Isolation, characterization and in-vitro antigenicity studies of outer membrane proteins (OMPs) of Salmonella gallinarum coated gold nanoparticles (AuNPs).

The development of biomimetic nanoparticles by combining the natural cellular material with synthetic nanoparticles has inspired innovative therapeutic strategies as compared to traditional line of therapeutics. Therefore, the research was conducted to prepare the outer membrane proteins (OMPs) from Salmonella gallinarum and coated on to the surface of synthetic gold nanoparticles (AuNPs). The size of the final product was characterized by Zeta sizer and Zeta potential. The coating of outer membrane proteins onto the gold nanoparticles (AuNPs) was confirmed by transmission electron microscopy (TEM). The sterility, stability and safety of the prepared antigen was confirmed by standard culture media, in biological buffer solution and in-vitro pyrogenic testing, respectively. In-vitro antigenicity of outer membrane protein alone and coated with gold nanoparticles were observed through macrophage phagocytosis assay. On the basis of results, it was anticipated that bacterial outer membrane protein has immunogenic antigenic properties and coating of bacterial outer membrane protein on the surfaces of synthetic gold nanoparticles not only preserved the complex biological characteristics of bacteria but also provided greater immune responses as compared to outer membrane protein alone. So, it was concluded that the coating of bacterial outer membrane proteins on the surface of synthetic gold nanoparticles have synergistic effects to induce the immune responses and promising potential to develop the effective antibacterial vaccine against salmonellosis in poultry birds.

Encapsulation of Recombinant MOMP in Extended-Releasing PLGA 85:15 Nanoparticles Confer Protective Immunity Against a Chlamydia muridarum Genital Challenge and Re-Challenge.

Recently we reported the immune-potentiating capacity of a Chlamydia nanovaccine (PLGA-rMOMP) comprising rMOMP (recombinant major outer membrane protein) encapsulated in extended-releasing PLGA [poly (D, L-lactide-co-glycolide) (85:15)] nanoparticles. Here we hypothesized that PLGA-rMOMP would bolster immune-effector mechanisms to confer protective efficacy in mice against a Chlamydia muridarum genital challenge and re-challenge. Female BALB/c mice received three immunizations, either subcutaneously (SC) or intranasally (IN), before receiving an intravaginal challenge with C. muridarum on day 49 and a re-challenge on day 170. Both the SC and IN immunization routes protected mice against genital challenge with enhanced protection after a re-challenge, especially in the SC mice. The nanovaccine induced robust antigen-specific Th1 (IFN-γ, IL-2) and IL-17 cytokines plus CD4+ proliferating T-cells and memory (CD44high CD62Lhigh) and effector (CD44high CD62Llow) phenotypes in immunized mice. Parallel induction of antigen-specific systemic and mucosal Th1 (IgG2a, IgG2b), Th2 (IgG1), and IgA antibodies were also noted. Importantly, immunized mice produced highly functional Th1 avidity and serum antibodies that neutralized C. muridarum infectivity of McCoy fibroblasts in-vitro that correlated with their respective protection levels. The SC, rather than the IN immunization route, triggered higher cellular and humoral immune effectors that improved mice protection against genital C. muridarum. We report for the first time that the extended-releasing PLGA 85:15 encapsulated rMOMP nanovaccine confers protective immunity in mice against genital Chlamydia and advances the potential towards acquiring a nano-based Chlamydia vaccine.

Prime-Pull Immunization with a Bivalent M-Protein and Spy-CEP Peptide Vaccine Adjuvanted with CAF®01 Liposomes Induces Both Mucosal and Peripheral Protection from covR/S Mutant Streptococcus pyogenes.

