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Chlamydia abortus (C. abortus) is the causative agent of ovine enzootic abortion (OEA) and poses a zoonotic risk to pregnant women. Current live attenuated 1B vaccines are efficacious but cause disease in vaccinated animals and inactivated vaccines are only marginally protective. We tested the ability of a new C. abortus subunit vaccine candidate based on the conserved and immunogenic polymorphic membrane protein D (Pmp18D) formulated in CpG1826+FL (Fms-like tyrosine kinase 3 Ligand; Flt3L) or Vibrio cholerae ghosts (VCG) to induce innate and cross protective immunity against genital C. abortus infection. We found that delivery of rPmp18D with VCG was more effective than with CpG+FL in up-regulating the expression of molecules critically involved in T cell activation and differentiation, including MHC II, CD40, CD80, and CD86, activation of TLRs and NLRP3 inflammasome engagement, and secretion of IL-1β and TNF-α but not IL-10 and IL-4. rVCG-Pmp18D-immunized mice elicited more robust antigen-specific IFN-γ, IgA and IgG2c antibody responses compared to CpG+FL-delivered rPmp18D. Based on the number of mice with positive vaginal cultures, length of vaginal shedding, and number of inclusion forming units recovered following challenge with the heterologous C. abortus strain B577, vaccine delivery with VCG induced superior protective immunity than delivery with a combination of CpG1826 and FL, a nasal DC-targeting adjuvant. These results demonstrate that the ability of VCG to enhance protective immunity against genital C. abortus infection is superior to that of CpG+FL adjuvants., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The need to simultaneously target infections with epidemiological overlap in the population with a single vaccine provides the basis for developing combination vaccines. Vibrio cholerae ghosts (rVCG) offer an attractive approach for developing vaccines against a number of human and animal pathogens. In this study, we constructed a multisubunit vaccine candidate co-expressing the serovar D-derived Porin B and polymorphic membrane protein-D proteins of Chlamydia trachomatis and evaluated its ability to simultaneously induce broad-based chlamydial immunity and elicit a vibriocidal antibody response to the Vibrio carrier envelope. Intramuscular (IM) immunization with the vaccine candidate elicited high levels of antigen-specific genital mucosal and systemic Th1 cell-mediated and humoral immune responses against heterologous serovars and strains, including serovars E-H and L. Also, in addition to the multisubunit vaccine, the single subunit constructs conferred significant cross protection against the heterologous mouse strain, Chlamydia muridarum. Furthermore, all mice immunized with rVCG vaccine constructs responded with a significant rise in vibriocidal antibody titer, the surrogate marker for protection in cholera. These findings demonstrate the ability of the multisubunit vaccine to induce cross protective chlamydial as well as vibriocidal immunity and establish the possibility of developing a broadly efficacious Chlamydia-cholera combination vaccine., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We tested the hypothesis that intramuscular immunization with a multisubunit chlamydial vaccine candidate will induce long lasting immune responses in mice. Accordingly, groups of female C57BL/6 mice were immunized intramuscularly with Vibrio cholerae ghosts (VCG) expressing the Poring B and polymorphic membrane protein-D proteins of Chlamydia trachomatis or a control antigen. Humoral and cell-mediated immune responses were evaluated following immunization and after live chlamydial infection. Immunization induced an anamnestic response characterized by chlamydial-specific IgG2a and IgA antibodies in sera and vaginal lavage as well as specific genital and splenic T cell responses. The results also revealed that the local mucosal and systemic cellular and humoral immune effectors induced in mice following immunization with the vaccine candidate are long lasting. Vaccinated mice cleared intravaginal challenge with 10(5) chlamydial inclusion forming units within 12 days compared to control mice, which shed up to 2 × 10(3) IFUs at this time point. Moreover, rechallenge of mice 98 days after resolution of the primary infection resulted in the recall and retention of a relatively high frequency of chlamydial-specific Th1 cells and IgG2a in the genital mucosa. These results provide the first evidence that a VCG-based multisubunit chlamydial vaccine is capable of effectively stimulating anamnestic systemic and mucosal immune responses in mice. The data support further vaccine evaluation and testing for induction of long-term protective immunity., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A new paradigm for designing vaccines against certain microbial pathogens, including Chlamydia trachomatis, is based on the induction of local mucosal Th1 response. IL-10 is an anti-inflammatory cytokine that exerts negative immunoregulatory influence on Th1 response. This study investigated whether biochemical modulation of endogenous IL-10 expression at the level of APCs is a practical strategy for enhancing the specific Th1 response against pathogens controlled by Th1 immunity. The results revealed that the high resistance of genetically engineered IL-10-/- (IL-10KO) mice to genital chlamydial infection is a function of the predilection of their APCs to rapidly and preferentially activate a high Th1 response. Thus, in microbiological analysis, IL-10KO mice suffered a shorter duration of infection, less microbial burden, and limited ascending infection than immunocompetent wild-type mice. Also, IL-10KO were resistant to reinfection after 8 wk of the primary infection. Cellular and molecular immunologic evaluation indicated that IL-10KO mice induced greater frequency of chlamydial-specific Th1 response following C. trachomatis infection. Moreover, IL-10KO APCs or antisense IL-10 oligonucleotide-treated wild-type APCs were potent activators of Th1 response from naive or immune T cells. Furthermore, both Ag-pulsed dendritic cells from IL-10KO mice and IL-10 antisense-treated dendritic cells from wild-type mice were efficient cellular vaccines in adoptive immunotherapeutic vaccination against genital chlamydial infection. These findings may furnish a novel immunotherapeutic strategy for boosting the Th1 response against T cell-controlled pathogens and tumors, using IL-10-deficient APCs as vaccine delivery agents.

