Margherita Rosati

Effect of Plasmid DNA Vaccine Design and In Vivo Electroporation on the Resulting Vaccine-Specific Immune Responses in Rhesus Macaques

ABSTRACT Since human immunodeficiency virus (HIV)-specific cell-mediated immune (CMI) responses are critical in the early control and resolution of HIV infection and correlate with postchallenge outcomes in rhesus macaque challenge experiments, we sought to identify a plasmid DNA (pDNA) vaccine design capable of eliciting robust and balanced CMI responses to multiple HIV type 1 (HIV-1)-derived antigens for further development. Previously, a number of two-, three-, and four-vector pDNA vaccine designs were identified as capable of eliciting HIV-1 antigen-specific CMI responses in mice (M. A. Egan et al., Vaccine 24:4510-4523, 2006). We then sought to further characterize the relative immunogenicities of these two-, three-, and four-vector pDNA vaccine designs in nonhuman primates and to determine the extent to which in vivo electroporation (EP) could improve the resulting immune responses. The results indicated that a two-vector pDNA vaccine design elicited the most robust and balanced CMI response. In addition, vaccination in combination with in vivo EP led to a more rapid onset and enhanced vaccine-specific immune responses. In macaques immunized in combination with in vivo EP, we observed a 10- to 40-fold increase in HIV-specific enzyme-linked immunospot assay responses compared to those for macaques receiving a 5-fold higher dose of vaccine without in vivo EP. This increase in CMI responses translates to an apparent 50- to 200-fold increase in pDNA vaccine potency. Importantly, in vivo EP enhanced the immune response against the less immunogenic antigens, resulting in a more balanced immune response. In addition, in vivo EP resulted in an approximate 2.5-log10 increase in antibody responses. The results further indicated that in vivo EP was associated with a significant reduction in pDNA persistence and did not result in an increase in pDNA associated with high-molecular-weight DNA relative to macaques receiving the pDNA without EP. Collectively, these results have important implications for the design and development of an efficacious vaccine for the prevention of HIV-1 infection.

Persistent HIV-1 infection of natural killer cells in patients receiving highly active antiretroviral therapy.

We have identified a subset of CD56+CD3− human natural killer (NK) cells that express CD4 and the HIV coreceptors CCR5 and CXCR4. These cells can be productively infected in vitro by both CCR5- and CXCR4-using molecular clones of HIV-1 in a CD4-dependent manner. Analysis of HIV-infected persons showed that viral DNA is present in purified NK cells, and virus could be rescued from these cells after in vitro cultivation. Longitudinal analysis of the HIV-1 DNA levels in NK cells from patients after 1–2 years of highly active antiretroviral therapy indicated that NK cells remain persistently infected and account for a substantial amount of the viral DNA in peripheral blood mononuclear cells. These results demonstrate that a subset of non-T cells with NK markers are persistently infected and suggest that HIV infection of NK cells is important for virus persistence. The properties of the virus reservoir in these cells should be considered in attempts to further optimize antiretroviral therapies.

Intracellular Interaction of Interleukin-15 with Its Receptor α during Production Leads to Mutual Stabilization and Increased Bioactivity

We show that co-expression of interleukin 15 (IL-15) and IL-15 receptor α (IL-15Rα) in the same cell allows for the intracellular interaction of the two proteins early after translation, resulting in increased stability and secretion of both molecules as a complex. In the absence of co-expressed IL-15Rα, a large portion of the produced IL-15 is rapidly degraded immediately after synthesis. Co-injection into mice of IL-15 and IL-15Rα expression plasmids led to significantly increased levels of the cytokine in serum as well as increased biological activity of IL-15. Examination of natural killer cells and T lymphocytes in mouse organs showed a great expansion of both cell types in the lung, liver, and spleen. The presence of IL-15Rα also increased the number of CD44high memory cells with effector phenotype (CD44highCD62L-). Thus, mutual stabilization of IL-15 and IL-15Rα leads to remarkable increases in production, stability, and tissue availability of bioactive IL-15 in vivo. The in vivo data show that the most potent form of IL-15 is as part of a complex with its receptor α either on the surface of the producing cells or as a soluble extracellular complex. These results explain the reason for coordinate expression of IL-15 and IL-15Rα in the same cell and suggest that the IL-15Rα is part of the active IL-15 cytokine rather than part of the receptor.

