Search

In our previous studies, we have identified several in vivo-induced antigens and evaluated their potential as subunit vaccine candidates in a murine model, in which the recombinant protein GalT showed the most potent immunogenicity and immunoprotective efficacy against Actinobacillus pleuropneumoniae . To exploit a more efficient way of delivering GalT proteins, in this study, we employed the widely studied E. coli outer membrane vesicles (OMVs) as a platform to deliver GalT protein and performed the vaccine trial using the recombinant GalT-OMVs in the murine model. Results revealed that GalT-OMVs could elicit a highly-specific, IgG antibody titer that was comparable with the adjuvant GalT group. Significantly higher lymphocyte proliferation and cytokines secretion levels were observed in the GalT-OMVs group. 87.5% and 50% of mice were protected from a lethal dose challenge using A. pleuropneumoniae in active or passive immunization, respectively. Histopathologic and immunohistochemical analyses showed remarkably reduced pathological changes and infiltration of neutrophils in the lungs of mice immunized with GalT-OMVs after the challenge. Taken together, these findings confirm that OMVs can be used as a platform to deliver GalT protein and enhance its immunogenicity to induce both humoral and cellular immune responses in mice.

Background: Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the etiologic agent Anaplasma phagocytophilum. HGA was designated a nationally notifiable disease in the United States in 1998. Currently there are no vaccines available against HGA. Conserved membrane proteins that are subdominant in Anaplasma species, such as VirB9 and VirB10, may represent better vaccine targets than the variable immunodominant surface proteins. VirB9 and VirB10 are constituents of the Type 4 secretion system (T4SS) that is conserved amongst many intracellular bacteria and performs essential functions for invasion and survival in host cells., Results: Immunogenicity and contribution to protection, provided after intramuscular vaccination of plasmid DNA encoding VirB9-1, VirB9-2, and VirB10 followed by inoculation of homologous recombinant proteins, in a prime-boost immunization strategy was evaluated in a murine model of HGA. Recombinant VirB9-1-, VirB9-2-, and VirB10-vaccinated mice developed antibody responses that specifically reacted with A. phagocytophilum organisms. However, only the mice vaccinated with VirB10 developed a significant increase in IFN-γ CD4 + T cells and partial protection against challenge with A. phagocytophilum., Conclusions: This work provides evidence that A. phagocytophilum T4SS VirB10 is partially protective in a murine model against infection in an IFN-γ-dependent fashion and suggests that this protein may be a potential vaccine candidate against this and possibly other pathogenic bacteria with a T4SS.

Glycoconjugate vaccines obtained by chemical linkage of a carbohydrate antigen to a protein are part of routine vaccinations in many countries. Licensed antimicrobial glycan-protein conjugate vaccines are obtained by random conjugation of native or sized polysaccharides to lysine, aspartic or glutamic amino acid residues that are generally abundantly exposed on the protein surface. In the last few years, the structural approaches for the definition of the polysaccharide portion (epitope) responsible for the immunological activity has shown potential to aid a deeper understanding of the mode of action of glycoconjugates and to lead to the rational design of more efficacious and safer vaccines. The combination of technologies to obtain more defined carbohydrate antigens of higher purity and novel approaches for protein modification has a fundamental role. In particular, methods for site selective glycoconjugation like chemical or enzymatic modification of specific amino acid residues, incorporation of unnatural amino acids and glycoengineering, are rapidly evolving. Here we discuss the state of the art of protein engineering with carbohydrates to obtain glycococonjugates vaccines and future perspectives.

Brucellosis is a highly contagious and zoonotic disease and has a considerable impact on animal health and economy of a country, principally in Pakistan, where rural income largely depends upon livestock farming and dairy products. The disease burden is more in underdeveloped/developing countries due to the low economy and limited access to the diagnostic facilities. In Pakistan, the prevalence of Brucella abortus is very high, so it is the need of the hour to control this disease through more advanced methods. This study was designed with the aim to construct the DNA based vaccine of gene encoding antigenic surface protein (BCSP 31 ). For this purpose, the BCSP 31 gene was amplified, purified and ligated in pTZ57 R/T (cloning vector). Dubbed BCSP 31 -pTZ57 R/T vector was transformed into competent cells (DH5α). After plasmid extraction, the plasmid and pET-28a vector was restricted with EcoRI and BamHI. Again, ligation was done and dubbed pET-28a-BCSP 31 transformed into E. coli (BL21). After expression, the protein was purified and used for evaluation of immunogenic response. The protective and immunogenic efficacy of the vaccine was evaluated in rabbits (n = 20). The rabbits were divided into four equal groups. Groups A-C were given purified protein diluted in normal saline @ 750, 1500 and 3000 μg/0.2 mL, respectively through intraconjunctival route. Group D was given 0.2 mL normal saline through intraconjunctival route. Specific immunoglobulin G (IgG) responses were measured through indirect ELISA on a weekly basis. The titer of IgG against the antigen was significantly (p < 0.05) higher in vaccinated groups A-C as compared to group D (control group) in a dose dependent manner. Moreover, log units of protection produced by DNA based vaccine in the rabbits (3.02) also indicated the protective efficacy of the DNA vaccine against B. abortus challenge. The response of this vaccine in rabbit suggested its potential effectiveness against Brucella abortus in large animals., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Leptospirosis is known as a zoonotic disease of global importance originated from infection with the spirochete bacterium Leptospira. Although several leptospirosis vaccines have been tested, the vaccination is relatively unsuccessful in clinical application despite decades of research. Therefore, this study was conducted to construct a novel multi-epitope based vaccine against leptospirosis by using Hap1, LigA, LAg42, SphH and HSP58 antigens. T cell and IFN gamma epitopes were predicted from these antigens. In addition, to induce strong CD4 + helper T lymphocytes (HTLs) responses, Pan HLA DR-binding epitope (PADRE) and helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied. Moreover, for boosting immune response, Heparin-Binding Hemagglutinin (HBHA), a novel TLR4 agonist was added to the construct as an adjuvant. Finally, selected epitopes were linked together using EAAAK, GPGPG, AAY and HEYGAEALERAG linkers. Based on the predicted epitopes, a multi-epitope vaccine was construct with 490 amino acids. Physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility, and allergenicity of this vaccine construct were assessed by applying immunoinformatics analyses. A soluble, and non-allergic protein with a molecular weight of 53.476 kDa was obtained. Further analyses validated the stability of the chimeric protein and the predicted epitopes in the chimeric vaccine indicated strong potential to induce B-cell and T-cell mediated immune response. Therefore, immunoinformatics analysis showed that the modeled multi-epitope vaccine can properly stimulate the both T and B cells immune responses and could potentially be used for prophylactic or therapeutic usages., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Plasmid DNA encoding flagellin FlaB3 was used as a vaccination candidate for the evaluation of immunogenicity and protection against Treponema pallidum subsp. pallidum dissemination. First, intramuscular injection of the flagellin encoded by the plasmid DNA into New Zealand rabbits elicited both humoral and cellular immune responses. Total IgG production increased in response to flagellin. In addition, serum IFN-γ secretion and CD8+ cells were substantially greater in the rabbits immunized with the plasmid encoding flagellin FlaB3 than those in the rabbits immunized with recombinant flagellin. The flagellin encoded by the plasmid DNA induced significant upregulation of serum IL-6 and IL-8 compared to that of the control rabbits. Subsequently, intradermal challenge of the vaccinated New Zealand rabbits with 1 × 10 7 T. pallidum resulted in a significant reduction of the bacterial organ burden in the blood, liver, spleen, and testicles in the flagellin plasmid DNA-vaccinated rabbits. Furthermore, the histopathological analysis demonstrated that the rabbits immunized with the plasmid DNA-encoded flagellin (FlaB3) showed better immune protection. These findings provide evidence that plasmid DNA-encoded flagellin (FlaB3) may be useful as a potential immunization route for future development of a vaccine to inhibit T. pallidum dissemination in related animals.

