Viral infection engenders bona fide and bystander subsets of lung-resident memory B cells through a permissive mechanism.

Lung-resident memory B cells (MBCs) provide localized protection against reinfection in respiratory airways. Currently, the biology of these cells remains largely unexplored. Here, we combined influenza and SARS-CoV-2 infection with fluorescent-reporter mice to identify MBCs regardless of antigen specificity. We found that two main transcriptionally distinct subsets of MBCs colonized the lung peribronchial niche after infection. These subsets arose from different progenitors and were both class switched, somatically mutated, and intrinsically biased in their differentiation fate toward plasma cells. Combined analysis of antigen specificity and B cell receptor repertoire segregated these subsets into "bona fide" virus-specific MBCs and "bystander" MBCs with no apparent specificity for eliciting viruses generated through an alternative permissive process. Thus, diverse transcriptional programs in MBCs are not linked to specific effector fates but rather to divergent strategies of the immune system to simultaneously provide rapid protection from reinfection while diversifying the initial B cell repertoire.

Fcγ Receptor IIa (FCGR2A) Polymorphism Is Associated With Severe Respiratory Syncytial Virus Disease in Argentinian Infants.

Background: Most patients with respiratory syncytial virus (RSV) infection requiring hospitalization have no risk factors for severe disease. Genetic variation in the receptor for the Fc portion of IgG (FcγR) determines their affinity for IgG subclasses driving innate and adaptive antiviral immunity. We investigated the relationship between FcγRIIa-H131R polymorphism and RSV disease. Methods: Blood samples were collected from 182 infants ≤24-month-old (50 uninfected, 114 RSV-infected with moderate course and 18 suffering severe disease). FcγRIIa-H131R SNP genotypic frequencies (HH, HR, RR) and anti-RSV IgG1, IgG2 and IgG3 levels were studied. Results: Genotypic frequencies for FcγRIIa-H131R SNP were comparable between uninfected and RSV-infected infants. In contrast, we found a significant higher frequency of HH genotype in severe RSV-infected children compared to moderate patients. Among severe group, HH infants presented more factors associated to severity than HR or RR patients did. Furthermore, compared to moderate RSV-infected infants, severe patients showed higher levels of anti-RSV IgG1 and IgG3. Conclusions: We found an association between an FcγRIIa (H131) polymorphism and severe RSV disease, which points towards a critical role for interactions between FcγRs and immune complexes in RSV pathogenesis. This genetic factor could also predict the worse outcome and identify those infants at risk during hospitalization.

Impact of HIV-ART on the restoration of Th17 and Treg cells in blood and female genital mucosa.

The aim of this study was to evaluate the effectiveness of antiretroviral treatment (ART) on the proportion and functions of Th17 and Treg cells in peripheral blood and female genital tract (FGT) respectively. To this aim, samples from 41 HIV-neg, 33 HIV+ ART-naïve and 32 HIV+ ART+ subjects were obtained. In peripheral blood, altered Th17 and Th17/Treg proportions were normalized in HIV+ ART+, but certain abnormal Treg and activated T-cell proportions were still observed. In FGT, abnormal patterns of secretion for Th17-related cytokines were observed in cervical mononuclear cells (CMCs) from HIV+ women, even in those from HIV+ ART+, compared to the HIV-neg group. Moreover, these altered patterns of secretion were associated with diminished levels of CXCL5 and CXCL1 chemokines and with an immunoregulatory skew in the CCL17/CCL20 ratio in ectocervix samples of these women. Finally, ART did not restore proportions of Th17-precursor cells with gut-homing potential in PBMCs, and positive correlations between these cells and the levels of IL-17F and IL-21 production by CMCs may suggest that a better homing of these cells to the intestine could also imply a better restoration of these cells in the female genital tract. These results indicate that antiretroviral treatment did not restore Th17-related immune functions completely at the female mucosal level.

CD32 Ligation Promotes the Activation of CD4+ T Cells.

