Altered Immunoglobulin Repertoire and Decreased IgA Somatic Hypermutation in the Gut during Chronic HIV-1 Infection.

Humoral immune perturbations contribute to pathogenic outcomes in persons with HIV-1 infection (PWH). Gut barrier dysfunction in PWH is associated with microbial translocation and alterations in microbial communities (dysbiosis), and IgA, the most abundant immunoglobulin (Ig) isotype in the gut, is involved in gut homeostasis by interacting with the microbiome. We determined the impact of HIV-1 infection on the antibody repertoire in the gastrointestinal tract by comparing Ig gene utilization and somatic hypermutation (SHM) in colon biopsies from PWH (n = 19) versus age and sex-matched controls (n = 13). We correlated these Ig parameters with clinical, immunological, microbiome and virological data. Gene signatures of enhanced B cell activation were accompanied by skewed frequencies of multiple Ig Variable genes in PWH. PWH showed decreased frequencies of SHM in IgA and possibly IgG, with a substantial loss of highly mutated IgA sequences. The decline in IgA SHM in PWH correlated with gut CD4+ T cell loss and inversely correlated with mucosal inflammation and microbial translocation. Diminished gut IgA SHM in PWH was driven by transversion mutations at A or T deoxynucleotides, suggesting a defect not at the AID/APOBEC3 deamination step but at later stages of IgA SHM. These results expand our understanding of humoral immune perturbations in PWH that could have important implications in understanding mucosal immune defects in individuals with chronic HIV-1 infection. IMPORTANCE The gut is a major site of early HIV-1 replication and pathogenesis. Extensive CD4+ T cell depletion in this compartment results in a compromised epithelial barrier that facilitates the translocation of microbes into the underlying lamina propria and systemic circulation, resulting in chronic immune activation. To date, the consequences of microbial translocation on the mucosal humoral immune response (or vice versa) remains poorly integrated into the panoply of mucosal immune defects in PWH. We utilized next-generation sequencing approaches to profile the Ab repertoire and ascertain frequencies of somatic hypermutation in colon biopsies from antiretroviral therapy-naive PWH versus controls. Our findings identify perturbations in the Ab repertoire of PWH that could contribute to development or maintenance of dysbiosis. Moreover, IgA mutations significantly decreased in PWH and this was associated with adverse clinical outcomes. These data may provide insight into the mechanisms underlying impaired Ab-dependent gut homeostasis during chronic HIV-1 infection.

Interferon resistance of emerging SARS-CoV-2 variants.

The emergence of SARS-CoV-2 variants with enhanced transmissibility, pathogenesis, and resistance to vaccines presents urgent challenges for curbing the COVID-19 pandemic. While Spike mutations that enhance virus infectivity or neutralizing antibody evasion may drive the emergence of these novel variants, studies documenting a critical role for interferon responses in the early control of SARS-CoV-2 infection, combined with the presence of viral genes that limit these responses, suggest that interferons may also influence SARS-CoV-2 evolution. Here, we compared the potency of 17 different human interferons against multiple viral lineages sampled during the course of the global outbreak, including ancestral and five major variants of concern that include the B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta), and B.1.1.529 (omicron) lineages. Our data reveal that relative to ancestral isolates, SARS-CoV-2 variants of concern exhibited increased interferon resistance, suggesting that evasion of innate immunity may be a significant, ongoing driving force for SARS-CoV-2 evolution. These findings have implications for the increased transmissibility and/or lethality of emerging variants and highlight the interferon subtypes that may be most successful in the treatment of early infections.

High SARS-CoV-2 Seroprevalence and Rapid Neutralizing Antibody Decline among Agricultural Workers in Rural Guatemala, June 2020-March 2021.

