PotentVaccinotherapy Of ExperimentalRheumatoid Arthritis With A Novel Tolerizing DNA Vaccine

Rheumatoid arthritis (RA) is a chronic inflammatory disease, which causes progressive deformity and destruction of the joints, thus leading to disability and even premature death. The condition is characterized by the aggressive infiltration of the synovial lining of the joints by both monocytes and T lymphocytes, together with proliferation of fibroblastic lining cells. This hyperproliferative pannus invades and subsequently destroys the underlying cartilage and bone. RA inflicts significant morbidity on approximately 1% of the population. Joint destruction and extra-articular inflammation result in a long-term reduction in independence and an increased mortality compared with the general population. It was estimated that up to 50% of patients are no longer in full-time employment after 10 years of their illness, and population studies have suggested that the standardized mortality ratio of RA patients is up to 5.5 times that of the general population.Autoimmunity of type II collagen (CII) has been suggested to play an important role in the pathogenesis of rheumatoid arthritis. Immunization of susceptible rats with CII leads to the development of autoimmune polyarthritis, and the pathological and clinical features are quite similar to those of human RA. Antibodies directed toward CII have been found in the sera, synovial fluid, and cartilage in patients with RA, but the clinical significance of these antibodies needs to be more extensively studied. Cll, a homotrimer composed of a1(II) chains, is the most abundant fibrillar protein found in articular cartilage and constitutes 80-85% of the total collagen content. During embrogenesis Cll, as other cartilage collagens, can be found in noncartilaginous tissues, although only for a limited tine. Arthritis is initiated by complement fixing antibodies that bind to type II collagen in autologous cartilage.Strong theoretical reasons suggest that patients with autoimmune arthritis should benefit from treatment with cartilaginous proteins, such as type II collagen (CII). The destruction of cartilage that occurs in rheumatoid arthritis (RA) involves an autoimmune response to cartilage-specific proteins. Therefore, inducing the state of tolerance to these autoantigens should down-regulate the inflammatory T- cell cytokine and antibody response, regulating in decreased joint inflammation and cartilage destruction. In fact, the induction of tolerance to CII by parenteral, intranasal or oral administration of antigen caused a profound suppression of arthritis in the animal model of collagen-induced arthritis (CIA). Recently, it was published the results of a double-blind control study in which oral administration of low doses of CII to RA patients resulted in improvement in their disease activity, presumably by induction of tolerance to CII. In addition, it was reported that oral administration of CII was beneficial to patients with juvenile rheumatoid arthritis (JRA) in an open-labelled trial. Although conflicting results were obtained in a study of early RA patients, none of these studies reported any appreciable toxicity following oral administration of Cll. These data suggest that further studies are warranted to clarify the possible efficacy of Cll in the therapy of patients with RA and JRA.However, Cll protein therapy has several limitations, since systemic administration of these proteins exert adverse effects, and daily or weekly administration, due to the short half-life, is necessary to obtain therapeutic effects.Thus, gene therapy is currently being applied as a new approach in the treatment of RA in an experimental model. In this therapy, a vector is used for gene transfer. CIA is an experimental animal model that encompasses clinical and pathologic features similar to RA. In the present study, we investigated the effect of gene transfer by using a plasmid encoding CCII in a murine CIA models.In 2002, the CCIl sequence had been declared invention patent of China, and the acceptance number was 02149315.8. The corresponding gene sequence of CCII had also been deposited in Genbank databases (the accession numbers were AY046949 and AF452711, respectively).Based on previous study, 3 objectives of the present study were carefully set:(1) Construction of eukaryotic expressing plasmid and expression in COS-7 cells; (2) Establishment of CIA models and Findings in therapeutic effects; (3) Mechanism of gene vaccine effecting on CIA rats. (1) Construction of eukaryotic expressing plasmid and expression in COS-7 cellsDNA vaccines (pcDNA3.1-CCOL2A1) consisting of CMV promoter and CCII gene were