A potent subset of Mycobacterium tuberculosis glycoproteins as relevant candidates for vaccine and therapeutic target.

Tuberculosis (TB) remains one of the most afflictive bacterial infections globally. In high burden TB countries, surveillance, diagnosis and treatment of drug resistant TB (RR and X/MDRTB) display a crucial public health challenge. Therefore, we need new TB vaccines; diagnostic and therapeutic strategies to briskly prevent disease promotion; reduce drug-resistant TB and protect everyone from disease. The study identified various potent membrane and cell wall M. tuberculosis glycolipoproteins that are relevant for diagnostics, drug and vaccine discovery. A M. tuberculosis Proskauer and Beck broth culture was extracted for total proteins by ammonium sulfate method. After ConA-Affinity Chromatography reputed glycoproteins were collected followed by 2DE gel electrophoresis and LC Mass spectrometry. A total of 293 glycoproteins were identified using GlycoPP and IEDB database. Probable conserved trans-membrane protein (Rv0954), LpqN (Rv0583), PPE68 (Rv3873), Phosphate-binding protein (Rv0932c), PPE61 (Rv3532) and LprA (Rv1270c), had the highest glycosylation percentage value with 13.86%, 11.84%, 11.68%, 11.1%, 10.59% and10.2%, respectively. Our study discloses several dominant glycoproteins that play roles in M. tuberculosis survival, and immunogenicity. These include glycoproteins involved in antigenicity, transport and biosynthesis of M. tuberculosis cell envelope, pathogen-host interaction and drug efflux pumps, which are considered as a feasible drug targets or TB new vaccine candidates.

Anticancer Activity of Kefir on Glioblastoma Cancer Cell as a New Treatment.

Kefir drink is one of the most important probiotic products, which is made using kefir microorganisms in fermenting the milk. Numerous investigation have been accomplished in the field of the therapeutic property of probiotic products. In the present study, we assessed the cytotoxic effect of kefir on the rate of growth and increase of glioblastoma cancer cell as the most severe form of brain tumors. In this experimental study, we used a U87 cancer cell line (glioblastoma). The interaction between cancer cells and different concentrations of kefir drink and supernatants at 24 and 48 hours was considered. The cell cytotoxicity of kefir and sedimentation of cell lysate and extract of kefir was assessed using the MTT test after 24 and 48 hours. The result of the MTT test, treatment of the cells with the 48-hour fermented drink, demonstrated the most cell cytotoxicity in comparison with the control group. Results showed that the toxicity effect in all groups was dose-dependent, and by increasing the concentration, cell survival decreased noticeably. The results indicated that the supernatant of fermented kefir drink as a probiotic product has more toxicity and lethality effect on the glioblastoma cancer cell. This product can be utilized as a replacement or a complementary therapy of cancer.

Construction and assessment of the immunogenicity and bactericidal activity of fusion protein porin A from Neisseria meningitidis serogroups A and B admixed with OMV adjuvant as a novel vaccine candidate.

OBJECTIVES: The porins A and B and also outer membrane vesicles (OMVs) of Neisseria meningitidis are used for vaccine purposes. In the present study, we aimed to design a new vaccine candidate based on a fusion of PorA of serogroups A and B of N. meningitidis admixed with OMV and evaluate it in an animal model. MATERIALS AND METHODS: After bioinformatic studies, a fusion protein composed of porin A from both serogroups A and B of N. meningitidis was constructed, expressed, and purified by nickel resins. Extraction of OMV of N. meningitidis was performed using a chemical method. The mice were vaccinated subcutaneously in different groups with mixtures of PorA proteins, OMV, and Freund's adjuvants. Then, the immune responses were measured using the ELISA method. Finally, serum bactericidal activity (SBA) procedure was applied to assay the activity of the immune responses in mice. RESULTS: Mice received the PorA protein plus Freund's adjuvant. Mice vaccinated with PorA fusion of serogroups A+B plus Freund's adjuvant produced more IgG, IgG1, and IgG2a than combinations admixed with OMV. Furthermore, the vaccinated mice tended to direct the IgG responses toward IgG1. Sera of the mice that received PorA+Freund's and those that received PorA+OMV produced higher bactericidal activity than the controls. CONCLUSION: Fusion protein porin A could be a valuable target for developing vaccines against N. meningitidis. Although, Freund's adjuvant induced the strongest IgG responses, given that Freund's adjuvant has no human use, and OMV is a human adjuvant, OMV could be considered in vaccine design against N. meningitidis.

