INTRODUCTION:Recombinant protein vaccines against infectious diseases, based on immunogenic antigen identification and employing polymeric nanoparticles as a delivery system, can provoke immune responses comparable to or better than traditional vaccines. The production of a safe and immunogenic vaccine against diphtheria was achieved by preparing sodium alginate nanoparticles containing recombinant diphtheria toxoid (CRM197). METHODS:Alginate nanoparticles loaded with CRM197 were prepared using the ionic-gelation method and thoroughly characterized. Safety and immunogenicity studies were conducted in an animal model for comparison with commercial vaccines. Antibody responses were evaluated using both qualitative and quantitative measurements, as determined by the toxin neutralization test (TNT) and indirect ELISA, respectively. IgG subclasses in the sera of immunized mice and possible pathological lesions in vital tissues of all immunized mouse groups were investigated. RESULTS:Nanoparticles with or without CRM197 were synthesized by the ionic gelation method. LE and LC measurements showed ˃80% and ˃20%, respectively, indicating stable and persistent release without a bursting pattern. In vivo studies showed safety and enhanced immunogenicity in mice immunized with the CRM197- loaded sodium alginate nanoparticles, with higher levels of total anti-CRM197 IgG and subclasses than those induced by conventional vaccines. DISCUSSION:Reducing antigen usage in vaccine production while increasing immunogenicity and safety compared with traditional vaccines are the goals of new vaccine development, which were achieved in the current study. CONCLUSION:Engineered alginate nanoparticles loaded with recombinant diphtheria antigen (CRM197) demonstrated in vitro controlled and slow release, as well as safety and immunogenicity profiles against diphtheria in vivo. Nanoparticles containing CRM197 antigens equivalent to adult and children doses showed high levels of IgG1 and IgG2a, confirming the combined responses of the humoral and cellular immune systems.

Objective: To investigate the safety and immunogenicity of the RAZI Cov Pars (RCP) vaccine in children and adolescents aged 5-17 years. Methods: In this open-label, single arm trial, 26 of the 68 registered volunteers met the inclusion criteria. The participants received RCP vaccine twice intramuscularly (on days 0 and 21) and intranasally on day 51. Safety was assessed up to 6 months after the second dose. Immunogenicity was assessed on days 35, 90, and 180 by measuring neutralizing antibody levels as well as anti-RBD and anti-S1 IgG antibodies. Results: Among the 26 volunteers, 22 were in the age group of 5-11 years, and 4 were in the age group of 12-17 years. No grade 3 or higher local or systemic adverse reactions were reported one week after vaccination. Six abnormal laboratory findings were observed after both vaccine doses, none of which were classified as grade 3 or higher. During a total follow-up period of 3 875 person- years, 31 adverse events were recorded (incidence rate: 0.008). The seroconversion rates for VNT, anti-RBD and anti-S1 IgG antibodies two weeks after receiving the second dose were 72.7%, 76.2% and 80.9%, respectively. In the 5-11 year age group, the seroconversion rates for VNT, anti-RBD and anti-S1 were 78.9%, 83.3% and 88.9%, respectively. Conclusions: Intramuscular and intranasal administration of the RCP vaccine did not lead to serious adverse events in any of the children or adolescents. The vaccine elicited a robust response in the 5-11 year age group two weeks after the second dose. Considering that this group received half of the adult vaccine dose, these results support the suitability of this dose for the study group.

Background and Objectives: The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has presented a challenging issue for global health in the 21st century. Frequent mutations in viral genomes have diminished the effectiveness of current vaccines against new variants. Messenger RNA (mRNA) vaccine is a promising platform for eliciting a robust T cell immune response. Materials and Methods: We evaluated the uptake of mRNA-LNPs into human monocyte-derived dendritic cells by measuring the intensity of enhanced eGFP expression in the transfected cells. Next, we assessed the effect of mRNA-LNPs on immune response induction in mice following a prime-boost immunization strategy, along with analyzing cytokine release. The safety of the vaccine candidate was examined through pyrogenicity and toxicity assays. Results: Upon intramuscular injection of mice, potent antibodies specific to viral S protein, robust Th1-biased cell-mediated immunity, and enhanced IFN-γ expression were induced. These observations indicate that mRNA-LNP was taken up and that it migrated to the lymph nodes. Furthermore, the vaccine candidate did not cause inflammation or local reactions after injection, as confirmed by biochemical, hematological, and histopathological examinations. Conclusion: Because of its ability to target immune cells, the mRNA vaccine candidate can potentially improve immune responses against circulating or emerging variants.

