Comprehensive characterization of bacterial glycoconjugate vaccines by liquid chromatography - mass spectrometry.

Bacterial pathogens can cause a broad range of infections with detrimental effects on health. Vaccine development is essential as multi-drug resistance in bacterial infections is a rising concern. Recombinantly produced proteins carrying O-antigen glycosylation are promising glycoconjugate vaccine candidates to prevent bacterial infections. However, methods for their comprehensive structural characterization are lacking. Here, we present a bottom-up approach for their site-specific characterization, detecting N-glycopeptides by nano reversed-phase liquid chromatography-mass spectrometry (RP-LC-MS). Glycopeptide analyses revealed information on partial site-occupancy and site-specific glycosylation heterogeneity and helped corroborate the polysaccharide structures and their modifications. Bottom-up analysis was complemented by intact glycoprotein analysis using nano RP-LC-MS allowing the fast visualization of the polysaccharide distribution in the intact glycoconjugate. At the glycopeptide level, the model glycoconjugates analyzed showed different repeat unit (RU) distributions that spanned from 1 to 21 RUs attached to each of the different glycosylation sites. Interestingly, the intact glycoprotein analysis displayed a RU distribution ranging from 1 to 28 RUs, showing the predominant species when the different glycopeptide distributions are combined in the intact glycoconjugate. The complete workflow based on LC-MS measurements allows detailed and comprehensive analysis of the glycosylation state of glycoconjugate vaccines.

Planting a chemical flag on antigens.

Next-generation live vaccines are created by autonomous production of nitrated antigens.

Synthesis of BLF1-containing trimethyl chitosan nanoparticles and evaluation of its immunogenicity and protection in syrian mice by oral and subcutaneous injections.

The bacterium Burkholderia pseudomallei is the cause of melioidosis infectious disease. In this bacterium, the BLF1 protein wide inhibits the synthesis of proteins in human cells. This disease is reported to cause a death rate of 40% in some parts of the world. Currently, no effective vaccine is available against this bacterial infection. In this study, therefore, a Nano vaccine was synthesized based on the trimethyl chitosan (TMC) polymer containing the BLF1 recombinant protein, and its immunogenicity and protection in Syrian mice were evaluated by oral and subcutaneous injections. The BLF1 recombinant protein expression was induced in Escherichia coli Bl21 (DE3) and purified by the affinity chromatography technique. Recombinant protein-containing nanoparticles (NPs) were then synthesized by the ionotropic gelation method. After oral and subcutaneous injections, antibody titration was assessed by the indirect ELISA assay. Finally, murine groups were challenged using the BLF1 toxin. The results indicated that the immune system showed more antibody titration in subcutaneous injection than in the oral form. However, the results were reversed in the challenge results, and the survival rate was more significant in the oral injection.

Immunoinformatics design of multi-epitope vaccine using OmpA, OmpD and enterotoxin against non-typhoidal salmonellosis.

BACKGROUND: Non-typhoidal Salmonella (NTS) is one of the important bacteria that cause foodborne diseases and invasive infections in children and elderly people. Since NTS infection is difficult to control due to the emergence of antibiotic-resistant species and its adverse effect on immune response, the development of a vaccine against NTS would be necessary. This study aimed to develop a multi-epitope vaccine against the most prevalent serovars of NTS (Salmonella Typhimurium, Salmonella Enteritidis) using an immunoinformatics approach and targeting OmpA, OmpD, and enterotoxin (Stn). RESULTS: Initially, the B cell and T cell epitopes were predicted. Then, epitopes and suitable adjuvant were assembled by molecular linkers to construct a multi-epitope vaccine. The computational tools predicted the tertiary structure, refined the tertiary structure and validated the final vaccine construct. The effectiveness of the vaccine was evaluated via molecular docking, molecular dynamics simulation, and in silico immune simulation. The vaccine model had good binding affinity and stability with MHC-I, MHC-II, and toll-like receptors (TLR-1, 2, 4) as well as activation of T cells, IgM, IgG, IFN-γ and IL-2 responses. Furthermore, after codon optimization of the vaccine sequence, this sequence was cloned in E. coli plasmid vector pET-30a (+) within restriction sites of HindIII and BamHI. CONCLUSIONS: This study, for the first time, introduced a multi-epitope vaccine based on OmpA, OmpD and enterotoxin (Stn) of NTS that could stimulate T and B cell immune responses and produced in the prokaryotic system. This vaccine was validated in-silico phase which is an essential study to reduce challenges before in vitro and in vivo studies.

