MAdCAM-1 Co-stimulation Combined with Retinoic Acid and TGF-ß Induces Blood CD8+ T cells to Adopt a Gut CD101+ TRM Phenotype.

Resident memory T cells (TRMs) help control local immune homeostasis and contribute to tissue protective immune responses. The local cues that guide their differentiation and localization are poorly defined. We demonstrate that MAdCAM-1, a ligand for the gut homing receptor α4ß7 integrin, in the presence of retinoic acid and TGF-ß provide a costimulatory signal that induces blood CD8+ T cells to adopt a TRM -like phenotype. These cells express CD103 (integrin αE) and CD69, the two major TRM cell surface markers, along with CD101. They also express CCR5, CCR9 and α4ß7, three receptors associated with gut homing. A subset also express E-cadherin, a ligand for αEß7. Fluorescent lifetime imaging indicated an αEß7 and E-cadherin cis interaction on the plasma membrane. This report advances our understanding of the signals that drive the differentiation of CD8+ T cells into TRMs and provides a means to expand these cells in vitro, thereby affording an avenue to generate more effective tissue specific immunotherapies.

The V2 domain of HIV gp120 mimics an interaction between CD4 and integrin ⍺4ß7.

The CD4 receptor, by stabilizing TCR-MHC II interactions, plays a central role in adaptive immunity. It also serves as the HIV docking receptor. The HIV gp120 envelope protein binds directly to CD4. This interaction is a prerequisite for viral entry. gp120 also binds to ⍺4ß7, an integrin that is expressed on a subset of memory CD4+ T cells. HIV tropisms for CD4+ T cells and gut tissues are central features of HIV pathogenesis. We report that CD4 binds directly to ⍺4ß7 in a dynamic way, consistent with a cis regulatory interaction. The molecular details of this interaction are related to the way in which gp120 interacts with both receptors. Like MAdCAM-1 and VCAM-1, two recognized ligands of ⍺4ß7, the binding interface on CD4 includes 2 sites (1° and accessory), distributed across its two N-terminal IgSF domains (D1 and D2). The 1° site includes a sequence in the G ß-strand of CD4 D2, KIDIV, that binds directly to ⍺4ß7. This pentapeptide sequence occurs infrequently in eukaryotic proteins. However, a closely related and conserved sequence, KLDIV, appears in the V2 domain of gp120. KLDIV mediates gp120-⍺4ß7 binding. The accessory ⍺4ß7 binding site on CD4 includes Phe43. The Phe43 aromatic ring protrudes outward from one edge of a loop connecting the C'C" strands of CD4 D1. Phe43 is a principal contact for HIV gp120. It interacts with conserved residues in the recessed CD4 binding pocket. Substitution of Phe43 abrogates CD4 binding to both gp120 and ⍺4ß7. As such, the interactions of gp120 with both CD4 and ⍺4ß7 reflect elements of their interactions with each other. These findings indicate that gp120 specificities for CD4 and ⍺4ß7 are interrelated and suggest that selective pressures which produced a CD4 tropic virus that replicates in gut tissues are linked to a dynamic interaction between these two receptors.

Microbiome analysis of Brazilian women cervix reveals specific bacterial abundance correlation to RIG-like receptor gene expression.

The relationship among microbiome, immunity and cervical cancer has been targeted by several studies, yet many questions remain unanswered. We characterized herein the virome and bacteriome from cervical samples and correlated these findings with innate immunity gene expression in a Brazilian convenience sample of HPV-infected (HPV+) and uninfected (HPV-) women. For this purpose, innate immune gene expression data were correlated to metagenomic information. Correlation analysis showed that interferon (IFN) is able to differentially modulate pattern recognition receptors (PRRs) expression based on HPV status. Virome analysis indicated that HPV infection correlates to the presence of Anellovirus (AV) and seven complete HPV genomes were assembled. Bacteriome results unveiled that vaginal community state types (CST) distribution was independent of HPV or AV status, although bacterial phyla distribution differed between groups. Furthermore, TLR3 and IFNαR2 levels were higher in the Lactobacillus no iners-dominated mucosa and we detected correlations among RIG-like receptors (RLR) associated genes and abundance of specific anaerobic bacteria. Collectively, our data show an intriguing connection between HPV and AV infections that could foster cervical cancer development. Besides that, TLR3 and IFNαR2 seem to create a protective milieu in healthy cervical mucosa (L. no iners-dominated), and RLRs, known to recognize viral RNA, were correlated to anaerobic bacteria suggesting that they might be related to dysbiosis.

