Protection against Shiga toxin 1 challenge by immunization of mice with purified mutant Shiga toxin 1

Shiga toxin 1 (Stx1) of enterohemorrhagic Escherichia coli O157:H7 was cloned, and four mutant Stx1s were constructed by site-directed mutagenesis with PCR. The wild-type and mutant Stx1s with amino acid replacements at positions 167 and 170 of the A subunit were purified by one-step affinity chromatography with commercially available Globotriose Fractogel, and the mutant Stxs were used for the immunization of mice. The mutant toxins were nontoxic to Vero cells in vitro and to mice in vivo and induced the immunoglobulin G antibody against the wild-type Stx1, which neutralized the cytotoxicity of Stx1. The induced antibody titers depended on the mutation at position 170 of the A subunit. The mice immunized with the mutant Stx1s were protected against a challenge of approximately 100 times the 50% lethal dose of the wild-type Stx1, suggesting that the mutant toxins are good candidates for toxoid vaccines for infection by Stx1-producing E. coli.     

Parenteral adjuvant activities of Escherichia coli heat-labile toxin and its B subunit for immunization of mice against gastric Helicobacter pylori infection

The heat-labile toxin (LT) of Escherichia coli is a potent mucosal adjuvant that has been used to induce protective immunity against Helicobacter felis and Helicobacter pylori infection in mice. We studied whether recombinant LT or its B subunit (LTB) has adjuvant activity in mice when delivered with H. pylori urease antigen via the parenteral route. Mice were immunized subcutaneously or intradermally with urease plus LT, recombinant LTB, or a combination of LT and LTB prior to intragastric challenge with H. pylori. Control mice were immunized orally with urease plus LT, a regimen shown previously to protect against H. pylori gastric infection. Parenteral immunization using either LT or LTB as adjuvant protected mice against H. pylori challenge as effectively as oral immunization and enhanced urease-specific immunoglobulin G (IgG) responses in serum as effectively as aluminum hydroxide adjuvant. LT and LTB had adjuvant activity at subtoxic doses and induced more consistent antibody responses than those observed with oral immunization. A mixture of a low dose of LT and a high dose of LTB stimulated the highest levels of protection and specific IgG in serum. Urease-specific IgG1 and IgG2a antibody subclass responses were stimulated by all immunization regimens tested, but relative levels were dependent on the adjuvant used. Compared to parenteral immunization with urease alone, LT preferentially enhanced IgG1, while LTB or the LT-LTB mixture preferentially enhanced IgG2a. Parenteral immunization using LT or LTB as adjuvant also induced IgA to urease in the saliva of some mice. These results show that LT and LTB stimulate qualitatively different humoral immune responses to urease but are both effective parenteral adjuvants for immunization of mice against H. pylori infection.     

Comparative phenotypic characterization of hybrid Shiga toxin-producing / uropathogenic Escherichia coli,canonical uropathogenic and Shiga toxin-producing Escherichia coli

Hybrid Shiga toxin (Stx)-producing Escherichia coli (STEC) and uropathogenic E. coli (UPEC) strains are phylogenetically positioned between STEC and UPEC and can cause both diarrhea and urinary tract infections (UTIs). However, their virulence properties and adaptation to different host milieu in comparison to canonical UPEC and STEC strains are unknown.We determined phenotypes of the STEC/UPEC hybrid with respect to virulence including acid resistance, motility, biofilm formation, siderophore production, and adherence to human colonic Caco-2 and bladder T24 cells and compared to phenotypes of commensal strain MG1655, UPEC strain 536, and STEC strains B2F1 and Sakai. Moreover, we assessed the adaptation of the hybrid to artificial urine medium (AUM) and simulated colonic environment medium (SCEM).Overall acid resistance at pH 2.5 was high except in strains B2F1 and hybrid 05−00787 which showed reduced and extremely low acid resistance, respectively. Motility was reduced in hybrid 05−00787 and 09−05501 but strong in the remaining hybrids. While some hybrids showed high biofilm formation in LB, overall biofilm formation in SCEM and AUM were low and non-existent, respectively. All strains tested showed siderophore activity at equilibrium. All strains except MG1655 adhered to Caco-2 cells with the hybrid having similar adherence when compared to 536 but exhibited 2 and 3 times lower adherence when compared to B2F1 and Sakai, respectively. All Stx-producing strains adhered stronger to T24 cells than strains 536 and MG1655. Overall growth in LB, SCEM and AUM was consistent within the hybrid strains, except hybrid 05−00787 which showed significantly different growth patterns.Our data suggest that the hybrid is adapted to both, the intestinal and extraintestinal milieu. Expression of phenotypes typical of intestinal and extraintestinal pathogens thereby supports its potential to cause diarrhea and UTI.     