constructed. When the recombinant plasmid pcDNA3.1-CCOL2A1 was cut with EcoR I and Hind III, the 4.3kb CII band and 5.4kb pcDNA3.1 band were observed, mean while when it was cut with Hind III, a about 9.7kb band was observed. From these results it was concluded that the recombinant plasmid pcDNA3.1-CCOL2A1 was constructed successfully.pcDNA3.1-CCOL2A1 were transfected into COS-7 cells (10⁶) in vitro and cultured for 2 days in DMEM medium. Then, the medium were harvested, concentrated, and dissolved in 01 mol/L acetic acid. The resultant supernatants were analyzed on 15% gradient SDS/polyacrylamide gels and transferred electrophoretically onto nitrocellulose members. The protein was detected by Western blotting with anti-chicken-CII polyclonal antibody.14.3kD band was observed, corresponding to the full-length CII and the synthetic polypeptide encoded by the DNA plasmid. No band was present in control samples transfected with pcDNA3.1, the control empty vector. When stained with anti-chicken-Cll antibody, the 14.3kD band was observed. From these results it was concluded that the pcDNA-CCII plasmid is properly expressing the achains of type II collagen which were secreted out of cell.CCII ptotein separates on SDS-PAGE gels as a well-defined protein band that can easily be excised from gels, Gel protein was analysed after 'in gel' trypsin digestion by mass spectrometry sequencing. The parts of the peptide sequence are compared to the database using software Mascot. It matched to alpha 1 type II procollagen [Gallus gallus]. According to the m/z of proline in fig, it showed that the proline in the peptide doesn't hydroxylated.(2) Establishment of CIA models and findings in therapeutic effects In rats treated with 200μg/kg of pcDNA3.1-CCOL2A1, the arthritis score was significantly lower on days 23-49 compared with untreated rats with CIA.CIA rats were divided into 5 groups 21days after they were primed. 3 groups received a single treatment intravenously (i.v) into the tail vein with 20μg/kg,200μg/kg,400μg/kg pcDNA3.1-CCOL2A1 respectively ; 1 group received MTX which were orally administered 0.32mg/ml once a day as a positive control and the last group received 200μg/kg empty vector pcDNA3.1 as a blank control. Administration of the empty control vector pcDNA3.1 and 20μg/kg of pcDNA3.1-CCOL2A1 had the same effects to CIA (they did not inhibit the development of CIA); Injection in 200μg/kg group and MTX group reduced the severity of the disease, compared with the untreated group. Interestingly, the milder exacerbation was detected in rats treated with 400μg/kg of pcDNA3.1-CCOL2A1 23-49 days after disease onset.Histological analysis using haematoxylin and eosin was performed on paws taken from untreated (200μg/kg pcDNA3.1), pcDNA3.1-CCOL2A1 (20μg/kg,200μg/kg,400μg/kg pcDNA3.1-CCOL2A1) and MTX-treated arthritis rats 14 days after disease onset, and sections were graded in a blinded manner. The percentage of animals exhibiting changes in bone/cartilage and synovitis was calculated. Sections from untreated and 20μg/kg pcDNA3.1-CCOL2A1 treated rats were damaged by rapidly expanding dynovial pannus. Mononuclear cell infiltration and thickening of the synovial membrane were apparent. But injection of 200μg/kg pcDNA3.1-CCOL2A1 suppressed inflammation in rat's hind joints, as shown by a reduced number of synovial histiocytes, a low amount of mononuclear cell infiltration, and less membrane thickening. Cartilage destruction and bone erosion were significantly reduced (p < 0.05), which showed almost same effects as MTX group. In contrast, section from rats, which had received 400μg/kg pcDNA3.1-CCOL2A1, showed extensive leucocyte infiltration within the joint space and synovial lining, accompanied by significant pannus formation and the thickening of synovial layer.Radiographic examination of the hind paws of CIA rats revealed severe paw swelling, severe bone matrix resorption and erosion that suggested active arthritis and joint destruction. Among the three doses groups, only CIA-200μg/kg pcDNA3.1-CCOL2A1 rats showed good therapeutic effects, reducing paw swelling and making articular cavity clear. Mean while the surface of articular cartilage and bone density in 20μg/kg pcDNA3.1-CCOL2A1 and 400μg/kg pcDNA3.1-CCOL2A1 groups had no significant changes versus vehicle control. The mean radiographic score in CIA-pcDNA3.1, CIA-20ug/kg pcDNA3.1-CCOL2A1, CIA-400ug/kg pcDNA3.1-CCOL2A1 rats were significantly higher than the mean score in CIA-20ug/kg pcDNA3.1-CCOI₂A1 rats and MTX-CIA rats (both P < 0.05 versus CIA-pcDNA3.1).