HIV-1 infection induces chronic oxidative stress. The resultant neurotoxicity has been associated with Tat protein. Here, we for the first time describe the induction of oxidative stress by another HIV-1 protein, reverse transcriptase (RT). Expression of HIV-1 RT in human embryonic kidney cells generated potent production of the reactive oxygen species (ROS), detected by the fluorescence-based probes. Quantitative RT-PCR demonstrated that expression of RT in HEK293 cells induced a 10- to 15-fold increased transcription of the phase II detoxifying enzymes human NAD(P)H:quinone oxidoreductase (Nqo1) and heme oxygenase 1 (HO-1), indicating the induction of oxidative stress response. The capacity to induce oxidative stress and stress response appeared to be an intrinsic property of a vast variety of RTs: enzymatically active and inactivated, bearing mutations of drug resistance, following different routes of processing and presentation, expressed from viral or synthetic expression-optimized genes. The total ROS production induced by RT genes of the viral origin was found to be lower than that induced by the synthetic/expression-optimized or chimeric RT genes. However, the viral RT genes induced higher levels of ROS production and higher levels of HO-1 mRNA than the synthetic genes per unit of protein in the expressing cell. The capacity of RT genes to induce the oxidative stress and stress response was then correlated with their immunogenic performance. For this, RT genes were administered into BALB/c mice by intradermal injections followed by electroporation. Splenocytes of immunized mice were stimulated with the RT-derived and control antigens and antigen-specific proliferation was assessed by IFN-γ/IL-2 Fluorospot. RT variants generating high total ROS levels induced significantly stronger IFN-γ responses than the variants inducing lower total ROS, while high levels of ROS normalized per unit of protein in expressing cell were associated with a weak IFN-γ response. Poor gene immunogenicity was also associated with a high (per unit of protein) transcription of antioxidant response element (ARE) dependent phase II detoxifying enzyme genes, specifically HO-1. Thus, we have revealed a direct link between the propensity of the microbial proteins to induce oxidative stress and their immunogenicity.

The efficacy of DNA vaccines is highly dependent on the methods used for their delivery and the choice of delivery sites/targets for gene injection, pointing at the necessity of a strict control over the gene delivery process. Here, we have investigated the effect of the injection site on gene expression and immunogenicity in BALB/c mice, using as a model a weak gene immunogen, DNA encoding firefly luciferase (Luc) delivered by superficial or deep injection with subsequent electroporation (EP). Immunization was assessed by monitoring the in vivo expression of luciferase by 2D- and 3D-bioluminescence imaging (BLI) and by the end-point immunoassays. Anti-Luc antibodies were assessed by ELISA, and T-cell response by IFN-γ and IL-2 FluoroSpot in which mouse splenocytes were stimulated with Luc or a peptide representing its immunodominant CD8+ T-cell epitope GFQSMYTFV. Monitoring of immunization by BLI identified EP parameters supporting the highest Luc gene uptake and expression. Superficial injection of Luc DNA followed by optimal EP led to a low level Luc expression in the mouse skin, and triggered a CD8+ T-cell response characterized by the peptide-specific secretion of IFN-γ and IL-2, but no specific antibodies. Intramuscular gene delivery resulted in a several-fold higher Luc expression and anti-Luc antibody, but induced low IL-2 and virtually no specific IFN-γ. Photon flux from the sites of Luc gene injection was inversely proportional to the immune response against GFQSMYTFV (p < 0.05). Thus, BLI permitted to control the accuracy of gene delivery and transfection with respect to the injection site as well as the parameters of electroporation. Further, it confirmed the critical role of the site of DNA administration for the type and magnitude of the vaccine-specific immune response. This argues for the use of luminescent reporters in the preclinical gene vaccine tests to monitor both gene delivery and the immune response development in live animals.

