Inactivated Whole-cell Vaccine Research Articles

Safety assessments of recombinant DTaP vaccines developed in South Korea.

Pertussis bacteria have many pathogenic and virulent antigens and severe adverse reactions have occurred when using inactivated whole-cell pertussis vaccines. Therefore, inactivated acellular pertussis (aP) vaccines and genetically detoxified recombinant pertussis (rP) vaccines are being developed. The aim of this study was to assess the safety profile of a novel rP vaccine under development in comparison to commercial diphtheria-tetanus-acellular pertussis (DTaP) vaccines. The two positive control DTaP vaccines (two- and tri-components aP vaccines) and two experimental recombinant DTaP (rDTaP) vaccine (two- and tri-components aP vaccines adsorbed to either aluminum hydroxide or purified oat beta-glucan) were used. Temperature histamine sensitization test (HIST), indirect Chinese hamster ovary (CHO) cell cluster assay, mouse-weight-gain (MWG) test, leukocytosis promoting (LP) test, and intramuscular inflammatory cytokine assay of the injection site performed for safety assessments. HIST results showed absence of residual pertussis toxin (PTx) in both control and experimental DTaP vaccine groups, whereas in groups immunized with tri-components vaccines, the experimental tri-components rDTaP absorbed to alum showed an ultra-small amount of 0.0066 IU/mL. CHO cell clustering was observed from 4 IU/mL in all groups. LP tests showed that neutrophils and lymphocytes were in the normal range in all groups immunized with the two components vaccine. However, in the tri-components control DTaP vaccine group, as well as two- and tri-components rDTaP with beta-glucan group, a higher monocyte count was observed 3 days after vaccination, although less than 2 times the normal range. In the MWG test, both groups showed changes less than 20% in body temperature and body weight before the after the final immunizations. Inflammatory cytokines within the muscle at the injection site on day 3 after intramuscular injection revealed no significant response in all groups. There were no findings associated with residual PTx, and no significant differences in both local and systemic adverse reactions in the novel rDTaP vaccine compared to existing available DTaP vaccines. The results suggest that the novel rDTaP vaccine is safe.

Recombinant outer membrane vesicles delivering eukaryotic expression plasmid of cytokines act as enhanced adjuvants against Helicobacter pylori infection in mice.

The widespread prevalence of Helicobacter pylori (H. pylori) infection remains a great challenge to human health. The existing vaccines are not ideal for preventing H. pylori infection; thus, exploring highly effective adjuvants may improve the immunoprotective efficacy of H. pylori vaccines. In a previous study, we found that the outer membrane vesicles (OMVs), a type of nanoscale particle spontaneously produced by Gram-negative bacteria, could act as adjuvants to boost the immune responses to vaccine antigens. In this study, we explored the potential application of OMVs as delivery vectors for adjuvant development. We constructed recombinant OMVs containing eukaryotic expression plasmid of cytokines, including interleukin 17A or interferon-γ, and evaluated their function as adjuvants in combination with inactivated whole-cell vaccine (WCV) or UreB as vaccine antigens. Our results showed that recombinant OMVs as adjuvants could induce stronger humoral and mucosal immune responses in mice than wild-type H. pylori OMVs and the cholera toxin (CT) adjuvant. Additionally, the recombinant OMVs significantly promoted Th1/Th2/Th17-type immune responses. Furthermore, the recombinant OMV adjuvant induced more potent clearance of H. pylori than CT and wild-type OMVs. Our findings suggest that the recombinant OMVs coupled with cytokines may become potent adjuvants for the development of novel and effective vaccines against H. pylori infection.

A comprehensive immunoinformatic analysis of chitin deacetylase's and MP88 for designing multi-epitope vaccines against Cryptococcus neoformans.