MAdCAM-1 costimulation in the presence of retinoic acid and TGF-ß promotes HIV infection and differentiation of CD4+ T cells into CCR5+ TRM-like cells.

CD4+ tissue resident memory T cells (TRMs) are implicated in the formation of persistent HIV reservoirs that are established during the very early stages of infection. The tissue-specific factors that direct T cells to establish tissue residency are not well defined, nor are the factors that establish viral latency. We report that costimulation via MAdCAM-1 and retinoic acid (RA), two constituents of gut tissues, together with TGF-ß, promote the differentiation of CD4+ T cells into a distinct subset α4ß7+CD69+CD103+ TRM-like cells. Among the costimulatory ligands we evaluated, MAdCAM-1 was unique in its capacity to upregulate both CCR5 and CCR9. MAdCAM-1 costimulation rendered cells susceptible to HIV infection. Differentiation of TRM-like cells was reduced by MAdCAM-1 antagonists developed to treat inflammatory bowel diseases. These finding provide a framework to better understand the contribution of CD4+ TRMs to persistent viral reservoirs and HIV pathogenesis.

Evidence of recurrent selection of mutations commonly found in SARS-CoV-2 variants of concern in viruses infecting immunocompromised patients.

Chronically immunosuppressed patients infected with SARS-CoV-2 often experience prolonged virus shedding, and may pave the way to the emergence of mutations that render viral variants of concern (VOC) able to escape immune responses induced by natural infection or by vaccination. We report herein a SARS-CoV-2+ cancer patient from the beginning of the COVID-19 pandemic whose virus quasispecies across multiple timepoints carried several immune escape mutations found in more contemporary VOC, such as alpha, delta and omicron, that appeared to be selected for during infection. We hypothesize that immunosuppressed patients may represent the source of VOC seen throughout the COVID-19 pandemics.

New infections by SARS-CoV-2 variants of concern after natural infections and post-vaccination in Rio de Janeiro, Brazil.

After a one-year rollout of the pandemic caused by SARS-CoV-2, the continuous dissemination of the virus has generated a number of variants with increased transmissibility and infectivity, called variants of concern (VOC), which now predominate worldwide. Concerns about the susceptibility of humans that have already been infected before or those already vaccinated to infection by VOC rise among scientists and clinicians. Herein, we assessed the prevalence of different VOC among recent infections at the Brazilian National Cancer Institute (Rio de Janeiro, Brazil). By using a Sanger-based sequencing approach targeting the viral S gene to identify VOC, we have analyzed 72 recent infections. The overall prevalence of VOC was 97%. Among the subjects analyzed, six had been vaccinated with the ChAdOx1-S/nCoV-19 (n = 4; one with two doses and three with one dose) or the CoronaVac (n = 2; both with 2 doses) vaccine, while five subjects represented reinfection cases, being two of them also part of the vaccinated group (each one with one vaccine type). All vaccinated and re-infected subjects carried VOC irrespective of the vaccine type taken, the number of doses taken, IgG titers or being previously infected during the first wave of the Brazilian pandemic. Importantly, all six vaccinees only had mild symptoms. We present here several examples of how natural infections or vaccination may not be fully capable of conferring sterilizing immunity against VOC.

SARS-CoV-2 genomic analyses in cancer patients reveal elevated intrahost genetic diversity.

