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The use of attenuated bacteria for oral delivery of DNA vaccines is a recent innovation. We designed and constructed the naked plasmid DNA vaccine pcDNA-CCOL2A1, which effectively prevented and treated a rheumatoid arthritis model by inducing immunotolerance. We aimed to ensure a reliable, controllable dosage of this oral DNA vaccine preparation and establish its stability. We transformed pcDNA-CCOL2A1 via electroporation into attenuated Salmonella typhimurium SL7207. A resistant plate assay confirmed the successful construction of the transformed strain of the SL7207/pcDNA-CCOL2A1 oral DNA vaccine. We verified its identification and stability in vitro and in vivo. Significant differences were observed in the characteristics of the transformed and blank SL7207 strains. No electrophoretic restriction patterns or direct sequencing signals were observed in the original extract of the transformed strain. However, target gene bands and sequence signals were successfully detected after PCR amplification. CCOL2A1 expression was detected in the ilea of BALB/c mice that were orally administered SL7207/pcDNA-CCOL2A1. The pcDNA-CCOL2A1 plasmid of the transformed strain was retained under the resistant condition, and the transformed strain remained stable at 4 °C for 100 days. The concentration of the strain harboring the pcDNA-CCOL2A1 plasmid was stable at 109 CFU/mL after 6–8 h of incubation. The results demonstrated that the transformed strain SL7207/pcDNA-CCOL2A1 can be expressed in vivo, has good stability, and may be used to prepare the oral DNA vaccine pcDNA-CCOL2A1 with a stable, controllable dosage and the capacity to provide oral immunization. This vehicle can effectively combine both oral immunotolerance and DNA vaccination. Key points: Attenuated Salmonella typhimurium SL7207 aids immunization as a DNA vaccine carrier. The transformed strain SL7207/pcDNA-CCOL2A1 was constructed as an oral DNA vaccine. The formulated oral DNA vaccine offers a reliable, stable, and adjustable dosage. [ABSTRACT FROM AUTHOR]

Background: Recent therapeutic-plasmid DNA vaccine strategies for rheumatoid arthritis (RA) have significantly improved. Our pcDNA-CCOL2A1 vaccine is the most prominent and the first antigen-specific tolerising DNA vaccine with potent therapeutic and prophylactic effects compared with methotrexate (MTX), the current "gold standard" treatment for collagen-induced arthritis (CIA). This study developed a highly efficient, cost-effective, and easy-to-operate system for the lab-scale production of endotoxin-free supercoiled plasmids with high quality and high yield. Based on optimised fermentation culture, we obtained a high yield of pcDNA-CCOL2A1 vaccine by PEG/MgCl2 precipitation and TRION-114. We then established a method for quality control of the pcDNA-CCOL2A1 vaccine. Collagen-induced arthritis (CIA) model rats were subjected to intramuscular injection of the pcDNA-CCOL2A1 vaccine (300 μg/kg) to test its biological activity. Results: An average yield of 11.81 ± 1.03 mg purified supercoiled plasmid was obtained from 1 L of fermentation broth at 670.6 ± 57.42 mg/L, which was significantly higher than that obtained using anion exchange column chromatography and a commercial purification kit. Our supercoiled plasmid had high purity, biological activity, and yield, conforming to the international guidelines for DNA vaccines. Conclusion: The proposed innovative downstream process for the pcDNA-CCOL2A1 vaccine can not only provide a large-scale high-quality supercoiled plasmid DNA for preclinical research but also facilitate further pilot-scale and even industrial-scale production of pcDNA-CCOL2A1 vaccine. [ABSTRACT FROM AUTHOR]

