Co-Administration of Molecular Adjuvants Expressing NF-Kappa B Subunit p65/RelA or Type-1 Transactivator T-bet Enhance Antigen Specific DNA Vaccine-Induced Immunity

DNA vaccine-induced immunity can be enhanced by the co-delivery of synthetic gene-encoding molecular adjuvants. Many of these adjuvants have included cytokines, chemokines or co-stimulatory molecules that have been demonstrated to enhance vaccine-induced immunity by increasing the magnitude or type of immune responses and/or protective efficacy. In this way, through the use of adjuvants, immune responses can be highly customizable and functionally tailored for optimal efficacy against pathogen specific (i.e., infectious agent) or non-pathogen (i.e., cancer) antigens. In the novel study presented here, we examined the use of cellular transcription factors as molecular adjuvants. Specifically the co-delivery of (a) RelA, a subunit of the NF-κB transcription complex or (b) T-bet, a Th1-specific T box transcription factor, along with a prototypical DNA vaccine expressing HIV-1 proteins was evaluated. As well, all of the vaccines and adjuvants were administered to mice using in vivo electroporation (EP), a technology demonstrated to dramatically increase plasmid DNA transfection and subsequent transgene expression with concomitant enhancement of vaccine induced immune responses. As such, this study demonstrated that co-delivery of either adjuvant resulted in enhanced T and B cell responses, specifically characterized by increased T cell numbers, IFN-γ production, as well as enhanced antibody responses. This study demonstrates the use of cellular transcription factors as adjuvants for enhancing DNA vaccine-induced immunity

Nonstructural Protein 2 (nsP2) of Chikungunya Virus (CHIKV) Enhances Protective Immunity Mediated by a CHIKV Envelope Protein Expressing DNA Vaccine

Chikungunya virus (CHIKV) is an important emerging mosquito-borne alphavirus, indigenous to tropical Africa and Asia. It can cause epidemic fever and acute illness characterized by fever and arthralgias. The epidemic cycle of this infection is similar to dengue and urban yellow fever viral infections. The generation of an efficient vaccine against CHIKV is necessary to prevent and/or control the disease manifestations of the infection. In this report, we studied immune response against a CHIKV-envelope DNA vaccine (pEnv) and the role of the CHIKV nonstructural gene 2 (nsP2) as an adjuvant for the induction of protective immune responses in a relevant mouse challenge model. When injected with the CHIKV pEnv alone, 70% of the immunized mice survived CHIKV challenge, whereas when co-injected with pEnv+pnsP2, 90% of the mice survived viral challenge. Mice also exhibited a delayed onset signs of illness, and a marked decrease in morbidity, suggesting a nsP2 mediated adjuvant effect. Co-injection of the pnsP2 adjuvant with pEnv also qualitatively and quantitatively increased antigen specific neutralizing antibody responses compared to vaccination with pEnv alone. In sum, these novel data imply that the addition of nsP2 to the pEnv vaccine enhances anti-CHIKV-Env immune responses and maybe useful to include in future CHIKV clinical vaccination strategies

HIV-1 Env DNA vaccine plus protein boost delivered by EP expands B- and T-cell responses and neutralizing phenotype in vivo.

An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs), and the elicitation of antibody-dependent cellular cytotoxicity (ADCC). Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP). However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted

Detection of antibody secreting cells (ASCs).

<p>Groups of mice (<i>n = 4</i>) were immunized with the multi-clade constructs. (A) 96-well plates were coated with goat anti-mouse IgG in PBS and blocked overnight at 4°C. Approximate number of IgG producing B-cells was determined by ELISpot assay. (B) Representative plots of two individual experiments are shown; error bars represent standard deviation of at least three replicate wells. (C) Correlation between the binding antibody titers and SFU obtained by B-cell ELISpot assay.</p

Serum IgG ELISA responses in guinea pig immunized with HIV-1 envelope.

<p>(A) Timeline for the DNA prime-protein boost immunization study in guinea pigs. Serum samples from the immunized and control guinea pigs were obtained as indicated. (B&C) Anti-gp120 antibody-binding titers were determined by ELISA two weeks after the first protein boost (<i>n = 5</i>). Data are presented as the mean endpoint titers ± D.</p

Characterization of antisera directed against HIV-1 Env.

<p>Binding of mouse antisera from DNA prime-protein boosts with subtypes A, B C and D envelope DNA and subtype B proteins. ELISA plates were coated with recombinant gp120 (subtype B) envelope glycoproteins. (A&B) End-point anti-gp120 IgG titers obtained from mice (<i>n = 4</i>) immunized with different Env immunogens as indicated, data shown titers at day 35, one week after the second protein boost. (C) Correlation between the binding antibody titers and SFU obtained by T-cell ELISpot assay.</p

HIV-1 Env vaccines are potent inducers of cell-mediated immune response.

<p>The antigen-specific T-cell responses from a single plasmid (A) or combined plasmid (B) formulation were assessed by the IFN-γ ELIspot. Splenic T-cells were stimulated with BALB/c immunodominant Env peptide and IFN-γ spot forming cells were enumerated after overnight incubation. Results shown are the mean number of spot forming cells (SFC) ± SD for four animals/group with control SFC counts with background peptide subtracted.</p

IFN-γ and IL-2 production in response to HIV-1 Env antigen.

<p>Intracellular cytokine staining with flow cytometry analysis of IFN-γ - and IL-2 expressing, Env-specific CD8⁺/CD4⁺ splenic T cells stimulated with Env peptides. Mice were immunized with multi-clade Env constructs and protein boost groups, and splenocytes were collected and cultured. (A) Representative flow cytometry data for splenocytes harvested from mice immunized with multi-clade DNA followed protein boost and stimulated for five hours with the envelope peptide. (B&C) Bar graph showing the number of Env-specific IFN-γ and IL-2 expressing (B) CD4⁺ and (C) CD8⁺T-cell responses generated by <i>in vitro</i> stimulation as described in Materials & Methods. Results are the mean ± SD for 4 mice per group (<i>n = 4</i>). Data presented in this figure are from one experiment representative of two performed.</p