Introduction:Heterologous vaccines enhance the immune response to new variants and allow flexibility in booster administration when the original vaccine is unavailable. Studies show that heterologous boosters can generate comparable or superior antibody responses compared to homologous boosters. Considering rare side effects is essential in evaluating COVID-19 vaccines, especially those associated with ChAdOx1-S (AstraZeneca) and Ad26.COV2.S (Janssen), including blood clotting and idiopathic thrombocytopenia. Severe side effects, such as myocarditis and pericarditis, may occur after Pfizer or Moderna boosters but are rare. Methods:This study administered two vaccines: the Sinopharm inactivated virus vaccine and the Razi-CoV-Pars (RCP) booster. Various evaluations included biochemical markers, coagulation factors, autoimmune antibodies, and antibodies against concerning variants. Results:All 90 participants exhibited a notable rise in antibody levels against the variant of concern (VOC). Participants receiving the Razi-CoV-Pars booster after Sinopharm/BBIBP-CorV showed significantly higher antibody levels (Wuhan ∼ 3.25 times, Delta ∼4 times, Omicron ∼ 14 times) compared to those receiving Sinopharm's homologous vaccine. No significant changes (P: <0.05) were found in LDH, CPK, CK-MB, ANA, and Anti-CCP levels. However, individuals receiving Sinopharm's booster after two doses showed a significant increase (4 cases) in D-Dimer levels. Conclusion:The Razi-CoV-Pars vaccine demonstrates a favorable safety profile and promising potential as an effective booster against current variants, particularly due to its significant protective titer against Omicron.

Background The immunity induced by primary vaccination is effective against COVID-19; however, booster vaccines are needed to maintain vaccine-induced immunity and improve protection against emerging variants. Heterologous boosting is believed to result in more robust immune responses. This study investigated the safety and immunogenicity of the Razi Cov Pars vaccine (RCP) as a heterologous booster dose in people primed with Beijing Bio-Institute of Biological Products Coronavirus Vaccine (BBIBP-CorV). Methods We conducted a randomized, double-blind, active-controlled trial in adults aged 18 and over primarily vaccinated with BBIBP-CorV, an inactivated SARS-CoV-2 vaccine. Eligible participants were randomly assigned (1:1) to receive a booster dose of RCP or BBIBP-CorV vaccines. The primary outcome was neutralizing antibody activity measured by a conventional virus neutralization test (cVNT). The secondary efficacy outcomes included specific IgG antibodies against SARS-CoV-2 spike (S1 and receptor-binding domain, RBD) antigens and cell-mediated immunity. We measured humoral antibody responses at 2 weeks (in all participants) and 3 and 6 months (a subgroup of 101 participants) after the booster dose injection. The secondary safety outcomes were solicited and unsolicited immediate, local, and systemic adverse reactions. Results We recruited 483 eligible participants between December 7, 2021, and January 13, 2022. The mean age was 51.9 years, and 68.1% were men. Neutralizing antibody titers increased about 3 (geometric mean fold increase, GMFI = 2.77, 95% CI 2.26–3.39) and 21 (GMFI = 21.51, 95% CI 16.35–28.32) times compared to the baseline in the BBIBP-CorV and the RCP vaccine groups. Geometric mean ratios (GMR) and 95% CI for serum neutralizing antibody titers for RCP compared with BBIBP-CorV on days 14, 90, and 180 were 6.81 (5.32–8.72), 1.77 (1.15–2.72), and 2.37 (1.62–3.47) respectively. We observed a similar pattern for specific antibody responses against S1 and RBD. We detected a rise in gamma interferon (IFN-γ), tumor necrosis factor (TNF-α), and interleukin 2 (IL-2) following stimulation with S antigen, particularly in the RCP group, and the flow cytometry examination showed an increase in the percentage of CD3 + /CD8 + lymphocytes. RCP and BBIBP-CorV had similar safety profiles; we identified no vaccine-related or unrelated deaths. Conclusions BBIBP-CorV and RCP vaccines as booster doses are safe and provide a strong immune response that is more robust when the RCP vaccine is used. Heterologous vaccines are preferred as booster doses. Trial registration This study was registered with the Iranian Registry of Clinical Trial at www.irct.ir , IRCT20201214049709N4. Registered 29 November 2021.

