Recombinant Toxoid Research Articles

Determination of degradation for sulfo-SIAB, SM(PEG)2, and sulfo-GMBS by RPLC-UV analysis

Bi-functional N-Hydroxysuccinimide (NHS) linkers are widely used in the conjugation processes linking an immunogen with a carrier protein capable of boosting immunity. A potential vaccine candidate against HIV-1, called fusion peptide (FP), is covalently linked to the recombinant tetanus toxoid heavy-chain fragment C (rTTHC) via this type of linker. A reversed-phase liquid chromatography (RPLC-UV) method was used to monitor the linker’s degradation kinetics in various buffers, mimicking the steps in the conjugation process. The kinetics of the reactivities of the linkers are revealed in this study and can provide a good guidance to help effective conjugation process before these linkers are completely hydrolyze to the inactive degradants. Three cross-linkers degradation pathways were evaluated: Sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (Sulfo-SIAB), PEGylated SMCC (SM(PEG)2), and N-γ-maleimidobutyryl-oxysulfosuccinimide ester (Sulfo-GMBS). We have reported kinetics for Sulfo-SIAB.

Immunogenicity of a pentavalent recombinant Escherichiacoli bacterin against enterotoxemia and botulism in sheep

IntroductionProducing commercial bacterins/toxoids against Clostridium spp. is laborious and hazardous. Conversely, developing prototype vaccines using purified recombinant toxoids, though safe and effective, is both laborious and costly for application in production animals. ObjectiveConsidering that inactivated recombinant Escherichiacoli (bacterin) is a simple, cost-effective, and to be safe solution, we evaluated, for the first time, a pentavalent formulation of recombinant bacterins containing the alpha, beta, and epsilon toxins of Clostridiumperfringens and C and D neurotoxins of Clostridiumbotulinum in sheep. MethodsSubcutaneously, 18 Texel sheep received two doses (200 μg of each antigen) of recombinant bacterin (n = 7) or purified recombinant antigens (n = 6) on days 0 and 28, while the control group (n = 5) did not receive an immunization. Sera samples from days 0 (before the 1st dose), 28 (before the 2nd dose), and 56, 84, and 112 were used for measuring IgG (indirect ELISA) and neutralizing antibodies (mouse serum neutralization). ResultsBoth formulations induced significant levels of IgG against all five toxins (p < 0.05) up to day 112, with peaks at days 28 and 56 post-immunization. The expected booster effect occurred only for the botulinum toxins. The neutralizing antibody titers were satisfactory against ETX (≥2 IU/ml for both formulations) and BoNT-D [5 IU/ml (bacterin) and 10 IU/ml (purified)]. ConclusionWhile adjustments are required, the recombinant bacterin platform holds great potential for polyvalent vaccines due to its straightforward, safe, and cost-effective production, establishing it as a user-friendly technology for the veterinary immunobiological industry.

Protective effects of immunization with a novel 4 recombinant pore-forming toxoid combination vaccine in a rabbit model of systemic methicillin-resistant Staphylococcus aureus infection.

Staphylococcus aureus is a Gram-positive bacterium that most frequently acquires antibiotic resistance. As an opportunistic pathogen, it can cause conditions such as bacteremia, sepsis, and myocarditis. Due to the social need for a vaccine against methicillin-resistant Staphylococcus aureus (MRSA), many research groups have been designing and studying vaccines for decades. In this study, we developed a multivalent vaccine and evaluated its efficacy by applying a novel adjuvant, β-glucan. A vaccine composed of four pore-forming toxins from S. aureus was administered to rabbits 3 times, after which they were challenged with S. aureus USA 300 LAC strain. We measured changes in the rabbits' body weight to monitor systemic adverse reactions and analyzed the total immunoglobulin G antibody titer against the four antigens using enzyme-linked immunosorbent assay. For each rabbit, the number of abscesses and colony-forming units (CFU) in the kidneys were measured. In all vaccinated groups, strong antibody responses against the four antigens were observed. After challenging with MRSA, the vaccinated groups showed less weight change compared to the non-vaccinated groups (average 5.7% versus 13.5%). Additionally, the number of renal abscesses was significantly lower in the vaccinated groups, with three individuals in group 1 (four antigens adjuvanted with β-glucan_PK1) showing no abscess formation. The number of bacteria identified in the kidneys was also statistically significantly lower in the vaccinated group compared to the non-vaccinated group. We demonstrated that the four toxoid antigens we selected can protect against S. aureus infection in a rabbit model and that β-glucan could be used as an immune enhancer. Overall, our study shows that new antigen combinations can induce protective immunity in animal models and that a toxin-based vaccine can help control bacterial colonization.

