Innate immune signatures to a partially-efficacious HIV vaccine predict correlates of HIV-1 infection risk.

The pox-protein regimen tested in the RV144 trial is the only vaccine strategy demonstrated to prevent HIV-1 infection. Subsequent analyses identified antibody and cellular immune responses as correlates of risk (CoRs) for HIV infection. Early predictors of these CoRs could provide insight into vaccine-induced protection and guide efforts to enhance vaccine efficacy. Using specimens from a phase 1b trial of the RV144 regimen in HIV-1-uninfected South Africans (HVTN 097), we profiled innate responses to the first ALVAC-HIV immunization. PBMC transcriptional responses peaked 1 day post-vaccination. Type I and II interferon signaling pathways were activated, as were innate pathways critical for adaptive immune priming. We then identified two innate immune transcriptional signatures strongly associated with adaptive immune CoR after completion of the 4-dose regimen. Day 1 signatures were positively associated with antibody-dependent cellular cytotoxicity and phagocytosis activity at Month 6.5. Conversely, a signature present on Days 3 and 7 was inversely associated with Env-specific CD4+ T cell responses at Months 6.5 and 12; rapid resolution of this signature was associated with higher Env-specific CD4+ T-cell responses. These are the first-reported early immune biomarkers of vaccine-induced responses associated with HIV-1 acquisition risk in humans and suggest hypotheses to improve HIV-1 vaccine regimens.

The transcriptome of HIV-1 infected intestinal CD4+ T cells exposed to enteric bacteria.

Global transcriptome studies can help pinpoint key cellular pathways exploited by viruses to replicate and cause pathogenesis. Previous data showed that laboratory-adapted HIV-1 triggers significant gene expression changes in CD4+ T cell lines and mitogen-activated CD4+ T cells from peripheral blood. However, HIV-1 primarily targets mucosal compartments during acute infection in vivo. Moreover, early HIV-1 infection causes extensive depletion of CD4+ T cells in the gastrointestinal tract that herald persistent inflammation due to the translocation of enteric microbes to the systemic circulation. Here, we profiled the transcriptome of primary intestinal CD4+ T cells infected ex vivo with transmitted/founder (TF) HIV-1. Infections were performed in the presence or absence of Prevotella stercorea, a gut microbe enriched in the mucosa of HIV-1-infected individuals that enhanced both TF HIV-1 replication and CD4+ T cell death ex vivo. In the absence of bacteria, HIV-1 triggered a cellular shutdown response involving the downregulation of HIV-1 reactome genes, while perturbing genes linked to OX40, PPAR and FOXO3 signaling. However, in the presence of bacteria, HIV-1 did not perturb these gene sets or pathways. Instead, HIV-1 enhanced granzyme expression and Th17 cell function, inhibited G1/S cell cycle checkpoint genes and triggered downstream cell death pathways in microbe-exposed gut CD4+ T cells. To gain insights on these differential effects, we profiled the gene expression landscape of HIV-1-uninfected gut CD4+ T cells exposed to bacteria. Microbial exposure upregulated genes involved in cellular proliferation, MAPK activation, Th17 cell differentiation and type I interferon signaling. Our findings reveal that microbial exposure influenced how HIV-1 altered the gut CD4+ T cell transcriptome, with potential consequences for HIV-1 susceptibility, cell survival and inflammation. The HIV-1- and microbe-altered pathways unraveled here may serve as a molecular blueprint to gain basic insights in mucosal HIV-1 pathogenesis.

Type I interferon signaling is required for the APOBEC3/Rfv3-dependent neutralizing antibody response but not innate retrovirus restriction.

BACKGROUND: APOBEC3/Rfv3 restricts acute Friend retrovirus (FV) infection and promotes virus-specific neutralizing antibody (NAb) responses. Classical Rfv3 studies utilized FV stocks containing lactate-dehydrogenase elevating virus (LDV), a potent type I interferon inducer. Previously, we showed that APOBEC3 is required for the anti-FV activity of exogenous IFN-alpha treatment. Thus, type I interferon receptor (IFNAR) signaling may be required for the APOBEC3/Rfv3 response. RESULTS: To test if the APOBEC3/Rfv3 response is dependent on type I IFN signaling, we infected IFNAR knockout versus IFNAR/APOBEC3 double-knockout mice with FV/LDV or LDV-free FV, and evaluated acute FV infection and subsequent NAb titers. We show that LDV co-infection and type I IFN signaling are not required for innate APOBEC3-mediated restriction. By contrast, removal of LDV and/or type I IFN signaling abrogated the APOBEC3-dependent NAb response. CONCLUSIONS: APOBEC3 can restrict retroviruses in a type I IFN-independent manner in vivo. By contrast, the ability of APOBEC3 to promote NAb responses is type I IFN-dependent. These findings reveal novel insights on the interplay between type I IFNs and APOBEC3 in vivo that may have implications for augmenting antiretroviral NAb responses.

