was associated with reduced risk of viral acquisition. Conclusion: Taken together, these data suggest that individual viral spike B-cell epitopes can be highly immunogenic and functional as isolated immunogens, although they might not be sufficient on their own to provide full protection against HIV/SIV infection.

Vaccine plus microbicide effective in preventing vaginal SIV transmission in macaques.

The human immunodeficiency virus epidemic continues in sub-Saharan Africa, and particularly affects adolescent girls and women who have limited access to antiretroviral therapy. Here we report that the risk of vaginal simian immunodeficiency virus (SIV)mac251 acquisition is reduced by more than 90% using a combination of a vaccine comprising V1-deleted (V2 enhanced) SIV envelope immunogens with topical treatment of the zinc-finger inhibitor SAMT-247. Following 14 weekly intravaginal exposures to the highly pathogenic SIVmac251, 80% of a cohort of 20 macaques vaccinated and treated with SAMT-247 remained uninfected. In an arm of 18 vaccinated-only animals without microbicide, 40% of macaques remained uninfected. The combined SAMT-247/vaccine regimen was significantly more effective than vaccination alone. By analysing immune correlates of protection, we show that, by increasing zinc availability, SAMT-247 increases natural killer cytotoxicity and monocyte efferocytosis, and decreases T-cell activation to augment vaccine-induced protection.

Effect of Passive Administration of Monoclonal Antibodies Recognizing Simian Immunodeficiency Virus (SIV) V2 in CH59-Like Coil/Helical or ß-Sheet Conformations on Time of SIVmac251 Acquisition.

The monoclonal antibodies (MAbs) NCI05 and NCI09, isolated from a vaccinated macaque that was protected from multiple simian immunodeficiency virus (SIV) challenges, both target an overlapping, conformationally dynamic epitope in SIV envelope variable region 2 (V2). Here, we show that NCI05 recognizes a CH59-like coil/helical epitope, whereas NCI09 recognizes a ß-hairpin linear epitope. In vitro, NCI05 and, to a lesser extent, NCI09 mediate the killing of SIV-infected cells in a CD4-dependent manner. Compared to NCI05, NCI09 mediates higher titers of antibody-dependent cellular cytotoxicity (ADCC) to gp120-coated cells, as well as higher levels of trogocytosis, a monocyte function that contributes to immune evasion. We also found that passive administration of NCI05 or NCI09 to macaques did not affect the risk of SIVmac251 acquisition compared to controls, demonstrating that these anti-V2 antibodies alone are not protective. However, NCI05 but not NCI09 mucosal levels strongly correlated with delayed SIVmac251 acquisition, and functional and structural data suggest that NCI05 targets a transient state of the viral spike apex that is partially opened, compared to its prefusion-closed conformation. IMPORTANCE Studies suggest that the protection against SIV/simian-human immunodeficiency virus (SHIV) acquisition afforded by the SIV/HIV V1 deletion-containing envelope immunogens, delivered by the DNA/ALVAC vaccine platform, requires multiple innate and adaptive host responses. Anti-inflammatory macrophages and tolerogenic dendritic cells (DC-10), together with CD14+ efferocytes, are consistently found to correlate with a vaccine-induced decrease in the risk of SIV/SHIV acquisition. Similarly, V2-specific antibody responses mediating ADCC, Th1 and Th2 cells expressing no or low levels of CCR5, and envelope-specific NKp44+ cells producing interleukin 17 (IL-17) also are reproducible correlates of decreased risk of virus acquisition. We focused on the function and the antiviral potential of two monoclonal antibodies (NCI05 and NCI09) isolated from vaccinated animals that differ in antiviral function in vitro and recognize V2 in a linear (NCI09) or coil/helical (NCI05) conformation. We demonstrate that NCI05, but not NCI09, delays SIVmac251 acquisition, highlighting the complexity of antibody responses to V2.

Anti-V2 antibodies virus vulnerability revealed by envelope V1 deletion in HIV vaccine candidates.

The efficacy of ALVAC-based HIV and SIV vaccines in humans and macaques correlates with antibodies to envelope variable region 2 (V2). We show here that vaccine-induced antibodies to SIV variable region 1 (V1) inhibit anti-V2 antibody-mediated cytotoxicity and reverse their ability to block V2 peptide interaction with the α4ß7 integrin. SIV vaccines engineered to delete V1 and favor an α helix, rather than a ß sheet V2 conformation, induced V2-specific ADCC correlating with decreased risk of SIV acquisition. Removal of V1 from the HIV-1 clade A/E A244 envelope resulted in decreased binding to antibodies recognizing V2 in the ß sheet conformation. Thus, deletion of V1 in HIV envelope immunogens may improve antibody responses to V2 virus vulnerability sites and increase the efficacy of HIV vaccine candidates.

Engagement of monocytes, NK cells, and CD4+ Th1 cells by ALVAC-SIV vaccination results in a decreased risk of SIVmac251 vaginal acquisition.