Cryptococcus neoformans causes life-threatening pneumonia and meningitis and is regarded as one of the leading killers of immunocompromised individuals. There is currently no vaccine against this pathogen. Recently, WHO placed it at the top among the critical priority groups in the fungal priority pathogens to accelerate the development of effective treatments. Numerous studies suggested the potential of subunit vaccines to overcome the challenges associated with live and inactivated whole-cell vaccines. Therefore, this study exploited integrated reverse vaccinology and immunoinformatic approach to construct and characterize multi-epitope vaccines targeting chitin deacetylases (Cda1, Cda2, Cda3) and MP88 of C. neoformans. 4 CTL, 8 HTL and 6 B cell epitopes were fused with different adjuvants and appropriate linkers to design two multi-epitope vaccines (VC1 and VC2). Both chimeric constructs were predicted to be highly antigenic, non-allergenic, non-toxic, soluble and had satisfactory physicochemical properties. Molecular docking and binding free energy calculation revealed strong binding interactions between vaccine constructs and human TLRs (TLR-2 and TLR-4). Classical MD Simulation and Normal mode analysis verified the stability of the vaccine-TLR complex in the biological environment. Codon adaptation, cloning and in silico expression suggested the efficient expression of recombinant vaccine proteins in E. coli. Both candidates also generated robust immune profiles comprising innate, adaptive and humoral immune responses. Taken together, experimental validations of our findings through extensive invitro and invivo testing might provide an effective vaccine for prophylactic control of C. neoformans.Communicated by Ramaswamy H. Sarma.

Efficacy of combined inactivated vaccines against Vibrio alginolyticus and Streptococcus agalactiae infections in Nile tilapia in Egypt

Vibrio alginolyticus and Streptococcus agalactiae are important bacterial pathogens that yielded high losses in Nile tilapia in Egypt. The present study aimed to check the protective efficacy of inactivated whole-cell bivalent vaccines against these pathogens using incomplete Freund’s adjuvant and Montanide™ IMS 1312 VG as adjuvants. The antibody titers have been determined at different weeks post-vaccination (WPV). Moreover, the protection levels against the challenged bacterial pathogens have also been examined in relation to the time-dependent protection at different WPV. The results revealed that serum antibodies were generated in all immunized fish at 1st WPV, peaked at 4th WPV, continued, and gradually decreased from 6th WPV to 14th WPV in all vaccinated groups. In addition, vaccines induced significantly higher protection of the immunized tilapia, manifested by higher survival rates. We noticed that the antibody levels and survival rates of the vaccinated fish by a vaccine adjuvanted by Montanide™ IMS 1312 VG were higher than those produced by a vaccine adjuvanted by incomplete Freund’s adjuvant at different time points. Moreover, no external clinical signs, visceral adhesions, or internal lesions were recorded in the vaccinated tilapia, demonstrating the safety of the formulated vaccines. According to the aforementioned findings, we could suggest that the prepared bivalent vaccines, using the two adjuvant types, are safe and highly protective and could be utilized as promising candidate vaccines to increase the resistance of Nile tilapia against V. alginolyticus and S. agalactiae infections. Moreover, Montanide™ IMS 1312 VG enhanced the immuno-protectivity and exhibited optimum immune response and earlier protection compared to the vaccine adjuvanted by incomplete Freund’s adjuvant, demonstrating its added value during the preparation of tilapia vaccines.

Development of a Serum-Free Medium To Aid Large-Scale Production of Mycoplasma-Based Therapies.

To assist in the advancement of the large-scale production of safe Mycoplasma vaccines and other Mycoplasma-based therapies, we developed a culture medium free of animal serum and other animal components for Mycoplasma pneumoniae growth. By establishing a workflow method to systematically test different compounds and concentrations, we provide optimized formulations capable of supporting serial passaging and robust growth reaching 60 to 70% of the biomass obtained in rich medium. Global transcriptomic and proteomic analysis showed minor physiological changes upon cell culture in the animal component-free medium, supporting its suitability for the production of M. pneumoniae-based therapies. The major contributors to growth performance were found to be glucose as a carbon source, glycerol, cholesterol, and phospholipids as a source of fatty acids. Bovine serum albumin or cyclodextrin (in the animal component-free medium) were required as lipid carriers to prevent lipid toxicity. Connaught Medical Research Laboratories medium (CMRL) used to simplify medium preparation as a source of amino acids, nucleotide precursors, vitamins, and other cofactors could be substituted by cysteine. In fact, the presence of protein hydrolysates such as yeastolate or peptones was found to be essential and preferred over free amino acids, except for the cysteine. Supplementation of nucleotide precursors and vitamins is not strictly necessary in the presence of yeastolate, suggesting that this animal origin-free hydrolysate serves as an efficient source for these compounds. Finally, we adapted the serum-free medium formulation to support growth of Mycoplasma hyopneumoniae, a swine pathogen for which inactivated whole-cell vaccines are available. IMPORTANCE Mycoplasma infections have a significant negative impact on both livestock production and human health. Vaccination is often the first option to control disease and alleviate the economic impact that some Mycoplasma infections cause on milk production, weight gain, and animal health. The fastidious nutrient requirements of these bacteria, however, challenges the industrial production of attenuated or inactivated whole-cell vaccines, which depends on the use of animal serum and other animal raw materials. Apart from their clinical relevance, some Mycoplasma species have become cellular models for systems and synthetic biology, owing to the small size of their genomes and the absence of a cell wall, which offers unique opportunities for the secretion and delivery of biotherapeutics. This study proposes medium formulations free of serum and animal components with the potential of supporting large-scale production upon industrial optimization, thus contributing to the development of safe vaccines and other Mycoplasma-based therapies.