Leptospirosis is a serious epidemic disease caused by pathogenic Leptospira species. The disease is endemic in most tropical and sub-tropical regions of the world. Currently, there is no effective polyvalent vaccine for prevention against most of the circulating serovars. Moreover, development of an efficient leptospiral vaccine capable of stimulating cross-protective immune responses against a wide range of serovars remains a daunting challenge. This, in part, is associated with the extensive diversity and variation of leptospiral serovars from region to region. In this study, a multi-epitope DNA vaccine encoding highly immunogenic epitopes from LipL32 and LipL41 was designed using in-silico approach. The DNA encoding antigenic epitopes was constructed from conserved pathogenic Leptospira genes (LipL32 and LipL41). Immunization of golden Syrian hamsters with the multi-epitope chimeric DNA vaccine resulted in the production of both agglutinating and neutralizing antibodies as evidence by MAT and in-vitro growth inhibition tests respectively. The antibodies produced reacted against eight different serovars and significantly reduced renal colonization following in vivo challenge. The vaccine was also able to significantly reduce renal colonization which is a very important factor responsible for persistence of leptospires among susceptible and reservoir animal hosts. In conclusion, the leptospiral multi-epitope chimeric DNA vaccine can serve as a potentially effective and safe vaccine against infection with different pathogenic leptospiral serovars., (Copyright © 2018. Published by Elsevier Ltd.)

The symptoms of Clostridium difficile infection (CDI) are attributed largely to two C. difficile toxins, TcdA and TcdB. Significant efforts have been devoted to developing vaccines targeting both toxins through parenteral immunization routes. However, C. difficile is an enteric pathogen, and mucosal/oral immunization would be particularly useful to protect the host against CDI, considering that the gut is the main site of disease onset and progression. Moreover, vaccines directed only against toxins do not target the cells and spores that transmit the disease. Previously, we constructed a chimeric vaccine candidate, mTcd138, comprised of the glucosyltransferase and cysteine proteinase domains of TcdB and the receptor binding domain of TcdA. In this study, to develop an oral vaccine that can target both C. difficile toxins and colonization/adhesion factors, we expressed mTcd138 in a nontoxigenic C. difficile (NTCD) strain, resulting in strain NTCD&#95;mTcd138. Oral immunization with spores of NTCD&#95;mTcd138 provided mice full protection against infection with a hypervirulent C. difficile strain, UK6 (ribotype 027). The protective strength and efficacy of NTCD&#95;mTcd138 were further evaluated in the acute CDI hamster model. Oral immunization with spores of NTCD&#95;mTcd138 also provided hamsters significant protection against infection with 2 × 10 4 UK6 spores, a dose 200-fold higher than the lethal dose of UK6 in hamsters. These results imply that the genetically modified, nontoxigenic C. difficile strain expressing mTcd138 may represent a novel mucosal vaccine candidate against CDI., (Copyright © 2018 American Society for Microbiology.)

Clostridium perfringens enterotoxin (CPE) is a common cause of food poisoning and hyperkalemia-associated death. Previously, we reported that fusion of pneumococcal surface protein A (PspA) to C-terminal fragment of CPE (C-CPE) efficiently bound mucosal epithelium so that PspA-specific immune responses could be provoked. In this study, we found that fusion of C-CPE with PspA augmented the antigenicity of C-CPE itself. These findings allowed us to hypothesize that fusion of C-CPE and another food poisoning vaccine act as a bivalent food poisoning vaccine. Therefore, we constructed an adjuvant-free bivalent vaccine against CPE and cholera toxin (CT), which is a major food poisoning in developing country, by genetically fusing CT B subunit to C-CPE. Because of the low antigenicity of C-CPE, immunization of mice with C-CPE alone did not induce C-CPE-specific immune responses. However, immunization with our vaccine induced both C-CPE- and CT-specific neutralizing antibody. The underlying mechanism of the augmented antigenicity of C-CPE included the activation of T cells by CTB. Moreover, neutralizing antibodies lasted for at least 48 weeks and the quality of the antibody was dependent on the binding activity of CTB-C-CPE to its receptors. These findings suggest that our fusion protein is a potential platform for the development of an adjuvant-free bivalent vaccine against CPE and CT.

