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Attenuated Salmonella typhimurium SV4089 as a potential carrier of oral DNA vaccine in chickens.

ABSTRACT: Attenuated Salmonella has been used as a carrier for DNA vaccine. However, in vitro and in vivo studies on the bacteria following transfection of plasmid DNA were poorly studied. In this paper, eukaryotic expression plasmids encoding avian influenza virus (AIV) subtype H5N1 genes, pcDNA3.1/HA, NA, and NP, were transfected into an attenuated Salmonella enteric typhimurium SV4089. In vitro stability of the transfected plasmids into Salmonella were over 90% after 100 generations. The attenuated Salmonella were able to invade MCF-7 (1.2%) and MCF-10A (0.5%) human breast cancer cells. Newly hatched specific-pathogen-free (SPF) chicks were inoculated once by oral gavage with 10(9) colony-forming unit (CFU) of the attenuated Salmonella. No abnormal clinical signs or deaths were recorded after inoculation. Viable bacteria were detected 3 days after inoculation by plating from spleen, liver, and cecum. Fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR) were carried out for confirmation. Salmonella was not detected in blood cultures although serum antibody immune responses to Salmonella O antiserum group D1 factor 1, 9, and 12 antigens were observed in all the inoculated chickens after 7 days up to 35 days. Our results showed that live attenuated S. typhimurium SV4089 harboring pcDNA3.1/HA, NA, and NP may provide a unique alternative as a carrier for DNA oral vaccine in chickens.

SUBMITTER: Jazayeri SD

PROVIDER: S-EPMC3373249 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Attenuated Salmonella typhimurium SV4089 as a potential carrier of oral DNA vaccine in chickens.

Jazayeri Seyed Davoud SD Ideris Aini A Zakaria Zunita Z Omar Abdul Rahman AR

Journal of biomedicine & biotechnology 20120604

Attenuated Salmonella has been used as a carrier for DNA vaccine. However, in vitro and in vivo studies on the bacteria following transfection of plasmid DNA were poorly studied. In this paper, eukaryotic expression plasmids encoding avian influenza virus (AIV) subtype H5N1 genes, pcDNA3.1/HA, NA, and NP, were transfected into an attenuated Salmonella enteric typhimurium SV4089. In vitro stability of the transfected plasmids into Salmonella were over 90% after 100 generations. The attenuated Sal ...[more]

PMID: 22701301

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Project description:In this research, Salmonella species were isolated from the animal, insect and human enteric sources in Faisalabad, Punjab, Pakistan. These species were characterized by different microbiological and molecular techniques including polymerase chain reaction (PCR) by amplification of the 16S rRNA gene. Furthermore, sequencing of the amplicons confirmed all ten isolates as Salmonella strains. The antigenic cross-reactivity was found maximum between the HB1 (strain isolated from honeybee) antiserum and its antigen with an antibody titer of 1:128, while the HB1 antiserum showed a cross-reactive titer range of 1:8 to 1:64. On the basis of the highest geometric mean titer (GMT) shown by the antiserum of the HB1 antigen, it was selected as the best candidate for a cross-reactive live Salmonella oral antigen. Moreover, the HB1 antigen was used a live oral antigen (1 × 1010 CFU/ml) in a safety test in rabbits and proved to be avirulent. During the animal trial, three different oral doses of the HB1 live oral antigen were evaluated in four different rabbits' groups (R1, R2, R3, and R4). The dose number 2 of 0.5 ml (two drops orally and repeated after one week) gave the best GMT measured by indirect hemagglutination (IHA) as compared to the other two doses, while R4 group was kept as control. Results of the challenge protection test also validated the efficacy of the double dose of the HB1 live vaccine, which gave the highest survival percentage. Results of this study lay the foundation for a potential cross-reactive live oral Salmonella vaccine that has proved to be immunogenic in rabbits.In this research, Salmonella species were isolated from the animal, insect and human enteric sources in Faisalabad, Punjab, Pakistan. These species were characterized by different microbiological and molecular techniques including polymerase chain reaction (PCR) by amplification of the 16S rRNA gene. Furthermore, sequencing of the amplicons confirmed all ten isolates as Salmonella strains. The antigenic cross-reactivity was found maximum between the HB1 (strain isolated from honeybee) antiserum and its antigen with an antibody titer of 1:128, while the HB1 antiserum showed a cross-reactive titer range of 1:8 to 1:64. On the basis of the highest geometric mean titer (GMT) shown by the antiserum of the HB1 antigen, it was selected as the best candidate for a cross-reactive live Salmonella oral antigen. Moreover, the HB1 antigen was used a live oral antigen (1 × 1010 CFU/ml) in a safety test in rabbits and proved to be avirulent. During the animal trial, three different oral doses of the HB1 live oral antigen were evaluated in four different rabbits’ groups (R1, R2, R3, and R4). The dose number 2 of 0.5 ml (two drops orally and repeated after one week) gave the best GMT measured by indirect hemagglutination (IHA) as compared to the other two doses, while R4 group was kept as control. Results of the challenge protection test also validated the efficacy of the double dose of the HB1 live vaccine, which gave the highest survival percentage. Results of this study lay the foundation for a potential cross-reactive live oral Salmonella vaccine that has proved to be immunogenic in rabbits.