Trial Evaluation of Protection and Immunogenicity of Piscine Bivalent Streptococcal Vaccine: From the Lab to the Farms.

Streptococcosis is one of the major diseases that causes devastation to farmed fish, leading to significant economic losses all around the world. Currently, two serotypes of Streptococcus agalactiae, serotype Ia and III, have been identified as virulent strains and major causative agents of the disease in farmed Nile tilapia (Oreochromis niloticus Linn.) in Thailand. Upon inactivated vaccine development, monovalent inactivated whole-cell vaccines demonstrated high specific antibody production against homologous serotypes and limited production with heterologous serotypes. However, for higher efficacy, a bivalent streptococcal vaccine was designed to maximize protective immunity to both serotypes. Interestingly, our bivalent vaccine could successfully induce specific antibody production against both serotypes with similar levels, and the response could extend over the 8 weeks of the experimental period. Evaluation of vaccines in the laboratory scale revealed relative percent survival (RPS) of vaccinated tilapia to serotype Ia (81.2 ± 9.4%) and serotype III (72.2 ± 4.8%), respectively. The efficacy of the bivalent vaccine showed significant RPS higher than the monovalent vaccine (p < 0.05) at 30 days, and the protection of all those vaccines was reduced thereafter. Evaluation of the vaccine in a farm trial in different locations in Thailand revealed the efficacy of the bivalent vaccine in increasing the production yield by greater than 80% in all tested farms in 2015 and 2021. Taken together, this study affirms the efficacy of the bivalent streptococcal vaccine in the prevention of streptococcus disease in Nile tilapia, which could be used in different areas. This vaccine development could be effectively applied in the tilapia culture industry.

TbpBY167A-Based Vaccine Can Protect Pigs against Glässer's Disease Triggered by Glaesserella parasuis SV7 Expressing TbpB Cluster I.

Glaesserella parasuis is the etiological agent of Glässer’s disease (GD), one of the most important diseases afflicting pigs in the nursery phase. We analyzed the genetic and immunological properties of the TbpB protein naturally expressed by 27 different clinical isolates of G. parasuis that were typed as serovar 7 and isolated from pigs suffering from GD. All the strains were classified as virulent by LS-PCR. The phylogenetic analyses demonstrated high similarity within the amino acid sequence of TbpB from 24 clinical strains all belonging to cluster III of TbpB, as does the protective antigen TbpBY167A. Three G. parasuis isolates expressed cluster I TbpBs, indicating antigenic diversity within the SV7 group of G. parasuis. The antigenic analysis demonstrated the presence of common epitopes on all variants of the TbpB protein, which could be recognized by an in vitro analysis using pig IgG induced by a TbpBY167A-based vaccine. The proof of concept of the complete cross-protection between clusters I and III was performed in SPF pigs immunized with the TbpBY167A-based vaccine (cluster III) and challenged with G. parasuis SV7, strains LM 360.18 (cluster I). Additionally, pigs immunized with a whole-cell inactivated vaccine based on G. parasuis SV5 (Nagasaki strain) did not survive the challenge performed with SV7 (strain 360.18), demonstrating the absence of cross-protection between these two serovars. Based on these results, we propose that a properly formulated TbpBY167A-based vaccine may elicit a protective antibody response against all strains of G. parasuis SV7, despite TbpB antigenic diversity, and this might be extrapolated to other serovars. This result highlights the promising use of the TbpBY167A antigen in a future commercial vaccine for GD prevention.

