Adjuvant Effect Of Costimulatory Molecule CD137L In Immune Responses To Recombinant HBsAg Vaccination In Mice

One of the main mechanisms of HBV persistent infection is that the specific cellular immune response is insufficient. But it is hoped that the therapeutic vaccine which induce effective cellular immunity will enhance the antiviral immune, and will be used as one of the effective means about comprehensive treatment of HBV infection. Therefore, to research of therapeutic vaccine, which is effective, sustainable and safety, is an important goal of chronic hepatitis B immunotherapy.The recombinant vaccine which combined with HBsAg and aluminum hydroxide adjuvant is effective preventive vaccine. But because of the insufficient of cellular immune response which be induced, it is generally not be used in antiviral treatment.CD137/CD137L (4-1BB/4-1BBL) is an important pair of costimulatory signal molecule, which is the important effect to enhance and keep immune responses, especially to proliferation, survival and mnemonic killer of specific T cell. It has potential clinical application of antiretroviral immune treatment.This study is to construct the eukaryotic expression plasmid of mouse CD137L at first. At second, the immunologic enhancement effect of CD137L recombinant plasmid and CD137L recombinant protein of the specific immune responses in mice inducted by the HBsAg recombinant protein and the hepatitis B vaccine combined with aluminum hydroxide adjuvant was observed. At last, the possibility of CD137L molecule as the adjuvant of recombinant hepatitis B vaccine to enhance the antiviral immune effect also was explored. Part1construction and identification of the eukaryotic expression plasmid of mouse CD137LObjectiveThis study will focus on the expression of mRNA and protein in NIH3T3cells by constructing the eukaryotic expression plasmid of CD137L, meanwhile, lay the experiment foundation for the further research of the animal study.Methods1. Construction of the recombinant plasmid of CD137LThe splenic cells of BALB/c mouse were isolated, and the total RNA from those cells which cultured in vitro and stimulated by ConA were extracted. Then the PCR products which had extracted and purified were recombinanted into pcDNA3.1(+).At last the recombinant plasmid was identified by PCR, enzyme digest and sequencing, respectively..2. Expression and examinations of the recombinant plasmid of CD137LEukaryotic expression vector containing the full length of mouse CD137L cDNA sequence (pcD137L) was transfected into NIH3T3cells using Lipofectamine2000, and then the expression of CD137L mRNA and protein in the transfected cells were detected by RT-PCR, flow cytometry and immunofluorescence method, respectively.Results1. A single band which amplified by PCR products of recombinant plasmid (pcD137L) is consistent with the target fragment. And the size of fragment which gained by enzyme digest is consistent with target fragment too.2. The result of sequencing proved that the nucleotide sequence of inserted fragment is consistent to mouse CD137L gene sequence in Genebank.3. A single band which amplified from pcD137L transfected cells by RT-PCR is consistent with target fragment.4. After pcD137L transfected cells stained by PE-anti mouse CD137L, the expression of CD137L molecular on the cell surface were detect by flow cytometry, and membranes of those cells showed fluorescence under fluorescence microscope. Conclusions1. An eukaryotic expression vector (pcD137L) containing mouse CD137L cDNA was successfully constructed.2. It was proved that the recombinant plasmid (pcD137L) can express CD137L mRNA and protein in eukaryotic cells successfully. Part2Adjuvant effect of CD137L recombinant plasmid and CD137L recombinant protein to recombinant HBsAg vaccineobjectiveMice were immunized with CD137L recombinant plasmid (pcD137L) or recombinant protein (rCD137L) combined with recombinant HBsAg, respectively. And the possibility of CD137L molecule as the adjuvant of recombinant HBsAg enhance the role of antiviral immune was explored.MethodsBALB/c mice were randomly divided into eight experimental