Condiciones de adsorción de un candidato vacunal basado en vesículas de membrana externa derivada de Salmonella Paratyphi A en adyuvantes de aluminio / Adsorption conditions of a vacunal candidate based on outer membrane vesicles of Salmonella Paratyphi A in aluminum adjuvants

La capacidad inmunoestimuladora de la mayoría de las vacunas es potenciada mediante la adsorción en adyuvantes que contienen aluminio. Variando las condiciones de adsorción (pH, tiempo de adsorción) cambia la cantidad de antígeno adsorbida y por lo tanto la capacidad de estimulación del sistema inmune. El Instituto Finlay de Vacunas investiga un nuevo candidato vacunal basado en vesículas de membrana externa de Salmonella Paratyphi A (VME-SPA). El objetivo de este trabajo fue determinar las condiciones de adsorción de las VME-SPA en dos adyuvantes de sales de aluminio (Al(OH)3 y AlPO4). Para ello, las VME-SPA fueron adsorbidas en ambos adyuvantes bajo diferentes condiciones de pH y tiempo. Mediante la construcción de una Isoterma de Langmuir se determinaron parámetros como la capacidad adsortiva (qm) y el coeficiente de adsorción (Kd). El lote de VME-SPA empleado estaba formado por poblaciones de nanoestructuras con un tamaño de partículas entre 60 y 100 nm. La adsorción de las VME-SPA en ambos adyuvantes, mostró valores ≥95 por ciento a pH neutro (6,5-7,0). Las VME-SPA en presencia de AlPO4 alcanzaron el estado de equilibrio en menor tiempo (99 por ciento a partir de 30 min) en comparación con Al(OH)3 (95 por ciento a partir de 3 h). Las isotermas evaluadas para ambos adyuvantes cumplieron con el modelo de Langmuir (R2≥0,99), con valores de qm y Kd diferentes entre los sistemas de adsorción. El estudio demostró que las VME-SPA se adsorbieron satisfactoriamente en ambos geles, proceso en el que están involucrados diferentes mecanismos de adsorción(AU)

The immunostimulation capacity of most vaccines is enhanced through antigen adsorption on aluminum adjuvants. The changes in adsorption conditions (pH, adsorption time), could change the amount of antigen adsorbed and therefore the ability to stimulate the immune system. The Finlay Institute of Vaccine researches a new vaccine candidate based on outer membrane vesicle from Salmonella Paratyphi A (OMV-SPA). The study aim was to determine adsorption condition of OMV-SPA with two aluminium adjuvants (Al(OH)3 and AlPO4). OMV-SPA was adsorbed in both adjuvants under differences conditions of pH and time. Parameters as adsorptive capacity (qm) and adsorption coefficient (Kd) were determined by construction of Langmuir Isotherm. The lot of OMV-SPA used is composed by population of nanostructure with a particle size between 60 and 100 nm. Adsorption of OMV-SPA in both adjuvants showed values ≥95 percent in neutral pH (6.5-7.0). OMV-SPA with AlPO4 got equilibrium state in less time (99 percent from 30 min) compared with Al(OH)3 (95 percent from 3 h). Isotherms from both adjuvants described Langmuir model (R2≥0.99), with qm and Kd values very different between adsorption systems. As conclusion, the study showed that OMV-SPA was adsorbed satisfactorily in both aluminium adjuvants, process in which are involved different adsorption mechanism(AU)

The role of rpoS, hmp, and ssrAB in Salmonella enterica Gallinarum and evaluation of a triple-deletion mutant as a live vaccine candidate in Lohmann layer chickens

Salmonella enterica Gallinarum (SG) causes fowl typhoid (FT), a septicemic disease in avian species. We constructed deletion mutants lacking the stress sigma factor RpoS, the nitric oxide (NO)-detoxifying flavohemoglobin Hmp, and the SsrA/SsrB regulator to confirm the functions of these factors in SG. All gene products were fully functional in wild-type (WT) SG whereas mutants harboring single mutations or a combination of rpoS, hmp, and ssrAB mutations showed hypersusceptibility to H2O2, loss of NO metabolism, and absence of Salmonella pathogenicity island (SPI)-2 expression, respectively. A triple-deletion mutant, SGDelta3 (SGDeltarpoSDeltahmpDeltassrAB), was evaluated for attenuated virulence and protection efficacy in two-week-old Lohmann layer chickens. The SGDelta3 mutant did not cause any mortality after inoculation with either 1 x 10(6) or 1 x 10(8) colony-forming units (CFUs) of bacteria. Significantly lower numbers of salmonellae were recovered from the liver and spleen of chickens inoculated with the SGDelta3 mutant compared to chickens inoculated with WT SG. Vaccination with the SGDelta3 mutant conferred complete protection against challenge with virulent SG on the chickens comparable to the group vaccinated with a conventional vaccine strain, SG9R. Overall, these results indicate that SGDelta3 could be a promising candidate for a live Salmonella vaccine against FT.