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, peri-plasma and internal lumen of the ghosts can be fully utilized. This extended recombinant ghost system represents a new strategy for adjuvant free combination vaccines.

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts have been produced from a great variety of bacteria and are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extents the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens, immunomodulators or other substances. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in bacterial candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying inserts of foreign epitopes of up to 600 amino acids within the flexible surface loop areas of the S-layer further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts do not need the addition of adjuvants to induce immunity in experimental animals they can also be used as carriers or targeting vehicles or as adjuvants in combination with subunit vaccines. Matrixes like dextran which can be used to fill the internal lumen of ghosts can be substituted with various ligands to bind the subunit or other materials of interest. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of ghosts and recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in the production of ghosts. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. As carriers of foreign antigens there is no limitation in the size of foreign antigens to be inserted and the capacity of all spaces including the membranes, periplasma and internal lumen of the ghosts can be fully utilized. Using the different building blocks and combining them into the recombinant ghost system represents a new strategy for adjuvant free combination vaccines.

Expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts have been produced from a variety of bacteria including Escherichia coli. Salmonella typhimurium, Salmonella enteritidis, Vibrio cholerae, Klebsiella pneumoniae, Actinobacillus pleuropneumoniae, Haemophilus influenzae, Pasteurella haemolytica, Pasteurella multocida, and Helicobacter pylori. Such ghosts are used as non-living candidate vaccines and represent an alternative to heat or chemically inactivated bacteria. In recombinant ghosts, foreign proteins can be inserted into the inner membrane prior to E-mediated lysis via specific N-, or C-, or N- and C-terminal anchor sequences. The export of proteins into the periplasmic space or the expression of recombinant S-layer proteins vastly extents the capacity of ghosts or recombinant ghosts as carriers of foreign epitopes or proteins. Oral, aerogenic or parenteral applications of (recombinant) ghosts in experimental animals induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of ghosts and recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in the production of ghosts used as vaccines or as carriers of relevant antigens. The inserted target antigens into the inner membrane or into S-layer proteins are not limited in size.

