The transcription factor CREB1 is a mechanistic driver of immunogenicity and reduced HIV-1 acquisition following ALVAC vaccination.

Development of effective human immunodeficiency virus 1 (HIV-1) vaccines requires synergy between innate and adaptive immune cells. Here we show that induction of the transcription factor CREB1 and its target genes by the recombinant canarypox vector ALVAC + Alum augments immunogenicity in non-human primates (NHPs) and predicts reduced HIV-1 acquisition in the RV144 trial. These target genes include those encoding cytokines/chemokines associated with heightened protection from simian immunodeficiency virus challenge in NHPs. Expression of CREB1 target genes probably results from direct cGAMP (STING agonist)-modulated p-CREB1 activity that drives the recruitment of CD4+ T cells and B cells to the site of antigen presentation. Importantly, unlike NHPs immunized with ALVAC + Alum, those immunized with ALVAC + MF59, the regimen in the HVTN702 trial that showed no protection from HIV infection, exhibited significantly reduced CREB1 target gene expression. Our integrated systems biology approach has validated CREB1 as a critical driver of vaccine efficacy and highlights that adjuvants that trigger CREB1 signaling may be critical for efficacious HIV-1 vaccines.

Antibody-dependent cellular cytotoxicity, infected cell binding and neutralization by antibodies to the SIV envelope glycoprotein.

Antibodies specific for diverse epitopes of the simian immunodeficiency virus envelope glycoprotein (SIV Env) have been isolated from rhesus macaques to provide physiologically relevant reagents for investigating antibody-mediated protection in this species as a nonhuman primate model for HIV/AIDS. With increasing interest in the contribution of Fc-mediated effector functions to protective immunity, we selected thirty antibodies representing different classes of SIV Env epitopes for a comparison of antibody-dependent cellular cytotoxicity (ADCC), binding to Env on the surface of infected cells and neutralization of viral infectivity. These activities were measured against cells infected with neutralization-sensitive (SIVmac316 and SIVsmE660-FL14) and neutralization-resistant (SIVmac239 and SIVsmE543-3) viruses representing genetically distinct isolates. Antibodies to the CD4-binding site and CD4-inducible epitopes were identified with especially potent ADCC against all four viruses. ADCC correlated well with antibody binding to virus-infected cells. ADCC also correlated with neutralization. However, several instances of ADCC without detectable neutralization or neutralization without detectable ADCC were observed. The incomplete correspondence between ADCC and neutralization shows that some antibody-Env interactions can uncouple these antiviral activities. Nevertheless, the overall correlation between neutralization and ADCC implies that most antibodies that are capable of binding to Env on the surface of virions to block infectivity are also capable of binding to Env on the surface of virus-infected cells to direct their elimination by ADCC.

High level of genomic divergence in orf-I p12 and hbz genes of HTLV-1 subtype-C in Central Australia.

BACKGROUND: Human T cell lymphotropic virus type 1 (HTLV-1) infection remains a largely neglected public health problem, particularly in resource-poor areas with high burden of communicable and non-communicable diseases, such as some remote populations in Central Australia where an estimated 37% of adults are infected with HTLV-1. Most of our understanding of HTLV-1 infection comes from studies of the globally spread subtype-A (HTLV-1a), with few molecular studies reported with the Austral-Melanesian subtype-C (HTLV-1c) predominant in the Indo-Pacific and Oceania regions. RESULTS: Using a primer walking strategy and direct sequencing, we constructed HTLV-1c genomic consensus sequences from 22 First Nations participants living with HTLV-1c in Central Australia. Phylogenetic and pairwise analysis of this subtype-C proviral gDNA showed higher levels of genomic divergence in comparison to previously published HTLV-1a genomes. While the overall genomic homology between subtypes was 92.5%, the lowest nucleotide and amino acid sequence identity occurred near the 3' end of the proviral genome coding regulatory genes, especially overlapping hbz (85.37%, 77.46%, respectively) and orf-I product p12 (82.00%, 70.30%, respectively). Strikingly, the HTLV-1c genomic consensus sequences uniformly showed a defective translation start codon for the immune regulatory proteins p12/p8 encoded by the HTLV-1A orf-I. Deletions in the proviral genome were detected in many subjects, particularly in the structural gag, pol and env genes. Similarly, using a droplet digital PCR assay measuring the copies of gag and tax per reference host genome, we quantitatively confirmed that provirus retains the tax gene region at higher levels than gag. CONCLUSIONS: Our genomic analysis of HTLV-1c in Central Australia in conjunction with earlier Melanesian HTLV-1c sequences, elucidate substantial differences with respect to the globally spread HTLV-1a. Future studies should address the impact these genomic differences have on infection and the regionally distinctive frequency of associated pulmonary disease. Understanding the host and virus subtype factors which contribute to the differential morbidity observed, is crucial for the development of much needed therapeutics and vaccine strategies against this highly endemic infection in remote First Nations communities in Central Australia.

