Carlos E. Hormaeche

The role of a stress‐response protein in Salmonella typhimurium virulence

We recently described the use of selective transposon mutagenesis to generate a series of a virulent mutants of a pathogenic strain of Salmonella typhimurium. Cloning and sequencing of the insertion sites from two of these mutants reveals that both have identical locations within an open reading frame that is highly homologous to a gene, htrA, encoding a heat‐shock protein in Escherichia coli. DNA sequence analysis of S. typhimurium htrA reveals the presence of a gene capable of encoding a protein with a calculated Mr of 49316 that has 88.7% protein:protein homology with its E. coli counterpart. In E. coli, lesions in this gene, also known as degP, reduce proteolytic degradation of aberrant periplasmic proteins. Characteristics of the S. typhimurium htrA mutants, 046 and 014, in vivo and in vitro suggested that they are avirulent because of impaired ability to survive and/or replicate in host tissues. In vitro, the S. typhimurium htrA mutants 046 and 014 are not temperature‐sensitive but were found to be more susceptible to oxidative stress than the parent, suggesting that they may be less able to withstand oxidative killing within macrophages.

Role of T cells, TNFα and IFNγ in recall of immunity to oral challenge with virulent salmonellae in mice vaccinated with live attenuated aro− salmonella vaccines☆

Abstract The SL3261 Salmonella typhimurium aro A live vaccine strain confers solid protection against oral challenge with virulent salmonellae, immunity persisting long after the vaccine has been cleared from the tissues. BALB/c mice immunized with SL3261 and later subjected to in vivo depletion of both CD4 + and CD8 + T cells had impaired recall of immunity to oral challenge with the virulent S. typhimurium C5, with increased mortality and higher bacterial loads in the reticuloendothelial system (RES). Selective depletion of CD4 + cells alone significantly impaired resistance both 8 and 14 weeks after vaccination as determined by estimation of bacterial numbers in organ homogenates. Depletion of CD8 + cells alone had less effect on immunity when performed at 8 weeks than at 14 weeks after immunization. Administration of anti-IFNγ or anti-TNFα antibodies also impaired recall of immunity, exacerbating a secondary infection in vaccinated mice. Challenge of T cell-depleted immune mice with virulent salmonellae caused hepatosplenomegaly with minute grossly visible focal lesions, and a marked increase in the number and severity of necrotic foci in spleen, liver and lymph nodes. A widespread mononuclear cell infiltrate was present. The histopathology in anti-IFNγ-treated mice was qualitatively similar to that seen in T-cell depleted mice. In contrast, in the anti-TNFα-treated mice splenomegaly was much less than in T cell-depleted mice. Granulomas were absent, no mononuclear infiltration was observed and there was severe necrosis; the lesions appeared similar to or worse than those seen in naive mice. Surprisingly, IFNγ was detectable in sera of both controls and T cell-depleted mice on day 8 of the secondary infection, as well as in sera of anti-TNFα-treated mice on day 6 of infection. The results indicate that T cells, IFNγ and TNFα are all important in the specific recall of immunity to virulent salmonellae conferred by immunization with live vaccines, with the effect of T cell and IFNγ depletion (marked macrophage infiltration) being qualitatively very different from that of TNFα neutralization (no mononuclear infiltrate or granuloma formation).

Evaluation of Salmonella typhimurium strains harbouring defined mutations in htrA and aroA in the murine salmonellosis model

Derivatives of the mouse-virulent Salmonella typhimurium strain SL1344 were constructed harbouring defined mutations in htrA, aroA or htrA aroA combined. When administered orally or intravenously to BALB/c mice, all the mutants were found to be highly attenuated. All mutants were able to confer significant protection against lethal challenge with SL1344 after a single oral dose of live organisms. SL1344 htrA mutants persisted in livers and spleens at a lower level than SL1344 aroA mutants after intravenous administration. SL1344 htrA aroA mutants persisted at an even lower level and were cleared from the livers and spleens of mice within 21 days of intravenous administration. Thus htrA and htrA aroA mutants can be considered as potential oral vaccines against salmonellosis.

Salmonella typhimuriumaroA mutants as carriers of the Escherichia coli heat-labile enterotoxin B subunit to the murine secretory and systemic immune systems

We investigated the ability of Salmonella typhimurium vaccines to deliver heterologous antigens to the systemic and secretory immune systems of the mouse, while retaining their immunogenicity against salmonellosis. S. typhimurium SL3261, an avirulent aroA mutant, or SL3261 carrying plasmid pBRD026, a pBR322 derivative encoding the gene for Escherichia coli LT-B were used to immunize BALB/c mice orally. Both immunizing strains invaded the mononuclear phagocyte system of the mice, grew slowly until approximately day 14 post-infection, and then were rapidly cleared. No salmonellae were detected in livers, spleens, mesenteric lymph nodes or Peyers patches by day 42. Mice immunized with either strain and challenged orally with the virulent parent strain, SL1344, several weeks after clearing the immunizing organism, were protected against the lethal S. typhimurium infection. Mice infected with SL3261 (pBRD026) developed substantial levels of IgG and IgA anti-LT-B antibodies 14 days post-infection in both serum and gut samples. The sera neutralized the effects of LT in an in vitro Vero cell assay. Thus, aroA mutants of S. typhimurium can deliver a heterologous antigen from a different enteric pathogen to the murine systemic and secretory immune systems without altering their efficacy against salmonellosis.

