Molecular Cloning and Immunogenicity Evaluation of PpiC, GelE, and VS87_01105 Proteins of Enterococcus faecalis as Vaccine Candidates

Background: Among the enterococci strains, Enterococcus faecalis is considered as one of the important nosocomial pathogens affecting immunocompromised patients. In this study, the immunogenicity of PpiC, GelE, and VS87_01105 proteins against enterococcal infection was investigated in a mice model. Methods: The genes encoding these proteins were cloned into pET21a expression vector, and the recombinant proteins were produced. Mice and rabbits were immunized with the purified recombinant proteins, and subsequently, mice were challenged with E. faecalis for the evaluation of their survival and bacterial clearances. The antibody responses to recombinant proteins were determined by ELISA assay, and opsonophagocytic activities of the antibodies were also measured. Passive immunization was performed using purified antibodies. Mice were challenged, and their survival and bacterial clearance were determined. Results: Immunized mice with PpiC, GelE, and VS87_01105 recombinant proteins showed 80%, 70%, and 40% survival rate, respectively. The survival rates among passively immunized mice that received 500 µg of IgG fraction in 100 µl PBS buffer of each of anti-PpiC, anti-GelE, and anti-VS87_01105 were 60%, 50%, and 20%, respectively. The rates of opsonization with anti-PpiC, anti-GelE, and anti-VS87_01105 antibodies at 1/10 dilution were 77%, 64%, and 23%, respectively. Conclusion: Based on our findings, PpiC, and GelE proteins can protect the mice against E. faecalis ATCC 29212 and effectively induce a protective antibody response. Thus, these proteins could be used as an additional therapeutic tool against enterococcal infections. Further studies to determine the role of PpiC in ligand binding and demonstration of epitope mapping may establish a credible target for vaccination.

Vaccine potential of LenA and LcpA proteins of Leptospira interrogans in combination with Escherichia coli heat-labile enterotoxin, B subunit (LTB).

BACKGROUND AND OBJECTIVES: Leptospirosis is a zooanthroponosis caused by the genus of Leptospira. It is an emerging public health problem due to its increasing incidence. The achievement to a vaccine that prevent from entrance of Leptospira interrogans to the deeper tissues of the host is needed. This study aimed to investigate the immunogenicity of LcpA (rLcpA) and LenA (rLenA) recombinant proteins in combination with LTB (rLTB) recombinant protein as an adjuvant against leptospiral infection in hamsters. MATERIALS AND METHODS: The genes encoding these proteins were cloned into pGH cloning vector and then lenA, lcpA and ltb genes subcloned into pET-15b and pET-28a expression vectors, respectively. The hamsters were immunized with the purified recombinant proteins and challenged with Leptospira interrogans for evaluation of their survival. The antibody responses to the recombinant proteins were determined by ELISA. Then, data entered into SPSS software. Statistical Kruskal-Wallis test was used to compare the significant differences among different groups. The groups with significant differences were further analyzed by post hoc tests. The p value < 0.05 statistically was considered significant. RESULTS: Immunized hamsters with rLenA-plus-rLTB, rLcpA-plus-rLTB and rLenA-plus-rLcpA-plus-rLTB proteins showed 60%, 74%, and 80% survival rates, respectively. A significant amount of interleukin-17 (IL-17), interleukin-4 (IL-4) and gamma interferon (IFNγ) cytokines were produced in immunized hamsters. CONCLUSION: Based on our findings, rLcpA and rLenA proteins in combination with rLTB can protect the hamsters against L. interrogans and effectively induce a protective antibody response. Thus, these proteins can be used as an additional prophylactic tool against leptospira.

CbpM and CbpG of Streptococcus Pneumoniae Elicit a High Protection in Mice Challenged with a Serotype 19F Pneumococcus.

Among many pneumococcal antigens, choline-binding proteins (CPBs) display a high immunogenicity in animal models. This study aims to determine the immunogenicity of CbpM, CbpG and CbpL proteins of Streptococcus pneumoniae in a mice model. The genes were cloned into pET21a expression vector and the recombinant proteins were produced. Mice were immunized with the purified recombinant proteins. Subsequently, the mice were challenged with S. pneumoniae ATCC 49619 (2×106 CFU) and their survival and bacterial clearances were followed 24 hours after infection. The antibody responses of the mice were determined by ELISA assay. The opsonophagocytosis assay was performed using rabbit's sera. Passive immunization was carried out using two doses of anti-CbPs antibodies. Finally, these mice were  experimentally infected with virulent bacteria and the protective effects of two doses of 10 and 100 µg/mL by monitoring the survival rate and bacterial clearance were determined at 2, 3 and 7 days after bacterial challenge. The mice actively immunized with CbpM, CbpG and CbpL recombinant proteins showed survival rate of 100%, 85% and 75%, respectively. The survival rates among passively immunized mice groups which received 100 µg/mL dose of anti-CbpM, anti-CbpG and anti- CbpL were 50%, 50% and 25%, respectively. The rates of opsonization with rabbit's antibodies against CbpM, CbpG, and CbpL  at 100 µg/mL doses was 45.6%, 14.7% and 82.3%, and at 10 µg/mL was 12.9%, 12.2% and 9.35%, respectively. Our findings suggest that the recombinant proteins particularly CbpM and CbpG can protect the mice against pneumococcus19F serotype and effectively induce a protective antibody response. Thus, CbpG and CbpM proteins might be used as suitable vaccine candidate in pneumococcal vaccine formulations.

Molecular characterization of the first community-acquired methicillin-resistant Staphylococcus aureus strains from Central Iran.

BACKGROUND: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has spread throughout the world with varying regional incidences and different staphylococcal cassette chromosome mec (SCCmec) elements in different genetic backgrounds. No information is available on CA-MRSA in Iran. A cross-sectional study was carried out among healthy students to investigate: (1) the prevalence of CA-MRSA in Central Iran, (2) the molecular epidemiology of such CA-MRSA strains, (3) the antimicrobial resistance patterns of the strains, and (4) the distribution of virulence genes in these CA-MRSA strains. METHODS: A total of 700 nasal swabs were collected and subjected to S. aureus and MRSA-specific isolation procedures. Antimicrobial resistance patterns were determined using the disk diffusion method, and molecular typing was carried out by multi-locus sequence typing (MLST), SCCmec typing, and Staphylococcus protein A (spa) typing for all CA-MRSA isolates. PCR was used to detect various virulence genes. RESULTS: One hundred fifty-four S. aureus strains were isolated from the anterior nares of 700 healthy students. According to the US Centers for Disease Control and Prevention definitions for CA-MRSA, seven (4.5%) isolates were confirmed as CA-MRSA. CA-MRSA isolates belonged to SCCmec types IV (n = 6) and V (n = 1). The predominant spa-type among the CA-MRSA isolates was t790 (n = 3), with single t660, t084, and t325 isolates; one isolate was not typeable. The predominant sequence type was ST22, t790, SCCmec IV in three isolates, and the four other sequence types were ST25, ST859, ST14, and ST15. CONCLUSIONS: Iranian CA-MRSA strains are genetically diverse with an elevated prevalence of t790/ST22 SCCmec IV isolates. These findings support the need for more effective infection control measures to reduce nasal carriage and prevent dissemination of CA-MRSA in Iran.