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.

Murine alveolar macrophages rapidly accumulate intranasally administered SARS-CoV-2 Spike protein leading to neutrophil recruitment and damage.

The trimeric SARS-CoV-2 Spike protein mediates viral attachment facilitating cell entry. Most COVID-19 vaccines direct mammalian cells to express the Spike protein or deliver it directly via inoculation to engender a protective immune response. The trafficking and cellular tropism of the Spike protein in vivo and its impact on immune cells remains incompletely elucidated. In this study, we inoculated mice intranasally, intravenously, and subcutaneously with fluorescently labeled recombinant SARS-CoV-2 Spike protein. Using flow cytometry and imaging techniques, we analyzed its localization, immune cell tropism, and acute functional impact. Intranasal administration led to rapid lung alveolar macrophage uptake, pulmonary vascular leakage, and neutrophil recruitment and damage. When injected near the inguinal lymph node medullary, but not subcapsular macrophages, captured the protein, while scrotal injection recruited and fragmented neutrophils. Widespread endothelial and liver Kupffer cell uptake followed intravenous administration. Human peripheral blood cells B cells, neutrophils, monocytes, and myeloid dendritic cells all efficiently bound Spike protein. Exposure to the Spike protein enhanced neutrophil NETosis and augmented human macrophage TNF-α (tumor necrosis factor-α) and IL-6 production. Human and murine immune cells employed C-type lectin receptors and Siglecs to help capture the Spike protein. This study highlights the potential toxicity of the SARS-CoV-2 Spike protein for mammalian cells and illustrates the central role for alveolar macrophage in pathogenic protein uptake.

TGF-ß blockade drives a transitional effector phenotype in T cells reversing SIV latency and decreasing SIV reservoirs in vivo.

HIV-1 persistence during ART is due to the establishment of long-lived viral reservoirs in resting immune cells. Using an NHP model of barcoded SIVmac239 intravenous infection and therapeutic dosing of anti-TGFBR1 inhibitor galunisertib (LY2157299), we confirm the latency reversal properties of in vivo TGF-ß blockade, decrease viral reservoirs and stimulate immune responses. Treatment of eight female, SIV-infected macaques on ART with four 2-weeks cycles of galunisertib leads to viral reactivation as indicated by plasma viral load and immunoPET/CT with a 64Cu-DOTA-F(ab')2-p7D3-probe. Post-galunisertib, lymph nodes, gut and PBMC exhibit lower cell-associated (CA-)SIV DNA and lower intact pro-virus (PBMC). Galunisertib does not lead to systemic increase in inflammatory cytokines. High-dimensional cytometry, bulk, and single-cell (sc)RNAseq reveal a galunisertib-driven shift toward an effector phenotype in T and NK cells characterized by a progressive downregulation in TCF1. In summary, we demonstrate that galunisertib, a clinical stage TGF-ß inhibitor, reverses SIV latency and decreases SIV reservoirs by driving T cells toward an effector phenotype, enhancing immune responses in vivo in absence of toxicity.

Bioorthogonal click labeling of an amber-free HIV-1 provirus for in-virus single molecule imaging.

Structural dynamics of human immunodeficiency virus 1 (HIV-1) envelope (Env) glycoprotein mediate cell entry and facilitate immune evasion. Single-molecule FRET using peptides for Env labeling revealed structural dynamics of Env, but peptide use risks potential effects on structural integrity/dynamics. While incorporating noncanonical amino acids (ncAAs) into Env by amber stop-codon suppression, followed by click chemistry, offers a minimally invasive approach, this has proved to be technically challenging for HIV-1. Here, we develope an intact amber-free HIV-1 system that overcomes hurdles of preexisting viral amber codons. We achieved dual-ncAA incorporation into Env on amber-free virions, enabling single-molecule Förster resonance energy transfer (smFRET) studies of click-labeled Env that validated the previous peptide-based labeling approaches by confirming the intrinsic propensity of Env to dynamically sample multiple conformational states. Amber-free click-labeled Env also enabled real-time tracking of single virion internalization and trafficking in cells. Our system thus permits in-virus bioorthogonal labeling of proteins, compatible with studies of virus entry, trafficking, and egress from cells.

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.

Identification of CD38, CD97, and CD278 on the HIV surface using a novel flow virometry screening assay.

