A Systems Biology Approach Identifies B Cell Maturation Antigen (BCMA) as a Biomarker Reflecting Oral Vaccine Induced IgA Antibody Responses in Humans.

Vaccines against enteric diseases could improve global health. Despite this, only a few oral vaccines are currently available for human use. One way to facilitate such vaccine development could be to identify a practical and relatively low cost biomarker assay to assess oral vaccine induced primary and memory IgA immune responses in humans. Such an IgA biomarker assay could complement antigen-specific immune response measurements, enabling more oral vaccine candidates to be tested, whilst also reducing the work and costs associated with early oral vaccine development. With this in mind, we take a holistic systems biology approach to compare the transcriptional signatures of peripheral blood mononuclear cells isolated from volunteers, who following two oral priming doses with the oral cholera vaccine Dukoral®, had either strong or no vaccine specific IgA responses. Using this bioinformatical method, we identify TNFRSF17, a gene encoding the B cell maturation antigen (BCMA), as a candidate biomarker of oral vaccine induced IgA immune responses. We then assess the ability of BCMA to reflect oral vaccine induced primary and memory IgA responses using an ELISA BCMA assay on a larger number of samples collected in clinical trials with Dukoral® and the oral enterotoxigenic Escherichia coli vaccine candidate ETVAX. We find significant correlations between levels of BCMA and vaccine antigen-specific IgA in antibodies in lymphocyte secretion (ALS) specimens, as well as with proportions of circulating plasmablasts detected by flow cytometry. Importantly, our results suggest that levels of BCMA detected early after primary mucosal vaccination may be a biomarker for induction of long-lived vaccine specific memory B cell responses, which are otherwise difficult to measure in clinical vaccine trials. In addition, we find that ALS-BCMA responses in individuals vaccinated with ETVAX plus the adjuvant double mutant heat-labile toxin (dmLT) are significantly higher than in subjects given ETVAX only. We therefore propose that as ALS-BCMA responses may reflect the total vaccine induced IgA responses to oral vaccination, this BCMA ELISA assay could also be used to estimate the total adjuvant effect on vaccine induced-antibody responses, independently of antigen specificity, further supporting the usefulness of the assay.

Immunogenicity noninferiority study of 2 doses and 3 doses of an Escherichia coli-produced HPV bivalent vaccine in girls vs. 3 doses in young women.

A new HPV-16/18 bivalent vaccine expressed by the Escherichia coli has been proven to be efficacious in adult women. A randomized, immunogenicity noninferiority study of this candidate vaccine was conducted in December 2015 in China. Girls aged 9-14 years were randomized to receive 2 doses at months 0 and 6 (n=301) or 3 doses at months 0, 1 and 6 (n=304). Girls aged 15-17 years (n=149) and women aged 18-26 years (n=225) received 3 doses. The objectives included noninferiority analysis of the IgG geometric mean concentration (GMC) ratio (95% CI, lower bound>0.5) to HPV-16 and HPV-18 at month 7 in girls compared with women. In the per-protocol set, the GMC ratio of IgG was noninferior for girls aged 9-17 years receiving 3 doses compared with women (1.76 (95% CI, 1.56, 1.99) for HPV-16 and 1.93 (95% CI, 1.69, 2.21) for HPV-18) and noninferior for girls aged 9-14 years receiving 2 doses compared with women (1.45 (95% CI, 1.25, 1.62) for HPV-16 and 1.17 (95% CI, 1.02, 1.33) for HPV-18). Noninferiority was also demonstrated for neutralizing antibodies. The immunogenicity of the HPV vaccine in girls receiving 3 or 2 doses was noninferior compared with that in young adult women.

The effect of J5 bacterins on clinical, behavioral, and antibody response following an Escherichia coli intramammary challenge in dairy cows at peak lactation.