Numerous factors have been identified to influence susceptibility to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and disease severity. Cancer patients are more prone to clinically evolve to more severe COVID-19 conditions, but the determinants of such a more severe outcome remain largely unknown. We have determined the full-length SARS-CoV-2 genomic sequences of cancer patients and healthcare workers (non-cancer controls) by deep sequencing and investigated the within-host viral population of each infection, quantifying intrahost genetic diversity. Naso- and oropharyngeal SARS-CoV-2+ swabs from 57 cancer patients and 14 healthcare workers from the Brazilian National Cancer Institute were collected in April to May 2020. Complete genome amplification using ARTIC network V3 multiplex primers was performed followed by next-generation sequencing. Assemblies were conducted in Geneious R11, where consensus sequences were extracted and intrahost single nucleotide variants were identified. Maximum likelihood phylogenetic analysis was performed using PhyMLv.3.0 and lineages were classified using Pangolin and CoV-GLUE. Phylogenetic analysis showed that all but one strain belonged to clade B1.1. Four genetically linked mutations known as the globally dominant SARS-CoV-2 haplotype (C241T, C3037T, C14408T and A23403G) were found in the majority of consensus sequences. SNV signatures of previously characterized Brazilian genomes were also observed in most samples. Another 85 SNVs were found at a lower frequency (1.4%-19.7%) among the consensus sequences. Cancer patients displayed a significantly higher intrahost viral genetic diversity compared to healthcare workers. This difference was independent of SARS-CoV-2 Ct values obtained at the diagnostic tests, which did not differ between the two groups. The most common nucleotide changes of intrahost SNVs in both groups were consistent with APOBEC and ADAR activities. Intrahost genetic diversity in cancer patients was not associated with disease severity, use of corticosteroids, or use of antivirals, characteristics that could influence viral diversity. Moreover, the presence of metastasis, either in general or specifically in the lung, was not associated with intrahost diversity among cancer patients. Cancer patients carried significantly higher numbers of minor variants compared to non-cancer counterparts. Further studies on SARS-CoV-2 diversity in especially vulnerable patients will shed light onto the understanding of the basis of COVID-19 different outcomes in humans.

Distinguishing SARS-CoV-2 bonafide re-infection from pre-existing minor variant reactivation.

Different groups have recently reported events of SARS-CoV-2 reinfection, where patients had a sequence of positive-negative-positive RT-PCR tests. However, such events could be explained by different scenarios such as intermittent viral shedding, bonafide re-infection or multiple infection with alternating predominance of different viruses. Analysis of minor variants is an important tool to distinguish between these scenarios. Using ARTIC network PCR amplification and next-generation sequencing, we obtained SARS-CoV-2 sequences from two timepoints (with a time span of 102 days) of a patient followed at the Brazilian National Cancer Institute. Within-host variant analysis evidenced three single nucleotide variants (SNVs) at the consensus viral sequence in the second timepoint that were already present in the first timepoint as minor variants. Another five SNVs found in the second timepoint were not detected in the first sample sequenced, suggesting an additional infection by a yet another new virus. Our observation shed light into the existence of different viral populations that are present in dynamic frequencies and fluctuate during the course of SARS-CoV-2 infection. The detection of these variants in distinct disease events of an individual highlights a complex interplay between viral reactivation from a pre-existing minority variant and reinfection by a different virus.

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.

HPV Induces Changes in Innate Immune and Adhesion Molecule Markers in Cervical Mucosa With Potential Impact on HIV Infection.

While most HPV infections are asymptomatic and clear spontaneously, persistent infection with high-risk HPVs is associated with cervical cancer and with increased risk of HIV acquisition. Although several hypotheses have been proposed to explain this phenomenon, none has been confirmed. Our aim was to investigate the expression of host factors involved in the susceptibility to HIV infection among HPV-infected women. Cervical samples were collected to characterize the expression levels of HIV susceptibility markers in the mucosa of HPV-infected compared with HPV-uninfected women. No differences in the frequency of CCR5+, integrin α4ß7+, activated and memory CD4+ T-cell were detected between the groups. We additionally evaluated the expression levels of genes involved in innate immune responses and in cell adhesion. HPV infected patients expressed higher levels of TLR9 and lower levels of pattern recognition receptors that recognize RNA (TLR3, TLR7, and MDA5/IFIH1). We also detected an impaired IFN pathway, with an increased Type I IFN and a decreased IFNα2 receptor expression. HPV+ samples displayed reduced expression of genes for adherens and tight junctions. Taken together, these results suggest that although HPV infection does not result in the recruitment/activation of susceptible CD4+ T-cell in the female genital tract, it leads to changes in the innate antiviral immune responses and in cell adhesion that are likely to favor HIV infection.

