Different Formulations of Inactivated SARS-CoV-2 Vaccine Candidates in Human Compatible Adjuvants: Potency Studies in Mice Showed Different Platforms of Immune Responses.

Several inactivated SARS-CoV-2 vaccines have been approved for human use, but are not highly potent. In this study, different formulations of the inactivated SARS-CoV-2 virus were developed in Alum, Montanide 51VG, and Montanide ISA720VG adjuvants, followed by assessment of immune responses. The SARS-CoV-2 virus was inactivated with formalin and formulated in the adjuvants. BALB/c mice were immunized subcutaneously with 4 µg of vaccines on days 0 and 14; (IL-4) and (IFN-g), cytotoxic T lymphocyte (CTL) activity, and specific immunoglobulin G (IgG) titer and IgG1, IgG2a, and IgG2a/IgG1 ratio, and anti-receptor-binding domain (RBD) IgG response were assessed 2 weeks after the final immunization. Immunization with SARS-CoV-2-Montanide ISA51VG showed a significant increase in the IFN-γ cytokine versus SARS-CoV-2-Alum, SARS-CoV-2-Montanide ISA720VG, and control groups (p < 0.0033). Cytokine IL-4 response in SARS-CoV-2-Alum group showed a significant increase compared with SARS-CoV-2-Montanide ISA51VG, SARS-CoV-2-Montanide ISA720VG, and control groups (p < 0.0206). In addition, SARS-CoV-2-Montanide ISA51VG vaccine induced the highest IFN-γ/IL-4 cytokine ratio versus other groups (p < 0.0004). CTL activity in SARS-CoV-2-Montanide ISA51VG and SARS-CoV-2-Montanide ISA720VG groups showed a significant increase compared with SARS-CoV-2-Alum and control groups (p < 0.0075). Specific IgG titer in SARS-CoV-2-Montanide ISA51 VG and SARS-CoV-2-Montanide ISA720VG showed a significant increase compared with SARS-CoV-2-Alum and control groups (p < 0.0143). Results from specific IgG1and IgG2a in SARS-CoV-2-Alum, SARS-CoV-2-Montanide ISA51VG, and SARS-CoV-2-Montanide ISA720VG vaccine showed a significant increase compared with phosphate buffer saline (PBS) group (p < 0.0001), but SARS-CoV-2-Montanide ISA51VG and SARS-CoV-2-Montanide ISA 720VG groups showed the highest IgG2a/IgG1 ratio and a significant increase compared with SARS-CoV-2-Alum group (p < 0.0379). Moreover, inactivated SARS-CoV-2+Alum and SARS-CoV-2-Montanide ISA 720VG groups demonstrated a significant increase in anti-RBD IgG response versus the SARS-CoV-2-Montanide ISA51VG group. It seems that the type of vaccine formulation is a critical parameter, influencing the immunologic pattern and vaccine potency and human-compatible oil-based adjuvants were more potent than Alum adjuvant in the vaccine formulation.

Genomic pattern analysis of Burkholderia mallei field isolates by pulsed-field gel electrophoresis (PFGE) discriminatory typing.

BACKGROUND AND OBJECTIVES: Glanders is a serious zoonotic disease caused by Burkholderia mallei. Prevention, control, and treatment strategies of glanders are prerequisites for microbial source tracking. The present study was aimed to analyze the genomic pattern of B. mallei Iranian field isolates by pulsed-field gel electrophoresis (PFGE) typing. MATERIALS AND METHODS: B. mallei isolates were aerobically cultured in nutrient broth/agar supplemented with glycerol 4% for 48 h at 37°C. API 20NE identification system was used for the biochemical characterization. Genomic DNA of bacterial isolates was extracted using OIE-recommended protocol. Molecular identification of bacterial isolates was done based on amplification of BimA and IS407-flip genes. PFGE was applied to prepare the genomic pattern of B. mallei isolates. The guinea pig was used as a suitable model for studying the histopathological characterization of B. mallei. RESULTS: In both enzymatic digestion patterns by using Af1II and VspI, we found three different clonal types; І) PFGE type of B. mallei Razi 325 strain, ІІ) PFGE type of Tiger, Kordan, and Oshnavieh strains, and ІІІ) PFGE type of Semirom strain. B. mallei Razi 325 was categorized as unrelated strain which was belonged to the different cluster differing more than four bands. CONCLUSION: PFGE showed more discriminatory power and considerable reproducibility for molecular typing of B. mallei strains in our study. It is standardized the approaches for outbreak detection, pathogen phylogeny, molecular epidemiology, and population studies.