Protective immunization of mice with an active-site mutant of subtilase cytotoxin of Shiga toxin-producing Escherichia coli

We have recently described a novel AB(5) cytotoxin produced by certain Shiga toxin-producing Escherichia coli strains. The A subunit of this toxin is a subtilase-like serine protease, while the B pentamer mediates binding to host cell glycolipid receptors. The subtilase cytotoxin is lethal for mice, causing extensive microvascular thrombosis as well as necrosis in the brain, kidneys, and liver. In the present study, we have immunized mice with a purified derivative of the toxin with a Ser272 --> Ala mutation in the A subunit which abolishes cytotoxicity. This elicited strong antibody responses, as judged by enzyme-linked immunosorbent assay, which conferred protection against intraperitoneal challenge with purified toxin. Immunized mice were also protected from weight loss resulting from oral challenge with an E. coli K-12 clone expressing the active toxin.     

Rapid detection of Shiga toxin-producing Escherichia coli by optical immunoassay

In a multi-health center study, a new rapid optical immunoassay (OIA) for the detection of Shiga toxin types 1 and 2, the BioStar OIA SHIGATOX kit (Inverness Medical Professional Diagnostics, Inc.), was used to prospectively screen 742 fresh fecal samples for Shiga toxins in parallel with the Premier enterohemorrhagic Escherichia coli (EHEC) kit (Meridian BioScience, Inc.) with and without enrichment of the specimens by incubation in MacConkey broth. Additionally, 85 previously tested frozen fecal samples were assessed as described above. All positive immunoassay results were confirmed by the Vero cell cytotoxicity assay. A further modification of the screening procedure was evaluated on 470 of the prospectively screened specimens. Swabs of growth from conventionally plated stool culture media were subjected to the OIA SHIGATOX, and results were compared with those obtained with the Premier EHEC kit following broth enrichment. Overall, the OIA SHIGATOX kit was significantly more sensitive than the Premier EHEC kit on fresh direct stool specimens (sensitivities, 96.8% and 83.9%, respectively; P < 0.05). The two assays performed equally well with each other on frozen and broth-enriched samples. The colony sweep method used in conjunction with the OIA kit was somewhat more effective at detection of Shiga toxins from growth on agar than the overnight broth enrichment procedure used with the Premier EHEC assay (sensitivities, 100% and 92%, respectively; P < 0.09). Overall, the OIA SHIGATOX kit provided rapid, easy-to-interpret results and was highly effective at detection of Shiga toxin-producing E. coli in fecal samples and overnight cultures.     

Protection against challenge with Escherichia coli heat-labile enterotoxin by immunization of rats with cholera toxin/toxoid.

Rats were used to study dose-response curves to intestinal challenge with cholera toxin or Escherichia coli heat-labile enterotoxin. Prior immunization with cholera toxin/toxoid induced significant and equal protection against both toxins.     

Protection of monkeys against Shiga toxin induced by Shiga toxin-liposome conjugates

BACKGROUND: We previously reported that the purified Shiga toxins (Stx) Stx1 and Stx2, when coupled with liposomes, induced substantial production of anti-Stx1 and anti-Stx2 IgG antibody, respectively, in mice. The levels of anti-Stx antibody in the sera of mice immune to Stx-liposome correlated well with the protection against subsequent challenge with Stx. Furthermore, mice immunized with a mixture of Stx1-liposome and Stx2-liposome were successfully protected against oral infection with cytotoxin-producing Escherichia coli O157:H7. METHODS: In this study, the induction of protection against Stx2 by Stx2-liposomes was evaluated in monkeys. RESULTS: Stx2-liposomes induced a substantial amount of anti-Stx2 IgG antibodies as well as Stx2 neutralizing antibodies in monkeys. Test monkeys were successfully protected against challenge with lethal doses of Stx2. Moreover, these monkeys showed no apparent symptoms, while nonimmunized control monkeys died within 4 days with hemorrhagic gastroenteritis and renal disorder. In addition, as shown by other cases involving antigen-liposome conjugates, Stx2-liposome did not induce anti-Stx2 IgE antibody production, though it stimulated the production of a substantial amount of anti-Stx2 IgG antibodies. CONCLUSION: These results suggest that Stx-liposome conjugates may serve as candidate vaccines to induce protection against death caused by cytotoxin-producing E. coli infection.     