Background: We conducted a phase III, non-inferiority trial comparing safety and efficacy of RCP recombinant spike protein Covid-19 vaccine to BBIBP (Sinopharm). Methods: Adult Iranian population received RCP or BBIBP in a randomized, double blind and an additional non-randomized open labeled trial arms. Eligible participants signed a written informed consent and received two intramuscular injections three weeks apart. In the randomized arm, an intranasal dose of vaccine or adjuvant-only preparation were given to the RCP and BBIBP recipients at day 51 respectively. Participants were actively followed for up to 4 months for safety and efficacy outcomes. Primary outcome was PCR + symptomatic Covid-19 disease two weeks after the second dose. The non-inferiority margin was 10% of reported BBIBP vaccine efficacy (HR = 1.36). Results: We recruited 23,110 participants (7224 in the randomized and 15,886 in the non-randomized arm). We observed 604 primary outcome events during 4 months of active follow-up including 121 and 133 in the randomized and 157 and 193 cases in the non-randomized arms among recipients of RCP and BBIBP respectively. Adjusted hazard ratios for the primary outcome in those receiving RCP compared with BBIBP interval were 0.91 (0.71–1.16) and 0.62 (0.49–0.77) in the randomized and non-randomized arms respectively. The upper boundary of 99.1% confidence interval of HR = 0.91 (0.67–1.22) remained below the margin of non-inferiority in the randomized arm after observing the early stopping rules using O'Brien Fleming method. Conclusion: Our study showed that the RCP efficacy is non-inferior and its safety profile is comparable to the BBIBP.

Protective antibodies in the upper respiratory tract prevent the spread of COVID-19 in the community. Intranasal vaccines could raise the specific secretory IgA and IgG levels. This is a single-center, randomized, double-blind, placebo-controlled clinical trial to evaluate the safety and immunogenicity of Razi Cov Pars (RCP) intranasal recombinant protein subunit COVID-19 vaccine as a booster in adults. We compared specific IgG and IgA levels in the intranasal RCP group (n = 97) versus placebo (n = 96) in serum, saliva, and nasal mucosal secretions on days 0 and 14 and reported their Geometric Mean Ratios (GMR) and 95% confidence intervals (CI). We showed significant increases in IgA and IgG anti-RBD in the nasal mucosa in the RCP group, but their increase was not detectable in the serum and saliva. Anti-spike IgA in the nasal mucosa also increased in the RCP group compared to the placebo. This increase against the COVID-19 variant Omicron was also similar to that of the Wuhan. We detected no serious adverse reactions or anaphylaxis and all adverse events resolved completely during the follow-up period and were similar in both groups. Intranasal RCP is safe, stimulates the respiratory mucosal immunity, and could be a booster on various COVID-19 vaccines and be effective against new virus variants.

Background & Aim: Diphtheria is an acute vaccine-controlled respiratory disease caused by the grampositive bacterium Corynebacterium diphtheria.Since the preparation, purification, and detoxification of the vaccine against this disease involves many and complex steps, the present study aimed reduction of time of various stages of toxoid purification.Materials & Methods: For this purpose, three factors were first optimized for the concentration of iron in the culture medium, incubation temperature and rotation speed to produce more toxins.In each case, the experiment was carried out in triplicate and Lf and Kf values were tested to evaluate toxin production.After toxin inactivation and production of the toxoid, step reductions were performed to purify the toxoid.For this, ultrafiltration, precipitation, dialysis and chromatography with Sephadex G-25 column are used.In step reduction conditions, dialysis, chromatography or both of them were removed in the first, second and third cases, respectively.The results of each method were evaluated by SDS-PAGE and the quantitative data was analyzed using Minitab 18 (p‹0.05). Results:The results for optimizing the culture medium showed that highest amounts of toxin were produced at an iron concentration of 1 mg/l, a temperature of 35 °C, and a rotation speed of 200 rpm(p‹0.05).In the reduction experiments related to toxoid purification steps, the best results were obtained in a series of purified samples with ultrafiltration, precipitation and column chromatography steps.Conclusion: Since time and cost are two key factors in industrial production, toxoid production for the diphtheria vaccine can be produced at a lower cost and in less time by eliminating the two-week dialysis phase.

