Anlan Dai

Combined effects of IL-12 and electroporation enhances the potency of DNA vaccination in macaques

DNA vaccines are a promising technology. Historically, however, the ability of DNA vaccines to induce high response rates and strong immune responses, especially antibody responses, in non-human primates and human clinical trials has proven suboptimal. Here, we performed a pilot study in rhesus macaques to evaluate whether we could improve the immunogenicity of DNA vaccines through the use of adjuvant technology and improved delivery systems. The study consisted of four groups of animals that received: DNA by intramuscular (IM) injection, DNA with plasmid-encoded IL-12 by IM injection, DNA by IM injection with in vivo electroporation (EP), and DNA with IL-12 by IM EP. Each group was immunized three times with optimized HIV gag and env constructs. Vaccine immunogenicity was assessed by IFNgamma ELISpot, CFSE proliferation, polyfunctional flow cytometry, and antibody ELISA. Similar to previous studies, use of IL-12 as an adjuvant increased the gag and env-specific cellular responses. The use of EP to enhance plasmid delivery resulted in dramatically higher cellular as well as humoral responses. Interestingly, the use of EP to administer the DNA and IL-12 adjuvant combination resulted in the induction of higher, more efficient responses such that a 10-fold increase in antigen-specific IFNgamma(+) cells compared to IM DNA immunization was observed after a single immunization. In addition to increases in the magnitude of IFNgamma production in the initial and memory responses, the combined approach resulted in enhancements in the proliferative capacity of antigen-specific CD8(+) T cells and the amount of polyfunctional cells capable of producing IL-2 and TNFalpha in addition to IFNgamma. These data suggest that adjuvant and improved delivery methods may be able to overcome previous immunogenicity limitations in DNA vaccine technology.

High antibody and cellular responses induced to HIV-1 clade C envelope following DNA vaccines delivered by electroporation

BACKGROUND Clade C is the predominant HIV-1 strain infecting people in sub-Saharan Africa, India, and China and there is a critical need for a vaccine targeted to these areas. In this study we tested a DNA based vaccine that encodes the SIVgag, SIVpol and HIV-1 envelope clade C. METHODS Rhesus macaques were immunized by electroporation with the DNA plasmid encoding optimized SIVgag, SIVpol and an HIV-1 env clade C with or without the adjuvant RANTES. Animals were monitored for immune responses and challenged following the final immunization with 25 animal infectious doses (AID) of SHIV-1157ipd3N4. RESULTS We found that the vaccine induced high levels of antigen specific IFN-γ producing effector cells and the capacity for CD4+ and CD8+ to proliferate upon antigen stimulation. Importantly, we found that the vaccine induced antibody titers as high as 1/4000. These antibodies were capable of neutralizing tier 1 HIV-1 viruses. Finally, when macaques were challenged with SHIV, viral loads were controlled in vaccinated groups. CONCLUSION We conclude that immunization with a simian/human immunodeficiency virus DNA-based vaccine delivered by electroporation can induce cellular and humoral immune responses that are able to control viral replication.

IL-15 as memory T-cell adjuvant for topical HIV-1 DermaVir vaccine

IL-7 and IL-15 are key cytokines involved in the generation and maintenance of memory CD8+ T-cells. We evaluated these cytokines as molecular adjuvants for topical HIV-1 DermaVir vaccine. We found that mice receiving DermaVir formulated with HIV-1 Gag plasmid in the presence of IL-7- or IL-15-encoding plasmid significantly enhanced Gag-specific central memory T-cells, as measured by a peptide-based cultured IFN-gamma ELISPOT. Additionally, IL-15 significantly improved DermaVir-induced Gag-specific effector memory CD8+ T-cell responses, measured by standard IFN-gamma ELISPOT. In a DermaVir prime/vaccinia vector boost regimen, the inclusion of IL-15 together with DermaVir significantly improved Gag-specific effector memory T-cell responses. Our study demonstrates that IL-15 is more potent than IL-7 in enhancing HIV-1-specific central memory T-cells induced by topical DermaVir. IL-15 adjuvanted DermaVir might be an alternative prime in a prophylactic vaccine regimen.