Temperature- and medium-dependent secretion of proteins by Shiga toxin-producing Escherichia coli.

Infections due to Shiga toxin-producing Escherichia coli (STEC) are responsible for severe diarrheal disease in humans and livestock, and these bacteria have recently emerged as a leading cause of renal failure in children. In this study, we have examined medium- and temperature-dependent production of secreted proteins from a STEC O26 serotype strain. Growth of bacteria in Luria broth led to the detection of secreted polypeptides of 104, 55, 54, and 37 kDa (p104, p55, p54, and p37, respectively). When grown in serum-free tissue culture medium, only p104, p37 and two additional polypeptides of 25 and 22 kDa (p25 and p22) were present in supernatant fluids. Production of these polypeptides was growth temperature dependent and induced in cultures grown at 37 degrees C. N-terminal amino acid sequencing revealed that p104 was homologous to the secreted p110 of enteropathogenic Escherichia coli (EPEC), and both proteins belong to a family of secreted proteins in pathogenic bacteria of which the immunoglobulin A protease of Neisseria gonorrhoeae is the prototype. The N-terminal amino acid sequences of p55 and p54 were unique to the STEC strain, while p37 and p25 were found to be highly homologous to the similarly sized EspA and EspB proteins, previously detected in culture supernatants of EPEC. Molecular cloning and sequencing of STEC espB alleles from two different serotypes showed that the encoded polypeptides were about 80% homologous. A monoclonal antibody raised against STEC EspB also cross-reacted with its EPEC analog and allowed us to demonstrate medium- and temperature-dependent production of this important virulence factor in STEC and EPEC strains of differing serotypes.     

Protection in rabbits immunized with a vaccine of Escherichia coli heat-stable toxin cross-linked to the heat-labile toxin B subunit.

Rabbits and rats were immunized with a vaccine consisting of synthetically produced Escherichia coli heat-stable toxin cross-linked by the carbodiimide reaction to the B subunit of biologically produced porcine heat-labile toxin. The vaccine contained 50% of each toxin component by weight and antigenicity; the toxicity of the heat-stable enterotoxin component was reduced by greater than 600-fold. Two or three peroral immunizations with vaccine containing 1,000 antigen units of each component raised greater-than-threefold increases in specific mucosal immunoglobulin A antitoxin titers to each component in all animal groups. Protection index values for challenge with either heat-labile or heat-stable toxins in ligated ileal loops were 3.4 to 4.0 in rats immunized by a parenteral primary immunization followed by two peroral booster immunizations, greater than 9 in rabbits immunized by these routes, and greater than 8 in rabbits given just three peroral immunizations. The antigenicity of the B-subunit component of the peroral vaccine was protected equally well against gastric acidity either by pretreatment with cimetidine or by delivery of the vaccine encapsulated in pH-dependent microspheres. The vaccine did not cause diarrhea when given perorally to any of the experimental animals or evoke fluid secretion when instilled into rabbit ligated ileal loops. These observations (i) confirm the effectiveness of this vaccine as an immunogen in a second animal model, (ii) establish that it is effective when given exclusively by the peroral route, and (iii) provide further evidence regarding its lack of toxicity.     

