Preliminary Assessment of the Safety and Immunogenicity of a New CTXΦ-Negative, Hemagglutinin/Protease-Defective El Tor Strain as a Cholera Vaccine Candidate

Vibrio cholerae 638 (El Tor, Ogawa), a new CTXΦ-negative hemagglutinin/protease-defective strain that is a cholera vaccine candidate, was examined for safety and immunogenicity in healthy adult volunteers. In a double-blind placebo-controlled study, no significant adverse reactions were observed in volunteers ingesting strain 638. Four volunteers of 42 who ingested strain 638 and 1 of 14 who received placebo experienced loose stools. The strain strongly colonized the human small bowel, as evidenced by its isolation from the stools of 37 of 42 volunteers. V. cholerae 638, at doses ranging from 4 × 10⁷ to 2 × 10⁹ vibrios, elicited significant serum vibriocidal antibody and anti-Ogawa immunoglobulin A antibody secreting cell responses.     

Safety, tolerability, and immunogenicity of a recombinant, genetically engineered, live-attenuated vaccine against canine blastomycosis

Blastomycosis is a severe, commonly fatal infection caused by the dimorphic fungus Blastomyces dermatitidis in dogs that live in the United States, Canada, and parts of Africa. The cost of treating an infection can be expensive, and no vaccine against this infection is commercially available. A genetically engineered live-attenuated strain of B. dermatitidis lacking the major virulence factor BAD-1 successfully vaccinates against lethal experimental infection in mice. Here we studied the safety, toxicity, and immunogenicity of this strain as a vaccine in dogs, using 25 beagles at a teaching laboratory and 78 foxhounds in a field trial. In the beagles, escalating doses of live vaccine ranging from 2 × 10⁴ to 2 × 10⁷ yeast cells given subcutaneously were safe and did not disseminate to the lung or induce systemic illness, but a dose of <2 × 10⁶ yeast cells induced less fever and local inflammation. A vaccine dose of 10⁵ yeast cells was also well tolerated in vaccinated foxhounds who had never had blastomycosis; however, vaccinated dogs with prior infection had more local reactions at the vaccine site. The draining lymph node cells and peripheral blood lymphocytes from vaccinated dogs demonstrated gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and granulocyte-macrophage colony-stimulating factor (GM-CSF) specifically in response to stimulation with Blastomyces antigens. Thus, the live-attenuated vaccine against blastomycosis studied here proved safe, well tolerated, and immunogenic in dogs and merits further studies of vaccine efficacy.     

Expression of an F1/V fusion protein in attenuatedSalmonella typhimuriumand protection of mice against plague

A novel approach to making fusions of F1 and V antigens, which may be incorporated into a live recombinant vaccine for plague, was developed. The nucleotide sequences encodingYersinia pestisV antigen (lcrV) and the mature form of F1 antigen (caf1) were amplified by PCR with primers which included tails. At the 3′ end ofcaf1and the 5′ end oflcrV, the tails encoded one of three six- or eight-amino acid linkers or their complementary sequences. The DNA overlap in each linker region was used to prime a second PCR to generate three F1/V fusions, which were cloned into pUC18. The resulting plasmids expressed fusion proteins consisting of F1 and V antigens, separated by the linkers Gly-Ser-Ile-Glu-Gly-Arg, Ser-Ala-Pro-Gly-Thr-Pro or Ser-Ala-Pro-Gly-Thr-Pro-Ser-Arg. As shown by Western blotting of bacterial cell lysates with anti-V and anti-F1 sera, the level of expression and degree of degradation of the three fusion proteins was similar. To investigate the immunogenicity of F1/V, one of the plasmids, placFV6 which encoded the Gly-Ser-Ile-Glu-Gly-Arg linker, was electroporated into the attenuatedSalmonella typhimuriumstrain SL3261 (aroA). Mice receiving two intravenous doses of 5 × 10⁶cfu SL3261/placFV6 developed serum anti-V and anti-F1 IgG titres, with similar IgG₁:IgG_2aisotype ratios, and T cell responses specific for V and F1 antigens. Six weeks after vaccination, mice were challenged subcutaneously with 7.4 × 10²or 7.4 × 10⁴LD₅₀s ofY. pestisstrain GB, and a significant degree of protection was demonstrated. These results demonstrate the potential of co-expressingY. pestisantigens as fusion proteins to develop a live recombinant vaccine against plague.     