Developing an Effective Glycan-Based Vaccine for Streptococcus Pyogenes.

Streptococcus pyogenes is a primary infective agent that causes approximately 700 million human infections each year, resulting in more than 500 000 deaths. Carbohydrate-based vaccines are proven to be one of the most promising subunit vaccine candidates, as the bacterial glycan pattern(s) are different from mammalian cells and show increased pathogen serotype conservancy than the protein components. In this Review we highlight reverse vaccinology for use in the development of subunit vaccines against S. pyogenes, and report reproducible methods of carbohydrate antigen production, in addition to the structure-immunogenicity correlation between group A carbohydrate epitopes and alternative vaccine antigen carrier systems. We also report recent advances used to overcome hurdles in carbohydrate-based vaccine development.

An integrated computational framework to design a multi-epitopes vaccine against Mycobacterium tuberculosis.

Tuberculosis (TB) is a highly contagious disease that mostly affects the lungs and is caused by a bacterial pathogen, Mycobacterium tuberculosis. The associated mortality rate of TB is much higher compared to any other disease and the situation is more worrisome by the rapid emergence of drug resistant strains. Bacillus Calmette-Guerin (BCG) is the only licensed attenuated vaccine available for use in humans however, many countries have stopped its use as it fails to confer protective immunity. Therefore, urgent efforts are required to identify new and safe vaccine candidates that are not only provide high immune protection but also have broad spectrum applicability. Considering this, herein, I performed an extensive computational vaccine analysis to investigate 200 complete sequenced genomes of M. tuberculosis to identify core vaccine candidates that harbor safe, antigenic, non-toxic, and non-allergic epitopes. To overcome literature reported limitations of epitope-based vaccines, I carried out additional analysis by designing a multi-epitopes vaccine to achieve maximum protective immunity as well as to make experimental follow up studies easy by selecting a vaccine that can be easily analyzed because of its favorable physiochemical profile. Based on these analyses, I identified two potential vaccine proteins that fulfill all required vaccine properties. These two vaccine proteins are diacylglycerol acyltransferase and ESAT-6-like protein. Epitopes: DSGGYNANS from diacylglycerol acyltransferase and AGVQYSRAD, ADEEQQQAL, and VSRADEEQQ from ESAT-6-like protein were found to cover all necessary parameters and thus used in a multi-epitope vaccine construct. The designed vaccine is depicting a high binding affinity for different immune receptors and shows stable dynamics and rigorous van der Waals and electrostatic binding energies. The vaccine also simulates profound primary, secondary, tertiary immunoglobulin production as well as high interleukins and interferons count. In summary, the designed vaccine is ideal to be evaluated experimentally to decipher its real biological efficacy in controlling drug resistant infections of M. tuberculosis.

Cattle and goats' humoral response to vaccination with Clostridium perfringens type D purified epsilon toxoids.

Herd vaccination is an important preventive measure against enterotoxemia in ruminants. Vaccination in goats should be performed every four months, and recent studies have shown that immunity in cattle lasts for less than one year. One of the mechanisms for increasing the duration of the immune response is to use purified toxoids as immunogens. The aim of the present study was to evaluate the humoral response in cattle and goats after vaccination with purified and semi-purified Clostridium perfringens type D epsilon toxoid. The following three different vaccines were used: vaccine 1 (V1), a semi-purified toxoid adsorbed to aluminum hydroxide; vaccine 2 (V2), a purified toxoid adsorbed to aluminum hydroxide; and vaccine (V3), a purified toxoid adsorbed on chitosan microparticles. Groups of cattle (n = 6-7) and goats (n = 6-7) were vaccinated on days 0 and 30, and serum samples for antitoxin titration were collected every 30 days for one-year post-vaccination. Goats were revaccinated on day 360, and their serum was evaluated on days 367 and 374. The antibody peaks ranged between 6.90 and 11.47 IU/mL in cattle and from 1.11 to 4.40 IU/mL in goats. In cattle administered with the V1 and V2 vaccines, we observed that the antibody titers were maintained above 0.2 IU/mL until the end of the experiment. In goats, V2 elicited long-lasting antibodies, and all animals maintained the protective titers for 210 days after the first dose. In conclusion, the purified toxoid vaccine with aluminum hydroxide adjuvant was able to induce strong and long-lasting humoral responses in both species and could be an alternative for improving the immunization schedule against enterotoxemia in goats and cattle.