DNA Vaccines Expressing Different Forms of Simian Immunodeficiency Virus Antigens Decrease Viremia upon SIVmac251 Challenge

ABSTRACT We have tested the efficacy of DNA immunization as a single vaccination modality for rhesus macaques followed by highly pathogenic SIVmac251 challenge. To further improve immunogenicity of the native proteins, we generated expression vectors producing fusion of the proteins Gag and Env to the secreted chemokine MCP3, targeting the viral proteins to the secretory pathway and to a β-catenin (CATE) peptide, targeting the viral proteins to the intracellular degradation pathway. Macaques immunized with vectors expressing the MCP3-tagged fusion proteins developed stronger antibody responses. Following mucosal challenge with pathogenic SIVmac251, the vaccinated animals showed a statistically significant decrease in viral load (P = 0.010). Interestingly, macaques immunized with a combination of vectors expressing three forms of antigens (native protein and MCP3 and CATE fusion proteins) showed the strongest decrease in viral load (P = 0.0059). Postchallenge enzyme-linked immunospot values for Gag and Env as well as gag-specific T-helper responses correlated with control of viremia. Our data show that the combinations of DNA vaccines producing native and modified forms of antigens elicit more balanced immune responses able to significantly reduce viremia for a long period (8 months) following pathogenic challenge with SIVmac251.

Circulating IL-15 exists as heterodimeric complex with soluble IL-15Rα in human and mouse serum

IL-15 is an important cytokine for the function of the immune system, but the form(s) of IL-15 produced in the human body are not fully characterized. Coexpression of the single-chain IL-15 and the IL-15 receptor alpha (IL-15Rα) in the same cell allows for efficient production, surface display, and eventual cleavage and secretion of the bioactive IL-15/IL-15Rα heterodimer in vivo, whereas the single-chain IL-15 is poorly secreted and unstable. This observation led to the hypothesis that IL-15 is produced and secreted only as a heterodimer with IL-15Rα. We purified human IL-15/IL-15Rα complexes from overproducing human cell lines and developed an ELISA specifically measuring the heterodimeric form of IL-15. Analysis of sera from melanoma patients after lymphodepletion revealed the presence of circulating IL-15/IL-15Rα complexes in amounts similar to the total IL-15 quantified by a commercial IL-15 ELISA that detects both the single-chain and the heterodimeric forms of the cytokine. Therefore, in lymphodepleted cancer patients, the serum IL-15 is exclusively present in its heterodimeric form. Analysis of the form of IL-15 present in either normal or lymphodepleted mice agrees with the human data. These results have important implications for development of assays and materials for clinical applications of IL-15.

Increased immune responses in rhesus macaques by DNA vaccination combined with electroporation

We used optimized DNA expression vectors to compare two gene delivery methodologies in rhesus macaques, namely direct DNA injection and in vivo adaptive constant-current electroporation via the intramuscular route. The use of in vivo electroporation increased levels of gene expression and immune responses. We used an optimized HIV gag expression plasmid to show the development of new cellular immune responses in SIV-infected animals controlling viremia. Furthermore, after vaccination with SIV expression plasmids the recall responses to the SIV antigens were very high, indicating that DNA is a strong boost in the presence of antiretroviral treatment in SIV-infected animals. There was substantial animal-to-animal variability in DNA expression, revealed by plasma measurements of IL-15 produced by co-injected IL-15 DNA. IL-15 expression levels correlated with peak immune responses. Electroporation led to an expansion of antigen-specific CD4+ and CD8+ T cells of both central and effector memory phenotype. These results indicate that improved gene delivery and expression by electroporation dramatically increases immunogenicity of DNA vaccines. Electroporation is thus an important method to improve the effectiveness of DNA vaccination.

DNA vaccination in rhesus macaques induces potent immune responses and decreases acute and chronic viremia after SIVmac251 challenge

Optimized plasmid DNAs encoding the majority of SIVmac239 proteins and delivered by electroporation (EP) elicited strong immune responses in rhesus macaques. Vaccination decreased viremia in both the acute and chronic phases of infection after challenge with pathogenic SIVmac251. Two groups of macaques were vaccinated with DNA plasmids producing different antigen forms, “native” and “modified,” inducing distinct immune responses. Both groups showed significantly lower viremia during the acute phase of infection, whereas the group immunized with the native antigens showed better protection during the chronic phase (1.7 log decrease in virus load, P = 0.009). Both groups developed strong cellular and humoral responses against the DNA vaccine antigens, which included Gag, Pol, Env, Nef, and Tat. Vaccination induced both central memory and effector memory T cells that were maintained at the day of challenge, suggesting the potential for rapid mobilization upon virus challenge. The group receiving the native antigens developed higher and more durable anti-Env antibodies, including neutralizing antibodies at the day of challenge. These results demonstrate that DNA vaccination in the absence of any heterologous boost can provide protection from high viremia comparable to any other vaccine modalities tested in this macaque model.