Evaluation of protective immunity responses against pneumococcal PhtD and its C-terminal in combination with outer-membrane vesicles as adjuvants.

Introduction. Streptococcus pneumoniae is a significant bacterial pathogen in humans. Currently, there are two types of pneumococcal vaccines, but there are concerns regarding their application.Aim. Since many pneumococcal proteins are serotype-independent, polyhistidine triad protein D (PhtD) has been selected as a vaccine candidate.Methodology. We prepared recombinant PhtD and its C-terminal fragment (PhtD-C) using alum and outer-membrane vesicles (OMVs) as adjuvants. The combinations were injected intraperitoneally into mice, and then total immunoglobulin G (IgG) and specific IgG, IgG1 and IgG2a were measured. A serum bactericidal assay and opsonophagocytosis were also performed as complementary tests. Meningococcal OMVs were used as an adjuvant.Results. The levels of specific IgG and IgG1 against combinations of PhtD and its C-terminal with OMVs and alum as adjuvants increased at the time of the third mouse immunization on day 35. Forty per cent and 60% of S. pneumoniae ATCC 6303 (serotype 3) as a virulent pneumococcal strain, respectively, were killed in the opsonophagocytosis test and these results could also be observed in the serum bactericidal assay. Mice mmunized iwith PhtD and its C-terminal with OMVs and alum as adjuvants survived after 10 days of pneumococcal challenge.Conclusion. The combination of PhtD and PhtD-C with alum produced optimal results, but the combination of PhtD and PhtD-C with OMVs produced minimal results by comparison. The survival rates were also measured, and these corresponded with the results of the immunological assessments. Our findings showed that mice receiving PhtD and PhtD-C plus OMV and alum had higher survival rates than the mice in the other groups.

Evaluation of immunological responses against outer membrane vesicles (OMV) of nontypeable Haemophilus influenzae using MPLA-CpG adjuvant as a vaccine candidate.

BACKGROUND AND OBJECTIVES: Nontypeable Haemophilus influenzae (NTHi) are major causes of non-invasive infections, including otitis media and sinusitis and it can also contribute to respiratory infections of all ages. Currently, there is no licensed vaccine against NTHi commercially available. Many studies have been conducted on the use of OMV as a vaccine against NTHi. The purpose of this study is to achieve an immunogenic vaccine against NTHi. MATERIALS AND METHODS: In this study, standard OMV Haemophilus (ATCC49766) with adjuncts CpG and MPLA was used and after infusion into BALB/c mice, the levels of antibodies and cytokines were measured on serum of immunized mice. RESULTS: The results showed that total IgG antibody and IgG1 and IgG2a isotypes in OMV immunized mice with mixture of CpG-MPLA adjuvant had a significant increase. Also, the results of cytokines (IL-10, IL-4 and IFN-γ) showed that IL-4 had the highest rate. CONCLUSION: These findings indicate that OMVs derived from NTHi strains have a high potential to act as a vaccine against NTHi infections.

In silico design, cloning, expression and immunologic evaluation of ED fusion protein of NT H. influenzae.

Infections due to nontypeable Haemophilus influenzae (NTHi) are important causes of child mortality throughout the world. Given the lack of effective vaccines for these strains and the spread and prevalence of these infections in the world, it is necessary to design novel vaccine candidates against these strains. D and E proteins are conserved membrane-specific lipoproteins among encapsulated and non-encapsulated H. influenza strains, which, according to the exposure surface and conservation degree between both strains, can be considered as vaccine candidates suitable for studies. This research was conducted to design a recombinant truncated fusion protein ED. Vaccination of BALB/c mice with recombinant truncated fusion protein ED showed high level of protective responses against NTHi. There were also strong responses of IgG and its subclasses (especially IgG1) as well as high titer levels of IL-4. A mixture of responses was observed considering IgG2a and INF-γ antibody titers, but the dominant response was toward Th2. According to the obtained results and the importance of humoral immunity in the immune system and vaccines production, it could be concluded that the produced recombinant construct can be used as a suitable vaccine candidate against NTHi or together with other carrier proteins.

