The authors have read the journal's policy and have the following conflicts: The authors note possible commercial conflicts associated with this work, which may include; Wyeth, Inovio, BMS, Virxsys, Ichor, Merck, VGXi & Aldeveron. Additionally, there is a patent pending on the vaccines described in the manuscript.
Conceived and designed the experiments: KLK. Performed the experiments: KLK RT AAG DJS. Analyzed the data: KLK. Contributed reagents/materials/analysis tools: DBW. Wrote the paper: KLK.
Current address: Division of Cancer Prevention, Cancer Prevention Fellowship Program, National Cancer Institute, Bethesda, Maryland, United States of America
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.
Perhaps the greatest challenge in vaccine development for Hepatitis C Virus is that unlike other hepatitis viruses, such as Hepatitis A and Hepatitis B, where successful antibody-based vaccines have been created, protection against HCV infection does not appear to be antibody mediated
While it is known that strong intrahepatic HCV-specific T cell responses are correlated with clearance of acute infection, most HCV infected individuals fail to either mount or sustain these responses resulting in the progression to chronic infection
Therefore, an effective T cell based HCV vaccine must be able to induce functional HCV-specific T cell responses within the immune modulatory environment of the liver. Previous studies from our laboratory and others have shown that vaccines targeting the NS3 region of the virus are able to induce functional T cell responses in the liver
The animals used in this study were maintained in accordance with the National Institutes of Health and the University of Pennsylvania Institutional Care and Use Committee (IACUC) guidelines. The protocol for this study was approved by the University of Pennsylvania's Animal Care and Use Committee (Permit Number: 801244).
The consensus sequences for NS4B and NS5B were generated from 174 different genotype 1a sequences and the consensus sequence for NS5B was generated from 259 genotype 1b sequences obtained from Los Alamos National Laboratory HCV Sequence Database (
An N-term IgE leader sequence and a C-term HA tag were added to each construct. The sequences were codon and RNA optimized using GeneOptimizer™ (GENEART, Germany). The final consensus sequences were synthesized, sequence verified and inserted in to the clinical expression vector pVAX (Invitrogen) by GENEART (Germany). The final constructs were named pConNS4B, pConNS5A and pConNS5B. The final DNA and protein sequences of pConNS4B, pConNS5A and pConNS5B can be found in
RD muscle cells were transfected with pConNS4B, pConNS5A and pConNS5B using Lipofectamine™ (Invitrogen) according to the manufacturer's guidelines for 48 hours. Expression of each protein was detected with an anti-HA polyclonal rabbit antibody (Invitrogen) followed by a Cy3 conjugated goat anti-rabbit secondary antibody (Invitrogen).
Six to eight week old female C57BL/6 mice were purchased from Jackson Laboratories and were maintained in accordance with the National Institutes of Health and the University of Pennsylvania Institutional Care and Use Committee (IACUC) guidelines. The protocol for this study was approved by the University of Pennsylvania's Animal Care and Use Committee (Permit Number: 801244).
Five mice were used per group. Each mouse received a total of two immunizations, two weeks apart and was sacrificed one week following the second immunization. Immunizations were given intramuscularly, followed by electroporation with the CELLECTRA® adaptive constant current electroporation device and electrode arrays. The final dosing for the constructs was as follows; pConNS4B, 12.5 µg; pConNS5A, 5 µg and pConNS5B, 12.5 µg.
Spleens were isolated and individually crushed with the use of a Stomacher device. Splenocytes were strained with a 40 µM cell strainer and treated 5 min with ACK lysis buffer (Biosource) to lyse RBCs. The splenocytes were resuspended in complete media (RPMI 1640 with 2 mM/L L-glutamine supplemented with 10% heat inactivated FBS, 1× anti-biotic/anti-mycotic, and 55 µM/L β-mercaptoethanol).
Livers were individually pulverized using a Stomacher machine, strained and treated 5 min with 10 ml ACK lysis buffer (Bioscience). The mixture was pelleted and the hepatocytes were separated from the lymphocytes through the use of a 35% percoll (GE Healthcare) gradient. The pelleted lymphocytes were resuspended in complete media. Experiments were performed with and without liver perfusion and no differences were observed.