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.

NK cells and monocytes modulate primary HTLV-1 infection.

We investigated the impact of monocytes, NK cells, and CD8+ T-cells in primary HTLV-1 infection by depleting cell subsets and exposing macaques to either HTLV-1 wild type (HTLV-1WT) or to the HTLV-1p12KO mutant unable to infect replete animals due to a single point mutation in orf-I that inhibits its expression. The orf-I encoded p8/p12 proteins counteract cytotoxic NK and CD8+ T-cells and favor viral DNA persistence in monocytes. Double NK and CD8+ T-cells or CD8 depletion alone accelerated seroconversion in all animals exposed to HTLV-1WT. In contrast, HTLV-1p12KO infectivity was fully restored only when NK cells were also depleted, demonstrating a critical role of NK cells in primary infection. Monocyte/macrophage depletion resulted in accelerated seroconversion in all animals exposed to HTLV-1WT, but antibody titers to the virus were low and not sustained. Seroconversion did not occur in most animals exposed to HTLV-1p12KO. In vitro experiments in human primary monocytes or THP-1 cells comparing HTLV-1WT and HTLV-1p12KO demonstrated that orf-I expression is associated with inhibition of inflammasome activation in primary cells, with increased CD47 "don't-eat-me" signal surface expression in virus infected cells and decreased monocyte engulfment of infected cells. Collectively, our data demonstrate a critical role for innate NK cells in primary infection and suggest a dual role of monocytes in primary infection. On one hand, orf-I expression increases the chances of viral transmission by sparing infected cells from efferocytosis, and on the other may protect the engulfed infected cells by modulating inflammasome activation. These data also suggest that, once infection is established, the stoichiometry of orf-I expression may contribute to the chronic inflammation observed in HTLV-1 infection by modulating monocyte efferocytosis.

The V2 loop of HIV gp120 delivers costimulatory signals to CD4+ T cells through Integrin α4ß7 and promotes cellular activation and infection.

Acute HIV infection is characterized by rapid viral seeding of immunologic inductive sites in the gut followed by the severe depletion of gut CD4+ T cells. Trafficking of α4ß7-expressing lymphocytes to the gut is mediated by MAdCAM, the natural ligand of α4ß7 that is expressed on gut endothelial cells. MAdCAM signaling through α4ß7 costimulates CD4+ T cells and promotes HIV replication. Similar to MAdCAM, the V2 domain of the gp120 HIV envelope protein binds to α4ß7 In this study, we report that gp120 V2 shares with MAdCAM the capacity to signal through α4ß7 resulting in CD4+ T cell activation and proliferation. As with MAdCAM-mediated costimulation, cellular activation induced by gp120 V2 is inhibited by anti-α4ß7 monoclonal antibodies (mAbs). It is also inhibited by anti-V2 domain antibodies including nonneutralizing mAbs that recognize an epitope in V2 that has been linked to reduced risk of acquisition in the RV144 vaccine trial. The capacity of the V2 domain of gp120 to mediate signaling through α4ß7 likely impacts early events in HIV infection. The capacity of nonneutralizing V2 antibodies to block this activity reveals a previously unrecognized mechanism whereby such antibodies might impact HIV transmission and pathogenesis.

Correction: ALVAC-HIV B/C candidate HIV vaccine efficacy dependent on neutralization profile of challenge virus and adjuvant dose and type.