Streamlining Peptide Mapping LC-MS Approach for Studying Fusion Peptide-Conjugated Vaccine Immunogens.

A newly introduced HIV-1 vaccination utilizes a fusion peptide (FP)-based immunogen-carrier conjugate system, where the FP is coupled to a protein carrier via a bifunctional linker. Such heterogeneous materials present a challenge for the routine product quality assessment. Peptide mapping LC-MS analysis has become an indispensable tool for assessing the site-specific conjugation ratio, estimating site occupancy, monitoring conjugation profiles, and analyzing post-translational modifications (PTMs) and disulfide bonds as well as high-order protein structures. To streamline the peptide mapping approach to match the needs of a fast-paced conjugate vaccine product characterization, a selection of signature fragment ions generated by MSE fragmentation was successfully applied to assess the product quality at the different stages of a conjugates' manufacturing process with an emphasis on monitoring the amount of a reactive linker. This technique was employed in different conjugation studies of the protein carriers, linkers, and FP compositions as well as the cross-linked species formed during stress-degradation studies. Multiple derivatives of the intermediate and final conjugated products formed during a multistaged synthesis were monitored by means of the sensitive extracted-ion chromatogram (XIC) profiling and were included in the estimation of the site-specific conjugation loads. Differentiation of the conjugates with various FP compositions was demonstrated. The conjugation site occupancy was evaluated with respect to the solvent exposure of Lys residues. The findings of these LC-MS studies greatly aided in choosing the best conjugation strategy to ensure that the final recombinant tetanus toxoid heavy chain (rTTHc) product is chemically inert and represents a safe vaccine candidate for clinical evaluation.

Scalable production and immunogenicity of a cholera conjugate vaccine

There is a need to develop cholera vaccines that are protective in young children under 5 years of age, which induce long-term immunity, and which can be incorporated into the Expanded Programme of Immunization (EPI) in cholera-endemic countries. The degree of protection afforded by currently available oral cholera vaccines (OCV) to young children is significantly lower than that induced by vaccination of older vaccine recipients. Immune responses that protect against cholera target the O-specific polysaccharide (OSP) of Vibrio cholerae, and young children have poor immunological responses to bacterial polysaccharides, which are T cell independent antigens. To overcome this, we have developed a cholera conjugate vaccine (CCV) containing the OSP of V. cholerae O1, the main cause of endemic and epidemic cholera. Here, we describe production of CCV through a scalable manufacturing process and preclinical evaluation of immunogenicity in the presence and absence of aluminum phosphate (alum) as an adjuvant. The vaccine displays V. cholerae O1 Inaba OSP in sun-burst display via single point attachment of core oligosaccharide to a recombinant tetanus toxoid heavy chain fragment (rTTHc). Two different pilot-scale production batches of non-GMP CCV were manufactured and characterized in terms of physico-chemical properties and immunogenicity. In preclinical testing, the vaccine induced OSP- and lipopolysaccharide (LPS)-specific IgG and IgM responses, vibriocidal responses, memory B cell responses, and protection in a V. cholerae O1 challenge model. The addition of alum to the administered vaccine increased OSP-specific immune responses. These results support evaluation of CCV in humans.

Humoral Immune Response Evaluation in Horses Vaccinated with Recombinant Clostridium perfringens Toxoids Alpha and Beta for 12 Months.