While numerous cellular proteins in the HIV envelope are known to alter virus infection, methodology to rapidly phenotype the virion surface in a high throughput, single virion manner is lacking. Thus, many human proteins may exist on the virion surface that remain undescribed. Herein, we developed a novel flow virometry screening assay to discover new proteins on the surface of HIV particles. By screening a CD4+ T cell line and its progeny virions, along with four HIV isolates produced in primary cells, we discovered 59 new candidate proteins in the HIV envelope that were consistently detected across diverse HIV isolates. Among these discoveries, CD38, CD97, and CD278 were consistently present at high levels on virions when using orthogonal techniques to corroborate flow virometry results. This study yields new discoveries about virus biology and demonstrates the utility and feasibility of a novel flow virometry assay to phenotype individual virions.

TGF-ß blockade drives a transitional effector phenotype in T cells reversing SIV latency and decreasing SIV reservoirs in vivo.

HIV-1 persistence during ART is due to the establishment of long-lived viral reservoirs in resting immune cells. Using an NHP model of barcoded SIVmac239 intravenous infection and therapeutic dosing of the anti-TGFBR1 inhibitor galunisertib (LY2157299), we confirmed the latency reversal properties of in vivo TGF-ß blockade, decreased viral reservoirs and stimulated immune responses. Eight SIV-infected macaques on suppressive ART were treated with 4 2-week cycles of galunisertib. ART was discontinued 3 weeks after the last dose, and macaques euthanized 6 weeks after ART-interruption(ATI). One macaque did not rebound, while the remaining rebounded between week 2 and 6 post-ATI. Galunisertib led to viral reactivation as indicated by plasma viral load and immunoPET/CT with the 64Cu-DOTA-F(ab')2-p7D3-probe. Half to 1 Log decrease in cell-associated (CA-)SIV DNA was detected in lymph nodes, gut and PBMC, while intact pro-virus in PBMC decreased by 3-fold. No systemic increase in inflammatory cytokines was observed. High-dimensions cytometry, bulk and single-cell RNAseq revealed a shift toward an effector phenotype in T and NK cells. In summary, we demonstrated that galunisertib, a clinical stage TGFß inhibitor, reverses SIV latency and decreases SIV reservoirs by driving T cells toward an effector phenotype, enhancing immune responses in vivo in absence of toxicity.

Human CD4-Binding Site Antibody Elicited by Polyvalent DNA Prime-Protein Boost Vaccine Neutralizes Cross-Clade Tier-2-HIV Strains.

The vaccine elicitation of HIV-neutralizing antibodies with tier-2-neutralization breadth has been a challenge. Here, we report the isolation and characteristics of a CD4-binding site specific monoclonal antibody, HmAb64, from a human volunteer immunized with a polyvalent gp120 DNA prime-protein boost vaccine. HmAb64 derived from heavy chain variable germline gene IGHV1-18, light chain germline gene IGKV1-39, and had a 3rd heavy chain complementarity determining region (CDR H3) of 15 amino acids. On a cross-clade panel of 208 HIV-1 pseudo-virus strains, HmAb64 neutralized 21 (10%), including tier-2 neutralization resistant strains from clades B, BC, C, and G. The cryo-EM structure of the antigen-binding fragment of HmAb64 bound to a conformation between prefusion closed and occluded open forms of envelope trimer, using both heavy and light CDR3s to recognize the CD4-binding loop, a critical component of the CD4-binding site. A gp120 subunit-based vaccine can thus elicit an antibody capable of tier 2-HIV neutralization.

Murine Alveolar Macrophages Rapidly Accumulate Intranasally Administered SARS-CoV-2 Spike Protein leading to Neutrophil Recruitment and Damage.

The trimeric SARS-CoV-2 Spike protein mediates viral attachment facilitating cell entry. Most COVID-19 vaccines direct mammalian cells to express the Spike protein or deliver it directly via inoculation to engender a protective immune response. The trafficking and cellular tropism of the Spike protein in vivo and its impact on immune cells remains incompletely elucidated. In this study we inoculated mice intranasally, intravenously, and subcutaneously with fluorescently labeled recombinant SARS-CoV-2 Spike protein. Using flow cytometry and imaging techniques we analyzed its localization, immune cell tropism, and acute functional impact. Intranasal administration led to rapid lung alveolar macrophage uptake, pulmonary vascular leakage, and neutrophil recruitment and damage. When injected near the inguinal lymph node medullary, but not subcapsular macrophages, captured the protein, while scrotal injection recruited and fragmented neutrophils. Wide-spread endothelial and liver Kupffer cell uptake followed intravenous administration. Human peripheral blood cells B cells, neutrophils, monocytes, and myeloid dendritic cells all efficiently bound Spike protein. Exposure to the Spike protein enhanced neutrophil NETosis and augmented human macrophage TNF-α and IL-6 production. Human and murine immune cells employed C-type lectin receptors and Siglecs to help capture the Spike protein. This study highlights the potential toxicity of the SARS-CoV-2 Spike protein for mammalian cells and illustrates the central role for alveolar macrophage in pathogenic protein uptake.