Infections with Streptococcus pyogenes and their sequelae are responsible for an estimated 18 million cases of serious disease with >700 million new primary cases and 500,000 deaths per year. Despite the burden of disease, there is currently no vaccine available for this organism. Here, we define a combination vaccine P*17/K4S2 comprising of 20-mer B-cell peptide epitopes, p*17 (a mutant derived from the highly conserved C3-repeat region of the M-protein), and K4S2 (derived from the streptococcal anti-neutrophil factor, Spy-CEP). The peptides are chemically conjugated to either diphtheria toxoid (DT) or a nontoxic mutant form of diphtheria toxin, CRM197. We demonstrate that a prime-pull immunization regimen involving two intramuscular inoculations with P*17/K4S2 adjuvanted with a two-component liposomal adjuvant system (CAF01; developed by Statens Serum Institut [SSI], Denmark), followed by an intranasal inoculation of unadjuvanted vaccine (in Tris) induces peptide- and S. pyogenes-binding antibodies and protects from mucosal and skin infection with hypervirulent covR/S mutant organisms. Prior vaccination with DT does not diminish the response to the conjugate peptide vaccines. Detailed Good Laboratory Practice (GLP) toxicological evaluation in male and female rats did not reveal any gross or histopathological adverse effects.IMPORTANCE A vaccine to control S. pyogenes infection is desperately warranted. S. pyogenes colonizes the upper respiratory tract (URT) and skin, from where it can progress to invasive and immune-mediated diseases. Global mortality estimates for S. pyogenes-associated diseases exceeds 500,000 deaths per year. S. pyogenes utilizes antigenic variation as a defense mechanism to circumvent host immune responses and thus a successful vaccine needs to provide strain-transcending and multicompartment (mucosal and skin) immunity. By defining highly conserved and protective epitopes from two critical virulence factors (M-protein and Spy-CEP) and combining them with a potent immunostimulant, CAF®01, we are addressing an unmet clinical need for a mucosally and skin-active subunit vaccine. We demonstrate that prime-pull immunization (2× intramuscular injections followed by intranasal immunization) promotes high sustained antibody levels in the airway mucosa and serum and protects against URT and invasive disease.

Protective response against Acinetobacter baumannii with ferric iron receptors HemTR-BauA in a murine sepsis model.

Aim: Iron uptake and metabolism pathways are promising targets in vaccine development as an alternative strategy for antibiotics. Methods & methods: HemTR, a putative heme receptor of Acinetobacter baumannii, was expressed and its protectivity against A. baumannii was determined singly or in combination with the siderophore receptor, BauA, in mice. Results: High level of IgG was elicited. There was a delay in mice mortality with reduced bacterial loads in internal organs in the sublethal challenge. Protection was better in the HemTR-BauA group in both lethal and sublethal challenges. Passive transfer of anti-HemTR and anti-BauA partially protected mice against A. baumannii infection. Conclusion: HemTR in combination with other iron receptors could contribute to the development of protective vaccines against A. baumannii.

Non-infectious outer membrane vesicles derived from Brucella abortus S19Δper as an alternative acellular vaccine protects mice against virulent challenge.

The prime human and animal safety issues accentuate the search of promising newer alternative vaccine candidates to resolve complications associated with the live attenuated Brucella abortus strain19 (S19) vaccine. Outer membrane vesicles (OMVs S19 Δper) extracted from Brucella abortus S19Δper (S19Δper) as an alternative subunit vaccine candidate has been explored in the present study as OMVs are endowed with immunogenic molecules, including LPS and outer membrane proteins (OMPs) and do not cause infection by virtue of being an acellular entity. The LPS defective S19Δper released a higher amount of OMVs than its parent strain S19. Under transmission electron microscopy (TEM), OMVs were seen as nano-sized outward bulge from the surface of Brucella. Dynamic light scattering analysis of OMVs revealed that OMVs S19Δper showed the less polydispersity index (PDI) than OMVs S19 pointing towards relatively more homogenous OMVs populations. Both OMVs S19Δper and OMVs S19 with or without booster dose and S19 vaccine were used for immunization of mice and subsequently challenged with 2 × 105 CFU virulent Brucella abortus strain 544 (S544) to assess protective efficacy of vaccines. The less splenic weight index and less S544 count in OMVs immunized mice in comparison to unimmunized mice after S544 challenge clearly indicated good protective efficacy of OMVs. OMVs S19 Δper induced relatively high titer of IgG than OMVs S19 but conferred nearly equal protection against brucellosis. An ELISA based determination of IgG and its isotype response, Cytometric Bead Array (CBA) based quantitation of serum cytokines and FACS based enumeration of CD4+ and CD8+ T cells revealed high titer of IgG, production of both Th1 (IgG2a) and Th2 (IgG1) related antibodies, stimulation of IL-2, TNF (Th1) and IL-4, IL-6, IL-10 (Th2) cytokines, and induced T cell response suggested that OMVs S19Δper elicited Th1 and Th2 type immune response and ensured protection against S544 challenge in murine model.