Vaccination against coliform mastitis has become part of mastitis control programs in the past 3 decades, as a means of reducing the severity of clinical mastitis. Our study objective was to evaluate the effect of 2 commercially available vaccines on clinical, behavioral, and antibody response following Escherichia coli intramammary challenge in cows near peak lactation. Cows (n = 12 per group) were vaccinated with vaccine 1 (V1) or vaccine 2 (V2) at dry-off, 21 d pre-calving, and 14 d post-calving. Twelve cows served as unvaccinated controls (CTL). Cows were challenged with E. coli in a rear quarter at approximately 100 d in milk. Milk samples were collected pre- and post-challenge to enumerate E. coli and determine somatic cell count. Serum was collected before each vaccination and at d 0, 1, 2, 3, 6, 30, and 60 relative to challenge, to study antibody response. Milk IgA and tumor necrosis factor-α concentrations were determined in whey. Vaginal temperature, cow activity, and milk yield and components were monitored post-challenge. Bacterial count, somatic cell score, milk yield and component decline, vaginal temperature, activity measures, and antibody and cytokine response were analyzed for treatment differences. The effects of parity, breed, and a repeated measure of time were also tested. Seven cows had to be removed from the study post-challenge for antibiotic treatment (CTL and V1, n = 3 each; V2, n = 1), 2 of which were euthanized (both CTL). Vaccinated cows exhibited fever (vaginal temperature ≥39.4°C) 3 h earlier than CTL cows, but we found no differences between treatments for bacterial count, somatic cell score, or milk yield reduction. Vaccinated cows spent more time lying per rest bout 2 d post-challenge, but total daily lying time was not different from CTL cows during the 7 d post-challenge. The vaccines differed in antibody response: V1 cows had greater serum IgG1 and IgG2 post-challenge. A parity effect was also evident: primiparous cows had lower bacterial counts, somatic cell score and a smaller milk yield decline than multiparous cows, but also had lower antibody production. Immunization with either J5 bacterin did not reduce clinical signs of mastitis in cows challenged at 100 d in milk, demonstrating that the effects of J5 vaccination had diminished at peak lactation.

Enterotoxigenic E. coli virulence gene regulation in human infections.

Enterotoxigenic Escherichia coli (ETEC) is a global diarrheal pathogen that utilizes adhesins and secreted enterotoxins to cause disease in mammalian hosts. Decades of research on virulence factor regulation in ETEC has revealed a variety of environmental factors that influence gene expression, including bile, pH, bicarbonate, osmolarity, and glucose. However, other hallmarks of the intestinal tract, such as low oxygen availability, have not been examined. Further, determining how ETEC integrates these signals in the complex host environment is challenging. To address this, we characterized ETEC's response to the human host using samples from a controlled human infection model. We found ETEC senses environmental oxygen to globally influence virulence factor expression via the oxygen-sensitive transcriptional regulator fumarate and nitrate reduction (FNR) regulator. In vitro anaerobic growth replicates the in vivo virulence factor expression profile, and deletion of fnr in ETEC strain H10407 results in a significant increase in expression of all classical virulence factors, including the colonization factor antigen I (CFA/I) adhesin operon and both heat-stable and heat-labile enterotoxins. These data depict a model of ETEC infection where FNR activity can globally influence virulence gene expression, and therefore proximity to the oxygenated zone bordering intestinal epithelial cells likely influences ETEC virulence gene expression in vivo. Outside of the host, ETEC biofilms are associated with seasonal ETEC epidemics, and we find FNR is a regulator of biofilm production. Together these data suggest FNR-dependent oxygen sensing in ETEC has implications for human infection inside and outside of the host.

A Plant-Produced Candidate Subunit Vaccine Reduces Shedding of Enterohemorrhagic Escherichia coli in Ruminants.

Enterohemorrhagic Escherichia coli (EHEC) are commonly present in the gastrointestinal tract of cattle and cause serious infectious disease in humans. Immunizing cattle against EHEC is a promising strategy to decrease the risk of food contamination; however, veterinary vaccines against EHEC such as Econiche have not been widely adopted by the agricultural industry, and have been discontinued, prompting the need for more cost-effective EHEC vaccines. The objective of this project is to develop a platform to produce plant-made antigens for oral vaccination of ruminants against EHEC. Five recombinant proteins were designed as vaccine candidates and expressed transiently in Nicotiana benthamiana and transplastomically in Nicotiana tabacum. Three of these EHEC proteins, NleA, Stx2b, and a fusion of EspA accumulated when transiently expressed. Transient protein accumulation was the highest when EHEC proteins were fused to an elastin-like polypeptide (ELP) tag. In the transplastomic lines, EspA accumulated up to 479 mg kg-1 in lyophilized leaf material. Sheep that were administered leaf tissue containing recombinant EspA shed less E. coli O157:H7 when challenged, as compared to control animals. These results suggest that plant-made, transgenic EspA has the potential to reduce EHEC shedding in ruminants.

