Nasal vaccination stimulates CD8(+) T cells for potent protection against mucosal Brucella melitensis challenge.

Brucellosis remains a significant zoonotic threat worldwide. Humans and animals acquire infection via their oropharynx and upper respiratory tract following oral or aerosol exposure. After mucosal infection, brucellosis develops into a systemic disease. Mucosal vaccination could offer a viable alternative to conventional injection practices to deter disease. Using a nasal vaccination approach, the ΔznuA B. melitensis was found to confer potent protection against pulmonary Brucella challenge, and reduce colonization of spleens and lungs by more than 2500-fold, with >50% of vaccinated mice showing no detectable brucellae. Furthermore, 10-fold more brucellae-specific, interferon-γ (IFN-γ)-producing CD8(+) T cells than CD4(+) T cells were induced in the spleen and respiratory lymph nodes. Evaluation of pulmonary and splenic CD8(+) T cells from mice vaccinated with ΔznuA B. melitensis revealed that these expressed an activated effector memory (CD44(hi)CD62L(lo)CCR7(lo)) T cells producing elevated levels of IFN-γ, tumor necrosis factor-α, perforin and granzyme B. To assess the relative importance of these increased numbers of CD8(+) T cells, CD8(-/-) mice were challenged with virulent B. melitensis, and they showed markedly increased bacterial loads in organs in contrast to similarly challenged CD4(-/-) mice. Only ΔznuA B. melitensis- and Rev-1-vaccinated CD4(-/-) and wild-type mice, not CD8(-/-) mice, were completely protected against Brucella challenge. Determination of cytokines responsible for conferring protection showed the relative importance of IFN-γ, but not interleukin-17 (IL-17). Unlike wild-type (wt) mice, IL-17 was greatly induced in IFN-γ(-/-) mice, but IL-17 could not substitute for IFN-γ's protection, although an increase in brucellae dissemination was observed upon in vivo IL-17 neutralization. These results show that nasal ΔznuA B. melitensis vaccination represents an attractive means to stimulate systemic and mucosal immune protection via CD8(+) T-cell engagement.

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Recombinant Salmonella vaccination technology and its application to human bacterial pathogens.

Salmonella enterica is a Gram-negative intracellular bacterial pathogen which causes salmonellosis in humans and animals. During the past several decades, extensive studies have shown that the attenuated Salmonella vaccine vector is an optimal vehicle for delivering passenger antigens to mucosal sites to induce humoral, cellular, and mucosal immunity. This immunity leads to protection against challenges with the wild-type pathogens from which the passenger antigens were derived. A myriad of studies have demonstrated that using attenuated Salmonella vaccines for recombinant multivalent vaccine construction has multiple advantages. In this review, we summarize these advantages and further evaluate the Salmonella-based vaccines against five bacterial diseases. Four of these are Gram-negative pathogens- Escherichia coli, Helicobacter pylori, Shigella dysenteriae, and Yersinia pestis-and one is a mycobacterial pathogen, Mycobacterium tuberculosis. Apart from H. pylori, the Salmonella-based vaccines against the other four pathogens exhibit excellent performance in safety, immunogenicity, and protection. These properties qualify them to be as a new generation of vaccines for preventing infections from bacterial pathogens.

Loss of sialic acid binding domain redirects protein σ1 to enhance M cell-directed vaccination.

Ovalbumin (OVA) genetically fused to protein sigma 1 (pσ1) results in tolerance to both OVA and pσ1. Pσ1 binds in a multi-step fashion, involving both protein- and carbohydrate-based receptors. To assess the relative pσ1 components responsible for inducing tolerance and the importance of its sialic binding domain (SABD) for immunization, modified OVA-pσ1, termed OVA-pσ1(short), was deleted of its SABD, but with its M cell targeting moiety intact, and was found to be immunostimulatory and enhanced CD4(+) and CD8(+) T cell proliferation. When used to nasally immunize mice given with and without cholera toxin (CT) adjuvant, elevated SIgA and serum IgG responses were induced, and OVA-pσ1(s) was more efficient for immunization than native OVA+CT. The immune antibodies (Abs) were derived from elevated Ab-forming cells in the upper respiratory tissues and submaxillary glands and were supported by mixed Th cell responses. Thus, these studies show that pσ1(s) can be fused to vaccines to effectively elicit improved SIgA responses.

Expression of Escherichia coli virulence usher protein attenuates wild-type Salmonella.