[This corrects the article DOI: 10.1371/journal.ppat.1008121.].

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.

Germinal epimutation of Fragile Histidine Triad (FHIT) gene is associated with progression to acute and chronic adult T-cell leukemia diseases.

BACKGROUND: Human T cell Leukemia virus type 1 (HTLV-I) is etiologically linked to adult T cell leukemia/lymphoma (ATL) and an inflammatory neurodegenerative disease called HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP). The exact genetic or epigenetic events and/or environmental factors that influence the development of ATL, or HAM/TSP diseases are largely unknown. The tumor suppressor gene, Fragile Histidine Triad Diadenosine Triphosphatase (FHIT), is frequently lost in cancer through epigenetic modifications and/or deletion. FHIT is a tumor suppressor acting as genome caretaker by regulating cellular DNA repair. Indeed, FHIT loss leads to replicative stress and accumulation of double DNA strand breaks. Therefore, loss of FHIT expression plays a key role in cellular transformation. METHODS: Here, we studied over 400 samples from HTLV-I-infected individuals with ATL, TSP/HAM, or asymptomatic carriers (AC) for FHIT loss and expression. We examined the epigenetic status of FHIT through methylation specific PCR and bisulfite sequencing; and correlated these results to FHIT expression in patient samples. RESULTS: We found that epigenetic alteration of FHIT is specifically found in chronic and acute ATL but is absent in asymptomatic HTLV-I carriers and TSP/HAM patients' samples. Furthermore, the extent of FHIT methylation in ATL patients was quantitatively comparable in virus-infected and virus non-infected cells. We also found that longitudinal HTLV-I carriers that progressed to smoldering ATL and descendants of ATL patients harbor FHIT methylation. CONCLUSIONS: These results suggest that germinal epigenetic mutation of FHIT represents a preexisting mark predisposing to the development of ATL diseases. These findings have important clinical implications as patients with acute ATL are rarely cured. Our study suggests an alternative strategy to the current "wait and see approach" in that early screening of HTLV-I-infected individuals for germinal epimutation of FHIT and early treatment may offer significant clinical benefits.

A Prime/Boost Vaccine Regimen Alters the Rectal Microbiome and Impacts Immune Responses and Viremia Control Post-Simian Immunodeficiency Virus Infection in Male and Female Rhesus Macaques.

An efficacious human immunodeficiency virus (HIV) vaccine will likely require induction of both mucosal and systemic immune responses. We compared the immunogenicity and protective efficacy of two mucosal/systemic vaccine regimens and investigated their effects on the rectal microbiome. Rhesus macaques were primed twice mucosally with replication-competent adenovirus type 5 host range mutant (Ad5hr)-simian immunodeficiency virus (SIV) recombinants and boosted twice intramuscularly with ALVAC-SIV recombinant plus SIV gp120 protein or with DNA for SIV genes and rhesus interleukin-12 plus SIV gp120 protein. Controls received empty Ad5hr vector and alum adjuvant only. Both regimens elicited strong, comparable mucosal and systemic cellular and humoral immunity. Prevaccination rectal microbiomes of males and females differed and significantly changed over the course of immunization, most strongly in females after Ad5hr immunizations. Following repeated low-dose intrarectal SIV challenges, both vaccine groups exhibited modestly but significantly reduced acute viremia. Male and female controls exhibited similar acute viral loads; however, vaccinated females, but not males, exhibited lower levels of acute viremia, compared to same-sex controls. Few differences in adaptive immune responses were observed between the sexes. Striking differences in correlations of the rectal microbiome of males and females with acute viremia and immune responses associated with protection were seen and point to effects of the microbiome on vaccine-induced immunity and viremia control. Our study clearly demonstrates direct effects of a mucosal SIV vaccine regimen on the rectal microbiome and validates our previously reported SIV vaccine-induced sex bias. Sex and the microbiome are critical factors that should not be overlooked in vaccine design and evaluation.IMPORTANCE Differences in HIV pathogenesis between males and females, including immunity postinfection, have been well documented, as have steroid hormone effects on the microbiome, which is known to influence mucosal immune responses. Few studies have applied this knowledge to vaccine trials. We investigated two SIV vaccine regimens combining mucosal priming immunizations and systemic protein boosting. We again report a vaccine-induced sex bias, with female rhesus macaques but not males displaying significantly reduced acute viremia. The vaccine regimens, especially the mucosal primes, significantly altered the rectal microbiome. The greatest effects were in females. Striking differences between female and male macaques in correlations of prevalent rectal bacteria with viral loads and potentially protective immune responses were observed. Effects of the microbiome on vaccine-induced immunity and viremia control require further study by microbiome transfer. However, the findings presented highlight the critical importance of considering effects of sex and the microbiome in vaccine design and evaluation.