Interferon Alpha Subtype-Specific Suppression of HIV-1 Infection In Vivo.

UNLABELLED: Although all 12 subtypes of human interferon alpha (IFN-α) bind the same receptor, recent results have demonstrated that they elicit unique host responses and display distinct efficacies in the control of different viral infections. The IFN-α2 subtype is currently in HIV-1 clinical trials, but it has not consistently reduced viral loads in HIV-1 patients and is not the most effective subtype against HIV-1 in vitro We now demonstrate in humanized mice that, when delivered at the same high clinical dose, the human IFN-α14 subtype has very potent anti-HIV-1 activity whereas IFN-α2 does not. In both postexposure prophylaxis and treatment of acute infections, IFN-α14, but not IFN-α2, significantly suppressed HIV-1 replication and proviral loads. Furthermore, HIV-1-induced immune hyperactivation, which is a prognosticator of disease progression, was reduced by IFN-α14 but not IFN-α2. Whereas ineffective IFN-α2 therapy was associated with CD8(+) T cell activation, successful IFN-α14 therapy was associated with increased intrinsic and innate immunity, including significantly higher induction of tetherin and MX2, increased APOBEC3G signature mutations in HIV-1 proviral DNA, and higher frequencies of TRAIL(+) NK cells. These results identify IFN-α14 as a potent new therapeutic that operates via mechanisms distinct from those of antiretroviral drugs. The ability of IFN-α14 to reduce both viremia and proviral loads in vivo suggests that it has strong potential as a component of a cure strategy for HIV-1 infections. The broad implication of these results is that the antiviral efficacy of each individual IFN-α subtype should be evaluated against the specific virus being treated. IMPORTANCE: The naturally occurring antiviral protein IFN-α2 is used to treat hepatitis viruses but has proven rather ineffective against HIV in comparison to triple therapy with the antiretroviral (ARV) drugs. Although ARVs suppress the replication of HIV, they fail to completely clear infections. Since IFN-α acts by different mechanism than ARVs and has been shown to reduce HIV proviral loads, clinical trials are under way to test whether IFN-α2 combined with ARVs might eradicate HIV-1 infections. IFN-α is actually a family of 12 distinct proteins, and each IFN-α subtype has different efficacies toward different viruses. Here, we use mice that contain a human immune system, so they can be infected with HIV. With this model, we demonstrate that while IFN-α2 is only weakly effective against HIV, IFN-α14 is extremely potent. This discovery identifies IFN-α14 as a more powerful IFN-α subtype for use in combination therapy trials aimed toward an HIV cure.

Interferon-α Subtypes in an Ex Vivo Model of Acute HIV-1 Infection: Expression, Potency and Effector Mechanisms.

HIV-1 is transmitted primarily across mucosal surfaces and rapidly spreads within the intestinal mucosa during acute infection. The type I interferons (IFNs) likely serve as a first line of defense, but the relative expression and antiviral properties of the 12 IFNα subtypes against HIV-1 infection of mucosal tissues remain unknown. Here, we evaluated the expression of all IFNα subtypes in HIV-1-exposed plasmacytoid dendritic cells by next-generation sequencing. We then determined the relative antiviral potency of each IFNα subtype ex vivo using the human intestinal Lamina Propria Aggregate Culture model. IFNα subtype transcripts from the centromeric half of the IFNA gene complex were highly expressed in pDCs following HIV-1 exposure. There was an inverse relationship between IFNA subtype expression and potency. IFNα8, IFNα6 and IFNα14 were the most potent in restricting HIV-1 infection. IFNα2, the clinically-approved subtype, and IFNα1 were both highly expressed but exhibited relatively weak antiviral activity. The relative potencies correlated with binding affinity to the type I IFN receptor and the induction levels of HIV-1 restriction factors Mx2 and Tetherin/BST-2 but not APOBEC3G, F and D. However, despite the lack of APOBEC3 transcriptional induction, the higher relative potency of IFNα8 and IFNα14 correlated with stronger inhibition of virion infectivity, which is linked to deaminase-independent APOBEC3 restriction activity. By contrast, both potent (IFNα8) and weak (IFNα1) subtypes significantly induced HIV-1 GG-to-AG hypermutation. The results unravel non-redundant functions of the IFNα subtypes against HIV-1 infection, with strong implications for HIV-1 mucosal immunity, viral evolution and IFNα-based functional cure strategies.