Introduction The Diphtheria vaccine is currently manufactured by chemical inactivation of Diphtheria toxin (DT) following large-scale cultivation of the pathogenic Corynebacterium diphtheriae. The development of non-toxic variants of DT in non-pathogenic recombinant hosts improves the process safety aspects considerably and is therefore desirable. The cytotoxicity of DT was exerted through ADP-ribosylation of elongation factor 2 (EF2) by the catalytic (C) domain of DT using NAD as the ADP donor. Aim We, therefore, aim to design DT variants unable to perform ADP-ribosylation of EF2. Methods We used molecular dynamics simulations (MDS) for docking of the C-domain of DT onto EF2 to identify interacting amino acid fluctuations using the root mean square fluctuation (RMSF). Amino acids of the C domain possessing low fluctuations and hence low flexibility were then specified and used for the mutant design. The C domain and the mutants were docked with NAD and ADENYLYL-3'-5'-PHOSPHO-URIDINE3'-MONOPHOSPHATE (APU) as a C domain inhibitor. G52E showing negligible NAD binding was also included in our study. Free binding energies of the complexes were calculated and used to select the desired mutants. Moreover, contact maps of the C domain and the selected mutants were compared to elucidate their structural differences. The SCRATCH tool was used to estimate their solubility upon recombinant expression in E. coli. ElliPro was further used to determine their B-cell epitopes. Results Our results indicated that amino acids Y20, V80, V81, V83, and Y149 within the C domain showed low flexibility. The constructed mutants, including Y20E and Y80E were able to bind APU, whereas Y149E was not able to bind it. Both Y20E and Y149E were unable to bind NAD. Thus, these mutants did not have ADP-ribosylation activity and were nontoxic. These were structurally different from the C domain. However, their solubility in E. coli and their conformational B-cell epitopes were similar to those of the C domain. Conclusion Therefore, Y20E and Y149E are applicable for the vaccine design with DT using a much safer process compared to the commercial process.

Objectives: This study aimed to determine the safety and immunogenicity of a combined intramuscular/intranasal recombinant spike protein COVID-19 vaccine (RCP). Methods: We conducted a randomized, double-blind, placebo-controlled, phase I trial. Three vaccine strengths were compared with an adjuvant-only preparation. It included two intramuscular and a third intranasal dose. Eligible participants were followed for adverse reactions. Specific IgG, secretory IgA, neutralizing antibodies, and cell-mediated immunity were assessed. Results: A total of 153 participants were enrolled (13 sentinels, 120 randomized, 20 non-randomized open-labeled for IgA assessment). No related serious adverse event was observed. The geometric mean ratios (GMRs) and 95% CI for serum neutralizing antibodies compared with placebo two weeks after the second injection were 5.82 (1.46–23.13), 11.12 (2.74–45.09), and 20.70 (5.05–84.76) in 5, 10, and 20 µg vaccine groups, respectively. The GMR for anti-RBD IgA in mucosal fluid two weeks after the intranasal dose was 23.27 (21.27–25.45) in the 10 µg vaccine group. The humoral responses were sustained for up to five months. All vaccine strengths indicated a strong T-helper 1 response. Conclusion: RCP is safe and creates strong and durable humoral and cellular immunity and good mucosal immune response in its 10 µg /200 µL vaccine strengths. Trial registration: IRCT20201214049709N1.