Vaccines are one of the most important methods for preventing infectious disease. Structural modification of lipopolysaccharide (LPS) provides a strategy for the development of live attenuated vaccines, either by altering the immunogenicity or by attenuating virulence of the bacteria. This review summarizes various approaches that utilize LPS mutants as whole-cell vaccines., (Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Otitis media (OM) is a common disease affecting humans, especially paediatric populations. OM refers to inflammation of the middle ear and can be broadly classified into two types, acute and chronic. Bacterial infection is one of the most common causes of OM. Despite the introduction of vaccines, the incidence of OM remains significantly high worldwide. In this mini-review article, we discuss the recent treatment modalities for OM, such as suspension gel, transcutaneous immunization, and intranasal and transtympanic drug delivery, including therapies that are currently undergoing clinical trials. We provide an overview of how these recent advancements in therapeutic strategies can facilitate the circumvention of current treatment challenges involving preadolescence soft palate dysfunction, biofilm formation, tympanic membrane (ear drum) barrier and the attainment of efficacious drug concentrations in the middle ear. While traditional first-line immunization strategies are generally not very efficacious against biofilms, new technologies that use transdermal or intranasal drug delivery via chitosan-PsaA nanoparticles have shown promising results in experimental animal models of OM. Sustained drug delivery systems such as penta-block copolymer poloxamer 407-polybutylphosphoester (P407-PBP) or poloxamer 407 (e.g. OTO-201, with the brand name 'OTIPRIO') have demonstrated that treatments can be reduced to a single topical application. The emergence of effective new treatment modalities opens up promising new avenues for the treatment of OM that could lead to improved quality of life for many children and their families.

Aims: The aim of this study was to screen vaccine candidates from virulence factors of Streptococcus iniae in flounder model., Methods and Results: The immunogenicity of recombinant phosphoglucomutase (rPGM) and rCAMP factor was confirmed by Western blot. The percentage of surface membrane immunoglobulin-positive (sIg + ) lymphocytes in peripheral blood leucocytes, the specific and total serum IgM and the activity of acid phosphatase (ACP) and peroxidase (POD) in flounder were determined with flow cytometry, ELISA and commercial enzyme activity kits, respectively, after intraperitoneal immunization with rPGM and rCAMP factor. The results showed that rPGM and rCAMP factor could induce significant rise in sIg + lymphocytes, specific serum IgM and activities of ACP and POD. Additionally, the relative percent survival rate of the vaccinated flounder was 64 and 54% in challenge experiment using S. iniae, respectively. These results indicated that rPGM and rCAMP factor could evoke humoural and innate immune response in flounder and provide high-efficiency immunoprotection against S. iniae infection., Conclusions: Phosphoglucomutase (PGM) and CAMP factor were promising vaccine candidates against S. iniae in flounder., Significance and Impact of the Study: Phosphoglucomutase and CAMP factor have the potential to be vaccine candidates, which provide important information for us to develop the effective subunit vaccines, especially the multivaccine, against S .iniae in aquaculture., (© 2018 The Society for Applied Microbiology.)

The epsilon toxin (Etx) produced by Clostridium perfringens type B and D causes severe enterotoxaemia associated with a general edema and neurological alterations, leading to subsequent death and is listed as one of the most lethal toxins. Currently employed vaccines against C. perfringens epsilon toxin include toxoid based vaccines. Use of peptide vaccines has become an interesting approach for vaccination after the successful licensing of peptide vaccines against Haemophilus influenza, Neisseria meningitides and Streptococcus pneumonia that have demonstrated the potential and effectiveness of these vaccines. Therefore, the present study was undertaken to develop a peptide based vaccine against epsilon toxin. Peptides were selected on the basis of epitope mapping by making 35 overlapping peptides of 15 amino acid residues in length specific to the primary amino acid sequence of the toxin, with a 7 amino acid residues overlaps between sequential peptides. Chemically synthesized peptides that were recognised by the antibody against the full length epsilon toxin were further assessed for vaccine potential. The selected peptides were chemically conjugated to partially reduced tetanus toxoid (TT) using of N-succinimidyl-3(2-pyridyldithio) propionate. Immunization of BALB/c mice with TT-peptide conjugates by sub-cutaneous route induced sustained high level mixed immune response as analyzed by antibody isotyping. Immunoblot analysis and ELISA clearly indicated generation of Etx-specific antibodies. Further, neutralization studies with the antisera generated against the TT-conjugated peptide(s) demonstrated that the antisera were able to neutralize the lethal dose of epsilon toxin in vitro demonstrating its potential as a promising vaccine candidate against enterotoxaemia., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Here, we used multilocus sequence typing (MLST) to evaluate 3 intergenic genes (16S rRNA, ospA, and 5S-23S IGS) in Borrelia isolated from South Korea to analyze the relationships between host, vector, and molecular background. We identified B. afzelii, B. yangtzensis, B. garinii, and B. bavariensis. This study is the first report for the identification of B. yangtzensis using MLST in South Korea.

It is widely accepted that live vaccines elicit higher immune protection than inactivated vaccines. However, the mechanisms are largely unknown. Here, an array with 64 recombinant outer membrane proteins of Vibrio parahemolyticus was developed to explore antibody responses of live and inactivated V. parahemolyticus post immunization of the 8th, 12th, 16th and 20th day. Among the 64 outer membrane proteins, 28 elicited antibody generation. They were all detected in live vaccine-induced immunity but only 15 antibodies were found in inactivated vaccine-induced immunity. Passive immunization showed that higher percent survival was detected in live than inactivated vaccine-induced immunities. Active immunization indicated that out of 19 randomly selected outer membrane proteins, 5 stimulated immune protection against V. parahemolyticus infection. Among them, antibodies to VP2309 and VPA0526 were shared in mice immunized by live or inactivated vaccines, whereas antibodies to VPA0548, VPA1745, and VP1667 were only found in mice immunized by live vaccine. In addition, live V. parahemolyticus stimulated earlier antibody response than inactivated bacteria. These results indicate that not all of the outer membrane proteins elicited antibody responses when they work together in the form of live or inactivated bacteria; live vaccine elicits more protective antibodies, which contribute to higher immune protection in live vaccine than inactivated vaccine. Notably, the recombinant proteins might be different from those separated from live bacteria, and they might be different in their immunogenic potencies.

