Viral transmission and pathogenesis in human tissues

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

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.