Development of an inactivated whole cell vaccine through immersion for immunoprophylaxis of Francisella orientalis infections in Nile tilapia (Oreochromis niloticus L.) fingerlings and juveniles

Francisella orientalis infections, known as francisellosis, are one of the most important diseases affecting the production of Nile tilapia, causing high mortality rates in the most susceptible fish stages: fingerlings and juveniles. Antibiotic therapy is the method of choice for treating the disease, as there are no commercially available vaccines. In this study, we developed an inactivated whole-cell vaccine using an isolate of F. orientalis in combination with the aqueous adjuvant Montanide IMS 1312 VG, which was administered to Nile tilapia through immersion. Two immunization trials (1 and 2) were conducted with fish at the fingerling and juvenile stages. For each trial, five different experimental groups were established: a complete vaccine (bacterin in combination with aqueous adjuvant), bacterin, aqueous adjuvant, and positive and negative controls. Thirty days after vaccination, an experimental challenge was performed through intraperitoneal injection of the same F. orientalis isolate. As a result, the vaccinated fingerlings were the only group in which mortality and progression of clinical signs of francisellosis were statistically significantly reduced, although relative percentage of survival (RPS) was low at 50%. In the juvenile group, RPS was higher at 63%, but not statistically significant. Nevertheless, an RPS of only 50% is acceptable for using vaccines in the field. The bacterin and adjuvant treatments alone were not effective, showing an RPS of 37% and 0%, respectively. Post-vaccination mortality was observed in the group exposed only to the adjuvant, which may indicate excessive immune stimulation at this stage. Interestingly, the immune response elicited by the vaccine was unable to eliminate the pathogen from the host; therefore, the surviving animals became carriers. Although the immune response elicited by the vaccine was unable to eliminate the pathogen from the host, this vaccine formulation could be a viable alternative for use in the field and serve as another means of controlling the mortality caused by the pathogen. Our study provides the first report of vaccination, using immersion, against francisellosis at the most susceptible stages of farmed Nile tilapia. Future studies should address the efficiency of immersion vaccines under field conditions.

The use of outer membrane vesicles as novel, mucosal adjuvants against intracellular bactiera

Abstract Many pathogens first enter the body via mucosal surfaces where they can then invade and disseminate systemically to cause disease. Despite this, most vaccines are given parenterally and are unable to induce mucosal immunity. Immunizing directly at the mucosa could solve this problem, however delivering vaccines at these surfaces often doesn’t invoke robust immunity. One way to alleviate this is to use adjuvants that can evoke an immune response. Most adjuvants, like aluminum salts, are unable to induce mucosal immunity and so novel adjuvants must be employed. Outer membrane vesicles (OMVs) from Burkholderia pseudomallei are potent immune mediators and have been shown to have adjuvant capabilities. The goal of this study is to highlight the role of OMVs as a novel adjuvant that can be used in the next generation of mucosal vaccines. To test this, we created an OMV-adjuvanted inactivated whole-cell vaccine against two intracellular pathogens – Salmonella Typhimurium and Francisella holarctica LVS that could be delivered mucosally. An oral vaccine against S. Typhimurium adjuvanted with OMVs showed protection against lethal challenge in addition to evoking antigen specific CD4 T cells, B cells, and anti-Salmonella antibodies. These antibodies induced greater bacterial killing in macrophages. We are currently exploring an OMV-adjuvanted oropharyngeally delivered vaccine against F. holarctica LVS. Immunity against Francisella requires both CD4 and CD8 T cells and we are determining how an OMV-adjuvanted vaccine will influence these immune cell populations. This study represents a novel approach to mucosal vaccines using OMVs as adjuvants. Supported by NIH U01 AI124289 NIH BAA HHSN72201800045C

Immune Responses to Irradiated Pneumococcal Whole Cell Vaccine.