Objectives: The rise in multidrug resistant Neisseria gonorrhoeae poses a threat to healthcare, while the development of an effective vaccine has remained elusive due to antigenic and phase variability of surface-expressed proteins. In the current study, we identified a fully conserved surface expressed protein and characterized its suitability as a vaccine antigen., Methods: An in silico approach was used to predict surface-expressed proteins and analyze sequence conservation and phase variability. The most conserved protein and its surface-exposed Loop 2, which was displayed as both a structural and linear epitope on the oligomerization domain of C4b binding protein, were used to immunize mice. Immunogenicity was subsequently analyzed by determination of antibody titers and serum bactericidal activity., Results: MtrE was identified as one of the most conserved surface-expressed proteins. Furthermore, MtrE and both Loop 2-containing fusion proteins elicited high protein-specific antibody titers and particularly the two Loop 2 fusion proteins showed high anti-Loop 2 titers. In addition, antibodies raised against all three proteins were able to recognize MtrE expressed on the surface of N. gonorrhoeae and showed high MtrE-dependent bactericidal activity., Conclusions: Our results show that MtrE and Loop 2 are promising novel conserved surface-expressed antigens for vaccine development against N. gonorrhoeae., (Copyright © 2018. Published by Elsevier Ltd.)

Vibriosis is a severe infection occurring in many commercially important marine fish species. In this study, vaccines containing Vibrio harveyi recombinant outer membrane protein K (rOmpK), outer membrane protein U (rOmpU) and rOmpK-OmpU fusion protein in addition to the metabolizable Montanide TM ISA 763 A VG adjuvant were developed and evaluated in the orange-spotted grouper. The results indicate that recombinant V. harveyi protein-based vaccines resulted in a remarkably higher expression of IL-1β and IL-8 at 24 hr, and greater antibody production, as early as 2 weeks postimmunization. Notably, enhanced immune responses and significant protective efficacy against V. harveyi infections were observed in the fusion protein vaccine-injected fishes with relative per cent survival value of 81.8%. Additionally, the rOmpK-OmpU antisera presented a high bactericidal effect on not only V. harveyi, but also Vibrio parahaermolyticus and Vibrio alginolyticus. Our results demonstrated that the fusion protein rOmpK-OmpU was an effective vaccine candidate that exhibited potentially great versatility for controlling vibrio infections., (© 2018 John Wiley & Sons Ltd.)

All Neisseria gonorrhoeae strains whose DNA sequences have been determined possess filamentous phage sequences representing their full genomes. The presence of filamentous phage DNA sequences in all sequenced N. gonorrhoeae strains suggest that purified phage particles might be used as a gonococcal vaccine. To test this hypothesis, we purified filamentous NgoΦfil phages and immunized rabbits subcutaneously. The elicited sera contained large quantities of anti-phage IgG and IgA antibodies that bound to the surface of N. gonorrhoeae cells, as shown by ELISA and flow cytometry. The elicited sera bound to the structural NgoΦ6fil proteins present in phage particles and to N. gonorrhoeae cells. The sera did not react with gonococcal outer membrane proteins. The sera also had bactericidal activity and blocked adhesion of gonococci to tissue culture cells. These data demonstrate that NgoΦfil phage particles can induce antibodies with anti-gonococcal activity and may be a candidate for vaccine development., Competing Interests: The authors have declared that no competing interests exist.

Borrelia burgdorferi responds to a variety of host-derived factors and appropriately alters its gene expression for adaptation under different host-specific conditions. We previously showed that various levels of acetate, a short-chain fatty acid (SCFA), altered the protein profile of B. burgdorferi In this study, we determined the effects of other physiologically relevant SCFAs in the regulation of metabolic/virulence-associated proteins using mutant borrelial strains. No apparent increase in the synthesis of outer surface protein C (OspC) was noted when a carbon storage regulator A ( csrA of B. burgdorferi , or csrA Bb ) mutant (mt) was propagated within dialysis membrane chambers implanted within rat peritoneal cavity, while the parental wild type (wt; B31-A3 strain) and csrA Bb cis -complemented strain (ct) had increased OspC with a reciprocal reduction in OspA levels. Growth rates of wt, mt, ct, 7D ( csrA Bb mutant lacking 7 amino acids at the C terminus), and 8S ( csrA Bb with site-specific changes altering its RNA-binding properties) borrelial strains were similar in the presence of acetate. Increased levels of propionate and butyrate reduced the growth rates of all strains tested, with mt and 8S exhibiting profound growth deficits at higher concentrations of propionate. Transcriptional levels of rpoS and ospC were elevated on supplementation of SCFAs compared to those of untreated spirochetes. Immunoblot analysis revealed elevated levels of RpoS, OspC, and DbpA with increased levels of SCFAs. Physiological levels of SCFAs prevalent in select human and rodent fluids were synergistic with mammalian host temperature and pH to increase the levels of aforementioned proteins, which could impact the colonization of B. burgdorferi during the mammalian phase of infection., (Copyright © 2018 American Society for Microbiology.)

Francisella tularensis, a highly infectious, intracellular bacterium possesses an atypical type VI secretion system (T6SS), which is essential for the virulence of the bacterium. Recent data suggest that the HSP100 family member, ClpB, is involved in T6SS disassembly in the subspecies Francisella novicida. Here, we investigated the role of ClpB for the function of the T6SS and for phenotypic characteristics of the human pathogenic subspecies holarctica and tularensis. The ∆clpB mutants of the human live vaccine strain, LVS, belonging to subspecies holarctica, and the highly virulent SCHU S4 strain, belonging to subspecies tularensis, both showed extreme susceptibility to heat shock and low pH, severely impaired type VI secretion (T6S), and significant, but impaired intracellular replication compared to the wild-type strains. Moreover, they showed essentially intact phagosomal escape. Infection of mice demonstrated that both ΔclpB mutants were highly attenuated, but the SCHU S4 mutant showed more effective replication than the LVS strain. Collectively, our data demonstrate that ClpB performs multiple functions in the F. tularensis subspecies holarctica and tularensis and its function is important for T6S, intracellular replication, and virulence.