Enhancement of HIV-1 infection and intestinal CD4+ T cell depletion ex vivo by gut microbes altered during chronic HIV-1 infection.

BACKGROUND: Early HIV-1 infection is characterized by high levels of HIV-1 replication and substantial CD4 T cell depletion in the intestinal mucosa, intestinal epithelial barrier breakdown, and microbial translocation. HIV-1-induced disruption of intestinal homeostasis has also been associated with changes in the intestinal microbiome that are linked to mucosal and systemic immune activation. In this study, we investigated the impact of representative bacterial species that were altered in the colonic mucosa of viremic HIV-1 infected individuals (HIV-altered mucosal bacteria; HAMB) on intestinal CD4 T cell function, infection by HIV-1, and survival in vitro. Lamina propria (LP) mononuclear cells were infected with CCR5-tropic HIV-1BaL or mock infected, exposed to high (3 gram-negative) or low (2 gram-positive) abundance HAMB or control gram-negative Escherichia coli and levels of productive HIV-1 infection and CD4 T cell depletion assessed. HAMB-associated changes in LP CD4 T cell activation, proliferation and HIV-1 co-receptor expression were also evaluated. RESULTS: The majority of HAMB increased HIV-1 infection and depletion of LP CD4 T cells, but gram-negative HAMB enhanced CD4 T cell infection to a greater degree than gram-positive HAMB. Most gram-negative HAMB enhanced T cell infection to levels similar to that induced by gram-negative E. coli despite lower induction of T cell activation and proliferation by HAMB. Both gram-negative HAMB and E. coli significantly increased expression of HIV-1 co-receptor CCR5 on LP CD4 T cells. Lipopolysaccharide, a gram-negative bacteria cell wall component, up-regulated CCR5 expression on LP CD4 T cells whereas gram-positive cell wall lipoteichoic acid did not. Upregulation of CCR5 by gram-negative HAMB was largely abrogated in CD4 T cell-enriched cultures suggesting an indirect mode of stimulation. CONCLUSIONS: Gram-negative commensal bacteria that are altered in abundance in the colonic mucosa of HIV-1 infected individuals have the capacity to enhance CCR5-tropic HIV-1 productive infection and depletion of LP CD4 T cells in vitro. Enhanced infection appears to be primarily mediated indirectly through increased expression of CCR5 on LP CD4 T cells without concomitant large scale T cell activation. This represents a novel mechanism potentially linking intestinal dysbiosis to HIV-1 mucosal pathogenesis.

Immunoglobulin VH gene diversity and somatic hypermutation during SIV infection of rhesus macaques.

B cell functional defects are associated with delayed neutralizing antibody development in pathogenic lentivirus infections. However, the timeframe for alterations in the antibody repertoire and somatic hypermutation (SHM) remains unclear. Here, we utilized the SIV/rhesus macaque (RM) model to investigate the dynamics of immunoglobulin V(H) gene diversity and SHM following infection. Three RMs were infected with SIVmac239, and V(H)1, V(H)3, and V(H)4 genes were amplified from peripheral blood at 0, 2, 6, 24, and 36 weeks postinfection for next-generation sequencing. Analysis of over 3.8 million sequences against currently available RM germline V(H) genes revealed a highly biased V(H) gene repertoire in outbred RMs. SIV infection did not significantly perturb the predominant IgG1 response, but overall immunoglobulin SHM declined during the course of SIV infection. Moreover, SHM at the AID deamination hotspot, WRC, rapidly decreased and was suppressed throughout SIV infection. In contrast, a transient increase in mutations at the APOBEC3G deamination hotspot, CCC, coincided with a spike in APOBEC3G expression during acute SIV infection. The results outline a timetable for altered V(H) gene repertoire and IgG SHM in the SIV/RM model and suggest a burst of APOBEC3G-mediated antibody SHM during acute SIV infection.

IFN-α treatment inhibits acute Friend retrovirus replication primarily through the antiviral effector molecule Apobec3.