Intestinal Shiga toxin-producing Escherichia coli bacteria mitigate bovine leukemia virus infection in experimentally infected sheep

Ruminants often carry gastrointestinal Shiga toxin (Stx)-producing Escherichia coli (STEC). Stxs belong to a large family of ribosome-inactivating proteins (RIPs), found in many plants and some bacteria. Plant RIPs, secreted into extracellular spaces, limit the spread of viruses through plant tissues by penetrating and killing virally infected cells. Previously, we showed Stx activity against bovine leukemia virus (BLV)-infected cells in vitro and hypothesized that STEC bacteria have antiviral activity in ruminant hosts. Here, we investigated the impact of STEC on the initial phases of BLV infection in sheep. Sheep were treated with biweekly oral doses of E. coli O157:H7 (an STEC) or an isogenic stx mutant strain. A different group of sheep were similarly treated with five naturally occurring ovine STEC isolates or stx-negative E. coli. Intestinal STEC bacteria were enumerated and identified by standard fecal culture and DNA hybridization. Oral STEC treatment did not always result in carriage of STEC, although many animals consistently presented with >10(4) CFU/g feces. BLV viremia was assessed by spontaneous lymphocyte proliferation (SLP) in cultures of blood mononuclear cells and by syncytium formation in cocultures of the same with F-81 indicator cells. SLP was lower (P < 0.05) and syncytia were fewer (P < 0.05) in STEC-treated sheep than in untreated sheep. Both lower SLP and fewer syncytia positively correlated with fecal STEC numbers. Average weight gain post-BLV challenge was higher in STEC-treated sheep than in untreated sheep (P < 0.05). These results support the hypothesis that in ruminants, intestinal STEC bacteria have antiviral activity and mitigate BLV-induced disease.     

Probiotic bifidobacteria protect mice from lethal infection with Shiga toxin-producing Escherichia coli O157:H7

The anti-infectious activity of probiotic Bifidobacteria against Shiga toxin-producing Escherichia coli (STEC) O157:H7 was examined in a fatal mouse STEC infection model. Stable colonization of the murine intestines was achieved by the oral administration of Bifidobacterium breve strain Yakult (naturally resistant to streptomycin sulfate) as long as the mice were treated with streptomycin in their drinking water (5 mg/ml). The pathogenicity of STEC infection, characterized by marked body weight loss and subsequent death, observed in the infected controls was dramatically inhibited in the B. breve-colonized group. Moreover, Stx production by STEC cells in the intestine was almost completely inhibited in the B. breve-colonized group. A comparison of anti-STEC activity among several Bifidobacterium strains with natural resistance to streptomycin revealed that strains such as Bifidobacterium bifidum ATCC 15696 and Bifidobacterium catenulatum ATCC 27539(T) did not confer an anti-infectious activity, despite achieving high population levels similar to those of effective strains, such as B. breve strain Yakult and Bifidobacterium pseudocatenulatum DSM 20439. The effective strains produced a high concentration of acetic acid (56 mM) and lowered the pH of the intestine (to pH 6.75) compared to the infected control group (acetic acid concentration, 28 mM; pH, 7.15); these effects were thought to be related to the anti-infectious activity of these strains because the combination of a high concentration of acetic acid and a low pH was found to inhibit Stx production during STEC growth in vitro.     

Shiga toxin-producing Escherichia coli infection and antibodies against Stx2 and Stx1 in household contacts of children with enteropathic hemolytic-uremic syndrome

Ninety-five household contacts (aged 2 months to 73 years) of patients with enteropathic hemolytic-uremic syndrome (HUS) were investigated for the presence of immunoglobulin (Ig) G antibodies to Shiga toxins Stx2 and Stx1 by Western blot assay. Thirty-one percent of the household contacts and 19% of 327 controls had anti-Stx2 IgG (heavy and light chain [H + L]), 5 and 8%, respectively, had anti-Stx1 IgG (H + L), and 3 and 2%, respectively, had both anti-Stx2 and anti-Stx1 IgG (H + L). The incidence of infections with Stx-producing Escherichia coli (STEC) was determined based on the following diagnostic criteria: STEC isolation, detection of stx gene sequences, free fecal Stx in stool filtrates, and serum IgM antibodies against E. coli O157 lipopolysaccharide. Evidence of STEC infection was observed in 25 household contacts, of whom 18 (72%) were asymptomatic and represented a potential source of infection. Six of 13 (46%) household contacts with Stx2-producing E. coli O157:H7 in stool culture developed anti-Stx2 IgG (H + L), compared to 71% of Stx2-associated HUS cases. In individuals showing anti-Stx2 IgG (H + L), the antibody response was directed against the B subunit in 69% of household contacts and 71% of controls, in contrast to 28% of HUS patients. In this investigation controls had a significant increase of the median of IgM antibodies to O157 lipopolysaccharide (LPS) with age, up to the fifth decade. The lack of disease in household contacts with B subunit-specific antibodies, as well as the significantly higher median of anti-O157 LPS IgM antibodies in controls beyond 4.9 years of age, suggests a protective role for anti-Stx and anti-O157 LPS antibodies.     

