Intranasal immunogenicity of a Delta cya Delta crp-pabA mutant of Salmonella enterica serotype Typhimurium for the horse.

The aim of this study was to investigate the intranasal immunogenicity for the horse of a Deltacya Deltacrp-pabA mutant (MGN-707) of Salmonella enterica serotype Typhimurium (S. typhimurium). MGN-707 caused no sign of disease, was not detected in feces and a single administration induced strong Salmonella-specific serum and nasal mucosal antibody responses. All ponies had made strong salmonella specific serum IgGa, IgGb, IgA and IgM antibody responses by day 25 after the first immunization. IgM responses to salmonella lipopolysaccharide (LPS) were short lived whereas salmonella specific serum IgGa and IgGb persisted at high levels in all ponies until 83 and 140 days, respectively. Specific nasal mucosal antibody responses dominated by IgA and IgM were evident by day 25 in all ponies except one in which only specific IgGa and IgGb were evident. Specific nasal mucosal IgA persisted in most ponies until day 69. A second immunization on day 140 boosted antibody responses, and stimulated a strong nasal mucosal IgA response in the pony that failed to make an IgA response after primary immunization. At the termination of the experiment, IgA and IgGb dominated jejunal antibody responses whereas vaginal responses were mainly IgA. The latter response unequivocally confirms the existence of a common mucosal immune system in equids. The results indicate that a S. typhimurium Deltacya Deltacrp-pabA mutant has potential as an intranasal vaccine against salmonellosis in the horse.

Intranasal immunogenicity of a Deltacya Deltacrp-pabA mutant of Salmonella enterica serotype Typhimurium for the horse.

The aim of this study was to investigate the intranasal immunogenicity for the horse of a Deltacya Deltacrp-pabA mutant (MGN-707) of Salmonella enterica serotype Typhimurium (S. typhimurium). MGN-707 caused no sign of disease, was not detected in feces and a single administration induced strong Salmonella-specific serum and nasal mucosal antibody responses. All ponies had made strong salmonella specific serum IgGa, IgGb, IgA and IgM antibody responses by day 25 after the first immunization. IgM responses to salmonella lipopolysaccharide (LPS) were short lived whereas salmonella specific serum IgGa and IgGb persisted at high levels in all ponies until 83 and 140 days, respectively. Specific nasal mucosal antibody responses dominated by IgA and IgM were evident by day 25 in all ponies except one in which only specific IgGa and IgGb were evident. Specific nasal mucosal IgA persisted in most ponies until day 69. A second immunization on day 140 boosted antibody responses, and stimulated a strong nasal mucosal IgA response in the pony that failed to make an IgA response after primary immunization. At the termination of the experiment, IgA and IgGb dominated jejunal antibody responses whereas vaginal responses were mainly IgA. The latter response unequivocally confirms the existence of a common mucosal immune system in equids. The results indicate that a S. typhimurium Deltacya Deltacrp-pabA mutant has potential as an intranasal vaccine against salmonellosis in the horse.

Expression and regulation of transcription factors GATA-4 and GATA-6 in developing mouse testis.

Previous studies have shown that transcription factors GATA-4 and GATA-6 are expressed in granulosa and thecal cells of the mouse ovary and that GATA-4 expression in ovarian tissue is regulated by gonadotropins. Given the emerging role of GATA-4 and GATA-6 in gonadal cells, we have now studied the expression and regulation of these factors in the mouse testis and testicular cell lines. In situ hybridization demonstrated GATA-4 messenger RNA (mRNA) in the fetal testis at 13.5 days postcoitum. Both GATA-4 and GATA-6 transcripts were observed in late fetal, neonatal, juvenile, and adult Sertoli cells. In addition, GATA-4 mRNA was detected in interstitial cells throughout development. Immunohistochemistry demonstrated GATA-4 protein in both Sertoli and Leydig cells in postnatal animals. The regulation of GATA-4 and GATA-6 expression was explored using established testicular cell lines. Treatment of Leydig tumor cell lines with hCG resulted in a modest, but statistically significant, increase in the steady state level of GATA-4 mRNA, comparable to the previously described effect of FSH on GATA-4 expression in Sertoli cell lines. Gonadotropin or androgen action was not, however, a prerequisite for the basal expression of GATA-4 or GATA-6 in the testis, as their presence in Sertoli and Leydig cells was demonstrated in genetically hypogonadal hpg mice, in rats treated with GnRH receptor antagonist, and in Sertoli cells after chemical abolition of Leydig cells. Cotransfection studies using a GATA-4 expression plasmid and an inhibin alpha promoter/reporter gene construct in Leydig and granulosa tumor cell lines revealed that the inhibin alpha promoter harboring essential GATA-binding sites can be trans-activated by GATA-4. In light of these results, we propose that transcription factors GATA-4 and GATA-6 play differing roles in the maturation and function of testicular somatic cells.