Mycoplasmas are potential contaminants that introduce undesirable changes in mammalian cell cultures. They frequently contaminate cell substrates and other starting materials used for manufacturing cell-derived biologics, such as vaccines and pharmaceutical products. Mycoplasma purity testing of live vaccines, active ingredients, raw material, and seed lots is required during vaccine production. Previously, testing using a time-consuming, costly 28-day culture assay, which lacks sensitivity for species that do not grow in culture, was required in the European Pharmacopoeia (Ph. Eur). But now nucleic acid amplification techniques (NATs) can be used. NATs provide rapid results and are sensitive. We evaluated the sensitivity and specificity of a commercially-available NAT to detect individual mycoplasma DNA in a veterinary modified live vaccine using five reference strains recommended by the Ph. Eur. Our results showed that this NAT-based method can be used to detect mycoplasma in spiked live vaccine, without interference from the vaccine components, with a limit of detection of 10 CFU/mL, as required by the Ph. Eur. Its specificity was demonstrated since no mycoplasmas were detected in non-spiked vaccine. This method is undergoing validation as a replacement for the conventional culture method in the production of veterinary live vaccines., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Francisella tularensis is the etiologic agent of tularemia, and subspecies tularensis (type A) is the most virulent subspecies. The live vaccine strain (LVS) of subspecies holarctica produces a capsule-like complex (CLC) that consists of a large variety of glycoproteins. Expression of the CLC is greatly enhanced when the bacteria are subcultured in and grown on chemically defined medium. Deletion of two genes responsible for CLC glycosylation in LVS results in an attenuated mutant that is protective against respiratory tularemia in a mouse model. We sought to further characterize the CLC composition and to determine if a type A CLC glycosylation mutant would be attenuated in mice. The CLCs isolated from LVS extracted with 0.5% phenol or 1 M urea were similar, as determined by gel electrophoresis and Western blotting, but the CLC extracted with urea was more water-soluble. The CLC extracted with either 0.5% phenol or 1 M urea from type A strains was also similar to the CLC of LVS in antigenic properties, electrophoretic profile, and by transmission electron microscopy (TEM). The solubility of the CLC could be further enhanced by fractionation with Triton X-114 followed by N-Lauroylsarcosine detergents; the largest (>250 kDa) molecular size component appeared to be an aggregate of smaller components. Outer membrane vesicles/tubules (OMV/T) isolated by differential centrifugation and micro-filtration appeared similar to the CLC by TEM, and many of the proteins present in the OMV/T were also identified in soluble and insoluble fractions of the CLC. Further investigation is warranted to assess the relationship between OMV/T and the CLC. The CLC conjugated to keyhole limpet hemocyanin or flagellin was highly protective against high-dose LVS intradermal challenge and partially protective against intranasal challenge. A protective response was associated with a significant rise in cytokines IL-12, IL-10, and IFN-γ. However, a type A CLC glycosylation mutant remained virulent in BALB/c mice, and immunization with the CLC did not protect mice against high dose respiratory challenge with type A strain SCHU S4.

Background: The live, temperature-attenuated vaccine strain 1B of Chlamydia abortus, the aetiological agent of ovine enzootic abortion (OEA), has been implicated in cases of vaccine breakdown. The aim of this study was to understand the nature of this attenuation through sequencing of the vaccine parent strain (AB7) and the derived mutant strains 1B and 1H, as well as to clarify the role of the vaccine strain in causing disease through comparative whole genome analysis., Methods: Whole genome sequencing was performed on: vaccine parent strain AB7; N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-induced temperature attenuated mutant strain 1B grown from the commercial live vaccines Cevac Chlamydia and Enzovax; strain 1H a reverted NTG mutant; and 5 strains isolated from cases of OEA originating from animals from the original vaccine safety trial (2 strains) or from vaccinated ewes or ewes exposed to vaccinated animals (3 strains)., Results: We confirmed that AB7 is in a different lineage from the reference strain S26/3. The genome of vaccine strain 1B contains ten single nucleotide polymorphisms (SNPs) created by the NTG treatment, which are identical to those found in strain 1H. The strains from OEA cases also cluster phylogenetically very tightly with these vaccine strains., Conclusions: The results show that C. abortus vaccine strain 1B has an identical genome sequence to the non-attenuated "reverted mutant" strain 1H. Thus, the protection of the 1B vaccine is unlikely to be due to the NTG induced SNPs and is more likely caused by the administration of high doses of C. abortus elementary bodies that stimulate protective immunity. Vaccine-identical strains were also isolated from cases of disease, as well as strains which had acquired 1-3 SNPs, including an animal that had not been vaccinated with either of the commercial live OEA vaccines, indicating that the 1B vaccine strain may be circulating and causing disease., (Copyright © 2018. Published by Elsevier Ltd.)

Although many naturally occurring proteins consist of multiple domains, most studies on protein folding to date deal with single-domain proteins or isolated domains of multi-domain proteins. Studies of multi-domain protein folding are required for further advancing our understanding of protein folding mechanisms. Borrelia outer surface protein A (OspA) is a β-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer β-sheet. Thus, OspA is particularly suited as a model system for studying the interplays of domains in protein folding. Here, we studied the equilibria and kinetics of the urea-induced folding-unfolding reactions of OspA probed with tryptophan fluorescence and ultraviolet circular dichroism. Global analysis of the experimental data revealed compelling lines of evidence for accumulation of an on-pathway intermediate during kinetic refolding and for the identity between the kinetic intermediate and a previously described equilibrium unfolding intermediate. The results suggest that the intermediate has the fully native structure in the N-terminal domain and the single layer β-sheet, with the C-terminal domain still unfolded. The observation of the productive on-pathway folding intermediate clearly indicates substantial interactions between the two domains mediated by the single-layer β-sheet. We propose that a rigid and stable intervening region between two domains creates an overlap between two folding units and can energetically couple their folding reactions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Streptococcus mitis (S. mitis) may transform into highly pathogenic bacteria. The aim of the present study was to identify potential antigen targets for designing an effective vaccine against the pathogenic S. mitis321A. The genome of S. mitis321A was sequenced using an Illumina Hiseq2000 instrument. Subsequently, Glimmer 3.02 and Tandem Repeat Finder (TRF) 4.04 were used to predict genes and tandem repeats, respectively, with DNA sequence function analysis using the Basic Local Alignment Search Tool (BLAST) in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Cluster of Orthologous Groups of proteins (COG) databases. Putative gene antigen candidates were screened with BLAST ahead of phylogenetic tree analysis. The DNA sequence assembly size was 2,110,680 bp with 40.12% GC, 6 scaffolds and 9 contig. Consequently, 1,944 genes were predicted, and 119 TRF, 56 microsatellite DNA, 10 minisatellite DNA and 154 transposons were acquired. The predicted genes were associated with various pathways and functions concerning membrane transport and energy metabolism. Multiple putative genes encoding surface proteins, secreted proteins and virulence factors, as well as essential genes were determined. The majority of essential genes belonged to a phylogenetic lineage, while 321AGL000129 and 321AGL000299 were on the same branch. The current study provided useful information regarding the biological function of the S. mitis321A genome and recommends putative antigen candidates for developing a potent vaccine against S. mitis.