In horses, Clostridium perfringens is associated with acute and fatal enterocolitis, which is caused by a beta toxin (CPB), and myonecrosis, which is caused by an alpha toxin (CPA). Although the most effective way to prevent these diseases is through vaccination, specific clostridial vaccines for horses against C. perfringens are not widely available. The aim of this study was to pioneer the immunization of horses with three different concentrations (100, 200 and 400 µg) of C. perfringens recombinant alpha (rCPA) and beta (rCPB) proteins, as well as to evaluate the humoral immune response over 360 days. Recombinant toxoids were developed and applied to 50 horses on days 0 and 30. Those vaccines attempted to stimulate the production of alpha antitoxin (anti-CPA) and beta antitoxin (anti-CPB), in addition to becoming innocuous, stable and sterile. There was a reduction in the level of neutralizing anti-CPA and anti-CPB antibodies following the 60th day; therefore, the concentrations of 200 and 400 µg capable of inducing a detectable humoral immune response were not determined until day 180. In practical terms, 200 µg is possibly the ideal concentration for use in the veterinary industry’s production of vaccines against the action of C. perfringens in equine species.

Investigation of Cysteine Modifications in Recombinant Protein Tetanus Toxoid Heavy Chain Fragment C.

For conjugated HIV-1 fusion peptide vaccine development, recombinant Tetanus toxoid heavy chain fragment C (rTTHC) was applied as a carrier protein to boost peptide immunogenicity. Understanding the characteristics of rTTHC is the first step prior to the peptide conjugation. A comprehensive mass spectrometry (MS) characterization was performed on E. coli expressed rTTHC during its purification process. Intact mass along with peptide mapping analysis discovered the existence of three cysteine modification forms: glutathionylation, trisulfide bond modification, and disulfide bond shuffling, in correlation to a three-peak profile during a hydrophobic interaction chromatography (HIC) purification step. Coexistence of these multiple oxidative forms indicated that the active thiols underwent redox reaction in the rTTHC material. Identity confirmation of the rTTHC carrier protein by MS analysis provided pivotal guidance to assess the purification step and helped ensure that vaccine development could proceed.

Parenteral Vaccination with a Cholera Conjugate Vaccine Boosts Vibriocidal and Anti-OSP Responses in Mice Previously Immunized with an Oral Cholera Vaccine.

.Oral cholera vaccination protects against cholera; however, responses in young children are low and of short duration. The best current correlates of protection against cholera target Vibrio cholerae O-specific polysaccharide (anti-OSP), including vibriocidal responses. A cholera conjugate vaccine has been developed that induces anti-OSP immune responses, including memory B-cell responses. To address whether cholera conjugate vaccine would boost immune responses following oral cholera vaccination, we immunized mice with oral cholera vaccine Inaba CVD 103-HgR or buffer only (placebo) on day 0, followed by parenteral boosting immunizations on days 14, 42, and 70 with cholera conjugate vaccine Inaba OSP: recombinant tetanus toxoid heavy chain fragment or phosphate buffered saline (PBS)/placebo. Compared with responses in mice immunized with oral vaccine alone or intramuscular cholera conjugate vaccine alone, mice receiving combination vaccination developed significantly higher vibriocidal, IgM OSP-specific serum responses and OSP-specific IgM memory B-cell responses. A combined vaccination approach, which includes oral cholera vaccination followed by parenteral cholera conjugate vaccine boosting, results in increased immune responses that have been associated with protection against cholera. These results suggest that such an approach should be evaluated in humans.

Quantification of trivalent non-replicating rotavirus vaccine antigens in the presence of aluminum adjuvant

Parenterally administered rotavirus vaccines may overcome the low efficacy observed in resource-poor regions that use live oral formulations. We have reported work on a trivalent nonreplicating rotavirus vaccine (NRRV) for parenteral administration consisting of the recombinant tetanus toxoid P2 CD4 epitope fused to a truncated VP8* fragment (P2-VP8*) for the P[4], P[6], and P[8] serotypes of rotavirus adjuvanted with aluminum. An essential part of developing this vaccine candidate was devising quantification methods for each antigen in the trivalent NRRV in the presence of aluminum adjuvant. This report describes the development of quantitative inhibition enzyme-linked immunosorbent assays (ELISAs) for in vitro antigenicity determination of the adjuvanted trivalent NRRV using serotype-specific monoclonal antibodies (mAbs) against each of the P2-VP8* antigens. Adjuvanted trivalent vaccine samples are titrated and incubated with a constant concentration of specific mAbs against each NRRV P2-VP8* antigen variant. Unbound mAbs are measured by ELISA to indirectly quantify the amount of each antigen present in the trivalent vaccine. Sensitive, specific, and reproducible inhibition ELISAs were developed and qualified for each antigen and used for final product quantification and release testing without desorption of the vaccine antigen.