Safety and Immunogenicity Assessment of an Oral Cholera Vaccine through Phase I Clinical Trial in Korea

The safety, tolerability and immunogenicity of an oral cholera vaccine (OCV) was assessed in adult Korean male through an open-label, non-comparative clinical study. Two doses of vaccine with an interval of 2 weeks were given to 20 healthy subjects. A total of 7 adverse events occurred in 6 subjects. However, no clinically significant change was observed in electrocardiograms, vital signs, physical examinations, and clinical laboratory tests. The immunogenicity of OCV was evaluated by serum vibriocidal assay where anti-Vibrio cholerae O1 and O139 antibodies were measured at day 0, 14, and 28 of vaccine administration. The antibody titers ranged from < 2.5-5,120 for V. cholerae O1 Inaba, < 2.5-10,240 for V. cholerae O1 Ogawa and < 2.5-480 for V. cholerae O139. In addition, the fold increase in antibody titers ranged from 1-4,096 for O1 Inaba, 1-8,192 for O1 Ogawa, and 1-384 for O139. The seroconversion rate was 95% and 45% for O1 and O139 antibodies, respectively. Our study clearly shows that administration of two doses of OCV at a 2 week-interval increases an appropriate level of antibody titer in the serum of healthy Korean adult males (Clinical Trial Number, NCT01707537).

Graphical Abstract

     

Immunogenic potential and protective efficacy of a sptP deletion mutant of Salmonella Enteritidis as a live vaccine for chickens against a lethal challenge

Salmonella Enteritidis (SE) is a highly adapted pathogen causing severe economic losses in the poultry industry worldwide. Chickens infected by SE are a major source of human food poisoning. Vaccination is an effective approach to control SE infections. This study evaluated the immunogenicity and protective efficacy of a SE sptP deletion mutant (C50336ΔsptP) as a live attenuated vaccine (LAV) candidate in chickens. 14 day-old specific pathogen-free (SPF) chickens were intramuscularly immunized with various doses of C50336ΔsptP. Several groups of chickens were challenged with the virulent wild-type SE strain Z-11 via the same route at 14 days post vaccination. Compared to the control group, the groups vaccinated with 1 × 10⁶, 1 × 10⁷ and 1 × 10⁸ colony-forming units (CFU) of C50336ΔsptP exhibited no clinical symptoms after immunization. Only slight pathological changes occurred in the organs of the 1 × 10⁹ CFU vaccinated group. C50336ΔsptP bacteria were cleared from the organs of immunized chickens within 14 days after vaccination. Lymphocyte proliferation and serum cytokine analyses indicated that significant cellular immune responses were induced after the vaccination of C50336ΔsptP. Compared to the control group, specific IgG antibody levels increased significantly in vaccinated chickens, and the levels increased markedly after the challenge. The 1 × 10⁷, 1 × 10⁸, and 1 × 10⁹ CFU vaccinated chickens groups showed no clinical symptoms or pathological changes, and no death after the lethal challenge. Whereas severe clinical signs of disease and pathological changes were observed in the control group chickens after the challenge. These results suggest that a single dose of C50336ΔsptP could be an effective LAV candidate to against SE infection in chickens.     

