Secretion of the Shiga toxin B subunit (Stx1B) via an autotransporter protein optimizes the protective immune response to the antigen expressed in an attenuated E. coli (rEPEC E22Δler) vaccine strain.

We previously developed attenuated rabbit enteropathogenic E. coli (rEPEC) strains which are effective oral vaccines against their parent pathogens by deleting ler, a global regulator of virulence genes. To use these strains as orally administered vectors to deliver other antigens we incorporated the B subunit of shiga-like toxin 1(Stx1) into the passenger domain of the autotransporter EspP expressed on a plasmid. Native EspP enters the periplasm where its passenger domain is exported to the bacterial surface through an outer membrane channel formed by its translocator domain, then cleaved and secreted. Since antigen localization may determine immunogenicity, we engineered derivatives of EspP expressing Stx1B- passenger domain fusions: 1. in cytoplasm 2. in periplasm, 3. surface-attached or 4. secreted. To determine which construct was most immunogenic, rabbits were immunized with attenuated O103 E. coli strain (E22 Δler) alone or expressing Stx1B in each of the above four cellular locations. IgG responses to Stx1B, and toxin-neutralizing antibodies were measured. Animals were challenged with a virulent rabbit Enterohemorrhagic E. coli (EHEC) strain of a different serogroup (O15) than the vaccine strain expressing Stx1 (RDEC-H19) and their clinical course observed. IgG responses to Stx1B subunit were induced in all animals vaccinated with the strain secreting Stx1B, in some vaccinated with surface-expressed Stx1B, but in not animals immunized with periplasmic or cytoplasmic Stx1B. Robust protection was observed only in the group immunized with the vaccine secreting Stx1B. Taken together, our data suggest that secretion of Stx1B, or other antigens, via an autotransporter, may maximize the protective response to live attenuated oral vaccine strains.

The secreted effector protein EspZ is essential for virulence of rabbit enteropathogenic Escherichia coli.

Attaching and effacing (A/E) pathogens adhere intimately to intestinal enterocytes and efface brush border microvilli. A key virulence strategy of A/E pathogens is the type III secretion system (T3SS)-mediated delivery of effector proteins into host cells. The secreted protein EspZ is postulated to promote enterocyte survival by regulating the T3SS and/or by modulating epithelial signaling pathways. To explore the role of EspZ in A/E pathogen virulence, we generated an isogenic espZ deletion strain (ΔespZ) and corresponding cis-complemented derivatives of rabbit enteropathogenic Escherichia coli and compared their abilities to regulate the T3SS and influence host cell survival in vitro. For virulence studies, rabbits infected with these strains were monitored for bacterial colonization, clinical signs, and intestinal tissue alterations. Consistent with data from previous reports, espZ-transfected epithelial cells were refractory to infection-dependent effector translocation. Also, the ΔespZ strain induced greater host cell death than did the parent and complemented strains. In rabbit infections, fecal ΔespZ strain levels were 10-fold lower than those of the parent strain at 1 day postinfection, while the complemented strain was recovered at intermediate levels. In contrast to the parent and complemented mutants, ΔespZ mutant fecal carriage progressively decreased on subsequent days. ΔespZ mutant-infected animals gained weight steadily over the infection period, failed to show characteristic disease symptoms, and displayed minimal infection-induced histological alterations. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining of intestinal sections revealed increased epithelial cell apoptosis on day 1 after infection with the ΔespZ strain compared to animals infected with the parent or complemented strains. Thus, EspZ-dependent host cell cytoprotection likely prevents epithelial cell death and sloughing and thereby promotes bacterial colonization.

A directed intimin insertion mutant of a rabbit enteropathogenic Escherichia coli (REPEC) is attenuated, immunogenic and elicits serogroup specific protection.