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.

ALVAC-HIV B/C candidate HIV vaccine efficacy dependent on neutralization profile of challenge virus and adjuvant dose and type.

The ALVAC-HIV clade B/AE and equivalent SIV-based/gp120 + Alum vaccines successfully decreased the risk of virus acquisition in humans and macaques. Here, we tested the efficacy of HIV clade B/C ALVAC/gp120 vaccine candidates + MF59 or different doses of Aluminum hydroxide (Alum) against SHIV-Cs of varying neutralization sensitivity in macaques. Low doses of Alum induced higher mucosal V2-specific IgA that increased the risk of Tier 2 SHIV-C acquisition. High Alum dosage, in contrast, elicited serum IgG to V2 that correlated with a decreased risk of Tier 1 SHIV-C acquisition. MF59 induced negligible mucosal antibodies to V2 and an inflammatory profile with blood C-reactive Protein (CRP) levels correlating with neutralizing antibody titers. MF59 decreased the risk of Tier 1 SHIV-C acquisition. The relationship between vaccine efficacy and the neutralization profile of the challenge virus appear to be linked to the different immunological spaces created by MF59 and Alum via CXCL10 and IL-1ß, respectively.

Functional properties and sequence variation of HTLV-1 p13.

Human T cell leukemia virus type-1 (HTLV-1) was the first retrovirus found to cause cancer in humans, but the mechanisms that drive the development of leukemia and other diseases associated with HTLV-1 infection remain to be fully understood. This review describes the functional properties of p13, an 87-amino acid protein coded by HTLV-1 open reading frame II (orf-II). p13 is mainly localized in the inner membrane of the mitochondria, where it induces potassium (K+) influx and reactive oxygen species (ROS) production, which can trigger either proliferation or apoptosis, depending on the ROS setpoint of the cell. Recent evidence indicates that p13 may influence the cell's innate immune response to viral infection and the infected cell phenotype. Association of the HTLV-1 transcriptional activator, Tax, with p13 increases p13's stability, leads to its partial co-localization with Tax in nuclear speckles, and reduces the ability of Tax to interact with the transcription cofactor CBP/p300. Comparison of p13 sequences isolated from HTLV-1-infected individuals revealed a small number of amino acid variations in the domains controlling the subcellular localization of the protein. Disruptive mutations of p13 were found in samples obtained from asymptomatic patients with low proviral load. p13 sequences of HTLV-1 subtype C isolates from indigenous Australian patients showed a high degree of identity among each other, with all samples containing a pattern of 5 amino acids that distinguished them from other subtypes. Further characterization of p13's functional properties and sequence variants may lead to a deeper understanding of the impact of p13 as a contributor to the clinical manifestations of HTLV-1 infection.

Essential Role of Human T Cell Leukemia Virus Type 1 orf-I in Lethal Proliferation of CD4+ Cells in Humanized Mice.