T- and B-Cell Immune Responses of Patients Who Had Undergone Colectomies to Oral Administration of Salmonella enterica Serovar Typhi Ty21a Vaccine

The capacity of an oral live attenuated Salmonella enterica serovar Typhi Ty21a vaccine to induce immune responses in patients who had undergone colectomies because of ulcerative colitis was evaluated, and these responses were compared with those of healthy volunteers. Purified CD4⁺ and CD8⁺ T cells from peripheral blood were stimulated in vitro by using the heat-killed Ty21a vaccine strain, and the proliferation and gamma interferon (IFN-γ) production were measured before and 7 or 8 days after vaccination. Salmonella-specific immunoglobulin A (IgA) and IgG antibody responses in serum along with IgA antibody responses in ileostomy fluids from the patients who had undergone colectomies were also evaluated. Three doses of vaccine given 2 days apart failed to induce proliferative T-cell responses in all the six patients who had undergone colectomies, and increases in IFN-γ production were found only among the CD8⁺ cells from three of the patients. In contrast, both proliferative responses and increased IFN-γ production were observed among CD4⁺ and CD8⁺ T cells from 3 and 6 of 10 healthy volunteers, respectively. Salmonella-specific IgA and/or IgG antibody responses in serum were observed for five (56%) of nine patients who had undergone colectomies and in 15 (88%) of 17 healthy volunteers. In ileostomy fluids, significant anti-Salmonella IgA antibody titer increases were detected in six (67%) of nine patients who had undergone colectomies. The impaired T- and B-cell immune responses found after vaccination in the circulation of patients who have undergone colectomies may be explained by a diminished colonization of the Ty21a vaccine strain due to the lack of a terminal ileum and colon.     

Molecular Characterization of the Aeromonas hydrophila aroA Gene and Potential Use of an Auxotrophic aroA Mutant as a Live Attenuated Vaccine

The aroA gene of Aeromonas hydrophila SO2/2, encoding 5-enolpyruvylshikimate 3-phosphate synthase, was cloned by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829, and the nucleotide sequence was determined. The nucleotide sequence of the A. hydrophila aroA gene encoded a protein of 440 amino acids which showed a high degree of homology to other bacterial AroA proteins. To obtain an effective attenuated live vaccine against A. hydrophila infections in fish, the aroA gene was inactivated by the insertion of a DNA fragment containing a kanamycin resistance determinant and reintroduced by allelic exchange into the chromosome of A. hydrophila AG2 by means of the suicide vector pSUP202. The A. hydrophila mutant AG2 aroA::Ka^r was highly attenuated when inoculated intraperitoneally into a rainbow trout, with a 50% lethal dose of >2 × 10⁸ CFU. The mutants were not recoverable from the internal organs after 48 h postinoculation. Immunohistochemical studies demonstrated that immunopositive materials, but not whole cells, reacting with a polyclonal antiserum against A. hydrophila were present in the kidney and spleen 9 days postinjection. Vaccination of rainbow trout with the AroA mutant as a live vaccine conferred significant protection against the wild-type strain of A. hydrophila.     

Effectiveness of a Vaccine Composed of Heat-Killed Candida albicans and a Novel Mucosal Adjuvant, LT(R192G), against Systemic Candidiasis

The incidence of fungal infections caused by the opportunistic yeast Candida albicans has increased significantly in recent years. The ability to vaccinate selected patients against the organism would be advantageous. In this paper we describe a potential anti-C. albicans vaccine consisting of heat-killed C. albicans (HK-CA) in combination with the novel mucosal adjuvant LT(R192G), a genetically detoxified form of the heat-labile toxin of enterotoxigenic Escherichia coli. Groups of male CBA/J mice were immunized intranasally on three occasions at weekly intervals with 2 × 10⁷ HK-CA per dose, alone or in conjunction with 10 μg of LT(R192G) per dose. Two weeks following the last application of antigen, some animals were challenged intravenously (i.v.) with 10⁴, 10⁵, or 10⁶ viable C. albicans to assess protection as measured by survival and/or culture. Some groups of animals were footpad tested with C. albicans mannan to assess delayed-type hypersensitivity (DTH), and all the animals were bled for antibody assays. In two independent studies, all the animals immunized with HK-CA plus LT(R192G) were able to eradicate 10⁴ C. albicans completely, as determined by kidney culture 4 weeks after challenge. Animals immunized with HK-CA only had reduced levels of C. albicans compared to the adjuvant or saline-only control. Greatly enhanced survival was observed when mice immunized with HK-CA plus LT(R192G) were challenged with 10⁵ live C. albicans as well. Animals immunized with HK-CA plus LT(R192G) developed a significant DH response, while those given HK-CA alone developed only marginal DH responses. High immunoglobulin G (IgG) levels to cytoplasmic antigens developed in mice immunized with HK-CA plus LT(R192G), but they were found only after i.v. challenge. Addition of adjuvant shifted the antibody isotype production in i.v.-challenged animals to a response dominated by IgG2a. Clearly, intranasal immunization with killed C. albicans in conjunction with LT(R192G) afforded significant levels of protection. This novel approach offers new possibilities for the development of an effective vaccine against candidiasis for use in humans.     

