SARS-CoV-2 Specific Humoral Immune Responses after BNT162b2 Vaccination in Hospital Healthcare Workers.

BACKGROUND: COVID-19 pandemic has led to a loss of human life in millions and devastating socio-economic consequences worldwide. So far, vaccination is the most effective long-term strategy to control and prevent severe COVID-19 disease. The aim of the current study was to evaluate the humoral immune responses raised against the BNT162b2 vaccine in hospital healthcare workers. METHODS: Total number of 173 healthcare workers enrolled in the study. Their blood samples were collected in three different time intervals after the second SARS-CoV-2 vaccination and evaluated by the ELISA method to detect anti-spike protein IgM and IgG antibodies. The baseline characteristics of all participants were collected using questionnaires and were evaluated for finding any significant data. RESULTS: Our results demonstrated that the levels of antibodies were higher in the young group (21-30 years old) and also among male participants. Moreover, the highest levels of antibodies were detected from the group that received the third shot vaccination. CONCLUSIONS: Our results indicate that age, gender and third-dose vaccination can affect the levels of humoral immune responses against the BNT162b2 vaccine in healthcare workers.

Filamentous Hemagglutinin of Bordetella pertussis Does Not Interact with the ß2 Integrin CD11b/CD18.

The pertussis agent Bordetella pertussis produces a number of virulence factors, of which the filamentous hemagglutinin (FhaB) plays a role in B. pertussis adhesion to epithelial and phagocytic cells. Moreover, FhaB was recently found to play a crucial role in nasal cavity infection and B. pertussis transmission to new hosts. The 367 kDa FhaB protein translocates through an FhaC pore to the outer bacterial surface and is eventually processed to a ~220 kDa N-terminal FHA fragment by the SphB1 protease. A fraction of the mature FHA then remains associated with bacterial cell surface, while most of FHA is shed into the bacterial environment. Previously reported indirect evidence suggested that FHA, or its precursor FhaB, may bind the ß2 integrin CD11b/CD18 of human macrophages. Therefore, we assessed FHA binding to various cells producing or lacking the integrin and show that purified mature FHA does not bind CD11b/CD18. Further results then revealed that the adhesion of B. pertussis to cells does not involve an interaction between the bacterial surface-associated FhaB and/or mature FHA and the ß2 integrin CD11b/CD18. In contrast, FHA binding was strongly inhibited at micromolar concentrations of heparin, corroborating that the cell binding of FHA is ruled by the interaction of its heparin-binding domain with sulfated glycosaminoglycans on the cell surface.

Multiple Sclerosis Patients and Disease Modifying Therapies: Impact on Immune Responses against COVID-19 and SARS-CoV-2 Vaccination.

This article reviews the literature on SARS-CoV-2 pandemic and multiple sclerosis (MS). The first part of the paper focuses on the current data on immunopathology of SARS-CoV-2 and leading vaccines produced against COVID-19 infection. In the second part of the article, we discuss the effect of Disease Modifying Therapies (DMTs) on COVID-19 infection severity or SARS-CoV-2 vaccination in MS patients plus safety profile of different vaccine platforms in MS patients.

Evaluation of Poly(I:C) and combination of CpG ODN plus Montanide ISA adjuvants to enhance the efficacy of outer membrane vesicles as an acellular vaccine against Brucella melitensis infection in mice.

Brucellosis is the most common zoonotic disease worldwide and still there is no vaccine for human use. The commercial animal vaccines also have major problems that limit their use. Therefore, there is a need for an effective Brucella vaccine which is multivalent and produces a good protective immunity with minimal disadvantages. Due to their heterogeneous composition and diverse functions, OMVs are promising acellular vaccine candidates against brucellosis. In the present study, the potential of Poly(I:C) or CpG ODN 1826+ Montanide ISA 70 VG adjuvant formulations were evaluated to enhance the immunity and protection levels conferred by OMVs against Brucella challenge in mice. The results indicated that both vaccine regimens were able to induce strong Th1-biased responses and confer protective levels significantly higher than REV.1 live vaccine. With regard to the results, it is concluded that OMVs in either adjuvant can be introduced as a new vaccine candidate against B. melitensis infection.

Comparison of the protective immunity elicited by a Brucella cocktail protein vaccine (rL7/L12+rTOmp31+rSOmp2b) in two different adjuvant formulations in BALB/c mice.