The mouse IFN-γ ELISpot assays were conducted as previously described
The following directly conjugated antibodies were used: anti-mouse CD3- allophycocyanin cyanine dye 7 (APC-Cy7) [clone 145-C11], anti-mouse CD4- fluorescein isothiocyanate (FITC) [clone H129.19], anti-mouse CD8- peridinin chlorophyll protein 5.5 (PerCP5.5) [clone 53-6.7], anti-mouse IFN-γ- phycoerythryin cyanine dye 7 (PE-Cy7) [clone XMG1.2] (all from BD Biosciences, San Jose, CA). Aqua Live/Dead fixable dead cell Stain Kit (Molecular Probes, Eugene, OR) was used according to manufacturer's protocol to identify live cells.
Samples were collected on a LSRII flow cytometer (BD Biosciences, Franklin Lakes, NJ). Data was analyzed using FlowJo software, version 8.7.1 for Mac, (Tree Star, Ashland, OR).
Splenocytes were resuspended in complete media at a concentration of 1×106 cells/100µl and plated in a round bottom 96-well plate. Splenocytes were stimulated with 100 µl of: 1) 2 µg/ml pConNS4B, pConNS5A or pConNS5B overlapping peptides, 2) 1 µg/ml Staphylococcus enterotoxin B (positive control; Sigma-Aldrich, St. Louis, MO) or 3) 0.1% dimethyl sulfoxide (negative control) all diluted in complete media supplemented with GolgiStop and GolgiPlug (BD Bioscience). Splenocytes were stimulated for 5 hours at 37°C, washed with PBS and stained for viability. Splenocytes were stained extracellularly for; anti- CD4, CD8 for 30 min at 4°C. Splenocytes were permeabilized and washed using BD Cytofix/Cytoperm Solution Kit (BD Bioscience) and stained intracellularly with anti- IFN-γ and CD3 for 45 min at 4°C. Splenocytes were stored at 4°C until analysis. Specific function was reported as the percent function of the peptide stimulated group minus the percent function of the 0.1% dimethyl sulfoxide stimulated group (negative control).
Liver biopsies were fixed in 2% paraformaldehyde followed by overnight cryoprotection in 30% sucrose. Biopsies were quick frozen in Tissue-Tek OCT (Bayer Corporation, Pittsburgh, PA). Tissue sections (6 µm) were mounted on Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA) and kept at 80°C until use. For staining slides were brought to room temperature (RT), washed (three 10 min washes in PBS), and blocked in PBS containing 10% serum and 0.1% Triton. Sections were incubated for 1 hour at RT or overnight at 4°C in primary reagents, washed and secondary reagents were applied for 30 min at RT. The sections were counterstained with DAPI (Invitrogen, Carlsbad, CA). Coverslips were mounted with Prolong Gold mounting media (Invitrogen, Carlsbad, CA). Monoclonal antibodies used were mouse anti-HA.11 (clone 16B12; Covance, Emeryville, CA). All images were obtained using a Zeiss Axiovert 100 inverted confocal microscope and Image J software (NIH, Rockville, MD) was used for analysis. For each group, three random images were captured for each animal (n = 5) by a microscopist blinded to the vaccine status of the animals. MFI values for NS4B, NS5A or NS5B (red) were calculated and normalized to the MFI value for DAPI (blue) for each image.