The Granuloma Response Controlling Cryptococcosis in Mice Depends on the Sphingosine Kinase 1–Sphingosine 1-Phosphate Pathway

Cryptococcus neoformans is a fungal pathogen that causes pulmonary infections, which may progress into life-threatening meningitis. In commonly used mouse models of C. neoformans infections, fungal cells are not contained in the lungs, resulting in dissemination to the brain. We have previously reported the generation of an engineered C. neoformans strain (C. neoformans Δgcs1) which can be contained in lung granulomas in the mouse model and have shown that granuloma formation is dependent upon the enzyme sphingosine kinase 1 (SK1) and its product, sphingosine 1-phosphate (S1P). In this study, we have used four mouse models, CBA/J and C57BL6/J (both immunocompetent), Tgε26 (an isogenic strain of strain CBA/J lacking T and NK cells), and SK^−/− (an isogenic strain of strain C57BL6/J lacking SK1), to investigate how the granulomatous response and SK1-S1P pathway are interrelated during C. neoformans infections. S1P and monocyte chemotactic protein-1 (MCP-1) levels were significantly elevated in the bronchoalveolar lavage fluid of all mice infected with C. neoformans Δgcs1 but not in mice infected with the C. neoformans wild type. SK1^−/− mice did not show elevated levels of S1P or MCP-1. Primary neutrophils isolated from SK1^−/− mice showed impaired antifungal activity that could be restored by the addition of extracellular S1P. In addition, high levels of tumor necrosis factor alpha were found in the mice infected with C. neoformans Δgcs1 in comparison to the levels found in mice infected with the C. neoformans wild type, and their levels were also dependent on the SK1-S1P pathway. Taken together, these results suggest that the SK1-S1P pathway promotes host defense against C. neoformans infections by regulating cytokine levels, promoting extracellular killing by phagocytes, and generating a granulomatous response.     

Virulence,Transmission, and Heterologous Protection of Four Isolates of Haemophilus parasuis

Haemophilus parasuis causes Glässer''s disease, a syndrome of polyserositis, meningitis, and arthritis in swine. Previous studies with H. parasuis have revealed virulence disparity among isolates and inconsistent heterologous protection. In this study, virulence, direct transmission, and heterologous protection of 4 isolates of H. parasuis (SW114, 12939, MN-H, and 29755) were evaluated using a highly susceptible pig model. In an initial experiment, isolates 12939, MN-H, and 29755 caused Glässer''s disease, while strain SW114 failed to cause any clinical signs of disease. One pig from each group challenged with MN-H or 29755 failed to develop clinical disease but was able to transmit H. parasuis to noninfected pigs, which subsequently developed Glässer''s disease. Pigs colonized with SW114, 29755, or MN-H that were free of clinical disease were protected from a subsequent challenge with isolate 12939. In a following experiment, pigs vaccinated with strain SW114 given as either a bacterin intramuscularly or a live intranasal vaccine were protected from subsequent challenge with isolate 12939; however, some pigs given live SW114 developed arthritis. Overall these studies demonstrated that pigs infected with virulent isolates of H. parasuis can remain healthy and serve as reservoirs for transmission to naive pigs and that heterologous protection among H. parasuis isolates is possible. In addition, further attenuation of strain SW114 is necessary if it is to be used as a live vaccine.     