The recombinant Canarypox ALVAC-HIV/gp120/alum vaccine regimen was the first to significantly decrease the risk of HIV acquisition in humans, with equal effectiveness in both males and females. Similarly, an equivalent SIV-based ALVAC vaccine regimen decreased the risk of virus acquisition in Indian rhesus macaques of both sexes following intrarectal exposure to low doses of SIVmac251. Here, we demonstrate that the ALVAC-SIV/gp120/alum vaccine is also efficacious in female Chinese rhesus macaques following intravaginal exposure to low doses of SIVmac251 and we confirm that CD14+ classical monocytes are a strong correlate of decreased risk of virus acquisition. Furthermore, we demonstrate that the frequency of CD14+ cells and/or their gene expression correlates with blood Type 1 CD4+ T helper cells, α4ß7+ plasmablasts, and vaginal cytocidal NKG2A+ cells. To better understand the correlate of protection, we contrasted the ALVAC-SIV vaccine with a NYVAC-based SIV/gp120 regimen that used the identical immunogen. We found that NYVAC-SIV induced higher immune activation via CD4+Ki67+CD38+ and CD4+Ki67+α4ß7+ T cells, higher SIV envelope-specific IFN-γ producing cells, equivalent ADCC, and did not decrease the risk of SIVmac251 acquisition. Using the systems biology approach, we demonstrate that specific expression profiles of plasmablasts, NKG2A+ cells, and monocytes elicited by the ALVAC-based regimen correlated with decreased risk of virus acquisition.

Expression of CD40L by the ALVAC-Simian Immunodeficiency Virus Vector Abrogates T Cell Responses in Macaques.

Immunization with recombinant ALVAC/gp120 alum vaccine provided modest protection from human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) acquisition in humans and macaques. Vaccine-mediated protection was associated with the elicitation of IgG against the envelope V2 loop and of envelope-specific CD4+ T cell responses. We hypothesized that the simultaneous expression of the costimulatory molecule CD40L (CD154) by the ALVAC-HIV vector could increase both protective humoral and cellular responses. We engineered an ALVAC-SIV coexpressing CD40L with SIVmac251 (ALVAC-SIV/CD40L) gag, pol, and env genes. We compared its immunogenicity in macaques with that of a canonical ALVAC-SIV, with both given as a vector-prime/gp120 in alum boost strategy. The ALVAC-SIV/CD40L was superior to the ALVAC-SIV regimen in inducing binding and tier 1 neutralizing antibodies against the gp120. The increase in humoral responses was associated with the expression of the membrane-bound form of the CD40L by CD4+ T cells in lymph nodes. Unexpectedly, the ALVAC-SIV/CD40L vector had a blunting effect on CD4+ Th1 helper responses and instead favored the induction of myeloid-derived suppressor cells, the immune-suppressive interleukin-10 (IL-10) cytokine, and the down-modulatory tryptophan catabolism. Ultimately, this strategy failed to protect macaques from SIV acquisition. Taken together, these results underlie the importance of balanced vaccine-induced activating versus suppressive immune responses in affording protection from HIV.IMPORTANCE CD40-CD40 ligand (CD40L) interaction is crucial for inducing effective cytotoxic and humoral responses against pathogens. Because of its immunomodulatory function, CD40L has been used to enhance immune responses to vaccines, including candidate vaccines for HIV. The only successful vaccine ever tested in humans utilized a strategy combining canarypox virus-based vector (ALVAC) together with an envelope protein (gp120) adjuvanted in alum. This strategy showed limited efficacy in preventing HIV-1/SIV acquisition in humans and macaques. In both species, protection was associated with vaccine-induced antibodies against the HIV envelope and CD4+ T cell responses, including type 1 antiviral responses. In this study, we tested whether augmenting CD40L expression by coexpressing it with the ALVAC vector could increase the protective immune responses. Although coexpression of CD40L did increase humoral responses, it blunted type 1 CD4+ T cell responses against the SIV envelope protein and failed to protect macaques from viral infection.

Myeloid Cell Crosstalk Regulates the Efficacy of the DNA/ALVAC/gp120 HIV Vaccine Candidate.

Vaccination with DNA-SIV + ALVAC-SIV + gp120 alum results in inflammasome activation, high levels of IL-1ß production, emergency myelopoiesis, and the egress of CXCR4+ CD14+ pre-monocytes from bone marrow. Previously we have shown that this vaccine-induced innate monocyte memory is associated with decreased risk of SIVmac251 acquisition. Because IL-1ß also promotes the propagation of monocyte-derived suppressor (M-MDSC)-like cells, here we extended our analysis to this negative regulator subset, characterizing its levels and functions in macaques. Interestingly, we found that DNA prime engages M-MDSC-like cells and their levels are positively associated with the frequency of CD14+ classical monocytes, and negatively with the levels of CD16+ monocytes, correlates of decreased and increased risk of SIV acquisition, respectively. Accordingly, M-MDSC frequency, arginase activity, and NO were all associated with decrease of CD8 T cells responses and worse vaccination outcome. DNA vaccination thus induces innate immunity by engaging three subsets of myeloid cells, M-MDSCs, CD14+ innate monocyte memory, and CD16+ monocytes all playing different role in protection. The full characterization of the immunological space created by myeloid cell crosstalk will likely provide clues to improve the efficacy of HIV vaccine candidates.

HIV vaccine candidate activation of hypoxia and the inflammasome in CD14+ monocytes is associated with a decreased risk of SIVmac251 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.