Kerry J. Lavender

Humans produce 12 distinct interferon alpha (IFN-α) subtypes that were previously thought to be functionally redundant. Recent studies have revealed that IFN-α subtypes differ in their ability to control different viral infections. However, little is known about how IFN-α subtypes differentially modulate immune cells to mediate these effects. This study compared the effects of IFN-α2 and IFN-α14, subtypes identified to differentially control viral infection, on T cells both in vivo in humanized TKO-BLT mice and in vitro using human peripheral blood mononuclear cells (PBMC). Treatment of humanized mice with the 2 IFN-α subtypes resulted in the differential modulation of genes associated with T-cell immunity, such as proliferation, activation and inhibition. Therefore, we assessed whether stimulation of PBMC in the presence of IFN-α2 or IFN-α14 resulted in subtype-specific effects on T cells in vitro. IFN-α14 decreased the proliferation of human CD4+ and CD8+ T cells significantly more than IFN-α2. Additionally, IFN-α14 increased expression of T-cell activation markers and the frequency of IL-2 producing T cells to a significantly greater extent compared to IFN-α2. However, the co-culture of CD8+ T cells with IFN-α-treated, autologous CD4+ T cells or IFN-α-matured monocyte-derived dendritic cells (mo-DC) only increased the expression of CD38 compared to no IFN-α treatment in a non-subtype specific manner. Overall, we identified IFN-α subtype-specific effects on the proliferation and activation of T cells. However, there were no significant differences detected directly between the IFN-α subtypes when the effects on CD8+ T cells arose through IFN-α pre-treatment of CD4+ T cells or mo-DC.

Type I IFN, including IFN-α, induces the expression of antiviral restriction factors that can interfere with multiple steps of the HIV-1 replication cycle. Humans have 13 IFN-α genes which encode 12 different IFN-α subtypes. Our previous work in HIV-1 infected humanized mice showed that IFN-α14 treatment more potently controlled HIV-1 than treatment with the clinically approved IFN-α2 subtype. However, the mechanisms behind the more potent control of HIV-1 by IFN-α14 are unknown. The IFN-α14 subtype is known to more potently induce the expression of the restriction factors MX2 and ISG15 and increased APOBEC3G signature mutations in vivo compared to IFN-α2. To study the importance of each of these restriction factors in mediating the potent control of HIV-1, we used a CRISPR-Cas9 lentivirus system to create stable knockouts in the MT4C5 cell line that is susceptible to HIV-1 but does not produce measurable amounts of endogenous IFN-α. Knock out of ISG15, but not MX2, eliminated differences in viral suppression after IFN-α14 and IFN-α2 treatment. Similarly, APOBEC3G deletion eliminated differences in viral suppression and the number of infectious particles produced after IFN-α14 and IFN-α2 treatment. Furthermore, APOBEC3G deletion resulted in significantly fewer GG→AG mutations in viral DNA isolated from target cells incubated with supernatant from IFN-α14 treated groups. However, APOBEC3G knock out did not result in significant increases in vDNA compared to the wild type in any experimental group. Overall, elimination of APOBEC3G and ISG15 impaired IFN-α14–mediated suppression of HIV-1, highlighting them as downstream effectors of IFN-α14’s more potent anti-HIV-1 activity. IMPORTANCE: This study uncovers the molecular basis for the more potent antiviral activity of IFN-α14 compared to the clinically used IFN-α2 subtype against HIV-1. Although interferons are known to induce numerous restriction factors, the mechanisms underlying subtype-specific antiviral potency remained unclear. By using CRISPR-Cas9 knockout MT4C5 cell lines, the study identifies ISG15 and APOBEC3G as key effectors mediating IFN-α14’s enhanced suppression of HIV-1 replication. Loss of either ISG15 or APOBEC3G abolished the differential antiviral effect between IFN-α14 and IFN-α2, demonstrating their essential roles in IFN-α14 driven viral restriction. These findings highlight that individual IFN-α subtypes engage distinct downstream pathways and that subtype diversity encodes functional specialization rather than redundancy. Overall, this work advances our understanding of innate immune control of HIV-1 and provides a foundation for developing targeted interferon-based therapies that exploit the unique mechanisms of potent subtypes like IFN- α14.