Evaluation of prevalence, immunogenicity and efficacy of FyuA iron receptor in uropathogenic Escherichia coli isolates as a vaccine target against urinary tract infection.

Uropathogenic Escherichia coli (UPEC) are among the most prevalent agents of urinary tract infections (UTIs). Antibiotic resistance reaches the need for alternative treatment approaches such as vaccination against UTIs. There is no ideal vaccine against UTIs, thus there is a need to evaluate different targets of uropathogens against UTIs. Ferric scavenger receptor FyuA in UPEC has the properties of an ideal vaccine candidate against UTIs. In the present study, the prevalence of FyuA among UPEC isolates, its immunogenicity with and without alum adjuvant, and its efficacy against experimental UTI were assessed. Totally, fyuA gene was present in 77% of the UPEC isolates tested. Alignments of FyuA exhibited a high degree of conservation among different submitted UPEC isolates in GenBank. The bioinformatics studies showed the high confidence value and stability of the FyuA structure. SDS-PAGE and Western blot confirmed the purification of FyuA with high yield by nickel resins. Mice vaccinated subcutaneously with the FyuA induced a significantly higher humoral response (total IgG, IgG1 and IgG2a) than control mice that alum enhanced these responses. The FuyA alone showed the ability to reduce the colonization of UPEC in bladder and kidney of mice as compared to the control group. But the addition of alum to FyuA increased the protection level against UPEC in these organs. Since, FyuA induced significant IgG1 (Th2) and IgG2a (Th1) responses and protected the mice against experimental UTI, it could be a promising target against UPEC infections.

Inflammation-Induced Adhesin-Receptor Interaction Provides a Fitness Advantage to Uropathogenic E. coli during Chronic Infection.

Uropathogenic E. coli (UPEC) is the dominant cause of urinary tract infections, clinically described as cystitis. UPEC express CUP pili, which are extracellular fibers tipped with adhesins that bind mucosal surfaces of the urinary tract. Here we identify the role of the F9/Yde/Fml pilus for UPEC persistence in the inflamed urothelium. The Fml adhesin FmlH binds galactose ß1-3 N-acetylgalactosamine found in core-1 and -2 O-glycans. Deletion of fmlH had no effect on UPEC virulence in an acute mouse model of cystitis. However, FmlH provided a fitness advantage during chronic cystitis, which is manifested as persistent bacteriuria, high bladder bacterial burdens, and chronic inflammation. In situ binding confirmed that FmlH bound avidly to the inflamed, but not the naive bladder. In accordance with its pathogenic profile, vaccination with FmlH significantly protected mice from chronic cystitis. Thus, UPEC employ separate CUP pili to adapt to the rapidly changing niche during bladder infection.

Multiepitope fusion antigen induces broadly protective antibodies that prevent adherence of Escherichia coli strains expressing colonization factor antigen I (CFA/I), CFA/II, and CFA/IV.

Diarrhea is the second leading cause of death in children younger than 5 years and continues to be a major threat to global health. Enterotoxigenic Escherichia coli (ETEC) strains are the most common bacteria causing diarrhea in developing countries. ETEC strains are able to attach to host small intestinal epithelial cells by using bacterial colonization factor antigen (CFA) adhesins. This attachment helps to initiate the diarrheal disease. Vaccines that induce antiadhesin immunity to block adherence of ETEC strains that express immunologically heterogeneous CFA adhesins are expected to protect against ETEC diarrhea. In this study, we created a CFA multiepitope fusion antigen (MEFA) carrying representative epitopes of CFA/I, CFA/II (CS1, CS2, and CS3), and CFA/IV (CS4, CS5, and CS6), examined its immunogenicity in mice, and assessed the potential of this MEFA as an antiadhesin vaccine against ETEC. Mice intraperitoneally immunized with this CFA MEFA exhibited no adverse effects and developed immune responses to CFA/I, CFA/II, and CFA/IV adhesins. Moreover, after incubation with serum of the immunized mice, ETEC or E. coli strains expressing CFA/I, CFA/II, or CFA/IV adhesins were significantly inhibited in adherence to Caco-2 cells. Our results indicated this CFA MEFA elicited antibodies that not only cross-reacted to CFA/I, CFA/II and CFA/IV adhesins but also broadly inhibited adherence of E. coli strains expressing these seven adhesins and suggested that this CFA MEFA could be a candidate to induce broad-spectrum antiadhesin protection against ETEC diarrhea. Additionally, this antigen construction approach (creating an MEFA) may be generally used in vaccine development against heterogenic pathogens.