We previously showed (Agin et al., 2005) that a truncated beta-intimin mutant of an O15 A/E REPEC strain, which does not contain the C terminal tir binding region of intimin but expresses the preceding immunodominant portion of the molecule in outer membranes, is attenuated, induces anti-intimin and anti whole cell antibody, and protects against challenge with a virulent strain of the same serogroup. Since the ability of this and other intimin mutants lacking the tir binding region to provide broad protection against challenge with REPEC strains of other serogroups is incompletely studied, we generated a targeted insertion/deletion intimin mutant in an O103 strain, immunized rabbit's orogastrically, then challenged them with the parent strain and a virulent strain of a different serogroup. We used λ red recombinase to generate an eae mutation in the prototypic rabbit A/E Escherichia coli strain E22 (O103) by replacing the 81 C-terminal (860-939) tir-binding amino acids of intimin with an inserted kanamycin resistance gene. This mutant did not express intimin in its outer membranes. A range of increasing immunizing doses (10(4)-10(7)CFU) was used for the first immunization in 4 groups of 6 rabbits. All 4 groups received a 2nd immunization with 10(7)CFU after 2weeks. At 4weeks, half of the rabbits in each group, and 6 control rabbits, were challenged with the parent O103 strain or with the O15 strain RDEC-H19A. All unimmunized rabbits exhibited characteristic weight loss with diarrhea and shedding of the challenge strain after challenge with E22 or RDEC-H19A. Rabbits challenged with the parent O103 E22, but not with the O15 RDEC-H19A, were protected against clinical signs of disease, maintained normal weight gain, had reduced fecal shedding of challenge organism. At sacrifice, CFU of E22, but not RDECH19, were decreased in ileum, cecum and colon. Serum antibodies to E22 somatic antigens, but not intimin, were detected in rabbits immunized with E22 Δeae860-939 and correlated with protection. An intimin insertion mutation replacing the tir binding region of O103 REPEC strain E22 with an antibiotic resistance gene was attenuated, induced antibody to whole bacteria but not to intimin, and yielded protection against challenge with the WT strain from which it was prepared but not against a virulent strain of another serogroup. These results suggest that intimin expression in outer membranes may be necessary to confer cross serogroup protection by inducing anti-intimin immunity.

Attenuated Escherichia coli strains expressing the colonization factor antigen I (CFA/I) and a detoxified heat-labile enterotoxin (LThK63) enhance clearance of ETEC from the lungs of mice and protect mice from intestinal ETEC colonization and LT-induced fluid accumulation.

Although enterotoxigenic Escherichia coli (ETEC) infections are important causes of infantile and traveler's diarrhea there is no licensed vaccine available for those at-risk. Our goal is to develop a safe, live attenuated ETEC vaccine. We used an attenuated E. coli strain (O157:H7, Δ-intimin, Stx1-neg, Stx2-neg) as a vector (ZCR533) to prepare two vaccine strains, one strain expressing colonization factor antigen I (ZCR533-CFA/I) and one strain expressing CFA/I and a detoxified heat-labile enterotoxin (ZCR533-CFA/I+LThK63) to deliver ETEC antigens to mucosal sites in BALB/c mice. Following intranasal and intragastric immunization with the vaccine strains, serum IgG and IgA antibodies were measured to the CFA/I antigen, however, only serum IgG antibodies were detected to the heat-labile enterotoxin. Intranasal administration of the vaccine strains induced respiratory and intestinal antibody responses to the CFA/I and LT antigens, while intragastric administration induced only intestinal antibody responses with no respiratory antibodies detected to the CFA/I and LT antigens. Mice immunized intranasally with the vaccine strains showed enhanced clearance of wild-type (wt) ETEC bacteria from the lungs. Mice immunized intranasally and intragastrically with the vaccine strains were protected from intestinal colonization following oral challenge with ETEC wt bacteria. Mice immunized intragastrically with the ZCR533-CFA/I+LThK63 vaccine strain had less fluid accumulate in their intestine following challenge with ETEC wt bacteria or with purified LT as compared to the sham mice indicating that the immunized mice were protected from LT-induced intestinal fluid accumulation. Thus, mice intragastrically immunized with the ZCR533-CFA/I+LThK63 vaccine strain were able to effectively neutralize the activity of the LT enterotoxin. However, no difference in intestinal fluid accumulation was detected in the mice immunized intranasally with the vaccine strain as compared to the sham mice as the immunized mice induced insufficient intestinal anti-LT antibody to neutralize the activity of the enterotoxin. These results show that our ETEC vaccine induced serum and mucosal antibody responses to CFA/I and LT after mucosal administration which then acted to protect the immunized mice against lung and intestinal colonization, as well as, intestinal fluid accumulation.