SARS-CoV-2 variants and seasonal coronaviruses continue to cause disease and coronaviruses in the animal reservoir pose a constant spillover threat. Importantly, understanding of how previous infection may influence future exposures, especially in the context of seasonal coronaviruses and SARS-CoV-2 variants, is still limited. Here we adopted a step-wise experimental approach to examine the primary immune response and subsequent immune recall toward antigenically distinct coronaviruses using male Syrian hamsters. Hamsters were initially inoculated with seasonal coronaviruses (HCoV-NL63, HCoV-229E, or HCoV-OC43), or SARS-CoV-2 pango B lineage virus, then challenged with SARS-CoV-2 pango B lineage virus, or SARS-CoV-2 variants Beta or Omicron. Although infection with seasonal coronaviruses offered little protection against SARS-CoV-2 challenge, HCoV-NL63-infected animals had an increase of the previously elicited HCoV-NL63-specific neutralizing antibodies during challenge with SARS-CoV-2. On the other hand, primary infection with HCoV-OC43 induced distinct T cell gene signatures. Gene expression profiling indicated interferon responses and germinal center reactions to be induced during more similar primary infection-challenge combinations while signatures of increased inflammation as well as suppression of the antiviral response were observed following antigenically distant viral challenges. This work characterizes and analyzes seasonal coronaviruses effect on SARS-CoV-2 secondary infection and the findings are important for pan-coronavirus vaccine design.

Small animal models that accurately model pathogenesis of SARS-CoV-2 variants are required for ongoing research efforts. We modified our human immune system mouse model to support replication of SARS-CoV-2 by implantation of human lung tissue into the mice to create TKO-BLT-Lung (L) mice and compared infection with two different variants in a humanized lung model. Infection of TKO-BLT-L mice with SARS-CoV-2 recapitulated the higher infectivity of the B.1.1.7 variant with more animals becoming infected and higher sustained viral loads compared to mice challenged with an early B lineage (614D) virus. Viral lesions were observed in lung organoids but no differences were detected between the viral variants as expected. Partially overlapping but distinct immune profiles were also observed between the variants with a greater Th1 profile in VIDO-01 and greater Th2 profile in B.1.1.7 infection. Overall, the TKO-BLT-L mouse supported SARS-CoV-2 infection, recapitulated key known similarities and differences in infectivity and pathogenesis as well as revealing previously unreported differences in immune responses between the two viral variants. Thus, the TKO-BLT-L model may serve as a useful animal model to study the immunopathobiology of newly emerging variants in the context of genuine human lung tissue and immune cells.

Significance: Type I interferons (IFN-I) exhibit various biological effects during viral infections, and they have been successfully used for clinical treatment of viral diseases. Humans express 12 IFNα subtypes, which strongly differ in their antiviral responses against different viruses. Here we analyzed the antiviral activity of all human IFNα subtypes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to identify the underlying immune signatures and explore their therapeutic potential. Our data provide a systemic pattern of antiviral host effector responses mediated by high antiviral IFN-I, which could help to identify key cellular effectors targeted in novel therapeutic approaches against SARS-CoV-2 infection.

Multiple humanized mouse models have been produced for the study of HIV-1 infection and treatment. Humanized mice produced using the bone marrow, liver, thymus (BLT) method particularly have well-reconstituted and functional human immune systems, providing an excellent model for HIV-1 cure strategies that aim to harness the human immune system as part of the cure approach. The TKO-BLT humanized mouse model is especially useful for long-term studies as it is highly resistant to the wasting syndrome and graft-versus-host disease (GVHD) that can limit the use of other BLT-models. Here we describe the methods used to induce latency in TKO-BLT mice, using both injectable and free-fed combination antiretroviral therapy (cART) regimens, for use in the study of HIV-1 latency and evaluation of HIV-1 cure interventions.

