Genetic control of immune response to recombinant antigens carried by an attenuated Salmonella typhimurium vaccine strain: Nramp1 influences T-helper subset responses and protection against leishmanial challenge

Attenuated strains of Salmonella typhimurium have been widely used as vehicles for delivery and expression of vaccine antigens in murine models of infectious disease. In mice, early bacterial replication following infection with S. typhimurium is controlled by the gene (Nramp1, formerly Ity/Lsh/Bcg) encoding the natural-resistance-associated macrophage protein (Nramp1). Nramp1 regulates macrophage activation and has multiple pleiotropic effects, including regulation of tumor necrosis factor alpha, interleukin 1beta (IL-1beta), and major histocompatibility complex class II molecules, all of which influence antigen processing and presentation. Nramp1 also has a direct effect on antigen processing, possibly by regulating the activity of proteases in the late endosomal compartment. Hence, there are multiple ways (regulation of bacterial load or recombinant antigen dose, class II molecule expression, costimulatory or adjuvant activity, and antigen processing) that Nramp1 might influence responses to recombinant salmonella vaccines. To test the hypothesis that Nramp1 influences responses to vaccination, congenic mouse strains have been used to analyze immune responses to recombinant antigens (tetanus toxoid antigen and leishmanial gp63) carried by live attenuated S. typhimurium aroA aroD mutants. Results show that congenic mice carrying the wild-type (S. typhimurium resistance) Nramp1 allele mount a predominantly T-helper-1 (IL-2 and gamma interferon) response to vaccination and show enhanced resolution of lesions following challenge infection with Leishmania major. In contrast, mice carrying mutant (S. typhimurium susceptibility) Nramp1 mount a T-helper-2 (immunoglobulin E and IL-4) response and show exacerbated lesion growth upon challenge.     

Comparison of the abilities of different attenuated Salmonella typhimurium strains to elicit humoral immune responses against a heterologous antigen

We compared the abilities of different Salmonella enterica var. Typhimurium (S. typhimurium) strains harboring mutations in the genes aroA, aroAD, purA, ompR, htrA, and cya crp to present the heterologous antigen, C fragment of tetanus toxin, to the mouse immune system. Plasmid pTETtac4, encoding C fragment, was transferred into the various S. typhimurium mutants, and the levels of antigen expression were found to be equivalent. After primary oral immunization of BALB/c mice, all attenuated strains were capable of penetrating the gut epithelium and colonizing the Peyer's patches and spleens of mice. Of all strains compared, the delta purA mutant colonized and persisted in the Peyer's patches at the lowest level, whereas the delta htrA mutant colonized and persisted in the spleen at the lowest level. The level of specific antibody elicited by the different strains against either S. typhimurium lipopolysaccharide or tetanus toxoid was strain dependent and did not directly correlate to the mutants' ability to colonize the spleen. The level of immunoglobulin G1 (IgG1) and IgG2a antibody specific for tetanus toxoid was determined in mice immunized with four S. typhimurium mutants. The level of antigen-specific IgG1 and IgG2a was significantly lower in animals immunized with S. typhimurium delta purA. Antigen-specific T-cell proliferation assays indicated a degree of variability in the capacity of some strains to elicit T cells to the heterologous antigen. Cytokine profiles (gamma interferon and interleukin-5) revealed that the four S. typhimurium mutants tested induced a Th1-type immune response. Mice were challenged with a lethal dose of tetanus toxin 96 days after oral immunization. With the exception of the S. typhimurium delta purA mutant, all strains elicited a protective immune response. These data indicate that the level of total Ig specific for the carried antigen, C fragment, does not correlate with the relative invasiveness of the vector, but it is determined by the carrier mutation and the background of the S. typhimurium strain.     

Safety and immunogenicity in humans of an attenuated Salmonella typhi vaccine vector strain expressing plasmid-encoded hepatitis B antigens stabilized by the Asd-balanced lethal vector system