Essential agricultural workers work under occupational conditions that may increase the risk of SARS-CoV-2 exposure and transmission. Data from an agricultural worker cohort in Guatemala, and anti-SARS-CoV-2 nucleocapsid IgG (anti-N IgG) testing were used to estimate past infections and analyze risk factors associated with seropositivity at enrollment and association with SARS-CoV-2 infection. The stability of neutralizing antibody (NAb) responses were assessed in a subset of participants. The adjusted relative risk (aRR) for seroprevalence at enrollment was estimated accounting for correlations within worksites. At enrollment, 616 (46.2%) of 1334 (93.2%) participants had anti-N IgG results indicating prior SARS-CoV-2 infection. A cough ≤ 10 days prior to enrollment (aRR = 1.28, 95% CI: 1.13−1.46) and working as a packer (aRR = 2.00, 95% CI: 1.67−2.38) or packing manager within the plants (aRR = 1.82, 95% CI: 1.36−2.43) were associated with increased risk of seropositivity. COVID-19 incidence density among seronegative workers was 2.3/100 Person-Years (P-Y), higher than seropositive workers (0.4/100 P-Y). Most workers with follow-up NAb testing (65/77, 84%) exhibited a 95% average decrease in NAb titers in <6 months. While participants seropositive at baseline were less likely to experience a symptomatic SARS-CoV-2 infection during follow-up, NAb titers rapidly waned, underscoring the need for multipronged COVID-19 prevention strategies in the workplace, including vaccination.

COVID-19 Serology Control Panel Using the Dried-Tube Specimen Method.

The dried-tube specimen (DTS) procedure was used to develop the COVID-19 serology control panel (CSCP). The DTS offers the benefit of shipping materials without a cold chain, allowing for greater access without deterioration of material integrity. Samples in the panel were sourced from COVID-19 convalescent persons from March to May 2020. The immunoglobulin subtypes (total Ig, IgM, and IgG) and their respective reactivity to severe acute respiratory syndrome coronavirus 2 nucleocapsid, spike, and receptor-binding domain antigens of the samples were delineated and compared with the WHO International Standard to elucidate the exact binding antibody units of each CSCP sample and ensure the CSCP provides adequate reactivity for different types of serological test platforms. We distribute the CSCP as a kit with five coded tubes to laboratories around the world to be used to compare test kits for external quality assurance, for harmonizing laboratory testing, and for use as training materials for laboratory workers.

Recovery from Acute SARS-CoV-2 Infection and Development of Anamnestic Immune Responses in T Cell-Depleted Rhesus Macaques.

Severe coronavirus disease 2019 (COVID-19) has been associated with T cell lymphopenia, but no causal effect of T cell deficiency on disease severity has been established. To investigate the specific role of T cells in recovery from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, we studied rhesus macaques that were depleted of either CD4+, CD8+, or both T cell subsets prior to infection. Peak virus loads were similar in all groups, but the resolution of virus in the T cell-depleted animals was slightly delayed compared to that in controls. The T cell-depleted groups developed virus-neutralizing antibody responses and class switched to IgG. When reinfected 6 weeks later, the T cell-depleted animals showed anamnestic immune responses characterized by rapid induction of high-titer virus-neutralizing antibodies, faster control of virus loads, and reduced clinical signs. These results indicate that while T cells play a role in the recovery of rhesus macaques from acute SARS-CoV-2 infections, their depletion does not induce severe disease, and T cells do not account for the natural resistance of rhesus macaques to severe COVID-19. Neither primed CD4+ nor CD8+ T cells appeared critical for immunoglobulin class switching, the development of immunological memory, or protection from a second infection. IMPORTANCE Patients with severe COVID-19 often have decreased numbers of T cells, a cell type important in fighting most viral infections. However, it is not known whether the loss of T cells contributes to severe COVID-19 or is a consequence of it. We studied rhesus macaques, which develop only mild COVID-19, similar to most humans. Experimental depletion of T cells slightly prolonged their clearance of virus, but there was no increase in disease severity. Furthermore, they were able to develop protection from a second infection and produced antibodies capable of neutralizing the virus. They also developed immunological memory, which allows a much stronger and more rapid response upon a second infection. These results suggest that T cells are not critical for recovery from acute SARS-CoV-2 infections in this model and point toward B cell responses and antibodies as the essential mediators of protection from re-exposure.

Recovery from acute SARS-CoV-2 infection and development of anamnestic immune responses in T cell-depleted rhesus macaques.