Background: This study explores the safety and immunogenicity of the Razi-Cov-Pars (RCP) SARS Cov-2 recombinant spike protein vaccine. Method: In a randomized, double-blind, placebo-controlled trial, adults aged 18-70 were randomly allocated to receive selected 10 mg/200 ml vaccine strengths or placebo (adjuvant). It included two intramuscular injections at days 0 and 21, followed by an intranasal dose at day 51. Immediate and delayed solicited local and systemic adverse reactions after each dose up to a week, and specific IgG antibodies against SARS Cov-2 spike antigens two weeks after the 2nd dose were assessed as primary outcomes. Secondary safety outcomes were abnormal laboratory findings and medically attended adverse events (MAAE) over six months follow up. Sec-ondary immunogenicity outcomes were neutralizing antibody activity and cell-mediated immune response. Result: Between May 27th and July 15th, 2021, 500 participants were enrolled. Participants' mean (SD) age was 37.8 (9.0), and 67.0 % were male. No immediate adverse reaction was observed following the intervention. All solicited local and systemic adverse events were moderate (Grade I-II). Specific IgG antibody response against S antigen in the vaccine group was 5.28 times (95 %CI: 4.02-6.94) the placebo group with a 75 % seroconversion rate. During six months of follow-up, 8 SAEs were reported, unrelated to the study intervention. The participants sustained their acquired humoral responses at the end of the sixth month. The vaccine predominantly resulted in T-helper 1 cell-mediated immunity, CD8+ cytotoxic T-cell increase, and no increase in inflammatory IL-6 cytokine. Conclusion: RCP vaccine is safe and creates strong and durable humoral and cellular immunity. Trial registration: (IRCT20201214049709N2). (c) 2023 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.

Several vaccine candidates for COVID-19 have been developed, and few vaccines received emergency approval with an acceptable level of efficacy and safety. We herein report the development of the first recombinant protein-based vaccine in Iran based on the recombinant SARS-CoV-2 spike protein in its monomeric (encompassing amino acid 1-674 for S1 and 685-1211 for S2 subunits) and trimer form (S-Trimer) formulated in the oil-in-water adjuvant system RAS-01 (Razi Adjuvant System-01). The safety and immunity of the candidate vaccine, referred to as RAZI-COV PARS, were evaluated in Syrian hamster, BALB/c mice, Pirbright guinea pig, and New Zeeland white (NZW) rabbit. All vaccinated animals received two intramuscular (IM) and one intranasal (IN) candidate vaccine at 3-week intervals (days 0, 21, and 51). The challenge study was performed intranasally with 5×106 pfu of SARS-CoV-2 35 days post-vaccination. None of the vaccinated mice, hamsters, guinea pigs, or rabbits showed any changes in general clinical observations; body weight and food intake, clinical indicators, hematology examination, blood chemistry, and pathological examination of vital organs. Safety of vaccine after the administration of single and repeated dose was also established. Three different doses of candidate vaccine stimulated remarkable titers of neutralizing antibodies, S1, Receptor-Binding Domain (RBD), and N-terminal domain (NTD) specific IgG antibodies as well as IgA antibodies compared to placebo and control groups (P<0.01). Middle and high doses of RAZI-COV PARS vaccine significantly induced a robust and quick immune response from the third-week post-immunization. Histopathological studies on vaccinated hamsters showed that the challenge with SARS-CoV-2 did not induce any modifications in the lungs. The protection of the hamster was documented by the absence of lung pathology, the decreased virus load in the lung, rapid clearance of the virus from the lung, and strong humoral and cellular immune response. These findings confirm the immunogenicity and efficacy of the RAZI-COV PARS vaccine. Of the three tested vaccine regimens, the middle dose of the vaccine showed the best protective immune parameters. This vaccine with heterologous prime-boost vaccination method can be a good candidate to control the viral infection and its spread by stimulating central and mucosal immunity.

