Expression and immunogenicity of pertussis toxin S1 subunit-tetanus toxin fragment C fusions in Salmonella typhi vaccine strain CVD 908.
ABSTRACT: Salmonella typhi vaccine strain CVD 908 can deliver heterologous antigens to the host immune system following mucosal immunization. Stable expression of foreign proteins in Salmonella cells often requires antigen-specific engineering strategies. Fusion of antigens to stabilizing proteins has proven to be a successful strategy for rescuing otherwise unstable proteins. We designed plasmids to allow the fusion of antigens to the amino terminus or carboxyl terminus of fragment C of tetanus toxin, separated by a 4-amino-acid hinge region. Towards the ultimate goal of developing a live oral diphtheria-pertussis-tetanus vaccine, we used these plasmids to stably express the S1 subunit of pertussis toxin in CVD 908. Driven by the anaerobically inducible nirB promoter, the S1 subunit alone was expressed poorly in Salmonella cytoplasm. In contrast, hybrid proteins with S1 fused to either the amino or carboxyl terminus of fragment C were expressed at a high level in CVD 908 and were recognized in Western blot (immunoblot) analysis by monoclonal antibodies directed to S1 and to fragment C. Mice were immunized by the oral or intranasal routes with CVD 908 derivatives harboring these recombinant plasmids. All fusion proteins elicited serum antibody responses to fragment C following intranasal immunization, whereas oral inoculation did not. The configuration of antigens constituting the fusion was critical; S1 fused to the amino terminus of fragment C was less effective than S1 fused to the carboxyl terminus in generating anti-fragment C antibodies. CVD 908 expressing truncated S1 fused to the carboxyl terminus of fragment C elicited neutralizing serum pertussis antitoxin following intranasal immunization of mice.
Project description:Two Salmonella enterica serovar Typhi strains that express and export a truncated version of Plasmodium falciparum circumsporozoite surface protein (tCSP) fused to Salmonella serovar Typhi cytolysin A (ClyA) were constructed as a first step in the development of a preerythrocytic malaria vaccine. Synthetic codon-optimized genes (t-csp1 and t-csp2), containing immunodominant B- and T-cell epitopes present in native P. falciparum circumsporozoite surface protein (PfCSP), were fused in frame to the carboxyl terminus of the ClyA gene (clyA::t-csp) in genetically stabilized expression plasmids. Expression and export of ClyA-tCSP1 and ClyA-tCSP2 by Salmonella serovar Typhi vaccine strain CVD 908-htrA were demonstrated by immunoblotting of whole-cell lysates and culture supernatants. The immunogenicity of these constructs was evaluated using a "heterologous prime-boost" approach consisting of mucosal priming with Salmonella serovar Typhi expressing ClyA-tCSP1 and ClyA-tCSP2, followed by parenteral boosting with PfCSP DNA vaccines pVR2510 and pVR2571. Mice primed intranasally on days 0 and 28 with CVD 908-htrA(pSEC10tcsp2) and boosted intradermally on day 56 with PfCSP DNA vaccine pVR2571 induced high titers of serum NANP immunoglobulin G (IgG) (predominantly IgG2a); no serological responses to DNA vaccination were observed in the absence of Salmonella serovar Typhi-PfCSP priming. Mice primed with Salmonella serovar Typhi expressing tCSP2 and boosted with PfCSP DNA also developed high frequencies of gamma interferon-secreting cells, which surpassed those produced by PfCSP DNA in the absence of priming. A prime-boost regimen consisting of mucosal delivery of PfCSP exported from a Salmonella-based live-vector vaccine followed by a parenteral PfCSP DNA boosting is a promising strategy for the development of a live-vector-based malaria vaccine.