Streptococcus pneumoniae (pneumococcus) can cause respiratory and systemic diseases. Recently, γ-irradiation-inactivated, non-encapsulated, intranasal S. pneumoniae (r-SP) vaccine has been introduced as a novel serotype-independent and cost-effective vaccine. However, the immunogenic mechanism of r-SP is poorly understood. Here, we comparatively investigated the protective immunity and immunogenicity of r-SP to the heat-(h-SP) or formalin-inactivated vaccine (f-SP) without adjuvants. Mice were intranasally immunized with each vaccine three times and then challenged with a lethal dose of S. pneumoniae TIGR4 strain and then subsequently evaluated for their immune responses. Immunization with r-SP elicited modestly higher protection against S. pneumoniae than h-SP or f-SP. Immunization with r-SP enhanced pneumococcal-specific IgA in the nasal wash and IgG in bronchoalveolar lavage fluid. Immunization with r-SP enhanced S. pneumoniae-specific IgG, IgG1, and IgG2b in the serum. r-SP more potently induced the maturation of dendritic cells in the cervical lymph nodes than h-SP or f-SP. Interestingly, populations of follicular helper T cells and IL-4-producing cells were potently increased in cervical lymph nodes of r-SP-immunized mice. Collectively, r-SP could be an effective intranasal, inactivated whole-cell vaccine in that it elicits S. pneumoniae-specific antibody production and follicular helper T cell activation leading to protective immune responses against S. pneumoniae infection.

Lack of evidence supporting a role of IFN-β and TGF-β in differential polarization of Bordetella pertussis specific-T cell responses

Bordetella Pertussis (BP) vaccine-induced immunity is waning worldwide despite excellent vaccine coverage. Replacement of the whole-cell inactivated vaccine (wP) by an acellular subunit vaccine (aP) is thought to play a major role and to be associated with the recurrence of whooping cough. Previously, we detected that the polarization towards a Th2 and Th1/Th17 response in aP and wP vaccinees, respectively, persists upon aP boosting in adolescents and adults. Additionally, IL-9 and TGF-β were found to be up-regulated in aP-primed donors and network analysis further identified IFN-β as a potential upstream regulator of IL-17 and IL-9. Based on these findings, we hypothesized that IFN-β produced following aP vaccination may lead to increased IL-9 and decreased IL-17 production. Also, due to the well characterized role of TGF-β in both Th17 and Th9 differentiation, we put forth that TGF-β addition to BP-stimulated CD4 + T cells might modulate IL-17 and IL-9 production. To test this hypothesis, we stimulated in vitro cultures of PBMC or isolated naive CD4 + T cells from aP vs wP donors with a pool of BP epitopes and assessed the effect of IFN-β or TGF-β in proliferative responses as well as in the cytokine secretion of IL-4, IL-9, IL-17, and IFN-γ. IFN-β reduced BP-specific proliferation in PBMC as well as cytokine production but increased IL-9, IL-4, and IFN-γ cytokines in naïve CD4 + T cells. These effects were independent of the childhood vaccination received by the donors. Similarly, TGF-β reduced BP-specific proliferation in PBMC but induced proliferation in naïve CD4 + T cells. However, stimulation was associated with a generalized inhibition of cytokine production regardless of the original aP or wP vaccination received by the donors. Our study suggests that key T cell functions such as cytokine secretion are under the control of antigen stimulation and environmental cues but molecular pathways different than the ones investigated here might underlie the long-lasting differential cytokine production associated with aP- vs wP-priming in childhood vaccination.

Development of Next Generation Streptococcus pneumoniae Vaccines Conferring Broad Protection.

Streptococcus pneumoniae is a major pathogen causing pneumonia with over 2 million deaths annually, especially in young children and the elderly. To date, at least 98 different pneumococcal capsular serotypes have been identified. Currently, the vaccines for prevention of S. pneumoniae infections are the 23-valent pneumococcal polysaccharide-based vaccine (PPV23) and the pneumococcal conjugate vaccines (PCV10 and PCV13). These vaccines only cover some pneumococcal serotypes and are unable to protect against non-vaccine serotypes and unencapsulated S. pneumoniae. This has led to a rapid increase in antibiotic-resistant non-vaccine serotypes. Hence, there is an urgent need to develop new, effective, and affordable pneumococcal vaccines, which could cover a wide range of serotypes. This review discusses the new approaches to develop effective vaccines with broad serotype coverage as well as recent development of promising pneumococcal vaccines in clinical trials. New vaccine candidates are the inactivated whole-cell vaccine strain (Δpep27ΔcomD mutant) constructed by mutations of specific genes and several protein-based S. pneumoniae vaccines using conserved pneumococcal antigens, such as lipoprotein and surface-exposed protein (PspA). Among the vaccines in Phase 3 clinical trials are the pneumococcal conjugate vaccines, PCV-15 (V114) and 20vPnC. The inactivated whole-cell and several protein-based vaccines are either in Phase 1 or 2 trials. Furthermore, the recent progress of nanoparticles that play important roles as delivery systems and adjuvants to improve the performance, as well as the immunogenicity of the nanovaccines, are reviewed.

