Characterization of four novel genomic regions of uropathogenic Escherichia coli highly associated with the extraintestinal virulent phenotype: a jigsaw puzzle of genetic modules

Extraintestinal pathogenic Escherichia coli (ExPEC) are a major cause of urinary tract infections, sepsis, and neonatal meningitis. A variety of virulence factors in these strains is encoded by mobile genetic elements, such as transposons or pathogenicity islands (PAIs). Using subtractive cloning of ExPEC genomes, we recently detected short DNA fragments, which were significantly associated with the extraintestinal virulent phenotype. In this study, we identified four novel genomic DNA regions of the highly virulent uropathogenic E. coli strain JS299 carrying these previously identified DNA fragments. Characterization of the partial sequences of the genomic DNA regions revealed complex DNA arrangements with variable genetic compositions regarding the G+C contents and codon usage patterns. The prevalence of 15 previously uncharacterized genes was determined in a collection of clinical ExPECs and commensal E. coli strains by means of DNA microarray analyses. From this, 13 novel DNA sequences were demonstrated to be significantly associated with extraintestinal virulent strains, and thus may represent new virulence traits. Beside genes predicted to play a role in metabolic functions, such as sucrose utilization (scr), we identified DNA sequences shared by both ExPEC and enteropathogenic E. coli (EPEC). These sequences were significantly more prevalent among ExPECs when compared to commensal E. coli isolates. Our results support the idea of a considerable genetic variability among ExPEC strains and suggest that the novel genomic determinants described in this study may contribute to the ExPEC virulence.     

Oral immunization of rabbits with enterotoxigenic Escherichia coli protects against intraintestinal challenge.

The development of a successful oral vaccine against enterotoxigenic Escherichia coli depends upon the identification of appropriate protective antigens which can be delivered effectively to intestinal mucosa. We have determined in a modified RITARD model the relative protection against intraintestinal challenge afforded by oral immunization with live enterotoxigenic E. coli carrying different candidate antigens. Studies were done with both wild-type strains and genetically manipulated strains of enterotoxigenic E. coli (parent strain E1392/75 2A) which carried plasmids containing intact heat-labile toxin (LT) gene sequences or various mutations of the LT genes. Immunizations were done by orogastric tube inoculation on days 0, 7, and 14; challenges were done on day 33. Protection against diarrhea with a homologous challenge was found to be 84 to 100% (P less than 0.01). Protection against diarrhea with challenges in which specific antigens could be tested included the following: (i) O and H antigens (O6:H16), 87 to 100% protection with different E. coli strains with identical O and H antigens (P less than 0.01) but no protection against a heterologous challenge; (ii) LT or the B subunit of LT only, approximately 50% protection (P less than 0.02). These findings suggest that O antigens are highly protective in this model but afford only serotype-specific protection and that the B subunit (with or without the A subunit) affords less protection but confers cross-protection against heterologous strains producing LT. This model should be useful in further defining appropriate protective antigens for candidate enterotoxigenic E. coli vaccine strains.     

A Burkholderia pseudomallei Outer Membrane Vesicle Vaccine Provides Protection against Lethal Sepsis

The environmental Gram-negative encapsulated bacillus Burkholderia pseudomallei is the causative agent of melioidosis, a disease associated with high morbidity and mortality rates in areas of Southeast Asia and northern Australia in which the disease is endemic. B. pseudomallei is also classified as a tier I select agent due to the high level of lethality of the bacterium and its innate resistance to antibiotics, as well as the lack of an effective vaccine. Gram-negative bacteria, including B. pseudomallei, secrete outer membrane vesicles (OMVs) which are enriched with multiple protein, lipid, and polysaccharide antigens. Previously, we demonstrated that immunization with multivalent B. pseudomallei-derived OMVs protects highly susceptible BALB/c mice against an otherwise lethal aerosol challenge. In this work, we evaluated the protective efficacy of OMV immunization against intraperitoneal challenge with a heterologous strain because systemic infection with phenotypically diverse environmental B. pseudomallei strains poses another hazard and a challenge to vaccine development. We demonstrated that B. pseudomallei OMVs derived from strain 1026b afforded significant protection against septicemic infection with B. pseudomallei strain K96243. OMV immunization induced robust OMV-, lipopolysaccharide-, and capsular polysaccharide-specific serum IgG (IgG1, IgG2a, and IgG3) and IgM antibody responses. OMV-immune serum promoted bacterial killing in vitro, and passive transfer of B. pseudomallei OMV immune sera protected naive mice against a subsequent challenge. These results indicate that OMV immunization provides antibody-mediated protection against acute, rapidly lethal sepsis in mice. B. pseudomallei-derived OMVs may represent an efficacious multivalent vaccine strategy against melioidosis.     