Objective: Interferon-alpha (IFN-α) has been associated with excessive immune activation and dysfunction during HIV-1 infection. However, evidence suggests specific IFN-α subtypes may be beneficial rather than detrimental. This study compared the effects of treatment with two different IFN-α subtypes on indicators of T-cell activation and dysfunction during HIV-1 infection. Design: Humanized mice were infected with HIV-1 for 5 weeks and then treated with two different IFN-α subtypes for an additional 3 weeks. Splenic T cells were assessed both immediately posttreatment and again 6 weeks after treatment cessation. Methods: HIV-1 infected triple-knockout bone marrow-liver-thymus mice received daily intraperitoneal injections of either IFN-α14 or the clinically approved subtype, IFN-α2. T cells were analysed directly ex vivo for indicators of activation and dysfunction or stimulated to determine their proliferative capacity and ability to produce functional mediators. Results: Unlike IFN-α2, IFN-α14 treatment reduced viremia and resulted in less activated CD4⁺ T cells and a lower naïve to effector CD8⁺ T-cell ratio. Despite exhibiting a reduced proliferative response, the frequency of CD8⁺ T cells from IFN-α14 treated mice that produced functional mediators and expressed markers of dysfunction was more similar to healthy controls than untreated and IFN-α2 treated mice. Frequencies of exhaustion marker expression remained higher in untreated and IFN-α2 treated mice 6 weeks posttreatment despite similar viral loads between groups at this timepoint. Conclusions: Treatment with different IFN-α subtypes had distinctive effects on T cells during HIV-1 infection. IFN-α14 was associated with fewer indicators of T-cell dysfunction whereas IFN-α2 treatment had little impact.

The Type I Interferons (IFN-Is) are innate antiviral cytokines that include 12 different IFNα subtypes and IFNβ that signal through the IFN-I receptor (IFNAR), inducing hundreds of IFN-stimulated genes (ISGs) that comprise the ‘interferome’. Quantitative differences in IFNAR binding correlate with antiviral activity, but whether IFN-Is exhibit qualitative differences remains controversial. Moreover, the IFN-I response is protective during acute HIV-1 infection, but likely pathogenic during the chronic stages. To gain a deeper understanding of the IFN-I response, we compared the interferomes of IFNα subtypes dominantly-expressed in HIV-1-exposed plasmacytoid dendritic cells (1, 2, 5, 8 and 14) and IFNβ in the earliest cellular targets of HIV-1 infection. Primary gut CD4 T cells from 3 donors were treated for 18 hours ex vivo with individual IFN-Is normalized for IFNAR signaling strength. Of 1,969 IFN-regulated genes, 246 ‘core ISGs’ were induced by all IFN-Is tested. However, many IFN-regulated genes were not shared between the IFNα subtypes despite similar induction of canonical antiviral ISGs such as ISG15, RSAD2 and MX1, formally demonstrating qualitative differences between the IFNα subtypes. Notably, IFNβ induced a broader interferome than the individual IFNα subtypes. Since IFNβ, and not IFNα, is upregulated during chronic HIV-1 infection in the gut, we compared core ISGs and IFNβ-specific ISGs from colon pinch biopsies of HIV-1-uninfected (n = 13) versus age- and gender-matched, antiretroviral-therapy naïve persons with HIV-1 (PWH; n = 19). Core ISGs linked to inflammation, T cell activation and immune exhaustion were elevated in PWH, positively correlated with plasma lipopolysaccharide (LPS) levels and gut IFNβ levels, and negatively correlated with gut CD4 T cell frequencies. In sharp contrast, IFNβ-specific ISGs linked to protein translation and anti-inflammatory responses were significantly downregulated in PWH, negatively correlated with gut IFNβ and LPS, and positively correlated with plasma IL6 and gut CD4 T cell frequencies. Our findings reveal qualitative differences in interferome induction by diverse IFN-Is and suggest potential mechanisms for how IFNβ may drive HIV-1 pathogenesis in the gut. The Type I Interferons (IFN-Is) serve as the first line of defense against viral infections. IFN-Is are evolutionarily diverse, with 12 distinct IFNα subtypes and IFNβ in humans. All IFN-Is bind to the same receptor, but it remains unclear whether distinct IFN-Is will trigger the same set of IFN-stimulated genes. Here, we provide evidence that diverse IFN-Is altered gene expression in gut CD4 T cells in different ways. Specifically, IFNβ induced a broader gene expression profile than individual IFNα subtypes. Genes uniquely induced by IFNβ in gut CD4 T cells ex vivo were downregulated in the gut during chronic HIV-1 infection. This downregulation correlated with markers of inflammation and immune dysfunction. Our data unravel qualitative differences between the IFN-Is and suggest a complex picture of how IFNβ may be driving HIV-1 pathogenesis in the gastrointestinal tract.