Consensus HIV-1 FSU-A Integrase Gene Variants Electroporated into Mice Induce Polyfunctional Antigen-Specific CD4+ and CD8+ T Cells

Abstract

Our objective is to create gene immunogens targeted against drug-resistant HIV-1, focusing on HIV-1 enzymes as critical components in viral replication and drug resistance. Consensus-based gene vaccines are specifically fit for variable pathogens such as HIV-1 and have many advantages over viral genes and their expression-optimized variants. With this in mind, we designed the consensus integrase (IN) of the HIV-1 clade A strain predominant in the territory of the former Soviet Union and its inactivated derivative with and without mutations conferring resistance to elvitegravir. Humanized IN gene was synthesized; and inactivated derivatives (with 64D in the active site mutated to V) with and without elvitegravir-resistance mutations were generated by site-mutagenesis. Activity tests of IN variants expressed in E coli showed the consensus IN to be active, while both D64V-variants were devoid of specific activities. IN genes cloned in the DNA-immunization vector pVax1 (pVaxIN plasmids) were highly expressed in human and murine cell lines (>0.7 ng/cell). Injection of BALB/c mice with pVaxIN plasmids followed by electroporation generated potent IFN-γ and IL-2 responses registered in PBMC by day 15 and in splenocytes by day 23 after immunization. Multiparametric FACS demonstrated that CD8+ and CD4+ T cells of gene-immunized mice stimulated with IN-derived peptides secreted IFN-γ, IL-2, and TNF-α. The multi-cytokine responses of CD8+ and CD4+ T-cells correlated with the loss of in vivo activity of the luciferase reporter gene co-delivered with pVaxIN plasmids. This indicated the capacity of IN-specific CD4+ and CD8+ T-cells to clear IN/reporter co-expressing cells from the injection sites. Thus, the synthetic HIV-1 clade A integrase genes acted as potent immunogens generating polyfunctional Th1-type CD4+ and CD8+ T cells. Generation of such response is highly desirable for an effective HIV-1 vaccine as it offers a possibility to attack virus-infected cells via both MHC class I and II pathways.

Influence of Drug Resistance Mutations on the Activity of HIV-1 Subtypes A and B Integrases: a Comparative Study

“…The reduced integration activity of the subtype B mutant IN B G118R had been explained by the reduced ability of the complex of this mutant with its DNA substrate to bind the DNA target [ 17 ]. As a result of natural polymorphism, INB contains Ser at position 119 and IN A contains Pro [ 21 ]. It should be noted that Ser119 is likewise present in drug-resistant strains of HIV-1 subtype C, which most often contain the G118R mutation [ 16 , 18 ].…”

“…In particular, information on resistance mutations caused by IN inhibitors in HIV-1 strain FSU-A is limited. To assess the impact of drug resistance mutations on the enzymatic properties of IN of HIV-1 subtype A, we prepared a consensus IN of the FSU-A strain, where RAL- and EVG-resistance mutations were introduced by site-directed mutagenesis [ 21 , 22 ]. The consensus IN sequence of HIV-1 strain FSU-A (INA) differs from the sequence of the best studied IN of HIV-1 subtype B (HXB-2) by substitutions of 16 amino acid residues, nine of which are located in the catalytic domain.…”

Codon optimization and improved delivery/immunization regimen enhance the immune response against wild-type and drug-resistant HIV-1 reverse transcriptase, preserving its Th2-polarity

“…(PLoS One [PONE-D-17-40839] [EMID: 2890cecde7221c73], accepted). Correlation of the loss of photon flux with the anti-Luc response was insignificant (data not shown) due to the low immunogenicity of Luc 31 , 41 , 63 . These observations established in vivo imaging as an animal-sparing method to evaluate the overall performance of DNA immunogens 31 , 41 , 63 .…”

“…Based on the prime-boost regimen, we designed experiments to evaluate the effector capacity of the immune response. We have repeatedly shown in previous studies that the cellular immune response can be indirectly monitored in vivo by introducing the DNA immunogen together with a reporter gene, such as firefly luciferase (Luc), with further monitoring of the loss of signal emitted by the reporter 31 , 41 , 63 . We found that the photon flux from the sites of immunogen/reporter gene co-injections is inversely correlated to the cellular immune response against the immunogen 31 , 41 , 63 and Petkov S. et al .…”

A multi-head intradermal electroporation device allows for tailored and increased dose DNA vaccine delivery to the skin

“…Multiple examples of published literature demonstrate the ability of the ID-EP platform to elicit robust immune responses in a spectrum of animal models 30,[34][35][36][37][38][39] and in the clinic. 28 The use of combination vaccine strategies has a number of clinical benefits.…”