In the present study, protective efficacy conferred by a cocktail protein consisted of Brucella L7/L12 ribosomal, truncated outer membrane protein 31 (TOmp31) and SOmp2b recombinant proteins in CpG ODN 1826+ Montanide ISA 70VG or Poly (I:C) adjuvants was evaluated and compared in BALB/c mice. Immunization of mice with both vaccine regimens elicited strong specific IgG responses (higher IgG2a titers over IgG1 titers), provided T helper1 (Th1) oriented immune responses and conferred protection levels compatible to the live vaccines against Brucella challenge. Vaccination of BALB/c mice with the cocktail protein in CpG ODN 1826+ Montanide ISA 70 V G adjuvants induced higher levels of antibody, IFN-γ/IL-2 and conferred more protection levels against B. melitenisis and B. abortus challenge than did the cocktail protein in Poly (I:C) formulation. In conclusion, both vaccine regimens are capable of stimulating specific Th1- biased immune responses and conferring cross protection against B. melitensis and B. abortus infections. Therefore, they could be introduced as new potential candidates for the development of subunit vaccines against Brucella infection.

In silico Design, and In vitro Expression of a Fusion Protein Encoding Brucella abortus L7/L12 and SOmp2b Antigens.

BACKGROUND: L7/L12 is a protective antigen conserved in main Brucella pathogens and is considered as potential vaccine candidate. Outer membrane protein 2b is an immunogen conserved in all Brucella pathogens. MATERIALS AND METHODS: The purpose of the current study was to in silico design a L7/L12-SOmp2b fusion protein and in vitro production of the chimera. Two possible fusion forms, L7/L12-SOmp2b and SOmp2b-L7/L12, were subjected to in silico modeling and analysis. Cloning and expression of the fusion protein has been done in the pET28a vector and Escherichia coli Bl21 (DE3), respectively. RESULTS: Analysis and validation of the fusion proteins three-dimensional models showed that both models are in the range of native proteins. However, L7/L12-SOmp2b structure was more valid than the SOmp2b-L7/L12 model and subjected to in vitro production. The major histocompatibility complex II (MHC-II) epitope mapping using Immune Epitope DataBase indicated that the model contained good MHC-II binders. The L7/L12-Omp2b coding sequence was cloned in pET28a vector. The fusion was successfully expressed in E. coli BL21 by induction with isopropyl-ß-d-thiogalactopyranoside. The rL7/L12-SOmp2b was purified with Ni-NTA column. The yield of the purified rL7/L12-SOmp2b was estimated by Bradford method to be 240 µg/ml of the culture. Western blot analysis revealed a specific reactivity with purified rL7/L12-SOmp2b produced in E. coli cells and showed the expression in the prokaryotic system. CONCLUSIONS: Our data indicates that L7/L12-SOmp2b fusion protein has a potential to induce both B- and T-cell-mediated immune responses and it can be evaluated as a new subunit vaccine candidate against brucellosis.

A review of Brucellosis in Iran: Epidemiology, Risk Factors, Diagnosis, Control, and Prevention

Brucellosis caused by species of Brucella is among the most prevalent zoonoses with the annual incidence of half a million cases globally. Most parts of Iran are endemic for brucellosis, and the annual incidence of the human and animal brucellosis is still high. At present, there is no safe and protective human vaccine against brucellosis, and the only preventive strategy is animal vaccination, which harbors significant disadvantages. Considering the identification of many immunogenic proteins in Brucella, several studies have recently been performed to evaluate the vaccine potency of such antigens as a new subunit vaccine candidate. This review represents an overview of brucellosis in Iran, including epidemiology, transmission routs, diagnosis, and treatment. Moreover, it mainly highlights the history of brucellosis control and prevention in Iran, including eradication programs, vast livestock vaccination programs, and subunit vaccine studies. It also discusses major problems that the country encounters with disease control. In recent years, Persian scientists have focused on evaluating the efficacy of best Brucella immunogens in vivo to introduce a new subunit vaccine. The results of some studies could demonstrate the vaccine potential of some immunogens.

Protein/Protein, DNA/DNA and DNA/Protein based vaccination strategies using truncated Omp2b against Brucella infection in BALB/c Mice.

The purpose of the present study was to evaluate the immunogenicity and protective efficacy of the truncated form of outer membrane protein 2b (TOmp2b) from Brucella abortus in BALB/c mice. Three immunization regimens Protein/Protein, DNA/DNA and DNA/Protein were used. Immunization of mice with all vaccine strategies elicited a strong specific IgG responses (IgG2a titers over IgG1) and provided T helper1 (Th1) oriented immune responses. Furthermore, Protein/Protein (Pro/Pro-) and DNA/Pro- vaccinated groups conferred protection levels against B. abortus challenge not significantly different from those induced by B. abortus RB51 vaccine stain. In conclusion, TOmp2b is potential to stimulate specific immune responses and to confer cross protection against B. melitensis and B. abortus infection. Therefore, TOmp2b could be introduced as a new subunit vaccine candidate against Brucella infection.