groups and five control groups with5in each group. And mice were immuned three times at the week of0,3,6and killed at one week after the last immunity.Experimental groups:(1) rHBsAg (S);(2) rHBsAg+pcDNA3.1(+)(S+pcD);(3) rHBsAg+pcD137L (S+pcD137L);(4) rHBsAg+aluminum hydroxide adjuvant (S+Al);(5) rHBsAg+aluminum hydroxide adjuvant+pcDNA3.1(+)(S+Al+pcD);(6) rHBsAg+aluminum hydroxide adjuvant+pcD137L (S+Al+pcD137L);(7) rHBsAg+rCD137L (S+rCD137L);(8) rHBsAg+aluminum hydroxide adjuvant+rCD137L (S+Al+rCD137L).Control groups:(1) NS;(2) pcDNA3.1(+)(pcD);(3) aluminum hydroxide adjuvant (Al);(4) pcD137L;(5) rCD137L.2. Serum IgG, IgGl and IgG2a of immunization mice were detected by ELISA.3. The expression level of IFN-γ/IL-4in splenic CD8+T cells was detected by flow cytometry.4. The percentage of CD44+high and CD127+CD44+higbin splenic memory CD8+T cells was analied by flow cytometry.5. The activity of apoptotic target cells which induced by specific CTL was analied by flow cytometry.Results1. Detection of serum specific antibodies of anti-HBs(1)pcD137L promoted the antibody response inducted by rHBsAg(The titer of total IgG,IgG2a and IgGlwere rised), but can not play regulatory role of antibody response to the hepatitis B vaccine combined with aluminum hydroxide adjuvant.(2)pcD137L enhanced the Thl antibody response inducted by the rHBsAg(The titer of total IgG and IgG2a were rised), and increased the Thl antibodies but decreased Th2antibodies inducted by the hepatitis B vaccine combined with aluminum hydroxide adjuvant(The titer of IgG2a was rised but IgGl was decreased).(3) Aluminum hydroxide adjuvant substantially increased the level of antibodies inducted by the rHBsAg (The titer of total IgG,IgG2a and IgGlwere rised).2. Detection of the percentage of IFN-y/IL-4in CD8+T cells(1) pcD137L elicited higher level of IFN-y secreted by CD8+T cell inducted by the rHBsAg, and enhanced the Tcl response inducted by hepatitis B vaccine combined with aluminum hydroxide adjuvant.(2) rCD137L can not played regulatory role of the Tcl cellular immune response inducted by the rHBsAg, but obviously promoted the Tel cellular immune response inducted by the hepatitis B vaccine combined with aluminum hydroxide adjuvant.(3) Aluminum hydroxide adjuvant enhanced the immunogenicity of the rHBsAg and improved the level of cellular immune response of Tcl.(4) Both pcD137L and rCD137L can not played an obvious regulatory role of the IL-4secreted by specific CD8+T cells which stimulated by the rHBsAg or the rHBsAg combined with aluminum hydroxide adjuvant.3. Analysis of the percentage of memory CD8+T cells in splenic cells(1) pCD137L increased productions of CD44+high CD8+and CD127+CD44+high CD8+T cells induced by the rHBsAg but not induced by the hepatitis B vaccine combined with aluminum hydroxide adjuvant.(2) rCD137L elicited higher level of memory CD8+T cells both induced by the rHBsAg and hepatitis B vaccine combined with aluminum hydroxide adjuvant.(3) Aluminum hydroxide adjuvant increased the number of memory CD8+T cells inducted by the rHBsAg.4. Analysis of the activity of apoptotic target cells induced by CTL(1) rCD137L enhanced specific CTL response which induced by the rHBsAg and the rHBsAg combined with aluminum hydroxide adjuvant.(2) Aluminum hydroxide adjuvant enhanced the activity of CTL induced by the rHBsAg.The differences of all the above have statistical significance.Conclusions1.CD137L recombinant plasmid enhanced the antibody responses, Tcl cellular responses and the production of memory CD8+T cells inducted by the rHBsAg in mice but not to by the rHBsAg combined with aluminum hydroxide adjuvant.2.CD137L recombinant protein enhanced the IgG2a (Thl) antibody response and cellular response characterized by secrete IFN-y which inducted by the rHBsAg in mice, the activity of specific CTL and the production of memory CD8+T cells. It obesrvely enhanced the type1cellular immune responses and humoral immune responses to the hepatitis B vaccine combined with aluminum hydroxide adjuvant. It may be an effective adjuvant for translating prophylactic vaccines into therapeutic vaccine to HBV.