Induction of intrahepatic HCV NS4B, NS5A and NS5B-specific cellular immune responses following peripheral immunization.

Numerous studies have suggested that an effective Hepatitis C Virus (HCV) vaccine must induce strong cytotoxic and IFN-γ+ T cell responses targeting the non-structural region of the virus. Most importantly, these responses must be able to migrate into and remain functional within the liver, an organ known to cause T cell tolerance. Using three novel HCV DNA vaccines encoding non-structural proteins NS4B, NS5A and NS5B, we assessed the ability of peripheral immunization to induce functional intrahepatic immunity both in the presence and absence of cognate HCV antigen expression within the liver. We have shown that these constructs induced potent HCV-specific CD4+ and CD8+ T cell responses in the spleen of C57BL/6 mice and that these responses were detected within the liver following peripheral immunization. Additionally, using a transfection method to express HCV antigen within the liver, we showed that intrahepatic HCV-specific T cells remained highly functional within the liver and retained the ability to become highly activated as evidenced by upregulation of IFN-γ and clearance of HCV protein expressing hepatocytes. Taken together, these findings suggest that peripheral immunization can induce potent HCV-specific T cell responses able to traffic to and function within the tolerant environment of the liver.

Hepatitis C virus NS3/NS4A DNA vaccine induces multiepitope T cell responses in rhesus macaques mimicking human immune responses [corrected].

Numerous studies have suggested that an effective hepatitis C virus (HCV) vaccine must induce a strong T helper 1 (Th1) T cell response. While several therapeutic vaccine candidates have shown promise in clinical trials, response rates have been low suggesting that further optimization is important. However, such optimization has been hindered by a lack of a benchmark animal model in which to test vaccine-induced immune responses before clinical evaluation. The goal of this study was to analyze the utility of the rhesus macaque vaccination model in assessing HCV vaccine-induced T cell responses. To test this, we employed the use of a novel HCV genotype 1a/1b consensus DNA vaccine encoding both HCV nonstructural protein 3 (NS3) and nonstructural protein 4A (NS4A) proteins. Following immunization, rhesus macaques mounted HCV-specific responses strikingly similar to those reported in resolving patients, including strong NS3-specific interferon-γ (IFN-γ) responses, robust CD4(+) and CD8(+) T cell proliferation, and induction of polyfunctional T cells. Additionally, fine epitope mapping revealed one animal that mounted a T cell response against a known HCV NS3 human leukocyte antigen A2 (HLA-A2) epitope previously identified in humans. Taken together our findings suggest that the rhesus macaque vaccination model is a useful tool in the evaluation of immune responses induced by HCV immunogens.

Peripheral immunization induces functional intrahepatic hepatitis C specific immunity following selective retention of vaccine-specific CD8 T cells by the liver.

It is believed that an effective HCV vaccine must induce strong HCV-specific cytotoxic IFN-γ⁺ CD8⁺ T cells able to migrate into and become fully activated within the liver, an organ known to suppress T cell responses and induce tolerance. Given the importance of intrahepatic HCV-specific T cells in the clearance of acute infection, the goal of this present study was to determine if peripheral immunization was able to induce functional intrahepatic HCV-specific T cell based immunity both in the presence and absence of HCV antigen expression within the liver. Using a novel HCV NS3/NS4A DNA vaccine, we show that peripheral immunization of C57BL/6 mice results in the formation of a large pool of fully functional HCV-specific cytotoxic IFN-γ⁺ CD8⁺ T cells within the liver and that these cells were highly enriched within the liver as compared to the spleen. Following hepatic expression of cognate HCV antigen using a previously described liver transfection method, we show that this pool of vaccine-induced HCV-specific CD8⁺ T cells retained its ability to become highly activated as shown by the upregulation of IFN-γ and CCR5 expression, as well as by the clearance of HCV NS3 expressing hepatocytes. Taken together, these findings suggest that T cell effector function is preserved within the liver and that selective recruitment of antigen-specific T cells to the liver may play a previously unappreciated role in the process of immune surveillance, which may be exploited for future T cell based HCV vaccines.

Enhanced control of pathogenic Simian immunodeficiency virus SIVmac239 replication in macaques immunized with an interleukin-12 plasmid and a DNA prime-viral vector boost vaccine regimen.

DNA priming has previously been shown to elicit augmented immune responses when administered by electroporation (EP) or codelivered with a plasmid encoding interleukin-12 (pIL-12). We hypothesized that the efficacy of a DNA prime and recombinant adenovirus 5 boost vaccination regimen (DNA/rAd5) would be improved when incorporating these vaccination strategies into the DNA priming phase, as determined by pathogenic simian immunodeficiency virus SIVmac239 challenge outcome. The whole SIVmac239 proteome was delivered in 5 separate DNA plasmids (pDNA-SIV) by EP with or without pIL-12, followed by boosting 4 months later with corresponding rAd5-SIV vaccine vectors. Remarkably, after repeated low-dose SIVmac239 mucosal challenge, we demonstrate 2.6 and 4.4 log reductions of the median SIV peak and set point viral loads in rhesus macaques (RMs) that received pDNA-SIV by EP with pIL-12 compared to the median peak and set point viral loads in mock-immunized controls (P < 0.01). In 5 out of 6 infected RMs, strong suppression of viremia was observed, with intermittent "blips" in virus replication. In 2 RMs, we could not detect the presence of SIV RNA in tissue and lymph nodes, even after 13 viral challenges. RMs immunized without pIL-12 demonstrated a typical maximum of 1.5 log reduction in virus load. There was no significant difference in the overall magnitude of SIV-specific antibodies or CD8 T-cell responses between groups; however, pDNA delivery by EP with pIL-12 induced a greater magnitude of SIV-specific CD4 T cells that produced multiple cytokines. This vaccine strategy is relevant for existing vaccine candidates entering clinical evaluation, and this model may provide insights into control of retrovirus replication.