Triple Combinations of AAV9-Vectors Encoding Anti-HIV bNAbs Provide Long-Term In Vivo Expression of Human IgG Effectively Neutralizing Pseudoviruses from HIV-1 Global Panel.

Anti-human immunodeficiency virus (HIV) broadly neutralizing antibodies (bNAbs) offer a promising approach for the treatment of HIV-1. The current paradigm for antibody therapy involves passive antibody transfer, requiring regular delivery of bNAbs in treating chronic diseases such as HIV-1. An alternative strategy is to use AAV-mediated gene transfer to enable in vivo production of desirable anti-HIV-1 antibodies. In this study, we investigated two sets of triple combinations of AAV9-vectors encoding different bNAbs: N6, 10E8, 10-1074 (CombiMab1), and VRC07-523, PGDM1400, 10-1074 (CombiMab2). We used CBAxC57Bl and C57BL/6 mouse models to characterize rAAV-induced antibody expression and to evaluate the neutralization capacity of mouse sera against a global panel of HIV-1 viral strains. rAAV9-mediated IgG expression varied between bNAb clones and mouse strains, with C57BL/6 mice exhibiting higher bNAb titers following rAAV delivery. Although CombiMab2 treatment elicited a higher IgG titer than CombiMab1, both combinations resulted in neutralization of all the viral strains from the global HIV-1 panel. Our data highlight the potential of AAV vectors as a long-term option for HIV-1 therapy.

Analysis of the Frequency of Mutations at Diagnostic Oligonucleotide Sites and Their Impact on the Efficiency of PCR for HIV-1.

The development of effective diagnostic kits for HIV-1 remains a pressing concern. We designed diagnostic oligonucleotides for HIV-1 real-time PCR to target the most conserved region of the HIV-1 genome and assessed the mutation frequency at annealing sites. Two databases of nucleotide sequences, Los Alamos and NCBI, were analyzed, revealing that more than 99% of the sequences either lack mutations or contain 1-2 mutations at the binding site of the forward and reverse primers. Additionally, 98.5% of the sequences either lack mutations or contain 1-2 mutations at the binding site of the TaqMan probe. To evaluate the efficiency of primers and the probe in real-time PCR in the case of mutations at their binding sites, we constructed several plasmids containing the most common mutations and, in a model experiment, showed how different mutations affect the efficiency of PCR. Our analysis demonstrated that about 98.5% of HIV-1 strains can be efficiently detected using a single pair of selected primers. For the remaining 1.5% of strains, a more careful selection of the second target is needed.