Attenuated Salmonella typhi organisms which express genes encoding protective antigens of other pathogens have been developed for use as experimental oral vaccines. A delta asd S. typhi strain attenuated by deletions in cya, crp, and cdt which contains hepatitis B core (HBc) and pre-S genes encoded on an Asd+ pBR-based plasmid vector was constructed. Healthy adult volunteers ingested a single dose of 5 x 10(5) to 5 x 10(8) CFU of strain chi4073 (delta cya delta crp delta cdt S. typhi Ty2), 6 x 10(7) or 1 x 10(9) CFU of strain chi4632(pYA3149), a further derivative of chi4073 deleted in asd and containing the Asd+ vector without the HBc-pre-S fusion, or 3 x 10(7) or 7 x 10(8) CFU of strain X4632(pYA3167), a derivative containing the vector with the HBc-pre-S fusion. Chi4073 was generally well tolerated by 22 volunteers. No volunteer had fever or positive blood cultures; 4 of 22 volunteers shed vaccine organisms in the stool in the first 48 h only. Two of 18 volunteers who received one of the plasmid-containing derivatives of chi4073 developed low-grade fevers on day 10 or 12 after ingestion. One of these volunteers had positive blood cultures on days 7 and 8. Seven of these 18 volunteers had vaccine organisms detected in their stools in the first 48 h only. Most volunteers developed S. typhi-specific serum responses and developed S. typhi-specific antibody-secreting cells. However, no volunteer developed serum antibody to hepatitis pre-S or pre-S-specific antibody-secreting cells. Although the parent strain chi4073 was well tolerated, induced immunoglobulin G seroconversion to S. typhi lipopolysaccharide in 80 to 100% of vaccinees and stimulated specific IgA-secreting lymphocytes in 80 to 100% of vaccinees given a single oral dose of 2 x 10(7) and 5 x 10(8) CFU, chi4073 derivatives containing the Asd+ vector with and without sequences encoding the HBc-pre-S fusion caused occasional febrile reactions at high doses and did not stimulate detectable immune responses to hepatitis B antigens.     

Expression of the murine interleukin-4 gene in an attenuated aroA strain of Salmonella typhimurium: persistence and immune response in BALB/c mice and susceptibility to macrophage killing

Cytokines are potentially useful in vaccination as adjuvants or modulators of the type of response induced. The work below describes the expression of a cloned cytokine gene for murine interleukin-4 (mIL-4) by a live vaccine vector, an attenuated aroA strain (SL7207) of Salmonella typhimurium, in a murine model system. SL7207 was used as a carrier for two different high-level expression vectors. Both resulting strains, designated SL7207(pOmpAmIL-4) and SL7207(pKKmIL-4), expressed the cloned gene product as monitored by both immunological and biological assays. However, SL7207(pOmpAmIL-4) produced mIL-4 at higher levels and was more stable in vitro than SL7207(pKKmIL-4). When SL7207(pOmpAmIL-4) was used as a live vaccine in BALB/c mice, this strain grew and survived at higher levels than the parental attenuated strain or empty plasmid-carrying strain in spleens, livers, and intestines. This difference in growth and survival did not appear to be caused by alterations in specific lymphocyte-mediated anti-Salmonella immune responses such as delayed-type hypersensitivity or serum antibody as measured by enzyme-linked immunosorbent assay; such alterations have been induced by IL-4 administration in other in vivo systems, and the lack of effect here may reflect the fact that IL-4 is not secreted from the bacteria in large quantities, most of the cytokine being in the cytoplasmic-membrane-bound fraction. Conversely, the ability of mouse macrophages to kill the bacteria in vitro was inhibited by bacterial production of mIL-4. This reduction in macrophage killing activity suggests that bacterial production of mIL-4 may be detrimental to host defense against Salmonella infection and may explain the enhanced bacterial growth and survival in vivo.     

Heat denaturation of egg-white proteins abrogates the induction of oral tolerance of specific Th2 immune responses in mice

BACKGROUND: Technetium Tc 99m hexamethyl propylene amine oxine (99mTc-HMPAO) has been used to radiolabel leukocytes with promising results for its clinical use in inflammatory bowel disease. During active ulcerative colitis, colonoscopy is indicated to determine the extent and the intensity of the disease for proper management. The aim of this prospective study was to determine whether 99mTc-HMPAO-labeled leukocyte scintigraphy can give information similar to that obtained with colonoscopy during acute attacks of ulcerative colitis. METHODS: Thirty-three consecutive patients with 50 acute episodes of ulcerative colitis underwent 99mTc-HMPAO scintigraphy and colonoscopy with biopsies. Scintigraphic determination of disease extent and intensity were compared with those obtained by colonoscopy with biopsies and clinicobiologic markers. RESULTS: The scintigraphic index of disease intensity was correlated with endoscopic index, Truelove index, biologic markers, and local release of interleukin-8. The extent measured by scintigraphy was well correlated to the endoscopic and histologic extent. CONCLUSIONS: 99mTc-HMPAO scintigraphy accurately determines the extent and the intensity of acute ulcerative colitis lesions. This noninvasive method can specify the extent and the intensity of an acute attack in patients with previously known ulcerative colitis.     