Evaluation of vaccine candidate potential of ΔaroA, ΔhtrA and ΔaroAΔhtrA mutants of Salmonella enterica subspecies enterica serovar Abortusequi in guinea pigs.

Salmonella enterica subspecies enterica serovar Abortusequi (S. Abortusequi), a host adapted Salmonella causes abortions, still births and foal mortality in equids. Though known since more than 100 years, it is still a problem in many of the developing countries including India. There is dearth of really good vaccine affording immunity lasting at least for one full gestation. In search of a potential vaccine candidate, three defined deletion mutants (ΔaroA, ΔhtrA and ΔaroAΔhtrA) of S. Abortusequi were tested in guinea pig model for attenuation, safety, immunogenicity, humoral immune response, protective efficacy and persistence in host. The ΔhtrA and ΔaroAΔhtrA mutants were found to be safe on oral inoculation in doses as high as 4.2×109 cfu/animal. Also through subcutaneous inoculation ΔaroAΔhtrA mutant did not induce any abortion in pregnant guinea pigs. All the three mutants did not induce any illness or death in 1-2 week-old baby guinea pigs except ΔhtrA mutant which caused mortality on intraperitoneal inoculation. Inoculation with mutants protected against challenge and increased breeding efficiency of guinea pigs. After >4.5 months of mutant inoculation, guinea pigs were protected against abortifacient dose of wild type S. Abortusequi and mother guinea pigs also conferred resistance to their babies to the similar challenge. Early humoral immune response of S. Abortusequi mutants was characteristic. Faecal excretion of ΔaroA and htrA mutants was detected up to 45 days of inoculation in guinea pigs while ΔaroAΔhtrA mutant could not be detected after 21 days of inoculation. The results indicated that the double deletion mutant (ΔaroAΔhtrA) was the most effective and safe candidate for vaccination against S. Abortusequi through mucosal route of inoculation.

Evaluation of systemic and mucosal immune responses in mice administered with novel recombinant Salmonella vaccines for avian pathogenic Esherichia coli / 대한수의학회지

Avian pathogenic Escherichia coli (APEC) is a causative agent for a number of extra intestinal diseases and account for significant losses to the poultry industry. Since protective immunity against APEC is largely directed to virulence antigens, we have individually expressed four different viulence antigens, papA, papG, IutA, and CS31A, using an attenuated Salmonella Typhimurium and a plasmid pBB244. Following oral immunization of mice with combination of two or four of these strains, serum IgG and mucosal IgA responses were elicited against each antigen represented in the mixture. The antigen-specific mucosal IgA responses were significantly higher in the group of mice immunized with the heat-labile Escherichia coli enterotoxin B subunit (LTB) strain than those in the group of mice immunized without the LTB strain. While, there was no significant difference between these two groups in antigen-specific serum IgG responses. The results showed that LTB could act as mucosal immune adjuvant. To assess the nature of immunity, the distribution of antigen-specific IgG isotypes was analyzed. All groups promoted Th1-type immunity as determined by the IgG2a/IgG1 ratio. Thus, our findings provided evidence that immunization with a combination of several vaccine strains is one of the strategies of developing effective vaccines against APEC.

Characterization of attenuated Salmonella C500 strain with a delta asd mutant and use as an Asd+ balanced-lethal host-vector system / 生物工程学报

