Showing papers by "James Arthos published in 2020"

Shedding-Resistant HIV-1 Envelope Glycoproteins Adopt Downstream Conformations That Remain Responsive to Conformation-Preferring Ligands.

Abstract: The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer of gp120-gp41 heterodimers mediates virus entry into CD4-positive (CD4+) cells. Single-molecule fluorescence resonance energy transfer (smFRET) has revealed that native Env on the surface of viruses predominantly exists in a pretriggered conformation (state 1) that is preferentially recognized by many broadly neutralizing antibodies (bNAbs). Env is activated by binding receptor CD4, which drives transitions through a default intermediate conformation (state 2) into the three-CD4-bound open conformation (state 3). The application of smFRET to assess the conformational state of existing Env constructs and ligand complexes recently revealed that all current high-resolution structures correspond to downstream states 2 and 3. The structure of state 1, therefore, remains unknown. We sought to identify conditions whereby HIV-1 Env could be stabilized in the pretriggered state 1 for possible structural characterization. Shedding of gp120, known to severely complicate structural studies, can be prevented by using the uncleaved gp160JR-FL precursor with alterations in the protease cleavage site (R508S/R511S) or by introducing a disulfide bridge between gp120 and gp41 designated "SOS" (A501C/T605C). smFRET demonstrated that both shedding-preventing modifications shifted the conformational landscape of Env downstream toward states 2 and 3. However, both membrane-bound Env proteins on the surface of intact viruses remained conformationally dynamic, responsive to state-stabilizing ligands, and able to be stabilized in state 1 by specific ligands such as the Bristol-Myers Squibb (BMS) entry inhibitors. The here-described identification of state 1-stabilizing conditions may enable structural characterization of the state 1 conformation of HIV-1 Env.IMPORTANCE The HIV-1 envelope glycoprotein (Env) opens in response to receptor CD4 binding from a pretriggered (state 1) conformation through a necessary intermediate to the three-CD4-bound conformation. The application of smFRET to test the conformational state of existing Env constructs and ligand complexes used for high-resolution structures recently revealed that they correspond to the downstream conformations. The structure of the pretriggered Env conformation, preferentially recognized by broadly neutralizing antibodies, remains unknown. Here, we identify experimental conditions that stabilize membrane-bound and shedding-resistant virus Env trimers in state 1, potentially facilitating structural characterization of this unknown conformational state.

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

Abstract: 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.

The V2 loop of HIV gp120 delivers costimulatory signals to CD4 + T cells through Integrin α 4 β 7 and promotes cellular activation and infection

Abstract: 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 α4β7-expressing lymphocytes to the gut is mediated by MAdCAM, the natural ligand of α4β7 that is expressed on gut endothelial cells. MAdCAM signaling through α4β7 costimulates CD4+ T cells and promotes HIV replication. Similar to MAdCAM, the V2 domain of the gp120 HIV envelope protein binds to α4β7 In this study, we report that gp120 V2 shares with MAdCAM the capacity to signal through α4β7 resulting in CD4+ T cell activation and proliferation. As with MAdCAM-mediated costimulation, cellular activation induced by gp120 V2 is inhibited by anti-α4β7 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 α4β7 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.

Signal peptide of HIV-1 envelope modulates glycosylation impacting exposure of V1V2 and other epitopes.

Abstract: HIV-1 envelope (Env) is a trimer of gp120-gp41 heterodimers, synthesized from a precursor gp160 that contains an ER-targeting signal peptide (SP) at its amino-terminus. Each trimer is swathed by ~90 N-linked glycans, comprising complex-type and oligomannose-type glycans, which play an important role in determining virus sensitivity to neutralizing antibodies. We previously examined the effects of single point SP mutations on Env properties and functions. Here, we aimed to understand the impact of the SP diversity on glycosylation of virus-derived Env and virus neutralization by swapping SPs. Analyses of site-specific glycans revealed that SP swapping altered Env glycan content and occupancy on multiple N-linked glycosites, including conserved N156 and N160 glycans in the V1V2 region at the Env trimer apex and N88 at the trimer base. Virus neutralization was also affected, especially by antibodies against V1V2, V3, and gp41. Likewise, SP swaps affected the recognition of soluble and cell-associated Env by antibodies targeting distinct V1V2 configurations, V3 crown, and gp41 epitopes. These data highlight the contribution of SP sequence diversity in shaping the Env glycan content and its impact on the configuration and accessibility of V1V2 and other Env epitopes.