Antigen-specific tolerizing DNA vaccines are one of the most promising strategies for rheumatoid arthritis (RA) treatment. They act by inducing potent immune tolerance instead of generalized immunosuppression. Recently, we developed a novel antigen-specific tolerizing DNA vaccine pcDNA-CCOL2A1 coding for chicken type II collagen (CCII) and confirmed its potent therapeutic efficacy in an established rat model of collagen-induced arthritis (CIA). Here we report the prophylactic vaccination efficacy of a single 300 μg/kg dose of pcDNA-CCOL2A1 against CIA incidence, severity, and onset. CCOL2A1 transcripts were detected in the blood of CIA rats 14–42 days after intramuscular injection by 300 μg/kg pcDNA-CCOL2A1. The expression of CCOL2A1 transcripts increased quickly on day 21, peaked at day 28, and then gradually decreased thereafter. Importantly, a single prophylactic vaccination of pcDNA-CCOL2A1 14 days before CIA establishment significantly reduced CIA incidence and severity, deferred its onset, and was as efficacious as the current gold standard drug, methotrexate. The marked effects on CIA incidence and severity closely corresponded to the expression of CCOL2A1. Furthermore, prophylactic vaccination with pcDNA-CCOL2A1 markedly decreased serum content of anti-type II collagen (CII) immunoglobulin G (IgG) antibodies, induced Th1-to-Th2 and Tc1-to-Tc2 shifts, and decreased the percentages of CD4+CD29+ and Th17 T cells. Prophylactic vaccination with pcDNA-CCOL2A1 also downregulated various Th1 cytokines, while upregulating both the Th2-type cytokine interleukin-10 and the Th3-type cytokine transforming growth factor β. Our results indicate that the pcDNA-CCOL2A1 DNA vaccine acts as a highly efficient inducer of specific immunotolerance that could be a promising option for RA treatment in the near future. [ABSTRACT FROM AUTHOR]

Abstract: Immunotherapy of rheumatoid arthritis (RA) using oral-dosed native chicken or bovine type II collagen (nCII) to induce specific immune tolerance is an attractive strategy. However, the majority of clinical trials of oral tolerance in human diseases including RA in recent years have been disappointing. Here, we describe a novel recombinant peptide rcCTE1-2 which contains only two tolerogenic epitopes (CTE1 and CTE2) of chicken type II collagen (cCII). These are the critical T-cell determinants for suppression of RA that were first developed and used to compare its suppressive effects with ncCII on the collagen-induced arthritis (CIA) model. The rcCTE1-2 was produced using the prokaryotic pET expression system and purified by Ni-NTA His affinity chromatography. Strikingly, our results showed clearly that rcCTE1-2 was as efficacious as ncCII at the dose of 50μg/kg/d. This dose significantly reduced footpad swelling, arthritic incidence and scores, and deferred the onset of disease. Furthermore, rcCTE1-2 of 50μg/kg/d could lower the level of anti-nCII antibody in the serum of CIA animals, decrease Th1-cytokine INF-γ level, and increase Th3-cytokine TGF-β1 produced level by spleen cells from CIA mice after in vivo stimulation with ncCII. Importantly, rcCTE1-2 was even more potent than native cCII, which was used in the clinic for RA. Equally importantly, the findings that the major T-cell determinants of cCII that are also recognized by H-2b MHC-restricted T cells have not previously been reported. Taken together, these results suggest that we have successfully developed a novel recombinant peptide rcCTE1-2 that can induce a potent tolerogenic response in CIA. [Copyright &y& Elsevier]

Tolerizing DNA vaccines encoding key autoantigens are one of emerging strategies for the treatment of rheumatoid arthritis (RA). Among these vaccines, the most representative is pcDNA- CCOL2A1 , an antigen-specific DNA vaccine encoding chicken type Ⅱ collagen (CCⅡ) with significant therapeutic and prophylactic efficacy in collagen-induced arthritis (CIA) rat models. We compared the in situ expression levels of CCOL2A1 -mRNA and CCⅡ protein and the protective efficacies against CIA after a single dose (300 μg/kg) of this vaccine via intramuscular (IM), subcutaneous (SC) and intravenous (IV) vaccinations. The IM vaccination routes resulted in good protective efficacies in terms of decreasing CIA incidence and severity and significantly improved radiographic and histopathologic findings and scores of joints. Furthermore, IM, SC, and IV vaccinations markedly decreased serum levels of anti-type Ⅱ collagen (CⅡ) IgG antibodies, but only IM vaccination significantly reduced serum levels of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibody. The vaccine exhibited a continuous CCOL2A1 -mRNA expression in the tail and abdominal subcutaneous tissue injection sites, but no CCOL2A1 -mRNA signal was observed in muscle. Strikingly, CCⅡ protein expression levels at the three injection sites were comparable with minimal variation. IM administration may be considered the preferred route for RA treatment in clinical practice. [Display omitted] • The pcDNA- CCOL2A1 is an antigen-specific DNA vaccine encoding type Ⅱ collagen. • IM-vaccination has good protective efficacies than SC- and IV-vaccination against CIA. • Intramuscular administration may be the preferred route for treating RA in clinics. [ABSTRACT FROM AUTHOR]