Severe COVID-19 has been associated with T cell lymphopenia 1,2, but no causal effect of T cell deficiency on disease severity has been established. To investigate the specific role of T cells in recovery from SARS-CoV-2 infections we studied rhesus macaques that were depleted of either CD4+, CD8+ or both T cell subsets prior to infection. Peak virus loads were similar in all groups, but the resolution of virus in the T cell-depleted animals was slightly delayed compared to controls. The T cell-depleted groups developed virus-neutralizing antibody responses and also class-switched to IgG. When re-infected six weeks later, the T cell-depleted animals showed anamnestic immune responses characterized by rapid induction of high-titer virus-neutralizing antibodies, faster control of virus loads and reduced clinical signs. These results indicate that while T cells play a role in the recovery of rhesus macaques from acute SARS-CoV-2 infections, their depletion does not induce severe disease, and T cells do not account for the natural resistance of rhesus macaques to severe COVID-19. Neither primed CD4+ or CD8+ T cells appeared critical for immunoglobulin class switching, the development of immunological memory or protection from a second infection.

Interferon Resistance of Emerging SARS-CoV-2 Variants.

The emergence of SARS-CoV-2 variants with enhanced transmissibility, pathogenesis and resistance to vaccines presents urgent challenges for curbing the COVID-19 pandemic. While Spike mutations that enhance virus infectivity or neutralizing antibody evasion may drive the emergence of these novel variants, studies documenting a critical role for interferon responses in the early control of SARS-CoV-2 infection, combined with the presence of viral genes that limit these responses, suggest that interferons may also influence SARS-CoV-2 evolution. Here, we compared the potency of 17 different human interferons against multiple viral lineages sampled during the course of the global outbreak, including ancestral and four major variants of concern. Our data reveal increased interferon resistance in emerging SARS-CoV-2 variants, suggesting that evasion of innate immunity may be a significant, ongoing driving force for SARS-CoV-2 evolution. These findings have implications for the increased lethality of emerging variants and highlight the interferon subtypes that may be most successful in the treatment of early infections.

Systemic Expression of a Viral RdRP Protects against Retrovirus Infection and Disease.

The innate immune system is normally programmed for immediate but transient upregulation in response to invading pathogens, and interferon (IFN)-stimulated gene (ISG) activation is a central feature. In contrast, chronic innate immune system activation is typically associated with autoimmunity and a broad array of autoinflammatory diseases that include the interferonopathies. Here, we studied retroviral susceptibility in a transgenic mouse model with lifelong innate immune system hyperactivation. The mice transgenically express low levels of a picornaviral RNA-dependent RNA polymerase (RdRP), which synthesizes double-stranded RNAs that are sensed by melanoma differentiation-associated protein 5 (MDA5) to trigger constitutive upregulation of many ISGs. However, in striking counterpoint to the paradigm established by numerous human and murine examples of ISG hyperactivation, including constitutive MDA5 activation, they lack autoinflammatory sequelae. RdRP-transgenic mice (RdRP mice) resist infection and disease caused by several pathogenic RNA and DNA viruses. However, retroviruses are sensed through other mechanisms, persist in the host, and have distinctive replication and immunity-evading properties. We infected RdRP mice and wild-type (WT) mice with various doses of a pathogenic retrovirus (Friend virus) and assessed immune parameters and disease at 1, 4, and 8 weeks. Compared to WT mice, RdRP mice had significantly reduced splenomegaly, viral loads, and infection of multiple target cell types in the spleen and the bone marrow. During chronic infection, RdRP mice had 2.35 ± 0.66 log10 lower circulating viral RNA than WT. Protection required ongoing type I IFN signaling. The results show that the reconfigured RdRP mouse innate immune system substantially reduced retroviral replication, set point, and pathogenesis.IMPORTANCE Immune control of retroviruses is notoriously difficult, a fundamental problem that has been most clinically consequential with the HIV-1 pandemic. As humans expand further into previously uninhabited areas, the likelihood of new zoonotic retroviral exposures increases. The role of the innate immune system, including ISGs, in controlling retroviral infections is currently an area of intensive study. This work provides evidence that a primed innate immune system is an effective defense against retroviral pathogenesis, resulting in reduced viral replication and burden of disease outcomes. RdRP mice also had considerably lower Friend retrovirus (FV) viremia. The results could have implications for harnessing ISG responses to reduce transmission or control pathogenesis of human retroviral pathogens.