CD8+ T Cells Directed Against a Peptide Epitope Derived From Peptidoglycan-Associated Lipoprotein of Legionella pneumophila Confer Disease Protection.

Legionella pneumophila, an intracellular bacterium, may cause life-threatening pneumonia in immunocompromised individuals. Mononuclear cells and antibodies have been reported to be associated with the host defense response against L. pneumophila. This study is to determine whether Legionella peptidoglycan-associated lipoprotein (PAL)-specific CD8+ T cells are directly associated with protection against L. pneumophila, with a focus on potential epitopes. Synthetic peptides derived from PAL of L. pneumophila were obtained and tested through in vitro and in vivo cytotoxic T lymphocyte (CTL) assays for immunogenicity. PAL DNA vaccines or a peptide epitope with or without CpG-oligodeoxynucleotides (ODN) was evaluated for protection against L. pneumophila infection in animal models. When mice were immunized with DNA vaccines expressing the PAL of L. pneumophila, they were significantly protected against a lethal challenge with L. pneumophila through induction of antigen-specific CD8+ CTLs. Of the 13 PAL peptides tested, PAL92-100 (EYLKTHPGA) was the most immunogenic and induced the strongest CTL responses. When mice were immunized with the PAL92-100 peptide plus CpG-ODN, they were protected against the lethal challenge, while control mice died within 3-6 days after the challenge. Consistent with lung tissue histological data, bacterial counts in the lungs of immunized mice were significantly lower than those in control mice. Also, the amino acid sequence of PAL92-100 peptides is conserved among various Legionella species. To our knowledge, this study is the first to demonstrate that PAL92-100-specific CD8+ T cells play a central role in the host defense response against L. pneumophila.

Expression of LamB Vaccine Antigen in Wolffia globosa (Duck Weed) Against Fish Vibriosis.

Vibriosis is a commonly found bacterial disease identified among fish and shellfish cultured in saline waters. A multitude of Vibrio species have been identified as the causative agents. LamB, a member of outer membrane protein (OMPs) family of these bacteria is conserved among all Vibrio species and has been identified as an efficient vaccine candidate against vibriosis. Rootless duckweed (Wolffia) is a tiny, edible aquatic plant possessing characteristics suitable for the utilization as a bioreactor. Thus, we attempted to express a protective edible vaccine antigen against fish vibriosis in nuclear-transformed Wolffia. We amplified LamB gene from virulent Vibrio alginolyticus and it was modified to maximize the protein expression level and translocate the protein to the endoplasmic reticulum (ER) in plants. It was cloned into binary vector pMYC under the control of CaMV 35S promoter and introduced into Wolffia globosa by Agrobacterium-mediated transformation. Integration and expression of the LamB gene was confirmed by genomic PCR and RT-PCR. Western blot analysis revealed accumulation of the LamB protein in 8 transgenic lines. The cross-protective property of transgenic Wolffia was evaluated by orally vaccinating zebrafish through feeding fresh transgenic Wolffia and subsequently challenging with virulent V. alginolyticus. High relative percent survival (RPS) of the vaccinated fish (63.3%) confirmed that fish immunized with transgenic Wolffia were well-protected from Vibrio infection. These findings suggest that Wolffia expressed LamB could serve as an edible plant-based candidate vaccine model for fish vibriosis and feasibility of utilizing Wolffia as bioreactor to produce edible vaccines.