SARS-CoV-2 genomic and quasispecies analyses in cancer patients reveal relaxed intrahost virus evolution.

Numerous factors have been identified to influence susceptibility to SARS-CoV-2 infection and disease severity. Cancer patients are more prone to clinically evolve to more severe COVID-19 conditions, but the determinants of such a more severe outcome remain largely unknown. We have determined the full-length SARS-CoV-2 genomic sequences of cancer patients and healthcare workers (HCW; non-cancer controls) by deep sequencing and investigated the within-host viral quasispecies of each infection, quantifying intrahost genetic diversity. Naso- and oropharyngeal SARS-CoV-2 + swabs from 57 cancer patients and 14 healthcare workers (HCW) from the Brazilian Cancer Institute were collected in April-May 2020. Complete genome amplification using ARTIC network V3 multiplex primers was performed followed by next-generation sequencing. Assemblies were conducted in Geneious R11, where consensus sequences were extracted and intrahost single nucleotide variants (iSNVs) were identified. Maximum likelihood phylogenetic analysis was performed using PhyMLv.3.0 and lineages were classified using Pangolin and CoV-GLUE. Phylogenetic analysis showed that all but one strain belonged to clade B1.1. Four genetically linked mutations known as the globally dominant SARS-CoV-2 haplotype (C241T, C3037T, C14408T and A23403G) were found in the majority of consensus sequences. SNV signatures of previously characterized Brazilian genomes were also observed in most samples. Another 85 SNVs were found at a lower frequency (1.4-19.7%). Cancer patients displayed a significantly higher intrahost viral genetic diversity compared to HCW (p = 0.009). Intrahost genetic diversity in cancer patients was independent of SARS-CoV-2 Ct values, and was not associated with disease severity, use of corticosteroids, or use of antivirals, characteristics that could influence viral diversity. Such a feature may explain, at least in part, the more adverse outcomes to which cancer/COVID-19 patients experience. AUTHOR SUMMARY: Cancer patients are more prone to clinically evolve to more severe COVID-19 conditions, but the determinants of such a more severe outcome remain largely unknown. In this study, phylogenetic and variation analysis of SARS-CoV-2 genomes from cancer patients and non-cancer healthcare workers at the Brazilian National Cancer Institute were characterized by deep sequencing. Viral genomes showed signatures characteristic of Brazilian viruses, consistent with the hypothesis of local, community transmission rather than virus importation from abroad. Despite most genomes in patients and healthcare workers belonging to the same lineage, intrahost variability was higher in cancer patients when compared to non-cancer counterparts. The intrahost genomic diversity analysis presented in our study highlights the relaxed evolution of SARS-CoV-2 in a vulnerable population of cancer patients. The high number of minor variations can result in the selection of immune escape variants, resistance to potential drugs, and/or increased pathogenicity. The impact of this higher intrahost variability over time warrants further investigation.

Racial differences in α4ß7 expression on CD4+ T cells of HIV-negative men and women who inject drugs.

INTRODUCTION: We performed a cross-sectional study of HIV-uninfected men and women who inject drugs from the ALIVE cohort to examine if black men and women who inject drugs have higher levels of CD4+ T cells expressing the integrin heterodimer α4ß7 compared to white men and women. MATERIALS AND METHODS: Flow cytometry was used to examine expression of α4ß7 and other markers associated with different functional CD4+ T cell subsets in both men and women who inject drugs. RESULTS: Higher levels of α4ß7, CCR5, and CCR6 were observed on CD4+ T cells from black participants compared with white participants. In a multivariable model, α4ß7 expression differed by race, but not sex, age, or other factors. DISCUSSION: Black men and women express higher percentages of α4ß7 expressing CD4+ T cells, which may play a role in HIV disease.

MAdCAM costimulation through Integrin-α4ß7 promotes HIV replication.