A live-attenuated Pseudomonas aeruginosa vaccine elicits outer membrane protein-specific active and passive protection against corneal infection

Pseudomonas aeruginosa can cause sight-threatening corneal infections in humans, particularly those who wear contact lenses. We have previously shown that a live-attenuated P. aeruginosa vaccine given intranasally protected mice against acute lethal pneumonia in a lipopolysaccharide (LPS) serogroup-specific manner. In the current study, we evaluated the protective and therapeutic efficacies, as well as the target antigens, of this vaccine in a murine corneal infection model. C3H/HeN mice were nasally immunized with the vaccine (an aroA deletion mutant of strain PAO1, designated PAO1DeltaaroA) or with Escherichia coli as a control and were challenged 3 weeks later by inoculating the scratch-injured cornea with P. aeruginosa. For passive prophylaxis and therapy, we utilized a serum raised in rabbits nasally immunized with PAO1DeltaaroA or E. coli. Outcome measures included corneal pathology scores and, in some experiments, reductions in total and internalized bacterial CFU. We found that both active and passive immunization reduced corneal pathology scores after challenge with a variety of P. aeruginosa strains, including several serogroup-heterologous strains. Even when given therapeutically starting as late as 24 h after infection, the rabbit antiserum to PAO1DeltaaroA was effective at reducing corneal pathology scores. Immunotherapy of established infections also reduced the numbers of total and internalized corneal P. aeruginosa bacteria. Experiments using absorbed sera showed that the protective antibodies are specific to outer membrane proteins. Thus, live-attenuated P. aeruginosa vaccines delivered nasally protect against corneal infections in mice and potentially can be used to prepare passive therapy reagents for the treatment of established P. aeruginosa corneal infections caused by diverse LPS serogroups.     

Immunization with a recombinant C-terminal fragment of Plasmodium yoelii merozoite surface protein 1 protects mice against homologous but not heterologous P. yoelii sporozoite challenge.

It has been reported previously that immunization with recombinant protein containing the two epidermal growth factor (EGF)-like modules from merozoite surface protein 1 (MSP-1) of Plasmodium yoelii (strain YM) protects mice against a lethal blood-stage challenge with the same parasite strain. Since MSP-1 is expressed in both liver- and blood-stage schizonts and on the surface of merozoites, we evaluated the effectiveness of immunization with recombinant proteins containing either the individual or the two combined EGF-like modules in producing a protective response against a sporozoite challenge. The recombinant protein expressing the combined EGF-like modules of the YM strain protected mice against a homologous sporozoite challenge, and sterile protection, as defined by the absence of detectable blood-stage parasites, was observed in the majority of the mice. In contrast, mice immunized with recombinant P. yoelii YM MSP-1 were not protected against a heterologous challenge with sporozoites from strain 265 BY of P. yoelii. The lack of protection may be explained by differences identified in the amino acid sequences of MSP-1 for the two strains. A recombinant protein containing the two EGF-like modules of MSP-1 from P. yoelii 265 BY was produced and used to immunize mice. These mice were protected against a homologous challenge with sporozoites of P. yoelii 265 BY. The results suggest that a recombinant MSP-1 has potential as a vaccine against malaria, but its efficacy may be limited by sequence polymorphism and selection of variants.     

What defines extraintestinal pathogenic Escherichia coli?