The alpha-toxin is one of the virulence factors of Clostridium perfringens for gas gangrene in humans and animals or necrotic enteritis in poultry. The C-terminal domain of this toxin ( cpa 247-370 ) was synthesized and cloned into pT1NX vector to construct the pT1NX-alpha plasmid. This surface-expressing plasmid was electroporated into Lactobacillus casei ATCC 393, generating the recombinant L. casei strain expressing alpha-toxoid (LC-α strain). Expression of this modified alpha-toxoid was confirmed by SDS-PAGE, immunoblotting, and direct immunofluorescence microscopy. BALB/c mice, immunized orally by the recombinant LC-α strain, elicited mucosal and significantly humoral immune responses (p < 0.05) and developed a protection against 900 MLD/mL of the standard alpha-toxin. This study showed that this recombinant LC-α strain could be a promising vaccine candidate against gas gangrene and necrotic enteritis.

Flagellin, a major structural component of bacteria flagella, is responsible for facilitating motility and invading host cells. The binding capacity of flagellin to toll-like receptor 5 (TLR5) and its trigger action for innate immunity make it an attractive vaccine adjuvant. In this work, we explored the efficacy of F18 + STEC flagellin as an adjuvant in a ghost vaccine candidate against porcine edema disease (ED). TLR5-stimulating activity and IL-8 cytokine expression produced via downstream signaling following ligand exposure was detected in HEK-Blue™-hTLR5 cells stimulated with flagellin expressed in the Salmonella Typhimurium (ST) ghost, JOL1485. Mice immunized with JOL1485 mixed with the vaccine candidate showed significantly increased secretory IgA specific to flagellin during the study period. JOL1485 efficiently induced maturation of naïve dendritic cells, resulting in production of immunomodulatory cytokines associated with Th1 and Th2 immunity. Particularly, marked increases in IL-6 inducing B-cell differentiation and IL-12 associated with Th1 immunity were observed in CD4 + T-cells stimulated with JOL1485 primed DCs. Furthermore, all mice immunized with JOL1485 mixed with the vaccine candidate showed higher protection efficacy against challenge and significantly enhanced clearance of the challenge strain, compared to mice immunized with only the vaccine candidate. Collectively, these results indicated that flagellin expression in JOL1485 may have immunostimulatory properties that serve as a potent adjuvant for the vaccines against ED., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Aims: Available bacterins, commercial or autogenous, for Actinobacillus pleuropneumoniae disease control have, thus far, shown debatable protective efficacy and only in homologous challenges. Our study sought to determine whether the addition of reombinant protein ApxIV to the multicomponent vaccine could enhance protection against homologous and heterologous challenge of A. pleuropneumoniae., Methods and Results: The virulence of ApxI, ApxII, ApxIV and OMP were cloned and expressed using a prokaryotic system; these recombinant proteins were combined with inactivated A. pleuropneumoniae serovar 1 to formulate different multicomponent vaccines. Immune response and protective efficacy of the vaccines were evaluated in mice and pigs. A protection rate of 67% was observed against heterologous challenge in mice vaccinated with the rApxIV formulation. Piglets vaccinated with vaccine containing ApxIV produced significantly higher antibody titre and provided complete protection and reduced gross lesions by 67% when compared with the nonimmunized group after homologous challenge. Additionally, flow cytometry analysis showed significant cellular immune response., Conclusions: The results of our vaccination experiments revealed that a combination of inactivated bacteria and the recombinant antigens rApxI, rApxII, rApxIV and rOMP can provide effective protection against heterologous A. pleuropneumoniae challenge., Significance and Impact of the Study: The addition of ApxIV to the multicomponent vaccine could enhance homologous and heterologous protection in mice and pigs, respectively, against challenge by A. pleuropneumoniae., (© 2018 The Society for Applied Microbiology.)

Lactic acid bacteria (LAB) have been utilized since the 1990s for therapeutic heterologous gene expression. The ability of LAB to elicit an immune response against expressed foreign antigens has led to their exploration as potential mucosal vaccine candidates. LAB vaccine vectors offer many attractive advantages: simple, noninvasive administration (usually oral or intranasal), the acceptance and stability of genetic modifications, relatively low cost, and the highest level of safety possible. Experimentation using LAB of the genus Lactobacillus has become popular in recent years due to their ability to elicit strong systemic and mucosal immune responses. This article reviews Lactobacillus vaccine constructs, including Lactobacillus species, antigen expression, model organisms, and in vivo immune responses, with a primary focus on viral and bacterial antigens., (Copyright © 2018 LeCureux and Dean.)

Francisella tularensis is the causative agent of tularemia and a Tier I bioterrorism agent. In the 1900s, several vaccines were developed against tularemia including the killed "Foshay" vaccine, subunit vaccines comprising F. tularensis protein(s) or lipoproteins(s) in an adjuvant formulation, and the F. tularensis Live Vaccine Strain (LVS); none were licensed in the U.S.A. or European Union. The LVS vaccine retains toxicity in humans and animals-especially mice-but has demonstrated efficacy in humans, and thus serves as the current gold standard for vaccine efficacy studies. The U.S.A. 2001 anthrax bioterrorism attack spawned renewed interest in vaccines against potential biowarfare agents including F. tularensis . Since live attenuated-but not killed or subunit-vaccines have shown promising efficacy and since vaccine efficacy against respiratory challenge with less virulent subspecies holarctica or F. novicida , or against non-respiratory challenge with virulent subsp. tularensis (Type A) does not reliably predict vaccine efficacy against respiratory challenge with virulent subsp. tularensis , the route of transmission and species of greatest concern in a bioterrorist attack, in this review, we focus on live attenuated tularemia vaccine candidates tested against respiratory challenge with virulent Type A strains, including homologous vaccines derived from mutants of subsp. holarctica, F. novicida , and subsp. tularensis , and heterologous vaccines developed using viral or bacterial vectors to express F. tularensis immunoprotective antigens. We compare the virulence and efficacy of these vaccine candidates with that of LVS and discuss factors that can significantly impact the development and evaluation of live attenuated tularemia vaccines. Several vaccines meet what we would consider the minimum criteria for vaccines to go forward into clinical development-safety greater than LVS and efficacy at least as great as LVS, and of these, several meet the higher standard of having efficacy ≥LVS in the demanding mouse model of tularemia. These latter include LVS with deletions in purMCD, sodB Ft , capB or wzy ; LVS Δ capB that also overexpresses Type VI Secretion System (T6SS) proteins; FSC200 with a deletion in clpB ; the single deletional purMCD mutant of F. tularensis SCHU S4, and a heterologous prime-boost vaccine comprising LVS Δ capB and Listeria monocytogenes expressing T6SS proteins.