Preliminary evaluation of the immunoenhancement potential of Newcastle disease vaccine formulated as a cationic liposome

This study evaluates the enhancement of immune response of birds to Newcastle disease (ND) vaccine encapsulated in 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)-based liposomes. The vesicles of the liposomal ND vaccine were physically characterized for shape, particle size and zeta potential. The results of the analyses showed that vesicles of the liposomal ND vaccine were spherical and tightly packed. The mean size distribution was below 100 nm. The mean zeta potential was 24 mV. Sixty experimental birds were then divided into an unvaccinated group, a liposomal ND vaccine group and a live La Sota^® vaccine group. Both the liposomal ND vaccine and live La Sota^® vaccine groups were vaccinated orally at 3 and 6 weeks of age. The mean antibody titres, total and differential white blood cell count, and blood chemistry, respectively, were assessed. Ten birds from each group were challenged by oral administration of 0.2 ml virulent Herts 33 strain at 9 weeks of age. The log₂ mean antibody titre induced by the liposomal ND vaccine after secondary immunization of the birds was 9.60±0.95 while that of the live La Sota ^® vaccine was 6.00±0.63. Nine of the 10 challenged birds in the unvaccinated group died while none died from the liposomal ND vaccine group or the live La Sota^® vaccine group. After the boost vaccination, the chickens vaccinated with the liposomal ND vaccine had a higher mean antibody titre, indicating that encapsulating ND vaccine in DOTAP-based liposome induced significantly higher immunity than the live La Sota^® vaccine.     

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.     

Effect of late administration of anti-TNα antibodies on a Salmonella infection in the mouse model

The effect of late administration of anti-TNFα antibodies on the course of a Salmonella infection in mice was evaluated. Administration of anti-TNFα antiserum as late as day 5 after challenge enhanced a sublethal primary infection with the virulent Salmonella typhimurium C5 in innately resistant (Ity^s) A/J mice by preventing the suppression of exponential bacterial growth in the reticuloendothelial system (RES) (plateau phase). When the anti-TNFα treatment was started well after the establishment of the plateau (day 7) a prompt relapse of the infection occurred, with the rapid resurgence of bacterial growth in the reticuloendothelial system leading to the death of the animals. In contrast, late administration of the antiserum did not affect the clearance from the tissues of an avirulent temperature-sensitive mutant of S. typhimurium C5 (C5TS).Innately susceptible (Ity^s) BALB/c mice immunized with the SL3261 aroA live vaccine acquire solid long-lasting protection from oral challenge with the virulent C5 strain, suppressing growth of the challenge in the RES. Administration of anti-TNFα antibodies on day 8 of a secondary oral infection with strain C5 abrogated vaccine induced protection, with a progressive increase of bacterial numbers in the RES leading to the death of the animals.The results indicate that TNFα is constantly required for the control of virulent salmonellae in the RES, both in a sublethal primary infection in innately resistant mice and also in a secondary infection in innately susceptible mice immunized with a live vaccine. TNFα may not be essential for bacterial clearance of avirulent organisms from the tissues.     

Identification of Three Highly Attenuated Salmonella typhimurium Mutants That Are More Immunogenic and Protective in Mice than a Prototypical aroA Mutant

A panel of Salmonella typhimurium 14028s mutants, which were previously shown to be highly attenuated in the BALB/c mouse model of infection, were analyzed for their potential as live Salmonella oral-vaccine candidates. A prototypical aroA mutant was chosen as a basis of comparison. From the panel of mutants initially chosen for this study, three mutants with comparable levels of attenuation elicited higher Salmonella-specific serum immunoglobulin G (IgG) and/or mucosal secretory-IgA antibody titers than the aroA vaccine strain. The three mutants, CL288, CL401, and CL554, also elicited a better protective immune response than the aroA control strain, after a single oral dose of 1 × 10⁹ to 2 × 10⁹ bacteria.     

Salmonella typhimurium infection in calves: protection and survival of virulent challenge bacteria after immunization with live or inactivated vaccines.

