Recombinant Pseudomonas Vaccine: Technological Aspects of Obtaining and Evaluating Quality Indicators

For an aim to prevent Pseudomonas aeruginosa, a candidate recombinant vaccine has been developed. This vaccine – (RPV) was based on two protective proteins of P. aeruginosa: the outer membrane protein F (OprF) and the recombinant truncated form of the Exotoxin A (toxoid) that were adsorbed on the aluminum hydroxide. The optimal immunization schedule for mice included two intraperitoneal administrations with a two-week interval. RPV promoted to increase survival rates in challenged immunized mice and stimulated humoral and innate immune responses. During preclinical studies, we confirmed the immunogenicity of the vaccine that had not pyrogenicity, acute and chronic toxicity, allergenicity and immunotoxicity. Keywords: Pseudomonas aeruginosa, outer membrane protein F (OprF), toxoid, Pseudomonas Recombinant Vaccine (PRV

Development of ELISA test for the quality control of Pseudomonas aeruginosa recombinant vaccine based on the hybrid recombinant protein

A hybrid recombinant protein containing the amino  acid sequences of the three  most significant Pseudomonas aeruginosa antigens  (membrane proteins OprF, OprI  and toxoid  aTox)  was incorporated into a vaccine against Pseudomonas infection. Quality control of a hybrid recombinant protein and appropriate vaccine includes  determination of authentity and completeness of adsorption upon aluminum hydroxide adjuvant. The aim of our study was to develop  techniques of quality  control for a vaccine  based on the hybrid  OprF-aToxOprI  recombinant protein specific  to  P. aeruginosa.  Hybridomas secreting  specific  monoclonal antibodies for OprF-aTox-OprI were derived  from the fusion of myeloma cells and murine spleen  cells immunized with recombinant proteins P. aeruginosa. To  produce sufficient  quantities of antibodies, the  hybrid  cells were in vivo cultured in BALB/c mice.  Supernates and ascite liquids were chromatographically purified  with immune sorbent. Conjugation of antibodies with  horseradish peroxidase was carried  out  according to  P.K.Nakane. The  hybrid  OprF-aTox-OprI recombinant protein was detected by the  solid-phase ELISA, using a panel  of monoclonal antibodies and  conjugates of monoclonal antibodies with  horseradish peroxidase. Monoclonal antibodies were specific for different  OprF-aTox-OprI epitopes. Titration assays containing OprF-aTox-OprI protein at 78 ng/ml to 5000 ng/ml were used as quantitative standards for calibration curves.To identify  the recombinant protein OprF-aTox-OprI, 55 variants  of of MAb pairs were tested.  Limits  of quantitative detection served  for selection of most  sensitive  and  specific  ELISA  variants.  The  quantitative detection limit was calculated for all 11 ELISA  variants.  Two ELISA  variants  with the highest  sensitivity were selected  for  quality  control of the  hybrid  recombinant protein. The  limits  of quantitative detection were, respectively, 2.9 and 13.6 ng/ml (0.0058  and 0.027% of the estimated antigen  content in the vaccine)  for the first and  second  ELISA  variants.  The  first variant  included a pair  of monoclonal antibodies specific  for the OprF  and OprI  epitopes, the second  variant  represented aTox and OprI  epitopes. Two variants  of ELISA  were developed to detect  the hybrid recombinant OprF-aTox-OprI protein. The first variant allows to determine the protein amount and to evaluate completeness of its adsorption on aluminum hydroxide. To confirm authenticity of the protein, both methods must be used, since they can detect all three antigens (OprF, aTox and OprI) which are present  in the fusion protein

A proposal for a CT driven classification of left colon acute diverticulitis

Computed tomography (CT) imaging is the most appropriate diagnostic tool to confirm suspected left colonic diverticulitis. However, the utility of CT imaging goes beyond accurate diagnosis of diverticulitis; the grade of severity on CT imaging may drive treatment planning of patients presenting with acute diverticulitis. The appropriate management of left colon acute diverticulitis remains still debated because of the vast spectrum of clinical presentations and different approaches to treatment proposed. The authors present a new simple classification system based on both CT scan results driving decisions making management of acute diverticulitis that may be universally accepted for day to day practice