Therapeutic administration of IFN-α in clinical trials significantly reduced HIV-1 plasma viral load and human T-lymphotropic virus type I proviral load in infected patients. The mechanism may involve the concerted action of multiple antiretroviral effectors collectively known as "restriction factors," which could vary in relative importance according to the magnitude of transcriptional induction. However, direct genetic approaches to identify the relevant IFN-α restriction factors will not be feasible in humans in vivo. Meanwhile, mice encode an analogous set of restriction factor genes and could be used to obtain insights on how IFN-α could inhibit retroviruses in vivo. As expected, IFN-α treatment of mice significantly upregulated the transcription of multiple restriction factors including Tetherin/BST2, SAMHD1, Viperin, ISG15, OAS1, and IFITM3. However, a dominant antiretroviral factor, Apobec3, was only minimally induced. To determine whether Apobec3 was necessary for direct IFN-α antiretroviral action in vivo, wild-type and Apobec3-deficient mice were infected with Friend retrovirus, then treated with IFN-α. Treatment of infected wild-type mice with IFN-α significantly reduced acute plasma viral load 28-fold, splenic proviral load 5-fold, bone marrow proviral load 14-fold, and infected bone marrow cells 7-fold, but no inhibition was observed in Apobec3-deficient mice. These findings reveal that IFN-α inhibits acute Friend retrovirus infection primarily through the antiviral effector Apobec3 in vivo, demonstrate that transcriptional induction levels did not predict the mechanism of IFN-α-mediated control, and highlight the potential of the human APOBEC3 proteins as therapeutic targets against pathogenic retrovirus infections.

Friend retrovirus drives cytotoxic effectors through Toll-like receptor 3.

BACKGROUND: Pathogen recognition drives host defense towards viral infections. Specific groups rather than single members of the protein family of pattern recognition receptors (PRRs) such as membrane spanning Toll-like receptors (TLRs) and cytosolic helicases might mediate sensing of replication intermediates of a specific virus species. TLR7 mediates host sensing of retroviruses and could significantly influence retrovirus-specific antibody responses. However, the origin of efficient cell-mediated immunity towards retroviruses is unknown. Double-stranded RNA intermediates produced during retroviral replication are good candidates for immune stimulatory viral products. Thus, we considered TLR3 as primer of cell-mediated immunity against retroviruses in vivo. RESULTS: Infection of mice deficient in TLR3 (TLR3(-/-)) with Friend retrovirus (FV) complex revealed higher viral loads during acute retroviral infection compared to wild type mice. TLR3(-/-) mice exhibited significantly lower expression levels of type I interferons (IFNs) and IFN-stimulated genes like Pkr or Ifi44, as well as reduced numbers of activated myeloid dendritic cells (DCs) (CD86(+) and MHC-II(+)). DCs generated from FV-infected TLR3(-/-) mice were less capable of priming virus-specific CD8(+) T cell proliferation. Moreover, cytotoxicity of natural killer (NK) cells as well as CD8(+) T cells were reduced in vitro and in vivo, respectively, in FV-infected TLR3(-/-) mice. CONCLUSIONS: TLR3 mediates antiretroviral cytotoxic NK cell and CD8(+) T cell activity in vivo. Our findings qualify TLR3 as target of immune therapy against retroviral infections.

Breaching peripheral tolerance promotes the production of HIV-1-neutralizing antibodies.

A subset of characterized HIV-1 broadly neutralizing antibodies (bnAbs) are polyreactive with additional specificities for self-antigens and it has been proposed immunological tolerance may present a barrier to their participation in protective humoral immunity. We address this hypothesis by immunizing autoimmune-prone mice with HIV-1 Envelope (Env) and characterizing the primary antibody response for HIV-1 neutralization. We find autoimmune mice generate neutralizing antibody responses to tier 2 HIV-1 strains with alum treatment alone in the absence of Env. Importantly, experimentally breaching immunological tolerance in wild-type mice also leads to the production of tier 2 HIV-1-neutralizing antibodies, which increase in breadth and potency following Env immunization. In both genetically prone and experimentally induced mouse models of autoimmunity, increased serum levels of IgM anti-histone H2A autoantibodies significantly correlated with tier 2 HIV-1 neutralization, and anti-H2A antibody clones were found to neutralize HIV-1. These data demonstrate that breaching peripheral tolerance permits a cross-reactive HIV-1 autoantibody response able to neutralize HIV-1.

Reassessment of murine APOBEC1 as a retrovirus restriction factor in vivo.

