Modifying the HIV-1 env gp160 gene to improve pDNA vaccine-elicited cell-mediated immune responses

Abstract

Plasmid DNA (pDNA) vaccines are effective at eliciting immune responses in a wide variety of animal model systems, however, pDNA vaccines have generally been incapable of inducing robust immune responses in clinical trials. Therefore, to identify means to improve pDNA vaccine performance, we compared various post-transcriptional and post-translational genetic modifications for their ability to improve antigen-specific CMI responses. Mice vaccinated using a sub-optimal 100 mcg dose of a pDNA encoding an unmodified primary isolate HIV-1 6101 env gp160 failed to demonstrate measurable env-specific CMI responses. In contrast, significant envspecific CMI responses were seen in mice immunized with pDNA expression vectors encoding env genes modified by RNA optimization or codon optimization. Further modification of the RNA optimized env gp160 gene by the addition of (i) a simian retrovirus type 1 constitutive RNA transport element; (ii) a murine intracisternal A-particle derived RNA transport element; (iii) a tissue plasminogen activator protein signal leader sequences; (iv) a beta-catenin derived ubiquitination target sequence; or (v) a monocyte chemotactic protein-3 derived signal sequence failed to further improve the induction of env-specific CMI responses. Therefore, modification of the env gp160 gene by RNA or codon optimization alone is necessary for high-level revindependent expression and results in robust env-specific CMI responses in immunized mice. Importantly, further modification(s) of the env gene to alter cellular localization or increase proteolytic processing failed to result in increased env-specific immune responses. These results have important implications for the design and development of an efficacious vaccine for the prevention of HIV-1 infection.

Comparison of immune responses generated by optimized DNA vaccination against SIV antigens in mice and macaques

“…It is likely that the structure and intracellular trafficking of these hybrid Env proteins is altered by the N-terminal modifications, affecting the processing of the precursor gp160. The HIV-1 env 6101 (clade B) expression plasmids producing the native and modified forms were previously described [44]. Similarly, we found that upon fusion of the tPA signal peptide to HIVenv 6101 less gp120 was produced and that the MCP-3 env fusion showed very poor gp120 production (data not shown), in agreement with the SIV Env results (see above).…”

Reconstitution of the Ancestral Glycoprotein of Human Endogenous Retrovirus K and Modulation of Its Functional Activity by Truncation of the Cytoplasmic Domain

“…Codon optimization has previously been successfully used to boost the expression of several other retroviral proteins, including envelope glycoproteins (18,45). Full-length retroviral genomes require a balanced expression of their proteins for optimal replication, and their DNA, RNA, and protein sequences are under a multitude of constraints and selective pressures.…”

T-Cell Vaccine Strategies for Human Immunodeficiency Virus, the Virus with a Thousand Faces

“…The pathway for these immunogens begins with the design of the mosaic protein sequences, followed by the design of a "humanized" gene to optimize their expression (65), the synthesis and expression of the genes, and then the study of the properties of the proteins, including binding to relevant antibodies. After this is accomplished, the immunogenicity of the constructs is assessed with mice.…”