Regulation by the H-2 gene complex of macrophage-lymphoid cell interactions in secondary antibody responses in vitro

The ability of antigen-bearing syngeneic and allogeneic peptone-induced peritoneal exudate macrophages to support development of primary and secondary antibody responses by murine lymphoid or spleen cells in vitro has been investigated. The antigen used was the terpolymer of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT). Syngeneic and allogeneic macrophages supported development of comparable primary antibody responses to GAT, indicating that genetic restrictions do not limit efficient macrophage-lymphocyte interactions in primary responses. By contrast, immunized spleen or lymphoid cells developed secondary antibody responses preferentially when stimulated in vitro with GAT on macrophages syngeneic to the macrophages used to present GAT during in vivo immunization. Thus, genetic restrictions regulate efficient macrophage-lymphocyte interactions in secondary antibody responses. These restrictions have been demonstrated from 2 to 8 wk after a single immunization with limiting quantities of GAT and are controlled by the H-2 gene complex. The implications that immune lymphocytes selectively recognize and respond to antigen presented in the context of the macrophage membrane-antigen complex which sensitized the lymphocytes initially are considered.     

Experimental in vivo passage of H2 strain of live attenuated vaccine of hepatitis A in common marmosets

Common marmosets with negative serum antibodies against hepatitis A virus (anti-HAV) and normal liver functions were used for the tests of passage of H2 vaccine strain of HAV to explore its residual virulence after attenuation and possibility of its reversion. Common marmosets were inoculated with attenuated live vaccine, and fecal suspension and/or proliferative liquid in tissue culture of the progeny of vaccine HAV excreted from their feces were passaged in common marmosets. Result of three serial passages in vivo indicated that there was no significant difference in serum anti-HAV titer, and no or low-level changes in activities of liver enzymes, no pathological changes in liver tissues observed, and detectable but minimal HAV was shed in stool of the all animals.     

Mutation of the htrB gene in a virulent Salmonella typhimurium strain by intergeneric transduction: strain construction and phenotypic characterization

The htrB gene product of Haemophilus influenzae contributes to the toxicity of the lipooligosaccharide. The htrB gene encodes a 2-keto-3-deoxyoctulosonic acid-dependent acyltransferase which is responsible for myristic acid substitutions at the hydroxy moiety of lipid A beta-hydroxymyristic acid. Mass spectroscopic analysis has demonstrated that lipid A from an H. influenzae htrB mutant is predominantly tetraacyl and similar in structure to lipid IV(A), which has been shown to be nontoxic in animal models. We sought to construct a Salmonella typhimurium htrB mutant in order to investigate the contribution of htrB to virulence in a well-defined murine typhoid model of animal pathogenesis. To this end, an r- m+ galE mutS recD strain of S. typhimurium was constructed (MGS-7) and used in inter- and intrastrain transduction experiments with both coliphage P1 and Salmonella phage P22. The Escherichia coli htrB gene containing a mini-Tn10 insertion was transduced from E. coli MLK217 into S. typhimurium MGS-7 via phage P1 and subsequently via phage P22 into the virulent Salmonella strain SL1344. All S. typhimurium transductants showed phenotypes similar to those described for the E. coli htrB mutant. Mass spectrometric analysis of the crude lipid A fraction from the lipopolysaccharide of the S. typhimurium htrB mutant strain showed that for the dominant hexaacyl form, a lauric acid moiety was lost at one position on the lipid A and a palmitic acid moiety was added at another position; for the less abundant heptaacyl species, the lauric acid was replaced with palmitoleic acid.     

DNA restriction and modification systems in Salmonella. SQ, a new system derived by recombination between the SB system of Salmonella typhimurium and the SP system of Salmonella potsdam

As the result of P1-mediated cotransduction with serB from Salmonella potsdam to the Escherichia coli/Salmonella typhimurium hybird 4617, one recombinant, L4004, was isolated which had a restriction-modification (R--M) system different from the SB and SP systems of its parents, and was designated SQ. The genes of SQ were allelic to those of the SB system of S. typhimurium and were shown by complementation experiments to be functionally related to those of the K system of E. coli. Evidence that the SQ system in L4004 arose as the result of a recombination event within the hsdS genes of SB and SP is discussed.     