Mix-and-Match System for the Enzymatic Synthesis of Enantiopure Glycerol-3-Phosphate-Containing Capsule Polymer Backbones from Actinobacillus pleuropneumoniae, Neisseria meningitidis, and Bibersteinia trehalosi.

Capsule polymers are crucial virulence factors of pathogenic bacteria and are used as antigens in glycoconjugate vaccine formulations. Some Gram-negative pathogens express poly(glycosylglycerol phosphate) capsule polymers that resemble Gram-positive wall teichoic acids and are synthesized by TagF-like capsule polymerases. So far, the biotechnological use of these enzymes for vaccine developmental studies was restricted by the unavailability of enantiopure CDP-glycerol, one of the donor substrates required for polymer assembly. Here, we use CTP:glycerol-phosphate cytidylyltransferases (GCTs) and TagF-like polymerases to synthesize the poly(glycosylglycerol phosphate) capsule polymer backbones of the porcine pathogen Actinobacillus pleuropneumoniae, serotypes 3 and 7 (App3 and App7). GCT activity was confirmed by high-performance liquid chromatography, and polymers were analyzed using comprehensive nuclear magnetic resonance studies. Solid-phase synthesis protocols were established to allow potential scale-up of polymer production. In addition, one-pot reactions exploiting glycerol-kinase allowed us to start the reaction from inexpensive, widely available substrates. Finally, this study highlights that multidomain TagF-like polymerases can be transformed by mutagenesis of active site residues into single-action transferases, which in turn can act in trans to build-up structurally new polymers. Overall, our protocols provide enantiopure, nature-identical capsule polymer backbones from App2, App3, App7, App9, and App11, Neisseria meningitidis serogroup H, and Bibersteinia trehalosi serotypes T3 and T15. IMPORTANCE Economic synthesis platforms for the production of animal vaccines could help reduce the overuse and misuse of antibiotics in animal husbandry, which contributes greatly to the increase of antibiotic resistance. Here, we describe a highly versatile, easy-to-use mix-and-match toolbox for the generation of glycerol-phosphate-containing capsule polymers that can serve as antigens in glycoconjugate vaccines against Actinobacillus pleuropneumoniae and Bibersteinia trehalosi, two pathogens causing considerable economic loss in the swine, sheep, and cattle industries. We have established scalable protocols for the exploitation of a versatile enzymatic cascade with modular architecture, starting with the preparative-scale production of enantiopure CDP-glycerol, a precursor for a multitude of bacterial surface structures. Thereby, our approach not only allows the synthesis of capsule polymers but might also be exploitable for the (chemo)enzymatic synthesis of other glycerol-phosphate-containing structures such as Gram-positive wall teichoic acids or lipoteichoic acids.

pH-Controlled Release of Antigens Using Mesoporous Silica Nanoparticles Delivery System for Developing a Fish Oral Vaccine.