Project description:Live attenuated bacterial strains expressing heterologous antigens represent an attractive vaccine development strategy. However, the use of drug resistance genes for the selection of expression plasmids introduced into live vectors poses theoretical health risks. Therefore, we developed a novel approach for plasmid selection based on immunity to the antimicrobial peptide microcin H47 (MccH47). Two expression plasmids encoding the reporter green fluorescent protein (GFPuv) were constructed; selection markers comprised either mchI, conferring immunity to MccH47 (pGEN222I), or bla (encoding beta-lactamase), conferring conventional resistance to ampicillin (pGEN222). GFPuv-specific serum immunoglobulin G (IgG) antibody responses were analyzed in mice immunized intranasally either with Salmonella enterica serovar Typhi CVD 908-htrA or Shigella flexneri 2a CVD 1208S live vector and were boosted parenterally with purified GFPuv. Similar IgG antibody responses were observed for both pGEN222 and pGEN222I when either CVD 1208S or CVD 908-htrA(pGEN222I) was used as the carrier. Interestingly, CVD 908-htrA(pGEN222I) elicited a significantly higher IgG response than CVD 908-htrA(pGEN222). We also compared the priming potential of homologous priming either with CVD 908-htrA(pGEN222I) or CVD 1208S(pGEN222I) to heterologous priming first with CVD 908-htrA(pGEN222I) and then with CVD 1208S(pGEN222I) and vice versa. Immunization with two unrelated live vectors significantly enhanced the IgG responses compared to responses engendered by homologous CVD 908-htrA(pGEN222I) but not to those of CVD 1208S(pGEN222I). MccH47 offers an alternate system for plasmid selection in bacterial live vectors that greatly improves their clinical acceptability. Furthermore, the success of the heterologous priming strategy supports the feasibility of the future development of multivalent live vector-based immunization strategies against multiple human pathogens.
Project description:After decades of containment, pertussis disease, caused by Bordetella pertussis seems to be re-emerging and still remains a major cause of reported vaccine-preventable deaths worldwide. The current licensed whole-cell vaccines display reactogenicity while acellular vaccines are expensive and do not induce Th1-type immune responses that are required for optimum protection against the disease. Thus, there is an urgent need to develop new vaccines and the recombinant technology seems to be the method of choice for this purpose. The present study was an attempt to develop a new, simplified, cost-effective and well-defined vaccine against Bordetella pertussis, with capacity to induce a Th1 response.A fusion DNA fragment encoding the N-terminal region of pertussis toxin S1 subunit and filamentous hemagglutinin type 1 immunodominant domain was constructed and the corresponding fusion protein (F1S1) was produced in Escherichia coli. F1S1 in conjunction with imiquimod was administered by subcutaneous (SC) and intranasal (IN) routes to BALB/c mice.This vaccine formulation could elicit high levels of IFN-γ, serum IgG (with higher IgG2a/IgG1 ratio) and lung IgA after the SC and, to a lesser extent, following the IN administration.Our results indicate that the above-mentioned important proteins of B. pertussis could be successfully produced in E. coli as a single fusion protein. Furthermore, this protein could induce proper systemic and mucosal immune responses after administration via SC or IN routes.
Project description:Salmonella enterica serovar Typhi vaccine strain CVD 908-htrA was genetically engineered for stable plasmid-based expression of protective antigen of anthrax toxin (PA83) fused with the export protein ClyA (ClyA-PA83). The priming potential of CVD 908-htrA expressing ClyA-PA83 was assessed in 12 rhesus and 20 cynomolgus macaques that were immunized mucosally (i.e., intranasally) on days 0 and 14. A parenteral booster with purified PA83 plus alum was given to rhesus macaques on days 42 and 225; cynomolgus monkeys received a booster with either PA or licensed anthrax vaccine (BioThrax; Emergent Biosolutions) only one time, 3 months after priming. Monkeys primed with S. Typhi expressing ClyA-PA83 developed high levels of serum toxin-neutralization activity (TNA) antibodies (50% effective dose [ED50], >1.3x10(3)), 7 days after receipt of the booster, whereas unprimed controls lacked serum TNA (ED50, 0). In nonhuman primates, the success of this anthrax vaccine strategy based on heterologous mucosal priming followed by a parenteral subunit vaccine booster paves the way for clinical trials.