Cationic Lipid-based Intracellular Delivery of Bacterial Effectors for Rewiring Malignant Cell Signaling.

The abundance of bacterial effectors have inspired us to explore their potential in rewiring malignant cell signaling. Their incapability for entering cells, however, hinders such application. Herein we developed a cationic lipid-based high throughput library screening platform for effective intracellular delivery of bacterial effectors. As the misregulated MAPK signaling is a hallmark of many types of cancer, we turned to the Shigella effector OspF which irreversibly inactivates ERK, the terminal component of MAPK cascade. We created a function-based screening assay to obtain AMPA-O16B lipid nanoparticles for effective OspF intracellular delivery, which inhibited the malignant MAPK signaling and tumor growth in vitro and in vivo. Furthermore, the optimized lipid nanoparticle formulation can deliver OspF to modulate the immunosuppressive responses in macrophages. Our work is a general strategy to explore the therapeutic potentials of naturally evolved bacterial effectors.

Sublingual Omp16-driven redirection of the allergic intestinal response in a pre-clinical model of food allergy.

BACKGROUND: IgE-mediated food allergy remains a significant and growing worldwide problem. Sublingual immunotherapy (SLIT) shows an excellent safety profile for food allergy, but the clinical efficacy needs to be improved. This study assessed the effects of the Toll-like receptor 4 agonist outer membrane protein (Omp) 16 from Brucella abortus combined with cow´s milk proteins (CMP) through the sublingual route to modulate cow's milk allergy in an experimental model. METHODS: Mice sensitized with cholera toxin and CMP were orally challenged with the allergen to elicit hypersensitivity reactions. Then, mice were treated with a very low amount of CMP along with Omp16 as a mucosal adjuvant, and finally, animals were re-exposed to CMP. Systemic and mucosal immune parameters were assessed in vivo and in vitro. RESULTS: We found that the sublingual administration of Omp16 + CMP induced a buccal Th1 immune response that modulated the intestinal allergic response with the suppression of symptoms, reduction of IgE and IL-5, and up-regulation of IgG2a and IFN-γ. The adoptive transfer of submandibular IFN-γ-producing α4ß7+ CD4+ and CD8+ cells conferred protection against allergic sensitization. The use of Omp16 + CMP promoted enhanced protection compared to CMP alone. CONCLUSION: In conclusion, Omp16 represents a promising mucosal adjuvant that can be used to improve the clinical and immune efficacy of SLIT for food allergy.

High protection of mice against Brucella abortus by oral immunization with recombinant probiotic Lactobacillus casei vector vaccine, expressing the outer membrane protein OMP19 of Brucella species.

Brucellosis is a zoonotic disease threatening the public health and hindering the trade of animals and their products, which has a negative impact on the economic development of a country. Vaccination is the most effective way to control brucellosis. The recombinant vector vaccines are promising candidates for immunization in humans and animals. In this study, the gene encoding OMP19 antigen was primarily amplified and cloned into an expression vector called pT1NX, and then transformed to L. casei cell via electroporation technique. The expression was confirmed using specific antibody against the recombinant protein via immunological screening tests such as western blot and immunofluorescence assay. Finally, recombinant L. casei was orally fed to mice and the results were further recorded, indicating that the mice group which received OMP19 through L. casei based vaccine represented a very good general and mucosal immune responses protective against challenges with virulent B. abortus 544 strain compared with negative control recipient groups. Therefore, the vaccine produced in this research plan can be a very good candidate for protection against brucellosis.

Broadly Protective Multivalent OspA Vaccine against Lyme Borreliosis, Developed Based on Surface Shaping of the C-Terminal Fragment.

