Cationic Chitosan Derivatives for the Inactivation of HIV-1 and SARS-CoV-2 Enveloped Viruses.

Cationic chitosan derivatives have been widely studied as potential antimicrobial agents. However, very little is known about their antiviral activity and mode of action against enveloped viruses. We investigated the ability of hydroxypropanoic acid-grafted chitosan (HPA-CS) and N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC) to inactivate enveloped viruses like the human immunodeficiency virus (HIV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The membrane-disrupting potential of the chitosan derivatives was initially investigated in a hemolysis assay. At 1.0 mg/mL, about 80% hemolysis was observed for the cationic chitosan derivatives, which was significant when compared to almost no membrane-disrupting activity by the unmodified chitosan. Virus inhibition was evaluated using the luciferase-based antiviral assay against the HIV-1 NL4.3 virus (400 TCID). The IC50 of HPA-CS was 4.109 mg/mL, while the HTCC showed a higher antiviral activity at an IC50 = 0.225 mg/mL. For practical application, the antiviral efficacies of the HTCC-coated and uncoated nonmedical masks were evaluated for SARS- CoV-2 virus capture. The coated masks demonstrated an almost excellent performance with nearly 100% viral inhibition compared to less than 60% inhibition by the uncoated masks. Molecular docking predictions suggest that the HTCC polymers interact with the viral spike protein, blocking the coronavirus interaction with the target host cell's angiotensin-converting enzyme 2 cellular receptors.

Development of a Multiplex HIV/TB Diagnostic Assay Based on the Microarray Technology.

Currently there are diagnostic tests available for human immunodeficiency virus (HIV) and tuberculosis (TB); however, they are still diagnosed separately, which can delay treatment in cases of co-infection. Here we report on a multiplex microarray technology for the detection of HIV and TB antibodies using p24 as well as TB CFP10, ESAT6 and pstS1 antigens on epoxy-silane slides. To test this technology for antigen-antibody interactions, immobilized antigens were exposed to human sera spiked with physiological concentrations of primary antibodies, followed by secondary antibodies conjugated to a fluorescent reporter. HIV and TB antibodies were captured with no cross-reactivity observed. The sensitivity of the slides was compared to that of high-binding plates. We found that the slides were more sensitive, with the detection limit being 0.000954 µg/mL compared to 4.637 µg/mL for the plates. Furthermore, stability studies revealed that the immobilized antigens could be stored dry for at least 90 days and remained stable across all pH and temperatures assessed, with pH 7.4 and 25 °C being optimal. The data collectively suggested that the HIV/TB multiplex detection technology we developed has the potential for use to diagnose HIV and TB co-infection, and thus can be developed further for the purpose.

Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9.

The hypersensitive response is elicited by Agrobacterium infiltration of Nicotiana benthamiana, including the induction and accumulation of pathogenesis-related proteins, such as proteases. This includes the induction of the expression of several cysteine proteases from the C1 (papain-like cysteine protease) and C13 (legumain-like cysteine protease) families. This study demonstrates the role of cysteine proteases: NbVPE-1a, NbVPE-1b, and NbCysP6 in the proteolytic degradation of Nicotiana benthamiana (glycosylation mutant ΔXTFT)-produced anti-human immunodeficiency virus broadly neutralizing antibody, CAP256-VRC26.25. Three putative cysteine protease cleavage sites were identified in the fragment crystallizable region. We further demonstrate the transient coexpression of CAP256-VRC26.25 with CRISPR/Cas9-mediated genome editing vectors targeting the NbVPE-1a, NbVPE-1b, and NbCysP6 genes which resulted in a decrease in CAP256-VRC26.25 degradation. No differences in structural features were observed between the human embryonic kidney 293 (HEK293)-produced and ΔXTFT broadly neutralizing antibodies produced with and without the coexpression of genome-editing vectors. Furthermore, despite the presence of proteolytically degraded fragments of plant-produced CAP256-VRC26.25 without the coexpression of genome editing vectors, no influence on the in vitro functional activity was detected. Collectively, we demonstrate an innovative in planta strategy for improving the quality of the CAP256 antibodies through the transient expression of the CRISPR/Cas9 vectors.