The encapsulation of enterotoxigenic Escherichia coli colonization factor CS3 in biodegradable microspheres enhances the murine antibody response following intranasal administration.

The aim of this study was to measure serum and mucosal antibody responses following intranasal administration of biodegradable poly(DL-lactide-co-glycolide) (PLGA) microspheres loaded with the CS3 colonization factor isolated from enterotoxigenic Escherichia coli (ETEC). The response was compared against that measured in mice similarly administered the native CS3 antigen and in mice co-administered, along with the CS3 antigen, a known mucosal adjuvant, the R192G mutant heat-labile enterotoxin (mLT). The integrity of the CS3 antigen released from the microspheres was maintained as determined by SDS-PAGE and immunoblotting. Native CS3 induced serum and mucosal (bronchoalveolar, small intestinal and faecal) IgG and IgA responses. The co-administration of the mLT mucosal adjuvant significantly enhanced (P<0.001) serum and mucosal antibody responses to the CS3 protein. Likewise, the CS3-loaded PLGA microspheres induced significantly greater (P<0.001) serum and mucosal antibody responses than native CS3, as well as inducing antibody responses superior to those of the CS3 plus mLT formulation. Following administration of CS3 plus mLT, the mice became distressed (loss of activity, increased huddling, ruffled fur), a situation not seen following administration of the CS3-loaded PLGA microspheres. The results in this trial show that the CS3-loaded PLGA microspheres when administered intranasally to mice caused no observable distress to the mice and significantly (P<0.001) enhanced the immunogenicity of the CS3 protein.

Long-term systemic and mucosal antibody responses measured in BALB/c mice following intranasal challenge with viable enterotoxigenic Escherichia coli.

The immunogenicity induced in BALB/c mice following intranasal challenge with a viable nonlethal dose (1.2 x 10(8) CFU) of enterotoxigenic Escherichia coli (ETEC) strain E23477A (O139:H28:CS1:CS3:LT+:ST+) was studied over a 140-day period. Serum IgG and IgM antibodies against coli surface antigen 3 (CS3), O139 lipopolysaccharide and heat-labile enterotoxin were measured by day 14 and remained at elevated levels out to day 140. The serum IgG response to the somatic antigens (CS3 and O139 lipopolysaccharide) was significantly greater (P < 0.05) than the IgG response to heat-labile enterotoxin, and the serum IgG response to CS3 was significantly greater (P < 0.05) than the IgG response to O139 lipopolysaccharide. The predominant serum IgG subclasses to CS3 were IgG1 and IgG2a, and they were significantly greater (P < 0.05) than IgG2b and IgG3. The predominant serum IgG subclass response to O139 lipopolysaccharide was initially IgG3 until day 56, after which IgG1 was predominant. The serum subclass response to CS3 indicated a mixed T helper 1/2 (Th1/Th2) profile, whereas the response to O139 lipopolysaccharide was primarily that of a Th2-type, at least over time. Fecal IgG and IgA responses to CS3 and O139 lipopolysaccharide were detected by day 14 and were measured out to day 140, with the CS3 fecal antibody responses being significantly greater (P < 0.05) than the O139 lipopolysaccharide and heat-labile enterotoxin fecal antibody responses. The aim of this study is the development of the intranasal mouse model that can aid in better understanding the immunopathology of ETEC infection and in screening of vaccine candidates prior to volunteer trials.