Vaccination with a live-attenuated small-colony variant improves the humoral and cell-mediated responses againstStaphylococcus aureus.

Staphylococcus aureus is known to produce persistent and chronic infections in both humans and animals. It is recognized that small-colony variants (SCVs), which produce higher levels of biofilm and that are capable of intracellular persistence, contribute to the chronicity or recurrence of infections and that this phenotype is inherent to the pathogenesis process. Prevention of S. aureus infections through vaccination has not yet met with considerable success. Some of the current vaccine formulations for S. aureus bovine mastitis consist of inactivated S. aureus bacteria, sometimes combined to E. coli J5. As such, the stimulation of cell-mediated immunity by these vaccines might not be optimal. With this in mind, we recently engineered a genetically stable double mutant SCV (ΔvraGΔhemB), which was highly attenuated in a mastitis model of infection. The present work describes the immune responses elicited in mice by various experimental vaccine compositions including the live-attenuated SCV double mutant and its inactivated form, combined or not with inactivated E. coli J5. The live-attenuated SCV was found to provoke a strong and balanced humoral response in immunized mice, as well as strong proliferation of ex-vivo stimulated splenocytes isolated from these animals. These splenocytes were also found to release high concentration of IL-17 and IFN-γ when compared to every other vaccination formulation. Inversely, the inactivated whole-cell vaccine, alone or in combination with the E. coli J5 bacterin, elicited lower antibody titers and failed to induce Th1 or Th17 cell-mediated responses in the splenocyte proliferation assay. Our results suggest that live-attenuated SCVs can trigger host immunity differently than inactivated bacteria and could represent a suitable vector for inducing strong humoral and cell-mediated immune responses, which are crucial for protection. This could represent an important improvement over existing vaccine formulations for preventing S. aureus bovine mastitis and other infections caused by this pathogen.

The immunoprotective effect of whole-cell lysed inactivated vaccine with SWCNT as a carrier against Aeromonas hydrophila infection in grass carp

Aeromonas hydrophila is a strong gram-negative bacterium that can cause a mass death of grass carp, and result in the huge economic loss. Development of practical vaccines is the best way to control the outbreak of this bacterial disease. In this study, a whole-cell inactivated vaccine was obtained via sonication, and then single-walled carbon nanotubes (SWCNTs) was used to link to the bacterial lysate (BL) for a novel vaccine (SWCNTs-BL). A total of 400 fish were vaccinated with BL and SWCNTs-BL via immersion (5, 10 mg L−1) or injection (5, 10 μg/fish) before challenge with live A. hydrophila at the 28 days post immunization (d.p.i.). The results showed that the antibody titer, enzymatic activity, expression of some immune-related genes (especially IgM and TNF-α) and RPS of fish in the injection groups were significantly increased compared to the control group after 28 d.p.i. For the immersion groups, immunological parameters were increased compared to the control group. Furthermore, the immuno-protective effects of SWCNTs-BL were better than BL. The above results indicated that BL of A. hydrophila can effectively induce specific immune response of grass carp, and BL linked with functionalized SWCNTs could enhance the protective effect of immersion immunization. Our results may provide a practical vaccine, with a simple production, to fight against bacterial diseases in aquaculture industry.

Immunogenicity of recombinant outer membrane porin protein and protective efficacy against lethal challenge with Bordetella bronchiseptica in rabbits.