The development of vaccines for prevention of diseases caused by pathogenic species can encounter major obstacles if high sequence diversity is observed between individual strains. Therefore, development might be restricted either to conserved antigens, which are often rare, or to multivalent vaccines, which renders the production more costly and cumbersome. In light of this complexity, we applied a structure-based surface shaping approach for the development of a Lyme borreliosis (LB) vaccine suitable for the United States and Europe. The surface of the C-terminal fragment of outer surface protein A (OspA) was divided into distinct regions, based primarily on binding sites of monoclonal antibodies (MAbs). In order to target the six clinically most relevant OspA serotypes (ST) in a single protein, exposed amino acids of the individual regions were exchanged to corresponding amino acids of a chosen OspA serotype. Six chimeric proteins were constructed, and, based on their immunogenicity, four of these chimeras were tested in mouse challenge models. Significant protection could be demonstrated for all four proteins following challenge with infected ticks (OspA ST1, OspA ST2, and OspA ST4) or with in vitro-grown spirochetes (OspA ST1 and OspA ST5). Two of the chimeric proteins were linked to form a fusion protein, which provided significant protection against in vitro-grown spirochetes (OspA ST1) and infected ticks (OspA ST2). This article presents the proof-of-concept study for a multivalent OspA vaccine targeting a wide range of pathogenic LB Borrelia species with a single recombinant antigen for prevention of Lyme borreliosis.

Elicitation of Th1/Th2 related responses in mice by chitosan nanoparticles loaded with Brucella abortus malate dehydrogenase, outer membrane proteins 10 and 19.

Brucella spp. is the causative agent of brucellosis, one of the worldwide diseases. The pathogen infects humans and animals mainly through the digestive or respiratory tract. Therefore, induction of mucosal immunity is required as the first line of defense. In this study, three Brucella abortus recombinant proteins, malate dehydrogenase (rMdh), outer membrane proteins (rOmp) 10 and 19 were loaded in mucoadhesive chitosan nanoparticles (CNs) and induction of mucosal and systemic immunity were investigated after intranasal immunization of BALB/c mice. These antigens were also coimmunized as cocktail (rCocktail) to evaluate multiple antigen specific vaccine candidates. At 6-weeks post-immunization (wpi), antigen specific total IgG was increased in all of the immunized groups, predominantly IgG1. In addition, spleenocyte from rMdh-, rOmp19-, and rCocktail-immunized groups significantly produced IFN-γ and IL-4 suggesting the induction of a mixed Th1-Th2 response. For mucosal immunity, anti-Mdh IgA from nasal washes and fecal excretions, and anti-Omps IgA from sera, nasal washes, genital secretions and fecal excretions were significantly increased in single antigen immunized groups. In the rCocktail-immunized group, anti-Mdh IgA were significantly increased while anti-Omps IgA was not. Collectively, this study indicates that comprise of B. abortus antigen-loaded CNs elicited the antigen-specific IgA with a Th2-polarized immune responses and combination of the highly immunogenic antigens elicited IgG specific to each type of antigen.

Type I IFN signalling is required for cationic adjuvant formulation (CAF)01-induced cellular immunity and mucosal priming.

Despite being in the midst of a global pandemic of infections caused by the pathogen Chlamydia trachomatis, a vaccine capable of inducing protective immunity remains elusive. Given the C. trachomatis mucosal port of entry, a formulation compatible with mucosal administration and capable of eliciting potent genital tract immunity is highly desirable. While subunit vaccines are considered safer and better tolerated, these are typically poorly immunogenic and require co-formulation with immune-potentiating adjuvants. However, of the adjuvants licensed for use in humans, very few drive robust cellular responses, a pre-requisite for protection against C. trachomatis infection. Recently, the cationic adjuvant formulations (CAF) have been shown to induce robust humoral and cellular immunity in pre-clinical models of chlamydia, malaria and tuberculosis (TB). Here, we demonstrate that CAF01 induces potent immune responses when combined with the major outer membrane protein (MOMP) of C. trachomatis following parenteral immunisation and also as part of a heterologous prime/boost regime. We show that a subcutaneous prime with CAF01-adjuvanted recombinant MOMP licenses antigen-specific immunity at distant mucosal sites which can be activated following oral antigen re-encounter in the absence of concomitant adjuvant stimulation. Finally, we shed light on the mechanism(s) through which CAF01 elicits robust antigen-specific immunity to co-formulated MOMP via type I interferon (IFN) signalling.

