Evaluation of impact of temperature and pH alterations on the size and antigenicity of meningococcal serogroup A and X polysaccharides and conjugates.

The changes in the recommended storage conditions of the glycoconjugate vaccines against Neisseria meningitidis (Men) serogroup A and serogroup X can affect its activity or potency. Elevated temperature and the change in pH may result in the physical instability leading to the size degradation of the polysaccharide (PS) and subsequent loss of PS epitopes. Moreover, high temperature may also result in protein aggregation and altered tertiary structure of the protein in the conjugate. Consequently, the construction of a potent glycoconjugate is dependent on optimal temperature and pH. The changes in both these conditions can also affect the production of a capsular polysaccharide (PS) and its conjugation to a protein carrier and may also affect the integrity of the vaccine molecule including the maintenance of the protective epitopes. In our study we have used inhibition ELISA as a tool to assess the impact of temperature and pH alterations on the antigenicity of N. meningitidis serogroup A and X, PS and conjugates and their correlation with the size distribution analysis using high pressure size exclusion chromatography. The studies on pH alterations from 5 to 9 led to minimal impact on size and antigenicity of all antigens, however, an elevated temperature adversely impacted the antigen size as well as antigenicity to varying extent. Results indicate the higher stability of MenX PS and conjugate as compared to that for MenA counterparts at elevated temperatures. Furthermore, both the MenA and MenX conjugates appears to be more stable as compared to the corresponding PSs.

Effects of prolonged exposure to moderate static magnetic field and its synergistic effects with alkaline pH on Enterococcus faecalis.

Static magnetic field (SMF) has been shown to biologically affect various microorganisms, but its effects on Enterococcus faecalis, which is associated with multiple dental infections, have not been reported yet. Besides, Enterococcus faecalis was found to be resistant to the alkaline environment provided by a major dental antimicrobial, calcium hydroxide. Therefore, the antibacterial activity of prolonged exposure to moderate SMF (170 mT) and its possible synergistic activity with alkaline pH (pH = 9) were evaluated in the study. The ability to form a biofilm under these conditions was examined by crystal violet assay. Real-time quantitative PCR was performed to evaluate the relative expression of stress (dnaK and groEL) and virulence (efaA, ace, gelE and fsrC) related genes. As the results indicated, cell proliferation was inhibited after 120 h of SMF exposure. What's more, the combined treatment of SMF and alkaline pH showed significantly improved antimicrobial action when compared to single SMF and alkaline pH treatment for more than 24 h and 72 h respectively. However, the ability to form a biofilm was also enhanced under SMF and alkaline pH treatments. SMF can induce stress response by up-regulating the expression of dnaK and elevate virulence gene expression (efaA and ace). These responses were more significant and more genes were up-regulated including groEL, gelE and fsrC when exposed to SMF and alkaline pH simultaneously. Hence, combination of SMF and alkaline pH could be a promising disinfection strategy in dental area and other areas associated with Enterococcus faecalis infections.

Pathogens Inactivated by Low-Energy-Electron Irradiation Maintain Antigenic Properties and Induce Protective Immune Responses.

Inactivated vaccines are commonly produced by incubating pathogens with chemicals such as formaldehyde or ß-propiolactone. This is a time-consuming process, the inactivation efficiency displays high variability and extensive downstream procedures are often required. Moreover, application of chemicals alters the antigenic components of the viruses or bacteria, resulting in reduced antibody specificity and therefore stimulation of a less effective immune response. An alternative method for inactivation of pathogens is ionizing radiation. It acts very fast and predominantly damages nucleic acids, conserving most of the antigenic structures. However, currently used irradiation technologies (mostly gamma-rays and high energy electrons) require large and complex shielding constructions to protect the environment from radioactivity or X-rays generated during the process. This excludes them from direct integration into biological production facilities. Here, low-energy electron irradiation (LEEI) is presented as an alternative inactivation method for pathogens in liquid solutions. LEEI can be used in normal laboratories, including good manufacturing practice (GMP)- or high biosafety level (BSL)-environments, as only minor shielding is necessary. We show that LEEI efficiently inactivates different viruses (influenza A (H3N8), porcine reproductive and respiratory syndrome virus (PRRSV), equine herpesvirus 1 (EHV-1)) and bacteria (Escherichia coli) and maintains their antigenicity. Moreover, LEEI-inactivated influenza A viruses elicit protective immune responses in animals, as analyzed by virus neutralization assays and viral load determination upon challenge. These results have implications for novel ways of developing and manufacturing inactivated vaccines with improved efficacy.