Evaluation of the adjuvant effect of Salmonella-based Escherichia coli heat-labile toxin B subunits on the efficacy of a live Salmonella-delivered avian pathogenic Escherichia coli vaccine.

The present study evaluated the adjuvant effect of live attenuated salmonella organisms expressing the heat-labile toxin of Escherichia coli B subunit (LTB) on the efficacy of an avian pathogenic Escherichia coli (APEC) vaccine. The Asd(+) (aspartate semialdehyde dehydrogenase) plasmid pMMP906 containing the LTB gene was introduced into a Salmonella enterica Typhimurium strain lacking the lon, cpxR and asd genes to generate the adjuvant strain. Live recombinant Salmonella-delivered APEC vaccine candidates were used for this study. The birds were divided into three groups: group A, non-vaccinated controls; group B, immunized with vaccine candidates only; and group C, immunized with vaccine candidates and the LTB strain. The immune responses were measured and the birds were challenged at 21 days of age with a virulent APEC strain. Group C showed a significant increase in plasma IgG and intestinal IgA levels and a significantly higher lymphocyte proliferation response compared with the other groups. Upon challenge with the virulent APEC strain, group C showed effective protection whereas group B did not. We also attempted to optimize the effective dose of the adjuvant. The birds were immunized with the vaccine candidates together with 1×107 or 1×108 colony-forming units of the LTB strain and were subsequently challenged at 3 weeks of age. The 1×107 colony-forming units of the LTB strain showed a greater adjuvant effect with increased levels of serum IgG, intestinal IgA and a potent lymphocyte proliferation response, and yielded higher protection against challenge. Overall, the LTB strain increased the efficacy of the Salmonella -delivered APEC vaccine, indicating that vaccination for APEC along with the LTB strain appears to increase the efficacy for protection against colibacillosis in broiler chickens.

Regional immune response to immunization with Escherichia coli O157:H7-derived intimin in cattle.

Escherichia coli O157:H7 is an enteric pathogen of animals and humans that can result in deadly sequelae. Cattle are asymptomatic carriers and shedders of the bacteria and serve as an important reservoir of human infection. E. coli O157:H7 colonizes the gastrointestinal tract, most frequently at the rectoanal junction mucosa in cattle. Vaccination is a potentially highly effective means of decreasing cattle colonization and shedding and thereby decreasing human infections. Currently available vaccines are administered subcutaneously or intramuscularly, and immune responses have been evaluated solely by systemic immunoglobulin responses. This study evaluated local and systemic lymphoproliferative responses in addition to immunoglobulin responses following subcutaneous or mucosal (rectal) immunization with E. coli O157:H7 outer membrane protein intimin over three trials. In all three trials, significant local and systemic lymphoproliferative responses (P < 0.05) occurred following immunization in the majority of animals, as well as significant immunoglobulin responses (P < 0.001) in all animals. Surprisingly, local responses in the mesorectal lymph nodes were very similar between the subcutaneous and mucosal immunization groups. Moreover, the responses in mesorectal lymph nodes appeared targeted rather than generalized, as minimal or no significant responses were observed in the associated prescapular lymph nodes of subcutaneously immunized animals. The results indicate that both subcutaneous and mucosal immunizations are effective methods of inducing immune responses against E. coli O157:H7 in cattle.

Early Treg suppression by a listeriolysin-O-expressing E. coli vaccine in heterologous prime-boost vaccination against cancer.