Human T cell leukemia virus type 1 (HTLV-1) is the ethological agent of adult T cell leukemia/lymphoma (ATLL) and a number of lymphocyte-mediated inflammatory conditions, including HTLV-1-associated myelopathy/tropical spastic paraparesis. HTLV-1 orf-I encodes two proteins, p8 and p12, whose functions in humans are to counteract innate and adaptive responses and to support viral transmission. However, the in vivo requirements for orf-I expression vary in different animal models. In macaques, the ablation of orf-I expression by mutation of its ATG initiation codon abolishes the infectivity of the molecular clone HTLV-1p12KO In rabbits, HTLV-1p12KO is infective and persists efficiently. We used humanized mouse models to assess the infectivity of both wild-type HTLV-1 (HTLV-1WT) and HTLV-1p12KO We found that NOD/SCID/γC-/- c-kit+ mice engrafted with human tissues 1 day after birth (designated NSG-1d mice) were highly susceptible to infection by HTLV-1WT, with a syndrome characterized by the rapid polyclonal proliferation and infiltration of CD4+ CD25+ T cells into vital organs, weight loss, and death. HTLV-1 clonality studies revealed the presence of multiple clones of low abundance, confirming the polyclonal expansion of HTLV-1-infected cells in vivo HTLV-1p12KO infection in a bone marrow-liver-thymus (BLT) mouse model prone to graft-versus-host disease occurred only following reversion of the orf-I initiation codon mutation within weeks after exposure and was associated with high levels of HTLV-1 DNA in blood and the expansion of CD4+ CD25+ T cells. Thus, the incomplete reconstitution of the human immune system in BLT mice may provide a window of opportunity for HTLV-1 replication and the selection of viral variants with greater fitness.IMPORTANCE Humanized mice constitute a useful model for studying the HTLV-1-associated polyclonal proliferation of CD4+ T cells and viral integration sites in the human genome. The rapid death of infected animals, however, appears to preclude the clonal selection typically observed in human ATLL, which normally develops in 2 to 5% of individuals infected with HTLV-1. Nevertheless, the expansion of multiple clones of low abundance in these humanized mice mirrors the early phase of HTLV-1 infection in humans, providing a useful model to investigate approaches to inhibit virus-induced CD4+ T cell proliferation.

Early T Follicular Helper Cell Responses and Germinal Center Reactions Are Associated with Viremia Control in Immunized Rhesus Macaques.

T follicular helper (TFH) cells are fundamental in germinal center (GC) maturation and selection of antigen-specific B cells within secondary lymphoid organs. GC-resident TFH cells have been fully characterized in human immunodeficiency virus (HIV) infection. However, the role of GC TFH cells in GC B cell responses following various simian immunodeficiency virus (SIV) vaccine regimens in rhesus macaques (RMs) has not been fully investigated. We characterized GC TFH cells of RMs over the course of a mucosal/systemic vaccination regimen to elucidate GC formation and SIV humoral response generation. Animals were mucosally primed twice with replicating adenovirus type 5 host range mutant (Ad5hr)-SIV recombinants and systemically boosted with ALVAC-SIVM766Gag/Pro/gp120-TM and SIVM766&CG7V gD-gp120 proteins formulated in alum hydroxide (ALVAC/Env) or DNA encoding SIVenv/SIVGag/rhesus interleukin 12 (IL-12) plus SIVM766&CG7V gD-gp120 proteins formulated in alum phosphate (DNA&Env). Lymph nodes were biopsied in macaque subgroups prevaccination and at day 3, 7, or 14 after the 2nd Ad5hr-SIV prime and the 2nd vector/Env boost. Evaluations of GC TFH and GC B cell dynamics including correlation analyses supported a significant role for early GC TFH cells in providing B cell help during initial phases of GC formation. GC TFH responses at day 3 post-mucosal priming were consistent with generation of Env-specific memory B cells in GCs and elicitation of prolonged Env-specific humoral immunity in the rectal mucosa. GC Env-specific memory B cell responses elicited early post-systemic boosting correlated significantly with decreased viremia postinfection. Our results highlight the importance of early GC TFH cell responses for robust GC maturation and generation of long-lasting SIV-specific humoral responses at mucosal and systemic sites. Further investigation of GC TFH cell dynamics should facilitate development of an efficacious HIV vaccine.IMPORTANCE The modest HIV protection observed in the human RV144 vaccine trial associated antibody responses with vaccine efficacy. T follicular helper (TFH) cells are CD4+ T cells that select antibody secreting cells with high antigenic affinity in germinal centers (GCs) within secondary lymphoid organs. To evaluate the role of TFH cells in eliciting prolonged virus-specific humoral responses, we vaccinated rhesus macaques with a combined mucosal prime/systemic boost regimen followed by repeated low-dose intrarectal challenges with SIV, mimicking human exposure to HIV-1. Although the vaccine regimen did not prevent SIV infection, decreased viremia was observed in the immunized macaques. Importantly, vaccine-induced TFH responses elicited at day 3 postimmunization and robust GC maturation were strongly associated. Further, early TFH-dependent SIV-specific B cell responses were also correlated with decreased viremia. Our findings highlight the contribution of early vaccine-induced GC TFH responses to elicitation of SIV-specific humoral immunity and implicate their participation in SIV control.