Background and Aim: Leptospirosis is one of the most common zoonotic diseases worldwide, occurring mostly in tropical, subtropical, temperate, and humid regions with heavy rainfall. It is important to diagnose this condition correctly and promptly. Loa22 is an outer membrane protein exposed to the surface in some Leptospira serovars. The purpose of this study was to determine the presence of the gene encoding Loa22 protein in Leptospira interrogans serovars. Materials and Methods: The present study was conducted on 23 pathogenic leptospira serovars and two non-pathogenic leptospira serovars. These serovars were prepared from the Reference Laboratory for Leptospira, Department of Microbiology, Razi Vaccine, and Serum Research Institute, Karaj, Iran. After genomic DNA extraction using the standard phenol-chloroform method, loa22 gene was amplified by specific primers. Results: PCR was performed on the loa22 gene by producing a 671-bp fragment. The results showed that the loa22 gene was present in all 23 pathogenic leptospira serovars but not in the non-pathogenic L. biflexa . The specificity of tested primers was confirmed as well. Conclusion: The loa22 gene is a specific gene for pathogenic leptospira serovars that is not found in saprophytic serovars, so it is suggested that this gene be used to detect leptospira pathogenic serovars.

This paper aims to investigate the preparation and characterisation of the alginate nanoparticles (NPs) as antigen delivery system loaded by diphtheria toxoid (DT). For this purpose, both the loading capacity (LC) and Loading efficiency (LE) of the alginate NPs burdened by DT are evaluated. Moreover, the effects of different concentrations of sodium alginate and calcium chloride on the NPs physicochemical characteristics are surveyed in addition to other physical conditions such as homogenization time and rate. To do so, the NPs are characterised using particle size and distribution, zeta potential, scanning electron microscopy, encapsulation efficiency, in vitro release study and FT-IR spectroscopy. Subsequently, the effects of homogenization time and rate on the NPs are assessed. At the meantime, the NPs LC and efficiency in several DT concentrations are estimated. The average size of the NPs was 400.7 and 276.6 nm for unloaded and DT loaded, respectively. According to the obtained results, the zeta potential of the blank and DT loaded NPs are estimated as -23.7 mV and -21.2 mV, respectively. Whereas, the LC and LE were >80% and >90%, in that order. Furthermore, 95% of the releasing DT loaded NPs occurs at 140 h in the sustained mode without any bursting release. It can be concluded that the features of NPs such as morphology and particle size are strongly depended on the calcium chloride, sodium alginate concentrations and physicochemical conditions in the NPs formation process. In addition, appropriate concentrations of the sodium alginate and calcium ions would lead to obtaining the desirable NPs formation associated with the advantageous LE, LC (over 80%) and sustained in vitro release profile. Ultimately, the proposed NPs can be employed in vaccine formulation for the targeted delivery, controlled and slow antigen release associated with the improved antigen stability.

Background: Outer membrane vesicles (OMVs) release from Gram-negative bacteria and are interesting alternatives that can replace those vaccines that contain naturally incorporated bacterial surface antigens, lipopolysaccharides (LPS) and outer membrane proteins (OMPs). Nanoparticles can be used to encapsulate vesicles for slow release and prevent macromolecular degradation. Objective: Therefore, encapsulation of OMVs of B. pertussis into sodium alginate nanoparticles was the main aim of the current study. Methods: The OMVs of B. pertussis extracted and characterized by particle sizer, electron microscopy, SDSPAGE and Western blot assays. The extracted OMVs were encapsulated in sodium alginate nanoparticles (OMV-NP) using unique gelation process and injected into BALB/c mice. Immunogenicity indices such as different classes of antibodies and interleukins related to different T cell lines were evaluated in immunized mice by ELISA. The respiratory challenge was evaluated in the groups of mice. The existence of pertussis toxin (PTX), filamentous haemagglutinin (FHA) and PRN (pertactin) in B. pertussis OMVs was verified using SDSPAGE and Western blot analysis. Results: TEM electron microscopy showed the size of these OMVs to be around 20-80 nm. The OMVs with appropriate quality were encapsulated into sodium alginate nanoparticles (OMV-NP), and the final size was about 500 nm after encapsulation. Immunity indices were significantly higher in the OMV-NP receiving group. In challenge tests, the OMV-NP vaccine was able to show the highest rate of lung clearance compared to the control groups (OMV and wPV) at the lowest injection dose. Conclusion: The results indicate the potential of OMV-NP as a novel vaccine delivery system.