Generation of a live attenuated vaccine for bacterial pathogens often requires prior knowledge of the pathogen's virulence factors. We hypothesized an alternative approach of heterologous gene expression would make a wild-type (wt) pathogen more susceptible to host cell killing, thus, resulting in immunization. As proof of concept, the heterologous expression of enterotoxigenic E. coli (ETEC) colonization factor antigen I (CFA/I) was tested to attenuate Salmonella. The overexpression of CFA/I resulted in significant attenuation of wt Salmonella. In-depth studies revealed the attenuation depended on the co-expression of chaperone (CfaA) and usher (CfaC) proteins. Remarkably, the CfaAC-attenuated Salmonella conferred protection against wt Salmonella challenge. Mechanistic study indicated CfaAC made Salmonella outer membranes permeable, causing Salmonella to be vulnerable to host destruction. Thus, enhancing bacterial permeability via CfaAC represents an alternative method to attenuate pathogens despite the presence of unknown virulence factors.

DNA vaccination of bison to brucellar antigens elicits elevated antibody and IFN-γ responses.

Brucella abortus remains a threat to the health and well-being of livestock in states bordering the Greater Yellowstone Area. During the past several years, cohabitation of infected wildlife with cattle has jeopardized the brucellosis-free status of Idaho, USA; Wyoming, USA; and Montana, USA. Current livestock B. abortus vaccines have not proven to be efficacious in bison (Bison bison) or elk (Cervus elaphus nelsoni). One problem with the lack of vaccine efficacy may stem from the failure to understand wildlife immune responses to vaccines. In an attempt to understand their immune responses, bison were vaccinated with eukaryotic DNA expression vectors encoding the Brucella periplasmic protein, bp26, and the chaperone protein, trigger factor (TF). These DNA vaccines have previously been shown to be protective against Brucella infection in mice. Bison were immunized intramuscularly at weeks 0, 2, and 4 with bp26 and TF DNA vaccines plus CpG adjuvant or empty vector (control) plus CpG. Blood samples were collected before vaccination and at 8, 10, and 12 wk after primary vaccination. The results showed that bison immunized with bp26 and TF DNA vaccines developed enhanced antibody, proliferative T cell, and interferon-gamma (IFN-γ) responses upon in vitro restimulation with purified recombinant bp26 or TF antigens, unlike bison immunized with empty vector. Flow cytometric analysis revealed that the percentages of CD4(+) and CD8(+) T lymphocytes from the DNA-vaccinated groups were significantly greater than they were for those bison given empty vector. These data suggest that DNA vaccination of bison may elicit strong cellular immune responses and serve as an alternative for vaccination of bison for brucellosis.

Protective live oral brucellosis vaccines stimulate Th1 and th17 cell responses.

Zoonotic transmission of brucellosis often results from exposure to Brucella-infected livestock, feral animals, or wildlife or frequently via consumption of unpasteurized milk products or raw meat. Since natural infection of humans often occurs by the oral route, mucosal vaccination may offer a means to confer protection for both mucosal and systemic tissues. Significant efforts have focused on developing a live brucellosis vaccine, and deletion of the znuA gene involved in zinc transport has been found to attenuate Brucella abortus. A similar mutation has been adapted for Brucella melitensis and tested to determine whether oral administration of ΔznuA B. melitensis can confer protection against nasal B. melitensis challenge. A single oral vaccination with ΔznuA B. melitensis rapidly cleared from mice within 2 weeks and effectively protected mice upon nasal challenge with wild-type B. melitensis 16M. In 83% of the vaccinated mice, no detectable brucellae were found in their spleens, unlike with phosphate-buffered saline (PBS)-dosed mice, and vaccination also enhanced the clearance of brucellae from the lungs. Moreover, vaccinated gamma interferon-deficient (IFN-γ(-/-)) mice also showed protection in both spleens and lungs, albeit protection that was not as effective as in immunocompetent mice. Although IFN-γ, interleukin 17 (IL-17), and IL-22 were stimulated by these live vaccines, only RB51-mediated protection was codependent upon IL-17 in BALB/c mice. These data suggest that oral immunization with the live, attenuated ΔznuA B. melitensis vaccine provides an attractive strategy to protect against inhalational infection with virulent B. melitensis.

DeltaznuADeltapurE Brucella abortus 2308 mutant as a live vaccine candidate.