The immunogenic potential of Vibrio cholerae ghosts (VCG) in comparison with heat-killed whole-cell vibrios (WCV) was evaluated after intraperitoneal immunization of adult mice. Swiss white mice received four doses of VCG or WCV intraperitoneally, consisting of 500 micrograms of lyophilized material in 200 microliters of phosphate-buffered saline (PBS), pH 7.4. The control group received 200 microliters of PBS. Serum samples were collected from all mice on the day of immunization and on days 14, 24, 35 and 62 postimmunization. Sera were examined for vibriocidal antibodies by the microtitre and tube-dilution methods and Vibrio-specific serum IgG antibodies were assessed by ELISA. IgG antibodies to intact WCV were detected in sera from all animals immunized with VCG or WCV. The response was specific and of high magnitude. Significantly higher antibody responses were obtained when sera from both VCG- and WCV-immunized mice were titrated against VCG. The immunogenicity of VCG in evoking serum IgG responses was higher than that of WCV. However, the immunogenicity of the two antigen preparations was comparable in terms of seroconversion for vibriocidal antibodies. These results demonstrate that VCG administered intraperitoneally evoke Vibrio-specific serum IgG responses as well as vibriocidal antibody activity in mice.

The protein E-specific lysis mechanism of the Escherichia coli-specific bacteriophage PhiX174 was employed to produce Vibrio cholerae ghosts (VCG). VCG consist of both rounded and collapsed cells that have lost their cytoplasmic contents through an E-specific hole in the cell envelope. These ghosts are proposed as non-living material for immunization against cholera. A specific membrane anchor sequence was used to insert the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) fusion protein into the cell envelope of V. cholerae. The identity of the expression products was confirmed by Western blot analysis employing an RT-specific monoclonal antibody. HIV-1 RT was chosen as a model for the purpose of evaluating heterologous gene expression in V. cholerae and the carrier potential of VCG. Intraperitoneal immunization of mice was used to evaluate the immunogenic potential of VCG. Preliminary results showed significant seroconversions to intact whole-cell vibrio antigens in mice immunized with VCG or a heat-killed whole-cell vibrio preparation.

In 1991 a cholera epidemic occurred in Nigeria. The features of this cholera outbreak in a single hospital in Cross River, Nigeria, were examined. Microbiologic techniques included the use of thiosulphate citrate bile-salts sucrose (TCBS) medium for culture of all stool specimens. Vibrio isolates from diarrheic patients included V. cholerae-O1 (75), V. cholerae non-O1 (10) and V. parahaemolyticus (21). The illnesses were diverse, ranging from mild to severe, and in most instances requiring hospitalization, rehydration as well as antibiotic treatment. Eighty patients were hospitalized and six died mainly from hypovolemic shock and acute renal failure arising from excessive fluid loss. The low vibrio-associated mortality observed in this outbreak may have been influenced by the proximity and easy transit access to the health care facilities offered by the teaching hospital. This contrasts with the high mortality figures reported by Health Centers in the rural areas during the same period. Some features of vibrio diarrheas were comparable with those of other enteric pathogens. Poorly developed water and sewage disposal systems, contact with sea water, consumption of fishery products and leftover foods were the main risk factors identified.

Clinical and environmental isolates of Vibrio parahaemolyticus were examined for their ability to agglutinate human and rabbit erythrocyte and to adhere to human buccal epithelial (HBE) cells in the presence or absence of mannose. All strains produced cell-associated haemagglutinins (HGs) after 3 h at 37 degrees C. Mannose-sensitive haemagglutination (MSHA) appeared to be a significant marker for differentiating between clinical and environmental isolates; 75% of clinical and 11% of environmental isolates exhibited MSHA with rabbit erythrocytes. All strains showed mannose-resistant adhesion (MRA) to HBE cells whose pattern had no relationship to the epidemiological source of the isolates. Adherence to HBE cells correlated with haemagglutination (HA) capability of environmental but not clinical isolates. This suggests that although intestinal adherence may be an essential step in vibrio colonization, it may not be a sufficient prerequisite for the subsequent expression of pathogenicity in Vibrio parahaemolyticus.

During a 3-year study (January 1986-December 1988), stools of 2200 diarrhoeal or dysenteric patients were examined by culturing and 108 (4.9%) were found positive for shigellae. Shigella flexneri was the commonest species isolated (54.6%), followed by Sh. dysenteriae (24.1%). Patients aged less than or equal to 15 years accounted for 51.4% of cases. Shigellae over the 3 years showed high and sometimes rising resistance to ampicillin, chloramphenicol, streptomycin and cotrimoxazole and complete resistance to tetracyclines and sulphonamides. Sh. sonnei strains isolated in 1986 and 1987 were almost invariably sensitive to all antimicrobial agents except ampicillin, while in 1988 strains were resistant to all. The isolation rate was higher (74.1%) during the dry season than in the rainy season (25.9%) (P less than 0.01). Low standards of community and personal hygiene and improper sewage disposal are the prevailing epidemiological factors identified.

