Intranasal Immunization With Nanoparticles Containing an Orientia tsutsugamushi Protein Vaccine Candidate and a Polysorbitol Transporter Adjuvant Enhances Both Humoral and Cellular Immune Responses.

Scrub typhus, a mite-borne infectious disease, is caused by Orientia tsutsugamushi. Despite many attempts to develop a protective strategy, an effective preventive vaccine has not been developed. The identification of appropriate Ags that cover diverse antigenic strains and provide long-lasting immunity is a fundamental challenge in the development of a scrub typhus vaccine. We investigated whether this limitation could be overcome by harnessing the nanoparticle-forming polysorbitol transporter (PST) for an O. tsutsugamushi vaccine strategy. Two target proteins, 56-kDa type-specific Ag (TSA56) and surface cell Ag A (ScaA) were used as vaccine candidates. PST formed stable nano-size complexes with TSA56 (TSA56-PST) and ScaA (ScaA-PST); neither exhibited cytotoxicity. The formation of Ag-specific IgG2a, IgG2b, and IgA in mice was enhanced by intranasal vaccination with TSA56-PST or ScaA-PST. The vaccines containing PST induced Ag-specific proliferation of CD8+ and CD4+ T cells. Furthermore, the vaccines containing PST improved the mouse survival against O. tsutsugamushi infection. Collectively, the present study indicated that PST could enhance both Ag-specific humoral immunity and T cell response, which are essential to effectively confer protective immunity against O. tsutsugamushi infection. These findings suggest that PST has potential for use in an intranasal vaccination strategy.

Protective efficacy of attenuated Salmonella Typhimurium strain expressing BLS, Omp19, PrpA, or SOD of Brucella abortus in goats.

BACKGROUND: Attenuated Salmonella strain can be used as a vector to transport immunogens to the host antigen-binding sites. OBJECTIVES: The study aimed to determine the protective efficacy of attenuated Salmonella strain expressing highly conserved Brucella immunogens in goats. METHODS: Goats were vaccinated with Salmonella vector expressing individually lipoprotein outer-membrane protein 19 (Omp19), Brucella lumazine synthase (BLS), proline racemase subunit A (PrpA), Cu/Zn superoxide dismutase (SOD) at 5 × 109 CFU/mL and challenge of all groups was done at 6 weeks after vaccination. RESULTS: Among these vaccines inoculated at 5 × 109 CFU/mL in 1 mL, Omp19 or SOD showed significantly higher serum immunoglobulin G titers at (2, 4, and 6) weeks post-vaccination, compared to the vector control. Interferon-γ production in response to individual antigens was significantly higher in SOD, Omp19, PrpA, and BLS individual groups, compared to that in the vector control (all p < 0.05). Brucella colonization rate at 8 weeks post-challenge showed that most vaccine-treated groups exhibited significantly increased protection by demonstrating reduced numbers of Brucella in tissues collected from vaccinated groups. Real-time polymerase chain reaction revealed that Brucella antigen expression levels were reduced in the spleen, kidney, and parotid lymph node of vaccinated goats, compared to the non-vaccinated goats. Besides, treatment with vaccine expressing individual antigens ameliorated brucellosis-related histopathological lesions. CONCLUSIONS: These results delineated that BLS, Omp19, PrpA, and SOD proteins achieved a definite level of protection, indicating that Salmonella Typhimurium successfully delivered Brucella antigens, and that individual vaccines could differentially elicit an antigen-specific immune response.

Protective efficacy of a Brucella vaccine using a Salmonella-based delivery system expressing Brucella Omp3b, BCSP31, and SOD proteins against brucellosis in Korean black goats.