OBTAINING FUSED RECOMBINANT PROTEINS OprF-ΔOprI, ΔOprF-ΔOprI AND OprF-aTox-ΔOprl OF PSEUDOMONAS AERUGINOSA

Aim. Obtaining fused recombinant proteins of Pseudomonas aeruginosa that have protective properties against experimental pseudomonas infection. Materials and methods. Fused sequences of P. aeruginosa genes oprF, oprl and deleted form of toxA were cloned in plasmids for the expression in Escherichia coli. The synthesized recombinant proteins were purified in Ni-sepharose columns. Recombinant proteins were administered to mice intraperitonealiy twice with a 2 week interval to evaluate protective properties. Virulent culture of P. aeruginosa strain PA103 was injected into the animals intraperitonealiy 2 weeks after the immunization course as experimental challenge. Results. 3 fused recombinant proteins were produced: 1. OprF-ΔOprl included full sequence of OprF protein and deletion variant of OprI (lacking first 20 amino acids); 2. AOprF-AOprl consisted of C-terminal region (192 - 342 amino acids) OprF and deletion variant of Oprl protein; 3. OprF-aTox-ΔOprI included full sequence of OprF protein, sequence of nontoxic variant of exotoxin A (without 106 C-terminal amino acids) and deletion variant of Oprl protein. Fused recombinant proteins OprF-AOprl and OprF-aTox-ΔOprI at immunization doses of 25 and 50 pg for the first and second protein, respectively, were shown to have the best protective properties. Conclusion. The results obtained open perspectives for further studies to create specific immune biological preparations based on fused recombinant proteins of P. aeruginosa

RECOMBINANT ANTIGENS OF PSEUDOMONAS AERUGINOSA: EFFECT ON IMMUNE RESPONSE IN MICE

Aim. Study the effect of recombinant antigens of P. aeruginosa on key effectors of the immune system. Materials and methods. Mice were immunized intraperiotoneally with 25 jig of OprF and 50 jig of anatoxin sorbed on aluminium hydroxide gel with a 2 week interval. 7 days after the last immunization spleen lymphocyte subpopulation structure was evaluated by flow cytometry. Cytokine levels in mice sera were studied after a single immunization with recombinant OprF and anatoxin at 4, 8, 24 hours and 14 days by flow cytometry using FlowCytomix Mouse Thl/Th2 10 plex. Results. OprF recombinant antigens and anatoxin affect molecular-cell mechanisms of immune response resulting in alteration of expression of differentiating and activating molecules as well as synthesis of Thl/Th2/Thl7/Th21/Th22 cytokines in mice that are necessary for effective presentation of the antigen. Conclusion. Complex of recombinant OprF and anatoxin facilitated formation of complete immune response against pseudomonas

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

Obtaining the recombinant fusion protein OprF-aTox of <i>Pseudomonas aeruginosa</i> whith protective properties

Into the cells of Escherichia coli (strain BL21(DE3)) two forms of the fusion protein Pseudomonas aeruginosa containing the full length outer membrane protein F (OprF) and nontoxic form of Exotoxin A (without 106 C-terminal amino acid residues) have been synthesized. Two recombinant genes were inserted into plasmid pET28 in different order: oprF-atox and atox-oprF. Only oprF-atox variant allowed to obtain the recombinant protein sufficient for purification by affinity chromatography on Ni-Sepharose. The recombinant fusion protein OprF-aTox showed a high specificity in interaction with the preparations of polyclonal immune rabbit serum to the recombinant OprF, the recombinant nontoxic form of Exotoxin A and the bacterial cells of P. aeruginosa. The purified recombinant fusion protein OprF-aTox after two immunizations protected the mice against toxigenic strain of P. aeruginosa (РА-103) being injected intraperitoneally. The index of efficiency of protective properties of OprF-aTox in the optimal dose (50 µg protein per mouse) was 3.5. This was more efficient than in cases when the recombinant OprF and the recombinant toxoid were injected separately. The indexes of efficiency of protective properties of OprF and the toxoid were 2.1 and 2.0

DEVELOPMENT OF ELISAS FOR THE QUALITY CONTROL OF A RECOMBINANT PSEUDOMONAS VACCINE

Aim. Development and optimization of enzyme immunoassays for quality control of pseudomonas recombinant vaccine. Materials and methods. Recombinant proteins in intermediate products and for completeness of adsorption control were detected in sandwich immunoassay using specific polyclonal and monoclonal antibodies. Detection of the antigen in the vaccine was carried out on the residual amount of antibodies specific to toxoid or OprF that did not bind to the vaccine preparation during the preincubation. Results. ELISAs have been developed and optimized for the quantitative determination of the components (toxoid and membrane protein OprF) of the Pseudomonas recombinant vaccine during the production process. It has been established that: the methods are specific for the determination of toxoid and OprF, the quantitative limit determination has acceptable reliability, the possibility of choosing interpolation of the calibration dependence within the analytical area is shown, the accuracy and precision meets the acceptance criteria, the technique is stable under the conditions of the analysis. Conclusion. Methods can be used to control the quality of the drug during its developing and storage