Mucosal priming of newborn mice with S. Typhi Ty21a expressing anthrax protective antigen (PA) followed by parenteral PA-boost induces B and T cell-mediated immunity that protects against infection bypassing maternal antibodies

The currently licensed anthrax vaccine has several limitations and its efficacy has been proven only in adults. Effective immunization of newborns and infants requires adequate stimulation of their immune system, which is competent but not fully activated. We explored the use of the licensed live attenuated S. Typhi vaccine strain Ty21a expressing Bacillus anthracis protective antigen [Ty21a(PA)] followed PA-alum as a strategy for immunizing the pediatric population. Newborn mice primed with a single dose of Ty21a(PA) exhibited high frequencies of mucosal IgA-secreting B cells and IFN-γ-secreting T cells during the neonatal period, none of which was detected in newborns immunized with a single dose of PA-alum. Priming with Ty21a(PA) followed by PA-boost resulted in high levels of PA-specific IgG, toxin neutralizing and opsonophagocytic antibodies and increased frequency of bone marrow IgG plasma cells and memory B cells compared with repeated immunization with PA-alum alone. Robust B and T cell responses developed even in the presence of maternal antibodies. The prime-boost protected against systemic and respiratory infection. Mucosal priming with a safe and effective S. Typhi-based anthrax vaccine followed by PA-boost could serve as a practical and effective prophylactic approach to prevent anthrax early in life.     

A comparison of immunogenicity and in vivo distribution of Salmonella enterica serovar Typhi and Typhimurium live vector vaccines delivered by mucosal routes in the murine model

We evaluated the immune responses elicited by attenuated Salmonella enterica serovar Typhi vaccine strain CVD 908-htrA and serovar Typhimurium strain SL3261 alone or as live vectors carrying a plasmid encoding fragment C of tetanus toxin (pTETnir15) in mice immunized intranasally and orogastrically, as well as the in vivo distribution of vaccine organisms following immunization. Higher serologic and proliferative responses against both vector and the foreign antigen were elicited when vaccines were delivered by intranasal route. Whereas both Salmonella strains were detected in the nasal tissue, lungs, and Peyer's patches following intranasal and orogastric immunization, larger numbers of vaccine organisms were recovered from these tissues when the vaccines were delivered intranasally.     

Oral immunisation with live aroA attenuated Salmonella enterica serovar Typhimurium expressing the Yersinia pestis V antigen protects mice against plague

Bubonic and pneumonic plague are caused by the bacterium Yersinia pestis. The V antigen of Y. pestis is a protective antigen against plague. In this study, an aroA attenuated strain of Salmonella enterica serovar Typhimurium (SL3261) has been used to deliver the Y. pestis V antigen as a candidate oral plague vaccine. SL3261 was transformed with the expression plasmid pTrc-LcrV, containing the lcrV gene encoding V antigen. Immunoblot analysis showed V antigen expression in SL3261 in vitro and intragastric immunisation of mice with the recombinant Salmonella resulted in the induction of V antigen-specific serum antibody responses and afforded protection against Y. pestis challenge. However, the antibody responses induced by the recombinant Salmonella did not correlate with the protection afforded, indicating that immune responses other than antibody may play a role in the protection afforded against plague by this candidate vaccine.     

Protection of rhesus macaques against inhalational anthrax with a Bacillus anthracis capsule conjugate vaccine

The efficacy of currently licensed anthrax vaccines is largely attributable to a single Bacillus anthracis immunogen, protective antigen. To broaden protection against possible strains resistant to protective antigen-based vaccines, we previously developed a vaccine in which the anthrax polyglutamic acid capsule was covalently conjugated to the outer membrane protein complex of Neisseria meningitidis serotype B and demonstrated that two doses of 2.5 μg of this vaccine conferred partial protection of rhesus macaques against inhalational anthrax . Here, we demonstrate complete protection of rhesus macaques against inhalational anthrax with a higher 50 μg dose of the same capsule conjugate vaccine. These results indicate that B. anthracis capsule is a highly effective vaccine component that should be considered for incorporation in future generation anthrax vaccines.     