This study aimed to assess the protective efficacy of a novel Brucella vaccine formulation in goats. Twenty black goats were separated into 2 groups. Group A was injected with 3.0 × 109 CFU (colony-forming units)/mL of a Salmonella-based delivery system harboring only vector (pMMP65). Group B was immunized with 3.0 × 109 CFU/mL of the vaccine, a mixture of 3 Brucella vaccine strains (using a Salmonella-based delivery system) expressing each recombinant B. abortus Omp3b, BCSP31, and SOD protein. No Salmonella delivery strain was isolated from all tested lymph nodes and parenchymal organs. Serum immunoglobulin (Ig) G titers and interferon gamma concentrations were significantly higher in group B than those in group A. After intraconjunctival challenge with virulent B. abortus strain 544, 40% of the vaccinated animals in group B were protected against B. abortus infection. The infection index and colonization of B. abortus in tested tissues was significantly lower in group B than group A. We conclude that this Brucella vaccine induces significant antigen-specific immune responses and provides effective protection against B. abortus infection in goats. Further studies are needed to enhance the protection rate of this Brucella vaccine and to discover its practical application in small ruminants.

La présente étude visait à évaluer l'efficacité protectrice d'une nouvelle formulation de vaccin contre Brucella chez les chèvres. Vingt chèvres noires furent séparées en deux groupes. Le Groupe A reçut par injection 3,0 × 109 unités formatrices de colonies (UFC)/mL de Salmonella servant de système de livraison ne contenant seulement que le vecteur (pMMP65). Le groupe B fut immunisé avec 3,0 × 109 UFC/mL du vaccin, un mélange de trois souches vaccinales de Brucella (utilisant le système de livraison à base de Salmonella) exprimant chaque protéine recombinante Omp3b, BCSP31, et SOD de B. abortus. Aucune bactérie Salmonella du système de livraison ne fut isolée des ganglions lymphatiques et organes testés. Les concentrations sériques d'immunoglobulines G (IgG) et d'interféron gamma étaient significativement plus élevées dans le groupe B que dans le groupe A. À la suite d'une infection défi par voie intra-conjonctivale avec une souche virulente de B. abortus (544), 40 % des animaux vaccinés dans le groupe B étaient protégés contre l'infection par B. abortus. L'index d'infection et de colonisation par B. abortus dans les tissus testés étaient significativement plus faible dans le groupe B comparativement au groupe A. Nous avons conclu que ce vaccin contre Brucella induisait des réponses immunes spécifiques d'antigène significatives et fournissait une protection efficace contre l'infection par B. abortus chez les chèvres. Des études additionnelles sont requises afin d'augmenter le taux de protection de ce vaccin (Brucella) et pour découvrir son application pratique chez les petits ruminants.(Traduit par Docteur Serge Messier).

Protective efficacy of a canine brucellosis vaccine candidate based on live attenuated Salmonella expressing recombinant Brucella BCSP31, Omp3b and SOD proteins in Beagles.

The aim of this study was to establish a proof-of-concept of protective efficacy of Salmonella-based B. abortus vaccine candidate in Beagles. Group A Beagles (n=10) were subcutaneously (SC) inoculated with S. Typhimurium delivery strain containing pMMP65 (vector to deliver antigens) only as vector control. Group B Beagles (n=10) were SC vaccinated with the mixture of the three Salmonella delivery strains expressing the recombinant B. abortus BCSP31, Outer membrane protein 3b (Omp3b), and superoxide dismutase (SOD) proteins, respectively. No Salmonella delivery strains were isolated from all tissues tested. Serum IgG, interleukin-4, tumor necrosis factor-alpha, and interferon-gamma concentrations were significantly higher in group B than in group A. Following intraconjunctival challenge with B. abortus 544, among 5 group B Beagles, the challenge strain was isolated from mandibular, and retropharyngeal lymph nodes of three Beagles, and no isolates were observed from all tissues of two Beagle. However, the challenge strains were detected from spleen, uterus (except two Beagles), and mandibular, prescapular, retropharyngeal, and superficial inguinal lymph nodes of all group A Beagles. These results suggest that the mixture of three S. Typhimurium delivery strains be a good vaccine candidate against brucellosis by B. abortus in dogs. Further investigations are needed to improve the protective efficacy of the Salmonella-based B. abortus vaccine candidate and explore its practical application in dogs.

Vaccination of goats with a combination Salmonella vector expressing four Brucella antigens (BLS, PrpA, Omp19, and SOD) confers protection against Brucella abortus infection.