Escherichia coli (E. coli) exhibits considerable physiological and metabolic versatility and includes a variety of non-pathogenic, commensal variants, which belong to the normal gut flora of humans and warm-blooded animals. Additionally, several pathogenic variants have been identified which cause various types of intestinal or extraintestinal infections in humans and animals. In contrast to intestinal pathogenic E. coli (IPEC), which are obligate pathogens, extraintestinal pathogenic E. coli (ExPEC) are facultative pathogens which belong to the normal gut flora of a certain fraction of the healthy population where they live as commensals. Comparative genomics and epidemiological studies have been applied to study genomic diversity, markers, and phenotypic traits that may support discrimination of different E. coli pathotypes. Whereas IPEC are often epidemiologically and phylogenetically distinct from ExPEC and non-pathogenic, commensal strains, many ExPEC and non-pathogenic E. coli share large genomic fractions. Furthermore, extraintestinal infections of elderly or immunocompromised patients can be caused by E. coli variants which differ in their geno- and phenotypes from archetypal ExPEC. Thus, strain typing based on the detection of a limited number of ExPEC virulence/fitness-related genes may be ambiguous. A limited number of ExPEC-dominated clonal complexes can be identified in the E. coli population by multi locus sequence typing. Nevertheless, ExPEC and non-pathogenic E. coli cannot be clearly discriminated by molecular epidemiological approaches. Increased knowledge of the phylogeny, virulence and fitness traits, and host factors contributing to host susceptibility of the different groups of ExPEC variants is required for a better understanding of the biological basis of ExPEC infections.     

Experimental vaccination against group B streptococcus, an encapsulated bacterium, with highly purified preparations of cell surface proteins Rib and alpha.

Encapsulated bacteria cause some of the most common diseases in humans. Although the polysaccharide capsules of these pathogens have attracted the most attention with regard to vaccine development, recent evidence suggests that bacterial surface proteins may also be used to confer protective immunity. We have analyzed this possibility in group B streptococcus (GBS), an encapsulated bacterium that is the major cause of invasive bacterial disease in the neonatal period. Previous work has shown that the majority of GBS strains causing invasive infections express the Rib protein, and that most strains lacking Rib express a protein designated alpha. Here we report that active immunization with highly purified preparations of Rib or alpha protected mice against lethal infection with strains expressing the corresponding protein. Vaccination with the Rib protein protected against two strains of capsular type III and two strains of type II, and vaccination with the alpha protein protected against one strain of type II and one strain of type Ib. The mice vaccinated with Rib or alpha showed a good immunoglobulin G response to the immunogen. These data suggest that a vaccine against GBS disease may be based on cell surface proteins and support the notion that proteins may be used for immunization against encapsulated bacteria.     

Mouse protection assay for group B streptococcus type III.

The mucin model for group B Streptococcus (GBS) type III was used to assay the protective effect of sera against a type III challenge in mice. Hyperimmune rabbit sera, prepared by the Lancefield method against the laboratory reference strain (SS620) and a clinical isolate (M732), protected against a lethal challenge with either strain of GBS type III. Absorption of the sera with either of these type III strains removed the protective effect. Neither normal rabbit sera nor heterologous antisera (anti-Ia, SS615) provided protection; however, protection was obtained with pooled human gamma globulin. Sera from adult volunteers were tested to assay protective levels in the mouse model. Human sera enhanced the mouse lethality of the clinical isolate, M732, but not the laboratory reference strain, SS620. Sera from adults vaccinated with type III polysaccharide of GBS were also tested. The murine-mucin-GBS model may be developed as a screening test to measure protective antibody levels in the pre- and postvaccine treatment period. The model may also be used to measure protective antibody in pooled human gamma globulin for use in the passive immunization of high-risk individuals.     

Correlation between lethal toxin-neutralizing antibody titers and protection from intranasal challenge with Bacillus anthracis Ames strain spores in mice after transcutaneous immunization with recombinant anthrax protective antigen

Transcutaneous immunization of mice with recombinant protective antigen (rPA) of Bacillus anthracis resulted in significantly higher lethal toxin-neutralizing antibody titers than did intramuscular injection of alum-adsorbed rPA. Immunized mice were partially protected against intranasal challenge with 235,000 (10 50% lethal doses) Ames strain B. anthracis spores. A highly significant correlation was observed between toxin-neutralizing antibody titer and survival after challenge. Future experiments with rabbits and nonhuman primates should confirm the significance of protection by this vaccine strategy.     