Influência da aplicação de irradiação por feixe de elétrons na qualidade microbiológica de filés de corvina (Micropogonias furnieri) refrigerados / Influence of irradiation application for electron beam on the microbiological quality of chilled croaker (Micropogonias furnieri) fillets

A adequada manipulação do pescado desde sua captura até seu processamento tecnológico, além da manutenção das condições higiênico-sanitárias, o que inclui a qualidade da água utilizada na cadeia, influencia na carga microbiana inicial apresentada. A fim de retardar o processo de deterioração, diminuir as perdas e os riscos iminentes à saúde coletiva, como a propagação de agentes etiológicos de doenças alimentares, são empregados diferentes métodos de conservação. Os feixes de elétrons são utilizados em vários países e levam à destruição dos microrganismos por alterações em suas estruturas, as quais ocorrem pela remoção de elétrons de seus átomos. Objetivou-se, no presente trabalho, contribuir para a avaliação da eficiência da irradiação por feixe de elétrons na qualidade microbiológica de filés de corvina (M. furnieri) refrigerados, desembarcados no município de Niterói - RJ, Brasil. Foram realizadas contagem de bactérias heterotróficas aeróbias mesófilas, de bactérias heterotróficas aeróbias psicrotróficas e enumeração de Enterococcus spp. e, posteriormente, comparadas as amostras do grupo controle com as dos grupos irradiados a doses de 0,7 e 1,0kGy. Os peixes inteiros foram adquiridos no cais de Itaipu, filetados no mercado, embalados a vácuo e mantidos a ±4°C. Embora não tenha sido encontrada diferença estatisticamente significativa entre os três grupos (P>0,05) em nenhuma das análises, concluiu-se que o processamento utilizado foi eficaz na redução do crescimento das três bactérias pesquisadas no dia zero de ambos os grupos irradiados.

The proper handling of fish from capture to technological processing, as well as maintenance of sanitary conditions and the quality of water, can influence the initial microbial load presented. In order to slow down the deterioration process, the reduction of losses and eminent risk to public health as the spread of etiological agents of foodborne illness are different methods used for storage. Electron beams are used in several countries and lead to the destruction of microorganisms by changes in their structures, which occur by removing electrons from their atoms. The objective in the present study contributes to the evaluation of electron beam irradiation efficiency in microbiological quality of chilled croaker fillets (M. furnieri), landed in Niterói - RJ, Brazil. Were performed Bacteria Count Heterotrophic aerobic mesophilic, psychrotrophic bacteria Heterotrophic Aerobic and enumeration of Enterococcus spp. and subsequently compared to the control group of samples with the groups irradiated at doses of 0.7 and 1.0kGy. Whole fish were purchased from the Itaipu pier, threaded on the market, vacuum packed and kept at ±4°C. Although there was no statistically significant difference among the three groups (P>0.05) in any of the analyzes, it was concluded that the application of this technology was effective in reducing the growth of the three bacteria surveyed on day zero of both irradiated groups.

Design and influence of gamma-irradiation on the biopharmaceutical properties of nanoparticles containing an antigenic complex from Brucella ovis.