Evaluation of the expression and immunogenicity of four versions of recombinant Clostridium perfringens beta toxin designed by bioinformatics tools

Beta toxins (CPB) produced by Clostridium perfringens type B and C cause various diseases in animals, and the use of toxoids is an important prophylactic measure against such diseases. Promising recombinant toxoids have been developed recently. However, both soluble and insoluble proteins expressed in Escherichia coli can interfere with the production and immunogenicity of these antigens. In this context, bioinformatics tools have been used to design new versions of the beta toxin, and levels of expression and solubility were evaluated in different strains of E. coli. The immunogenicity in sheep was assessed using the molecule with the greatest potential that was selected on analyzing these results. In silico analyzes, greater mRNA stability (−169.70 kcal/mol), solubility (−0.755), and better tertiary structure (−0.12) were shown by rCPB-C. None of the strains of E. coli expressed rFH8-CPB, but a high level of expression and solubility was shown by rCPB-C. Higher levels of total and neutralizing anti-CPB antibodies were observed in sheep inoculated with bacterins containing rCPB-C. Thus, this study suggests that due to higher productivity of rCPB-C in E. coli and immunogenicity, it is considered as the most promising molecule for the production of a recombinant vaccine against diseases caused by the beta toxin produced by C. perfringens type B and C.

Clostridium perfringens α and β recombinant toxoids in equine immunization

ABSTRACT: Clostridium perfringens is considered one of the main causative agents of superacute enterocolitis, usually fatal in the equine species, due to the action of the β toxin, and is responsible for causing severe myonecrosis, by the action of the α toxin. The great importance of this agent in the equine economy is due to high mortality and lack of vaccines, which are the main form of prevention, which guarantee the immunization of this animal species. The aim of this study was to evaluate three different concentrations (100, 200 and 400μg) of C. perfringens α and β recombinant toxoids in equine immunization and to compare with a group vaccinated with a commercial toxoid. The commercial vaccine was not able to stimulate an immune response and the recombinant vaccine was able to induce satisfactory humoral immune response in vaccinated horses, proving to be an alternative prophylactic for C. perfringens infection.

Generation and evaluation of specific activity and authenticity of recombinant vaccine designed preventing against Pseudomonas aeruginosa

The aim of the study was to obtain and characterize a recombinant vaccine for the immunoprophylaxis of infections caused by Pseudomonas aeruginosa. A set of two highly immunogenic P. aeruginosa recombinant proteins was used to create related vaccine. The first vaccine component was presented by recombinant outer membrane protein F (OprF), whereas the second one — by recombinant atoxic exotoxin A (toxoid). These antigens allowed to develop vaccine inducing immune response against the surface bacterial cues and promote production of neutralizing antibodies against exotoxin A, one of the most dangerous P. aeruginosa pathogenicity factors. Recombinant proteins were synthesized in Escherichia coli cells and isolated by two-step purification. In case of recombinant OprF protein, it was initially (stage 1) isolated as a precipitate containing hydrophobic fraction of producer cell-derived proteins, whereas for recombinant toxoid we purified inclusion bodies. At stage 2, a Ni Sepharose column affinity chromatography was performed. Next, purified recombinant proteins were dialyzed against buffer solution containing 50 mМ Tris-HCl (pH 9.0) and 0.01% Tween 20 followed by filtration sterilization. Three lots of the Pseudomonas Recombinant Vaccine (PRV) were obtained for the study, wherein the recombinant antigens were absorbed with aluminum hydroxide. Recombinant OprF and toxoid protein in vaccine formula were used at a dose of 25 μg and 50 μg, respectively. Absorption completeness of the recombinant antigens within the vaccine was evaluated by polyacrylamide gel electrophoresis with PRV preparations concentrated by ultrafiltration in spin columns. Authenticity of recombinant vaccine was assessed by using customized method by desorbing antigens followed by ultrafiltration concentration in spin columns. Final concentrated desorbed vaccine preparations were analyzed by polyacrylamide gel electrophoresis and immunoblotting, which allowed to confirm presence of specific recombinant antigens in the vaccine. The experimental PRV series demonstrated specific activity (protective properties) after inoculation in animals (mice) by using a dual vaccination protocol, wherein mice were immunized intraperitonially twice with a two-week interval. Next, two weeks later mice were infected by toxigenic P. aeruginosa strain PA-103 culture cells. The index of protective efficiency (IE) for experimental vaccine series was at least a value of three (IE: 3.0–3.3) that was by 1.5-fold higher than that for using single vaccine components (IE: 2.0–2.3). Thus, we confirmed an additive effect of using a set preparation to protect against infection caused by toxigenic Pseudomonas aeruginosa strains.