Project description:Salmonella enterica serovar Typhimurium genome encodes 13 fimbrial operons. Most of the fimbriae encoded by these operons are not produced under laboratory conditions but are likely to be synthesized in vivo We used an in vivo expression technology (IVET) strategy to identify four fimbrial operons, agf, saf, sti, and stc that are expressed in the spleen. When any three of these operons were deleted, the strain retained wild-type virulence. However, when all four operons were deleted, the resulting strain was completely attenuated, indicating that these four fimbriae play functionally redundant roles critical for virulence. In mice, oral doses of as low as 1 × 105 CFU of the strain with four fimbrial operons deleted provided 100% protection against challenge with 1 × 109 CFU of wild-type S Typhimurium. We also examined the possible effect of these fimbriae on the ability of a Salmonella vaccine strain to deliver a guest antigen. We modified one of our established attenuated vaccine strains, χ9088, to delete three fimbrial operons while the fourth operon was constitutively expressed. Each derivative was modified to express the Streptococcus pneumoniae antigen PspA. Strains that constitutively expressed saf or stc elicited a strong Th1 response with significantly greater levels of anti-PspA serum IgG and greater protective efficacy than strains carrying saf or stc deletions. The isogenic strain in which all four operons were deleted generated the lowest anti-PspA levels and did not protect against challenge with virulent S. pneumoniae Our results indicate that these fimbriae play important roles, as yet not understood, in Salmonella virulence and immunogenicity.IMPORTANCESalmonella enterica is the leading cause of bacterial food-borne infection in the United States. S. Typhimurium is capable of producing up to 13 distinct surface structures called fimbriae that presumably mediate its adherence to surfaces. The roles of most of these fimbriae in disease are unknown. Identifying fimbriae produced during infection will provide important insights into how these bacterial structures contribute to disease and potentially induce protective immunity to Salmonella infection. We identified four fimbriae that are produced during infection. Deletion of all four of these fimbriae results in a significant reduction in virulence. We explored ways in which the expression of these fimbriae may be exploited for use in recombinant Salmonella vaccine strains and found that production of Saf and Stc fimbriae are important for generating a strong immune response against a vectored antigen. This work provides new insight into the role of fimbriae in disease and their potential for improving the efficacy of Salmonella-based vaccines.

Project description:Attenuated Salmonella enterica serovar Typhimurium expressing recombinant antigens from other pathogens elicits primarily a Th1-type dominant immune response to both recombinant and Salmonella antigens. The immunogenicity and appropriate subcellular location of the recombinant antigen in the Salmonella vaccine strain may contribute to augmenting immune responses by facilitating adequate exposure of recombinant antigen to antigen-presenting cells for processing. To allow for secretion from gram-negative bacteria and overexpression of antigen, a DNA fragment encoding a highly antigenic alpha-helical region of PspA (pneumococcal surface protein A) was subcloned downstream from the beta-lactamase signal sequence in the multicopy Asd(+) pYA3493 vector to create pYA3494. pYA3493 was derived from a class of Asd(+) vectors with reduced expression of Asd to minimize selective disadvantage and enhance immunization of expressed recombinant antigens. The S. enterica serovar Typhimurium vaccine strain was constructed by the introduction of deletion mutations Delta crp-28 and Delta asdA16. Approximately 50% of the recombinant PspA (rPspA) expressed in a Salmonella strain harboring pYA3494 was detected in the combined supernatant and periplasmic fractions of broth-grown recombinant Salmonella. After a single oral immunization in BALB/c mice with 10(9) CFU of the recombinant Salmonella vaccine strain carrying pYA3494, immunoglobulin G (IgG) antibody responses were stimulated to both the heterologous antigen rPspA and Salmonella lipopolysaccharide (LPS) and outer membrane proteins (OMPs). About half, and even more at later times after immunization, of the antibodies induced to rPspA were IgG1 (indicating a Th2-type response), whereas 60 to 70% of the antibodies to LPS and 80 to 90% of those to OMPs were IgG2a (indicating a Th1-type response). A sublethal infection with Streptococcus pneumoniae WU2 boosted PspA antibody levels and maintained IgG2a/IgG1 ratios similar to those seen before the challenge. Oral immunization with Salmonella-PspA vaccine protected 60% of immunized mice from death after intraperitoneal challenge with 50 times the 50% lethal dose of virulent S. pneumoniae WU2.

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Attenuated Salmonella typhimurium SV4089 as a potential carrier of oral DNA vaccine in chickens.

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Attenuated Salmonella typhimurium SV4089 as a potential carrier of oral DNA vaccine in chickens.

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