Haemophilus parasuis is the causative agent of the Glässer's disease (GD), one of the most important bacterial diseases that affect young pigs worldwide. GD prevention based on vaccination is a major concern due to the limited cross-protection conferred by the inactivated whole cell vaccines used currently. In this study, vaccines based on two mutant recombinant proteins derived from transferrin binding protein B of H. parasuis (Y167A-TbpB and W176A-TbpB) were formulated and evaluated in terms of protection against lethal challenge using a serovar 7 (SV7) H. parasuis in a high susceptibility pig model. Our results showed that H. parasuis strain 174 (SV7) is highly virulent in conventional and colostrum-deprived pigs. The Y167A-TbpB and W176A-TbpB antigens were immunogenic in pigs, however, differences in terms of antigenicity and functional immune response were observed. In regard to protection, animals immunized with Y167A-TbpB antigen displayed 80% survival whereas the W176A-TbpB protein was not protective. In conjunction with previous studies, our results demonstrate, (a) the importance of testing engineered antigens in an in vivo pig challenge model, and, (b) that the Y167A-TbpB antigen is a promising antigen for developing a broad-spectrum vaccine against H. parasuis infection.

Bacillus anthracis, Yersinia pestis, and Francisella tularensis are the causative agents of Tier 1 Select Agents anthrax, plague, and tularemia, respectively. Currently, there are no licensed vaccines against plague and tularemia and the licensed anthrax vaccine is suboptimal. Here we report F. tularensis LVS ΔcapB (Live Vaccine Strain with a deletion in capB)- and attenuated multi-deletional Listeria monocytogenes (Lm)-vectored vaccines against all three aforementioned pathogens. We show that LVS ΔcapB- and Lm-vectored vaccines express recombinant B. anthracis, Y. pestis, and F. tularensis immunoprotective antigens in broth and in macrophage-like cells and are non-toxic in mice. Homologous priming-boosting with the LVS ΔcapB-vectored vaccines induces potent antigen-specific humoral and T-cell-mediated immune responses and potent protective immunity against lethal respiratory challenge with all three pathogens. Protection against anthrax was far superior to that obtained with the licensed AVA vaccine and protection against tularemia was comparable to or greater than that obtained with the toxic and unlicensed LVS vaccine. Heterologous priming-boosting with LVS ΔcapB- and Lm-vectored B. anthracis and Y. pestis vaccines also induced potent protective immunity against lethal respiratory challenge with B. anthracis and Y. pestis. The single vaccine platform, especially the LVS ΔcapB-vectored vaccine platform, can be extended readily to other pathogens.

Background: Recombinant subunit vaccines have been extensively evaluated as promising alternatives against leptospirosis. Here, we evaluated two proteins in formulations containing the adjuvant AddaVax™ as vaccine candidates for prevention and control of leptospirosis., Methods: Recombinant proteins rErp Y-like and rLemA were characterized by ELISA to assess their ability to bind extracellular matrix (ECM) components and fibrinogen. Groups of eight hamsters were immunized intramuscularly with rErp Y-like or rLemA mixed with a squalene-based adjuvant (AddaVax), and then vaccine efficacy was determined in terms of protection against a lethal challenge. The humoral immune response was determined by ELISA, and the evidence of sub-lethal infection was evaluated by histopathology and kidney culture., Results: rLemA protein binds laminin, fibrinogen, and collagen type IV, while rErp Y-like interacts with fibrinogen. Significant protection was achieved for rLemA and rErp Y-like vaccines, which showed 87.5% and 62.5% survivals, respectively. On day 28, the humoral immune response was significantly greater in the vaccine groups as compared to that in the control group, and the response was predominantly based on IgG2/3. The surviving animals showed negative results in culture isolation but presented with tissue lesions in the lungs and kidneys., Conclusion: Cumulatively, our findings suggest that LemA and Erp Y-like proteins act as adhesins and are able to protect against mortality, but not against tissue lesions. Moreover, AddaVax is a novel adjuvant with potential for improving the immunogenicity of leptospiral vaccines., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Vaccine development efforts against Streptococcus suis serotype 2 (S. suis 2) are often constrained by strain/serotype antigen variability. Bioinformatics analyses revealed two highly conserved S. suis 2 factors, EF-Tu and FtsZ. Murine immunization with recombinant proteins emulsified in white oil adjuvant or eukaryotic DNA vaccine vectors provided significant protection against lethal S. suis 2 challenge. Immune responses elicited by recombinant protein immunization revealed the robust generation of humoral immune responses, with a mixed induction of Th1-type and Th2-type responses. Furthermore, the antiserum from mice immunized with recombinant proteins significantly inhibited the growth of S. suis 2 in healthy pig whole blood, suggesting the triggering of a strong opsonizing response. Histological examination found that immunizing mice with purified recombinant proteins reduced neutrophil and macrophage accumulation in brain and lung tissues after challenge with virulent S. suis. Taken together, these findings reveal that EF-Tu and FtsZ may be promising targets for subunit and DNA vaccine candidates against S. suis 2 infection., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Catfish is the largest aquaculture industry in the United States. Edwardsiellosis is considered one of the most significant problems affecting this industry. Edwardsiella piscicida is a newly described species within the genus Edwardsiella, and it was previously classified as Edwardsiella tarda. It causes gastrointestinal septicaemia, primarily in summer months, in farmed channel catfish in the south-eastern United States. In the current study, we adapted gene deletion methods used for Edwardsiella to E. piscicida strain C07-087, which was isolated from a disease outbreak in a catfish production pond. Four genes encoding structural proteins in the type III secretion system (T3SS) apparatus of E. piscicida were deleted by homologous recombination and allelic exchange to produce in-frame deletion mutants (EpΔssaV, EpΔesaM, EpΔyscR and EpΔescT). The mutants were phenotypically characterized, and virulence and vaccine efficacy were evaluated. Three of the mutants, EpΔssaV, EpΔyscR and EpΔesaM, were significantly attenuated compared to the parent strain (p < .05), but EpΔescT strain was not. Vaccination of catfish with the four mutant strains (EpΔssaV, EpΔesaM, EpΔyscR and EpΔescT) provided significant protection when subsequently challenged with wild-type strain. In conclusion, we report methods for gene deletion in E. piscicida and development of vaccine candidates derived from a virulent catfish isolate., (© 2018 John Wiley & Sons Ltd.)