Synthesis of fluorescence labelled aptamers for use as low-cost reagents in HIV/AIDS research and diagnostics.

Aptamers are nucleic acids selected by systematic evolution of ligands by exponential enrichment. They have potential as alternatives to antibodies in medical research and diagnostics, with the advantages of being non-immunogenic and relatively inexpensive to produce. In the present study, gp120 aptamers conjugated with fluorescein isothiocyanate (FITC) were generated, which could interact with HIV-1 gp120. A previously isolated gp120 aptamer, CSIR 1.1, was conjugated with FITC by incubation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and imidazole. The conjugation and binding to the glycoprotein were confirmed by flow cytometry. FITC conjugated aptamers showed an increase in fluorescence emission 24-fold higher than baseline, and this difference was statistically significant (P=0.0016). Compared with a commercially available biotinylated anti-gp120 antibody, detected using FITC conjugated streptavidin, the emission of fluorescence obtained from the FITC-conjugated aptamer was 8-fold higher, suggesting a stronger interaction with gp120. In addition, the FITC conjugated aptamer neutralized HIV-1 pseudoviruses with an average IC50 of 21.3 nM, similar to the parent aptamer that had an IC50 of 19.2 nM. However, the difference in inhibition between the two aptamers was not statistically significant (P=0.784). These results indicate that the FITC-conjugated aptamer generated in the present study could potentially be used as a low-cost reagent in HIV/AIDS research and diagnostics.

Modification and optimization of the inhibition of HIV-1 cell-to-cell transmission assay.

HIV-1 infection is caused by cell-free and cell-associated viruses. Currently most of the assays used to screen potential HIV-1 entry inhibitors focus on the inhibition of cell-free viruses. One assay that is widely employed is the TZM-bl neutralization assay that uses pseudotyped viruses. However, a study by Abela et al. showed that many inhibitors that potently inhibit cell-free HIV-1 in this assay can be less effective against the cell-to-cell transmission of the virus. These researchers then designed a method to screen entry inhibitors for activity against cell-associated HIV-1, using pseudotyped viruses. The main limitation of this method, however, was that it can only be reliably employed against viruses that cannot infect target cells as cell-free virion in the absence of a polycation supplement such as DEAE (diethylaminoethyl). Thus, in the current study we provide modifications to this method that solves the problem and makes it possible to study entry inhibitors against cell-to-cell infection of both polycation depend and independent viruses. The main modification involves the introduction of the relative light unit (RLU) vs. virus producing 293-T cells / corresponding supernatants graph. This graph is used to select a virus input that only allows for the detection of cell-associated viruses infection.•The method is a modification of the cell-to-cell transmission assay published by Abela et al.•The method allows for the study of the inhibition of cell-to-cell transmission of both polycation dependent and independent HIV-1 pseudoviruses.

Comparison of the antiviral activity of the microbicide candidate griffithsin and its tandemers derivatives against different modes of HIV-1 transmission.

Tandemers 2MG, 2MG3, 3MG and 4MG are derivatives of the potent anti-HIV-1 microbicide candidate griffithsin (GRFT). We compared these compounds anti-HIV-1 activity to GRFT using the viruses CAP206.08 and CAAN5342.A2 that have decreased sensitivity to this lectin. The 2MG and 2MG3 tandemers had similar activity to GRFT against cell-free and cell-associated viruses, while 3MG and 4MG were significantly more potent. Furthermore, the restoration of the 234N or 295N glycan in these viruses, known to increase sensitivity to GRFT, also increased sensitivity to 2MG and 2MG3, and not to 3MG and 4MG. In addition, GRFT resistant viruses generated in-vitro were equally resistant to 2MG and 2MG3 while they had considerably low resistance to 3MG and 4MG. Lastly, all five compounds showed increased inhibitory activity in seminal and vaginal simulants although the effect was more pronounced in the former. These data support further studies of tandemers as potential microbicides.