A Salmonella enterica serovar Typhi vaccine expressing Yersinia pestis F1 antigen on its surface provides protection against plague in mice

A recombinant strain of attenuated Salmonella enterica serovar Typhi surface-expressing Yersinia pestis F1 antigen was generated by transforming strain BRD1116 (aroA aroC htrA) with plasmid pAH34L encoding the Y. pestis caf operon. BRD1116/pAH34L was stable in vitro and in vivo. An immunisation regimen of two intranasal doses of 1 x 10(8) cfu of BRD1116/pAH34L given intranasally to mice 7 days apart induced the strongest immune response compared to other regimens and protected 13 out of 20 mice from lethal challenge with Y. pestis. Intranasal immunisation of mice constitutes a model for oral immunisation with Salmonella vaccines in humans. Thus, the results demonstrate that attenuated strains of S. enterica serovar Typhi which express Y. pestis F1 antigen may be developed to provide an oral vaccine against plague suitable for use in humans.     

Core-linked LPS expression of Shigella dysenteriae serotype 1 O-antigen in live Salmonella Typhi vaccine vector Ty21a: preclinical evidence of immunogenicity and protection

Shigella dysenteriae serotype 1 (S. dysenteriae 1) causes severe shigellosis that is typically associated with high mortality. Antibodies against Shigella serotype-specific O-polysaccharide (O-Ps) have been shown to be host protective. In this study, the rfb locus and the rfp gene with their cognate promoter regions were PCR-amplified from S. dysenteriae 1, cloned, and sequenced. Deletion analysis showed that eight rfb ORFs plus rfp are necessary for biosynthesis of this O-Ps. A tandemly-linked rfb-rfp gene cassette was cloned into low copy plasmid pGB2 to create pSd1. Avirulent Salmonella enterica serovar Typhi (S. Typhi) Ty21a harboring pSd1 synthesized S. Typhi 9, 12 LPS as well as typical core-linked S. dysenteriae 1 LPS. Animal immunization studies showed that Ty21a (pSd1) induces protective immunity against high stringency challenge with virulent S. dysenteriae 1 strain 1617. These data further demonstrate the utility of S. Typhi Ty21a as a live, bacterial vaccine delivery system for heterologous O-antigens, supporting the promise of a bifunctional oral vaccine for prevention of shigellosis and typhoid fever.     

Anthrax capsule vaccine protects against experimental infection

Efficacy of a poly-gamma-D-glutamic acid anthrax capsule vaccine was assessed in a mouse model of infection. Capsule by itself was protective against lethal challenge with a toxin(-), capsule(+) Bacillus anthracis strain. Conjugation of capsule to bovine serum albumin resulted in enhanced IgG anti-capsule antibodies measured by ELISA, but completely abrogated the protection. The protective unconjugated capsule vaccine elicited significantly higher IgM titers and opsonic activity than did the non-protective capsule conjugate. When tested against a fully virulent toxin(+), capsule(+) B. anthracis strain, neither capsule nor protective antigen alone was protective. However, the combination of the two protected against a lethal challenge. These results suggest that capsule may enhance the protection afforded by protective antigen vaccines against anthrax if opsonizing antibodies are produced. Surprisingly, some protection was also observed when protective antigen was conjugated to itself.     

Monitoring of ELISA-reactive antibodies against anthrax protective antigen (PA), lethal factor (LF), and toxin-neutralising antibodies in serum of individuals vaccinated against anthrax with the PA-based UK anthrax vaccine

The human anthrax vaccines currently licensed contain the protective antigen (PA) of Bacillus anthracis as main antigen together with traces of some other bacillus components, e.g. lethal factor (LF). The present study aimed at monitoring the course of specific antibody titres against PA and LF by enzyme linked immunosorbent assays (ELISA), as well as the levels of toxin-neutralising antibodies, in 11 volunteers vaccinated with the human anthrax vaccine UK. After an initial seroconversion in all vaccinees, a significant reduction of both antibody titres against PA and LF, and of neutralising antibodies, was detected just prior to a vaccine boost 6 months after completion of the basic immunisation. Following the booster injection, titres increased again to levels comparable to those after the fourth immunisation. ELISA titres against PA correlated significantly with neutralising antibodies (r=0.816, p<0.001). Therefore, the less work- and time-consuming ELISA should be favoured to monitor the efficacy of an anthrax vaccination.     

