The Research Of Therapeutic Effect On Multiple Sclerosis And Immunopharmacologic Mechanism Of Kv1.3 Polypeptide Vaccine

Part Ⅰ Preparation of Kv1.3 polypeptide vaccine and identification of the specific antibodiesObjective:Kv1.3 potassium channel, a voltage-gated channel, is mainly found in the immunocyte, including T and B lymphocyte, macrophage and microglia; and regulates several physiological functions of immunocyte, including activation, differentiation, proliferation and migration. Hence, Kv1.3 has be attracted increasing attention as a promising new therapeutic target for autoimmune diseases. In this study, we would utilize the key peptide of Kv1.3 selected in the previous experiment as a B cell epitope, coupled with PADRE to generate a novel polypeptide vaccine targeting Kv1.3 channel, and identify whether the Kv1.3 antibodies can sepcifically combine with Kv1.3 channels of rats.Methods:Kv1.3 peptide selected in the previous experiment linked with PADRE to produce antibody in immunized Lewis rats and BABL/c mice. The titers of antibodies in serum were detected by ELSA. The rats’antisera were harvested and antibodies were purified by ammonium sulfate saturation and immunoaffinity chromatography. We obtained HEK293 cell line that stably express rKvl.3 protein using plasmid transfection. We detected the specific binding of anti-Kvl.3 antibodies with rKv1.3 using Western Blot and immunofluorescence.Results:By immunizing rats and mice with PADRE-hKv1.3 polypeptide vaccine, we generated antibodies against Kvl.3 and the titer of antibodies in rats was higher than mice. We obtained purified anti-Kvl.3 antibodies using ammonium sulfate saturation and immunoaffinity chromatography, and observed the specific recognition of rKvl.3 protein by Western Blot and their binding to Kvl.3 expressed in HEK/rKvl.3 cell by immmunostaining.Conclusion:The polypeptide vaccine, generated by coupled the key peptide of hKvl.3 with PADRE, successfully induced Kvl.3-specific antibodies and the antibodies can specifically recognize rKvl.3 channel.Part Ⅱ Biological safety evaluation of Kv1.3 polypeptide vaccineObjective:The aim of this experiment is to investigate whether the PADRE-hKv1.3 polypeptide vaccine has adverse effects on the experimental animals, and to detect the underlying subchronic toxicity.Methods:Rats were immunized with PADRE-hKv1.3 polypeptide vaccine for three times at two-week intervals, and during the immunization process, the rats were inspected daily regarding their body weight changes and their general condition. Two weeks after last immunization, electrocardiogram, chest X-ray radiation, routine blood examination and blood biochemistry were monitored. During necropsy, the organs were dissected and all organs were visually inspected and weighed directly. All organs were fixed in standard fomalin and following by paraffin embedded for further histological examine using HE staining.Results:During the immunization period, the general conditions of rats were well, weight gain was similar and rats had no infection lesions in both body surface and lung in each group (normal, sham immunization and PADRE-hKv1.3 immunization). PADRE-hKv1.3 polypeptide vaccine immunization had no side effects on the morphology and ultrastructure of heart and the ECG. PADRE-hKv1.3 polypeptide vaccine immunization did not cause damage on liver, spleen, kidney and brain of experimental animals, while slight congestion was observed in the lungs of each group rat. PADRE-hKv1.3 polypeptide vaccine had no adverse effects on circulating blood cells and blood biochemistry parameter.Conclusion:PADRE-hKvl.3 polypeptide vaccine has no toxicity effects on organs of experimental animals and dose not influence normal physiological function.Part III The study of therapeutic effect on experimental autoimmune encephalomyelitis and immunopharmacologic mechanism of Kvl.3 polypeptide vaccineObjective:PADRE-hKvl.3 polypeptide vaccine has inhibiting effect on pathogenic immunocyte through blockade of Kvl.3 channel. The objective of this study was to investigate the therapeutic effect on autoimmune disease of PADRE-hKv1.3 polypeptide vaccine and its immunopharmacologic mechanism using the animal disease model of MS-EAE.Methods:After three immunizations with PADRE-hKv1.3 polypeptide vaccine, Lewis rats were immunized with homogenates extracted from the spinal cord of guinea pigs for induction of EAE. Rats were scored for clinical signs of EAE and weighed daily, and the onset day of EAE, morbidity and mortality were also recorded. The rats were euthanized on day 14, the spinal cord was collected and then stained with HE and Luxol fast blue for histopathological evaluation of inflammation and demyelination, respectively. The infiltration and activation of T cells and microglia/mcrophage were detected by immunofluorescence histochemistry and the phenotype of microglia/mcrophage was detected using double immunofluorescence histochemistry marked with CD68 and iNOS or Arg-1. On day 14, single-cell suspensions were made from spleens and CNS tissue and flow cytometry was performed to detect the phenotype of T cells. We also assayed the level of related cytokines gene transcripts in spleens and CNS tissue using RT-PCR. On the 28 day after immunization, the spinal cord was collected and immunofluorescence histochemistry marked with MBP and NeuN was used to detect the degree of MBP degradation and neuronal damage. Furthermore, Bielschowsky staining was used to identify and quantify areas of axonal loss in the spinal cord.Results:EAE was successfully induced using homogenates extracted from the spinal cord of guinea pigs as immunizing antigen and morbidity was 100% in all groups. Compared with EAE and sham immunization +EAE groups, Previous immunization with PADRE-hKv 1.3 polypeptide vaccine improved clinical symptoms, notably reduced pathologic scores and attenuated demyelination in Lewis rats with EAE. In addition, PADRE-hKv1.3 polypeptide vaccine decreased T-cell infiltration in spinal cord and promoted T helper cell differentiate into CD4+IL-10+Treg cell while inhitied the differentiation of Thl and Th17 in spleens and CNS tissue. In EAE and sham immunization +EAE groups, levels of the mRNA transcripts for pro-inflammatory cytokines of IFN-y, IL-17A and IL-1β were elevated in the spleens and CNS tissue while were reduced by treatment with PADRE-hKv1.3 polypeptide vaccine. In contrast, compared with EAE and sham immunization+EAE groups, levels of the mRNA transcripts for anti-inflammatory cytokines of IL-10 and FoxP3 were elevated in the CNS tissue, while only level of IL-10 was increased in the spleens of rats treated with PADRE-hKv1.3 polypeptide vaccine. Furthermore, PADRE-hKvl.3 polypeptide vaccine inhibits microglia/macrophages activation and promoted microglia/macrophages polarizes into M2 phenotype. PADRE-hKv1.3 polypeptide vaccine attenuated MBP degradation, axonal loss and neuronal death in recovery phase of EAE.Conclusion:PADRE-hKvl.3 polypeptide vaccine influences the degree of inflammatory cells infiltration and their phenotype. PADRE-hKvl.3 polypeptide vaccine ameliorates the microenvironment of inflammatory in the spinal cord and significantly alleviates the pathologic changes, and then plays a beneficial role on EAE/MS and improves the clinical symptoms.