Evaluation of immunological responses to recombinant Porin A protein (rPoA) from native strains of Neisseria meningitidis serogroups A and B using OMV as an adjuvant in BALB/c mice.

Neisseria meningitidis is one of the main causes of sepsis and meningitis, which are two serious life-threatening diseases in both children and adolescents. Porin A (porA) from both serogroup A and B were cloned into the pET28a plasmid and expressed in E. coli BL21 (DE3). The protein was expressed in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western blot analysis. BALB/c mice were subcutaneously injected three times with 25 µg of the recombinant PorA. Specific total IgG antibodies and isotypes were evaluated using ELISA assay. Opsonophagocytic assay (OPA) and Serum Bactericidal assay (SBA) were performed. Results showed that vaccinated mice exhibited higher levels of anti-Porin A (p < 0.05) with a predominant IgG1 response compared to the control group. Results from in vitro experiments indicated that N. meningitidis was opsonized with immunized-mice sera, and compared to non-immunized mice, immunized mice displayed significantly increased phagocytic uptake and effective intracellular killing. In this study, serogroup B N. meningitidis OMV of strain CSBPI G-245 and complete and incomplete Freund's adjuvant were used. Results demonstrated that Porin A could be a valuable target for the development of immunotherapeutic strategies against N. meningitidis.

Distribution of Class I Integron and smqnr Resistance Gene Among Stenotrophomonas maltophilia Isolated from Clinical Samples in Iran.

BACKGROUND: Stenotrophomonas maltophilia (S. maltophilia) is a multiple-antibiotic-resistant opportunistic pathogen that is being isolated with increasing frequency from patients with health-care-associated infections. S. maltophilia is inherently resistant to most of the available antimicrobial agents. Spread of resistant strains has been attributed, in part, to class I integrons. In vitro susceptibility studies have shown trimethoprim-sulfamethoxazole and new floroquinolones as two important agents with activity against these organisms. METHODS: 150 isolates of S. maltophilia were isolated from clinical samples such as respiratory discharges, sputum, and catheter and hospital environments. These isolates were also subjected to susceptibility testing and polymerase chain reaction for four groups of genes including int encoding integron elements, sulI and sulII encoding trimethoprim-sulfamethoxazole resistance and smqnr encoding quinolone resistance. RESULTS: The rate of resistance to trimethoprim-sulfamethoxazole was up to 27 (18%) and the highest resistance to quinolone family belonged to ofloxacin (20%) and the lowest rate was for gatifloxacin (16%). The results showed that 14% of isolates contained integron elements concomitantly with sulI and sulII genes. CONCLUSION: Resistance rate of S. maltophilia to co-trimoxazole and fluoroquinolones and detection of integron elements between isolates in this study showed that this rate corresponded to other data obtained from other studies.

Recombinant truncated E protein as a new vaccine candidate against nontypeable H. influenzae: Its expression and immunogenic evaluation.

Protein E (PE) is a conserved entity observed in both nontypeable Haemophilus influenzae (NTHi) and encapsulated H. influenzae. This is a small surface lipoprotein, consisting of only 160 amino acids, involved in the adhesion of H. influenzae to various types of epithelial cells. A 384-bp-long fragment from NTHi PE was cloned into the prokaryotic expression vector pBAD-gIIIA. The recombinant protein was expressed with arabinose and then purified by affinity purification on an Ni-NTA agarose matrix. BALB/c mice were immunized by subcutaneous injection with purified recombinant truncated PE mixed with an alum adjuvant. Serum antibody response and the functional activity of antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA) and serum bactericidal assay (SBA), respectively. Colony PCR, double digestion, and sequencing were used to verify successful cloning of truncated PE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses indicated the presence of a ∼15-kDa recombinant protein. Serum IgG, IgG1, and IgG2a levels were significantly higher in the group immunized by recombinant truncated PE mixed with an alum adjuvant, compared to the non-vaccinated control group. Development of a strong bactericidal effect against NTHi was observed in the serum samples from immunized animals. Our findings suggest that recombinant truncated PE is a potential vaccine candidate for NTHi.

A comparative study of various methods for detection of IL28B rs12979860 in chronic hepatitis C.