Project description:BACKGROUNDThe live attenuated BPZE1 vaccine candidate induces protection against B. pertussis and prevents nasal colonization in animal models. Here we report on the responses in humans receiving a single intranasal administration of BPZE1.METHODSWe performed multiple assays to dissect the immune responses induced in humans (n = 12) receiving BPZE1, with particular emphasis on the magnitude and characteristics of the antibody responses. Such responses were benchmarked to adolescents (n = 12) receiving the complete vaccination program of the currently used acellular pertussis vaccine (aPV). Using immunoproteomics analysis, potentially novel immunogenic B. pertussis antigens were identified.RESULTSAll BPZE1 vaccinees showed robust B. pertussis-specific antibody responses with regard to significant increase in 1 or more of the following parameters: IgG, IgA, and memory B cells to B. pertussis antigens. BPZE1-specific T cells showed a Th1 phenotype, and the IgG exclusively consisted of IgG1 and IgG3. In contrast, all aPV vaccines showed a Th2-biased response. Immunoproteomics profiling revealed that BPZE1 elicited broader and different antibody specificities to B. pertussis antigens as compared with the aPV that primarily induced antibodies to the vaccine antigens. Moreover, BPZE1 was superior at inducing opsonizing antibodies that stimulated ROS production in neutrophils and enhanced bactericidal function, which was in line with the finding that antibodies against adenylate cyclase toxin were only elicited by BPZE1.CONCLUSIONThe breadth of the antibodies, the Th1-type cellular response, and killing mechanisms elicited by BPZE1 may hold prospects of improving vaccine efficacy and protection against B. pertussis transmission.TRIAL REGISTRATIONClinicalTrials.gov NCT02453048, NCT00870350.FUNDINGILiAD Biotechnologies, Swedish Research Council (Vetenskapsrådet), Swedish Heart-Lung Foundation.
Project description:The voltage-gated potassium channel Kv1.5 belongs to the Shaker superfamily. Kv1.5 is composed of four subunits, each comprising 613 amino acids, which make up the N terminus, six transmembrane segments (S1-S6), and the C terminus. We recently demonstrated that, in HEK cells, extracellularly applied proteinase K (PK) cleaves Kv1.5 channels at a single site in the S1-S2 linker. This cleavage separates Kv1.5 into an N-fragment (N terminus to S1) and a C-fragment (S2 to C terminus). Interestingly, the cleavage does not impair channel function. Here, we investigated the role of the N terminus and S1 in Kv1.5 expression and function by creating plasmids encoding various fragments, including those that mimic PK-cleaved products. Our results disclosed that although expression of the pore-containing fragment (Frag(304-613)) alone could not produce current, coexpression with Frag(1-303) generated a functional channel. Immunofluorescence and biotinylation analyses uncovered that Frag(1-303) was required for Frag(304-613) to traffic to the plasma membrane. Biochemical analysis revealed that the two fragments interacted throughout channel trafficking and maturation. In Frag(1-303)+(304-613)-coassembled channels, which lack a covalent linkage between S1 and S2, amino acid residues 1-209 were important for association with Frag(304-613), and residues 210-303 were necessary for mediating trafficking of coassembled channels to the plasma membrane. We conclude that the N terminus and S1 of Kv1.5 can attract and coassemble with the rest of the channel (i.e. Frag(304-613)) to form a functional channel independently of the S1-S2 linkage.
Project description:A vaccine based on outer membrane vesicles of pertussis (omvPV) is protective in a mouse-challenge model and induces a broad antibody and mixed Th1/Th2/Th17 response against multiple antigens following subcutaneous immunization. However, this route did not result in mucosal immunity and did not prevent nasopharyngeal colonization. In this study, we explored the potential of intranasal immunization with omvPV. Only intranasal immunization induced strong mucosal immune responses that encompasses enhanced pulmonary and nasal IgA antibody levels, mainly directed against Vag8 and LPS. Furthermore, high numbers of IgA- and IgG-producing plasma cells were detected as well as lung-resident IgA memory B-cells. Finally, only intranasal immunization induced pulmonary Th1/Th17-related cytokine responses. The magnitude and type of systemic immunity was comparable between both routes and included high systemic IgG antibody levels, strong IgG-producing plasma cell responses, memory B-cells residing in the spleen and systemic Th1/Th2/Th17-related cytokine responses. Importantly, only intranasal immunization prevented colonization in both the lungs and the nasal cavity. In conclusion, intranasal omvPV immunization induces mucosal IgA and Th17-mediated responses without influencing the systemic immunity profile. These responses resulted in prevention of Bordetella pertussis colonization in the respiratory tract, including the nasal cavity, thereby potentially preventing transmission.