Long-Term Programming of Antigen-Specific Immunity from Gene Expression Signatures in the PBMC of Rhesus Macaques Immunized with an SIV DNA Vaccine

While HIV-1-specific cellular immunity is thought to be critical for the suppression of viral replication, the correlates of protection have not yet been determined. Rhesus macaques (RM) are an important animal model for the study and development of vaccines against HIV/AIDS. Our laboratory has helped to develop and study DNA-based vaccines in which recent technological advances, including genetic optimization and in vivo electroporation (EP), have helped to dramatically boost their immunogenicity. In this study, RMs were immunized with a DNA vaccine including individual plasmids encoding SIV gag, env, and pol alone, or in combination with a molecular adjuvant, plasmid DNA expressing the chemokine ligand 5 (RANTES), followed by EP. Along with standard immunological assays, flow-based activation analysis without ex vivo restimulation and high-throughput gene expression analysis was performed. Strong cellular immunity was induced by vaccination which was supported by all assays including PBMC microarray analysis that identified the up-regulation of 563 gene sequences including those involved in interferon signaling. Furthermore, 699 gene sequences were differentially regulated in these groups at peak viremia following SIVmac251 challenge. We observed that the RANTES-adjuvanted animals were significantly better at suppressing viral replication during chronic infection and exhibited a distinct pattern of gene expression which included immune cell-trafficking and cell cycle genes. Furthermore, a greater percentage of vaccine-induced central memory CD8+ T-cells capable of an activated phenotype were detected in these animals as measured by activation analysis. Thus, co-immunization with the RANTES molecular adjuvant followed by EP led to the generation of cellular immunity that was transcriptionally distinct and had a greater protective efficacy than its DNA alone counterpart. Furthermore, activation analysis and high-throughput gene expression data may provide better insight into mechanisms of viral control than may be observed using standard immunological assays.

Augmentation of SIV DNA vaccine-induced cellular immunity by targeting the 4-1BB costimulatory molecule.

DNA vaccines are effective at inducing antigen-specific cellular immune responses. Approaches to improve these responses, however, are needed. We examined the effect of stimulating 4-1BB, an activation-inducible T-cell costimulatory receptor, by intravenously co-administering anti-human 4-1BB monoclonal antibody (mAb) in DNA-immunized cynomolgus macaques. Three groups of six cynomolgus macaques were immunized intramuscularly with a DNA vaccine encoding SIV Gag antigen (pSIVgag) at weeks 0, 4 and 8. At days 12, 15, and 19, six macaques received anti-4-1BB 4E9 mAb and six macaques received anti-4-1BB 10C7 mAb. Treatment with 10C7 mAb led to a significant augmentation of SIV Gag-specific IFN-gamma, granzyme B and perforin responses. Treatment with humanized 4E9 mAb also resulted in an enhancement of SIV Gag-specific cellular responses but the magnitude was lower compared to animals receiving 10C7 mAb. These responses persisted up to week 40 and were mostly mediated by CD8(+) T cells. Treatment with anti-4-1BB mAb was more effective in driving the CD8(+) T cells toward a more differentiated CCR7(-)/CD45RA(+) effector state. This study demonstrates that targeting the 4-1BB molecule in vivo results in an enhanced and long-lasting cellular immune response. 4-1BB stimulation may be a promising approach to enhance the effectiveness of DNA vaccines.

High dose of plasmid IL-15 inhibits immune responses in an influenza non-human primates immunogenicity model

Interleukin (IL)-15, is a cytokine that is important for the maintenance of long-lasting, high-avidity T cell response to invading pathogens and has, therefore, been used in vaccine and therapeutic platforms as an adjuvant. In addition to pure protein delivery, plasmids encoding the IL-15 gene have been utilized. However, it is critical to determine the appropriate dose to maximize the adjuvanting effects. We immunized rhesus macaques with different doses of IL-15 expressing plasmid in an influenza non-human primate immunogenicity model. We found that co-immunization of rhesus macaques with a Flu DNA-based vaccine and low doses of plasmid encoding macaque IL-15 enhanced the production of IFN-gamma (0.5 mg) and the proliferation of CD4(+) and CD8(+) T cells, as well as T(CM) levels in proliferating CD8(+) T cells (0.25 mg). Whereas, high doses of IL-15 (4 mg) decrease the production of IFN-gamma and the proliferation of CD4(+) and CD8(+) T cells and T(CM) levels in the proliferating CD4(+) and CD8(+) T cells. In addition, the data of hemagglutination inhibition (HI) antibody titer suggest that although not significantly different, there appears to be a slight increase in antibodies at lower doses of IL-15. Importantly, however, the higher doses of IL-15 decrease the antibody levels significantly. This study demonstrates the importance of optimizing DNA-based cytokine adjuvants.