Protective efficacy of a dengue 2 DNA vaccine in mice and the effect of CpG immuno-stimulatory motifs on antibody responses

A recently described DNA vaccine for dengue (DEN) type 2 was shown to elicit high levels of neutralizing antibodies in mice. The vaccine candidate consists of the PreM and 92% of the envelope genes of DEN 2 New Guinea C strain. We further evaluated this DNA vaccine candidate by examining the effect of immuno-stimulatory CpG DNA motifs on antibody response and by studying the protective efficacy of the vaccine. The results showed that CpG motifs present in pUC 19 significantly improved the antibody response to a suboptimal dose of 3.1 micrograms of the DEN DNA vaccine. In a lethal mouse intracerebral challenge model, the vaccine provided a significant level of protection. Sixty percent of the mice immunized with the DEN DNA vaccine plus pUC 19 survived the challenge compared to only 10% in the control group that received vector plus pUC. These studies illustrate that nucleic acid immunization is a viable approach to developing a DEN vaccine and that immuno-stimulatory CpG DNA motifs can be used to lower the minimum dose required to produce an antibody response.     

A model of peptide-induced lupus autoimmune B cell epitope spreading is strain specific and is not H-2 restricted in mice

Anti-Sm is a common and specific autoantibody found in systemic lupus erythematosus. The peptide PPPGMRPP from Sm B/B' is an early target of the autoimmune response in some anti-Sm-positive human patients. After immunization with this peptide on a MAP backbone, rabbits develop anti-Sm autoantibodies with B cell epitope spreading of the autoimmune response as well as other features of lupus autoimmunity. Various strains of inbred mice have been immunized with peptide PPPGMRPP or PSQQVMTP (nonantigenic region of Sm B/B') in Freund's adjuvant or with no peptide. All peptide-immunized mouse strains eventually develop high titers of specific anti-peptide of immunization Abs. Mice immunized with Freund's adjuvant alone have no measurable Ab binding to the PPPGMRPP peptide. With time, nearly half the mouse strains tested develop Abs that react with additional regions of Sm B/B' and Sm D. All the regions bound by mouse serum are major epitopes of the human systemic lupus erythematosus anti-Sm response. These same strains also develop significant anti-Sm and anti-nuclear ribonucleoprotein titers. In addition, some of these strains demonstrate positive anti-nuclear Abs and anti-dsDNA Abs. Experiments with congenic H-2 mice demonstrate that the H-2 region does not play a role in spreading the immune response from the peptide of immunization to other epitopes of the spliceosome. These results present a new murine model of B cell epitope spreading and lupus autoimmunity induced by peptide immunization that is strain specific and not apparently dependent upon the loci at H-2.     

Cell division inhibition in Salmonella typhimurium histidine-constitutive strains: an ftsI-like defect in the presence of wild-type penicillin-binding protein 3 levels

Histidine-constitutive (Hisc) strains of Salmonella typhimurium undergo cell division inhibition in the presence of high concentrations of a metabolizable carbon source. Filaments formed by Hisc strains show constrictions and contain evenly spaced nucleoids, suggesting a defect in septum formation. Inhibitors of penicillin-binding protein 3 (PBP3) induce a filamentation pattern identical to that of Hisc strains. However, the Hisc septation defect is caused neither by reduced PBP3 synthesis nor by reduced PBP3 activity. Gross modifications of peptidoglycan composition are also ruled out. D-Cycloserine, an inhibitor of the soluble pathway producing peptidoglycan precursors, causes phenotypic suppression of filamentation, suggesting that the septation defect of Hisc strains may be caused by scarcity of PBP3 substrate.     

Construction of a stable attenuated Shigella sonnei DeltavirG vaccine strain, WRSS1, and protective efficacy and immunogenicity in the guinea pig keratoconjunctivitis model

Construction of a stable Shigella sonnei vaccine has been complicated by the instability of the virulence phenotype caused by the spontaneous loss of the invasion plasmid. To select a suitable candidate for vaccine construction, 16 S. sonnei strains were screened for stability of the virulence phenotype. A stable strain, S. sonnei Mosely, was selected for further work. pDeltavirG2, a deletion derivative of the virG gene in the sacB suicide vector pCVD442, was used to generate an S. sonnei virG deletion strain, WRSS1, which was invasive in HeLa cells but negative in the Sereny test. WRSS1 was found to be both immunogenic and protective in the guinea pig keratoconjunctivitis model.     