Objective(s) Bacillus anthracis is the cause of the fatal anthrax. Available anthrax vaccines have low stability and require multiple injections in order to be effective. Poly lactic acid (PLA) has been approved as a biodegradable and biocompatible polymer for drug and vaccine delivery applications. The purpose of this study is to evaluate the antibody titer against the protective antigen recombinant protein (PA63) encapsulated by the mPEG-PLA double-block copolymers and to compare with the non-encapsulated PA63.Materials and Methods To attain this purpose, to start, the desired protein was purified and confirmed and then PA63 was encapsulated with mPEG-PLA double-block copolymers using a water- oil- water solvent evaporation method. Produced nanoparticles was characterized in terms of morphological specifications using scanning electron microscopy, size and polydispersity index using dynamic light scattering and zeta potential using a zeta seizer. The synthesized nanoparticle antigenic content and also its antigen release profile was measured. In the following, the nanoparticles containing antigens (PA63-NPs), blank nanoparticles (mPEG-PLA- NPs), PA63 and adjuvant control were injected subcutaneously to mice and the IgG polyclonal antibody titr was measured by indirect ELISA. Finally to evaluate biocompatibility and toxicity, synthesized nanoparticles were investigated by cell culture testing.Results The results of this study showed that the synthesized nanoparticles are of good quality. ELISA results showed that antibody production titr in mice receiving PA63-NPs was higher than those receiving the PA63 (P<0.05). Cell culture results revealed that the synthesized nanoparticles have no toxicity.Conclusion The findings of the study indicated that the obtained nano vaccine formulations had a higher ability than non-encapsulated recombinant proteins to stimulate the immune system of animal, and that PLA could be used as an appropriate carrier for an effective, stable, safe and biodegradable engineered recombinant vaccine against anthrax.

Background:Pertussis is a current contagious bacterial disease caused by Bordetella pertussis (Bp). Given the prevalence of pertussis, development of new vaccines is important. This study was attempted to evaluate the expression of main virulence factors (pertussis toxin [PTX], PRN [pertactin], and filamentous hemagglutinin [FHA]) from Bp predominant strains and also compare the expression of these factors in the outer membrane vesicles (OMVs) obtained from predominant circulating Bp isolate.Methods:The physicochemical features of the prepared OMVs were analyzed by electron microscopy and SDS-PAGE. The presence of the mentioned virulence factors was confirmed by Western blotting. BALB/c mice (n = 21) immunized with characterized OMVs were challenged intranasally with sublethal doses of Bp, to examine their protective capacity.Results:Electron microscopic examination of the OMVs indicated vesicles within the range of 40 to 200 nm. SDS-PAGE and Western blotting demonstrated the expression of all three main protective immunogens (PTX, PRN, and FHA), prevalent in the predominant, challenge, and vaccine strains, and OMVs of the predominant IR37 strain and BP134 vaccine strain. Significant differences were observed in lung bacterial counts between the immunized mice with OMV (30 CFU/lung) compared to the negative control group ((6 104 CFU/lung; p < 0.001). In mice immunized with OMVs (3 µg), the number of lungs recovered colonies after five days dropped at least five orders of magnitude compared to the control group.Conclusion:OMVs obtained from circulating isolates with the predominant profile may constitute a highly promising vaccine quality. They also can be proposed as a potential basic material for the development of new pertussis vaccine candidate.