Effect of Passive Administration of Monoclonal Antibodies Recognizing Simian Immunodeficiency Virus (SIV) V2 in CH59-Like Coil/Helical or ß-Sheet Conformations on Time of SIVmac251 Acquisition.

The monoclonal antibodies (MAbs) NCI05 and NCI09, isolated from a vaccinated macaque that was protected from multiple simian immunodeficiency virus (SIV) challenges, both target an overlapping, conformationally dynamic epitope in SIV envelope variable region 2 (V2). Here, we show that NCI05 recognizes a CH59-like coil/helical epitope, whereas NCI09 recognizes a ß-hairpin linear epitope. In vitro, NCI05 and, to a lesser extent, NCI09 mediate the killing of SIV-infected cells in a CD4-dependent manner. Compared to NCI05, NCI09 mediates higher titers of antibody-dependent cellular cytotoxicity (ADCC) to gp120-coated cells, as well as higher levels of trogocytosis, a monocyte function that contributes to immune evasion. We also found that passive administration of NCI05 or NCI09 to macaques did not affect the risk of SIVmac251 acquisition compared to controls, demonstrating that these anti-V2 antibodies alone are not protective. However, NCI05 but not NCI09 mucosal levels strongly correlated with delayed SIVmac251 acquisition, and functional and structural data suggest that NCI05 targets a transient state of the viral spike apex that is partially opened, compared to its prefusion-closed conformation. IMPORTANCE Studies suggest that the protection against SIV/simian-human immunodeficiency virus (SHIV) acquisition afforded by the SIV/HIV V1 deletion-containing envelope immunogens, delivered by the DNA/ALVAC vaccine platform, requires multiple innate and adaptive host responses. Anti-inflammatory macrophages and tolerogenic dendritic cells (DC-10), together with CD14+ efferocytes, are consistently found to correlate with a vaccine-induced decrease in the risk of SIV/SHIV acquisition. Similarly, V2-specific antibody responses mediating ADCC, Th1 and Th2 cells expressing no or low levels of CCR5, and envelope-specific NKp44+ cells producing interleukin 17 (IL-17) also are reproducible correlates of decreased risk of virus acquisition. We focused on the function and the antiviral potential of two monoclonal antibodies (NCI05 and NCI09) isolated from vaccinated animals that differ in antiviral function in vitro and recognize V2 in a linear (NCI09) or coil/helical (NCI05) conformation. We demonstrate that NCI05, but not NCI09, delays SIVmac251 acquisition, highlighting the complexity of antibody responses to V2.

An intact amber-free HIV-1 system for in-virus protein bioorthogonal click labeling that delineates envelope conformational dynamics.

The HIV-1 envelope (Env) glycoprotein is conformationally dynamic and mediates membrane fusion required for cell entry. Single-molecule fluorescence resonance energy transfer (smFRET) of Env using peptide tags has provided mechanistic insights into the dynamics of Env conformations. Nevertheless, using peptide tags risks potential effects on structural integrity. Here, we aim to establish minimally invasive smFRET systems of Env on the virus by combining genetic code expansion and bioorthogonal click chemistry. Amber stop-codon suppression allows site-specifically incorporating noncanonical/unnatural amino acids (ncAAs) at introduced amber sites into proteins. However, ncAA incorporation into Env (or other HIV-1 proteins) in the virus context has been challenging due to low copies of Env on virions and incomplete amber suppression in mammalian cells. Here, we developed an intact amber-free virus system that overcomes impediments from preexisting ambers in HIV-1. Using this system, we successfully incorporated dual ncAAs at amber-introduced sites into Env on intact virions. Dual-ncAA incorporated Env retained similar neutralization sensitivities to neutralizing antibodies as wildtype. smFRET of click-labeled Env on intact amber-free virions recapitulated conformational profiles of Env. The amber-free HIV-1 infectious system also permits in-virus protein bioorthogonal labeling, compatible with various advanced microscopic studies of virus entry, trafficking, and egress in living cells. Amber-free HIV-1 infectious systems actualized minimal invasive Env tagging for smFRET, versatile for in-virus bioorthogonal click labeling in advanced microscopic studies of virus-host interactions.