Intragastric immunization with recombinant Lactobacillus casei expressing flagellar antigen confers antibody-independent protective immunity against Salmonella enterica serovar Enteritidis

A recombinant Lactobacillus casei expressing a flagellar antigen from Salmonella enterica serovar Enteritidis was constructed and evaluated as a mucosal vaccine. Intragastric immunization of the recombinant strain conferred protective immunity against Salmonella infection in mice. This immunization did not result in antigen-specific antibody in either feces or sera but induced the release of IFN-gamma on restimulation of primed lymphocytes ex vivo. The results suggested that the protective efficacy provided by flagellin-expressing L. casei is mainly attributable to cell-mediated immune responses. In addition, an adjuvant-type effect of the antigen delivery system with L. casei was also observed.     

Recombinant Bacillus anthracis spore proteins enhance protection of mice primed with suboptimal amounts of protective antigen

Inactivated Bacillus anthracis spores given with protective antigen (PA) contribute to immunity against anthrax in several animal models. Antiserum raised against whole irradiated B. anthracis spores has been shown to have anti-germination and opsonic activities in vitro. Based on these observations, we hypothesized that surface-exposed spore proteins might serve as supplemental components of a PA-based anthrax vaccine. The protective anti-spore serum was tested for reactivity with recombinant forms of 30 proteins known, or believed to be, present within the B. anthracis exosporium. Eleven of those proteins were reactive with this antiserum, and, subsequently a subset of this group was used to generate rabbit polyclonal antibodies. These sera were evaluated for recognition of the immunogens on intact spores generated from Sterne strain, as well as from an isogenic mutant lacking the spore surface protein Bacillus collagen-like antigen (BclA). The data were consistent with the notion that the antigens in question were located beneath BclA on the basal surface of the exosporium. A/J mice immunized with either the here-to-for hypothetical protein p5303 or the structural protein BxpB, each in combination with subprotective levels of PA, showed enhanced protection against subcutaneous spore challenge. While neither anti-BxpB or anti-p5303 antibodies reduced the rate of spore germination in vitro, both caused increased uptake and lead to a higher rate of destruction by phagocytic cells. We conclude that by facilitating more efficient phagocytic clearance of spores, antibodies against individual exosporium components can contribute to protection against B. anthracis infection.     

DNA vaccination against anthrax in mice-combination of anti-spore and anti-toxin components

The predominant antigen in human anthrax vaccines is the protective antigen (PA) of Bacillus anthracis. To address the question whether immune responses against B. anthracis spores can improve the protectivity of a PA-based DNA vaccine against anthrax, we designed a eucaryotic expression plasmid encoding the exosporium antigen BclA, which is a collagen-like surface protein. This plasmid, pSecTag BclA, carries a secretion signal for the recombinant antigen. Using NMRI mice we compared the effects of immunisation with a combination of PA- and BclA-encoding plasmid DNA, to immunisations with either PA- or BclA-encoding pDNA. After three immunisations the mice were infected with 25 x LD(50) of B. anthracis Ames spores. The plasmid pSecTag BclA, induced high BclA-specific antibody responses. Vaccination with a combination of PA- and BclA-encoding pDNA led to significantly better survival than immunisation with only PA- or only BclA-encoding plasmids.     

Passive transfer of protection against Bacillus anthracis infection in a murine model

Passive transfer of lymphocytes and sera from mice immunised using two different formulations containing recombinant protective antigen (rPA) have been used to further elucidate the mechanism of protection against Bacillus anthracis infection. The results demonstrated that an antibody response maybe important in protection against B. anthracis infection, under the conditions tested. The results provide further data for the development of an improved anthrax vaccine.     

Efficacious, nontoxigenic Bacillus anthracis spore vaccines based on strains expressing mutant variants of lethal toxin components

We reported previously on the development of a Bacillus anthracis vaccine strain expressing high levels of recombinant protective antigen (rPA) [Cohen et al., Infec Immun 2000;68(8):4549-58]. To further explore the potential of the B. anthracis platform, we generated several attenuated strains expressing lethal toxin components PA and LF, which are biologically inactive, yet retain their antigenic properties. A single injection of 5 x 10(7) spores of one of these strains, carrying PA mutation at a site involved in effector translocation (residues 313-314) was shown to resemble wild type PA in inducing production of high levels of anti-PA neutralizing antibodies and producing effective protective immunity for 12 months. Long-term protection and persistence of functional antibody titers was observed after the gradual elimination of spores from guinea pig tissues 3 months after injection and in the measurable absence of bacteria in tissues. The mutant toxin components could, thus be an effective alternatives to their native counterparts when presented to the immune system in context of a live B. anthracis strain. These live vaccine prototypes may serve as a platform for future multi-component vaccines.     