Studies have shown that an enhanced CD8+ T cell response and better tumor protection can be achieved by heterologous prime-boost vaccination in mice. Such heterologous vaccination can be more immunogenic than the homologous setting. We previously demonstrated that a listeriolysin-O (LLO)-expressing E. coli vaccine can enhance CD8-cytotoxic T cell (CTL) responses by reducing regulatory T cell (Treg)-directed suppression. In the present study, we assessed the combination of this approach with plasmid DNA vaccination, in a prime-boost immunization strategy. E. coli-LLO bacteria expressing ovalbumin (OVA) and plasmid pcDNA-encoding OVA were used to vaccinate naive or B16-OVA tumor-bearing C57B6 mice. The anticancer activity was measured in a tumor prevention or therapeutic model. Higher OVA-specific CD8+ T cell responses and greater tumor inhibition were seen in the bacterial-prime/plasmid-boost setting than with the homologous and reversed sequences. This tumor protection effect from heterologous prime-boost remained in the therapeutic model. When examining the Treg effect during the prime-boost immunization, we found that only early Treg-suppression/depletion could lead to better antigen-specific CTL and tumor response. Our studies offer the first evidence that a listeriolysin-O E. coli vaccine can induce an enhanced antitumor effect in conjunction with DNA in a heterologous prime-boost protocol, and suggest that early Treg inhibition is crucial to a successful immunization against cancer.

Safety of live, attenuated oral vaccines in HIV-infected Zambian adults: oral vaccines in HIV.

BACKGROUND: Current recommendations are that HIV-infected persons should not be given live vaccines. We set out to assess potential toxicity of three live, attenuated oral vaccines (against rotavirus, typhoid and ETEC) in a phase 1 study. METHODS: Two commercially available oral vaccines against rotavirus (Rotarix) and typhoid (Vivotif) and one candidate vaccine against Enterotoxigenic Escherichia coli (ACAM2017) were given to HIV seropositive (n=42) and HIV seronegative (n=59) adults. Gastrointestinal symptoms were sought actively by weekly interview up to 1 month of vaccination. In rotavirus vaccine recipients, intestinal biopsies were collected by endoscopy and evaluated for expression of IL-8 and pro-inflammatory cytokines. RESULTS: No difference was observed between symptoms in HIV infected and HIV uninfected vaccinees, except for diarrhoea reported more than 7 days after the last dose of vaccine. If only diarrhoea episodes within 7 days of vaccination are included, diarrhoea was not more frequent in HIV seropositive than in HIV seronegative vaccinees (OR 6.7, 95% CI 1.2-67; P=0.09). However, if later episodes of diarrhoea are included, a significant increase in diarrhoea was demonstrated (OR 5.3, 95% CI 0.98-53; P=0.04). All episodes were mild and transient. IL-8 was slowly up-regulated over the week following vaccination (P=0.02), but IL-ß, IFNγ or TNFα were not. CONCLUSIONS: No evidence was found of adverse events following administration of these three vaccines, except for late episodes of diarrhoea which may not be attributable to vaccination. Our data do not support the need for a prohibition on oral administration of live, attenuated vaccines to all HIV infected adults, though further work on severely immunocompromised adults and children are required.

Modified heat-stable toxins (hSTa) of enterotoxigenic Escherichia coli lose toxicity but display antigenicity after being genetically fused to heat-labile toxoid LT(R192G).

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrhea in humans and animals. Heat-stable (STa) and heat-labile (LT) enterotoxins produced by ETEC disrupt fluid homeostasis in host small intestinal epithelial cells and cause fluid and electrolyte hyper-secretion that leads to diarrhea. ETEC strains producing STa or LT are sufficiently virulent to cause diarrhea, therefore STa and LT antigens must be included in ETEC vaccines. However, potent toxicity and poor immunogenicity (of STa) prevent them from being directly applied as vaccine components. While LT toxoids, especially LT(R192G), being used in vaccine development, STa toxoids have not been included. A recent study (IAI, 78:316-325) demonstrated porcine-type STa toxoids [pSTa(P12F) and pSTa(A13Q)] elicited protective anti-STa antibodies after being fused to a porcine-type LT toxoid [pLT(R192G)]. In this study, we substituted the 8th, 9th, 16th, or the 17th amino acid of a human-type STa (hSTa) and generated 28 modified STa peptides. We tested each STa peptide for toxicity and structure integrity, and found nearly all modified STa proteins showed structure alteration and toxicity reduction. Based on structure similarity and toxic activity, three modified STa peptides: STa(E8A), STa(T16Q) and STa(G17S), were selected to construct LT(192)-STa(-toxoid) fusions. Constructed fusions were used to immunize mice, and immunized mice developed anti-STa antibodies. Results from this study provide useful information in developing toxoid vaccines against ETEC diarrhea.