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.

Blockade of TGF-ß signaling reactivates HIV-1/SIV reservoirs and immune responses in vivo.

TGF-ß plays a critical role in maintaining immune cells in a resting state by inhibiting cell activation and proliferation. Resting HIV-1 target cells represent the main cellular reservoir after long-term antiretroviral therapy (ART). We hypothesized that releasing cells from TGF-ß-driven signaling would promote latency reversal. To test our hypothesis, we compared HIV-1 latency models with and without TGF-ß and a TGF-ß type 1 receptor inhibitor, galunisertib. We tested the effect of galunisertib in SIV-infected, ART-treated macaques by monitoring SIV-env expression via PET/CT using the 64Cu-DOTA-F(ab')2 p7D3 probe, along with plasma and tissue viral loads (VLs). Exogenous TGF-ß reduced HIV-1 reactivation in U1 and ACH-2 models. Galunisertib increased HIV-1 latency reversal ex vivo and in PBMCs from HIV-1-infected, ART-treated, aviremic donors. In vivo, oral galunisertib promoted increased total standardized uptake values in PET/CT images in gut and lymph nodes of 5 out of 7 aviremic, long-term ART-treated, SIV-infected macaques. This increase correlated with an increase in SIV RNA in the gut. Two of the 7 animals also exhibited increases in plasma VLs. Higher anti-SIV T cell responses and antibody titers were detected after galunisertib treatment. In summary, our data suggest that blocking TGF-ß signaling simultaneously increases retroviral reactivation events and enhances anti-SIV immune responses.

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.

Blocking α4ß7 integrin delays viral rebound in SHIVSF162P3-infected macaques treated with anti-HIV broadly neutralizing antibodies.

Anti-HIV broadly neutralizing antibodies (bNAbs) may favor development of antiviral immunity by engaging the immune system during immunotherapy. Targeting integrin α4ß7 with an anti-α4ß7 monoclonal antibody (Rh-α4ß7) affects immune responses in SIV/SHIV-infected macaques. To explore the therapeutic potential of combining bNAbs with α4ß7 integrin blockade, SHIVSF162P3-infected, viremic rhesus macaques were treated with bNAbs only (VRC07-523LS and PGT128 anti-HIV antibodies) or a combination of bNAbs and Rh-α4ß7 or were left untreated as a control. Treatment with bNAbs alone decreased viremia below 200 copies/ml in all macaques, but seven of eight macaques (87.5%) in the bNAbs-only group rebounded within a median of 3 weeks (95% CI: 2 to 9). In contrast, three of six macaques treated with a combination of Rh-α4ß7 and bNAbs (50%) maintained a viremia below 200 copies/ml until the end of the follow-up period; viremia in the other three macaques rebounded within a median of 6 weeks (95% CI: 5 to 11). Thus, there was a modest delay in viral rebound in the macaques treated with the combination antibody therapy compared to bNAbs alone. Our study suggests that α4ß7 integrin blockade may prolong virologic control by bNAbs in SHIVSF162P3-infected macaques.

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.

Anti-V2 antibodies virus vulnerability revealed by envelope V1 deletion in HIV vaccine candidates.

The efficacy of ALVAC-based HIV and SIV vaccines in humans and macaques correlates with antibodies to envelope variable region 2 (V2). We show here that vaccine-induced antibodies to SIV variable region 1 (V1) inhibit anti-V2 antibody-mediated cytotoxicity and reverse their ability to block V2 peptide interaction with the α4ß7 integrin. SIV vaccines engineered to delete V1 and favor an α helix, rather than a ß sheet V2 conformation, induced V2-specific ADCC correlating with decreased risk of SIV acquisition. Removal of V1 from the HIV-1 clade A/E A244 envelope resulted in decreased binding to antibodies recognizing V2 in the ß sheet conformation. Thus, deletion of V1 in HIV envelope immunogens may improve antibody responses to V2 virus vulnerability sites and increase the efficacy of HIV vaccine candidates.

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.