Intranasal immunization of mice with group B streptococcal protein rib and cholera toxin B subunit confers protection against lethal infection

Intranasal immunization of mice with Rib, a cell surface protein of group B streptococcus (GBS), conjugated to or simply coadministered with the recombinant cholera toxin B subunit, induces systemic immunoglobulin G (IgG) and local IgA antibody responses and confers protection against lethal GBS infection. These findings have implications for the development of a human GBS vaccine.     

Modulation of host cell signalling by enteropathogenic and Shiga toxin-producing Escherichia coli.

The majority of Escherichia coli strains are harmless symbionts in the intestinal tract. However, there are several pathogenic forms, which are responsible for various diseases in humans and live stock. In this review we discuss the interactions between Shiga toxin-producing E. coli and enteropathogenic E. coli and their target host cells, describing their strategies to activate specific cellular signalling pathways which lead to subversion of critical physiological functions. We mainly concentrate on those pathogenic mechanisms that are dependent on a functional type III secretion system, but we also briefly discuss additional factors that contribute to the specific pathogenic profiles of Shiga toxin-producing E. coli and enreropathogenic E. coli.     

Pathogenic mechanism of mouse brain damage caused by oral infection with Shiga toxin-producing Escherichia coli O157:H7

In a previous study, we showed that infection with Shiga toxin (Stx)-producing Escherichia coli O157:H7 (strain Sm(r)N-9) caused neurologic symptoms in malnourished mice with positive immunoreactions of Stx2 in brain tissues. The present study explores the mechanism of how Stx injures the vascular endothelium to enter the central nervous system in mice. Oral infection with strain Sm(r)N-9 elicited a tumor necrosis factor alpha (TNF-alpha) response in the blood as early as 2 days after infection, while Stx was first detected at 3 days postinfection. In the brain, TNF-alpha was detected at day 3, and its quantity was increased over the next 3 days. Frozen sections of the brains from moribound mice contained high numbers of apoptotic cells. Glycolipids recognized by an anti-Gb3 monoclonal antibody were extracted from the brain, and purified Stx2 was able to bind to the glycolipids. In human umbilical vascular endothelial cells (HUVEC) cultured with fluorescein-labeled Stx2 (100 ng/ml), TNF-alpha (20 U/ml) significantly facilitated the intracellular compartmentalization of fluorescence during 24 h of incubation, suggesting the enhanced intracellular processing of Stx2. Consequently, higher levels of apoptosis in HUVEC were found at 48 h. Short-term exposure of HUVEC to Stx2 abrogated their apoptotic response to subsequent incubation with TNF-alpha alone or TNF-alpha and Stx2. In contrast, primary exposure of HUVEC to TNF-alpha followed by exposure to Stx2 alone or TNF-alpha and Stx2 induced apoptosis at the same level as obtained after 48-h incubation with these two agents. These results suggest that the rapid production of circulating TNF-alpha after infection induces a state of competence in vascular endothelial cells to undergo apoptosis, which would be finally achieved by subsequent elevation of Stx in the blood. In this synergistic action, target cells must be first exposed to TNF-alpha. Such cell injury may be a prerequisite to brain damage after infection with Stx-producing E. coli O157:H7.     

Isolation,genotyping and antimicrobial resistance of Shiga toxin-producing Escherichia coli

Shiga toxin-producing Escherichia coli (STEC) is an enteric pathogen linked to outbreaks of human gastroenteritis with diverse clinical spectra. In this review, we have examined the currently methodologies and molecular characterization techniques for assessing the phenotypic, genotypic and functional characteristics of STEC O157 and non-O157. In particular, traditional culture and isolation methods, including selective enrichment and differential plating, have enabled the effective recovery of STEC. Following recovery, immunological serotyping of somatic surface antigens (O-antigens) and flagellum (H-antigens) are employed for the classification of the STEC isolates. Molecular genotyping methods, including multiple-locus variable-number tandem repeat analysis, arrays, and whole genome sequencing, can discriminate the isolate virulence profile beyond the serotype level. Virulence profiling is focused on the identification of chromosomal and plasmid genes coding for adhesins, cytotoxins, effectors, and hemolysins to better assess the pathogenic potential of the recovered STEC isolates. Important animal reservoirs are cattle and other small domestic ruminants. STEC can also be recovered from other carriers, such as mammals, birds, fish, amphibians, shellfish and insects. Finally, antimicrobial resistance in STEC is a matter of growing concern, supporting the need to monitor the use of these agents by private, public and agricultural sectors. Certain antimicrobials can induce Shiga toxin production and thus promote the onset of severe disease symptoms in humans. Together, this information will provide a better understanding of risks associated with STEC and will aid in the development of efficient and targeted intervention strategies.     