Intranasal immunization of BALB/c mice with enterotoxigenic Escherichia coli colonization factor CS6 encapsulated in biodegradable poly(DL-lactide-co-glycolide) microspheres.

Mice were intranasally administered enterotoxigenic Escherichia coli colonization factor CS6 encapsulated in poly(DL-lactide-co-glycolide) microspheres (CS6-PLG), with immune response measured and compared to that of similarly administered native CS6 and CS6 plus mutant heat-labile enterotoxin mucosal adjuvant (CS6+mLT). Native CS6 and the CS6-PLG microspheres administered intranasally to mice induced serum IgG responses, with the CS6-PLG microspheres inducing a significantly greater (P<0.001) response than native CS6. Following intranasal administration of native CS6, no fecal IgG and IgA responses were measured; however, the CS6-PLG microspheres induced significantly greater (P<0.001) fecal IgG and IgA responses than native CS6. The coadministration of the mLT mucosal adjuvant with CS6 induced significantly greater serum (P<0.001) and fecal (P<0.01) responses than the CS6-PLG microspheres. However, following intranasal administration of the mLT adjuvant, the mice showed definite signs of distress, indicating an adverse reaction to the mLT. Thus, this brings into question the safety of the mLT and its use as an intranasal adjuvant. In contrast, the PLG-microspheres administered intranasally caused no noticeable distress to the mice. The results obtained in this study indicate that the encapsulation of CS6 in PLG-microspheres administered intranasally to mice acted in an adjuvant capacity to enhance the CS6 immune response.

Microencapsulated subunit vaccine approach to enterotoxigenic Escherichia coli and other mucosal pathogens.

Infections of the intestinal, urogenital, and respiratory tracts are serious health problems worldwide from both a morbidity and mortality perspective. Mucosal pathogens attach to surfaces of mucosa as a prerequisite for colonization and subsequent pathogenesis. By expressing various surface adhesins (colonization factors, CF) they are able to bind to specific mucosal receptors. Enterotoxigenic Escherichia coli (ETEC) can express numerous CF that allow them to attach to a variety of hosts. Mucosal immunity directed against pathogenic microorganisms is critical in host protection with secretory IgA being particularly important in preventing microoganisms from colonizing host cells. M cells likewise have an important immunological function in the small intestines by binding and transporting antigens to lymphocytes and macrophages thus enhancing the immune response. The use of subunit vaccines, such as antigen encapsulated microspheres, can act to effectively deliver specific antigens so as to optimize their immunological response. With the threat of bioterrorism becoming a reality in recent years, the miroencapsulation of antigens from potential bioterrorist agents may be an effective method of delivery so as to induce a level of protection in at risk individuals. The encapsulation of ETEC colonization factors in microspheres and their subsequent administration in small animals and humans has been conducted for many years. Evidence suggests that this type of delivery system for ETEC antigens may enhance their immunogenicity and provide protection against this microorganism.

Murine antibody response to intranasally administered enterotoxigenic Escherichia coli colonization factor CS6.

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of bacterial diarrhea worldwide and is an important cause of infant morbidity and mortality in developing nations. ETEC colonization factors (CF) are virulence determinants that appear to be protective antigens in humans and are the major target of vaccine efforts. One of the most prevalent CF, CS6, is expressed by about 30% of ETEC worldwide. This study was designed to compare the immunogenicity between encapsulated CS6 (CS6-PLG) and unencapsulated CS6. Recombinant CS6 was purified and encapsulated in biodegradable poly(DL-lactide-co-glycolide) (PLG) microspheres using current Good Manufacturing Practices (cGMP). CS6-PLG and CS6 were administered intranasally (IN) to BALB/c mice in three vaccinations 4 weeks apart. Enzyme linked immunosorbent assay (ELISA) was used to measure the anti-CS6 response in serum and mucosal secretions following each of the three inoculations. Mice vaccinated with two or three doses of CS6-PLG demonstrated a significantly greater rise in serum anti-CS6 IgG and mucosal IgA titer values than those immunized with two or three doses of CS6 alone. Three doses of CS6-PLG led to anti-CS6 serum IgG and mucosal IgA titer values 14-fold and 4.4-fold greater, respectively, than three doses of CS6 (P<0.02). IN administered CS6 to mice is safe and highly immunogenic either alone or when encapsulated in microspheres. PLG microsphere encapsulation of CS6 significantly augments the antibody response to that antigen when administered to a mucosal surface.