The outer membrane porin protein (OMPP) of Bordetella bronchiseptica is an important adhesion factor and protective immunogen. The aim of this study was to verify the immunogenicity of recombinant OMPP and its protective efficacy against a lethal challenge with B. bronchiseptica in rabbits. Soluble rOMPP was successfully expressed in Escherichia coli, and the purified recombinant protein was mixed with the ISA 201 VG adjuvant to prepare a subunit vaccine for B. bronchiseptica. Rabbits were immunized with the rOMPP subunit vaccine and then infected with the virulent B. bronchiseptica strain QDBb01. Rabbits immunized with the subunit vaccine were completely protected compared to the control group, and the protective effect was obviously better than that ofthe inactivated whole-cell vaccine. Moreover, analysis of the immunization duration showed that the rOMPP subunit vaccine provided immune protection for at least 4months after the second immunization. The rOMPP subunit vaccine completely protected rabbits from a subsequent B. bronchiseptica challenge. The results will provide key information for the development of a safe and effective recombinant subunit vaccine against B. bronchiseptica in rabbits.

Cellular and Mucosal Immune Responses Following Vaccination with Inactivated Mutant of Escherichia coli O157:H7

Shiga toxin-producing Escherichia coli O157:H7 (O157) can cause mild to severe gastrointestinal disease in humans. Cattle are the primary reservoir for O157, which colonizes the intestinal tract without inducing any overt clinical symptoms. Parenteral vaccination can reduce O157 shedding in cattle after challenge and limit zoonotic transmission to humans, although the impact of vaccination and vaccine formulation on cellular and mucosal immune responses are undetermined. To better characterize the cattle immune response to O157 vaccination, cattle were vaccinated with either water-in-oil-adjuvanted, formalin-inactivated hha deletion mutant of Shiga toxin 2 negative (stx2−) O157 (Adj-Vac); non-adjuvanted (NoAdj-Vac); or non-vaccinated (NoAdj-NoVac) and peripheral T cell and mucosal antibody responses assessed. Cattle in Adj-Vac group had a higher percentage of O157-specific IFNγ producing CD4+ and γδ+ T cells in recall assays compared to the NoAdj-Vac group. Furthermore, O157-specific IgA levels detected in feces of the Adj-Vac group were significantly lower in NoAdj-Vac group. Extracts prepared only from Adj-Vac group feces blocked O157 adherence to epithelial cells. Taken together, these data suggest parenteral administration of adjuvanted, inactivated whole-cell vaccines for O157 can induce O157-specific cellular and mucosal immune responses that may be an important consideration for a successful vaccination scheme.

Efficacy of an inactivated whole-cell injection vaccine for nile tilapia, Oreochromis niloticus (L), against multiple isolates of Francisella noatunensis subsp. orientalis from diverse geographical regions

Francisellosis, induced by Francisella noatunensis subsp. orientalis (Fno), is an emerging bacterial disease representing a major threat to the global tilapia industry. There are no commercialised vaccines presently available against francisellosis for use in farmed tilapia, and the only available therapeutic practices used in the field are either the prolonged use of antibiotics or increasing water temperature. Recently, an autogenous whole cell-adjuvanted injectable vaccine was developed that gave 100% relative percent survival (RPS) in tilapia challenged with a homologous isolate of Fno. In this study, we evaluated the efficacy of this vaccine against challenge with heterologous Fno isolates. Healthy Nile tilapia, Oreochromis niloticus (∼15 g) were injected intraperitoneally (i.p.) with the vaccine, adjuvant-alone or phosphate buffer saline (PBS) followed by an i.p. challenge with three Fno isolates from geographically distinct locations. The vaccine provided significant protection in all groups of vaccinated tilapia, with a significantly higher RPS of 82.3% obtained against homologous challenge, compared to 69.8% and 65.9% with the heterologous challenges. Protection correlated with significantly higher specific antibody responses, and western blot analysis demonstrated cross-isolate antigenicity with fish sera post-vaccination and post-challenge. Moreover, a significantly lower bacterial burden was detected by qPCR in conjunction with significantly greater expression of IgM, IL-1 β, TNF-α and MHCII, 72 h post-vaccination (hpv) in spleen samples from vaccinated tilapia compared to fish injected with adjuvant-alone and PBS. The Fno vaccine described in this study may provide a starting point for development a broad-spectrum highly protective vaccine against francisellosis in tilapia.