Salmonella enterica serovar Choleraesuis strain C500 is a live, attenuated vaccine that has been used in China for over 40 years to prevent piglet paratyphoid. The objective of this study was to evaluate the potential of attenuated Salmonella enterica serovar Choleraesuis C500 strain with a delta asd mutant as an effective live vaccine vector by the Asd+ balanced-lethal host-vector system. Here, we compared the characteristics of S. enterica serovar Choleraesuis delta asdC500 strain with the parent C500 strain, including phenotype, growth rate, virulence, safety, and expression for heterologous antigen. The mean generation times of delta asdC500 mutant, the vector control delta asdC500 (pYA3493), and the parent avirulent C500 vaccine strain in Luria broth were 30.7, 28.1, and 27.9 min, respectively. The fermentation patterns of theses three strains on different carbohydrates, and the levels of production of H2S, were similar. The O and H antigens of delta asdC500 mutant, delta asdC500 (pYA3493) and delta asdC500 (pYA-F1P2) were 6,7:C:1,5, identical to the parent strain C500. By the method of Reed and Muench, groups of mice were challenged by the intraperitoneal route with different amounts of delta asdC500 (pYA3493) or the parent C500 strain, and the virulence of delta asdC500 (pYA3493) with LD50 of 1.1 x 10(7) CFU was a little lower than C500 with LD50 of 4.4 x 10(6) CFU. All piglets inoculated with delta asdC500 (pYA3493) or C500 survived, and no signs of disease were observed during the entire experimental period. No major differences were found in these two groups. In addition, the recombinant pYA-F1P2 plasmid was very stable in the recombinant delta asdC500 (pYA-F1P2) strain, which expressed secretorily a large amount of the recombinant filamentous hemagglutinin type I domain and pertactin region 2 domain antigen (rF1P2) of Bordetella bronchiseptica. In this study, we have shown that the delta asdC500 mutant had a series of biological characteristics similar to the parent vaccine strain C500. Furthermore, the strain could express secretorily a large amount of heterologous antigen. It is likely that this Salmonella expression and delivery system could be easily adapted to develop multivalent recombinant Salmonella vaccines against infectious agents using the Asd+ balanced-lethal host-vector system.

Construction of a dual-promoter expression plasmid delivered by Salmonella choleraesuis C500 / 生物工程学报

Salmonella choleraesuis C500 strain is an attenuated vaccine preventing piglet from paratyphoid and can also be used as a live vector of other DNA vaccines. Through mucosal immunization, immune response to specific antigens carried by it can be induced. To enhance the immune efficiency of DNA vaccine it carried, promoter Ptrc was inserted into the down stream of the human cytomegalovirus (CMV) immediate early promoter of eukaryotic expression plasmid pEGFP-C1. Then transcription terminator rrnbT1T2 was inserted into down stream of the multiple clone sites of pEGFP-C1, and the dual-promoter expression vector pEGFPPtrcR was constructed. Using 1xTSS method, we transformed the recombinant plasmid into C500, and obtained C500/pEGFPPtrcR. We used SDS-PAGE and Western blotting to detect the expression of report gene EGFP. Strong green fluorescence was observed under fluorescent microscope. The stable passages of this recombinant bacterium were at least 20 generations in vitro. Using liposome we transfected plasmid pEGFPPtrcR into Vero cell. After 24 h, green fluorescent was observed, showing the expression of EGFP in nuclei and endochylema. The construction of dual-promoter expression vector pEGFPPtrcR was successful. The foreign gene was expressed in Salmonella strain C500 and somatocytes, resulting in increased antigen expression. This research provides a foundation for the research of new DNA vaccines which use Salmonella C500 as carrier.

Anti-flagellin antibody responses elicited in mice orally immunized with attenuated Salmonella enterica serovar Typhimurium vaccine strains

In the present study we investigated the flagellin-specific serum (IgG) and fecal (IgA) antibody responses elicited in BALB/c mice immunized with isogenic mutant derivatives of the attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) SL3261 strain expressing phase 1 (FliCi), phase 2 (FljB), or no endogenous flagellin. The data reported here indicate that mice orally immunized with recombinant S. Typhimurium strains do not mount significant systemic or secreted antibody responses to FliCi, FljB or heterologous B-cell epitopes genetically fused to FliCi. These findings are particularly relevant for those interested in the use of flagellins as molecular carriers of heterologous antigens vectored by attenuated S. Typhimurium strains.

Construction and characterization of delta crp delta asd mutant host-vector balanced lethal system of Salmonella choleraesuis C500 strain / 生物工程学报

Salmonella choleraesuis C500 strain was an attenuated vaccine strain to prevent piglet paratyphoid, attenuated by chemical method. Although the vaccine has good immunogenicity, it remains some residual virulence. In order to develop a safer vaccine strain and exploit C500 as a live vaccine vector for mucosal immunization, delta crp delta asd double deletion mutant was constructed. Firstly, the recombination suicide vector with 320 bp-deleted crp (cAMP receptor protein) gene and sacB (sucrose-sensitive gene) gene was constructed and conjugated with C500. The unmarked crp deleted strain without resistance was selected by two-step method and crp deletion on the genome was determined by PCR. Then the asd (beta-aspartic semialdehyde dehydrogenase) gene was further deleted in the delta crp strain by the same method. Foreign DAP (diaminopimelic acid) must be supplied for delta crp delta asd mutant to grow. The phenotype, growth properties and virulence in mice of delta crp mutant were further characterized. In conclusion, the delta crp delta asd double-deletion mutant was successfully constructed. The delta crp delta asd mutant can be used as a live vector to express foreign genes and to develop potential oral multivalent vaccines.