Comparative ability of various plasmid-based cytokines and chemokines to adjuvant the activity of HIV plasmid DNA vaccines.

The effectiveness of plasmid DNA (pDNA) vaccines can be improved by the co-delivery of plasmid-encoded molecular adjuvants. We evaluated pDNAs encoding GM-CSF, Flt-3L, IL-12 alone, or in combination, for their relative ability to serve as adjuvants to augment humoral and cell-mediated immune responses elicited by prototype pDNA vaccines. In Balb/c mice we found that co-administration of plasmid-based murine GM-CSF (pmGM-CSF), murine Flt-3L (pmFlt-3L) or murine IL-12 (pmIL-12) could markedly enhance the cell-mediated immune response elicited by an HIV-1 env pDNA vaccine. Plasmid mGM-CSF also augmented the immune response elicited by DNA vaccines expressing HIV-1 Gag and Nef-Tat-Vif. In addition, the use of pmGM-CSF as a vaccine adjuvant appeared to markedly increase antigen-specific proliferative responses and improved the quality of the resulting T-cell response by increasing the percentage of polyfunctional memory CD8(+) T cells. Co-delivery of pmFlt-3L with pmGM-CSF did not result in a further increase in adjuvant activity. However, the co-administration of pmGM-CSF with pmIL-12 did significantly enhance env-specific proliferative responses and vaccine efficacy in the murine vaccinia virus challenge model relative to mice immunized with the env pDNA vaccine adjuvanted with either pmGM-CSF or pmIL-12 alone. These data support the testing of pmGM-CSF and pmIL-12, used alone or in combination, as plasmid DNA vaccine adjuvants in future macaque challenge studies.

Long-lasting humoral and cellular immune responses and mucosal dissemination after intramuscular DNA immunization.

Naïve Indian rhesus macaques were immunized with a mixture of optimized plasmid DNAs expressing several SIV antigens using in vivo electroporation via the intramuscular route. The animals were monitored for the development of SIV-specific systemic (blood) and mucosal (bronchoalveolar lavage) cellular and humoral immune responses. The immune responses were of great magnitude, broad (Gag, Pol, Nef, Tat and Vif), long-lasting (up to 90 weeks post third vaccination) and were boosted with each subsequent immunization, even after an extended 90-week rest period. The SIV-specific cellular immune responses were consistently more abundant in bronchoalveolar lavage (BAL) than in blood, and were characterized as predominantly effector memory CD4(+) and CD8(+) T cells in BAL and as both central and effector memory T cells in blood. SIV-specific T cells containing Granzyme B were readily detected in both blood and BAL, suggesting the presence of effector cells with cytolytic potential. DNA vaccination also elicited long-lasting systemic and mucosal humoral immune responses, including the induction of Gag-specific IgA. The combination of optimized DNA vectors and improved intramuscular delivery by in vivo electroporation has the potential to elicit both cellular and humoral responses and dissemination to the periphery, and thus to improve DNA immunization efficacy.

DNA and virus particle vaccination protects against acquisition and confers control of viremia upon heterologous simian immunodeficiency virus challenge

We have previously shown that macaques vaccinated with DNA vectors expressing SIVmac239 antigens developed potent immune responses able to reduce viremia upon high-dose SIVmac251 challenge. To further improve vaccine-induced immunity and protection, we combined the SIVmac239 DNA vaccine with protein immunization using inactivated SIVmac239 viral particles as protein source. Twenty-six weeks after the last vaccination, the animals were challenged intrarectally at weekly intervals with a titrated dose of the heterologous SIVsmE660. Two of DNA-protein coimmunized macaques did not become infected after 14 challenges, but all controls were infected by 11 challenges. Vaccinated macaques showed modest protection from SIVsmE660 acquisition compared with naïve controls (P = 0.050; stratified for TRIM5α genotype). Vaccinees had significantly lower peak (1.6 log, P = 0.0048) and chronic phase viremia (P = 0.044), with 73% of the vaccinees suppressing viral replication to levels below assay detection during the 40-wk follow-up. Vaccine-induced immune responses associated significantly with virus control: binding antibody titers and the presence of rectal IgG to SIVsmE660 Env correlated with delayed SIVsmE660 acquisition; SIV-specific cytotoxic T cells, prechallenge CD4+ effector memory, and postchallenge CD8+ transitional memory cells correlated with control of viremia. Thus, SIVmac239 DNA and protein-based vaccine protocols were able to achieve high, persistent, broad, and effective cellular and humoral immune responses able to delay heterologous SIVsmE660 infection and to provide long-term control of viremia. These studies support a role of DNA and protein-based vaccines for development of an efficacious HIV/AIDS vaccine.