Project description:We constructed a food-grade expression system harboring a F1S1 fusion protein of Bordetella pertussis to be produced in Lactococcus lactis NZ3900 as a new oral vaccine model against whooping cough, caused by B. pertussis. F1S1 was composed of N-terminally truncated S1 subunit of pertussis toxin and type I immunodominant domain of filamentous hemagglutinin which are both known as protective immunogens against pertussis. The recombinant L. lactis was administered via oral or intranasal routes to BALB/c mice and the related specific systemic and mucosal immune responses were then evaluated. The results indicated significantly higher levels of specific IgA in the lung extracts and IgG in sera of mucosally-immunized mice, compared to their controls. It was revealed that higher levels of IgG2a, compared to IgG1, were produced in all mucosally-immunized mice. Moreover, immunized mice developed Th1 responses with high levels of IFN-? production by the spleen cells. These findings provide evidence for L. lactis to be used as a suitable vehicle for expression and delivery of F1S1 fusion protein to mucosa and induction of appropriate systemic and mucosal immune responses against pertussis.
Project description:Bacterial live-vector vaccines aim to deliver foreign antigens to the immune system and induce protective immune responses, and surface-expressed or secreted antigens are generally more immunogenic than cytoplasmic constructs. We hypothesize that an optimum expression system will use an endogenous export system to avoid the need for large amounts of heterologous DNA encoding additional proteins. Here we describe the cryptic chromosomally encoded 34-kDa cytolysin A hemolysin of Salmonella enterica serovar Typhi (ClyA) as a novel export system for the expression of heterologous antigens in the supernatant of attenuated Salmonella serovar Typhi live-vector vaccine strains. We constructed a genetic fusion of ClyA to the reporter green fluorescent protein and showed that in Salmonella serovar Typhi CVD 908-htrA, the fusion protein retains biological activity in both domains and is exported into the supernatant of an exponentially growing live vector in the absence of detectable bacterial lysis. The utility of ClyA for enhancing the immunogenicity of an otherwise problematic antigen was demonstrated by engineering ClyA fused to the domain 4 (D4) moiety of Bacillus anthracis protective antigen (PA). A total of 11 of 15 mice immunized intranasally with Salmonella serovar Typhi exporting the protein fusion manifested fourfold or greater rises in serum anti-PA immunoglobulin G, compared with only 1 of 16 mice immunized with the live vector expressing cytoplasmic D4 (P = 0.0002). In addition, the induction of PA-specific gamma interferon and interleukin 5 responses was observed in splenocytes. This technology offers exceptional versatility for enhancing the immunogenicity of bacterial live-vector vaccines.
Project description:Little is known about the neutralizing epitopes in turkey coronavirus (TCoV). The spike (S) protein gene of TCoV was divided into 10 fragments to identify the antigenic region containing neutralizing epitopes. The expression and antigenicity of S fragments was confirmed by immunofluorescence antibody (IFA) assay using an anti-histidine monoclonal antibody or anti-TCoV serum. Polyclonal antibodies raised against expressed S1 (amino acid position 1 to 573 from start codon of S protein), 4F/4R (482-678), 6F/6R (830-1071), or Mod4F/Epi4R (476-520) S fragment recognized native S1 protein and TCoV in the intestines of TCoV-infected turkey embryos. Anti-TCoV serum reacted with recombinant 4F/4R, 6F/6R, and Mod4F/Epi4R in a western blot. The results of a virus neutralization assay indicated that the carboxyl terminal region of the S1 protein (Mod4F/Epi4R) or the combined carboxyl terminal S1 and amino terminal S2 protein (4F/4R) possesses the neutralizing epitopes, while the S2 fragment (6F/6R) contains antigenic epitopes but not neutralizing epitopes.