Fusion to Flaviviral Leader Peptide Targets HIV-1 Reverse Transcriptase for Secretion and Reduces Its Enzymatic Activity and Ability to Induce Oxidative Stress but Has No Major Effects on Its Immunogenic Performance in DNA-Immunized Mice

Abstract

Reverse transcriptase (RT) is a key enzyme in viral replication and susceptibility to ART and a crucial target of immunotherapy against drug-resistant HIV-1. RT induces oxidative stress which undermines the attempts to make it immunogenic. We hypothesized that artificial secretion may reduce the stress and make RT more immunogenic. Inactivated multidrug-resistant RT (RT1.14opt-in) was N-terminally fused to the signal providing secretion of NS1 protein of TBEV (Ld) generating optimized inactivated Ld-carrying enzyme RT1.14oil. Promotion of secretion prohibited proteasomal degradation increasing the half-life and content of RT1.14oil in cells and cell culture medium, drastically reduced the residual polymerase activity, and downmodulated oxidative stress. BALB/c mice were DNA-immunized with RT1.14opt-in or parental RT1.14oil by intradermal injections with electroporation. Fluorospot and ELISA tests revealed that RT1.14opt-in and RT1.14oil induced IFN-γ/IL-2, RT1.14opt-in induced granzyme B, and RT1.14oil induced perforin production. Perforin secretion correlated with coproduction of IFN-γ and IL-2 (R = 0,97). Both DNA immunogens induced strong anti-RT antibody response. Ld peptide was not immunogenic. Thus, Ld-driven secretion inferred little change to RT performance in DNA immunization. Positive outcome was the abrogation of polymerase activity increasing safety of RT-based DNA vaccines. Identification of the molecular determinants of low cellular immunogenicity of RT requires further studies.

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

“…S1 ). Effective inactivation was demonstrated in our in vitro studies 43 , 55 – 58 . Parental viral and newly generated synthetic RT genes were cloned into the eukaryotic expression vector pVax1 under the control of the immediate early CMV promotor.…”

“…Against this background, the introduction of the inactivating mutations gave little additional effect. Introduction of inactivating mutations gave no added value, supporting the notion that protein that is overexpressed in eukaryotic cells might lose its enzymatic activity due to aggregation 43 . Confirming this option, the aggregated protein was not secreted: the relative amount of the inactive RT1.14 in the cell lysate was 4–5-fold higher than in the secreted protein fraction (Supplementary Fig.…”

“…1a–d,g ). In previous work, while studying the expression of RT chimeras targeted for secretion, we detected RT in the cell culture fluids of RT-expressing cells 43 . Here, too, we found both drug-resistant and wild-type RTs in the lysates and cell culture fluids in approximately equal amounts (Supplementary Fig.…”

“…However, artificial secretion conferred only minor changes to RT immunogenic performance. The cellular immune response induced by the secreted RT variant was still weak 43 . As the single approach-oriented tactics had failed, we performed the systematic optimization of all parameters defining gene immunogenicity, including gene design and the process of immunization.…”

“…A significant improvement in immunogenic performance was achieved only in the case of RT retargeting to the lysosome 46 . We also tested whether RT can be made more immunogenic by expression optimization and artificial secretion, which we thought would help to overcome RT-induced oxidative stress, potentially toxic to the expressing cells 43 . However, artificial secretion conferred only minor changes to RT immunogenic performance.…”