Despite vaccination campaigns, brucellosis is still one of the most common bacterial zoonosis in the world. This work describes the development of a novel formulation strategy to the delivery of the Brucella ovis antigenic extract (HS) into ovine mucosal surfaces. Thus, HS was entrapped in conventional and mannosylated poly(anhydride) nanoparticles by the solvent displacement method, and the resulting nanosystems were gamma-irradiated to accomplish the sterilization required for the ophthalmic administration route. Sterilization, at either 10 kGy or 25 kGy, did not modify the size, morphology and antigen content of the nanoparticles. Similarly, the integrity and antigenicity of the entrapped antigen were not affected by gamma-irradiation. The 25 kGy gamma-irradiation dose seemed to influence negatively the HS release from the carriers. However, and in accordance with the Pearson's correlation, all the release patterns followed a similar tendency. Furthermore, the stability of the vaccine systems on lachrymal and nasal ovine fluids, showed that gamma-irradiation had no significant effects on the vaccine systems. Since all the vaccine systems accomplished the pharmacopoeial biological tests required for gamma-irradiation doses under 25 kGy, these results are highly suggestive for the use of HS loaded poly(anhydride) nanoparticles as an efficient vaccine delivery system for brucellosis immunoprophylaxis, especially for ophthalmic administration.

Development of an irradiated vaccine that protects against enterotoxigenic Escherichia coli diarrhoea.

In the pathogenesis of diarrhoea in man bacteria adhesion to enterocytes is mediated by specific CFA/I or CFA/II antigens. A perorally administered vaccine was prepared from E. coli H10407 (078:H11) by irradiation with electrons with high energy (EHE). Two hours after cimetidine administration rats were immunized per os with 5 irradiated vaccine doses at 4-day intervals. Seven days after the last immunization animals were infected by inoculating 1 x 10(9) germs in the ligated intestinal loop. Reduction of the intestinal secretion by over 50% 18 hours after inoculation was considered an efficient protection marker. The obtained results have proved a significant reduction of the intestinal secretion in immunized animals infected with serotypes 078:H11(63 +/- 4%) and 078:H12(59 +/- 5%) as compared to non-immunized animals. Experimental induction of the intestinal protection against Escherichia coli enterotoxigenic (ETEC) strains points to the possibility of using this type of irradiated vaccine in the prophylaxis of diarrhoea in man.

[Effect of gamma irradiation on the immunological properties of the brucellar protective antigen]. / Vliianie gamma-oblucheniia na immunologicheskie svoistva brutselleznogo protektivnogo antigena.

It was shown that the splenocytic response to mitogens in guinea pigs was activated 7 days following immunization thereof with a gamma-irradiated brucellar protective agent (gamma-BPA), while nonirradiated BPA inhibited lymphocyte proliferation under the effect of mitogens. gamma-BPA, as compared with BPA, circulated in blood for a longer time, induced a more rapid and prolonged synthesis of antibodies, and provided the development of a more intensive immunity.

Alterations in the chemical composition and antigenicity of Escherichia coli O89 lipopolysaccharide and lipid A after 60Co gamma irradiation.

The chemical composition and the antigenicity of Escherichia coli lipopolysaccharide (LPS) and lipid A were investigated after irradiation with 150 kGy 60Co-gamma ray. Compared to the original preparations, the irradiated LPS showed a significant reduction in glucosamine, glucose and galactose and a decrease in the phosphate content. The fatty acid components were reduced to a smaller degree. Irradiation induced a reduction in the antigenicity of the polysaccharide part. The amount of glucosamine and phosphate decreased in the irradiated lipid A. The fatty acid content was significantly reduced. The alteration in the chemical composition was not paralleled by changes in the antigenicity of irradiated lipid A.

Dose-dependent changes in the antigenicity of bacterial endotoxin exposed to ionizing radiation.