Distinct susceptibility of HIV vaccine vector-induced CD4 T cells to HIV infection.

The concerns raised from adenovirus 5 (Ad5)-based HIV vaccine clinical trials, where excess HIV infections were observed in some vaccine recipients, have highlighted the importance of understanding host responses to vaccine vectors and the HIV susceptibility of vector-specific CD4 T cells in HIV vaccination. Our recent study reported that human Ad5-specific CD4 T cells induced by Ad5 vaccination (RV156A trial) are susceptible to HIV. Here we further investigated the HIV susceptibility of vector-specific CD4 T cells induced by ALVAC, a canarypox viral vector tested in the Thai trial RV144, as compared to Ad5 vector-specific CD4 T cells in the HVTN204 trial. We showed that while Ad5 vector-specific CD4 T cells were readily susceptible to HIV, ALVAC-specific CD4 T cells in RV144 PBMC were substantially less susceptible to both R5 and X4 HIV in vitro. The lower HIV susceptibility of ALVAC-specific CD4 T cells was associated with the reduced surface expression of HIV entry co-receptors CCR5 and CXCR4 on these cells. Phenotypic analyses identified that ALVAC-specific CD4 T cells displayed a strong Th1 phenotype, producing higher levels of IFN-γ and CCL4 (MIP-1ß) but little IL-17. Of interest, ALVAC and Ad5 vectors induced distinct profiles of vector-specific CD8 vs. CD4 T-cell proliferative responses in PBMC, with ALVAC preferentially inducing CD8 T-cell proliferation, while Ad5 vector induced CD4 T-cell proliferation. Depletion of ALVAC-, but not Ad5-, induced CD8 T cells in PBMC led to a modest increase in HIV infection of vector-specific CD4 T cells, suggesting a role of ALVAC-specific CD8 T cells in protecting ALVAC-specific CD4 T cells from HIV. Taken together, our data provide strong evidence for distinct HIV susceptibility of CD4 T cells induced by different vaccine vectors and highlight the importance of better evaluating anti-vector responses in HIV vaccination.

Select gp120 V2 domain specific antibodies derived from HIV and SIV infection and vaccination inhibit gp120 binding to α4ß7.

The GI tract is preferentially targeted during acute/early HIV-1 infection. Consequent damage to the gut plays a central role in HIV pathogenesis. The basis for preferential targeting of gut tissues is not well defined. Recombinant proteins and synthetic peptides derived from HIV and SIV gp120 bind directly to integrin α4ß7, a gut-homing receptor. Using both cell-surface expressed α4ß7 and a soluble α4ß7 heterodimer we demonstrate that its specific affinity for gp120 is similar to its affinity for MAdCAM (its natural ligand). The gp120 V2 domain preferentially engages extended forms of α4ß7 in a cation -sensitive manner and is inhibited by soluble MAdCAM. Thus, V2 mimics MAdCAM in the way that it binds to α4ß7, providing HIV a potential mechanism to discriminate between functionally distinct subsets of lymphocytes, including those with gut-homing potential. Furthermore, α4ß7 antagonists developed for the treatment of inflammatory bowel diseases, block V2 binding to α4ß7. A 15-amino acid V2 -derived peptide is sufficient to mediate binding to α4ß7. It includes the canonical LDV/I α4ß7 binding site, a cryptic epitope that lies 7-9 amino acids amino terminal to the LDV/I, and residues K169 and I181. These two residues were identified in a sieve analysis of the RV144 vaccine trial as sites of vaccine -mediated immune pressure. HIV and SIV V2 mAbs elicited by both vaccination and infection that recognize this peptide block V2-α4ß7 interactions. These mAbs recognize conformations absent from the ß- barrel presented in a stabilized HIV SOSIP gp120/41 trimer. The mimicry of MAdCAM-α4ß7 interactions by V2 may influence early events in HIV infection, particularly the rapid seeding of gut tissues, and supports the view that HIV replication in gut tissue is a central feature of HIV pathogenesis.