Low affinity receptors for the Fc portion of IgG (FcγRs) represent a critical link between innate and adaptive immunity. Immune complexes (ICs) are the natural ligands for low affinity FcγRs, and high levels of ICs are usually detected in both, chronic viral infections and autoimmune diseases. The expression and function of FcγRs in myeloid cells, NK cells and B cells have been well characterized. By contrast, there are controversial reports about the expression and function of FcγRs in T cells. Here, we demonstrated that ~2% of resting CD4+ T cells express cell surface FcγRII (CD32). Analysis of CD32 expression in permeabilized cells revealed an increased proportion of CD4+CD32+ T cells (~9%), indicating that CD4+ T cells store a CD32 cytoplasmic pool. Activation of CD4+ T cells markedly increased the expression of CD32 either at the cell surface or intracellularly. Analysis of CD32 mRNA transcripts in activated CD4+ T cells revealed the presence of both, the stimulatory FcγRIIa (CD32a) and the inhibitory FcγRIIb (CD32b) isoforms of CD32, being the CD32a:CD32b mRNA ratio ~5:1. Consistent with this finding, we found not only that CD4+ T cells bind aggregated IgG, used as an IC model, but also that CD32 ligation by specific mAb induced a strong calcium transient in CD4+ T cells. Moreover, we found that pretreatment of CD4+ T cells with immobilized IgG as well as cross-linking of CD32 by specific antibodies increased both, the proliferative response of CD4+ T cells and the release of a wide pattern of cytokines (IL-2, IL-5, IL-10, IL-17, IFN-γ, and TNF-α) triggered by either PHA or anti-CD3 mAb. Collectively, our results indicate that ligation of CD32 promotes the activation of CD4+ T cells. These findings suggest that ICs might contribute to the perpetuation of chronic inflammatory responses by virtue of its ability to directly interact with CD4+ T cells through CD32a, promoting the activation of T cells into different inflammatory profiles.

Induction of HIF-1α by HIV-1 Infection in CD4+ T Cells Promotes Viral Replication and Drives Extracellular Vesicle-Mediated Inflammation.

Chronic immune activation and inflammation are hallmarks of HIV-1 infection and a major cause of serious non-AIDS events in HIV-1-infected individuals on antiretroviral treatment (ART). Herein, we show that cytosolic double-stranded DNA (dsDNA) generated in infected CD4+ T cells during the HIV-1 replication cycle promotes the mitochondrial reactive oxygen species (ROS)-dependent stabilization of the transcription factor hypoxia-inducible factor 1α (HIF-1α), which in turn, enhances viral replication. Furthermore, we show that induction of HIF-1α promotes the release of extracellular vesicles (EVs). These EVs foster inflammation by inducing the secretion of gamma interferon by bystander CD4+ T cells and secretion of interleukin 6 (IL-6) and IL-1ß by bystander macrophages through an HIF-1α-dependent pathway. Remarkably, EVs obtained from plasma samples from HIV-1-infected individuals also induced HIF-1α activity and inflammation. Overall, this study demonstrates that HIF-1α plays a crucial role in HIV-1 pathogenesis by promoting viral replication and the release of EVs that orchestrate lymphocyte- and macrophage-mediated inflammatory responses.IMPORTANCE Human immunodeficiency virus type 1 (HIV-1) is a very important global pathogen that preferentially targets CD4+ T cells and causes acquired immunodeficiency syndrome (AIDS) if left untreated. Although antiretroviral treatment efficiently suppresses viremia, markers of immune activation and inflammation remain higher in HIV-1-infected patients than in uninfected individuals. The hypoxia-inducible factor 1α (HIF-1α) is a transcription factor that plays a fundamental role in coordinating cellular metabolism and function. Here we show that HIV-1 infection induces HIF-1α activity and that this transcription factor upholds HIV-1 replication. Moreover, we demonstrate that HIF-1α plays a key role in HIV-1-associated inflammation by promoting the release of extracellular vesicles which, in turn, trigger the secretion of inflammatory mediators by noninfected bystander lymphocytes and macrophages. In summary, we identify that the coordinated actions of HIF-1α and extracellular vesicles promote viral replication and inflammation, thus contributing to HIV-1 pathogenesis.