Mycobacterium avium subsp. paratuberculosis and associated risk factors for inflammatory bowel disease in Iranian patients.

BACKGROUND: Inflammatory bowel disease (IBD) is described as a relapsing condition with high morbidity and uncertain complex pathogenesis. The association of Mycobacterium avium ssp. paratuberculosis (MAP) with Crohn's disease (CD) in human has been debated for decades, however there is no confirmed data to verify such relations in Iran. The aim of this study was to investigate risk factors and a possible role of MAP in Iranian patients with CD. METHODS: Anti-MAP antibodies were detected in serum of IBD patients and subjects without IBD (nIBD) through ELISA; MAP DNA and viable MAP cells were identified in patients' biopsies through nested PCR and direct culture methods, respectively. Principal component analysis (PCA) was used to investigate the risk factors in relation to IBD and MAP infection. RESULTS: Positivity for IS900 PCR was detected in 64% (n = 18) of CD, 33% (n = 10) of ulcerative colitis (UC) and 9.7% (n = 6) of nIBD samples. Live MAP cells were isolated from biopsies of 2 CD patients only. Among 28 patients with CD, 46% (n = 13) and 39% (n = 11) were positive for antibodies against MAP3865c133-141 and MAP3865c125-133 peptides, respectively, whereas much lower seroreactivity was detected in UC subjects accounting for 3% (n = 1) for MAP3865c133-141 and 16.7% (n = 5) for MAP3865c125-133. A high immune reactivity to MAP epitopes among CD patients was positively correlated with consumption of fast food meals and IBD familiarity. For both CD and UC, breastfeeding period and consumption of fruit/vegetables presented negative correlation with the presence of anti-MAP antibodies. CONCLUSIONS: This study provided evidences that high prevalence of MAP DNA and anti-MAP antibodies in CD patients is significantly associated with the development of CD. Despite the role of several factors contributing to IBD, the presence of MAP DNA and anti-MAP antibodies in Iranian CD patients highlights a possible transmission of MAP from animal-derived products to humans.

Detection of Mycobacterium avium subsp. paratuberculosis in Iranian patients with type 1 diabetes mellitus by PCR and ELISA.

INTRODUCTION: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis or Johne's disease in ruminants. Its role in triggering autoimmunity, including type 1 diabetes mellitus (T1DM), has been reported in recent years. Due to the high contamination rate of MAP in Iran's livestock and the increasing outbreak of T1DM, we investigated this association in a small group of patients with T1DM in Iran. METHODOLOGY: Blood samples of 29 T1DM patients and 29 healthy control subjects were tested through enzyme-linked immunosorbent assay (ELISA) to detect antibodies against MAP3865c and ZnT8 homologous epitopes and the presence of MAP DNA. Blood samples were also cultured in mycobacterial growth indicator tubes and Herrold's egg yolk medium containing mycobactin J. RESULTS: The results of ELISA showed a significant difference between T1DM patients and healthy group. IS900 was also detected in 51.72% of T1DM patients but in none of the control group. None of the samples grew in culture media. CONCLUSIONS: Due to the presence of MAP DNA and antibodies against MAP peptides in a significant number of T1DM patients compared with healthy control subjects, we may consider MAP as a possible trigger of T1DM in Iran. This indicates that exposure to MAP occurred in the positive subjects. Identifying the sources of contamination and routes of MAP transmission to humans seems necessary to prevent and reduce the burden of MAP infection in Iran.

Extensive Genetic Diversity among Clinical Isolates of Mycobacterium tuberculosis in Central Province of Iran.

Human tuberculosis caused by Mycobacterium tuberculosis (Mtb) remains a significant disease in many countries. According to Iran's borders with Afghanistan and Pakistan, which are among the 22 high burden countries around the world, this study was conducted to analyze the current molecular epidemiology of tuberculosis and survey genetic diversity of Mtb strains in Markazi Province in center of Iran. In this experimental study, 75 sputum specimens and one gastric lavage from all smear-positive TB patients admitted to the public hospitals across the Markazi Province were cultured on specific mycobacterial culture media. Genomic DNA was digested by PvuII and transferred to positively charged nylon membrane by southern blotting method and hybridization by PGRS and DR probes. Genotyping of the isolates by PGRS-RFLP and DR-RFLP displayed a wide range of genetic diversity as 25 and 26 genotypes were identified, respectively. Generally speaking, despite the relatively limited number of isolates in the study, high age of patients and also large heterogeneity found in the setting are both in opposition to active circulation of Mtb strains between patients under study either Iranian or Afghan nationals. Thus, it seems that reactivation of latent infection has had the main role in the spread of tuberculosis.