Porcine anti-human lymphocyte immunoglobulin depletes the lymphocyte population to promote successful kidney transplantation.

Introduction: Porcine anti-human lymphocyte immunoglobulin (pALG) has been used in kidney transplantation, but its impacts on the lymphocyte cell pool remain unclear. Methods: We retrospectively analyzed 12 kidney transplant recipients receiving pALG, and additional recipients receiving rabbit anti-human thymocyte immunoglobulin (rATG), basiliximab, or no induction therapy as a comparison group. Results: pALG showed high binding affinity to peripheral blood mononuclear cells (PBMCs) after administration, immediately depleting blood lymphocytes; an effect that was weaker than rATG but stronger than basiliximab. Single-cell sequencing analysis showed that pALG mainly influenced T cells and innate immune cells (mononuclear phagocytes and neutrophils). By analyzing immune cell subsets, we found that pALG moderately depleted CD4+T cells, CD8+T cells, regulatory T cells, and NKT cells and mildly inhibited dendritic cells. Serum inflammatory cytokines (IL-2, IL-6) were only moderately increased compared with rATG, which might be beneficial in terms of reducing the risk of untoward immune activation. During 3 months of follow-up, we found that all recipients and transplanted kidneys survived and showed good organ function recovery; there were no cases of rejection and a low rate of complications. Discussion: In conclusion, pALG acts mainly by moderately depleting T cells and is thus a good candidate for induction therapy for kidney transplant recipients. The immunological features of pALG should be exploited for the development of individually-optimized induction therapies based on the needs of the transplant and the immune status of the patient, which is appropriate for non-high-risk recipients.

[Immunogenicity of attenuated Salmonella choleraesuis vaccine strain expressing immunogenic genes of Mycoplasma hyopneumoniae in mice].

OBJECTIVE: The study was carried out to construct and characterize Salmonella choleraesuis vaccine strain expressing immunogenic genes of Mycoplasma hyopneumoniae and to test its immunogenicity in mice. METHOD: We made p36, p46, p65 and p97R1-Nrdf, the main immunogenic genes of Mycoplasma hyopneumoniae, to insert into the prokaryotic expression plasmid pYA3493. Then these recombinant plasmids and pYA3493 were electroporated into C500 asd-mutant, resulting in the recombinant Salmonella choleraesuis vaccine strains C36 (pYA-36), C46 (pYA-46), C65 (pYA-65), C97R1-Nrdf(pYA-97R1-Nrdf) and CpYA(pYA3493). We characterized these recombinant Salmonella choleraesuis vaccine strains and tested the immunogenicity in mice by intramuscular injection or orally immunized. RESULT: The results of the immunogenicity in mice indicated that the group orally immunized with C36, C46, C65, C97R1-Nrdf showed significantly higher Mycoplasma pneumoniae antibody than both the group orally immunized with C36, C46, C65 and the group intramuscular injected with the Mycoplasma hyopneumoniae bacterin (M + PAC) (P < 0.01). The group intramuscular injected with C36, C46, C65 showed higher IFN-gamma production than the group injected with the Mycoplasma hyopneumoniae bacterin (M + PAC) (P < 0.05), but there was no significant difference between the group orally immunized with C36, C46, C65 and the group orally immunized with C36, C46, C65, C97R1-Nrdf (P > 0.05). The highest level of IL-4 was found in the group orally immunized with C36, C46, C65; higher levels of IL-4 was observed in the group orally immunized with C36, C46, C65, C97R1-Nrdf than the group injected with the Mycoplasma hyopneumoniae bacterin (M + PAC); and the lowest IL-4 level was found in the group injected with C36, C46, C65. There were no significant differences among them (P > 0.05). The Mycoplasma pneumoniae antibody, IFN-gamma or IL-4 production of the each group was obviously higher than the control group (P < 0.01). CONCLUSION: The attenuated Salmonella choleraesuis vaccine strain expressing immunogenic genes of Mycoplasma hyopneumoniae which has immunogenicity in mice especially by intramuscular injection could probably serve as a vaccine against mycoplasmal pneumonia of swine.

Attenuated Actinobacillus pleuropneumoniae as a bacterial vector for expression of Mycoplasma hyopneumoniae P36 gene.

BACKGROUND: Actinobacillus pleuropneumoniae and Mycoplasma hyopneumoniae are causative agents of porcine pneumonia. Over the last few years, attenuated A. pleuropneumoniae live vaccines have been shown to provide protection against A. pleuropneumoniae infection. We postulated that attenuated A. pleuropneumoniae could additionally be used as a vaccine vector for protection against M. hyopneumoniae. METHODS: A mutant strain of A. pleuropneumoniae, SLW36, was constructed by replacing the urease structural gene of mutant strain SLW03 of A. pleuropneumoniae with the L-lactate dehydrogenase gene (p36) of M. hyopneumoniae by transconjugation and counter selection. The urease function and the growth kinetics of SLW36 were measured. Protein expression of P36 was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and western blotting. The attenuated virulence and immunity of SLW36 were analyzed in a mouse model. RESULTS: The mutant strain SLW36 was urease negative and four-fold less virulent than the parental strain SLW03. There were no differences in expression levels of p36 at different culture time-points and the foreign gene was stable after in vitro passage. Immunoglobulin G responses against p36 antigen and M. hyopneumoniae whole-cell antigen were detected. CONCLUSIONS: The mutant strain SLW36 can induce antibody against p36 and M. hyopneumoniae. The mutant strain SLW36 has the potential to be used as a live vaccine for protection against A. pleuropneumoniae and M. hyopneumoniae. Studies in pigs are needed to confirm protective levels of antibodies and to check for rare side-effects of the vaccine.

[Construction and characterization of an attenuate strain apxIC-/P36+ of Actinobacillus pleuropneumoniae serovar 10].

OBJECTIVE: To construct an attenuate Actinobacillus pleuropneumoniae serovar 10 strain apxIC-/P36+ for new vaccine development. METHODS: The mutant was constructed by transconjugation and counter-selection and then verified by PCR, western blot and sequence analysis. A transconjugation plasmid pEICALDH was constructed and transformed into donor strain Escherichia coli X7213. After mixing the donor cells with A. pleuropneumoniae acceptor cells, we cultivated the mixture for 6 hours and plated on solid medium containing chloromycetin. Then the Cm(R) positive clones were picked and inoculated into liquid medium without any antibiotic. Cultures were pelleted, plated on sucrose plates and incubated overnight. Finally, Sucrose-resistant colonies (SucB(R)) were selected and considered as mutant. RESULTS: Compared with parental strain, the mutant have the same growth rate in vitro and reduced virulence in mice; additionally, the animal experiment indicated that the mutant strain can successfully induce as good immune response as the parental strain, despite of deletion of apxIC gene. CONCLUSION: In conclusion, we successfully constructed the attenuate strain apxIC-/P36+ of Actinobacillus pleuropneumoniae serovar 10, and this mutation system will facilitate development of live attenuated vaccines.