Immunization of a novel bivalent outer membrane protein simultaneously resisting Aeromonas hydrophila, Edwardsiella anguillarum and Vibrio vulnificus infection in European eels (Angullia angullia).

In cultivated European eels, Aeromonas hydrophila, Edwardsiella anguillarum and Vibrio vulnificus are three important bacterial pathogens. In this study, European eels (Anguilla anguilla) were immunized by the bivalent expression products of the outer membrane protein (Omp) gene from A. hydrophila (OmpⅡ) and E. anguillarum (OmpA), and the effects of the bivalent protein (rOmpⅡ-A) on the immune function of the European eel were detected. Three hundred eels were divided average into three groups of PBS, adjuvant and rOmp. Eels of three goups were injected intraperitoneal with 0.2 mL of PBS (0.01 mol/L, pH7.4), PBS + F (PBS mixed equal volume of freund's uncomplete adjuvant) or rOmpⅡ-A (1 mg mL-1 rOmpⅡ-A mixed equal volume of freund's uncomplete adjuvant). Four immune-related genes expression, proliferation of whole blood cells, serum and skin mucus antibody titer, superoxide dismutase (SOD) activity and the relative percent of survival (RPS) were studied at different days (or hours) post the immunization. The results showed that the igm, lysC, mhc2 and sod gene in the liver, spleen, kidney and intestine tract were significant increased in the Omp group; On the 28 day post the immunization (dpi), blood cell proliferation was increased in the Omp group, and on the 14, 21, 28 and 42 dpi, antibody titers in serum and mucus of the Omp group were significantly higher than that of the PBS and adjuvant group, regardless of coating with bacteria or Omp antigen. The SOD activity of Omp group increased significantly in liver, kidney, skin mucus and serum from 14 to 42 dpi, especially in serum. Eels chanllenged by A. hydrophila, E. anguillarum and V. vulnificus in the bivalent Omp group showed the RPS were 83.33%, 55.56% and 44.44%, respectively. The results of this study showed that immunization of the bivalent Omp could effectively improve the immune function of European eels, and produced effectively protection to A. hydrophila and E. anguillarum infection. Simultaneously, the bivalent Omp also produced distinct cross-protection to the eels challenged by V. vulnificus.

Evaluation of the protective immunity of Riemerella anatipestifer OmpA.

Riemerella anatipestifer is responsible for an economically important disease of commercially raised ducks. No or only few cross-protection was observed between different serotypes of R. anatipestifer strains, and so far no protective antigen in this bacterium has been identified. OmpA is a predominant immunogenic protein of R. anatipestifer, and within the 1467 bp ompA ORF (ompA1467), there is another 1164 bp ORF (ompA1164) with the same C-terminal. In this study, our results showed that the full sequence of ompA1467 from some R. anatipestifer strains with different serotypes shared the same amino acid sequence. Animal experiments showed that the soluble recombinant protein rOmpA1164, but not rOmpA1467, could provide partial protective immunity against challenge. Moreover, there was no significant difference in protective immunity between ducklings immunized with Th4△ompA bacterin and those immunized with Th4 bacterin. In addition, OmpA1467 was the main existing form of OmpA in R. anatipestifer cells by gel electrophoresis and western blot analyses. The results suggested that OmpA1467 was not a protective antigen of R. anatipestifer, and antibodies against proteins other than OmpA play a critical role in the process of anti-R. anatipestifer infection.

Immunization of a novel outer membrane protein from Aeromonas hydrophila simultaneously resisting A. hydrophila and Edwardsiella anguillarum infection in European eels (Angullia angullia).