Construction of prokaryotic expression system of Salmonella paratyphi A spaO gene and immunogenicity and immunoprotection of the expressed product / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the immunogenicity and immunoprotection of the recombinant expressing product (rSpaO) of S. paratyphi A spaO gene, and to demonstrate the frequencies of spaO gene carrying and expressing in S. paratyphi A isolates.</p><p><b>METHODS</b>The spaO gene of a clinical S. paratyphi A strain JH01 was amplified and then cloned. After sequencing of the cloned spaO gene, a prokaryotic expression system of the gene was constructed. SDS-PAGE were applied to examine the rSpaO expression. Ni-NTA affinity chromatography was performed to collect rSpaO. Immunogenicity of rSpaO was determined by Western blot assay. A PCR assay and an ELISA were established to respectively detect the carrying and expressing frequencies of the spaO genes in 98 S. paratyphi A isolates. The immunoprotective effects of rSpaO in S. paratyphi A strain 50001 infected mice were observed.</p><p><b>RESULTS</b>In comparison with the reported corresponding sequences, the nucleotide and putative amino acid sequence homologies of the cloned spaO gene were 99.45%-99.89% and 99.01%-100%, respectively. The expression output of rSpaO was approximately 75% of the total bacterial proteins. S. paratyphi A antiserum could recognize as well as combine with rSpaO. rSpaO could efficiently induce rabbits to produce specific antibody. 94.9% (93/98) of the S. paratyphi A isolates had spaO gene and 91.4% (85/93) of the spaO+ strains could express SpaO. 58.3% and 50.0% of the mice that oral-taken or subcutaneous injected with 500 microg of rSpaO for immunization were survival after challenged by lethal dose of S. paratyphi A strain 50001. When co-immunized with 5 microg rLTB, the survival rates of the mice increased to 88.3% and 75.0%, respectively.</p><p><b>CONCLUSION</b>The S. paratyphi isolates had relatively high carrying and expressing frequencies of spaO gene. rSpaO showed a fine immunogenicity and a certain immunoprotective effect, which could be used as an antigen candidate for developing genetic engineering vaccine of S. paratyphi.</p>

Protective efficacy of maternal antibodies induced by Salmonella toxoid (vaccine).

An attempt was made to evaluate the protective efficacy of maternal antibodies in chicks against salmonellosis. Layer chicks ageing 21 days were individually vaccinated with 100 microg of Salmonella enterica subspecies enterica serovar Weltevreden (BM 1643) toxoid adjuvanted with vitamin E subcutaneously. After 90 days of the primary vaccination the birds were given booster dose of the vaccine. The saline extract of the yolk of eggs laid by the vaccinated birds yielded agglutination and ELISA titres ranging from 43.2 +/- 5.33 to 75.2 +/- 6.26 and 4.987 x 10(3) +/- 0.54 to 5.89x103 +/- 0.56, respectively. Sera of chicks hatched from eggs laid by the vaccinated layers were also subjected to agglutination and ELISA. Agglutination and ELISA titres on the 5th day--post hatching (dph) were 21.6 +/- 1.75 and 4.025 x 10(2) +/- 0.59, while on the 10th dph titers were 13.6 +/- 1.65 and 1.21 x 10(2) +/- 0.60, respectively. It was also observed that only one out of 6 chicks died when challenged with 2 x 10(9) CFU of S. serovar Gallirarum at the age of 7 days showing 83.33% protection. Thus it can be concluded that passive immunity confided by Salmonella enterica subspecies enterica serovar Weltevreden (BM 1643) toxoid can protect chicks against salmonellosis during their early days of life.

Comparison of the immunogenicity and safety of two different brands of Salmonella typhi Vi capsular polysaccharide vaccine.