Expression, Purification and Functional Assessment of Smallest Isoform of Human Interleukin-24 in Escherichia coli

ABSTRACT Interleukin-24 (IL-24) is a novel tumor-suppressor gene that has different alternative splice isoforms. It has been shown that new smallest isoform of human IL-24 gene, lacking three exons, induces higher levels of cytotoxicity than all the isoforms, indicating shortest isoform of IL-24 may be a new promising anti-cancer agent. In this study, we aimed to provide a reproducible method for recombinant production of the smallest isoform of IL-24 (sIL-24). The Structure of sIL-24 was analyzed using bioinformatics tools (I-TASSER, Prosa, RAMPAGE and SPDBV version 4.1). The DNA sequence encoding sIL-24 was chemically synthesized and sub-cloned into the pET-32a (+) vector for further protein expression in Escherichia coli BL21 (DE3) strain. Upon IPTG induction, sIL-24 peptide was expressed as a thioredoxin fusion protein. The recombinant sIL-24 was released from the fusion by TEV protease cleavage followed by nickel affinity chromatography. The yield of the purified sIL-24 was estimated about 380 μg/ml. MTT assay showed that sIL-24 peptide inhibited the proliferation of PC-3 cancer cells more effectively than full length IL-24 protein, while none affect the survival of MRC-5 normal cells. These results indicate that the presented expression system is an efficient system for the production of small functional recombinant sIL-24 peptide.This functional peptide may have cancer therapeutic application.

Comparison of potential protection conferred by three immunization strategies (protein/protein, DNA/DNA, and DNA/protein) against Brucella infection using Omp2b in BALB/c Mice.

In the present study, immunogenicity and protective efficacy of the Brucella outer membrane protein 2b (Omp2b) was evaluated in BALB/c mice using Protein/Protein, DNA/DNA and DNA/Protein vaccine strategies. Immunization of mice with three vaccine regimens elicited a strong specific IgG response (higher IgG2a titers over IgG1 titers) and provided Th1-oriented immune response. Vaccination of BALB/c mice with the DNA/Pro regimen induced higher levels of IFN-γ/IL-2 and conferred more protection levels against B. melitenisis and B. abortus challenge than did the protein or DNA alone. In conclusion, Omp2b is able to stimulate specific immune responses and to confer cross protection against B. melitensis and B. abortus infection. Therefore, it could be introduced as a new potential candidate for the development of a subunit vaccine against Brucella infection.

In silico analysis of Brucella abortus Omp2b and in vitro expression of SOmp2b.

PURPOSE: At present, there is no vaccine available for the prevention of human brucellosis. Brucella outer membrane protein 2b (Omp2b) is a 36 kD porin existed in common Brucella pathogens and it is considered as priority antigen for designing a new subunit vaccine. MATERIALS AND METHODS: In the current study, we aimed to predict and analyze the secondary and tertiary structures of the Brucella abortus Omp2b protein, and to predict T-cell and B-cell epitopes with the help of bioinformatics tools. Subsequently, cloning and expression of the short form of Omp2b (SOmp2b) was performed using pET28a expression vector and Escherichia coli BL21 host, respectively. The recombinant SOmp2b (rSOmp2b) was purified with Ni-NTA column. RESULTS: The recombinant protein was successfully expressed in E. coli host and purified under denaturation conditions. The yield of the purified rSOmp2b was estimated by Bradford method and found to be 220 µg/mL of the culture. CONCLUSION: Our results indicate that Omp2b protein has a potential to induce both B-cell- and T-cell-mediated immune responses and it can be evaluated as a new subunit vaccine candidate against brucellosis.

In silico analysis of Shiga toxins (Stxs) to identify new potential vaccine targets for Shiga toxin-producing Escherichia coli.

Shiga toxins belong to a family of structurally and functionally related toxins serving as the main virulence factors for pathogenicity of the Shiga toxin-producing Escherichia coli (STEC) associating with Hemolytic uremic syndrome (HUS). At present, there is no effective treatment or prevention for HUS. The aim of the present study was to find conserved regions within the amino acid sequences of Stx1, Stx2 (Shiga toxin) and their variants. In this regard, In-silico identification of conformational continuous B cell and T-cell epitopes was performed in order to introduce new potential vaccine candidates. 93-100% Homology was observed in Stx1 and its variants. In Stx2 and its variants, 69-100% homology was shown. By sequence alignment with Stx1 and Stx2, 54% homology was detected. T-cell epitope identification in Stx1A and Stx2A epitopes with highest binding affinity for each HLA (human leukocyte antigen) was demonstrated with 100% identity among all Stxs. B-cell epitope prediction was resulted in finding of four common epitopes between Stxs. In silico analysis of Stxs was resulted to identification of new peptide targets that could be used in development of new epitope vaccine candidates or in immunodiagnostic tests.