Dampening of IL-2 Function in Infants With Severe Respiratory Syncytial Virus Disease.

Background: FOXP3+ regulatory T cells (Tregs) restrain the destructive potential of the immune system. We have previously reported a pronounced reduction in circulating Tregs in infants with severe respiratory syncytial virus (RSV) disease. Because interleukin-2 (IL-2) is critical for Treg growth, survival, and activity, we here analyzed IL-2 production and function in RSV-infected infants. Methods: Phenotype, proliferation, IL-2 production, and IL-2 signaling in CD4+ T cells were analyzed by flow cytometry. Serum soluble CD25 levels were quantified by ELISA. Results: CD4+ T cells from RSV-infected infants produced lower amounts of IL-2 and showed a reduced proliferative response compared with healthy infants. IL-2 increased CD4+ T-cell proliferation and FOXP3 expression in both healthy and RSV-infected infants. However, although IL-2 induced a similar pattern of STAT5 phosphorylation, the proliferative response of CD4+ T cells and the expression of FOXP3+ remained significantly lower in RSV-infected infants. Interestingly, we found a negative correlation between disease severity and both the production of IL-2 by CD4+ T cells and the ability of exogenous IL-2 to restore the pool of FOXP3+CD4+ T cells. Conclusions: A reduced ability to produce IL-2 and a limited response to this cytokine may affect the function of CD4+ T cells in RSV-infected infants.

Biomarkers of Progression after HIV Acute/Early Infection: Nothing Compares to CD4⁺ T-cell Count?

Progression of HIV infection is variable among individuals, and definition disease progression biomarkers is still needed. Here, we aimed to categorize the predictive potential of several variables using feature selection methods and decision trees. A total of seventy-five treatment-naïve subjects were enrolled during acute/early HIV infection. CD4⁺ T-cell counts (CD4TC) and viral load (VL) levels were determined at enrollment and for one year. Immune activation, HIV-specific immune response, Human Leukocyte Antigen (HLA) and C-C chemokine receptor type 5 (CCR5) genotypes, and plasma levels of 39 cytokines were determined. Data were analyzed by machine learning and non-parametric methods. Variable hierarchization was performed by Weka correlation-based feature selection and J48 decision tree. Plasma interleukin (IL)-10, interferon gamma-induced protein (IP)-10, soluble IL-2 receptor alpha (sIL-2Rα) and tumor necrosis factor alpha (TNF-α) levels correlated directly with baseline VL, whereas IL-2, TNF-α, fibroblast growth factor (FGF)-2 and macrophage inflammatory protein (MIP)-1ß correlated directly with CD4⁺ T-cell activation (p < 0.05). However, none of these cytokines had good predictive values to distinguish "progressors" from "non-progressors". Similarly, immune activation, HIV-specific immune responses and HLA/CCR5 genotypes had low discrimination power. Baseline CD4TC was the most potent discerning variable with a cut-off of 438 cells/µL (accuracy = 0.93, κ-Cohen = 0.85). Limited discerning power of the other factors might be related to frequency, variability and/or sampling time. Future studies based on decision trees to identify biomarkers of post-treatment control are warrantied.

Deletion of A44L, A46R and C12L Vaccinia Virus Genes from the MVA Genome Improved the Vector Immunogenicity by Modifying the Innate Immune Response Generating Enhanced and Optimized Specific T-Cell Responses.

