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Principal Investigator: Leonid B. Margolis
Organization: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
Fiscal Year: 2021
Award: $2,104,621
Funding agency: Eunice Kennedy Shriver National Institute of Child Health and Human Development
1. Various factors are hypothesized to trigger persistent immune activation in HIV-1-infected individuals. Here, we used human ex-vivo lymphoid tissue to test several hypotheses: (i)Immune activation may be the result of pro-inflammatory effects of certain antiretroviral drugs themselves; (ii)HIV-1-triggered initial cytokine storm could modify the normal cytokine homeostasis, which remains disregulated even after HIV-1 replication is suppressed; (iii)HIV-1 infection reactivates endogenous viruses, in particular, HHVs that may continue to replicate and induce immune activation after HIV is suppressed; (iv)HIV-1 proteins that continue to be released, in spite of ART, activate the immune system; (v)immune activation is supported by defective virions or extracellular vesicles that carry viral molecules rather than residual infectious HIV-1. We tested these hypotheses evaluating tissue immune activation as measured by upregulation of cytokine release. (i) None of the used drugs, RTV, a protease inhibitor, a combination of two NRTIs (ZDV and 3TC), or NVP, an NNRTI, increased cytokine release by human lymphoid tissues ex vivo. Thus, the effect of drugs themselves did not explain persistent immune activation; (ii) Treating ex vivo tissues with combinations of the exogenous cytokines that were the most upregulated after HIV-1 infection did not result in sustained immune activation either. (iii) We found, however, that after HIV-1 replication was inhibited by RTV in ex-vivo tissues, CMV reactivation continued, albeit at a lower level; (iv) Neither Nef, nor Tat and gp120, were capable of eliciting a sustained release of pro-inflammatory cytokines in ex-vivo lymphoid tissues; (v) In contrast, both defective virions and extracellular vesicles isolated from ex-vivo lymphoid tissues, treated or not with RTV or NVP, upregulated the production of cytokines when transferred to an untreated tissue culture. Thus, these vesicles support residual immune regulations in tissues after HIV-1 is suppressed by ART.
2. The vaginal microbiota, dominated by Lactobacillus spp., plays a key role in preventing HIV-1 transmission. We investigated whether the anti-HIV effect of lactobacilli is mediated by extracellular vesicles (EVs) released by these bacteria. Human cervico-vaginal and tonsillar tissues ex vivo, and cell lines were infected with HIV-1 and treated with EVs released by lactobacilli isolated from the vagina of healthy women. EVs released by L. crispatus BC3 and L. gasseri BC12 largely protected in dose-dependent manner human tissues ex vivo and T cells from HIV-1 infection. Virions pretreated with EVs released by L. crispatus BC3 and L. gasseri BC12, were no longer recognized by the PG9 antibody that specifically binds functional trimeric gp120. In contrast, the pretreatment of cells with bacterial EVs did not affect HIV-1 infection. Bacterial EV-mediated HIV-1 inhibition is therefore the consequence of EVs affecting the infectivity of virions rather than cell functions. Principal component analysis of metabolomics indicated that EVs active toward HIV-1 infection were mainly associated with high amounts of amino acids. Proteomic analysis showed that EVs that inhibited HIV-1 replication are characterized by known bioactive molecules including enolase 2, 60kDa chaperonin, elongation factor Tu, ATP synthase gamma chain, foldase protein PrsA 1, ATP synthase subunit delta, and triosephosphate isomerase. Thus, the protective effect of vaginal Lactobacillus against HIV-1 transmission is, in part, mediated by bacterial EVs that inhibit HIV-1 attachment/entry to the target cells due to diminished exposure of viral Env. These findings may lead to new strategies to prevent male-to-female sexual HIV-1 transmission, for example by use of EVs derived from symbiotic bacteria.
3. Investigating the mechanisms of SARS CoV-2 tissue pathogenesis in vivo requires the development of an adequate system of human tissue culture under laboratory-controlled conditions. We developed such a system. Specifically, blocks of lung tissue are cultured at the air-liquid interface. Flow cytometry of cells from these blocks confirmed their viability and expression of ACE-2 receptor, while histology revealed well preserved structural elements. Inoculation of these blocks with SARS CoV-2 resulted in sustained viral replication and viral release into the culture medium. Also, we used retroviral-based SARS CoV-2 pseudoviruses expressing S and other viral proteins in different combinations. These one-cycle viruses infected cell lines expressing ACE-2. We found that M, N E and O proteins did not significantly affect the ability of the viruses to enter cell. In contrast, mutations in S protein that were identified in vivo changed the efficiency of pseudoviruses to enter cells. Furthermore, SARS-CoV-2 pseudoviruses enter primary human trophoblasts, expressing ACE2 and TMPRSS2 indicating the intra-trophoblast restriction factors that spare human fetus from SARS-CoV-2 infection.