Subcutaneous and intranasal immunization with Stx2B-Tir-Stx1B-Zot reduces colonization and shedding of Escherichia coli O157:H7 in mice.

The type III secretion system of Escherichia coli O157:H7 is involved in colonization of mammalian hosts by the organism. The translocated intimin receptor (Tir) is inserted into the mammalian host cell plasma membrane in a hairpin loop topology with the central loop of the molecule exposed to the host cell surface and accessible for interaction with an LEE-encoded bacterial outer membrane adhesin called intimin. Shiga toxin type 1 and 2 produced by E. coli O157:H7 are responsible for hemolytic uremic syndrome and able to promote intestinal colonization. Zonula occludens toxin (Zot) is a single polypeptide chain encoded by the filamentous bacteriophage CTXφ of Vibrio cholerae. Zot binds a receptor on intestinal epithelial cells and increases mucosal permeability by affecting the structure of epithelial tight junctions. Because of these properties, Zot is a promising tool for mucosal drug and antigen (Ag) delivery. In the current study, we constructed a novel fusion protein carrying both of the immunogenic B subunits derived from the two toxins, Tir and Zot, designated Stx2B-Tir-Stx1B-Zot, expressed in the E. coli BL21 and harvested the purified protein by a simple GST·Bind Resin chromatography method. We used a streptomycin-treated mouse model to evaluate the efficacy of subcutaneous vs. intranasal administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding. Immune responses against Stx2B-Tir-Stx1B-Zot, Stx2B-Tir-Stx1B and control agent (GST/PBS) were also monitored. Subcutaneous immunization of mice with Stx2B-Tir-Stx1B-Zot induced significant Stx2B-Tir-Stx1B-Zot-specific serum IgG antibodies but did not significantly induce any antigen-specific IgA in feces, whereas intranasal immunization elicited significant Stx2B-Tir-Stx1B-Zot-specific serum IgG antibodies with some animals developing antigen-specific IgA in feces. Mice that were immunized intranasally with Stx2B-Tir-Stx1B-Zot showed dramatically decreased E. coli O157:H7 shedding compared to those of Stx2B-Tir-Stx1B and control agent following experimental infection. Mice immunized subcutaneously with Stx2B-Tir-Stx1B-Zot or Stx2B-Tir-Stx1B both showed reduced shedding in feces, moreover, Stx2B-Tir-Stx1B-Zot did better. These results demonstrate the perspective for the use of Stx2B-Tir-Stx1B-Zot to prevent colonization and shedding of E. coli O157:H7.

Protection of mice against enterotoxigenic E. coli by immunization with a polyvalent enterotoxin comprising a combination of LTB, STa, and STb.

Currently available enterotoxigenic Escherichia coli (ETEC) vaccines are based on colonization factors and/or the heat-labile enterotoxin B subunit (LTB). However, the induction of antitoxic responses against heat-stable enterotoxin a (STa) and b (STb) has merit as these two poorly immunogenic toxins are frequently associated with ETEC strains. In this study, we genetically constructed a trivalent enterotoxin fusion protein (STa-LTB-STb, abbreviated to SLS) in an effort to develop a single toxoid containing these three enterotoxins for vaccination against ETEC. Mutagenesis at one disulfide-bridge-forming cysteine in STa led to a dramatic reduction in the STa toxicity of SLS; however, the fusion peptide retained the STb-associated toxicity. Immunization of mice with SLS protein elicited significant antibody responses to LTB, STa, and STb. Significantly, the mice antisera were able to neutralize the biological activity of both STa and STb. In the experiment to assess the protective effect of SLS immunization, the mortality of mice receiving SLS was significantly lower than their control cohorts (P < 0.01) after intraperitoneal challenge with ETEC. These results show that the trivalent fusion enterotoxin SLS has the potential to serve as a useful toxin-based vaccine against ETEC-induced diarrheal disease via a single immunogen.