Salmonella is an intracellular pathogen with a cellular infection mechanism similar to that of Brucella, making it a suitable choice for use in an anti-Brucella immune boost system. This study explores the efficacy of a Salmonella Typhimurium delivery-based combination vaccine for four heterologous Brucella antigens (Brucella lumazine synthase, proline racemase subunit A, outer-membrane protein 19, and Cu/Zn superoxide dismutase) targeting brucellosis in goats. We inoculated the attenuated Salmonella delivery-based vaccine combination subcutaneously at two different inoculation levels; 5 × 109 colony-forming unit (CFU)/mL (Group B) and 5 × 1010 CFU/mL (Group C) and challenged the inoculations with virulent Brucella abortus at 6 weeks post-immunization. Serum immunoglobulin G titers against individual antigens in Salmonella immunized goats (Group C) were significantly higher than those of the non-immunized goats (Group A) at 3 and 6 weeks after vaccination. Upon antigenic stimulation, interferon-γ from peripheral blood mononuclear cells was significantly elevated in Groups B and C compared to that in Group A. The immunized goats had a significantly higher level of protection as demonstrated by the low bacterial loads in most tissues from the goats challenged with B. abortus. Relative real-time polymerase chain reaction results revealed that the expression of Brucella antigens was lower in spleen, kidney, and lung of immunized goats than of non-immunized animals. Also, treatment with our combination vaccine ameliorated histopathological lesions induced by the Brucella infection. Overall, the Salmonella Typhimurium delivery-based combination vaccine was effective in delivering immunogenic Brucella proteins, making it potentially useful in protecting livestock from brucellosis.

Brucella abortus lysed cells using GI24 induce robust immune response and provide effective protection in Beagles.

The aim of the present study is to estimate the protective efficacy of Brucella abortus lysed cells by GI24 against brucellosis in Beagles. Group A was subcutaneously (sc) immunized with sterile phosphate-buffered saline, and group B was sc immunized with approximately 3 × 109 of the lysed cells. Brucella-LPS-specific serum IgG titers and IL-4, TNF-α and IFN-γ concentrations were investigated by enzyme linked immunosorbent assay. All dogs were intraconjunctivally challenged with B. abortus strain 544 at 6 weeks post-prime immunization. The serum IgG titers were considerably higher in group B than in group A. The levels of IL-4, TNF-α and IFN-γ in group B than in group A were significantly higher. Following challenge, no challenge strain was observed from all tissues of three dogs of group B. However, challenge strain was detected from spleen, uterus (except one Beagle) and inguinal and retropharyngeal lymph nodes of all group A Beagles. The results of this study demonstrated that sc immunization with the lysed cells induced robust antibody and cell-mediated immune responses in Beagles. The lysed cells also conferred protection against infection with B. abortus. These results suggest that sc immunization with B. abortus lysed cells by GI24 is a good vaccine candidate against brucellosis in dogs.

Protective efficacy of a Salmonella Typhimurium ghost vaccine candidate constructed with a recombinant lysozyme-PMAP36 fusion protein in a murine model.

A Salmonella Typhimurium ghost vaccine was constructed with the use of a recombinant fusion protein consisting of lysozyme and porcine myeloid antimicrobial peptide 36 expressed by the Escherichia coli overexpression system. After confirmation of its effectiveness by transmission electron microscopy the vaccine was evaluated in a murine model. Of the 60 BALB/c mice equally divided into 4 groups, group A mice were intramuscularly inoculated with 100 µL of sterile phosphate-buffered saline, and the mice in groups B, C, and D were intramuscularly inoculated with approximately 1.0 × 104, 1.0 × 105, or 1.0 × 106 cells of the S. Typhimurium ghost vaccine, respectively, in 100-µL amounts. The serum IgG titers against S. Typhimurium outer membrane proteins were significantly higher in groups B to D than in group A, as were the concentrations of interleukin-10 and interferon gamma in supernatants of harvested splenocytes. After challenge with wild-type S. Typhimurium, all the vaccinated groups showed significant protection compared with group A, notably perfect protection in groups C and D. Overall, these results show that intramuscular vaccination with 1.0 × 105 cells of this ghost vaccine candidate provided efficient protection against systemic infection with virulent S. Typhimurium.