Interleukin-28B (IL28B) single-nucleotide polymorphisms (SNPs) constitute important host-related factors influencing the response rate to Hepatitis C virus (HCV) standard antiviral therapy. In the last few years, several new technologies for SNP detection have been developed. However, the sensitivity and specificity of various methods are different and needs evaluation. Five different methods (resolution melting curve [RMC], polymerase chain reaction-restriction fragment length polymorphism [PCR-RFLP], PCR-sequencing analysis, amplification refractory mutation system [ARMS], and zip nucleic acid probe-based real-time PCR [ZNA]) were developed for genotyping rs12979860 associated with IL28B. In this study, limit of detection (LD), costs and turnaround time of these methods were compared in 350 subjects. As for IL28B rs12979860 polymorphisms, 348/350 (99.4%) samples were consistent among the five methods, while results for 2/350 (0.57%) samples were concordant by ZNAs and PCR-sequencing, and discordant by other methods. Without considering the cost of DNA extraction, the price of each reaction for ARMS-PCR, RMC, PCR-RFLP, ZNA and PCR-sequencing were respectively: US$3.10, US$5.0, US$5.50, US$8.50 and US$17.0. RMC was the fastest method, while the ZNA method was easy to use, reliable and effective. Lower LD was determined to be 50-60 copies/µL for the PCR-RFLP, RMC and ARMS-PCR assays; whilst ZNA assay was able to detect 2-3 copies/µL. In conclusion, in the current study, all four methods are suitable for IL28B rs12979860 genotyping, but the ZNA assay can be a reliable tool. Due to its lower LD for SNP identification, this method is better than others for detecting this type of polymorphism.

Vaccine Candidates against Nontypeable Haemophilus influenzae: a Review

Nonencapsulated, nontypeable Hemophilus influenzae (NTHi) remains an important cause of acute otitis and respiratory diseases in children and adults. NTHi bacteria are one of the major causes of respiratory tract infections, including acute otitis media, cystic fibrosis, and community-acquired pneumonia among children, especially in developing countries. The bacteria can also cause chronic diseases such as chronic bronchitis and chronic obstructive pulmonary disease in the lower respiratory tract of adults. Such bacteria express several outer membrane proteins, some of which have been studied as candidates for vaccine development. Due to the lack of effective vaccines as well as the spread and prevalence of NTHi worldwide, there is an urgent need to design and develop effective vaccine candidates against these strains.

Prevalence of Hepatitis G Virus Among Hemodialysis and Kidney Transplant Patients in Khuzestan Province, Iran.

BACKGROUND: Hepatitis G virus (HGV) is a member of Flaviviridae. Prevalence of HGV in healthy people is very low, but this virus is more prevalent in patients with hepatitis. Besides, relative frequency of HGV in patients undergoing hemodialysis, and kidney recipients is very high. The role of HGV in pathogenesis is not clear. Since this virus cannot be cultivated, molecular techniques such as Revers Transcription Polymerase Chain Reaction (RT-PCR) is applied to detect HGV. OBJECTIVES: The current study aimed to investigate the prevalence of HGV using determination of E2, viral envelope antigen, antibodies and the RNA by Enzyme Linked Immunosorbent Assay (ELISA) and RT-PCR techniques. The rational of the study was to determine the prevalence of HGV in patients undergoing hemodialysis and kidney transplantation in Khuzestan province, Iran. PATIENTS AND METHODS: Five hundred and sixteen serum samples of the patients undergoing hemodialysis and kidney transplantation from various cities of Khuzestan province were collected. Anti-hepatitis G E2 antibodies were investigated by ELISA method. RNAs were extracted from serums and Hepatitis G RNA was detected by RT-PCR. RESULTS: Of the 516 samples, 38 (7.36%) specimens were positive for anti-HGV by ELISA. All of these ELISA positive samples were negative for HGV genome by RT-PCR. Of the remaining 478 ELISA negative samples, 16 (3.14%) samples were positive by RT-PCR. CONCLUSIONS: Hepatitis G Virus was not prevalent in the patients undergoing hemodialysis and kidney transplantation in Khuzestan province. Although reports indicated high frequency of co-infection of HGV with hepatitis B and C viruses, in the current research, co-infection of HGV with B and C was not considerable. Since different groups and subtypes of HGV are reported, periodic epidemiologic evaluation of HGV and its co-infection with other hepatitis viruses is suggested in other populations such as the patients with thalassemia; however, periodic epidemiologic monitoring of HGV may be helpful to control future potential variations of the virus.