Immunosuppressive effects of Marek's disease virus (MDV) and herpesvirus of turkeys (HVT) in broiler chickens and the protective effect of HVT vaccination against MDV challenge

Much of the impact of Marek's disease in broiler chickens is considered to be due to immunosuppression induced by Marek's disease virus (MDV). The present study evaluates the effects of an Australian isolate of pathogenic MDV (strain MPF 57) and a non-pathogenic vaccinal strain of herpesvirus of turkeys (HVT) (strain FC 126) on the immune system of commercial broiler chickens for 35 days following challenge at days 0 or 3 of age. It also investigates the extent of protection provided by HVT vaccine against MDV-induced immunosuppression. Immune system variables, including relative lymphoid organ weight, blood lymphocyte phenotype (CD45+/CD3+, putatively T, and CD45+/LC+, putatively B) and antibody production following vaccination against infectious bronchitis (IB) at hatch, were used to assess the immune status of chickens. Immunosuppression was also assessed by susceptibility to secondary challenge with pathogenic Escherichia coli on day 29 post-MDV challenge. MDV infection reduced the weight of the thymus and bursa of Fabricius, the numbers of circulating T lymphocytes and B lymphocytes, and IB antibody titre. The timing of these effects varied. MDV infection greatly increased susceptibility to E. coli infection. HVT alone caused mild depletion of T and B lymphocytes but no effect on immune organ weight or IB titre. Vaccination with HVT provided good protection against most of the immunosuppressive effects of MDV but not against MDV-induced growth impairment and reduced responsiveness to IB vaccination, suggesting that recent Australian strains of MDV may be evolving in virulence to overcome the protective effects of HVT.     

Salmonella flagellin is not a dominant protective antigen in oral immunization with attenuated live vaccine strains

We found that oral immunization with flagellum-defective mutant strains of Salmonella enterica serovar Typhimurium with the ClpXP-deficient background protected mice against oral challenge with the virulent strain. These data indicate that Salmonella flagellin is not a dominant protective antigen in oral immunization with attenuated live vaccine strains.     

Experimental vaccination of young chickens with a live, non-pathogenic strain of Escherichia coli

A non-pathogenic, piliated strain of Escherichia coli (BT-7; Frommer et al., 1990), isolated from a meat-type chicken flock, was studied as a candidate for a live vaccine to protect chickens from E. coli infection. Active immunization provided substantial protection of chicks vaccinated at 14 or 21 days of age, resulting in better resistance to challenge than in those vaccinated at 1 or 7 days. Chicks vaccinated at 21 days of age and challenged 1 week later with pathogenic E. coli strains 01-.K1, 02:K1 or 078:K80, exhibited good protection for at least 2 weeks against all strains. Three vaccination routes were found to give the highest resistance to challenge with pathogenic E. coli strain 078:K80. Intramuscular (i.m.) at 7 and 21 days of age, i.m. at 21 days of age and spray at 7, followed by per os at 21 days of age. Vaccination per os once at 7 or twice at 7 and 21 days resulted in good protection. Chicks exhibiting high antibody titres by ELISA were well-protected against challenge.     

Antibody production and protection against influenza virus in immunodeficient mice.

The roles of T and B cells in the immune response to influenza virus were studied by using mice deficient in either T cells (athymic nude) or immunoglobulin production (CBA/N). The serological responses of these mice to either whole or disrupted A/Aichi/2/68 influenza virus vaccines were examined, and the protective effect of these inoculations was tested by challenge infection with mouse-adapted A/Aichi/2/68 influenza virus. In contrast to normal mice, neither strain of immunodeficient mouse produced detectable serum antibody after inoculation with either type of vaccine. CBA/N mice immunized with intact virus vaccine were protected, however, against subsequent lethal challenge. CBA/N mice inoculated with disrupted virus vaccine and nude mice inoculated with either disrupted or whole virus vaccine were not protected against viral challenge. Evidence of immunological memory was observed in CBA/N and nude mice that had survived live virus challenge after immunization with inactivated vaccine.     