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

Abstract: 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 α4β7+, 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.

Selective transmission of some HIV-1 subtype C variants might depend on Envelope stimulating dendritic cells to secrete IL-10

Abstract: Envelope (Env) phenotype(s) that provide transmitted founders (TF) with a selective advantage during HIV-1 transmission would be the ideal target for preventative therapy. We generated Env clones from four individuals infected with a single virus and one participant infected with multiple variants at transmission and compared phenotype with matched Envs from chronic infection (CI). When we determined whether pseudovirus (PSV) of the five TF and thirteen matched CI Env clones differed in their ability to 1) enter TZM-bl cells, 2) bind DC-SIGN, and 3) trans-infect CD4+ cells there was no association between time post-infection and variation in Env phenotype. However, when we compared the ability of PSV to induce monocyte-derived dendritic cells (MDDCs) to secrete Interleukin-10 (IL-10), we found that only TF Envs from single variant transmission cases induced MDDCs to secrete either higher or similar levels of IL-10 as the CI clones. Furthermore, interaction between MDDC DC-SIGN and Env was required for secretion of IL-10. When variants were grouped according to time post-infection, TF PSV induced the release of higher levels of IL-10 than their CI counterparts although this relationship varied across MDDC donors. The selection of variants during transmission is therefore likely a complex event dependent on both virus and host genetics. Our findings suggest that, potentially due to overall variation in N-glycosylation across variants, nuanced differences in binding of TF Env to DC-SIGN might trigger alternative DC immune responses (IRs) in the female genital tract (FGT) that favour HIV-1 survival and facilitate transmission.

Racial differences in α4β7 expression on CD4+ T cells of HIV-negative men and women who inject drugs.

Abstract: 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 α4β7 compared to white men and women. Materials and methods Flow cytometry was used to examine expression of α4β7 and other markers associated with different functional CD4+ T cell subsets in both men and women who inject drugs. Results Higher levels of α4β7, CCR5, and CCR6 were observed on CD4+ T cells from black participants compared with white participants. In a multivariable model, α4β7 expression differed by race, but not sex, age, or other factors. Discussion Black men and women express higher percentages of α4β7 expressing CD4+ T cells, which may play a role in HIV disease.

HIV-1 subtype C transmitted founders modulate dendritic cell inflammatory responses

Abstract: Heterosexual transmission remains the main route of HIV-1 transmission and female genital tract (FGT) inflammation increases the risk of infection. However, the mechanism(s) by which inflammation facilitates infection is not fully understood. In rhesus macaques challenged with simian immunodeficiency virus, dendritic cell (DC) mediated recruitment of CD4+ T cells to the FGT was critical for infection. The aim of this study was to delineate the mechanisms underlying DC-mediated HIV infection by comparing chemokine and pro-inflammatory cytokine production in response to transmitted founder (TF) and chronic infection (CI) Envelope (Env) pseudotyped viruses (PSV). Monocyte-derived DCs (MDDCs) were stimulated with PSV and recombinant gp140 representing matched TF and CI pairs of four individuals and cytokine secretion measured by multiplex immuno-assay. We found that 4/9 Env induced robust MDDC inflammatory responses and of those, three were cloned from TFs. Overall, TF Env induced MDDCs from healthy donors to secrete higher concentrations of inflammatory cytokines and chemokines than those from CI, suggesting TF Env were better inducers of inflammation. Assessing the signalling pathway associated with inflammatory cytokines, we found that PSV of matched TF and CI variants and a gp140 clone activated ERK and JNK to similar levels. Recombinant soluble DC-SIGN inhibited cytokine release and activation of ERK by PSV, suggesting that Env-DC-SIGN binding was partly involved in MDDC stimulation. Therefore, Env clones might differentially stimulate MDDC immune responses via alternative, yet unidentified signalling pathways. Overall, this could suggest that the genetics of the virus itself influences inflammatory responses during HIV infection. In the absence of pre-existing infections, induction of greater inflammatory response by TFs might favour virus survival within the healthy FGT by driving an influx of target cells to sites of infection while suppressing immune responses via IL-10.