Un vaccin fantôme dirigé contre Salmonella Typhimurium a été construit en utilisant une protéine de fusion recombinante composée de lysozyme et du peptide myéloïde antimicrobien 36 d'origine porcine exprimée par le système de surexpression d'Escherichia coli. Après confirmation de son efficacité par microscopie électronique à transmission, le vaccin a été évalué dans un modèle murin. Soixante souris BALB/c ont été séparées en quatre groupes. Les souris du groupe A ont été inoculées par voie intramusculaire (IM) avec 100 µL de saline tamponnée stérile, alors que les souris des groupes B, C, et D ont été inoculées IM avec approximativement 1,0 × 104, 1,0 × 105, ou 1,0 × 106 cellules du vaccin fantôme S. Typhimurium, respectivement, dans des volumes de 100 µL. Les titres d'IgG sériques contre les protéines de la membrane externe de S. Typhimurium étaient significativement plus élevés dans les groupes B à D que dans le groupe A, de même que les concentrations d'interleukine-10 et d'interféron gamma dans les surnageants de splénocytes récoltés. Suite à une infection défi avec une souche sauvage de S. Typhimurium, les animaux de tous les groupes vaccinés étaient protégés de manière significative comparativement à ceux du groupe A, notamment une protection parfaite pour les groupes C et D. De manière générale, ces résultats montrent que la vaccination IM avec 1,0 × 105 de ce vaccin fantôme candidat fourni une protection efficace contre une infection systémique par une souche virulente de S. Typhimurium.(Traduit par Docteur Serge Messier).

Protective efficacy by various doses of a new brucellosis vaccine candidate based on Salmonella strains expressing Brucella abortus BSCP31, Omp3b and superoxide dismutase against brucellosis in murine model.

Brucella species are important etiological agents of zoonotic diseases. Attenuated Salmonella strains expressing Brucella abortus BCSP31, Omp3b and superoxide dismutase proteins were tested as vaccine candidates in this study. In order to determine the optimal dose for intraperitoneal (IP) inoculation required to obtain effective protection against brucellosis, mice were immunized with various doses of a mixture of the three vaccine strains. Fifty BALB/c mice were divided into five equal groups (groups A-E). Group A mice were intraperitoneally inoculated with 100 µL of sterile phosphate-buffered saline. Group B, C, D and E mice were intraperitoneally immunized with approximately 1.2 × 105 colony-forming units (CFU) mL-1 of Salmonella containing pMMP65 in 100 µL and with 1.2 × 104 CFU mL-1, 1.2 × 105 CFU mL-1 and 1.2 × 106 CFU mL-1 of the mixture of the three strains in 100 µL, respectively. Serum IgG, tumor necrosis factor alpha and interferon gamma concentrations were significantly higher in group E than in groups A-D. Following challenge with B. abortus 544, the challenge strain was not detected in the spleen of any mouse from group E. Thus, IP immunization with 1.2 × 106 CFU mL-1 of the mixture of the three vaccine strains induced immune responses and provided effective protection against brucellosis in mice.

Protection efficacy of the Brucella abortus ghost vaccine candidate lysed by the N-terminal 24-amino acid fragment (GI24) of the 36-amino acid peptide PMAP-36 (porcine myeloid antimicrobial peptide 36) in murine models.

Brucella abortus cells were lysed by the N-terminal 24-amino acid fragment (GI24) of the 36-amino acid peptide PMAP-36 (porcine myeloid antimicrobial peptide 36). Next, the protection efficacy of the lysed fragment as a vaccine candidate was evaluated. Group A mice were immunized with sterile PBS, group B mice were intraperitoneally (ip) immunized with 3 × 108 colony-forming units (CFUs) of B. abortus strain RB51, group C mice were immunized ip with 3 × 108 cells of the B. abortus vaccine candidate, and group D mice were orally immunized with 3 × 109 cells of the B. abortus vaccine candidate. Brucella lipopolysaccharide (LPS)-specific serum IgG titers were considerably higher in groups C and D than in group A. The levels of interleukin (IL)-4, IL-10, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) were significantly higher in groups B-D than in group A. After an ip challenge with B. abortus 544, only group C mice showed a significant level of protection as compared to group A. Overall, these results show that ip immunization with a vaccine candidate lysed by GI24 can effectively protect mice from systemic infection with virulent B. abortus.