Animals were tail vein injected with 100 µg of pConNS4B, pConNS5A or pConNS5B diluted in 300 µl of PBS. Animals were sacrificed 48 hours following injection. This method for inducing expression of foreign antigen within the liver has been previously described
Due to its ability to rapidly mutate, HCV is highly variable in sequence and is currently classified into 6 different genotypes with more than 50 subtypes. Genotype 1 is the most prevalent in North America and Europe and is by far the most difficult to treat. It is estimated that genotype 1 accounts for 73 percent of all HCV infections in the US, with genotype 1a and 1b accounting for up to 51.6 and 26.5 percent of all cases, respectively
Once expression was confirmed, the cellular immunogenicity of the constructs was determined. Animals were separated into three dosing groups for each construct with five animals per group. The animals were immunized intramuscularly with 5 µg, 12.5 µg or 25 µg of pConNS4B, pConNS5A or pConNS5B, followed by electroporation. The animals received two immunizations, two weeks apart and were sacrificed one week following the second immunization. Immunogenicity was determined by IFN-γ ELISpot assays, the results of which can be seen in
Once the dosing was determined, a more detailed analysis of the cellular immune responses was performed. Dosing for the constructs in all subsequent experiments was as follows; pConNS4B, 12.5 µg; pConNS5A, 5 µg and pConNS5B, 12.5 µg. In order to determine the relative contributions of CD8+ and CD4+ T cell responses for each construct, splenocytes were intracellularly stained for IFN-γ and visualized with flow cytometry,
Given the importance of intrahepatic T cell responses in the clearance of HCV infection, once we had determined that our constructs were able to induce strong T cell responses in the periphery, we wanted to determine whether these responses could be detected within the liver. Mice were immunized as previously described. Liver lymphocytes were isolated from each animal and HCV-specific T cells were identified by IFN-γ expression and flow cytometry. Interestingly, HCV-specific T cells were identified in the livers of all immunized mice,
Values are reported as the average percent ± SE of HCV-specific
Next, we sought to determine whether liver-specific expression of either NS4B, NS5A or NS5B proteins could activate the HCV-specific T cells detected within the liver. In order to induce liver expression of NS4B, NS5A and NS5B, the hepatocytes of immunized mice were transfected by administering a tail vein injection of pConNS4B, pConNS5A or pConNS5B. This method for inducing expression of foreign antigen within the liver has been previously described
Lymphocytes from each animal (n = 5) were isolated and individually analyzed for NS4B-, NS5A- or NS5B-specific T cell responses. The isolated lymphocytes were intracellularly stained for IFN-γ and analyzed with flow cytometry.
Next, we determined whether this response was able to clear HCV protein expressing hepatocytes. To test this, a lobe of liver from each animal was stained for expression of NS4B, NS5A or NS5B. Clearance was assessed by measuring the expression of NS4B, NS5A or NS5B in hepatocytes of immunized mice versus naïve controls. Representative confocal images for each group are shown in
Numerous studies support the idea that a successful HCV vaccine must be able to induce strong HCV-specific T cell responses able to target the non-structural region and that these cells must be able to function within the liver
However, the most important question was whether peripheral immunization with pConNS4B, pConNS5A and pConNS5B could result in functional HCV-specific immunity within the liver. Following immunization, not only were NS4B-, NS5A- and NS5B- specific CD4+ and CD8+ T cell responses detected in the liver following peripheral immunization, but these cells were found at similar percentages within the liver as observed in the spleen suggesting that peripheral immunization with these constructs was in fact able to result in a large pool of vaccine-specific T cells within the liver. Likewise, this pool of vaccine-specific T cells was shown to be fully functional as evidenced by IFN-γ production in flow cytometric analysis.
However, since it has been reported that the liver has the ability to specifically delete activated T cells
Therefore, our study is to the first to suggest that peripheral immunization targeting the HCV non-structural proteins NS4B, NS5A and NS5B can result in the formation of a large pool HCV-specific T cells within the liver. Likewise, we have shown that this pool of HCV-specific T cells remains fully functional within the liver. Given that it has been previously reported that T cell infiltration into the liver is not observed until 72 hours following liver transfection
While this study is an important first step in showing that peripheral immunization targeting the non-structural NS4B-NS5B region of HCV can produce potent and function T cell responses within the liver, there were several limitations to our study that merit further study. First, these experiments were performed in one strain of inbred mice, additional studies are needed in order to assess whether these findings would be replicated in a population of outbred humans. Second, our study was limited in that vaccine-specific IFN-γ secretion was used as the main marker of immunogenicity. Additional studies are needed in order to assess the ability of these constructs to induce other forms of immunity including polyfunctional T cell responses. And lastly, additional longer-term memory studies will have to be undertaken to determine exactly how long these vaccine-specific T cells persist and remain functional within the liver.
In conclusion, our study has shown that DNA immunization with non-structural HCV proteins NS4B, NS5A and NS5B is able to induce potent HCV-specific T cell responses within the murine liver. Taken together, these findings may have important implications for future HCV vaccine development in humans.
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