A unique algorithm for the determination of peptide-carrier protein conjugation ratio by amino acid analysis using intrinsic internal standard

An N-terminal peptide of the HIV-1 fusion peptide (FP) with eight amino acid residues (FP8) was conjugated to a recombinant Tetanus Toxoid Heavy Chain Fragment C (rTTHc) as a carrier protein to help boosting immunogenicity against HIV-1. In this rapid communication, a unique algorithm to determine FP-rTTHc conjugation ratio was developed based off the amino acid analysis. Five well recovered amino acids (present in both FP and rTTHc) were used to calculate the conjugation ratio, while proline (present only in rTTHc) was identified and utilized as the intrinsic internal standard for normalization. With this calculation, the assay variability was minimized (<20%), especially for conjugates with moderate to low conjugation ratios as being compared to previously reported methods. The approach offers a reliable tool to determine the efficiency of the conjugation reactions for in-process monitoring and for final conjugate product characterization.

Preclinical Development of a Fusion Peptide Conjugate as an HIV Vaccine Immunogen

The vaccine elicitation of broadly neutralizing antibodies against HIV-1 is a long-sought goal. We previously reported the amino-terminal eight residues of the HIV-1-fusion peptide (FP8) – when conjugated to the carrier protein, keyhole limpet hemocyanin (KLH) – to be capable of inducing broadly neutralizing responses against HIV-1 in animal models. However, KLH is a multi-subunit particle derived from a natural source, and its manufacture as a clinical product remains a challenge. Here we report the preclinical development of recombinant tetanus toxoid heavy chain fragment (rTTHC) linked to FP8 (FP8-rTTHC) as a suitable FP-conjugate vaccine immunogen. We assessed 16 conjugates, made by coupling the 4 most prevalent FP8 sequences with 4 carrier proteins: the aforementioned KLH and rTTHC; the H. influenzae protein D (HiD); and the cross-reactive material from diphtheria toxin (CRM197). While each of the 16 FP8-carrier conjugates could elicit HIV-1-neutralizing responses, rTTHC conjugates induced higher FP-directed responses overall. A Sulfo-SIAB linker yielded superior results over an SM(PEG)2 linker but combinations of carriers, conjugation ratio of peptide to carrier, or choice of adjuvant (Adjuplex or Alum) did not significantly impact elicited FP-directed neutralizing responses in mice. Overall, SIAB-linked FP8-rTTHC appears to be a promising vaccine candidate for advancing to clinical assessment.