The antigenic properties of the highly purified US reference standard endotoxin (RSE) exposed to varying doses of ionizing radiation were studied with double immunodiffusion, immunoelectrophoresis and immunoblotting. Rabbit RSE antisera identified 2 distinct major antigenic components for untreated RSE: one related to the O-polysaccharide side chain ("O-antigenic specificity"), the other to the R-core. Based on a serologic cross-reactivity of R-core of RSE (Escherichia coli 0113) with the R-core of the lipopolysaccharide from E. coli 0111, the core type of E. coli 0113 was identified as coli R3. Increasing exposure of RSE to ionizing radiation progressively destroyed all antigenic reactivities: at lower doses of radiation the rate of elimination differed for the 2 antigen classes. The O-polysaccharide was more sensitive to gamma-radiation than the R-core and the O-antigenicity was lost before that of the R-core. Endotoxin molecules containing incomplete R-core (radiation-induced or mutant) did not react with the RSE antiserum.

[Immunogenicity of a chemical typhus vaccine and of a corpuscular radioantigen obtained from Rickettsia prowazekii]. / Immunogennost' khimicheskoi sypnotifoznoi vaktsiny i korpuskuliarnogo radioantigena, poluchennogo iz Rickettsia prowazekii.

The protective activity of chemical typhus vaccine and R. prowazekii corpuscular radioantigen (CRA) was studied. Guinea pigs were immunized with doses of 32 and 48 antigenic units. Antibody production was assayed in the complement fixation test. On days 7, 15, 21, 30 and 60 after immunization the animals were challenged with R. prowazekii introduced in an amount of 10(5) minimum embryonal infective doses (MEID). On day 30 some of the animals were challenged with 10(3) MEID of R. typhi. The results demonstrated that both preparations were highly immunogenic and capable of protecting most of the animals from 10(5) MEID of R. prowazekii. Immunity developed earlier after immunization with CRA. The guinea pigs immunized with CRA, purified in percoll density gradient, and challenged with 10(3) MEID of R. typhi on day 30 showed a high level of cross immunity. In all control animals high fever and periorchitis were observed.

[Structural changes in brucella chemical vaccine after gamma-irradiation]. / Strukturnye izmeneniia brutselleznoi khimicheskoi vaktsiny posle gamma-oblucheniia.

It was shown that gamma-irradiation of Brucella strain chemical vaccine stimulated phospholipid peroxidation therein: the content of extractable total phospholipids in the exposed vaccine decreased mainly due to diminution of phosphatidylcholines and phosphatidyl-ethanolamines. A relative content of high- and low-molecular weight protein components increased in the gamma-irradiated vaccine.

[Effect of gamma radiation on the immunobiological and immunochemical properties of cholera exotoxin. III. The serological activity and immunochemical properties of irradiated unpurified toxin]. / Vliianie gamma-izlucheniia na immunobiologicheskie i immunokhimicheskie svoistva kholernogo ékzotoksina. Soobshchenie III. Serologicheskaia aktivnost' i immunokhimicheskie svoistva obluchennogo neochischennogo toksina.

The immunochemical properties and serological activity of irradiated preparations of crude cholera exotoxin have been studied. This study has revealed that with the increase of the dose of ionizing radiation changes occur in the physico-chemical properties of the preparations of the toxin, which leads to an increase in the electrophoretic motility of the protein components of the toxin, to the aggregation and polymerization of individual fragments. The preparations of antigen exotoxins have been shown to retain their serological activity within the range of radiation doses under study (10-350 kGy).

Effect of ionizing radiation of the antigenic composition of typhoid bacteria.

Changes in the antigenic composition of typhoid bacteria occurring during the exposure of microbial suspension to different doses of gamma irradiation [Co60] ranging between 0.5 and 3.0 Mrad were studied. Immunoelectrophoresis in agar was used to determine the antigenic composition of different samples of irradiated bacteria. The antigenic composition of bacteria irradiated with doses up to 2.5 Mrad was found to be similar to that of non-irradiated bacteria. Antigens demonstrated by means of Vi, H and O ontisera are preserved in these bacteria. However, all irradiated bacteria in general slightly differ from non-irradiated bacteria; this is manifest in a different configuration and position of the precipitation lines in the cathodic part of the immunophoreograms. The content of the component migrating rapidly towards the cathode, evidently the O antigen in the R form, in the irradiated bacteria increases with the dose of radiation. No new serologically active substances, non-existent in non-irradiated bacteria, were found to appear in the process of irradiation.