Histomorphometric evaluation of intestinal cellular immune responses in pigs immunized with live oral F4ac+ non-enterotoxigenic E. coli vaccine against postweaning colibacillosis.

Enterotoxigenic Escherichia coli (ETEC) infection is the most common type of porcine postweaning colibacillosis (PWC). Among fimbriae of porcine ETEC strains the best studied family of fimbriae are the members of F4 adhesins, existing in at least three variants: ab, ac, ad. Active immunization against porcine PWC is difficult due to: i) ETEC strains are only one of the essential predisposing factors, ii) the success of vaccinal antigen uptake depends on the presence of enterocyte receptors for F4 adhesins, iii) the intestinal immune system may react with tolerance or hypersensitivity to the same antigens depending on the dose and form of the vaccinal immunogen, and iv) kinetics of the specific immune responses may be different in the case of F4 (earlier) and the other ETEC adhesins, particularly F18 (later). The aim of this study was to test the effectiveness of a live attenuated F4ac+ non-ETEC vaccine against porcine PWC by analyzing quantitative differences in the small intestinal lymphoid and myeloid cell subsets of immunized (with or without levamisole given as an adjuvant) vs control non-immunized pigs. Four week-old pigs were intragastrically immunized with a vaccine candidate F4ac+ non-ETEC strain 2407 at day 0, challenged 7 days later with a virulent F4ac+ strain ETEC 11-800/1/94, euthanatized at day 13 and sampled for immunohistology. Non-immunized pigs received saline at day 0 and were processed as the principals. Immuno-phenotypes of lymphoid and myeloid cell subsets were demonstrated within jejunal and ileal mucosa by immunohistochemical avidin-biotin complex method and corresponding morphometric data were analyzed using software program Lucia G for digital image analyses. Monoclonal antibodies reactive with surface molecules on porcine immune cells such as CD3, CD45RA, CD45RC, CD21 and SWC3 enabled clear insight into distribution patterns and amount of these cells within the gut-associated lymphoid tissues (GALT) examined. The numbers of jejunal and ileal cell subsets tested were significantly increased (at P<0.5 or lower) in both principal groups (vaccinated or levamisole primed-vaccinated) of pigs, compared to those recorded in the control non-vaccinated pigs. Based on the histomorphometric quantification of porcine intestinal immune cells from the GALT compartments tested, it is possible to differentiate the responses of pigs immunized by an experimental mucosal vaccine from those of non-immunized pigs.

Mucosal immunization with iron receptor antigens protects against urinary tract infection.

Uncomplicated infections of the urinary tract, caused by uropathogenic Escherichia coli, are among the most common diseases requiring medical intervention. A preventive vaccine to reduce the morbidity and fiscal burden these infections have upon the healthcare system would be beneficial. Here, we describe the results of a large-scale selection process that incorporates bioinformatic, genomic, transcriptomic, and proteomic screens to identify six vaccine candidates from the 5379 predicted proteins encoded by uropathogenic E. coli strain CFT073. The vaccine candidates, ChuA, Hma, Iha, IreA, IroN, and IutA, all belong to a functional class of molecules that is involved in iron acquisition, a process critical for pathogenesis in all microbes. Intranasal immunization of CBA/J mice with these outer membrane iron receptors elicited a systemic and mucosal immune response that included the production of antigen-specific IgM, IgG, and IgA antibodies. The cellular response to vaccination was characterized by the induction and secretion of IFN-gamma and IL-17. Of the six potential vaccine candidates, IreA, Hma, and IutA provided significant protection from experimental infection. In immunized animals, class-switching from IgM to IgG and production of antigen-specific IgA in the urine represent immunological correlates of protection from E. coli bladder colonization. These findings are an important first step toward the development of a subunit vaccine to prevent urinary tract infections and demonstrate how targeting an entire class of molecules that are collectively required for pathogenesis may represent a fundamental strategy to combat infections.

Escherichia coli O157:H7 vaccine dose-effect in feedlot cattle.