In cultivated European eels, Aeromonas hydrophila, Edwardsiella anguillarum and Vibrio vulnificus are three important bacterial pathogens. In this study, an expressed recombinant Outer membrane proteinⅡ (rOmpⅡ) from A. hydrophila was intraperitoneally injected into European eels (Angullia angullia). All examined eels were equally divided into three groups. One group was injected with PBS only (PBS group), one group was injected with 1:1 mixture of PBS and Freund's incomplete adjuvant (PBS + F, adjuvant group), and the third group was injected with 1:1 mixture of 1 mg mL-1 rOmpⅡ and Freund's incomplete adjuvant (rOmpⅡ+F, OmpⅡ group). The immunogenicity of OmpⅡ was studied by detecting the expression of 4 immune-related genes, stimulation index (SI) of the whole blood cell, serum antibody titer, lysozyme and Superoxide Dismutase (SOD) activity, and relative percent of survival (RPS) rate. The results showed that gene expression of MHC-Ⅱ, LysC, SOD and IgM in the OmpⅡ group significantly increased in liver, spleen, kidney and intestine. At 28 days post the immunization (dpi), the SI of whole blood cells in the OmpⅡ group increased significantly; at 14, 21, 28 and 42 dpi, the serum antibody titers against A. hydrophila and E. anguillarum in the OmpⅡ group were significantly higher than that of the PBS and the adjuvant group; the SOD in the OmpⅡ group was found increased significantly in liver, kidney, mucus and serum. On the 28 dpi, eels were challenged by A. hydrophila, E. anguillarum and V. vulnificus for cross protection study. The results showed that the RPS of the OmpⅡ group were 83.33%, 55.56% and 33.33% respectively. These results showed that the expressed OmpⅡ from A. hydrophila significantly improve the immune function of Europena eels and their resistance to the infection of A. hydrophila and E. anguillarum simultaneously.

Population dynamics and antigenic drift of Bordetella pertussis following whole cell vaccine replacement, Barcelona, Spain, 1986-2015.

Among the factors associated with the resurgence of whooping cough, special emphasis has been given to pathogen adaptation after the introduction of the acellular vaccine (ACV). To assess the impact of the vaccine transition strategy from whole-cell vaccine (WCV) to ACV on population dynamics of Bordetella pertussis in Barcelona (Spain), we studied 339 isolates collected from 1986 to 2015 by PFGE and multi-locus variable-number tandem repeat analysis (MLVA). Additionally, allelic variants for the pertussis toxin and its promoter, pertactin, type 3 fimbriae and fimbrial serotyping were assessed to determine its antigenic drift. A shift was observed in the B. pertussis population as well as in its antigenic profile concurrently with the introduction of ACV in Barcelona. Four out of the five most prevalent PFGE profiles were replaced by new profiles following the ACV introduction. MLVA type 27 was the dominant genotype, and its frequency increased from 25% to 79.3% after WCV replacement. Antigen typing demonstrated the emergence of prn2, ptxP3, fim3-2 and a shift from the fimbriae 3 to the fimbriae 2 serotypes after the ACV introduction. Our findings support the presence of population and antigenic dynamic changes in B. pertussis likely driven by the introduction of ACV.

Bordetella Colonization Factor A (BcfA) Elicits Protective Immunity against Bordetella bronchiseptica in the Absence of an Additional Adjuvant.