Pasteurella multocida inactivated with ferric chloride and adjuvanted with bacterial DNA is a potent and efficacious vaccine in Balb/c mice.

Pasteurella multocida (P. multocida) is a principal pathogen of domestic animals and an opportunistic pathogen of humans. It is the causative agent of pneumonia and haemorrhagic septicaemia in cattle, sheep and goats, fowl cholera in chickens and progressive atrophic rhinitis in swine. In this study, we investigated the humoral and cellular immune responses and protective immunity conferred by an iron-inactivated vaccine with bacterial DNA (IIV+bDNA) as an adjuvant in mice. P. multocida was grown in BHI broth, inactivated with formalin and FeCl3 and adjuvanted with alum and bDNA. Mice were immunized with two whole-cell inactivated vaccine doses 2 weeks apart. The animals were challenged 4 weeks after booster immunization. Immunogens (vaccines and bDNA) posed no safety problems when mice were injected subcutaneously (s/c) with these preparations. The serum antibody titres were tested by ELISA. At 28 days post immunization, cell-mediated immunity responses were determined. The responses were measured by assay of IL-6 and IL-12 in lymphocyte spleen culture supernatants. ELISA results showed that the levels of antibodies in iron inactivated with bDNA adjuvant groups were higher than in the formalin inactivated with alum adjuvant vaccine group. The protection rate of IIV+bDNA adjuvant vaccine was superior to that of the other vaccines and it protected 100 % of the challenge group mice. Following immunization, bDNA promoted increased production of interleukins compared to the control groups. These studies indicate that bDNA is effective as an immune adjuvant, and along with stimulatory bDNA represent promising new humoral and cellular immune enhancers for vaccination applications. In addition, this vaccine is able to provide long-term protection against infection.

Development of a vaccine against Streptococcus agalactiae in fish based on truncated cell wall surface anchor proteins.

Streptococcus agalactiae is an important fish pathogen and a leading cause of major economic losses to the aquaculture industry worldwide. In the present study, the two truncated recombinant proteins of cell wall surface anchor family of S agalactiae, CWSAP465 and CWSAP1035, were expressed in Escherichia coli, and their immunogenicity and efficacy against the bacterium were evaluated in tilapia and turbot. The results showed that the prokaryotic expression of the two constructs, p32a-CWSAP465 and p32a-CWSAP1035, gave rise to a high yield of soluble proteins with good immunogenicity. The immunisation-challenge study revealed that tilapia and turbot immunised with recombinant truncated proteins produced high levels of antibodies with a peak at four weeks after immunisation and were protected from a challenge by a virulent S agalactiae at a dose of 1×109 colony forming units/ml. The recombinant truncated proteins had higher efficacy than the whole-cell inactivated vaccine. Therefore, the study demonstrated that CWSAP465 and CWSAP1035 are two viable vaccine candidates against S agalactiae in fish.

Lactococcus garvieae outbreaks in Brazilian farms Lactococcosis in Pseudoplatystoma sp. - development of an autogenous vaccine as a control strategy.

This study evaluated the control of streptococcosis outbreaks in Brazil, isolated from diseased sorubim and identified as Lactococcus garvieae by genetic sequencing. This report determined the potential for lactococcosis control in sorubim Pseudoplatystoma sp. with two vaccines: an aqueous-based, whole-cell inactivated vaccine (bacterin) and an oil-adjuvanted bacterin. Their efficacy was evaluated at 30days post-vaccination (d.p.v.) by challenge with L.garvieae, and the antibody production response at 15, 30 and 60 d.p.v. and the non-specific immune response were compared amongst treatments. High protection levels (P<0.05) were achieved with the oil-adjuvanted vaccine with a relative percentage survival value of 81.7% at 30 d.p.v. Additionally, the oil-adjuvanted vaccine increased the immunogenicity of the bacterin as indicated by greater agglutination antibody titres from 15 until 60 d.p.v. This is the first report of a positive effect of vaccine administration on the specific immunity of sorubim, and the study showed that a specific antibody plays an important role in sorubim defence against lactococcosis because the innate immune responses were similar in all of the studied animals. These results demonstrated that oil-adjuvanted vaccinecan be an effective alternative for the protection of sorubim from L.garvieae disease.