The development of effective vaccines and delivery systems in aquaculture is a long-term challenge for controlling emerging and reemerging infections. Cost-efficient and advanced nanoparticle vaccines are of tremendous applicability in prevention of infectious diseases of fish. In this study, dihydrolipoamide dehydrogenase (DLDH) antigens of Vibrio alginolyticus were loaded into mesoporous silica nanoparticles (MSN) to compose the vaccine delivery system. Hydroxypropyl methylcellulose phthalate (HP55) was coated to provide protection of immunogen. The morphology, loading capacity, acid-base triggered release were characterized and the toxicity of nanoparticle vaccine was determined in vitro. Further, the vaccine immune effects were evaluated in large yellow croaker via oral administration. In vitro studies confirmed that the antigen could be stable in enzymes-rich artificial gastric fluid and released under artificial intestinal fluid environment. In vitro cytotoxicity assessment demonstrated the vaccines within 120 µg/ml have good biocompatibility for large yellow croaker kidney cells. Our data confirmed that the nanoparticle vaccine in vivo could elicit innate and adaptive immune response, and provide good protection against Vibrio alginolyticus challenge. The MSN delivery system prepared may be a potential candidate carrier for fish vaccine via oral administration feeding. Further, we provide theoretical basis for developing convenient, high-performance, and cost-efficient vaccine against infectious diseases in aquaculture.

Structural Modeling of the Treponema pallidum Outer Membrane Protein Repertoire: a Road Map for Deconvolution of Syphilis Pathogenesis and Development of a Syphilis Vaccine.

Treponema pallidum, an obligate human pathogen, has an outer membrane (OM) whose physical properties, ultrastructure, and composition differ markedly from those of phylogenetically distant Gram-negative bacteria. We developed structural models for the outer membrane protein (OMP) repertoire (OMPeome) of T. pallidum Nichols using solved Gram-negative structures, computational tools, and small-angle X-ray scattering (SAXS) of selected recombinant periplasmic domains. The T. pallidum "OMPeome" harbors two "stand-alone" proteins (BamA and LptD) involved in OM biogenesis and four paralogous families involved in the influx/efflux of small molecules: 8-stranded ß-barrels, long-chain-fatty-acid transporters (FadLs), OM factors (OMFs) for efflux pumps, and T. pallidum repeat proteins (Tprs). BamA (TP0326), the central component of a ß-barrel assembly machine (BAM)/translocation and assembly module (TAM) hybrid, possesses a highly flexible polypeptide-transport-associated (POTRA) 1-5 arm predicted to interact with TamB (TP0325). TP0515, an LptD ortholog, contains a novel, unstructured C-terminal domain that models inside the ß-barrel. T. pallidum has four 8-stranded ß-barrels, each containing positively charged extracellular loops that could contribute to pathogenesis. Three of five FadL-like orthologs have a novel α-helical, presumptively periplasmic C-terminal extension. SAXS and structural modeling further supported the bipartite membrane topology and tridomain architecture of full-length members of the Tpr family. T. pallidum's two efflux pumps presumably extrude noxious small molecules via four coexpressed OMFs with variably charged tunnels. For BamA, LptD, and OMFs, we modeled the molecular machines that deliver their substrates into the OM or external milieu. The spirochete's extended families of OM transporters collectively confer a broad capacity for nutrient uptake. The models also furnish a structural road map for vaccine development. IMPORTANCE The unusual outer membrane (OM) of T. pallidum, the syphilis spirochete, is the ultrastructural basis for its well-recognized capacity for invasiveness, immune evasion, and persistence. In recent years, we have made considerable progress in identifying T. pallidum's repertoire of OMPs. Here, we developed three-dimensional (3D) models for the T. pallidum Nichols OMPeome using structural modeling, bioinformatics, and solution scattering. The OM contains three families of OMP transporters, an OMP family involved in the extrusion of noxious molecules, and two "stand-alone" proteins involved in OM biogenesis. This work represents a major advance toward elucidating host-pathogen interactions during syphilis; understanding how T. pallidum, an extreme auxotroph, obtains a wide array of biomolecules from its obligate human host; and developing a vaccine with global efficacy.

Fight bacteria with bacteria: Bacterial membrane vesicles as vaccines and delivery nanocarriers against bacterial infections.