SAMHD1 suppresses innate immune responses to viral infections and inflammatory stimuli by inhibiting the NF-κB and interferon pathways.

Sterile alpha motif and HD-domain-containing protein 1 (SAMHD1) blocks replication of retroviruses and certain DNA viruses by reducing the intracellular dNTP pool. SAMHD1 has been suggested to down-regulate IFN and inflammatory responses to viral infections, although the functions and mechanisms of SAMHD1 in modulating innate immunity remain unclear. Here, we show that SAMHD1 suppresses the innate immune responses to viral infections and inflammatory stimuli by inhibiting nuclear factor-κB (NF-κB) activation and type I interferon (IFN-I) induction. Compared with control cells, infection of SAMHD1-silenced human monocytic cells or primary macrophages with Sendai virus (SeV) or HIV-1, or treatment with inflammatory stimuli, induces significantly higher levels of NF-κB activation and IFN-I induction. Exogenous SAMHD1 expression in cells or SAMHD1 reconstitution in knockout cells suppresses NF-κB activation and IFN-I induction by SeV infection or inflammatory stimuli. Mechanistically, SAMHD1 inhibits NF-κB activation by interacting with NF-κB1/2 and reducing phosphorylation of the NF-κB inhibitory protein IκBα. SAMHD1 also interacts with the inhibitor-κB kinase ε (IKKε) and IFN regulatory factor 7 (IRF7), leading to the suppression of the IFN-I induction pathway by reducing IKKε-mediated IRF7 phosphorylation. Interactions of endogenous SAMHD1 with NF-κB and IFN-I pathway proteins were validated in human monocytic cells and primary macrophages. Comparing splenocytes from SAMHD1 knockout and heterozygous mice, we further confirmed SAMHD1-mediated suppression of NF-κB activation, suggesting an evolutionarily conserved property of SAMHD1. Our findings reveal functions of SAMHD1 in down-regulating innate immune responses to viral infections and inflammatory stimuli, highlighting the importance of SAMHD1 in modulating antiviral immunity.

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.

Tetherin/BST-2: Restriction Factor or Immunomodulator?

BACKGROUND: Cell-mediated immune (CMI) responses are critical for the control of HIV-1 infection and their importance was highlighted by the existence of viral proteins, particularly Vpu and Nef, that antagonize these responses. Pandemic HIV-1 Vpu counteracts Tetherin/BST-2, a host factor that could prevent the release of HIV-1 virions by tethering virions on the cell surface, but a link between Tetherin and HIV-1 CMI responses has not yet been demonstrated in vivo. In vitro, the virological and immunological impact of Tetherin-mediated accumulation of virions ranged from enhanced or diminished cell-to-cell spread to enhanced recognition by virus-specific antibodies for natural killer cellmediated lysis. However, Tetherin-restricted virions could be internalized through an endocytosis motif in the Tetherin cytoplasmic tail. METHODS: Given the uncertainties on which in vitro results manifest in vivo and the dearth of knowledge on how Tetherin influences retroviral immunity, in vivo retrovirus infections in mice encoding wild-type, null and endocytosis-defective Tetherin were performed. Here, we review and highlight the results from these in vivo studies. RESULTS: Current data suggests that endocytosis-defective Tetherin functions as a potent innate restriction factor. By contrast, endocytosis-competent Tetherin, the form found in most mammals including humans and the form counteracted by HIV-1 Vpu, was linked to stronger CMI responses in mice. CONCLUSION: We propose that the main role of endocytosis-competent Tetherin is not to directly restrict retroviral replication, but to promote a more effective CMI response against retroviruses.

Tetherin/BST-2 promotes dendritic cell activation and function during acute retrovirus infection.