Optimization of DNA vaccine delivery improves the potency of the immune response and is crucial to clinical success. Here, we inquired how such optimization impacts the magnitude of the response, its specificity and type. BALB/c mice were DNA-immunized with two model immunogens, HIV-1 protease and reverse transcriptase by intramuscular or intradermal injections with electroporation. DNA immunogens were co-delivered with DNA encoding luciferase. Delivery and expression were monitored by in vivo bioluminescence imaging (BLI). The endpoint immune responses were assessed by IFN-γ/IL-2 FluoroSpot, multiparametric flow cytometry and antibody ELISA. Expression and immunogenicity were compared in relation to the delivery route. Regardless of the route, protease generated mainly IFN-γ, and reverse transcriptase, IL-2 and antibody response. BLI of mice immunized with protease- or reverse transcriptase/reporter plasmid mixtures, demonstrated significant loss of luminescence over time. The rate of decline of luminescence strongly correlated with the magnitude of immunogen-specific response, and depended on the immunogenicity profile and the immunization route. In vitro and in vivo BLI-based assays demonstrated that intradermal delivery strongly improved the immunogenicity of protease, and to a lesser extent, of reverse transcriptase. Immune response polarization and epitope hierarchy were not affected. Thus, by changing delivery/immunogen expression sites, it is possible to modulate the magnitude, but not the type or fine specificity of the induced immune response.

Recent guidelines recommend antiretroviral therapy (ART) to be administered as early as possible during HIV-1 infection. Few studies addressed the immunological benefit of commencing ART during the acute phase of infection. We used mass cytometry to characterize blood CD4 ⁺ T cells from HIV-1–infected patients who initiated ART during acute or chronic phase of infection. Using this method, we analyzed a large number of markers on millions of individual immune cells. The results revealed that CD4 ⁺ T cell clusters with high expression of CD27, CD28, CD127, and CD44, whose function involves T cell migration to inflamed tissues and survival, are more abundant in healthy controls and patients initiating ART during the acute phase; on the contrary, CD4 ⁺ T cell clusters in patients initiating ART during the chronic phase had reduced expression of these markers. The results are suggestive of a better preserved immune function in HIV-1–infected patients initiating ART during acute infection.

Direct-acting antivirals (DAAs) revolutionized treatment of hepatitis C virus (HCV) infection. Resistance-associated substitutions (RASs) present at the baseline impair response to DAA due to rapid selection of resistant HCV strains. NS5A is indispensable target of the current DAA treatment regimens. We evaluated prevalence of RASs in NS5A in DAA-naïve patients infected with HCV 1a (n = 19), 1b (n = 93), and 3a (n = 90) before systematic DAA application in the territory of the Russian Federation. Total proportion of strains carrying at least one RAS constituted 35.1% (71/202). In HCV 1a we detected only M28V (57.9%) attributed to a founder effect. Common RASs in HCV 1b were R30Q (7.5%), L31M (5.4%), P58S (4.4%), and Y93H (5.4%); in HCV 3a, A30S (31.0%), A30K (5.7%), S62L (8.9%), and Y93H (2.2%). Prevalence of RASs in NS5A of HCV 1b and 3a was similar to that worldwide, including countries practicing massive DAA application, i.e., it was not related to treatment. NS5A with and without RASs exhibited different co-variance networks, which could be attributed to the necessity to preserve viral fitness. Majority of RASs were localized in polymorphic regions subjected to immune pressure, with selected substitutions allowing immune escape. Altogether, this explains high prevalence of RAS in NS5A and low barrier for their appearance in DAA-inexperienced population.

We analyzed the whole transcriptome characteristics of blood CD4+ T naive (T-N) cells isolated from HIV-1 infected patients starting ART at acute (early ART = EA; n = 13) or chronic (late ART = LA; n = 11) phase of infection and controls (C; n = 15). RNA sequencing revealed 389 differentially expressed genes (DEGs) in EA and 810 in LA group in relation to controls. Comparison of the two groups of patients showed 183 DEGs. We focused on DEGs involved in apoptosis, inflammation and immune response. Clustering showed a poor separation of EA from C suggesting that these two groups present a similar transcriptomic profile of CD4+ T-N cells. The comparison of EA and LA patients resulted in a high cluster purity revealing that different biological dysfunctions characterize EA and LA patients. The upregulated expression of several inflammatory chemokine genes distinguished the patient groups from C; CCL2 and CCL7, however, were downregulated in EA compared to LA patients. BCL2, an anti-apoptotic factor pivotal for naive T cell homeostasis, distinguished both EA and LA from C. The expression of several DEGs involved in different inflammatory processes (TLR4, PTGS2, RAG1, IFNA16) was lower in EA compared LA. We conclude that although the transcriptome of CD4+ T-N cells isolated from patients initiating ART at acute infection reveals a more quiescent phenotype, the survival profile of these cells still appears to be affected. Our results show that the detrimental process of inflammation is under more efficient control in EA patients.