Despite the availability of a vaccine, pertussis is still a worldwide health problem. Outer membrane vesicles (OMVs) in gram-negative bacteria can stimulate the immune system due to several outer membrane proteins and are very good candidates in vaccine development. OMVs obtained from Bordetella pertussis contain several antigens, which are considered immunogenic, and could make them a potential candidate for vaccine production. The current study aimed to compare the current OMV extraction method (with ultracentrifuge) and a modified extraction method (without ultracentrifuge) and to evaluate the physicochemical properties as well as the expression of their main virulence factors. Vaccinal strain BP134 grown on Bordet Gengo agar were inoculated in Modified Stainer-Scholte medium for mass cultivation. OMVs were prepared using two different methods. They were then stained and examined with a transmission electron microscope. Protein contents were measured by the Bradford method, and then the protein profile was evaluated by SDS-PAGE. The presence of immunogenic antigens was detected by Western blotting. The size and shape of the OMVs obtained from the modified method without the use of ultracentrifuge were similar to the current method and had a size between 40 and 200 nm. The total protein yields of the OMV isolated using the current and modified methods were 800 and 600 µg/ml, respectively. Evaluating the protein profile of extracted OMVs showed the presence of different proteins. Finally, the presence of PTX, PRN, and FHA was observed in OMVs extracted from both methods. Comparison of the two OMV extraction methods showed that the obtained vesicles have a suitable and similar shape and size as well as the expression of three important pathogenic factors as immunogens. Despite the relatively low reduction in protein yield as the modified method does not require ultracentrifuge, this extraction method can be used as a suitable alternative for extracting the outer membrane vesicles from B. pertussis, especially in developing countries. It should be noted that further experiments including immunogenicity determination of OMVs obtained as vaccine candidates in animal models are required.

BACKGROUND AND OBJECTIVES:The re-emergence of pertussis still is being reported all over the world. Pathogen adaptation and antigenic divergence of circulating isolates from vaccine strains are the main reasons of infection resurgence. Waning immunity is also an important factor contributing to resurgence of pertussis.MATERIALS AND METHODS:The genetic diversity and evolutionary characteristics of circulating Iranian isolates of Bordetella pertussis during February 2015 to October 2018 was investigated by pulsed-field gel electrophoresis (PFGE) and subsequently ptxA, ptxP and fim3 alleles were characterized. The next generation genome sequencing was then used to compare the genomics of ptxP1 and ptxP3 of selected isolates from PFGE dendrogram.RESULTS:PFGE differentiated 62 clinical isolates and vaccine and reference strains into 19 PFGE profiles, indicating the higher level of heterogeneity in the population during 2015-2018. The predominant B. pertussis genotype harbored pertussis toxin promoter allele, ptxP3 and the expansion of ptxA1 isolates, were also observed in our population.CONCLUSION:No changes in allelic profile of predominant clone in recent years was observed but antigenic divergence between recently circulating isolates and the vaccine strain has been progressed and significantly was higher than previous studies. The comparative genomic analysis of the ptxP3 and ptxP1 isolates indicate that changes in ptxP3 genome structure including 32 unique SNPs and three unique indels may have contributed to the expansion of the ptxP3 clone. We compared ptxP3 and ptxP1 isolates in pathogenicity-associated genes and found five of them were specific for the ptxP3 isolates. The polymorphisms in pathogenicity-associated genes suggest structural adaptations for these virulence factors.

Background: Pertussis is still one of the major public health problems. The increase of the disease emerged in recent decades due to the replacement of the reactogenic whole cell vaccine with the safer acellular vaccine and the genetic diversity of the bacterium. As outer membrane vesicles (OMVs) obtained from Bordetella pertussis contains surface immunogenic antigen in its structure, it has an acceptable characteristic that could be considered as a good candidate for pertussis vaccine. Materials & Methods: Vaccinal strain BP134 strain of B. pertussis was cultured under standard conditions and OMVs were isolated by modifying the method without the use of ultracentrifuge. The isolated vesicles were examined by transmission electron microscopy and protein content was measured by the Bradford method. The expression of virulence factors was confirmed by SDS-PAGE and protein expression was confirmed by Western immunoblot. Pyrogenicity test and abnormal toxicity test were performed on extracted vesicles. Results: The morphology of the vesicles was confirmed in the range of 40 to 200 nm. The protein concentration of the extracted vesicles was determined as 600 μg. Expression analysis by SDS-PAGE and western blot confirmed the presence of virulence factors, pertussis toxin, filamentous hemagglutinin, and pertactin using monoclonal antibodies in OMVs of the vaccinal strain. Pyrogenicity test and abnormal toxicity test were negative. Conclusion: The proposed method is a simple and efficient method for isolation of the B. pertussis OMVs. The OMVs extracted from B. pertussis could be a candidate for a new generation of pertussis vaccine alone or in combination with adjuvants to design future acellular vaccines.