Terms: <2-Phospho-D-Glycerate Hydrolase><2-Phospho-D-glycerate hydro-lyase><2019 novel corona virus><2019 novel coronavirus><2019-nCoV><3TC><ACE2><AIDS Virus><AZT><Acquired Immune Deficiency Syndrome Virus><Acquired Immunodeficiency Syndrome Virus><Affect><Air><Amino Acids><Anti-Retroviral Agents><Antibodies><Antiproteases><Antiretroviral Agents><Autoregulation><Azidothymidine><Bacteria><Binding><Body Tissues><CMV><COVID-19 infection><COVID-19 virus><COVID19 infection><COVID19 virus><Cell Body><Cell Communication><Cell Function><Cell Interaction><Cell Line><Cell Process><Cell physiology><Cell-to-Cell Interaction><CellLine><Cells><Cellular Function><Cellular Physiology><Cellular Process><Chaperonin Family><CoV-2><CoV2><Collaborations><Communication><Cytomegalovirus><Development><Dose><Drug usage><Drugs><E protein><Elements><Elongation Factor Tu><Endopeptidase Inhibitors><Epitheliasin Gene><Female><Fetus><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Genetic Alteration><Genetic Change><Genetic defect><Goals><Gram-Positive Bacteria><HCMV><HIV><HIV Envelope Glycoprotein gp120><HIV Envelope Protein gp120><HIV env Protein gp120><HIV-1><HIV-I><HIV1><HTLV-III gp120><Health Promotion><Histology><Homeostasis><Human><Human Immunodeficiency Virus Type 1><Human Immunodeficiency Viruses><Human Pathology><Human immunodeficiency virus 1><Immune Cell Activation><Immune system><Immunomodulation><Individual><Infection><Inflammatory><LAV-HTLV-III><Laboratories><Lactobacillus><Lamivudine><Lead><Lipids><Liquid substance><Lung><Lung Parenchyma><Lung Respiratory System><Lung Tissue><Lymphadenopathy-Associated Virus><Lymphatic Tissue><Lymphoid Tissue><Measures><Mediating><Medication><Modern Man><Molecular><Molecular Interaction><Mothers><Mutation><NIH><NNRTI><National Institutes of Health><Nucleic Acids><PRSS10><Pathogenesis><Pathology><Patients><Pb element><Peptidase Inhibitors><Peptide Elongation Factor Tu><Peptide Hydrolase Inhibitors><Peptide Peptidohydrolase Inhibitors><Pharmaceutic Preparations><Pharmaceutical Preparations><Phosphopyruvate Hydratase><Phosphotriose Isomerase><Physiological Homeostasis><Play><Principal Component Analyses><Principal Component Analysis><Production><Protease Antagonists><Protease Inhibitor><Protein Synthesis Elongation Factor Tu><Proteinase Inhibitors><Proteins><Proteomics><Receptor Protein><Residual><Residual state><Role><SARS corona virus 2><SARS-CoV-2><SARS-CoV-2 infection><SARS-CoV-2 pathogenesis><SARS-CoV2><SARS-CoV2 infection><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Salivary Gland Viruses><Salutogenesis><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 infection><Severe acute respiratory syndrome related corona virus 2><Sexual Transmission><Strains Cell Lines><Structure><Structure of parenchyma of lung><Subcellular Process><System><T-Cells><T-Lymphocyte><TMPRSS2><TMPRSS2 gene><Testing><Tissues><Tonsillar Tissue><Transmission><Triose-Phosphate Isomerase><Triosephosphate Isomerase><Triosephosphate Mutase><United States National Institutes of Health><Up-Regulation><Upregulation><Vagina><Vaginal><Variant><Variation><Vesicle><Viral><Viral Gene Products><Viral Gene Proteins><Viral Pathogenesis><Viral Proteins><Virion><Virus><Virus Particle><Virus Replication><Virus-HIV><Woman><Work><Wuhan coronavirus><ZDV><Zidovudine><allergic/immunologic body system><allergic/immunologic organ system><aminoacid><angiotensin converting enzyme 2><angiotensin converting enzyme II><anti-retroviral><anti-retroviral therapy><anti-retroviral treatment><antiretroviral><antiretroviral therapy><antiretroviral treatment><azidodeoxythymidine><base><cervicovaginal><chaperonin><coronavirus disease 2019 infection><coronavirus disease 2019 virus><cultured cell line><cytokine><cytokine release syndrome><cytokine storm><cytomegalovirus group><developmental><drug use><drug/agent><enolase><extracellular vesicles><flow cytophotometry><fluid><genome mutation><gp120><gp120 ENV Glycoprotein><gp120(HIV)><hCoV19><heavy metal Pb><heavy metal lead><human pathogen><human tissue><immune activation><immune modulation><immune regulation><immunologic reactivity control><immunomodulatory><immunoregulation><immunoregulatory><in vivo><infected with COVID-19><infected with COVID19><infected with SARS-CoV-2><infected with SARS-CoV2><infected with coronavirus disease 2019><infected with severe acute respiratory syndrome coronavirus 2><liquid><male><metabolism measurement><metabolomics><metabonomics><microbial consortia><microbial flora><microbiota><microflora><multispecies consortia><nCoV2><non-nucleoside RT inhibitors><non-nucleoside reverse transcriptase inhibitors><nonnucleoside reverse transcriptase inhibitors><particle><pathogen><phosphoglycerate dehydratase><phosphoglycerate hydro lyase><preservation><prevent><preventing><promoting health><protective effect><pulmonary><receptor><severe acute respiratory syndrome coronavirus 2 pathogenesis><sexually transmitted><social role><thymus derived lymphocyte><tissue culture><transmission process><trophoblast><vagina microbiota><vaginal flora><vaginal lactobacilli><vaginal microbial community><vaginal microbiota><vaginal microflora><vesicle release><vesicular release><viral multiplication><viral replication><viral transmission><virus multiplication><virus pathogenesis><virus protein><virus 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