Bacterial membrane vesicles (MVs) are particles secreted by bacteria with diameter of 20-400 nm. The pathogen-associated molecular patterns (PAMPs) present on the surface of MVs are capable of activating human immune system, leading to non-specific immune response and specific immune response. Due to the immunostimulatory properties and proteoliposome nanostructures, MVs have been increasingly explored as vaccines or delivery systems for the prevention and treatment of bacterial infections. Herein, the recent progresses of MVs for antibacterial applications are reviewed to provide an overview of MVs vaccines and MVs-related delivery systems. In addition, the safety issues of bacterial MVs are discussed to demonstrate their potential for clinical translation. In the end of this review, the challenges of bacterial MVs as vaccines and delivery systems for clinical applications are highlighted with the purpose of predicting future research directions in this field.

Simultaneous purification and depolymerization of Streptococcus pneumoniae serotype 2 capsular polysaccharides by trifluoroacetic acid.

Development of an effective purification process in order to provide low cost and high-quality vaccine is the necessity of glycoconjugate vaccine manufacturing industries. In the present study, we have attempted to develop a method for simultaneous purification and depolymerization process for capsular polysaccharides (CPS) derived from Streptococcus pneumoniae serotype 2. Trifluoroacetic acid (TFA) was used to precipitate impurities which were then removed by centrifugation. It was observed that the TFA treatment could simultaneously depolymerize the CPS and purify it. The purified and depolymerized CPS was analyzed for its purity, structural identity and conformity, molecular size, antigenicity to meet desired quality specifications. The obtained results showed that the purification and depolymerization of S. pneumoniae serotype 2 CPS did not affect the antigenicity of CPS.

Alginate-chitosan microcapsules improve vaccine potential of gamma-irradiated Listeria monocytogenes against listeriosis in murine model.

Listeria monocytogenes is a cause of infectious food-borne disease in humans, characterized by neurological manifestations, abortion, and neonatal septicemia. It is intracellular bacterium, which limits the development of protective inactivated vacines. Adjuvants capable of stimulating cellular immune response are important tools for developing novel vaccines against intracellular bacteria. The aim of this study was to evaluate the vaccine potential of L. monocytogenes inactivated by gamma irradiation (KLM-γ) encapsulated in alginate microcapsules associated or not with chitosan against listeriosis in the murine model. At the fourth day after challenge there was a reduction in bacterial recovery in mice vaccinated with KLM-γ encapsulated with alginate or alginate-chitosan, with lower bacterial loads in the spleen (10 fold) and liver (100 fold) when compared to non-vaccinated mice. In vitro stimulation of splenocytes from mice vaccinated with alginate-chitosan-encapsulated KLM-γ resulted in lymphocyte proliferation, increase of proportion of memory CD4+ and CD8+ T cell and production of IL-10 and IFN-γ. Interestingly, the group vaccinated with alginate-chitosan-encapsulated KLM-γ had increased survival to lethal infection with lower L. monocytogenes-induced hepatic inflammation and necrosis. Therefore, KLM-γ encapsulation with alginate-chitosan proved to have potential for development of novel and safe inactivated vaccine formulations against listeriosis.

Immunogenicity evaluating of the SLNs-alginate conjugate against Pseudomonas aeruginosa.

P. aeruginosa is of particular importance due to its numerous pathogens and the spread of its multidrug-resistant strains around the world. Hence there is a need to develop an effective vaccine to prevent the diseases with P. aeruginosa. The aim of present study was to evaluate the immunogenicity of alginate (Alg) antigen in conjugation with SLN as a candidate for nanovaccine against P. aeruginosa in mouse model. Alginate is a weak immunogen, but the immune responses produced by alginate are effective in killing Pseudomonas bacteria. To increase the immunogenicity of alginate, SLN was used that is useful in drug delivery and can boost prolonged effectiveness. The results of ELISA and opsonophagocytosis tests showed that Alg-SLN conjugate has a better ability to stimulate the immune system to produce more immunoglobulins with better performance compared to alginate antigen alone. The challenge test also demonstrated that the Alg-SLN treated mice showed a higher level of immunity than the mice treated with pure alginate against infections caused by P. aeruginosa. Overally the findings showed the efficacy of new prepared vaccine to induce immunogenicity, and therefore it can be considered as a candidate for a strong vaccine against P. aeruginosa.