DEVELOPMENT OF THE VACCINE BASED ON THE RECOMBINANT ANTIGENS OF PSEUDOMONAS AERUGINOSA

Aim. The aim is obtaining, investigation and selection of recombinant antigens for inclusion theirs into the against Pseudomonas vaccine. Materials and methods. The genes encoding of the outer membrane proteins F, L and I and Exotoxin A were synthesized by PCR with the genomic DNA of Pseudomonas aeruginosa. The amplified sequences were cloned into plasmid vectors for expression in cells of Escherichia coli. As the result of expression were the synthesized recombinant proteins that were purified in columns with a nickel-activated sorbent. The authenticity of the recombinant antigens was assessed by electrophoresis and immunoblotting. For assessing the immunogenicity of the recombinant proteins,they were sorbed on aluminum hydroxide and used for intraperitoneal immunization of mice. After a course of immunization, mice were injected intraperitoneally with a live virulent culture or еxotoxin A. Results. The obtained recombinant outer membrane proteins OprF, OprL and OprI, as well as the deletion variant of еxotoxin A (toxoid) stimulated immune reactions and protected the experimental animals from the virulent culture of P. aeruginosa. Using of the complexes of the recombinant proteins, as well as immunization with the fusion proteins consisting from sequences of two or three recombinant antigens, produced an additive increase in protective effects. The combination of the recombinant OprF protein and the recombinant toxoid (efficiency index of protective properties (EI 3.0) and two recombinant fusion proteins (EI 3.5) were the most effective. The first recombinant fusion protein (OprF-aTox-OprI) consisted from fused polypeptide sequences of OprF, toxoid and OprI. The second recombinant fusion protein (OprF-OprI) consisted from fused polypeptide sequences of OprF and OprI. Conclusion. The data obtained showed the fundamental possibility of using recombinant fusion proteins OprF-aTox-OprI and OprF-OprI as well as the complex of the recombinant OprF protein and the recombinant toxoid as the candidated vaccines against Pseudomonas aeruginosa.

PRECLINICAL STUDIES OF RECOMBINANT PSEUDOMONAS VACCINE

Aim. Evaluation of the efficacy and safety of the three experimental lots of Recombinant Pseudomonas Vaccine. Materials and methods. The preparation contained the recombinant proteins OprF and toxoid that were purified by chromatography in nickel-sepharose. Aluminum hydroxide was used as an adjuvant. The authenticity of the vaccine components was confirmed by electrophoresis and immunoblotting. The concentration of endotoxin in the vaccine was determined by LAL test. The abnormal toxicity was evaluated in mice and cavy. The anaphylactic activity was evaluated in cavy. The delayed-type hypersensitivity reaction was evaluated in mice. Evaluation of the immunogenicity was carried out in an experiment with on double immunization of mice with following intraperitoneally infection by a live virulent culture of P. aeruginosa (PA-103 strain). Results. The authenticity of the vaccine, sterility, non-pyrogenicity and non-toxicity were confirmed after the studying of the quality indicators of the three lots of Recombinant Pseudomonas Vaccine. In animal experiments, the vaccine did not possess allergic properties and it was shown that it protected mice against Pseudomonas infection with Index of efficiency 3.0 and more. Conclusion. The efficacy, the safety, and the standardization of three experimental lots of the recombinant vaccine, which is intended to prevent infections caused by P. aeruginosa, have been shown.

ELISA for Quantitative Detection of Recombinant Toxoid in the Composition of Complex Vaccine against Pseudomonas Infection

ELISA for Quantitative Detection of Recombinant Toxoid in the Composition of Complex Vaccine against Pseudomonas Infection

Decreased STEC shedding by cattle following passive and active vaccination based on recombinant Escherichia coli Shiga toxoids

The principal virulence factor of Shiga toxin (Stx)-producing Escherichia coli (STEC), the eponymous Stx, modulates cellular immune responses in cattle, the primary STEC reservoir. We examined whether immunization with genetically inactivated recombinant Shiga toxoids (rStx1MUT/rStx2MUT) influences STEC shedding in a calf cohort. A group of 24 calves was passively (colostrum from immunized cows) and actively (intra-muscularly at 5th and 8th week) vaccinated. Twenty-four calves served as unvaccinated controls (fed with low anti-Stx colostrum, placebo injected). Each group was divided according to the vitamin E concentration they received by milk replacer (moderate and high supplemented). The effective transfer of Stx-neutralizing antibodies from dams to calves via colostrum was confirmed by Vero cell assay. Serum antibody titers in calves differed significantly between the vaccinated and the control group until the 16th week of life. Using the expression of activation marker CD25 on CD4+CD45RO+ cells and CD8αhiCD45RO+ cells as flow cytometry based read-out, cells from vaccinated animals responded more pronounced than those of control calves to lysates of STEC and E. coli strains isolated from the farm as well as to rStx2MUT in the 16th week. Summarized for the entire observation period, less fecal samples from vaccinated calves were stx1 and/or stx2 positive than samples from control animals when calves were fed a moderate amount of vitamin E. This study provides first evidence, that transfer to and induction in young calves of Stx-neutralizing antibodies by Shiga toxoid vaccination offers the opportunity to reduce the incidence of stx-positive fecal samples in a calf cohort.