First multi-epitope subunit vaccine against extraintestinal pathogenic Escherichia coli delivered by a bacterial type-3 secretion system (T3SS)

Infections due to extraintestinal pathogenic E. coli (ExPEC) are very common in humans as well as in animals. In humans ExPEC infections include urinary tract infections (UTI), septicemia, and wound infections, which result in significant morbidity, mortality, and substantial healthcare costs. In view of the increasing number of ExPEC infections caused by more and more resistant strains, effective prevention would be desirable. Given the rising treatment costs, a vaccine may be cost-effective in selected patient groups, such as women with recurrent UTI, patients with neurologic disorders impairing bladder function and men with prostate hyperplasia. Previous vaccine studies used single target proteins or whole inactivated ExPEC cells. Here, we describe a vaccine system for oral application based on artificial multiple subunit vaccine proteins. Those multi-epitope proteins are composed of predicted epitopes derived from ExPEC virulence-associated proteins. As ExPEC are known to form intracellular biofilms in the urothelium and can also resist killing by non-activated macrophages, T-cell responses are supposed to be an important measure to counteract these stages of ExPEC during infection. Therefore, a live bacterial antigen delivery system based upon the Salmonella type-III secretion system (T3SS) was used in this study to directly deliver the vaccine proteins into the cytoplasm of the host cells. Epitope-rich domains of the proteins FyuA, IroN, ChuA, IreA, Iha, and Usp were expressed in an attenuated Salmonella enterica serovar Typhimurium strain and translocated into target cells for extended periods of time inducing a strong T-cell response. No significant antibody titre increase against the secreted vaccine proteins could be detected in vaginal wash or serum. Despite that, one of the vaccine proteins was able to significantly reduce bacterial load in the challenge model of intraperitoneal sepsis. This study shows that a vaccine encompassing distinct epitopes of virulence-associated ExPEC proteins (i) can be applied for a T3SS-dependent vaccination strategy, (ii) elicits T-cell responses and (iii) confers protection after a single application.     

Cloning and characterization of Treponema hyodysenteriae antigens and protection in a CF-1 mouse model by immunization with a cloned endoflagellar antigen.

We cloned genes that code for Treponema hyodysenteriae antigens into Escherichia coli with the purpose of identifying protective antigens for vaccine development. Three different genomic libraries were screened with various antisera reactive with T. hyodysenteriae antigens. The cloned antigens and corresponding native T. hyodysenteriae antigens were analyzed for molecular size, serum reactivity, solubility in sarcosine, and segregation during phase partitioning with the nonionic detergent Triton X-114. The results from these analyses suggested that the gene products were components of either the cytoplasmic membrane, periplasm, or endoflagella of T. hyodysenteriae. The cloned antigens were tested as vaccine candidates in a CF-1 mouse model of T. hyodysenteriae infection and immunity. Intraperitoneal injection of crude E. coli extracts containing cloned antigens did not protect mice from challenge. However, serum from mice injected with a crude extract of an E. coli clone which expressed an endoflagellar antigen killed T. hyodysenteriae in vitro. Partially purified preparations of this cloned endoflagellar antigen protected mice against oral challenge with both the homologous serotype (B204) and a heterologous serotype (B234) of T. hyodysenteriae. These results suggest that the endoflagellar proteins could be used as an effective subunit vaccine against T. hyodysenteriae.     

Protection against Leptospira interrogans sensu lato challenge by DNA immunization with the gene encoding hemolysin-associated protein 1

The use of DNA constructs encoding leptospiral proteins is a promising new approach for vaccination against leptospirosis. In previous work we determined that immunization with hemolysis-associated protein 1 (Hap1) (LipL32) expressed by adenovirus induced significant protection against a virulent Leptospira challenge in gerbils. To avoid the use of the adenovirus vector, we checked for clinical protection against lethal challenge by DNA vaccination. A DNA vaccine expressing Hap1 was designed to enhance the direct gene transfer of this protein into gerbils. A challenge was performed 3 weeks after the last immunization with a virulent strain of serovar canicola. Our results show that the cross-protective effect with pathogenic strains of Leptospira, shared by Hap1, could be mediated by the DNA plasmid vector. This finding should facilitate the design and development of a new generation of vaccines against bacteria, particularly Leptospira interrogans sensu lato.     

Protection against experimental Pseudomonas aeruginosa infection by recombinant P. aeruginosa lipoprotein I expressed in Escherichia coli.