Assessment of immune response of the B subunit of Shiga toxin fused to AAF adhesin of enteroaggregative Escherichia coli

Shiga toxin is a member of AB toxin family and is composed of an A subunit which mediated toxicity and a homopentameric protein responsible for toxin binding and internalization into target cells. Another group of diarrheagenic Escherichia coli, enteroaggregative E. coli (EAEC) is a group of E. coli with aggregative adherence to epithelial cells, which play an important role in its pathogenesis. In the present investigation, the immune response of recombinant hybrid peptide composed of B subunit of Shiga toxin (StxB) and Aggregative Adherence Fimbriae (AAF) of EAEC (B-AAF/I, B-AAF/II) that elicited protective response was further characterized. The assessment of IgG subclasses (IgG1 and IgG2a) and cytokine production by these peptides indicated that although the hybrid peptides could induce immune response, but two adhesins behave differently in this regard. Lymphocyte proliferation assay and IFN-γ production were highly significant for B-AAF/II. Overall, based on the data obtained from this study it seems that mixed population of Th1-Th2 type of immune responses were induced by these hybrid peptides, which probably lead to observed protective response. In the present study, it is shown that the two hybrid peptides i.e. B-AAF/I and B-AAF/II, could be a promising strategy to make more effective and powerful vaccine.     

Real-time fluorescence PCR assays for detection and characterization of Shiga toxin,intimin, and enterohemolysin genes from Shiga toxin-producing Escherichia coli

PCR assays have proved useful for detecting and characterizing Shiga toxin-producing Escherichia coli (STEC). Recent advances in PCR technology have facilitated the development of real-time fluorescence PCR assays with greatly reduced amplification times and improved methods for the detection of amplified target sequences. We developed and evaluated two such assays for the LightCycler instrument: one that simultaneously detects the genes for Shiga toxins 1 and 2 (stx(1) and stx(2)) and another that simultaneously detects the genes for intimin (eae) and enterohemolysin (E-hly). Amplification and sequence-specific detection of the two target genes were completed within 60 min. Findings from the testing of 431 STEC isolates of human and animal origin, 73 isolates of E. coli negative for stx genes, and 118 isolates of other bacterial species with the LightCycler PCR (LC-PCR) assays were compared with those obtained by conventional block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the stx(1), eae, and E-hly genes and 96 and 100%, respectively, for the stx(2) gene. No stx(2) genes were detected from 10 stx(2f)-positive isolates because of significant nucleotide differences in their primer annealing regions. Melting curve analyses of the amplified Shiga toxin genes revealed sequence variation within each of the tested genes that correlated with described and novel gene variants. The performance characteristics of the LC-PCR assays, such as their speed, detection method, and the potential subtyping information available from melting curve analyses, make them attractive alternatives to block cycler PCR assays for detecting and characterizing STEC strains.     

Molecular mechanisms that mediate colonization of Shiga toxin-producing Escherichia coli strains

Shiga toxin-producing Escherichia coli (STEC) is a group of pathogens which cause gastrointestinal disease in humans and have been associated with numerous food-borne outbreaks worldwide. The intimin adhesin has been considered for many years to be the only colonization factor in these strains. However, the rapid progress in whole-genome sequencing of different STEC serotypes has accelerated the discovery of other adhesins (fimbrial and afimbrial), which have emerged as important contributors to the intestinal colonization occurring during STEC infection. This review summarizes recent progress to identify and characterize, at the molecular level, novel adhesion and colonization factors in STEC strains, with an emphasis on their contribution to virulence traits, their host-pathogen interactions, the regulatory mechanisms controlling their expression, and their role as targets eliciting immune responses in the host.