Neutrophil Vaccination Dynamics and Their Capacity To Mediate B Cell Help in Rhesus Macaques.

Neutrophils are the most abundant leukocyte and play a critical role in the initial response to an Ag. Recently, their ability to contribute to adaptive immunity has been highlighted. We evaluated the ability of neutrophils from blood to contribute to the adaptive immune response in a preclinical rhesus macaque SIV vaccine trial. Replication-competent adenovirus-SIV recombinants induced neutrophil activation, B cell help markers, and enhanced ability to generate reactive oxygen species. Boosting with SIV vaccines (adjuvant together with ALVAC or DNA plus envelope protein) elicited significant neutrophil responses. Serum cytokine and chemokine levels induced correlated with the frequency of neutrophil subsets expressing IL-21, myeloperoxidase, and CD64. Post-SIV infection, neutrophils exhibited dysfunction, both phenotypically and functionally. B cells from protected and infected macaques cocultured with autologous polymorphonuclear cells, consisting primarily of neutrophils, were activated, underwent class switching, and produced Abs. This B cell help was not aided by addition of IL-10 and was largely contact dependent. Numerous genes associated with inflammation, Ab production, and chemotaxis were upregulated in the cocultured B cells. We conclude that immune stimulation by vaccination or antigenic exposure imparts a greater ability of neutrophils to contribute to the adaptive immune response. Harnessing this granulocytic response has the potential to improve vaccine efficacy.

Role of HTLV-1 orf-I encoded proteins in viral transmission and persistence.

The human T cell leukemia virus type 1 (HTVL-1), first reported in 1980 by Robert Gallo's group, is the etiologic agent of both cancer and inflammatory diseases. Despite approximately 40 years of investigation, the prognosis for afflicted patients remains poor with no effective treatments. The virus persists in the infected host by evading the host immune response and inducing proliferation of infected CD4+ T-cells. Here, we will review the role that viral orf-I protein products play in altering intracellular signaling, protein expression and cell-cell communication in order to escape immune recognition and promote T-cell proliferation. We will also review studies of orf-I mutations found in infected patients and their potential impact on viral load, transmission and persistence. Finally, we will compare the orf-I gene in HTLV-1 subtypes as well as related STLV-1.

p30 protein: a critical regulator of HTLV-1 viral latency and host immunity.

The extraordinarily high prevalence of HTLV-1 subtype C (HTLV-1C) in some isolated indigenous communities in Oceania and the severity of the health conditions associated with the virus impress the great need for basic and translational research to prevent and treat HTLV-1 infection. The genome of the virus's most common subtype, HTLV-1A, encodes structural, enzymatic, and regulatory proteins that contribute to viral persistence and pathogenesis. Among these is the p30 protein encoded by the doubly spliced Tax-orf II mRNA, a nuclear/nucleolar protein with both transcriptional and post-transcriptional activity. The p30 protein inhibits the productive replication cycle via nuclear retention of the mRNA that encodes for both the viral transcriptional trans-activator Tax, and the Rex proteins that regulate the transport of incompletely spliced viral mRNA to the cytoplasm. In myeloid cells, p30 inhibits the PU-1 transcription factor that regulates interferon expression and is a critical mediator of innate and adaptive immunity. Furthermore, p30 alters gene expression, cell cycle progression, and DNA damage responses in T-cells, raising the hypothesis that p30 may directly contribute to T cell transformation. By fine-tuning viral expression while also inhibiting host innate responses, p30 is likely essential for viral infection and persistence. This concept is supported by the finding that macaques, a natural host for the closely genetically related simian T-cell leukemia virus 1 (STLV-1), exposed to an HTLV-1 knockout for p30 expression by a single point mutation do not became infected unless reversion and selection of the wild type HTLV-1 genotype occurs. All together, these data suggest that inhibition of p30 may help to curb and eventually eradicate viral infection by exposing infected cells to an effective host immune response.