Independence of Granzyme B Secretion and Interferon-γ Production during Acute Simian Immunodeficiency Virus Infection

BACKGROUND Quantification of interferon (IFN)-gamma by enzyme-linked immunospot (ELISPOT) assay is currently used as a surrogate measurement of cytotoxic T lymphocyte (CTL) activity in nonhuman primates, particularly in simian immunodeficiency virus (SIV) models. Given that noncytotoxic cells and natural killer cells can also release IFN-gamma, quantification of granzyme B (GrB), a molecule secreted predominantly by activated CD8+ T cells, may represent an additional surrogate measurement of CTL activity. METHODS We evaluated, by ELISPOT assay, GrB activity in response to 3 overlapping SIV Gag peptide pools in 18 rhesus macaques with acute SIVmac251 infection and analyzed its correlation with IFN-gamma ELISPOT responses and plasma viral load. RESULTS SIV Gag-specific GrB activity increased from 3.9- to 14.4-fold after infection, compared with that observed before infection. GrB secretion did not correlate directly with IFN-gamma production. Importantly, SIV Gag-specific IFN- gamma production was negatively correlated with plasma viral load, whereas GrB activity was not. However, the peak of GrB activity coincided with the lowest plasma viral load detected after infection, whereas the magnitude of IFN-gamma production was 1.8-fold lower than the GrB response; these results illustrate that the responses differ. CONCLUSION Our data support the concept that the GrB and IFN-gamma ELISPOT assays measure immune responses in different immune-cell populations with unique specificities.

Sustained suppression of SHIV89.6P replication in macaques by vaccine-induced CD8+ memory T cells.

Objective:We previously demonstrated that a strategy of co-immunizing cynomologous macaques with a simian/human immunodeficiency virus DNA-based vaccine and a plasmid encoding macaque interleukin (IL)-15 induces a strong CD8+ and CD4+ effector T-cell response that, upon subsequent challenge with SHIV89.6P, controls viral replication and protects immunized animals against ongoing infection. In this follow-up study, we measured viral replication 2 years after vaccination challenge and determined the mechanism by which antigen-specific CD8+ T cells suppress viral replication. Method:From the original group of 18, we assessed the immune response in the 13 surviving animals. In addition, using cM-T807, we depleted CD8 lymphocytes to assess the role CD8 cells play in suppression of viral replication. Result:We found that peripheral blood mononuclear cells from vaccinated animals had a robust simian immunodeficiency virus Gag-specific IFN-γ response. In addition, in the DNA and IL-15 group, we observed higher levels of simian immunodeficiency virus Gag-specific, proliferating CD8+ T cells. The profile of these cells revealed more central memory than effector cells. When we transiently depleted animals of CD8+ T cells, plasma viral load increased, and peak viral load was lower in the DNA and IL-15 group compared with the DNA alone and control groups. As CD8+ T cells recovered, viral replication was controlled and we observed an increase in the number of antigen-specific effector CD8+ T cells. Conclusion:We conclude that co-immunization with a simian/human immunodeficiency virus DNA-based vaccine and IL-15 achieves sustained viral suppression and that vaccine-induced CD8+ memory T cells, which differentiate into effector cells, are central to that suppression.

CLTA-4 blockade in vivo promotes the generation of short-lived effector CD8+ T cells and a more persistent central memory CD4+ T cell response

Background  Previously, we examined the effects of in vivo CTLA‐4 blockade using a fully human monoclonal antibody as a part of a DNA vaccination regimen in cynomolgus macaques (Macaca fascicularis). We observed that while the antibody had little effect on the IFN‐γ ELISpot response, CTLA‐4 blockade enhanced antigen‐specific cellular proliferation in both CD4+ and CD8+T‐cell compartments.

IL-4 and IFN-γ induced by human immunodeficiency virus vaccine in a schistosome infection model

The co-infection of HIV and helminth parasites, such as Schistosoma spp, has increased in sub-Saharan Africa. Many HIV vaccine candidate studies have been completed or are in ongoing clinical trials, but it is not clear how HIV vaccines might affect the course of schistosome infections. In this study, we immunized S. mansoni-infected mice with an efficient DNA vaccine that included HIV gag. Using this model, we found that Th2 cytokines, such as IL-4 and IL-13, were highly induced after schistosome infection. Treatment of infected mice with the HIV DNA vaccine resulted in a significant attenuation of this rise in IL-13 expression and an increase in expression of the Th1 cytokine, TNF-α. However, vaccine administration did not significantly influence the expression of IL-4, or IFN-γ, and did not affect T cell proliferative capacity. Interestingly, the IL-4+IFN-γ+ phenotype appears in schistosome-infected mice that received HIV vaccination, and is associated with the expression of transcription factors GATA3+T-bet+ in these mice. These studies indicate that DNA vaccination can have an impact on ongoing chronic infection.