APOBEC1 is a cytidine deaminase involved in cholesterol metabolism that has been linked to retrovirus restriction, analogous to the evolutionarily-related APOBEC3 proteins. In particular, murine APOBEC1 was shown to inhibit Friend retrovirus (FV) in vitro, generating high levels of C-to-T and G-to-A mutations. These observations raised the possibility that FV infection might be altered in APOBEC1-null mice. To examine this question directly, we infected wild-type and APOBEC1-null mice with FV complex and evaluated acute infection levels. Surprisingly, APOBEC1-null mice exhibited similar cellular infection levels and plasma viremia relative to wild-type mice. Moreover, next-generation sequencing analyses revealed that in contrast to APOBEC3, APOBEC1 did not enhance retroviral C-to-T and G-to-A mutational frequencies in genomic DNA. Thus, APOBEC1 neither inhibited nor significantly drove the molecular evolution of FV in vivo. Our findings reinforce that not all retrovirus restriction factors characterized as potent in vitro may be functionally relevant in vivo.

Fv1 restriction and retrovirus vaccine immunity in Apobec3-deficient 129P2 mice.

Understanding the host genetics of the immune response in retrovirus infection models could provide insights for basic HIV vaccine discovery. In Friend retrovirus (FV) infection of mice, Fv1 differentially inhibits N-tropic versus B-tropic FV infection by mediating a capsid-dependent post-entry block, Fv2 susceptibility governs splenomegaly induction, and Rfv3 resistance primes a stronger neutralizing antibody response due to more potent Apobec3 activity. Apobec3 polymorphisms in inbred mouse strains correlate with Rfv3 resistance and susceptibility, with one unresolved exception. The 129/OlaHsd (129P2) mouse strain is Fv2 and Rfv3 susceptible based on genotyping, but infection of 129P2 mice with B-tropic FV resulted in strong neutralizing antibody responses and no splenomegaly. Here we confirm that 129P2 mice are Fv1(nr/nr), explaining its resistance to B-tropic FV. Infection of 129P2 mice with NB-tropic FV, which can efficiently infect mice independent of Fv1 genotype, resulted in severe splenomegaly, high levels of viremia and weak neutralizing antibody responses regardless of Apobec3 status. Notably, high-dose B-tropic FV infection of 129P2 Apobec3-deficient mice induced significant adaptive immune responses and conferred high levels of protection following challenge with pathogenic NB-tropic FV. This immunological protection complemented previous studies that N-tropic FV can act as a live-attenuated vaccine in Fv1 (b/b) mice. Altogether, the results obtained in 129P2 mice strengthen the conclusion that Rfv3 is encoded by Apobec3, and highlight Fv1 incompatibility as a retroviral vaccine paradigm in mice. Due to its susceptibility to disease that allows for pathogenic challenge studies, B-tropic FV infection of 129P2 mice may be a useful model to study the immunological pathways induced by retroviral capsid restriction.

Humoral immunity in the Friend retrovirus infection model.

Major conceptual roadblocks impede the development of an HIV-1 vaccine that can stimulate a potent neutralizing antibody response. Animal models that support HIV-1 replication and allow for host genetic manipulation would be an ideal platform for testing various immunological hypotheses, but progress on this research front has been slow and disappointing. In contrast, many valuable concepts emerged from more than 50 years of studying the Friend retrovirus model. This was recently exemplified by the identification of an innate restriction gene, Apobec3, that could promote the retrovirus-specific neutralizing antibody response. Here we review both classical and recent data on humoral immunity against Friend retrovirus infection, and highlight the potential of this model for unraveling novel aspects of the retrovirus-specific antibody response that may guide HIV-1 vaccine development efforts.

Ribonuclease L is not critical for innate restriction and adaptive immunity against Friend retrovirus infection.

Ribonuclease L (RNase L) is a type I interferon regulated factor that can significantly inhibit retroviruses in vitro and may activate cytoplasmic sensing pathways to augment adaptive immunity. However, the antiretroviral activity of RNase L remains to be validated in vivo. We investigated the role of RNaseL in counteracting Friend retrovirus (FV) infection relative to a well-described restriction factor, Apobec3. C57BL/6 wild-type (WT) and RNaseL knock-out (KO) mice exhibited similar acute FV infection levels despite significant transcriptional induction of oligoadenylate synthetase 1, which produces activators of RNase L. Apobec3 KO mice showed higher FV infection levels relative to WT mice, but deletion of RNaseL in Apobec3 KO mice did not augment FV infection. Moreover, RNaseL did not influence FV-specific IgG responses and recovery from viremia by 28 days post-infection. The results suggest that RNase L is not an evolutionarily-conserved host defense mechanism to counteract retroviruses in vivo.