Assessment of Protective Properties of the Recombinant Complex of the Outer Membrane Protein F and the Toxoid of Pseudomonas aeruginosa

Pseudomonas aeruginosa induces the complications after burns, injuries, surgical interventions and appears to be one of the main causative agents of nosocomial infections. This pathogen has the high resistance to the antibacterial preparations, therefore the immunoprophylaxis is considered as one of the major approaches to reduce Pseudomonas infection. Objective: The aim of our investigation is to study the protective properties of the recombinant complex of the outer membrane protein F (OprF) and a non-toxic variant of the exotoxin A (toxoid) against Pseudomonas infection. Methods: The recombinant proteins which contained the additional histidine residues were synthesized into Escherichia coli with isopropyl-βD-thyogalactopyranoside (IPTG). The recombinant proteins were purified by affinity chromatography on Ni-Sepharose. The preparations of recombinant proteins were injected intraperitoneally into the mice. Aluminum hydroxide was used as an adjuvant. For an experimental infection in mice, animals were challenged intraperitoneally by a live virulent culture of P. aeruginosa (PA-103 strain). Results: The best protective effect for the complex containing 25 μg OprF and 50 μg toxoid was identified when we used the double immunization of mice (Index of efficiency of the protective properties in this case was 4.0). Indexes of efficiency of separated recombinant proteins which were injected twice in the same doses were 2.0 for OprF и 2.3 for toxoid. The triple immunization of animals was inefficient for separated recombinant proteins in the same doses. The injection of doses which were lowered twice (12.5 μg for OprF and 25 μg for toxoid) resulted in increased survival of mice immunized by individual proteins (indexes of efficiency: 3 for OprF and и 3,5 for toxoid). However when we administered to the complex of proteins with the same doses Index of efficiency was 2.8. Conclusion: It was shown that the maximum protective effect in a short time is achieved by the combination of double immunization and the mixture of the recombinant proteins OprF and the 25 and 50 μg doses of recombinant toxoid .

A New Immunoassay for Detecting All Subtypes of Shiga Toxins Produced by Shiga Toxin-Producing E. coli in Ground Beef.

BackgroundShiga toxin (Stx) is a common virulence factor of all Shiga toxin producing E. coli (STEC) that cause a wide spectrum of disease, including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Although several commercial kits are available for detection of Stx produced by STEC, none of them are capable of recognizing all subtypes of Stxs, which include three subtypes of Stx1 and seven subtypes of Stx2.Methods and FindingsNew monoclonal and polyclonal antibodies against Stx1 and Stx2 were developed. A universal sandwich ELISA capable of detecting all known subtypes of Stx1 and Stx2 was established using a pool of newly developed antibodies. To precisely monitor the sensitivity of the assay for each subtype of Stxs, recombinant toxoids were created and used as standards in ELISAs. Because of the high affinity of the antibodies incorporated, the ELISA assay is highly sensitive with a limit of detection for the different subtypes of Stx1a and Stx2a between 10 and 50 pg/mL in phosphate buffered saline (PBS). The assay was also able to identify STEC based on the production of Stxs using the supernatants of culture fluids or even single colonies on agar plates without lengthy enrichment in liquid medium. When applied to ground beef samples, this newly developed ELISA was capable of distinguishing beef samples spiked with a single bacterial cell.ConclusionsA highly sensitive and universal assay for all subtypes of Stx1 and Stx2 was developed. It has significantly improved upon the current technologies by avoiding false negative results due to the narrow detection range of the assay. The assay developed in this study can be useful for prompt detection of new and emerging serotypes and screening ground beef samples for contamination of STEC at an early stage in the food supply chain, thus avoiding the need for possible recall.