BACKGROUND: The recent emergence of multi-drug-resistant Salmonella strains highlights the need for better preventive measures, including vaccination. Safe and immunologic vaccines have been developed based on purified Vi polysaccharide. OBJECTIVE: To compare the immune response elicited by two different brands of Salmonella Vi capsular polysaccharide vaccine (ViCPS). SETTING AND DESIGN: Double blind, randomized (3:1), controlled, parallel, phase III study was conducted at two centres in India to compare the safety and immunogenicity of Typbar, the investigational vaccine with an already marketed vaccine "X", in healthy subjects aged between 12 -25 years. MATERIAL AND METHODS: A sample size of 184 subjects was calculated. Subjects were randomly distributed in two groups, immunized with single dose of Typbar or Vaccine "X". Serum samples were taken before 7 days and 4 weeks after immunization for the determination of antibodies to Vi polysaccharide, by ELISA method. Safety was assessed by physical examination, laboratory parameters before and after vaccination and by monitoring adverse events. Statistics: The geometric mean antibody titre (GMT) 4 weeks after vaccination was compared from respective pre-vaccination values by Wilcoxon signed rank test. Geometric mean of antibody levels before and after immunization and the ratio between them (Mann-Whitney test), the Seroconversion rates (Z test of proportions) and the adverse events (Fisher's exact test and Chi square test), were compared between two groups. P value < 0.05 was considered statistically significant. P values and 95% confidence intervals were estimated in two-tailed fashion. RESULTS: 153 subjects (Typbar =116 and Vaccine "X" =37) were studied. 71.6% (95% CI=63.4%-79.8%) and 75.7% (95% CI=64.9% - 89.5%) were the seroconversion rates with Typbar and vaccine "X" respectively. The GMT values for Vi antibodies induced after Typbar and vaccine "X" were 10.23 Typbar and 13.46 mg/mL respectively and these values showed high significance when compared to their respective pre-immunization GMT values (P<0.0001) at 95% CI (-10.49 to -7.19 mg/mL for Typbar and -14.69 to -8.86 mg/mL for Vaccine "X"). The induction of antibody response appeared to be slightly stronger (P=0.032) with vaccine "X" when compared to that of Typbar. This is justifiable as the same group also had high pre-immunization GMT values (P=0.021). CONCLUSION: The immunogenicity and safety of the investigational vaccine Typbar was found to be similar to that of already marketed brand of Vi CPS, Vaccine "X". The availability of a single dose of vaccine that is safe and effective enhances the prospective for control of typhoid fever.

Anti-angiogenesis effect on glioma of attenuated Salmonella typhimurium vaccine strain with flk-1 gene / 华中科技大学学报（医学）（英德文版）

To investigate the anti-vasculature effects and the anti-glioma effects of attenuated Salmonella typhimurium vaccine strain expressing VEGFR2 (flk-1) gene, plasmid pcDNA3.1-flk1 was constructed and electro-transfected into live attenuated Salmonella typhimurium strain SL7207. Mouse models of intracranial G1261 glioblastoma were treated with an orally administered attenuated Salmonella typhimurium expressing flk-1 gene. The survival period was recorded and vessel density was observed by immunofluorescence. CTLs activity was measured by MTT assay. Our results showed that attenuated Salmonella typhimurium vaccine strain expressing flk-1 gene could significantly inhibit glioblastoma growth, reduce vessel density, prolong the survival period and improve the survival rate in these mice. The flk-1 specific CTLs activity was increased obviously after the vaccination. Our study showed that attenuated Salmonella typhimurium vaccine strain expressing flk-1 gene could break peripheral immune tolerance a in glioma gainst this self-antigen and kill endothelial cells by the orally administered vaccine and can be used for both prophylactic and therapeutic purposes.

Seguridad e inmunogenicidad de una vacuna de polisacárido Vi de salmonella typhi en jóvenes cubanos / Security and immunogenecity of a vaccine of polyssacharide Vi from salmonella typhi in cuban youths

Se realizó un estudio aleatorizado, controlado y a doble ciegas, en adultos jóvenes de 18 a 20 años, con el objetivo de evaluar la reactogenicidad y la inmunogenicidad de vax-TyViâ, vacuna de polisacárido Vi de Salmonella typhi. Se distribuyeron en 3 grupos; inmunizados con una dosis de vax-TyViâ (Instituto Finlay), TYPHIM ViTM (Pasteur-Mérieux) o vax-TETâ (toxoide tetánico). Se tomaron muestras de suero antes y 21 d después de la inmunización. La inmunogenicidad se evaluó en 323 voluntarios mediante un ELISA indirecto. La seroconversión de los que recibieron vax-TyViâ fue 81,97 por ciento y de 65,05 por ciento para TYPHIM ViTM. Los títulos medios geométricos posvacunales fueron 7,41 U/mL (5,92û9,27 U/mL) y 5,41 U/mL (4,35û6,72 U/mL) respectivamente. La seroconversión con vax-TETâ fue 0 por ciento. La reactogenicidad de ambas vacunas polisacarídicas fue baja. Se concluyó que la inmunogenicidad de vax-TyViâ no fue inferior a la de TYPHIM ViTM y su reactogenicidad resultó similar