A clinical trial conducted during the summer of 2006 evaluated effects of two- and three-dose regimens of an Escherichia coli O157 vaccine product on the probability of detecting E. coli O157:H7 in feces and colonization of the terminal rectum. The three-dose regimen significantly reduced the probability for cattle to shed E. coli O157:H7 in feces 65% compared to placebo-treated cattle. A dose-effect was demonstrated because a two-dose regimen of the vaccine product was intermediate in effect. These results are consistent with previous estimates of vaccine efficacy against fecal shedding, and agree with our previous finding that efficacy of two or three doses of this vaccine product exhibit a dose-response.

A randomized longitudinal trial to test the effect of regional vaccination within a cattle feedyard on Escherichia coli O157:H7 rectal colonization, fecal shedding, and hide contamination.

We tested the efficacy of vaccinating all cattle within a region of a cattle feedlot using a two-dose regimen of a vaccine against type III secreted proteins of Escherichia coli O157:H7. Cattle (n = 504) were randomly assigned to 63 pens (8 steers/pen) within 3 treatment regions of the feedyard. All pens within each region were assigned: (1) two doses of vaccine (ALLVAC), (2) two doses of adjuvant as placebo (NOVAC), or (3) commingled vaccination (HALFVAC), four of eight cattle in each pen receiving two doses of vaccine, and the others adjuvant. Binary outcomes were (1) fecal shedding of E. coli O157:H7 42, 63, and 84 days postvaccination (dpv), (2) hide contamination 42, 63, and 84 dpv, and at the abattoir 85 dpv, and (3) colonization of the terminal rectal mucosa at the abattoir 85 dpv. For each outcome, multilevel logistic regression tested the effect of regional vaccination (ALLVAC vs. NOVAC), and compared commingled vaccinated versus placebo-treated cattle within HALFVAC pens. For fecal shedding, regional vaccine efficacy of ALLVAC compared to NOVAC pens was 63% (OR = 0.34, p = 0.0009), similar to vaccine efficacy of 52% for vaccinated cattle compared to placebo-treated cattle within HALFVAC pens (OR = 0.48, p = 0.014). For hide contamination, vaccine efficacy was 55% for regional vaccination of cattle in ALLVAC pens compared to NOVAC pens (OR = 0.43, p = 0.014). However, commingling vaccinated and placebo-treated cattle was not protective of hide contamination (OR = 0.67, p = 0.33). Colonization of cattle at the abattoir was not different among vaccinated and placebo-treated cattle (p = 0.63). We concluded that the two-dose vaccine regimen effectively reduced E. coli O157:H7 fecal shedding and hide contamination, and that vaccination of cattle within regions of the feedyard provided greater protection against hide contamination than commingling vaccinates and nonvaccinates.

Split immune response after oral vaccination of mice with recombinant Escherichia coli Nissle 1917 expressing fimbrial adhesin K88.

Enterotoxigenic Escherichia coli (ETEC) are a leading cause of diarrhoea in piglets and newborn calves. Massive efforts have therefore been made to develop a vaccine for the induction of protective mucosal immunity against ETEC. Since it has been shown that the probiotic strain E. coli Nissle 1917 (EcN) can serve as a safe carrier for targeted delivery of recombinant molecules to the intestinal mucosa, we constructed the recombinant strain EcN pMut2-kanK88 (EcN-K88) stably expressing the determinant for the K88 fimbrial adhesin of ETEC on the bacterial surface. After oral application of EcN-K88 to mice for one week, EcN-K88 as well as wild-type EcN and EcN mock-transformed with the plasmid vector only could be detected in faecal samples for a minimum of 7 days after the last feeding, indicating that EcN can transiently colonise the murine intestine. Oral application of EcN-K88 resulted in significant IgG serum titres against K88 as early as 7 days after the initial feeding with EcN-K88, but no significant IgA titres. In contrast, we failed to detect any specific T cell responses towards the K88 antigen both in spleen and mesenteric lymph nodes. Although dendritic cells readily upregulated maturation and activation markers in response to K88 stimulation, accompanied by secretion of interleukin (IL)-12, IL-6, IL-10, and tumour necrosis factor, restimulation of T cells from mice having received EcN-K88 with K88-loaded dendritic cells did not result in detectable T cell proliferation and IL-2 secretion, but rather induced an IL-10 bias. While the serum antibody responses clearly demonstrate that K88 is recognized by the humoral immune system, our findings indicate that oral application of probiotic EcN expressing the K88 fimbrial adhesin does not induce a selective T cell response towards the antigen.