PEGylated nano-Rehmannia glutinosa polysaccharide induces potent adaptive immunity against Bordetella bronchiseptica.

Vaccines, in many cases, stimulate only too weak immunogenicity to prevent infection. Therefore, adjuvants are required during their preparation to boost the immune response. We herein developed a PEGylated nano-adjuvant based on Rehmannia glutinosa polysaccharide (RGP). The addition of PEG layer exhibits enhanced immune performance of the nano-RGP. Stimulation of dendritic cells (DCs) with PEGylated nano-RGP (pRL) led to increased proliferation and cytokine production (IL-6, IL-12, IL-1ß and TNF-α). The pRL was internalized into DCs via a rapid and efficient method. The mice immunized with pRL exhibited enhanced antigen-specific serum IgG and Th1-(IFN-γ), Th2-(IL-4), and Th17-(IL-17, IL-6) cytokine production, contributing to a good anti-infection performance. Furthermore, the pRL could effectively deliver the antigen to the lymph nodes (LNs), activate DC in the LN and produce enhanced CD4+and CD8+ T-cells-derived memory (CD44high CD62Lhigh), and effector (CD44high CD62Llow) as well as functional phenotypes. Our results revealed that pRL can act as a promising adjuvant with targeted delivery of antigen due to its effective activation and robust adaptive immunity induction of DCs.

Size-Dependent Antibacterial Immunity of Staphylococcus aureus Protoplast-Derived Particulate Vaccines.

BACKGROUND: Vaccination provides a viable alternative to antibiotics for the treatment of drug-resistant bacterial infection. Bacterial protoplasts have gained much attention for a new generation vaccine due to depleting toxic outer wall components. PURPOSE: The objective of this study was to reveal the effects of bacterial protoplast-derived nanovesicles (PDNVs) size on antibacterial immunity. METHODS: Herein, we prepared bacterial PDNVs with different sizes by removing the cell wall of Staphylococcus aureus (S. aureus) to generate multi-antigen nanovaccines. Furthermore, we investigated the ability of PDNVs in different sizes to activate dendritic cells (DCs) and trigger humoral and cellular immune responses in vivo. RESULTS: We obtained particles of ∼200 nm, 400 nm, and 700 nm diameters and found that all the PDNVs readily induce efficient maturation of DCs in the draining lymph nodes of the vaccinated mice. Dramatically, the activation of DCs was increased with decreasing particle sizes. In addition, vaccination with PDNVs generated elevated expression levels of specific antibody and the production of INF-γ, especially the smaller ones, indicating the capability of inducing strong humoral immunity and Th1 biased cell responses against the source bacteria. CONCLUSION: These observed results provide evidence for size-dependent orchestration of immune responses of PDNVs and help to rationally design and develop effective antibacterial vaccines.

Chemoenzymatic synthesis of arabinomannan (AM) glycoconjugates as potential vaccines for tuberculosis.

Mycobacteria infection resulting in tuberculosis (TB) is one of the top ten leading causes of death worldwide in 2018, and lipoarabinomannan (LAM) has been confirmed to be the most important antigenic polysaccharide on the TB cell surface. In this study, a convenient synthetic method has been developed for synthesizing three branched oligosaccharides derived from LAM, in which a core building block was prepared by enzymatic hydrolysis in flow chemistry with excellent yield. After several steps of glycosylations, the obtained oligosaccharides were conjugated with recombinant human serum albumin (rHSA) and the ex-vivo ELISA tests were performed using serum obtained from several TB-infected patients, in order to evaluate the affinity of the glycoconjugate products for the human LAM-antibodies. The evaluation results are positive, especially compound 21 that exhibited excellent activity which could be considered as a lead compound for the future development of a new glycoconjugated vaccine against TB.

Computational design of a novel multi-epitope vaccine against Coxiella burnetii.

