The Pharmacokinetics in Mice and Cell Uptake of Thymus Immunosuppressive Pentapeptide Using LC-MS/MS Analysis.

Thymus immunosuppressive pentapeptide (TIPP) is a novel anti-inflammatory peptide with high efficacy and low toxicity. This study aims to establish a selective LC-MS/MS method for analyzing the analyte TIPP in biological samples, laying the foundation for further PK and PD studies of TIPP. Protein precipitation was conducted in acetonitrile supplemented with 2% formic acid and 25 mg/mL dithiothreitol as a stabilizer, which was followed by backwashing the organic phase using dichloromethane. The chromatographic separation of TIPP was achieved on a C18 column with a gradient elution method. During positive electrospray ionization, TIPP was analyzed via multiple-reaction monitoring. The linear relationships between the concentration of TIPP and peak area in murine plasma cell lysates, supernatants, and the final cell rinse PBS were established within the ranges of 20−5000 ng/mL, 1−200 ng/mL, 10−200 µg/mL, and 0.1−20 ng/mL, respectively (r2 > 0.99). Validated according to U.S. FDA guidelines, the proposed method was proved to be acceptable. Such a method had been successfully applied to investigate the pharmacokinetics of TIPP in mice via subcutaneous injection. The plasma half-life in mice was 5.987 ± 1.824 min, suggesting that TIPP is swiftly eliminated in vivo. The amount of TIPP uptake by RBL-2H3 cells was determined using this method, which was also visually verified by confocal. Furthermore, the effective intracellular concentration of TIPP was deduced by comparing the intracellular concentration of TIPP and degrees of inflammation, enlightening further investigation on the intracellular target and mechanism of TIPP.

Vaccination of Mice with Listeria ivanovii Expressing the Truncated M Protein of Porcine Reproductive and Respiratory Syndrome Virus Induces both Antigen-Specific CD4+ and CD8+ T Cell-Mediated Immunity.

Porcine reproductive and respiratory syndrome (PRRS), a serious disease of swine caused by the PRRS virus (PRRSV), had a severe economic impact worldwide. As commonly used PRRS vaccines, the attenuated or inactivated vaccines, provide unsatisfactory immune protection, a new PRRS vaccine is urgently needed. In this study, a part of the PRRSV ORF6 gene (from 253 to 519 bp) encoding the hydrophilic domain of PRRSV M protein was integrated into two Listeria strains via homologous recombination to generate two PRRS vaccine candidates, namely LI-M' and LM-ΔactAplcB-M'. Both candidate vaccines showed similar growth rate as their parent strains in culture media, but presented different bacterial loads in target organs. As the integrated heterogenous gene was not expressed, LM-ΔactAplcB-M' was excluded from the immunological test. In a mouse model, LI-M' provoked both CD4+ and CD8+ T cell-mediated immunity. In addition, LI-M' boosting dramatically enhanced CD8+ T cell-mediated immunity without affecting the response intensity of CD4+ T cell-mediated immunity. All of these data suggest that LI-M' is a promising PRRS vaccine candidate.

The Protein Expression Level of a Heterogeneous Gene Inserted in LIPI-1 of the Listeria ivanovii Genome Relies on Its Insertion Orientation.

Due to its capability to multiply in either phagocytic or nonphagocytic cells, and to subsequently elicit a robust cellular immune response, Listeria ivanovii (LI) is thought to be feasible for developing bacteria-based live attenuated vaccines. We previously generated several recombinant LI strains expressing Mycobacterium tuberculosis antigens. Since the expression level of heterogeneous protein was sometimes very low, we attempted to elucidate the principle of heterogeneous protein expression in such recombinant LI strains. In this study, we inserted the M. tuberculosis antigen gene Rv0129c into LI strains at the same site as the genome but with a different insertion orientation. RT-qPCR and Western blot showed that when the insertion orientation of the heterogeneous gene was opposite to the LIorfXYZ gene in the Listeria pathogenicity island 1 in the bacterial genome, the heterogeneous gene could be transcribed well but the protein expression level seemed limited, both in vitro and in vivo. When inserted at an orientation consistent with LIorfXYZ at the same site in the genome, the expected 43-kD protein was observed in vitro as well as in a mouse model. Bacterial virulence was found to have decreased after recombination. This work confirms that the protein expression level of the heterogenous gene in such genome-recombinant LI-based vaccines is related to its inserted orientation in the bacterial genome, and a foreign gene inserted at this position of LIPI-1 will abolish Listeria virulence without affecting its growth.

[Prediction and Analysis of Epitopes in clpP2 of Mycobacterium tuberculosis].