A live recombinant avirulent oral Salmonella vaccine expressing pneumococcal surface protein A induces protective responses against Streptococcus pneumoniae.

A live oral recombinant Salmonella vaccine strain expressing pneumococcal surface protein A (PspA) was developed. The strain was attenuated with Deltacya Deltacrp mutations. Stable expression of PspA was achieved by the use of the balanced-lethal vector-host system, which employs an asd deletion in the host chromosome to impose an obligate requirement for diaminopimelic acid. The chromosomal Deltaasd mutation was complemented by a plasmid vector possessing the asd+ gene. A portion of the pspA gene from Streptococcus pneumoniae Rx1 was cloned onto a multicopy Asd+ vector. After oral immunization, the recombinant Salmonella-PspA vaccine strain colonized the Peyer's patches, spleens, and livers of BALB/cByJ and CBA/N mice and stimulated humoral and mucosal antibody responses. Oral immunization of outbred New Zealand White rabbits with the recombinant Salmonella strain induced significant anti-PspA immunoglobulin G titers in serum and vaginal secretions. Polyclonal sera from orally immunized mice detected PspA on the S. pneumoniae cell surface as revealed by immunofluorescence. Oral immunization of BALB/cJ mice with the PspA-producing Salmonella strain elicited antibody to PspA and resistance to challenge by the mouse-virulent human clinical isolate S. pneumoniae WU2. Immune sera from orally immunized mice conferred passive protection against otherwise lethal intraperitoneal or intravascular challenge with strain WU2.

Hybrid hepatitis B virus core antigen as a vaccine carrier moiety. II. Expression in avirulent Salmonella spp. for mucosal immunization.

Hepatitis B virus (HBV) core antigen (HBcAg) is a highly immunogenic subviral particle. We and others have defined insertion sites for heterologous epitopes and successfully used hybrid particles to generate B and T cell immunity (reviewed in: Schödel et al. 1994a, 1995). Here we shall review recent progress in constructing avirulent Salmonella spp. expressing hybrid HBcAg particles carrying different epitopes. Hybrid HBcAg particles carrying virus neutralizing epitopes of the hepatitis B virus pre-S region or repeat epitopes of plasmodial circumsporozoite antigens were previously described (Schödel et al. 1992, 1994b). Salmonella spp. can be attenuated by defined genetic means so that they become avirulent, yet preserve invasiveness after oral uptake. Hybrid HBcAg-pre-S particles were expressed in Salmonella typhimurium and S. typhi vaccine strains. A single oral immunization of mice with such live recombinant S. typhimurium strains elicited a high titered serum anti-pre-S1 IgG response. Similarly, circumsporozoite repeat epitopes of three different malaria parasites were expressed as HBcAg-CS hybrids in recombinant S. spp. and were found to be highly immunogenic after oral immunization. To analyze mucosal immune responses, BALB/c mice were immunized with recombinant phoPc S. typhimurium expressing HBcAg by various mucosal routes (Hopkins et al., 1995). All routes of immunization resulted in high titered serum and local antibodies against HBcAg and S. typhimurium LPS. However, nasal immunization was most efficient in generating pulmonary IgA and rectal immunization in eliciting rectal IgA, suggesting some compartmentalization of the mucosal immune response.

Oral immunization with attenuated Salmonella expressing human sperm antigen induces antibodies in serum and the reproductive tract.

Induction of immune responses in the reproductive tract will be crucial for a functional gamete antigen-based antifertility vaccine. Here we describe the construction and development of an avirulent Salmonella as an oral vaccine delivery vector to elicit sperm-specific immune responses in reproductive tract secretions. A cDNA sequence encoding the human sperm antigen SP10 was cloned on an asd+vector and expressed to a high level in an avirulent delta cya, delta crp, and delta asd vaccine strain of Salmonella typhimurium. Oral immunization of female BALB/c mice with this recombinant Salmonella elicited high-titer anti-SP10 IgG antibodies in serum and IgA antibodies in vaginal secretions. Anti-SP10 antibody titers could be increased by secondary and tertiary oral administrations of the recombinant Salmonella. Induction of sperm-specific antibodies in the reproductive tract following oral administration of a recombinant Salmonella could lead to the development of a simple, safe, efficient, and easy-to-use antifertility vaccine.

Development of recombinant Salmonellae expressing hybrid hepatitis B virus core particles as candidate oral vaccines.