Live oral Salmonella vaccines: potential use of attenuated strains as carriers of heterologous antigens to the immune system

Summary Live attenuated strains of salmonellae are showing promise as live oral vaccines against human typhoid fever and other Salmonella infections of man and animals. Attenuation can be achieved by introducing genetically defined, non‐reverting mutations into specific genes on the Salmonella chromosome. Mutations in the gal E or aro A genes of Salmonella inhibit the ability of the bacteria to grow in vivo, and strains carrying such lesions are effective vaccines against salmonellosis. Genetic determinants encoding for the expression of potentially protective antigens from heterologous, non‐Salmonella pathogens can be readily introduced into these attenuated Salmonella strains. Expression of the heterologous antigen does not affect the ability of the Salmonella host to be used as a Salmonella vaccine. Mice infected orally with a Salmonella typhimurium aro A vaccine expressing the Escherichia coli heat‐labile toxin B subunit developed both a secretory and serum antibody response to this antigen. These serum antibodies were able to neutralise the activity of E. coli heat‐labile toxin in tissue culture assays. A humoral and cell‐mediated (DTH) immune response was detected against beta galactosidase, an intracellular antigen, in mice infected with an aro A vaccine expressing this cloned antigen. The prospects for the development of live Salmonella vaccines as a method for delivering heterologous antigens derived from bacteria, viruses and parasites is discussed.

Correlates of protection induced by live Aro- Salmonella typhimurium vaccines in the murine typhoid model

Live attenuated salmonella vaccines generally confer better protection than killed vaccines. The immune responses in BALB/c mice elicited by immunization with a live attenuated Aro Salmonella typhimurium vaccine given orally, intravenously or subcutaneously were compared with those elicited by killed whole‐cell vaccines (acetone or heat‐treated) given subcutaneously. Live vaccines given by all routes elicited higher interleukin‐2 (IL‐2) and interferon‐γ (IFN‐γ) responses in spleen cells against an alkali‐treated whole‐cell salmonella lysate than did killed vaccines. Live and killed vaccines elicited high total antibody levels to smooth lipopolysaccharide (LPS) (enzyme‐linked immunosorbent assay), but all live vaccine regimes elicited higher IgG2a, suggesting a Th1 response. Oral and intravenous vaccination with live organisms elicited IgA against smooth LPS which subcutaneous vaccination with live or killed salmonellae failed to evoke. Western blots using rough whole‐cell lysates showed that all vaccines elicited a varied anti‐protein response; however, all groups immunized with live organisms recognized three unidentified bands of MW 52 000, 46 000 and 18 000 which were consistently absent in groups immunized with killed organisms. The results indicate that immunization with live aroA salmonellae elicited a Th1 type of response, including bystander T‐cell help to LPS, and a response to proteins not seen in mice that received killed vaccines.

Protection against oral challenge three months after i.v. immunization of BALBc mice with live Aro Salmonella typhimurium and Salmonella enteritidis vaccines is serotype (species)-dependent and only partially determined by the main LPS O antigen

The role of the main LPS O antigen in the specificity of protection as mediated by systemic mechanisms following immunization with live attenuated Aro Salmonella vaccines was studied in mice. Innately Salmonella-susceptible (Itys) BALB/c mice were immunized intravenously with a single dose of either Salmonella typhimurium SL3261 aroA (LPS O4,5,12) or Salmonella enteritidis Se795aroA (LPS O1,9,12), and challenged orally 2-3 months later with either S. typhimurium C5 or S. enteritidis Thirsk. Nearly isogenic transductants of the two challenge strains expressing either their own LPS or that of the other serotype (S. typhimurium C5 O4 or O9, and S. enteritidis Thirsk O9 or O4) were also used. Both vaccines conferred similar high protection against the virulent strain of the homologous serotype expressing its own LPS. There was no protection against the heterologous serotype expressing its own LPS. However, when vaccinated mice were challenged with either the same serotype as the vaccine but expressing the heterologous LPS, or with the heterologous serotype expressing the LPS of the vaccine, protection was always lower than protection against the fully homologous serotype. Anti-smooth LPS antibodies showed higher titres against the homologous LPS, but with significant crossreactivity with the heterologous LPS. Antibodies to O-rough S. typhimurium and S. enteritidis LPS were present following immunization with either of the two vaccine strains. The LPS alone cannot fully account for the specificity of protection in this model; other (protein) antigens may be responsible. It remains to be seen whether there is a T-cell mediated component to the specificity of protection conferred by live Salmonella vaccines.

