The barrier functions of crude cervical mucus plugs against HIV-1 infection in the context of cell-free and cell-to-cell transmission

Abstract

Objective: The cervical mucus plugs are enriched with proteins of known immunological functions. We aimed to characterize the anti-HIV-1 activity of the cervical mucus plugs against a panel of different HIV-1 strains in the contexts of cell-free and cell-associated virus. Design: A cohort of consenting HIV-1-negative and HIV-1-positive pregnant women in labour was recruited from Mthatha General Hospital in the Eastern Cape province of South Africa, from whom the cervical mucus plugs were collected in 6 M guanidinium chloride with protease inhibitors and transported to our laboratories at −80 °C. Methods: Samples were centrifuged to remove insoluble material and dialysed before freeze--drying and subjecting them to the cell viability assays. The antiviral activities of the samples were studied using luminometric reporter assays and flow cytometry. Time-of-addition and BlaM-Vpr virus-cell fusion assays were used to pin-point the antiviral mechanisms of the cervical mucus plugs, before proteomic profiling using liquid chromatography-tandem mass spectrometry. Results: The proteinaceous fraction of the cervical mucus plugs exhibited anti-HIV-1 activity with inter-individual variations and some degree of specificity among different HIV-1 strains. Cell-associated HIV-1 was less susceptible to inhibition by the potent samples whenever compared with the cell-free HIV-1. The samples with high antiviral potency exhibited a distinct proteomic profile when compared with the less potent samples. Conclusion: The crude cervical mucus plugs exhibit anti-HIV-1 activity, which is defined by a specific proteomic profile.

Pharmacological inhibition of bromodomain and extra-terminal proteins induces NRF-2-mediated inhibition of SARS-CoV-2 replication and is subject to viral antagonism

“…Fig. 3C) and subjected them to liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) as described elsewhere (Mhlekude et al, 2021). PCA from the proteomic data demonstrated sample clustering according to their experimental groups, reflecting similar proteomic profiles between individual samples in each group (Sup.…”

“…Post-treatment, cells were subjected to viability assays using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, Germany) for Calu-3 cells and CellTiter-Glo 3-D Cell Viability Assay Kit (Promega, Germany) for hBAECs according to the manufacturer’s protocol. The raw data from the test samples were background subtracted, normalised to naïve cells, and analysed using GraphPad Prism v9 (LaJolla, CA, USA) as previously described (Mhlekude et al, 2021).…”

“…Treated but uninfected and naïve cells served as references. Preparation of cell lysate samples and protein quantification were conducted as mentioned above, from which 50 μg of proteins per sample were prepared and resolved on 7.5% linear SDS-PAGE gels as described elsewhere (Mhlekude et al, 2021). The in-gel protein digestion and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) experiments were performed as previously described (Jochim et al, 2011).…”

“…Genes identified as targets for NRF2 signalling were identified based on a previous report (Olagnier et al, 2020). LC-MS/MS data analysis was conducted as previously described (Mhlekude et al, 2021).…”

Ex Vivo Evaluation of Mucosal Responses to Vaccination with ALVAC and AIDSVAX of Non-Human Primates

“…However, the TZM-bl assay is not fully predictive of the neutralization potency in mucosal tissues [50]. This model might not be able to fully recapitulate the activity of mucus-associated proteins, such as mucins, [51] which inhibit HIV via several mechanisms, including, among others: (i) hindering of the viral particle movement [52]; (ii) fusion inhibitory activity [53]; and (iii) inhibition of HIV trans-infection of CD4+T cells by binding to DC-SIGN [54]. Mucins have also been shown to interact with antibodies, leading to Ig accumulation in the mucus and, potentially, to increased local activity.…”