Improved immunogenicity and protective efficacy of a divalent DNA vaccine encoding Brucella L7/L12-truncated Omp31 fusion protein by a DNA priming and protein boosting regimen.

Brucellosis is one of the most common zoonotic diseases caused by species of Brucella. At present, there is no commercially available vaccine for the human brucellosis. Brucella melitensis and Brucella abortus are the main causes of human brucellosis, worldwide. The outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved among human Brucella pathogens. The purpose of the current study was to evaluate and compare the immunogenicity and protective efficacy of the L7/L12-TOmp31 construct administered as DNA/DNA and DNA/Pro vaccine regimens. Vaccination of BALB/c mice with the DNA/Pro regimen provided more protection levels against B. melitenisis and B. abortus challenge than did the DNA/DNA regimen. IgG1 and IgG2a titers were higher in the sera from DNA/Pro-immunized mice than in those from mice immunized with DNA alone. Moreover, splenocytes from DNA/Pro-immunized mice produced significantly higher levels of IFN-γ than did those from mice given DNA alone. The pcDNA-L7/L12-TOmp31 priming followed by rL7/L12-TOmp31 boosting led to improved protection against B. abortus or B. melitensis infection.

Vaccination with recombinant L7/L12-truncated Omp31 protein induces protection against Brucella infection in BALB/c mice.

Brucellosis is the most common bacterial zoonotic disease worldwide and no vaccine is available for the prevention of human brucellosis. In humans, brucellosis is mostly caused by Brucella melitensis and Brucella abortus. The Outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved in human Brucella pathogens. In the present study, we evaluated the humoral and cellular immune responses induced by a fusion protein designed based on the Truncated form of Omp31 (TOmp31) and L7-L12 antigens. Vaccination of BALB/c mice with the recombinant fusion protein (rL7/L12-TOmp31) provided the significant protection level against B. melitensis and B. abortus challenge. Moreover, rL7/L12-TOmp31 elicited a strong specific IgG response (higher IgG2a titers) and significant IFN-γ/IL2 production and T-cell proliferation was also observed. The T helper1 (Th1) oriented response persisted for 12 weeks after the first immunization. The rL7/L12-TOmp31 could be a new potential antigen candidate for the development of a subunit vaccine against B. melitensis and B. abortus.

In silico design, cloning and high level expression of L7/L12-TOmp31 fusion protein of Brucella antigens.

Globally, Brucella melitensis and B. abortus are the most common cause of human brucellosis. The outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved in human Brucella pathogens which are considered as potential vaccine candidates. We aimed to design the fusion protein from Brucella L7/L12 and truncated Omp31proteins, in silico, clone the fusion in pET28a vector, and express it in Escherichia coli host. Two possible fusion forms, L7/L12-TOmp31 and TOmp31-L7/L12 were subjected to in silico modeling and analysis. Analysis and validation of the fusion proteins with three dimensional (3D) models showed that both models are in the range of native proteins. However, L7/L12-Tomp31 structure was more valid than the TOmp31-L7/L12 model and subjected to in vitro production. The major histocompatibility complex (MHC II) epitope mapping using IEDB database indicated that the model contained good MHC II binders. The L7/L12-TOmp31 coding sequence was cloned in pET28a vector. The integrity of the construct was confirmed by polymerase chain reaction, restriction enzyme mapping, and sequencing. The fusion was successfully expressed in E. coli BL21 (DE3) by induction with isopropyl ß-D-thiogalactopyranoside. The rL7/L12-TOmp31 was purified with Ni-NTA column. The yield of the purified rL7/L12-TOmp31 was estimated by Bradford method and found to be 40 mg/L of the culture. Western blotting with anti-His antibody revealed a specific reactivity with purified rL7/L12-TOmp31 produced in E. coli and showed the functional expression in the prokaryotic system. In this study, a new protein vaccine candidate against brucellosis was constructed with the help of bioinformatics tools and the construct was expressed in the bacterial host. Studies evaluating the immunogenicity and cross-protection of this fusion protein against B. melitensis and B. abortus are underway.