Immunogenicity of a subunit vaccine against Bacillus anthracis

The current approved vaccine against anthrax is based on protective antigen (PA) of Bacillus anthracis, requires six injections over an 18-month period and has a known history of side effects. Therefore, there is significant effort towards developing an improved vaccine against B. anthracis. Here we separately engineered and expressed domain 4 of PA (PAD4) and domain 1 of lethal factor (LFD1) as fusions to lichenase (LicKM), a thermostable enzyme from Clostridium thermocellum, and transiently expressed these fusions in Nicotiana benthamiana. Plant-produced antigens were combined and immunogenicity was evaluated in mice. All animals that received the experimental vaccine developed high antibody titers that were predominantly IgG1 and were able to neutralize the effects of LeTx in vitro.     

Efficacy of a capsule conjugate vaccine against inhalational anthrax in rabbits and monkeys

Bacillus anthracis, the causative agent of anthrax, is recognized as one of the most serious bioterrorism threats. The current human vaccines are based on the protective antigen component of the anthrax toxins. Concern about possible vaccine resistant strains and reliance on a single antigen has prompted the search for additional immunogens. Bacterial capsules, as surface-expressed virulence factors, are well-established components of several licensed vaccines. In a previous study we showed that an anthrax vaccine consisting of the B. anthracis poly-γ-d-glutamic acid capsule covalently conjugated to the outer membrane protein complex of Neisseria meningitidis serotype B protected mice against parenteral B. anthracis challenge. Here we tested this vaccine in rabbits and monkeys against an aerosol spore challenge. The vaccine induced anti-capsule antibody responses in both species, measured by ELISA and a macrophage opsono-adherence assay. While rabbits were not protected against a high aerosol challenge dose, significant protection was observed in monkeys receiving the capsule conjugate vaccine. The results confirm that the capsule is a protective immunogen against anthrax, being the first non-toxin antigen shown to be efficacious in monkeys and suggest that addition of capsule may broaden and enhance the protection afforded by protective antigen-based vaccines.     

Enhanced immune response by amphotericin B following NS1 protein prime-oral recombinant Salmonella vaccine boost vaccination protects mice from dengue virus challenge

A recombinant vaccine strain SL3261/pLT105 of attenuated aroA Salmonella enterica serovar Typhimurium SL3261 strain expressing a secreted dengue virus type 2 non-structural NS1 and Yersinia pestis F1 (Caf1) fusion protein, rNS1:Caf1, was generated. Immunological evaluation was performed by prime-boost vaccine regimen. Oral immunization of mice with 1 x 10(9)cfu of SL3261/pLT105 only induced low levels of NS1-specific antibody response and protective immunity following dengue virus challenge. The parenteral NS1 protein priming-oral Salmonella boosting protocol enhanced both NS1-specific serum IgG response and protective efficacy as compared to mice immunized with each type vaccine alone. Addition of an antifungal antibiotic amphotericin B (AmB) to Salmonella vaccine further enhanced the synergic effects of prime-boost vaccine regimen on the elicited NS1-specific serum IgG response and the protective efficacy. Together, the results demonstrated that the rNS1:Caf1 producing Salmonella SL3261/pLT105 strain fails to provide effective protection as an oral vaccine alone despite co-administration of AmB as an adjuvant capable of enhancing the immune responses, and moreover, the protein priming-oral Salmonella vaccine boosting approach in combination with AmB as an immunization regimen may have the potential to be further explored as an alternative approach for dengue vaccine development.     