Serum TNFα in mouse typhoid and enhancement of a salmonella infection by anti-TNFα antibodies

Tumour necrosis factor α (TNFα) was detected by the L929 cell assay in the sera of mice 1 h after large i.v. inocula of virulent Salmonella typhimurium C5. TNFα was not detectable in sera from innately susceptible BALB/c mice during the course of a lethal infection commencing from a low inoculum, or from resistant A/J mice during the course of a lethal or sublethal infection, but only 1 h after i.v. challenge with large numbers of organisms. Administration of a single dose of rabbit polyclonal anti-TNFα antiserum on day 1 had no effect on the early course of a lethal infection in A/J mice. However, the same treatment exacerbated a sublethal infection in A/J mice. Anti-TNFα treatment did not accelerate the early bacterial net growth rate in the RES. Instead, the cfu count in treated mice continued to increase past the point at which the host response suppressed a further increase in bacterial numbers (the plateau phase) in normal controls. A second dose of anti-TNFα antiserum on day 4 together with a higher but still sublethal challenge caused a lethal infection in A/J mice. The results indicate that TNFα is important in mediating the plateau phase in a salmonella infection, and its effect may be local.

Immunity conferred by Aro− salmonella live vaccines

The specificity of protection conferred by Aro- salmonellae was studied in BALB/c mice challenged 3 months after intravenous (i.v.) vaccination, more than 1 month after the vaccine had been cleared. Oral challenge showed better protection than i.v. challenge. Salmonella typhimurium aroA SL3261 conferred very good protection against wild-type S. typhimurium C5 (over 10,000 x LD50). Cross protection experiments were performed using S. typhimurium, S. enteritidis and S. dublin for vaccination and challenge, including variants of S. typhimurium and S. enteritidis of similar virulence differing in the main LPS antigen (O-4 or O-9). Salmonella typhimurium aroA conferred solid protection against S. typhimurium (O-4), but no protection against wild-type S. enteritidis (O-9). However challenge with LPS variant strains showed that although protection was generally better to strains of the homologous LPS type, specificity of protection was determined more by the parent strain background (S. typhimurium or S. enteritidis) of the challenge than by O-factors 4 or 9, suggesting that other antigens are involved. The nature of the protective antigen(s) in this model is unclear, but it does not appear to be the main O-specific antigen. A S. enteritidis Se795 aroA vaccine gave good protection against wild-type S. enteritidis Se795 2 weeks after vaccination, but much less at 3 months (approximately 10-200 x LD50), although the persistence of the S. enteritidis aroA vaccine in the liver and spleen was similar to that of the S. typhimurium vaccine, and the wild-type Se795 challenge strain was of similar virulence to S. typhimurium C5. A S. dublin aroA vaccine conferred similar protection against wild-type S. dublin (approximately 300 x LD50).

A Salmonella typhimurium htrA live vaccine expressing multiple copies of a peptide comprising amino acids 8–23 of herpes simplex virus glycoprotein D as a genetic fusion to tetanus toxin fragment C protects mice from herpes simplex virus infection

Multiple tandem copies of an immunogenic epitope comprising amino acids 8–23 of glycoprotein D of herpes simplex virus (HSV) were expressed as C‐terminal fusions to tetanus toxin fragment C (TetC) in different Salmonella typhimurium live vaccine strains. Expression of the longer fusions was best in strains harbouring a lesion in htrA, a stress protein gene. SL3261, an aroA strain, did not effectively express the longer fusions. Mice immunised with an S. typhimurium C5 htrA mutant expressing fusions with two or four copies of the peptide made an antibody response to both the peptide and TetC, whereas constructs expressing one copy of the peptide only elicited antibody to TetC. A non‐immunogenic octameric fusion underwent rearrangements in vivo resulting in a predominantly monomeric fusion. In contrast, the S. typhimurium SL3261 aroA vaccine expressing the TetC‐tetrameric fusion did not elicit antibody to the peptide. Sera from mice immunised with a single dose of the dimer and tetramer fusions in the htrA strain neutralised HSV in vitro, and the mice were protected from HSV infection as measured by a reduction in virus load in the ear pinna. We have previously shown that mice vaccinated with salmonella expressing TetC are protected against tetanus toxin and virulent salmonella challenge. These results suggest that it may be possible to develop a multivalent vaccine against salmonellosis, tetanus and HSV.