MVA is an attenuated vector that still retains immunomodulatory genes. We have previously reported its optimization after deleting the C12L gene, coding for the IL-18 binding-protein. Here, we analyzed the immunogenicity of MVA vectors harboring the simultaneous deletion of A44L, related to steroid synthesis and A46R, a TLR-signaling inhibitor (MVAΔA44L-A46R); or also including a deletion of C12L (MVAΔC12L/ΔA44L-A46R). The absence of biological activities of the deleted genes in the MVA vectors was demonstrated. Adaptive T-cell responses against VACV epitopes, evaluated in spleen and draining lymph-nodes of C57Bl/6 mice at acute/memory phases, were of higher magnitude in those animals that received deleted MVAs compared to MVAwt. MVAΔC12L/ΔA44L-A46R generated cellular specific memory responses of higher quality characterized by bifunctionality (CD107a/b⁺/IFN-γ⁺) and proliferation capacity. Deletion of selected genes from MVA generated innate immune responses with higher levels of determining cytokines related to T-cell response generation, such as IL-12, IFN-γ, as well as IL-1ß and IFN-ß. This study describes for the first time that simultaneous deletion of the A44L, A46R and C12L genes from MVA improved its immunogenicity by enhancing the host adaptive and innate immune responses, suggesting that this approach comprises an appropriate strategy to increase the MVA vaccine potential.

Env-Specific IgA from Viremic HIV-Infected Subjects Compromises Antibody-Dependent Cellular Cytotoxicity.

UNLABELLED: Elucidating the factors that modulate HIV-specific antibody-dependent cellular cytotoxicity (ADCC) will help in understanding its role in HIV immunity. The aim of this study was to determine whether IgA could modify the magnitude of ADCC in HIV infection, abrogating its protective role. Plasma samples from 20 HIV-positive (HIV(+)) subjects enrolled during primary HIV infection (PHI), 10 chronically infected subjects (chronic), and 7 elite controllers (EC) were used. ADCC was determined by using a fluorometric ADCC assay, before and after removal of plasma IgA. Data were analyzed by using nonparametric statistics. ADCC was documented in 80% of PHI enrollment samples and in 100% of PHI 12-month, chronic, and EC samples; it peaked after acute infection, reached a plateau in chronic infection, and decreased after initiation of antiretroviral treatment (ART). Significant associations between ADCC and disease progression were found only after removal of plasma IgA from 12-month PHI samples: the magnitude of ADCC not only increased after IgA removal but also correlated with CD4(+) T-cell preservation. This work provides evidence that gp120-specific IgA was capable of modifying ADCC responses during natural HIV infection for the first time and adds to similar evidence provided in other settings. Furthermore, it underscores the complexity of the ADCC phenomenon and will help in an understanding of its underlying mechanisms. IMPORTANCE: Although the induction of ADCC-mediating antibodies in HIV-infected subjects has been extensively documented, the association of these antibodies with protection from disease progression is poorly understood. Here, we demonstrate that plasma IgA is a factor capable of modifying the magnitude of IgG-mediated ADCC in HIV infection, mitigating its beneficial effect. These results help in understanding why previous studies failed to demonstrate correlations between ADCC and disease progression, and they also contribute to the notion that an HIV vaccine should stimulate the production of ADCC-mediating IgG antibodies but not IgA.

Th17 and Th17/Treg ratio at early HIV infection associate with protective HIV-specific CD8(+) T-cell responses and disease progression.

The aim of this study was to analyze Th17 and Treg subsets and their correlation with anti-HIV T-cell responses and clinical parameters during (acute/early) primary HIV infection (PHI) and up to one year post-infection (p.i). Samples from 14 healthy donors (HDs), 40 PHI patients, 17 Chronics, and 13 Elite controllers (ECs) were studied. The percentages of Th17 and Treg subsets were severely altered in Chronics, whereas all HIV-infected individuals (including ECs) showed Th17/Treg imbalance compared to HDs, in concordance with higher frequencies of activated CD8(+) T-cells (HLA-DR(+)/CD38(+)). Better clinical status (higher CD4 counts, lower viral loads and activation) was associated with higher Th17 and lower Treg levels. We found positive correlations between Th17 at baseline and anti-HIV CD8(+) T-cell functionality: viral inhibitory activity (VIA) and key polyfunctions (IFN-γ(+)/CD107A/B(+)) at both early and later times p.i, highlighting the prognostic value of Th17 cells to preserve an effective HIV T-cell immunity. Th17/Treg ratio and the IL-17 relative mean fluorescence intensity (rMFI of IL-17) were also positively correlated with VIA. Taken together, our results suggested a potential link between Th17 and Th17/Treg ratio with key HIV-specific CD8(+) T-cell responses against the infection.