Lipoprotein I (OprI) is one of the major proteins of the outer membrane of Pseudomonas aeruginosa. OprI is a candidate for a vaccine against P. aeruginosa, because it cross-reacts antigenically in all serotype strains of the International Antigenic Typing Scheme. We recently cloned and expressed the gene coding for OprI in Escherichia coli. This heterologously expressed OprI was used successfully to immunize mice against P. aeruginosa. In addition, OprI from serogroup 12 of P. aeruginosa was highly purified by preparative isoelectric focusing and used for immunization of mice. Both vaccines protected the mice against a challenge with a four- to fivefold 50% lethal dose of P. aeruginosa.     

Specific lung mucosal and systemic immune responses after oral immunization of mice with Salmonella typhimurium aroA, Salmonella typhi Ty21a, and invasive Escherichia coli expressing recombinant pertussis toxin S1 subunit.

Pertussis toxin (PT) is considered an essential protective component for incorporation into new generation vaccines against Bordetella pertussis, the causative agent of whooping cough. Traditionally, antipertussis vaccination has employed an intramuscular route. An alternative to this approach is to stimulate mucosal and systemic immune responses by oral immunization with live vaccine carrier strains of Salmonella spp. or Escherichia coli. Recombinant S1 subunit of pertussis toxin was expressed in the attenuated aroA mutant of Salmonella typhimurium, SL3261, in the human typhoid vaccine strain Salmonella typhi Ty21a, and in E. coli CAG629 containing the Shigella flexneri plasmid pWR110, which encodes bacterial invasiveness of epithelial cells. Expression of recombinant PT S1 subunit (rPT-S1) did not affect in vitro invasiveness of the tested strains, which retained the ability to adhere to and invade the embryonic human intestinal cell line HI-407. Following oral immunization of mice with the live vaccine strains expressing rPT-S1, immunoglobulin G (IgG), IgA, and IgM responses were monitored. IgG specific to PT was detected in serum samples of mice, while IgG and IgA specific to PT were detected in lung washes after oral immunization with living Salmonella spp. or E. coli (pWR110) expressing rPT-S1. Utilization of live oral vaccines expressing B. pertussis antigens, which stimulate both a systemic and lung mucosal response, may provide an attractive alternative to purified component vaccines against whooping cough.     

Protective immunity induced by Bacillus anthracis toxin-deficient strains.

The two toxins secreted by Bacillus anthracis are composed of binary combinations of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). Six mutant strains that are deficient in the production of one or two of these toxin components have been previously constructed and characterized (C. Pezard, E. Duflot, and M. Mock, J. Gen. Microbiol. 139:2459-2463, 1993). In this work, we examined the antibody response to the in vivo production of PA, LF, and EF in mice immunized with spores of strains producing these proteins. High titers of antibody to PA were observed after immunization with all strains producing PA, while titers of antibodies to EF and LF were weak in animals immunized with strains producing only EF or LF. In contrast, immunization with strains producing either PA and EF or PA and LF resulted in an increased antibody response to EF or LF, respectively. The differing levels of protection from a lethal anthrax challenge afforded to mice immunized with spores of the mutant strains not only confirm the role of PA as the major protective antigen in the humoral response but also indicate a significant contribution of LF and EF to immunoprotection. We observed, however, that PA-deficient strains were also able to provide some protection, thereby suggesting that immune mechanisms other than the humoral response may be involved in immunity to anthrax. Finally, a control strain lacking the toxin-encoding plasmid was unable to provide protection or elicit an antibody response against bacterial antigens, indicating a possible role for pXO1 in the survival of B. anthracis in a host.     

Protection against Experimental Melioidosis following Immunization with Live Burkholderia thailandensis Expressing a manno-Heptose Capsule

Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei. It is highly resistant to antibiotic treatment, and there is currently no licensed vaccine. Burkholderia thailandensis is a close relative of Burkholderia pseudomallei but is essentially avirulent in mammals. In this report, we detail the protective efficacy of immunization with live B. thailandensis E555, a strain which has been shown to express an antigenic capsule similar to that of B. pseudomallei. Immunization with E555 induced significant protection against a lethal intraperitoneal B. pseudomallei challenge in a mouse model of infection, with no mice succumbing to infection over the course of the study, even with challenges of up to 6,000 median lethal doses. By comparison, mice immunized with B. thailandensis not expressing a B. pseudomallei-like capsule had significantly decreased levels of protection. E555-immunized mice had significantly higher levels of IgG than mice immunized with noncapsulated B. thailandensis, and these antibody responses were primarily directed against the capsule.