Human gut-associated lymphoid tissues (GALT) play a key role in the acute phase of HIV infection. The propensity of HIV to replicate in these tissues, however, is not fully understood. Access and migration of naive and memory CD4+ T cells to these sites is mediated by interactions between integrin α4ß7, expressed on CD4+ T cells, and MAdCAM, expressed on high endothelial venules. We report here that MAdCAM delivers a potent costimulatory signal to naive and memory CD4+ T cells following ligation with α4ß7. Such costimulation promotes high levels of HIV replication. An anti-α4ß7 mAb that prevents mucosal transmission of SIV blocks MAdCAM signaling through α4ß7 and MAdCAM-dependent viral replication. MAdCAM costimulation of memory CD4+ T cells is sufficient to drive cellular proliferation and the upregulation of CCR5, while naive CD4+ T cells require both MAdCAM and retinoic acid to achieve the same response. The pairing of MAdCAM and retinoic acid is unique to the GALT, leading us to propose that HIV replication in these sites is facilitated by MAdCAM-α4ß7 interactions. Moreover, complete inhibition of MAdCAM signaling by an anti-α4ß7 mAb, an analog of the clinically approved therapeutic vedolizumab, highlights the potential of such agents to control acute HIV infection.

Heterosexual transmission of human immunodeficiency virus type 1 subtype C in southern Brazil.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) subtype B predominates in Brazil, but in the southern region subtype C is the most frequent, followed by subtypes B, F1 and recombinant forms. In southern Brazil, these subtypes co-circulate in subjects with homogeneous demographic and clinical features, enabling a better understanding of the role of HIV-1 subtypes on the characteristics of infection. OBJECTIVES: To evaluate the prevalence of different HIV-1 subtypes in subjects with recent diagnosis for HIV infection in the extreme south of Brazil, and to study their association with demographic, behavioral, clinical and laboratorial characteristics. STUDY DESIGN: We have determined the genetic sequence of viral protease and reverse transcriptase (polymerase, connection and RNase H domains) isolated from studied subjects. Viral subtype was inferred by comparison with reference HIV sequences, and recombination was determined with Simplot analysis. The association of HIV-1 subtypes with studied characteristics was evaluated by chi-square, Fisher's exact, Student's t and Kruskal-Wallis tests. RESULTS: Two hundred and forty-five HIV isolates were molecularly characterized, and the association with variables was studied for 233 (95.1%) patients. Of those, 46.8% followed AIDS defining criteria. HIV-1C was responsible for 56.3% of infections, and was associated with heterosexual transmission (p=0.001) and with higher CD4(+) T-cell counts (p=0.02). CONCLUSIONS: The molecular epidemiology of HIV-1 in the southernmost Brazil is currently steady with predominance of HIV-1C. This is the first study showing a robust association of the infection by this subtype and heterosexual transmission in the state of Rio Grande do Sul, Brazil.

Primary HIV-1 drug resistance in the C-terminal domains of viral reverse transcriptase among drug-naïve patients from Southern Brazil.

BACKGROUND: Major and accessory drug resistance mutations have been recently characterized in the C-terminal RT subdomains of HIV-1, connection and RNase H. However, their presence in treatment-naïve patients infected with HIV-1 non-B subtypes remains largely unknown. OBJECTIVES: To characterize the patterns of primary resistance at the C-terminal RT subdomains of HIV-1 infecting subjects in the southern region of Brazil, where HIV-1 subtypes B and C co-circulate. STUDY DESIGN: Plasma viral RNA was extracted from patients recently diagnosed for HIV infection (2005-2008). The protease and reverse transcriptase regions were PCR-amplified and sequenced. Infecting HIV subtypes were assigned by phylogenetic inference and drug resistance mutations were determined following the IAS consensus and recent reports on C-terminal RT mutations. RESULTS: The major mutation to NNRTI T369I/V was found in 1.8% of patients, while A376S was present in another 8.3%. In the RNase H domain, the compensatory mutation D488E was more frequently observed in subtype C than in subtype B (p=0.038), while the inverse was observed for mutation Q547K (p<0.001). The calculated codon genetic barrier showed that 22% of subtype B isolates, but no subtype C, carried T360, requiring two transitions to change into the resistance mutation 360V. CONCLUSIONS: Major resistance-conferring mutations to NNRTI were detected in 10% of RT connection domain viral sequences from treatment-naïve subjects. We showed for the first time that the presence of specific polymorphisms can constrain the acquisition of definite resistance mutations in the connection and RNase H subdomains of HIV-1 RT.