To create a new, safe brucellosis live vaccine, a double mutant strain was constructed from Brucella abortus 2308. Using the DeltaznuA B. abortus 2308 mutant, a second mutation was introduced by deleting purE gene. The DeltaznuA DeltapurE B. abortus 2308 strain was less capable of surviving in macrophages. When evaluated in vivo, it was cleared within 8 weeks (wks) from mice, causing significantly less inflammation than spleens obtained from wild-type B. abortus 2308-infected mice. Furthermore, two doses of DeltaznuA DeltapurE B. abortus 2308 conferred 0.79 log protection, similar to S19 as did a single dose of DeltaznuA B. abortus 2308. Thus, this study shows the DeltaznuA DeltapurE B. abortus 2308 strain to be a potential livestock vaccine candidate.

Nasal immunization with recombinant Brucella melitensis bp26 and trigger factor with cholera toxin reduces B. melitensis colonization.

bp26 and trigger factor (Tf) DNA vaccines have previously been shown to protect against Brucella infection. In this study, purified bp26 and Tf proteins were tested in BALB/c mice for immunity and protection. The results showed that intranasal (i.n.) immunization with bp26 and Tf in conjunction with cholera toxin (CT) adjuvant elicit both elevated mucosal and systemic immune responses. While nasal immunization with either bp26 or Tf elicited elevated antibody responses, co-immunization with both enhanced anti-Tf immunity, suggesting bp26 adjuvant activity. Evaluation of serum IgG subclass responses showed elevated IgG1 titers. Further analysis to discern the source of immune B cells revealed effective immunization of respiratory tissues. However, Tf stimulated a significantly higher level of cytokine-forming cells (CFC) than bp26. These results imply that co-immunization of bp26 and Tf proteins elicits synergistic cooperation to stimulate the immune system. When immunized mice were challenged with B. melitensis 16M, bp26-plus Tf-immunized mice showed no difference in splenic weights but harbored three-fold less bacterial CFU when compared to sPBS-immunized control mice.

Oral vaccination with salmonella simultaneously expressing Yersinia pestis F1 and V antigens protects against bubonic and pneumonic plague.

The gut provides a large area for immunization enabling the development of mucosal and systemic Ab responses. To test whether the protective Ags to Yersinia pestis can be orally delivered, the Y. pestis caf1 operon, encoding the F1-Ag and virulence Ag (V-Ag) were cloned into attenuated Salmonella vaccine vectors. F1-Ag expression was controlled under a promoter from the caf1 operon; two different promoters (P), PtetA in pV3, PphoP in pV4, as well as a chimera of the two in pV55 were tested. F1-Ag was amply expressed; the chimera in the pV55 showed the best V-Ag expression. Oral immunization with Salmonella-F1 elicited elevated secretory (S)-IgA and serum IgG titers, and Salmonella-V-Ag(pV55) elicited much greater S-IgA and serum IgG Ab titers than Salmonella-V-Ag(pV3) or Salmonella-V-Ag(pV4). Hence, a new Salmonella vaccine, Salmonella-(F1+V)Ags, made with a single plasmid containing the caf1 operon and the chimeric promoter for V-Ag allowed the simultaneous expression of F1 capsule and V-Ag. Salmonella-(F1+V)Ags elicited elevated Ab titers similar to their monotypic derivatives. For bubonic plague, mice dosed with Salmonella-(F1+V)Ags and Salmonella-F1-Ag showed similar efficacy (>83% survival) against approximately 1000 LD(50) Y. pestis. For pneumonic plague, immunized mice required immunity to both F1- and V-Ags because the mice vaccinated with Salmonella-(F1+V)Ags protected against 100 LD(50) Y. pestis. These results show that a single Salmonella vaccine can deliver both F1- and V-Ags to effect both systemic and mucosal immune protection against Y. pestis.

Deletion of znuA virulence factor attenuates Brucella abortus and confers protection against wild-type challenge.

znuA is known to be an important factor for survival and normal growth under low Zn(2+) concentrations for Escherichia coli, Haemophilus spp., Neisseria gonorrhoeae, and Pasteurella multocida. We hypothesized that the znuA gene present in Brucella melitensis 16 M would be similar to znuA in B. abortus and questioned whether it may also be an important factor for growth and virulence of Brucella abortus. Using the B. melitensis 16 M genome sequence, primers were designed to construct a B. abortus deletion mutant. A znuA knockout mutation in B. abortus 2308 (DeltaznuA) was constructed and found to be lethal in low-Zn(2+) medium. When used to infect macrophages, DeltaznuA B. abortus showed minimal growth. Further study with DeltaznuA B. abortus showed that its virulence in BALB/c mice was attenuated, and most of the bacteria were cleared from the spleen within 8 weeks. Protection studies confirmed the DeltaznuA mutant as a potential live vaccine, since protection against wild-type B. abortus 2308 challenge was as effective as that obtained with the RB51 or S19 vaccine strain.