Integration of minitransposons for expression of the Escherichia coli elt genes at a preferred site in Salmonella typhimurium identifies a novel putative fimbrial locus

An asd-complementing mini-Tn5 transposon was constructed for random insertion of the Escherichia coli LT enterotoxin genes (elt) into the genome of Deltaasd attenuated strains of Salmonella typhimurium. Transfer of the minitransposon to different S. typhimurium strains resulted in random integration only in strain chi4072, while in strain chi3987, which harbours the virulence plasmid, over 20% of the insertions occurred at the same site. Expression of elt was found to be highest in Salmonella isolates carrying the mini-Tn5 integrated at the preferred site, which was mapped to an uncharacterised region of the virulence plasmid. Sequence analysis of the integration site showed that it lies within an open reading frame with sequence similarity to E. coli leuO and contiguous to a novel fimbrial locus.     

Localization of the somatostatin receptor SST2A in rat brain using a specific anti-peptide antibody

Biological actions of somatostatin are exerted via a family of receptors, for which five genes recently have been cloned. However, none of these receptor proteins has been visualized yet in the brain. In the present-study, the regional and cellular distribution of the somatostatin sst2A receptor was investigated via immunocytochemistry in the rat central nervous system by using an antibody generated against a unique sequence of the receptor protein. Specificity of the antiserum was demonstrated by immunoblot and immunocytochemistry on rat brain membranes and/or on cells transfected with cDNA encoding the different sst receptor subtypes. In rat brain sections, sst2A receptor immunoreactivity was concentrated either in perikarya and dendrites or in axon terminals distributed throughout the neuropil. Somatodendritic labeling was most prominent in the olfactory tubercle, layers II-III of the cerebral cortex, nucleus accumbens, pyramidal cells of CA1-CA2 subfields of the hippocampus, central and cortical amygdaloid nuclei, and locus coeruleus. Labeled terminals were detected mainly in the endopiriform nucleus, deep layers of the cortex, claustrum, substantia innominata, subiculum, basolateral amygdala, medial habenula, and periaqueductal gray. Electron microscopy confirmed the association of sst2A receptors with perikarya and dendrites in the former regions and with axon terminals in the latter. These results provide the first characterization of the cellular distribution of a somatostatin receptor in mammalian brain. The widespread distribution of the sst2A receptor in cerebral cortex and limbic structures suggests that it is involved in the transduction of both pre- and postsynaptic effects of somatostatin on cognition, learning, and memory.     

The CorA Mg2+ transport protein of Salmonella typhimurium. Mutagenesis of conserved residues in the third membrane domain identifies a Mg2+ pore

The CorA transport system is the major Mg2+ influx pathway for bacteria and the Archaea. CorA contains three C-terminal transmembrane segments. No conserved charged residues are apparent within the membrane, suggesting that Mg2+ influx does not involve electrostatic interactions. We have mutated conserved residues within the third transmembrane segment to identify sites involved in transport. Mutation of conserved aromatic residues at either end of the membrane segment to alternative aromatic amino acids did not affect total cation uptake or cation affinity. Mutation to alanine greatly diminished uptake with little change in cation affinity implying that the conserved aromatic residues play a structural role in stabilizing this membrane segment of CorA at the interface between the bilayer and the aqueous environment. In contrast, mutation of Tyr292, Met299, and Tyr307 greatly altered the transport properties of CorA. Y292F, Y292S, Y292C, or Y292I mutations essentially abolished transport, without effect on expression or membrane insertion. M299C and M299A mutants exhibited a decrease in cation affinity for Mg2+, Co2+, or Ni2+ of 10-50-fold without a significant change in uptake capacity. Mutations at Tyr307 had no significant effect on cation uptake capacity; however, the affinity of Y307F and Y307A mutations for Mg2+ and Co2+ was decreased 3-10-fold, while affinity for Ni2+ was unchanged compared with the wild type CorA. In contrast, the affinity of the Y307S mutant for all three cations was decreased 2-5-fold. Projection of the third transmembrane segment as an alpha-helix suggests that Tyr292, Met299, and Tyr307 all reside on the same face of the alpha-helix. We interpret the transport data to suggest that a hydroxyl group is important at Tyr307, and that these three residues interact with Mg2+ during transport, forming part of the cation pore or channel within CorA.