Signal Peptide of HIV-1 Envelope Functions to Modulate Glycosylation Impacting Exposure of V1V2 Epitopes

Abstract: HIV-1 envelope (Env) is a trimer of gp120-gp41 heterodimers, synthesized from a precursor gp160 that contains an ER-targeting signal peptide (SP) at its amino-terminus. Each trimer is swathed by ~90 N-linked glycans, comprising complex-type and oligomannose-type glycans, which play an important role in determining virus sensitivity to neutralizing antibodies. We previously examined the effects of single point SP mutations on Env properties and functions. Here, we aimed to understand the impact of the SP diversity on glycosylation of virus-derived Env and virus neutralization by swapping SPs. Analyses of site-specific glycans revealed that SP swapping altered Env glycan content and occupancy on multiple N-linked glycosites, including the conserved N156 and N160 glycans in the V1V2 region at the Env trimer apex. Virus neutralization was also affected, especially by antibodies against the V2i, V2p and V2q epitopes. Likewise, SP swaps affected the recognition of soluble and cell-associated Env by antibodies targeting distinct V1V2 configurations. These data highlight the contribution of SP sequence diversity in shaping the Env glycan content and its impact on the configuration and accessibility of V1V2 epitopes on Env.

Signal Peptide of HIV-1 Envelope Modulates Glycosylation Impacting Exposure of V1V2 Epitopes

Abstract: HIV-1 envelope (Env) is a trimer of gp120-gp41 heterodimers, synthesized from a precursor gp160 that contains an ER-targeting signal peptide (SP) at its amino-terminus. Each trimer is swathed by ∼90 N-linked glycans, comprising complex-type and oligomannose-type glycans, which play an important role in determining virus sensitivity to neutralizing antibodies. We previously examined the effects of single point SP mutations on Env properties and functions. Here, we aimed to understand the impact of the SP diversity on glycosylation of virus-derived Env and virus neutralization by swapping SPs. Analyses of site-specific glycans revealed that SP swapping altered Env glycan content and occupancy on multiple N-linked glycosites, including the conserved N156 and N160 glycans in the V1V2 region at the Env trimer apex. Virus neutralization was also affected, especially by antibodies against the V2i, V2p and V2q epitopes. Likewise, SP swaps affected the recognition of soluble and cell-associated Env by antibodies targeting distinct V1V2 configurations. These data highlight the contribution of SP sequence diversity in shaping the Env glycan content and its impact on the configuration and accessibility of V1V2 epitopes on Env. Author Summary HIV-1 Env glycoprotein is produced by a precursor gp160 that has a signal peptide at its N-terminus. The SP is highly diverse among the HIV-1 isolates and no two SP are same. This study presents site-specific analyses of N-linked glycosylation on HIV-1 envelope glycoproteins from infectious viruses produced with different envelope signal peptides. We show that signal peptide swapping alters the envelope glycan shield, including the conserved N156 and N160 located in the V1V2 region on the trimer apex, to impact Env recognition and virus neutralization by antibodies, particularly those targeting the the V1V2 region. The data offer crucial insights into the role of signal peptide in the interplay between HIV-1 and antibodies and its potential utility to control Env glycosylation in the development of Env-based HIV-1 vaccine.