Partially assembled K99 fimbriae are required for protection.

Antibodies to K99 fimbriae afford protection to F5+ bovine enterotoxigenic Escherichia coli (ETEC). Previous studies show that murine dams immunized with Salmonella vaccine vectors stably expressing K99 fimbriae confer protection to ETEC-challenged neonatal pups. To begin to address adaptation of the K99 scaffold to display heterologous B- and T-cell epitopes, studies were conducted to determine how much of the assembled K99 fimbria is required to maintain protective immunity. Sequential deletions in the K99 gene clusters were made, resulting in diminished localization of the K99 fimbrial subunit in the outer membrane. As placement of the K99 fimbrial subunit became progressively contained within the vaccine vector, diminished immunoglobulin A (IgA) and IgG1 antibody titers, as well as diminished Th2-type cytokine responses, were observed in orally immunized mice. Deletion of fanGH, which greatly reduced the export of the fimbrial subunit to the outer membrane, showed only partial reduction in protective immunity. By contrast, deletion of fanDEFGH, which also reduced the export of the fimbrial subunit to the outer membrane but retained more subunit in the cytoplasm, resulted in protective immunity being dramatically reduced. Thus, these studies showed that retention of K99 fimbrial subunit as native fimbriae or with the deletion of fanGH is sufficient to confer protection.

Selection of protective epitopes for Brucella melitensis by DNA vaccination.

The Brucella melitensis 16M genome was examined for proteins in excess of 100 amino acids and for immunogenicity-associated genes. One subset of 32 annotated genes or open reading frames was identified, and each of these were cloned into the eukaryotic vector pcDNA3.1. Purified recombinant plasmids were used to intramuscularly (i.m.) immunize BALB/c mice. After challenge with B. melitensis 16M strain, two protective antigens were found: the periplasmic protein, bp26, and the chaperone protein, trigger factor (TF). Protective efficacy was confirmed with DNA vaccines for these two B. melitensis proteins and, when combined, protection against wild-type challenge was significantly enhanced. Both proteins were found to be immunogenic since elevated serum immunoglobulin G (IgG) antibodies without a specific IgG subclass bias were induced subsequent to i.m. DNA immunization. Antigen-restimulation assays revealed that bp26 and TF stimulated gamma interferon and only bp26 induced interleukin-4 (IL-4), IL-5, and IL-6 cytokines as measured by cytokine enzyme-linked immunospot assay. These collective results suggest that both bp26 and TF are excellent candidates for use in future vaccination studies against brucellosis.

Enhanced immunoglobulin A response and protection against Salmonella enterica serovar typhimurium in the absence of the substance P receptor.

The development of the neurokinin-1 receptor-deficient (NK1R(-/-)) mouse permitted inquiry into the regulation of secretory immunoglobulin A (S-IgA) responses by substance P (SP) after oral immunization with a Salmonella enterica serovar Typhimurium vector expressing colonization factor antigen I (CFA/I) from enterotoxigenic Escherichia coli. In NK1R(-/-) mice, mucosal and serum IgA anti-CFA/I fimbrial responses were augmented, while secreted IgG anti-CFA/I fimbrial responses remained unaffected compared to those of BALB/c (NK1R(+/+)) mice. Supportive antibody-forming cells were present in the small intestinal lamina propria and spleen. To gain insight as to why the augmented S-IgA responses occurred, minimally, the responses were not attributed to differences in vaccine colonization of Peyer's patch (PP) and spleen or in their respective tissue weights. However, these S-IgA responses were supported by increased numbers of PP CD4(+) T helper (Th) cells secreting interleukin-5 (IL-5) and IL-6 and splenic CD4(+) Th cells secreting IL-6 compared to NK1R(+/+) mice. Challenge of naive NK1R(-/-) mice with wild-type Salmonella showed improved median survival compared to naive NK1R(+/+) mice. Data from peritoneal macrophage infection studies suggest that this survival is in part contributed by increased IL-10 production. Oral vaccination with Salmonella CFA/I or Salmonella vector showed no significant differences in conferred protection against wild-type challenge for either NK1R(-/-) or NK1R(+/+) mice. Thus, these studies suggest that SP mediation contributes to proinflammatory responses to Salmonella infections.