Novel mucosal DNA-MVA HIV vaccination in which DNA-IL-12 plus cholera toxin B subunit (CTB) cooperates to enhance cellular systemic and mucosal genital tract immunity.

Induction of local antiviral immune responses at the mucosal portal surfaces where HIV-1 and other viral pathogens are usually first encountered remains a primary goal for most vaccines against mucosally acquired viral infections. Exploring mucosal immunization regimes in order to find optimal vector combinations and also appropriate mucosal adjuvants in the HIV vaccine development is decisive. In this study we analyzed the interaction of DNA-IL-12 and cholera toxin B subunit (CTB) after their mucosal administration in DNA prime/MVA boost intranasal regimes, defining the cooperation of both adjuvants to enhance immune responses against the HIV-1 Env antigen. Our results demonstrated that nasal mucosal DNA/MVA immunization schemes can be effectively improved by the co-delivery of DNA-IL-12 plus CTB inducing elevated HIV-specific CD8 responses in spleen and more importantly in genital tract and genito-rectal draining lymph nodes. Remarkably, these CTL responses were of superior quality showing higher avidity, polyfunctionality and a broader cytokine profile. After IL-12+CTB co-delivery, the cellular responses induced showed an enhanced breadth recognizing with higher efficiency Env peptides from different subtypes. Even more, an in vivo CTL cytolytic assay demonstrated the higher specific CD8 T-cell performance after the IL-12+CTB immunization showing in an indirect manner its potential protective capacity. Improvements observed were maintained during the memory phase where we found higher proportions of specific central memory and T memory stem-like cells T-cell subpopulations. Together, our data show that DNA-IL-12 plus CTB can be effectively employed acting as mucosal adjuvants during DNA prime/MVA boost intranasal vaccinations, enhancing magnitude and quality of HIV-specific systemic and mucosal immune responses.

IL-12 and GM-CSF in DNA/MVA immunizations against HIV-1 CRF12_BF Nef induced T-cell responses with an enhanced magnitude, breadth and quality.

In Argentina, the HIV epidemic is characterized by the co-circulation of subtype B and BF recombinant viral variants. Nef is an HIV protein highly variable among subtypes, making it a good tool to study the impact of HIV variability in the vaccine design setting. We have previously reported a specific cellular response against NefBF with low cross-reactivity to NefB in mice. The aim of this work was to analyze whether the co-administration of IL-12 and GM-CSF, using DNA and MVA vaccine vectors, could improve the final cellular response induced. Mice received three DNA priming doses of a plasmid that express NefBF plus DNAs expressing IL-12 and/or GM-CSF. Afterwards, all the groups were boosted with a MVAnefBF dose. The highest increase in the magnitude of the NefBF response, compared to that induced in the control was found in the IL-12 group. Importantly, a response with higher breadth was detected in groups which received IL-12 or GM-CSF, evidenced as an increased frequency of recognition of homologous (BF) and heterologous (B) Nef peptides, as well as a higher number of other Nef peptide pools representing different viral subtypes. However, these improvements were lost when both DNA cytokines were simultaneously administered, as the response was focused against the immunodominant peptide with a detrimental response towards subdominant epitopes. The pattern of cytokines secreted and the specific-T-cell proliferative capacity were improved in IL-12 and IL-12+GM-CSF groups. Importantly IL-12 generated a significant higher T-cell avidity against a B heterologous peptide.This study indicates that the incorporation of DNA expressing IL-12 in DNA/MVA schemes produced the best results in terms of improvements of T-cell-response key properties such as breadth, cross-reactivity and quality (avidity and pattern of cytokines secreted). These relevant results contribute to the design of strategies aimed to induce T-cell responses against HIV antigens with higher quality.