This paper provides a review on the development of hepatitis core antigen as a vaccine carrier moiety and the use of recombinant Salmonella vaccine strains expressing hybrid HBcAg particles as live oral vaccines. Salmonella spp. can be attenuated by defined genetic means so that they become avirulent, yet preserve invasiveness after oral uptake. Oral immunization of mice with such avirulent candidate Salmonella typhimurium vaccine strains elicited serum antibody responses against a limited number of bacterial antigens. A highly immunogenic viral nucleocapsid antigen, hepatitis B virus core antigen (HBcAg) that can be expressed in prokaryotes was used as a carrier moiety for B-cell epitopes. Insertion sites with an enhanced immunogenicity for the carried epitopes were defined using HBV envelope protein virus neutralizing epitopes. An internal insertion site in HBcAg was found that drastically enhanced the immunogenicity of the foreign (pre-S1) epitope while reducing the immunogenicity of the carrier protein. Internally fused HBc/pre-S hybrid particles were expressed in Salmonella typhimurium and S. typhi vaccine strains. A single oral immunization of mice with such live recombinant S. typhimurium strains elicited a high titred serum anti-pre-S1 IgG response. Similarly, circumsporozoite repeat epitopes of three different malaria parasites were expressed as HBcAg/CS hybrids in recombinant S. spp. and were found to be highly immunogenic.

Recombinant Salmonella vectors in vaccine development.

A diversity of means are available for the attenuation of Salmonella which can be used to immunize animals and humans orally to elicit mucosal, humoral and cellular immune responses. Avirulent Salmonellae can be genetically engineered to express foreign antigens and the recombinant avirulent Salmonellae are capable of stable, high-level expression of the foreign antigen in the orally immunized animal or human host. The resulting vaccines are safe, efficacious, and are easy and economical to use.

Virulence of Salmonella typhimurium mutants for White Leghorn chicks.

A strain of Salmonella typhimurium that is highly virulent for 1-day-old white leghorn chicks was genetically modified by deletion (delta) of the adenylate cyclase (cya) and cyclic AMP receptor protein (crp) genes or by removal (curing) of the 91-kilobase virulence plasmid. These mutants were then compared with the wild-type S. typhimurium strain for virulence in 1-day-old chicks and for their ability to colonize chicks of various ages. The plasmid-cured mutant showed a slight reduction in virulence, whereas the delta cya delta crp mutant was completely avirulent. The wild-type strain and both mutant strains were capable of colonizing various organs within the chicks. At all time points, the delta cya delta crp strain colonized chicks at lower levels than the wild-type strain. Titers of the plasmid-cured strain increased more slowly in visceral organs than did those of the wild type.

Isolation of the replication and partitioning regions of the Salmonella typhimurium virulence plasmid and stabilization of heterologous replicons.

Although the virulence plasmid of Salmonella typhimurium has a copy number of one to two per chromosome, plasmid-free segregants are produced at a rate less than 10(-7) per cell per generation. Three regions appear to be involved in the maintenance of this virulence plasmid. The first two, repB and repC, are functional replicons hybridizing with IncFII and IncFI plasmids, respectively, neither exhibiting the segregational stability of the parent virulence plasmid. The third region, par, cloned as a 3.9-kilobase Sau3A fragment, is not a functional replicon but exhibits incompatibility with the virulence plasmid. Subsequent tests revealed the ability of this 3.9-kilobase par insert to increase the stability of pACYC184 in S. typhimurium from less than 34% to 99% plasmid-containing cells after 50 generations. In addition, the par region increased the stability of oriC, R388, and repC replicons in both S. typhimurium and Escherichia coli hosts. The par region encodes 44,000- and 40,000-molecular-weight proteins essential for the Par+ phenotype but not for the Inc+ phenotype. Although actual sequestering of plasmids within the cell was not demonstrated, all results indicate that the par region described is an actual partitioning locus, similar in organization to those described for plasmids F, P1, and NR1.

Conservation of Salmonella typhimurium virulence plasmid maintenance regions among Salmonella serovars as a basis for plasmid curing.

The association of large plasmids with virulence in invasive Salmonella serovars has led to a number of studies designed to uncover the role of these plasmids in virulence. This study addresses two aspects of virulence-associated plasmids. The first is the distribution of the replication and maintenance regions among the plasmids of different Salmonella serovars, and the second is the use of the conserved virulence plasmid par region to provide a rapid method for eliminating the virulence plasmids specifically. Colony blots revealed that the par and repB regions of the S. typhimurium virulence plasmid hybridized with 80% of the isolates of S. choleraesuis, S. dublin, S. enteritidis, S. gallinarum, S. pullorum, and S. typhimurium, while the repC region was not detected in any of the isolates of S. dublin, S. gallinarum, or S. pullorum. None of these maintenance regions was found in any of the 30 additional serovars tested. The large plasmids of those serovars that hybridized with par were labeled with a Kmr insert within parA via P22HTint or P1L4 transduction, which destabilized the plasmids and allowed the rapid isolation of plasmid-free derivatives for all of the serovars, except for S. dublin, which exhibited weak homology with par.