Plasmid-based vaccination with candidate anthrax vaccine antigens induces durable type 1 and type 2 T-helper immune responses

The current anthrax vaccine imparts protective immunity by generating a humoral immune response against a single antigen, the PA exotoxin subunit. While this response neutralizes the two anthrax exotoxins and protects the recipient from toxin-related mortality, the recipient is not protected from spore germination, infection, and/or bacteremia. Moreover, protective immunity against PA must be generated via a lengthy injection schedule and maintained by a yearly booster. In an effort to improve upon the current vaccine formulation, we screened six of seven known virulence factors encoded by Bacillus anthracis epigenetic elements pXO1 and pXO2 as well as the major surface proteins EA1 and SAP. Screening was carried out in conjunction with a plasmid-based technology known for its ability to generate type 1 and type 2 T-helper responses. Long-term high level antibody titers were generated against the products of eag (EA1), sap (SAP), and the capA capsule synthesis subunit in vivo. Further analysis of PA- and EA1-vaccinated mice demonstrated antigen-specific type 1 helper responses including IFN-gamma secretion and lysis of EA1- or PA-loaded macrophages; further, an EA1 T-cell epitope was identified. The results demonstrate that anthrax antigens other than PA might be suitable for the generation of durable immune responses against anthrax.     

N-fragment of edema factor as a candidate antigen for immunization against anthrax

The nontoxic N-terminal fragment of Bacillus anthracis edema factor (EF) was evaluated as a candidate antigen in an anthrax vaccine using a replication-incompetent adenoviral vector. An E1/E3 deleted adenovirus (Ad/EFn) encoding the N-terminal region 1-254 amino acids of the edema factor (EFn) was constructed using the native DNA sequence of EFn. Intramuscular immunization three times with 10(8) plaque forming units (pfu)/dose of Ad/EFn in A/J mice resulted in 37% and 57% protection against a subcutaneous challenge with B. anthracis Sterne strain spores at a dosage of 200 x LD50 and 100 x LD50, respectively. EF-specific serum IgG responses (including total IgG, IgG1, and IgG2a isotype titers) were robust in the Ad/EFn immunized animals. Interestingly, anti-EF antibodies cross-reacted with anthrax lethal factor (LF), and had a neutralizing capability against both anthrax lethal toxin (Letx) and edema toxin (Edtx), as demonstrated by in vitro toxin neutralization assays using J774A.1 mouse macrophage and Chinese hamster ovary cell (CHO), respectively. Our data suggest that EF plays a role in eliciting protective immunity against anthrax, and that it should be included in a new generation multi-component subunit vaccine.     

Recombinant Salmonella enterica serovar Typhi in a prime-boost strategy

This study investigated the utility of attenuated Salmonella enterica serovar Typhi strain CVD 908-htrA (908 h) in a heterologous prime-boost strategy. Mice primed intranasally (i.n.) with 908 h expressing fragment C (Frag C) of tetanus toxin and boosted intramuscularly (i.m.) with tetanus toxoid (TT) mounted enhanced and accelerated serum IgG anti-Frag C responses in comparison to unprimed, vector-primed and homologously-primed and boosted mice. Serum antitoxin responses were also determined; mice that were vaccinated following a heterologous prime-boost regimen exhibited the highest levels of Frag C-specific toxin neutralizing antibodies 1 week after boosting. Mice primed and boosted i.m. with TT developed a significantly greater proportion of serum IgG1 antibodies and weaker IFN-gamma levels in contrast to those primed intranasally (i.n.) with rS. Typhi that were homologously or heterologously boosted. These encouraging pre-clinical data provide a rational basis for undertaking a pilot clinical trial to evaluate this strategy. An ability to stimulate enhanced, accelerated responses to parenteral vaccination following mucosal priming may be advantageous in the immunoprophylaxis of many infectious diseases, including those of biodefense importance.     

Sortase-conjugation generates a capsule vaccine that protects guinea pigs against Bacillus anthracis

Capsules protect bacteria against phagocytic clearance. Capsular polysaccharides or polyglutamates have evolved also to resist antigen presentation by immune cells, thereby interfering with the production of opsonophagocytic antibodies. Linking capsular material to a carrier protein stimulates its presentation to the immune system. For many conjugate vaccines this is achieved by a process of random chemical cross-linking. Here we describe a new technology, designated sortase-conjugation, which generates a single amide bond between the C-terminal end of a carrier protein and the capsular material. Sortase-conjugation was used to link the poly-D-γ-glutamic acid (PDGA) capsule of Bacillus anthracis to the receptor binding domain (D4) of protective antigen (PagA). When used as a vaccine, PDGA-D4 conjugate elicited robust antibody responses against both capsule and D4. Immunization with PDGA-D4 afforded guinea pigs complete protection against anthrax challenge with wild-type or pagA mutant B. anthracis Ames.