The prevalence, clinical profiles and virulence factors of Plesiomonas shigelloides were determined in patients attending the University of Calabar Teaching Hospital, Microbiology Laboratory. P. shigelloides was isolated from 12 (1.4%) of 880 patients with diarrhoea and from none of the controls (P less than 0.05). Isolates were mostly from febrile children less than or equal to 10 years with most of the infections occurring during the rainy months. Although our findings suggest the significance of Plesiomonas in acute diarrhoea in this environment, our isolates did not seem to show any of the proxy indicators of virulence usually associated with other enteric pathogens.

The microbiological and morbidity profiles of acute diarrhoeal episodes were studied in 881 patients seen at the Out-Patients Department of the University of Calabar Teaching Hospital (UCTH), Calabar, between January and December, 1988. Of a total of 108 (12.3%) culturally confirmed bacterial diarrhoeas, 47 (43.5%) were due to Escherichia coli, 33 (30.6%) to vibrios (Vibrio cholerae-01; classical and E1 Tor biotypes and V. parahaemolyticus), while shigella spp. and salmonella. spp. accounted for 29 (17.7%) and 9 (8.3%) episodes respectively. Twenty (64.5%) of the patients with vibrio diarrhoeas were children less than or equal to 10 years. The only case of diarrhoea-associated death observed, involved an 8-month old infant with kwashiorkor and V. parahaemolyticus infection. Bimodal peaks of cholera episodes occurred during the dry season and appeared to coincide with acute water shortage periods in the municipality. The significance of some prevailing ecological factors in stabilizing a focus of cholera and halophilic vibrio diarrhoea endemicity in this region is discussed.

The prevalence of Aeromonas spp. and other enteric pathogens in stool specimens from diarrheic and non-diarrheic patients was studied over a 12 month period (January to December, 1986). Except for the absence of fever, all the clinical features in Aeromonas diarrhea were comparable to those associated with other diarrheagenic agents. These features included abdominal pain (30%), vomiting (24.5%), fever (31.5%), dehydration (9.5%) and hematochezia (19.5%). Aeromonas spp. were more frequently isolated from patients with gastroenteritis (2.5%) than from control patients (1.0%) (P less than 0.05). Isolates were recovered more often during the dry months (66.7%), than during the wet months (33.3%). Among the enteric pathogens isolated, Aeromonas spp. (2.5%) ranked next to Esch. coli (14.5%) and Shigella spp. (6.3%) in prevalence. Other bacterial isolates included Plesiomonas shigelloides (1.5%) Vibrio spp. (1.0%), Yersinia enterocolitica (1.0%) and Salmonella spp. (1.8%).

During a 12-month study period (January-December 1986), 12 Aeromonas strains (eight A. hydrophila and four A. sobria) were isolated from the 400 diarrhoeic and 200 non-diarrhoeic stools examined. Isolates were characterized for frequency of phenotypic properties associated with virulence using the suckling mouse test for enterotoxin assay; haemolysis of rabbit erythrocytes for haemolysin production, and haemagglutination of human group A cells (HA) for presence of colonization factor antigens (CFA). All seven A. hydrophila and three A. sobria strains isolated from diarrhoeic stools produced enterotoxin. All, except one A. sobria strain from a diarrhoeic stool, produced haemolysin in titres greater than 4. Fifty per cent of strains from diarrhoeic stools and 100% of strains from non-diarrhoeic stools were HA positive. All HA reactions were sensitive to mannose and galactose. The frequency of enterotoxin-producing aeromonads was significantly higher (P less than 0.05) in diarrhoeic patients than in non-diarrhoeic controls. There was no significant difference (P greater than 0.05) in the frequency of haemolysin and HA production between diarrhoeic and non-diarrhoeic strains.