OBJECTIVES: To predict and analyze the antigenic epitopes in Mycobacterium tuberculosis protein caseinolytic protease P2 (clpP2), and explore its possibility to be applied as a new tuberculosis (TB) vaccine and drug development target. METHODS: Secondary structure of clpP2 based on nucleic sequence was predicted by DNA Star software. The homologous sequence conformation were analyzed by Swiss-Model online software. T cells antigenic epitopes were predicted through VaxiPred, and B cell epitopes were predicted by combining use of several different prediction programs, such as ABCpred, COBEPro and BepiPredPred. The immune characteristics of clpP2 were analyzed by DNA Star, SignalP, TMHMM online software and were searched through NCBI database. RESULTS: clpP2protein was diverse in structure, composing with a great deal of CTL and Th cell epitopes. clpP2 was also predicted to comprise rich potential liner and discontinuous B-cell epitopes. These epitopes were accessible on the protein surface, located in flexible and hydrophilic regions. CONCLUSION: clpP2 is prompted to induce immune responses and developes a novel target in surveillance, treatment and vaccine.

Listeria ivanovii Infection in Mice: Restricted to the Liver and Lung with Limited Replication in the Spleen.

Listeria monocytogenes (LM) vectors have shown much promise in delivery of viral and tumor antigens for the development of vaccines. L. ivanovii (LI) is a closely related bacterium with a similar intracellular life cycle that may offer advantages over LM because it is not a human pathogen, but can infect other animal species. Recent studies show that recombinant LI expressing Mycobacterium tuberculosis antigens is effective in inducing protective immunity in mouse models, demonstrating the potential of LI as a live vaccine vector. However, a key barrier in the development of LI into a live vaccine vector is that its pathogenic and immunogenic characteristics have yet to be fully understood. Therefore, in this research, C57BL/6J mice were inoculated with LM or LI intravenously or intranasally, and bacterial loads, histopathologic changes, and cytokine production were determined at indicated days post inoculation. Results showed that after intravenous infection with LM or LI, bacteria were found proliferating in the liver, spleen, and lung. However, LI could only reach a heavy burden in the liver and its ability to multiply and to resist host immunity seemed limited in the spleen and lung. After intranasal inoculation with LI, bacteria were mainly localized in the lung and failed to infect liver or spleen, while LM could. In organs with heavy LI burden, lesions were isolated, localized and densely packed, compared to lesions caused by LM, which were invasive. In the liver of intravenously inoculated mice and lung of intranasally inoculate mice, LI was able to elicit comparable cytokine production with LM and cause less severe histopathologic damages, and thus could be considered as a vector for treating or preventing hepatic or pulmonary diseases.

[Genetic Recombiniation and Protein Expression Detection of Listeria-based tuberculosis Vaccine Candidates.]

OBJECTIVES: Genetic construction of tuberculosis vaccine candidates based on Listeria(L.) monocytogenes,L.ivanovii,and evaluation their protein expression,in order to provide a novel method for research on tuberculosis controlling. METHODS: Two kinds of gene cassettes carrying tuberculosis antigen encoding gene Rv3875 or Rv0129c were inserted into targeting vector harboring L.monocytogenes,L.ivanovii homologous sequences via genetic connection methods and plasmid transformation technology in vitro.Targeting plasmids were electroporated into L.monocytogenes,L.ivanovii,and the recombinant strains were experienced serial passage at 42 °C and 30 °C.Subsequently,the tuberculosis antigen gene cassettes in targeting plasmids were integrated into L.monocytogenes and L.ivanovii attenuated strain (knocking out of virulence gene actA and plcB) and L.ivanovii wild type strain by homologous recombination and gene targeting technology.The recombinant strains were screened by blue-white spot and antibiotic resistance test;the intracellular and extracellular proteins of the recombinant strains were tested by Western blot. RESULTS: Five recombination strains carried antigen gene cassette were constructed,and the recombinant genome were confirmed by PCR and sequencing.No erythromycin resistance gene was found in 5 strains,which was coincident to expection.Recombination strains Li-Rv0129c,Li-ΔactAplcB-Rv0129c and Li-ΔactAplcB-Rv3875 expressed Mycobacterium tuberculosis antigenic protein,Ag85C or ESAT-6,as expected.But L.monocytogenes strains did not express proper antigenic protein. CONCLUSIONS: Three novel L.ivanovii-based tuberculosis vaccine candicates,carrying Mycobacterium tuberculosis Rv0129c antigen gene cassette (coding for Ag85C) or Rv3875 gene cassette (coding for ESAT-6),and expressing relevant antigenic proteins have been successfully selected.