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

Abstract

DNA vaccines require a considerable enhancement of immunogenicity. Here, we optimized a prototype DNA vaccine against drug-resistant HIV-1 based on a weak Th2-immunogen, HIV-1 reverse transcriptase (RT). We designed expression-optimized genes encoding inactivated wild-type and drug-resistant RTs (RT-DNAs) and introduced them into mice by intradermal injections followed by electroporation. RT-DNAs were administered as single or double primes with or without cyclic-di-GMP, or as a prime followed by boost with RT-DNA mixed with a luciferase-encoding plasmid (“surrogate challenge”). Repeated primes improved cellular responses and broadened epitope specificity. Addition of cyclic-di-GMP induced a transient increase in IFN-γ production. The strongest anti-RT immune response was achieved in a prime-boost protocol with electroporation by short 100V pulses done using penetrating electrodes. The RT-specific response, dominated by CD4+ T-cells, targeted epitopes at aa 199–220 and aa 528–543. Drug-resistance mutations disrupted the epitope at aa 205–220, while the CTL epitope at aa 202–210 was not affected. Overall, multiparametric optimization of RT strengthened its Th2- performance. A rapid loss of RT/luciferase-expressing cells in the surrogate challenge experiment revealed a lytic potential of anti-RT response. Such lytic CD4+ response would be beneficial for an HIV vaccine due to its comparative insensitivity to immune escape.

Expression of the Reverse Transcriptase Domain of Telomerase Reverse Transcriptase Induces Lytic Cellular Response in DNA-Immunized Mice and Limits Tumorigenic and Metastatic Potential of Murine Adenocarcinoma 4T1 Cells

“…We used a method to assess the integral immune response against TERT which we named “antigen challenge” [ 47 ]. Specifically, we primed mice with TERT DNA or TERT-HA DNA, and then boosted with the same plasmids mixed with DNA encoding firefly luciferase (Luc DNA) ( Figure 2 A).…”

“…We have earlier shown that immune response induced by DNA immunogen in prime eliminates cells coexpressing DNA immunogen and Luc after the boost with plasmid mixture. This results in a rapid loss of bioluminescence signal from the sites of booster injections in DNA-immunized mice compared to control mice [ 47 ]. Here, boosting of TERT or TERT-HA DNA-immunized mice with plasmids encoding TERT/TERT-HA and Luc resulted in a significant loss of bioluminescence signal in comparison to that in the control mice primed with pVAX1 and boosted with pVAX1 mixed with Luc DNA ( p < 0.05; Figure 2 B,C).…”

“…Here, we have developed and tested a new DNA vaccine targeting TERT based on the amino acid sequence of rat TERT, expected to overcome potential tolerance in preclinical trials in mice and potential clinical trials. Rat TERT and its variant with the C-terminal HA-tag encoded by synthetic expression-optimized genes, were used to immunize mice using an optimized protocol consisting of repeated intradermal injections followed by electroporation [ 47 ]. The effector potential of integral anti-TERT immune response was assessed in the “antigen challenge” experiment.…”

“…Plasmids were administered with 29G-needle insulin syringes. Injections were followed by electroporation using CUY21EditII (BEX Co., Tokyo, Japan) in vivo electroporator with fork-plate (CUY663-5 × 10) electrode (BEX Co., Tokyo, Japan) with a poration pulse of 400 V (0.1 ms with a 20 ms break) followed by 8 altering polarity (+/−) driving pulses of 10 ms performed at 100 V with 20 ms intervals [ 47 ].…”

“…Bioluminescence from the sites of injections of a mixture of DNA-immunogen or vector DNA and pVaxLuc2 was measured on days 1, 2, 5, 7, 9, and 12 after the boost by in vivo imaging (Spectrum; Perkin Elmer, Waltham, MA, USA) as described previously [ 47 , 48 ]. Prior to capturing of the luminescent signal, mice were injected intraperitoneally with a solution of XenoLight D-Luciferin potassium salt (Perkin Elmer) in PBS at a dose of 150 µg/g body weight.…”