Induction of Intrahepatic HCV NS4B, NS5A and NS5B-Specific Cellular Immune Responses following Peripheral Immunization

<div><p>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.</p> </div

Clearance of NS4B, NS5A or NS5B transfected hepatocytes.

<p><b>A</b>) Confocal microscopy. Representative confocal image of hepatocyte expression of NS4B, NS5A or NS5B in each group. Expression of each construct was detected with an anti-HA antibody (red). Nuclei were stained with DAPI (blue). <b>B, C</b> and <b>D</b>) Graph of MFI ratio of expression of <b>B</b>) NS4B, <b>C</b>) NS5A or <b>D</b>) NS5B as normalized to DAPI. For each group, three images were captured for each animal (n = 5). MFI values for NS4B, NS5A or NS5B (red) were calculated and normalized to the MFI value for DAPI (blue) for each image. The values shown are the averaged response ± SE of five individual animals from both the naïve and immunized groups. Significance was determined by Student's <i>t</i> test (*p<0.05, **p<0.005 and ***p<0.0005).</p

Flow cytometric analysis of the percentage of HCV-specific IFN-γ+ T cell responses from isolated liver lymphocytes.

<p>Values are reported as the average percent ± SE of HCV-specific <b>A</b>) CD4+ IFN-γ+ or <b>B</b>) CD8+ IFN-γ+ T cell responses of each animal (n = 5) from each group. Significance was determined by Student's <i>t</i> test (*p<0.05, **p<0.005 and ***p<0.0005).</p

Expression and immunogenicity of pConNS4B, pConNS5A and pConNS5B.

<p><b>A</b>) Human RD muscle cells were transiently transfected with each construct. Expression of each gene product was detected using an anti-HA monoclonal antibody and confocal microscopy (250×). <b>B</b>) IFN-γ ELISpot dose response. Animals (n = 5 per group) were immunized with 5 µg, 12.5 µg or 25 µg of pConNS4B, pConNS5A and pConNS5B. Animals received a total of two intramuscular immunizations, two weeks apart followed by electroporation. Animals were sacrificed one week following the last immunization after which splenocytes were isolated and analyzed. The response of each animal to each dose was determined with IFN-γ ELISpot assays. Splenocytes were isolated and individually analyzed for NS4B-, NS5A- or NS5B-specific T cell responses. <b>C, D, E</b>) Splenocytes were intracellularly stained for IFN-γ and analyzed with flow cytometry. <b>C</b>) Representative animal from each group. The values shown are the averaged response of five individual animals from each group. <b>D</b>) and <b>E</b>) graphical representation of percent HCV-specific IFN-γ+ T cell responses from isolated splenocytes. Values are reported as the average percent ± SE of the <b>D</b>) CD4+ IFN-γ+ or <b>E</b>) CD8+ IFN-γ+ T cell responses of each animal (n = 5) from each group. Significance was determined by Student's <i>t</i> test (*p<0.05, **p<0.005 and ***p<0.0005).</p

IL-28B/IFN-λ3 Drives Granzyme B Loading and Significantly Increases CTL Killing Activity in Macaques

Type III/λ interferons (IFNs) were discovered less than a decade ago and are still in the process of being characterized. Although previous studies have focused on the function of IFN-λ3 (also known as interleukin (IL)-28B) in a small animal model, it is unknown whether these functions would translate to a larger, more relevant model. Thus in the present study, we have used DNA vaccination as a method of studying the influence of IFN-λ3 on adaptive immune responses in rhesus macaques. Results of our study show for the first time that IFN-λ3 has significant influence on antigen-specific CD8+ T-cell function, especially in regards to cytotoxicity. Peripheral CD8+ T cells from animals that were administered IFN-λ3 showed substantially increased cytotoxic responses as gauged by CD107a and granzyme B coexpression as well as perforin release. Moreover, CD8+ T cells isolated from the mesenteric lymph nodes (MLN) of animals receiving IFN-λ3 loaded significant amounts of granzyme B upon extended antigenic stimulation and induced significantly more granzyme B-mediated cell death of peptide pulsed targets. These data suggest that IFN-λ3 is a potent effector of the immune system with special emphasis on CD8+ T-cell killing functions which warrants further study as a possible immunoadjuvant