(3) Mechanism of gene vaccine effecting on CIA ratsAs we know, the periphery tolerance including:â‘ anergy;â‘¡delete;â‘¢the suppressing mediated by cytokinesâ‘£Th1/ Th2 balance.So, we detected these aspects: Production of anti-CII IgG antibodies in peripheral blood serum of rats; Cytokine of TNF-α, TGF-β, IL-10, IL-4 level in supernatant of cultures of splencytes of rats; Effect of different dose with DNA vaccine on the T cell proliferative response to CM and PHA; Frequency of CD4⁺T cell subsets in peripheral blood from CIA model, therapeutic rats and control rats; Changes of CD4⁺CD25⁺ regulatory cells in peripheral blood from CIA model, therapeutic rats and control rats; Percentage of dendritic cells (DCs) in peripheral blood from CIA model, therapeutic rats and control rats.To examine the effect of pcDNA3.1-CCOL2A1 administered intravenously (i.v) into the tail vein in the production of anti-CII IgG antibodies, serum samples were collected on day 20 after primary immunization, and anti-CII IgG levels were measured by ELISA. The levels of anti-CII IgG antibodies in rats treated with 200μg/kg pcDNA-CCII and MTX were significantly lower compared with those in rats treated with pcDNA3.1 (P < 0.05). In addition, anti-CII IgG level in rats treated with 20μg/kg pcDNA3.1-CCOL2A1 have no statistically significant difference compared with those in rats treated with pcDNA3.1 (P > 0.05). But, anti-CII IgG level in rats treated with 400μg/kg pcDNA3.1-CCOL2A1 was significantly higher compared with those in rats treated with pcDNA3.1 (P = 0.047).The concentrations of TGF-βand IL-10 in 200μg/kg pcDNA3.1-CCOL2A1 -treated CIA rats were significantly higher than those in pcDNA3.1-treated CIA rats (P < 0.05). The TGF-βlevel in 400μg/kg pcDNA3.1-CCOL2A1-treated CIA rats were higher than those in pcDNA3.1-treated CIA rat, but there was no statistically significant difference compared with those in rats treated with pcDNA3.1 (P > 0.05). The TGF-βlevels in 20μg/kg pcDNA3.1-CCOL2A1 -treated CIA rats and MTX-treated group both did not reach significance (P = 0.09 and P = 0.08, respectively) compared to pcDNA3.1 group. The concentrations of TNF-αin 200μg/kg pcDNA3.1-CCOL2A1-treated CIA rats and MTX-treated group were both lower than that in pcDNA3.1 group (P < 0.05). Lymphocyte proliferative response to Cll in different concentrations was significantly lower in 200μg/kg group than in control group (200μg empty vector) (P<0.05) .However, lymphocyte proliferative response to PHA 400μg/kg was higher than in control group, but statistical analysis showed no significant differences (P>0.05) .whereas, lymphocyte proliferative responses to PHA in all groups showed no significant differences (P>0.05) .The mean percentage of Th1 cells among CD4⁺ T cell subsets in empty vector, 20μg/kg, 200μg/kg, 400μg/kg, MTX groups was higher than that in normal group, whereas the mean percentage of Th2 cells among CD4⁺ T cell subsets were not different from that in normal group. The mean Th1:Th2 ratio was 0.06 in normal (rang 0.03-0.09), 0.08 in 200μg/kg group (0.06-0.11), and 0.08 in MTX group (0.05-0.16), they all are significantly lower than that in control group (P<0.05) .To identify any possible involvement of particular T cell subsets that are known to exert immune suppressive function, we compared the proportion of CD4⁺CD25⁺ T lymphocytes among normal, therapeutic and control groups. The proportion of CD4⁺CD25.+ cells in 200μg pcDNA3.1-CCOL2A1 and MTX groups were higher than that in other groups (P<0.05) . The proportion of CD4+CD25+ cells in 20μg pcDNA3.1-CCOL2A1 group remains unchanged compared with control group. In 400μg pcDNA3.1-CCOL2A1, the proportion of CD4⁺CD25⁺ cells was also higher than that in control group, but it showed no significant differences.The mean percentage of OX-62 positive DC in blood mononuclear cell in normal group was 0.93±0.35%, and the mean percentage of OX-62 positive DC in blood mononuclear cell in all therapeutic group were 1.48±0.70%, 1.02±0.61%, 1.18±0.31%, 1.07±0.25% and 1.04±0.33%, respectively. The therapeutic groups showed no significance compared to normal group (P>0.05) except for 200μg pcDNA3.1-CCOL2A1 groups (P<0.05) .In summary, the present study is the first to demonstrate that delivery of the plasmid vector encoding CCII by systemic administration has an in vivo therapeutic effect in CIA model. Taken as a whole these results suggest that the CCII plasmid may act as a potent immunosuppressive antiarthritis agent in the treatment of RA, and efforts to better understand the molecular mechanism by which they inhibit inflammatory reactions, which demonstrated that the therapeutic effects may has relationship with the depressing of specific T cells, production of suppressing cytokines, offset of Th1/Th2 and increasing of CD4⁺CD25⁺ T regular cells.