Bordetella bronchiseptica is an etiologic agent of respiratory diseases in animals and humans. Despite the widespread use of veterinary B. bronchiseptica vaccines, there is limited information on their composition and relative efficacy and on the immune responses that they elicit. Furthermore, human B. bronchiseptica vaccines are not available. We leveraged the dual antigenic and adjuvant functions of Bordetella colonization factor A (BcfA) to develop acellular B. bronchiseptica vaccines in the absence of an additional adjuvant. BALB/c mice immunized with BcfA alone or a trivalent vaccine containing BcfA and the Bordetella antigens FHA and Prn were equally protected against challenge with a prototype B. bronchiseptica strain. The trivalent vaccine protected mice significantly better than the canine vaccine Bronchicine and provided protection against a B. bronchiseptica strain isolated from a dog with kennel cough. Th1/17-polarized immune responses correlate with long-lasting protection against bordetellae and other respiratory pathogens. Notably, BcfA strongly attenuated the Th2 responses elicited by FHA and Prn, resulting in Th1/17-skewed responses in inherently Th2-skewed BALB/c mice. Thus, BcfA functions as both an antigen and an adjuvant, providing protection as a single-component vaccine. BcfA-adjuvanted vaccines may improve the efficacy and durability of vaccines against bordetellae and other pathogens.

Long-Term Efficacy and Safety of Immunomodulatory Therapy for Atherosclerosis.

BACKGROUND AND AIMS: The long-term effect of immune tolerance has not been explored so far in atherosclerosis. In the present study, we assessed the effect of mucosal tolerance to a multi antigenic construct expressing three peptides from ApoB, HSP60, and outer membrane protein from Chlamydia pneumonia (AHC) for 30 weeks at every 6-week interval to understand the kinetics of immune modulation in disease progression. The safety profile of the molecule was also evaluated in mice. METHODS: Apobtm2SgyLdlrtm1Her/J mice (5-6 weeks) were orally dosed with multi antigenic construct (AHC) molecule on alternate days, followed by high-fat diet feeding to initiate atherosclerosis. RESULTS: Treated animals showed an efficient reduction in plaque growth and lipid accumulation at 6 weeks (49%, p < 0.01) and 12 weeks (42.3%, p < 0.01) which decreased to 29% (p = 0.0001) at 18 weeks and at later time points. Macrophage accumulation was significantly lower at all time points (53% at 12 weeks to 27% at 30 weeks). Regulatory T cells increased in the spleen following treatment until 12 weeks (week 0 (2.57 ± 0.18 vs. 6.36 ± 0.03, p = 0.02), week 6 (4.52 ± 0.2 vs. 8.87 ± 0.32, p = 0.02), and week 12 (8.74 ± 0.37 vs. 15.4 ± 0.27, p = 0.02)) but showed a decline later. A similar trend was observed with tolerogenic dendritic cells. We observed an increase in antibody levels to low-density lipoprotein and oxidized LDL at later stages. AHC molecule was found to be safe in acute and repeated dose toxicity studies. CONCLUSIONS: Our results suggest that immune tolerance to AHC protein by oral administration is able to provide efficient atheroprotection up to 18 weeks and moderately at later stages. Apart from immune regulatory cells, protective antibodies may also have a role in controlling atherosclerosis.

Immunization with a recombinant antigen composed of conserved blocks from TSA56 provides broad genotype protection against scrub typhus.

Scrub typhus is an acute febrile disease caused by Orientia tsutsugamushi infection. Despite the wide range of approaches explored during the last seventy years, an effective prophylactic vaccine is not yet available. Here, we developed a novel recombinant antigen derived from conserved regions of 56 kDa type-specific antigen (TSA56), a major outer membrane protein responsible for genetic heterogeneity and antigenicity, and evaluated it as a protective vaccine antigen. Our findings demonstrate that immunization with conserved blocks of TSA56 (cTSA56) not only provides protective immunity against lethal challenges with the homologous genotype, but also confers significantly better protection against heterologous genotypes than TSA56. Adoptive transfer of CD4+ or CD8+ T cells from immunized mice provided significantly enhanced protection against lethal challenge, whereas immune B cells failed to do so, indicating that cellular immunity against the conserved epitopes plays a protective role. Moreover, immunization with a 10-mer peptide mixture, screened from CD8+ T cell epitopes within the conserved region of TSA56, provided enhanced protection against lethal challenge with O. tsutsugamushi. Therefore, this novel recombinant antigen is a promising candidate for scrub typhus vaccine against a wide range of O. tsutsugamushi genotypes.