Tetherin/BST-2 is a host restriction factor that inhibits retrovirus release from infected cells in vitro by tethering nascent virions to the plasma membrane. However, contradictory data exists on whether Tetherin inhibits acute retrovirus infection in vivo. Previously, we reported that Tetherin-mediated inhibition of Friend retrovirus (FV) replication at 2 weeks post-infection correlated with stronger natural killer, CD4+ T and CD8+ T cell responses. Here, we further investigated the role of Tetherin in counteracting retrovirus replication in vivo. FV infection levels were similar between wild-type (WT) and Tetherin KO mice at 3 to 7 days post-infection despite removal of a potent restriction factor, Apobec3/Rfv3. However, during this phase of acute infection, Tetherin enhanced myeloid dendritic cell (DC) function. DCs from infected, but not uninfected, WT mice expressed significantly higher MHC class II and the co-stimulatory molecule CD80 compared to Tetherin KO DCs. Tetherin-associated DC activation during acute FV infection correlated with stronger NK cell responses. Furthermore, Tetherin+ DCs from FV-infected mice more strongly stimulated FV-specific CD4+ T cells ex vivo compared to Tetherin KO DCs. The results link the antiretroviral and immunomodulatory activity of Tetherin in vivo to improved DC activation and MHC class II antigen presentation.

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.

Requirement for Fc effector mechanisms in the APOBEC3/Rfv3-dependent neutralizing antibody response.

Antiretroviral neutralizing antibody (NAb) responses are often evaluated in the absence of Fc-dependent immune effectors. In murine Friend retrovirus infection, Apobec3/Rfv3 promotes a potent polyclonal NAb response. Here, we show that the Apobec3/Rfv3-dependent NAb response correlated with virus-specific IgG2 titers and that the in vivo neutralization potency of Apobec3/Rfv3-resistant antisera was dependent on activating Fcγ receptors but not complement. The data strengthen retroviral vaccine strategies aimed at eliciting NAbs that activate specific Fcγ receptors.

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.

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.

Tetherin promotes the innate and adaptive cell-mediated immune response against retrovirus infection in vivo.

Tetherin/BST-2 is a host restriction factor that could directly inhibit retroviral particle release by tethering nascent virions to the plasma membrane. However, the immunological impact of Tetherin during retrovirus infection remains unknown. We now show that Tetherin influences antiretroviral cell-mediated immune responses. In contrast to the direct antiviral effects of Tetherin, which are dependent on cell surface expression, the immunomodulatory effects are linked to the endocytosis of the molecule. Mice encoding endocytosis-competent C57BL/6 Tetherin exhibited lower viremia and pathology at 7 d postinfection with Friend retrovirus (FV) compared with mice encoding endocytosis-defective NZW/LacJ Tetherin. Notably, antiretroviral protection correlated with stronger NK cell responses. In addition, Friend retrovirus infection levels were significantly lower in wild-type C57BL/6 mice than in Tetherin knockout mice at 2 wk postinfection, and antiretroviral protection correlated with stronger NK cell and virus-specific CD8+ T cell responses. The results demonstrate that Tetherin acts as a modulator of the cell-mediated immune response against retrovirus infection in vivo.

Immunoglobulin somatic hypermutation by APOBEC3/Rfv3 during retroviral infection.

Somatic hypermutation (SHM) is an integral process in the development of high-affinity antibodies that are important for recovery from viral infections and vaccine-induced protection. Ig SHM occurs predominantly in germinal centers (GC) via the enzymatic activity of activation-induced deaminase (AID). In contrast, the evolutionarily related apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 3 (APOBEC3) proteins are known to restrict retroviruses, including HIV-1. We previously reported that mouse APOBEC3 encodes Recovery from Friend virus 3 (Rfv3), a classical resistance gene in mice that promotes the neutralizing antibody response against retrovirus infection. We now show that APOBEC3/Rfv3 complements AID in driving Ig SHM during retrovirus infection. Analysis of antibody sequences from retrovirus-specific hybridomas and GC B cells from infected mice revealed Ig heavy-chain V genes with significantly increased C-to-T and G-to-A transitions in wild-type as compared with APOBEC3-defective mice. The context of the mutations was consistent with APOBEC3 but not AID mutational activity. These findings help explain the role of APOBEC3/Rfv3 in promoting the neutralizing antibody responses essential for recovery from retroviral infection and highlight APOBEC3-mediated deamination as a previously unidentified mechanism for antibody diversification in vivo.

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.

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.