Query fever is a zoonotic disease caused by Coxiella burnetii. There is no universal method for the prevention of this disease. Recombinant vaccine is a potent strategy that can be utilized for this purpose. The current study was conducted to develop a multi-epitope vaccine against Coxiella burnetii. Hence, OmpA, Tuf2, GroEL, Mip and sucB antigens were used for the prediction of epitopes. Then, a multi-epitope vaccine was developed based on a molecular adjuvant and fragments that contained the best MHCI, B cell, MHCII and IFN-γ epitopes. The features of the developed vaccine including physicochemical parameters, antigenicity and protein structures were assessed. Also, interaction between the developed vaccine and TLR4/MD2 receptor along with molecular dynamics of the ligand-receptor complex were investigated. Finally, the codon adaptation and cloning were conducted for the developed vaccine. According to the results, molecular weight, instability index, antigenicity and random coil percentage of the developed vaccine were 54.4 kDa, 32.84, 1.1936 and 34.92%, respectively. Besides, residues distribution in core region of the refined model was 85%. The results demonstrated that the developed vaccine could dock to its receptor with the lowest energy of -976.7 as well as RMSD value of the complex was between 0.15 and 0.22 nm. Also, the results showed that CIA index of the codon adapted sequence was 0.95. Finally, cloning results revealed that nucleotide sequence of the developed vaccine could be successfully cloned into pET-21a (+). Based on these results, it seems that the developed vaccine can be a suitable candidate to prevent Coxiella burnetii.

Improvement in the Growth and α-toxin Production of Clostridium septicum by Magnesium Sulfate.

Clostridium septicum, the anaerobic toxigenic bacterium is the agent that causes dangerous disease in man and animals. There is a lethal toxin of the bacterium namely alpha toxin. The ɑ-toxin has hemolytic, necrotic and lethal activities. Today, Razi Vaccine and Serum Research Institute of Iran produced the C. septicum vaccine in the form of bacterin/toxoid. Because of some problems, the vaccine needs to improve on an industrial scale. The study is going to find an appropriate supplement to improve growth and ɑ-toxin production. Three strains of C. septicum (vaccine, NH1 and NH8 strains) were cultured in the basic vaccine media. Magnesium sulfate, Copper, Ferrous, yeast extract, and trace elements plus vitamins' solution were added to the basic vaccine media in different cultures. The effect of the ingredients on the growth was measured by a spectrophotometer and the α-toxin secretion was assayed by hemolysin test. Growth of the bacterium and α-toxin secretion were increased by Magnesium (80 mg/l) in NH8 and vaccine strains significantly. The black precipitate was difficult to dissolve in magnesium media that must be solved. Trace elements plus vitamins solution mildly influence on NH1strain growth and toxin secretion. Other supplements (Cu, Fe, yeast extract) were not showen any significant changes in the growth and α-toxin production of C. septicum. Overflowing peptone (4%) in the vaccine media, fixes essentials of proteolysis activity, allows the sufficient growth and toxin production without Cu, Fe, and yeast extract. Due to essentially of Mg for growth, extra magnesium was added for improvement of media culture. The study suggests for Magnesium addition in the C. septicum vaccine media during production procedure after precipitation solving problem.

Dynamic aspects of pressure and temperature-stabilized intermediates of outer surface protein A.

Structural characterization of alternatively folded and partially disordered protein conformations remains challenging. Outer surface protein A (OspA) is a pivotal protein in Borrelia infection, which is the etiological agent of Lyme disease. OspA exists in equilibrium with intermediate conformations, in which the central and the C-terminal regions of the protein have lower stabilities than the N-terminal. Here, we characterize pressure- and temperature-stabilized intermediates of OspA by nuclear magnetic resonance spectroscopy combined with paramagnetic relaxation enhancement (PRE). We found that although the C-terminal region of the intermediate was partially disordered, it retains weak specific contact with the N-terminal region, owing to a twist of the central ß-sheet and increased flexibility in the polypeptide chain. The disordered C-terminal region of the pressure-stabilized intermediate was more compact than that of the temperature-stabilized form. Further, molecular dynamics simulation demonstrated that temperature-induced disordering of the ß-sheet was initiated at the C-terminal region and continued through to the central region. An ensemble of simulation snapshots qualitatively described the PRE data from the intermediate and indicated that the intermediate structures of OspA may expose tick receptor-binding sites more readily than does the basic folded conformation.