[Effect of physical and chemical factors on the antigenic activity of the cell walls of Brucella abortus 99]. / Vliianie na niakoi fizichni i khimichni faktori vurkhu aktivnostta na antigena ot kletuchni steni na Brucella abortus 99.

Studied was the effect of some physical and chemical factors--heating, acidity, treatment with ultraviolet rays, and enzyme treatment--on the activity of the Brucella abortus 99 cell wall antigen. The activity of the antigen was determined through the microreaction of complement-fixing after Kolmer. It was found that the antigen was most sensitive to acid treatment and treatment with ultraviolet rays, and was more slightly sensitive to the effect of alkaline agents and pronase. Besides, the antigen proved heat-resistant and did not lose its activity after treatment with trypsin and alph- and beta-amylase. The chemical nature of the epitopes (the determinant groups), substantiating the activity of the antigen is briefly discussed.

[Serologic properties of mutants of Rhizobium trifolii and their interrelationship with effectiveness]. / Serologicheskie svoistva mutantov Rhizobium trifolii i vzaimosviaz' ikh s éffektivnost'iu.

UV and gamma irradiation of Rhizobium trifolii, strains 347a and 311a, produced their mutants with changed antigenic properties and effectiveness. The extent of changes of antigens did not depend on the dose of irradiation because mutants that completely lost antigens typical of the parent strains were obtained at different doses of UV and gamma rays. No correlation was established between the antigenic properties of the mutants of Rhizobium trifolii and their effectiveness.

[Changes in the toxic, antigenic and immunogenic properties of the O-antigen of Salmonella gallinarum-pullorum irradiated with gamma rays]. / Promeni v toksichnite, antigennite i immunogennite svoistva na O-antigena na Salmonella gallinarum-pullorum, obluchen s gama luchi.

Studied was the effect of gamma-rays at rates of 1 to 20 MR on the toxic, antigenic, and immunogenic properties of the O-antigen extracted from Salmonella gallinarum-pullorum by the method of Boivin. It was found that the irradiation of aqueous solutions of the O-antigen lowered the toxic, antigenic, and immunogenic capacity with the increase in the dose of treatment. Strongest detoxication of the endotoxin was produced through irradiation at the rate of 10 MR, and especially at 20 MR. The antigenic activity, established through the hyperimmunization of rabbits was expressed best at irradiation rates of 1 and 5 MR. At such treatments the immunogenic capacity remained intact as followed up by the murine protection test.

Experimental scrub typhus immunogens: gamma-irradiated and formalinized rickettsiae.

Scrub typhus immunogens were prepared by exposing infected yolk sac suspensions of Rickettsia tsutsugamushi to various doses of gamma radiation. Mouse lethality was abolished at doses greater than 200 krads, whereas immunogenicity of the suspensions, as shown by mouse protection tests, was diminished relatively little by radiation doses in the 200- to 400-krad range. Using a 300-krad gamma dose to provide a safety factor, immunogens were prepared and their protective capacity was contrasted with formalinized scrub typhus immunogens prepared by conventional techniques. Formalinized suspensions afforded mice only partial protection against intraperitoneal challenge with 1,000 50% mouse lethal doses of the virulent homologous strain and no significant protection against similar challenge with an equally virulent heterologous strain. Using the same strains, radiation-inactivated preparations provided 100% protection against 10,000 50% mouse lethal doses of the homologous strain and 70% protection against challenge with the same doses of a heterologous strain. Neither immunogen was a potent stimulator of antibody production as measured by the complement-fixation test. Cell-transfer studies using inbred mice indicated a role for cell-mediated immunity after vaccination with gamma-irradiated immunogens, but no cell-mediated protection could be demonstrated after vaccination with formalin-inactivated rickettsiae.