Mucosal immunization of BALB/c mice using enterotoxigenic Escherichia coli colonization factors CFA/I and CS6 administered with and without a mutant heat-labile enterotoxin.

Mice (BALB/c) were intranasally (IN) and intragastrically (IG) administered the ETEC colonization factors (CF), CFA/I and CS6, with and without the R192G mutant heat-labile enterotoxin (mLT), and immunogenicity and efficacy measured. The IN administration of CFA/I to mice induced strong serum and fecal IgG and IgA responses. The IG administration of CFA/I to mice induced serum IgG and fecal IgA responses, but only when mLT was co-administered with CFA/I were serum IgA titers detected. The IN administration of CS6 to mice induced serum IgG antibodies, and mLT, when co-administered with CS6, enhanced the serum IgG response. Only when the mLT was co-administered with CS6, were serum and fecal IgA responses detected. The IG administration of CS6 plus mLT induced serum IgG and fecal IgA responses. Partial protection against lethal challenge with ETEC strain H10407 was seen in the mice IN administered the CFA/I plus mLT (P<0.01), and H10407 was cleared from the lungs of CFA/I plus mLT-immunized mice at a significantly greater rate than from the control mice (P<0.05). CFA/I and CS6 administered IN and IG induced mixed Th1/Th2 immune responses with the Th2 type being predominant as evidenced by IgG1>IgG2a. The administration of colonization factors to mice, particularly by the IN route, potentially serves as a useful way to measure the serum and mucosal immune responses to these antigens prior to their use in volunteers.

Pathogenicity and immune response measured in mice following intranasal challenge with enterotoxigenic Escherichia coli strains H10407 and B7A.

The pathogenicity and immunogenicity induced in BALB/c mice by intranasal (i.n.) inoculation of enterotoxigenic Escherichia coli (ETEC) strains H10407 (O78:H11:CFA/I:LT(+):ST(+)) and B7A (O148:H28:CS6:LT(+):ST(+)) (two ETEC strains previously used in human challenge trials) were studied. The i.n. inoculation of BALB/c mice with large doses of ETEC strains H10407 and B7A caused illness and death. The H10407 strain was found to be consistently more virulent than the B7A strain. Following i.n. challenge with nonlethal doses of H10407 and B7A, the bacteria were cleared from the lungs of the mice at a steady rate over a 2-week period. Macrophages and neutrophils were observed in the alveoli and bronchioles, and lymphocytes were observed in the septa, around vessels, and in the pleura of the lungs in mice challenged with H10407 and B7A. In mice i.n. challenged with H10407, serum immunoglobulin G (IgG) and IgM antibodies were measured at high titers to the CFA/I and O78 lipopolysaccharide (LPS) antigens. In mice i.n. challenged with B7A, low serum IgG antibody titers were detected against CS6, and low serum IgG and IgM antibody titers were detected against O148 LPS. The serum IgG and IgM antibody titers against the heat-labile enterotoxin were equivalent in the H10407- and B7A-challenged mice. The CFA/I and O78 LPS antigens gave mixed T-helper cell 1-T-helper cell 2 (Th1-Th2) responses in which the Th2 response was greater than the Th1 response (i.e., stimulated primarily an antibody response). These studies indicate that the i.n. challenge of BALB/c mice with ETEC strains may provide a useful animal model to better understand the immunogenicity and pathogenicity of ETEC and its virulence determinants. This model may also be useful in providing selection criteria for vaccine candidates for use in primate and human trials.