Salmonella typhimurium SL1479, an auxotrophic mutant strain having a complete block in the aromatic biosynthetic pathway and therefore requiring p-aminobenzoic acid and 2,3-dihydroxybenzoate not available in mammalian tissues, was given orally in a dose of 10(10) live bacteria to 4- to 5-week-old calves. Only a mild transient fever response was seen. Strain SL1479 was unable to colonize and persist in the calves for more than 2 weeks. In a vaccination experiment, groups of six calves were (i) orally vaccinated with the live S. typhimurium SL1479 strain, (ii) subcutaneously vaccinated with a heat-inactivated S. typhimurium SVA1232 strain with aluminum hydroxide as adjuvant, or (iii) not vaccinated, to serve as controls. Calves were orally challenged with the live, calf-virulent S. typhimurium SVA44 strain: either 10(6) bacteria (equivalent to 100 25% lethal doses [LD25S]) or 10(9) bacteria (100,000 LD25S doses). The live oral vaccine gave significantly better protection than the heat-vaccinated, subcutaneously injected vaccine since (i) only control calves and calves given the killed vaccine developed profuse diarrhea, (ii) clinically, the mild fever responses seen after challenge in calves given the live vaccine were significantly lower (P less than 0.0005), (iii) autopsies performed 21 days after the challenge infection revealed normal findings in calves given the live vaccine, whereas calves given the killed vaccine showed signs of acute enteritis and chronic salmonellosis, (iv) all 12 calves given either 100 X or 100,000 X the LD25 survived the 21-day observation period; the mean survival time in nonvaccinated calves was 8.0 days; in calves given heat-inactivated vaccine and 100 X the LD25 it was 21.0 days, and in calves given 100,000 X the LD25 it was 11.5 days, (v) the fecal bacterial counts of the challenge S. typhimurium SVA44 strain were significantly lower (P less than 0.0005) in both groups given the live vaccine, and (vi) upon autopsy followed by culture, the qualitative recovery of the challenge strain from the alimentary canals and tissues of calves given the live vaccine was significantly lower (P less than 0.005).     

Augmentation of protective immune responses against sendai virus infection by fungal polysaccharide schizophyllan

When treated with fungal polysaccharide schizophyllan, mice survived otherwise lethal Sendai virus infection. Both intraperitoneal and oral administrations were effective when sonicated schizophyllan with a relative molecular mass (M_r) of 4.6 × 10⁵ was used. Antiviral antibody in the serum could be detected at an earlier time after virus infection and virus spread in the lung was more efficiently inhibited in schizophyllan-treated mice than in untreated controls. Schizophyllan also augmented protective immune responses induced by low doses of a live Sendai virus vaccine that were insufficient to confer complete protection against challenge infection with a virulent strain. On the other hand, schizophyllan did not influence interferon production in mice whether or not infected with Sendai virus. The present results suggest that schizophyllan confers better protection against virus infection through augmentation of antiviral immune responses and can be used as an immune enhancer.     

Highly attenuated Bordetella pertussis strain BPZE1 as a potential live vehicle for delivery of heterologous vaccine candidates

Bordetella pertussis, the causative agent of whooping cough, is a promising and attractive candidate for vaccine delivery via the nasal route, provided that suitable attenuation of this pathogen has been obtained. Recently, the highly attenuated B. pertussis BPZE1 strain has been described as a potential live pertussis vaccine for humans. We investigated here the use of BPZE1 as a live vehicle for heterologous vaccine candidates. Previous studies have reported the filamentous hemagglutinin (FHA), a major B. pertussis adhesin, as a carrier to express foreign antigens in B. pertussis. In this study, we also examined the BrkA autotransporter as a surface display system. Three copies of the neutralizing peptide SP70 from enterovirus 71 (EV71) were fused to FHA or in the passenger domain of BrkA, and each chimera was expressed in BPZE1. The FHA-(SP70)₃ and BrkA-(SP70)₃ chimeras were successfully secreted and exposed at the bacterial surface, respectively. Nasal administration of the live recombinant strains triggered a strong and sustained systemic anti-SP70 antibody response in mice, although the titers and neutralizing activities against EV71 were significantly higher in the sera of mice immunized with the BrkA-(SP70)₃-producing strain. These data indicate that the highly attenuated BPZE1 strain is a potential candidate for vaccine delivery via the nasal route with the BrkA autotransporter as an alternative to FHA for the presentation of the heterologous vaccine antigens.