Objective: Mechanisms underlying mucosal transmission of HIV-1 are incompletely understood. We describe the anti-HIV-1 activity of human β-defensins (hBD), small cationic molecules that provide protection at mucosal surfaces. Methods and results: HIV-1 induced expression of hBD-2 and -3 mRNA (but not that of hBD-1) 4- to 78-fold, respectively, above baseline in normal human oral epithelial cells. HIV-1 failed to infect these cells, even after 5 days of exposure. Recombinant hBD-1 had no antiviral activity, while rhBD-2 and rhBD-3 showed concentration-dependent inhibition of HIV-1 replication without cellular toxicity. Inhibition was greater against CXCR4-tropic than against the CCR5-tropic HIV-1 isolates. hBD-2 and hBD-3 induced an irreversible effect on virion infectivity, with electron microscopy confirming binding of hBDs to viral particles. Finally, hBD-2 and -3 induced downmodulation of the HIV-1 coreceptor CXCR4 (but not CCR5) in peripheral blood mononuclear cells and T lymphocytic cells as shown by confocal microscopy and flow cytometry. Conclusions: This study shows for the first time that HIV-1 induces β-defensin expression in human oral epithelial cells and that β-defensins block HIV-1 replication via a direct interaction with virions and through modulation of the CXCR4 coreceptor. These properties may be exploited as strategies for mucosal protection against HIV-1 transmission.

The frequency of Candida infections has increased in recent years and it has been accompanied by a significant rise in morbidity and mortality. The secretion of aspartic proteases by Candida spp. was demonstrated to be one of the virulence determinants. Candida albicans is classified as the major human pathogen in the genus Candida. However, other species of this genus have been found to cause an increasing number of candidiases. We isolated secreted aspartic proteases (Saps) of C. albicans (Sap2p), C. tropicalis (Sapt1p), C. parapsilosis (Sapp1p), and C. lusitaniae (Saplp) from culture media. All the isolated proteases were N‐terminally sequenced. Their specific proteolytic activities and sensitivity to series of peptidomimetic inhibitors modified in the type of scissile bond replacement as well as in the N‐ and C‐termini were analyzed. The most divergent substrate specificity was observed for the Sap of C. tropicalis. The specificity of Sap of C. lusitaniae is most closely related to that of Sap of C. parapsilosis. We designed and prepared an inhibitor containing phenylstatine isoster that was equipotent towards all four proteases within the range of 10⁻¹⁰−10⁻⁹ m. The HIV‐1 protease inhibitors ritonavir, saquinavir, indinavir, and nelfinavir were also tested for the inhibition of four Saps. Only ritonavir and saquinavir inhibited Sap2p, Sapt1p, Sapp1p, and Saplp in micromolar concentrations.

HIV-1 coreceptor tropism assays are required to rule out the presence of CXCR4-tropic (non-R5) viruses prior treatment with CCR5 antagonists. Phenotypic (e.g., Trofile™, Monogram Biosciences) and genotypic (e.g., population sequencing linked to bioinformatic algorithms) assays are the most widely used. Although several next-generation sequencing (NGS) platforms are available, to date all published deep sequencing HIV-1 tropism studies have used the 454™ Life Sciences/Roche platform. In this study, HIV-1 co-receptor usage was predicted for twelve patients scheduled to start a maraviroc-based antiretroviral regimen. The V3 region of the HIV-1 env gene was sequenced using four NGS platforms: 454™, PacBio® RS (Pacific Biosciences), Illumina®, and Ion Torrent™ (Life Technologies). Cross-platform variation was evaluated, including number of reads, read length and error rates. HIV-1 tropism was inferred using Geno2Pheno, Web PSSM, and the 11/24/25 rule and compared with Trofile™ and virologic response to antiretroviral therapy. Error rates related to insertions/deletions (indels) and nucleotide substitutions introduced by the four NGS platforms were low compared to the actual HIV-1 sequence variation. Each platform detected all major virus variants within the HIV-1 population with similar frequencies. Identification of non-R5 viruses was comparable among the four platforms, with minor differences attributable to the algorithms used to infer HIV-1 tropism. All NGS platforms showed similar concordance with virologic response to the maraviroc-based regimen (75% to 80% range depending on the algorithm used), compared to Trofile (80%) and population sequencing (70%). In conclusion, all four NGS platforms were able to detect minority non-R5 variants at comparable levels suggesting that any NGS-based method can be used to predict HIV-1 coreceptor usage.

Healthcare-acquired infections by pathogenic microorganisms including viruses represent significant health concern worldwide. Next to direct transmission from person-to-person also indirect transmission from contaminated surfaces is well documented and important route of infections. Here, we tested antiviral properties of hybrid coating containing silver, copper and zinc cations that was previously shown to be effective against pathogenic bacteria including methicillin-resistant Staphylococcus aureus. Hybrid coatings containing silver, copper and zinc cations were prepared through radical polymerization via sol-gel method and applied on glass slides or into the wells of polymethylmethacrylate plates. A 10 μl droplet of several viruses such as human immunodeficiency virus type 1 (HIV-1), influenza, dengue virus, herpes simplex virus, and coxsackievirus was added to coated and uncoated slides or plates, incubated usually from 5 to 240 min and followed by titer determination of recovered virus. Scanning electron microscopy analysis showed better adhesion of coatings on glass surfaces, which resulted in 99.5–100 % HIV-1 titer reduction (3.1 ± 0.8 log10TCID50, n = 3) already after 20 min of exposure to coatings, than on coated polymethylmethacrylate plates with 75–100 % (1.7 ± 1.1 log10TCID50, n = 3) and 98–100 % (2.3 ± 0.5 log10TCID50, n = 3) HIV-1 titer reduction after 20 and 120 min of exposure, respectively. Slower virucidal kinetics was observed with other enveloped viruses, where 240 min exposure to coated slides lead to 97 % (dengue), 100 % (herpes simplex) and 77 % (influenza) reduction in virus titers. Interestingly, only marginal reduction in viral titer after 240 min of exposure was noticed for non-enveloped coxsackie B3 virus. Our hybrid coatings showed virucidal activity against HIV and other enveloped viruses thus providing further findings towards development of broad-spectrum antimicrobial coating suitable for surfaces in healthcare settings.

The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) resistance mutation K65R confers intermediate levels of resistance to several RT inhibitors, including a three- to fourfold reduction of tenofovir susceptibility. Here, we have used for the first time primary HIV-1 isolates from individuals who developed the K65R mutation while enrolled in a clinical trial of tenofovir to analyze the impact of this mutation on HIV-1 replicative fitness. A marked impairment in replicative fitness was observed in association with the selection of viruses carrying the K65R mutation in all patients. The mean replicative fitness among these viruses was 20% relative to the corresponding baseline wild-type virus, ranging from 10 to 32% depending on the accompanying RT mutations. These results support a reduction in in vivo replication for K65R mutant viruses.