We previously developed a stable and marker-free Lactobacillus casei strain (PPαT Δupp) that contained a chromosomally integrated expression cassette (PPαT) that enabled the surface expression of the Clostridium perfringens alpha toxin. To measure immune responses against the alpha toxin, specific-pathogen-free BALB/c mice were inoculated with L. casei PPαT Δupp by oral gavage. Then, specific immunoglobulin A (IgA) and immunoglobulin G (IgG) antibodies and cytokines were measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FCM). The results showed that alpha toxin-specific IgA and IgG antibodies and cytokines were markedly increased following immunization. Natural alpha toxin challenge and neutralization tests were performed. The results showed that immunized mice can fully resist 1.5 minimum lethal doses of toxin. These results indicated that the immunized mice can produce not only humoral immunity, but also cellular immunity. These results provide a new pathway for the development of a safe, effective, and food-grade vaccine., (Copyright © 2018. Published by Elsevier Ltd.)

Urinary tract infections (UTI) caused by Proteus mirabilis are prevalent among the catheterized patients. There is no effective vaccine to reduce the frequency of UTIs caused by P. mirabilis. In the present study, the immune responses and effectiveness of different combinations of MrpA and flagellin (FliC) of P. mirabilis were assessed intranasally in the mice model. The addition of FliC as adjuvant to MrpA in fusion form significantly raised the mucosal IgA and cellular (IFN-γ and IL-17) responses and maintained the serum IgG responses for 180 days after the first vaccination. Furthermore, MrpA in fusion form with FliC significantly increased the systemic, mucosal and IFN-γ responses of the FliC alone. In a bladder challenge assay with P. mirabilis, the fusion MrpA.FliC and the mixture of MrpA and FliC significantly decreased the colony count of the bacteria in the bladder and kidneys of mice in comparison to the control mice. It suggests a complex of the systemic, mucosal and cellular responses are needed for protection of the bladder and kidneys against P. mirabilis UTI. In our knowledge, the adjuvant property of the recombinant P. mirabilis flagellin was evaluated for the first time in a vaccine combination administered by an intranasal route. Our results suggest the recombinant flagellin of P. mirabilis could be used as an intranasal adjuvant in combination with other potential antigens against UTIs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The incidence of tick-borne zoonoses such as Lyme disease has steadily increased in the southeastern United States. Southeastern states accounted for 1500 of over 28,000 confirmed cases of Lyme disease reported in the United States during 2015. Borrelia burgdorferi, the etiologic agent of Lyme disease, is maintained in small mammal reservoirs and vectored to new hosts by ixodid ticks. This study examined ecological relationships of the B. burgdorferi/vector/reservoir system in order to understand the dynamics of Lyme disease risk in Kentucky. Small mammals were captured using live traps from November 2014 to October 2015. Ticks were removed and blood and tissue collected from small mammals were screened for B. burgdorferi DNA by PCR with primers specific to the OspA gene. Prevalence of B. burgdorferi (21.8%) in Kentucky small mammals was comparable to the lowest recorded prevalence in regions where Lyme disease is endemic. Moreover, infestation of small mammals by Ixodes scapularis, the primary vector of B. burgdorferi, was rare, while Dermacentor variabilis comprised the majority of ticks collected. These findings provide ecological insight into the relative paucity of Lyme disease in Kentucky.

The temperature-sensitive (ts+) Mycoplasma synoviae vaccine strain MS-H harbors a non-synonymous mutation which results in Glycine to Arginine substitution at position 123 in the highly conserved glycine-rich motif of Obg-fold in the GTP-binding protein Obg. In-silico analysis of the wild-type and mutant Obgs of M. synoviae has indicated that this amino acid substitution affects structure of the protein, potentially leading to abrogation of Obg function in vivo. Present study was conducted to develop the first expression vector for M. synoviae and to investigate the potential effect(s) of complementation of MS-H vaccine with the wild-type obg from 86079/7NS, the parent strain of MS-H. An oriC vector, pKS-VOTL, harboring the 86079/7NS obg gene, downstream of the variable lipoprotein haemagglutinin (vlhA) gene promoter, also cloned from 86079/7NS, was used to transform MS-H. The plasmid was localised at the chromosomal oriC locus of MS-H without any detectable integration at the chromosomal obg locus. Analysis of the MS-H transformants revealed abundant obg transcripts as well as Obg protein, when compared to the MS-H transformed with a similar vector, pMAS-LoriC, lacking obg coding sequence. The MS-H transformants complemented with wild-type Obg maintained their original temperature-sensitivity phenotype (consistent with MS-H vaccine) but, when compared to the MS-H transformed with pMAS-LoriC, had significantly higher (p < 0.05) growth rate and viability at the permissive (33°C) and non-permissive temperature (39.5°C), respectively. Analysis of Obg expression in MS-H and its wild-type parent strain revealed comparatively lower levels of Obg in MS-H. These results indicate that not only the mutation in Obg, but also the level of Obg expression, can confer functional abnormalities in the bacterial host. Furthermore, with the construction of first expression vector for M. synoviae, this study has set foundation for the development of recombinant vaccine(s) based on MS-H.

We inactivated a virulence determinant, ArcA, in an Actinobacillus pleuropneumoniae quadruple deletion mutant SLW06 (ΔapxICΔapxIICΔorf1ΔcpxAR, serovar 1), and a quintuple deletion mutant SLW07 was generated. SLW07 showed decreased adherence to and invasion of host cells, compared to its parent strain SLW06. SLW07 was more sensitive in RAW264.7 macrophage-mediated phagocytosis and clearance. SLW07 was less virulent in mice. An immunization assay indicated that both SLW07 and SLW06 preferentially stimulated T helper cell type 2 response in mice. Live vaccines induced the production of interleukin-6 and tumor necrosis factor-α by splenic lymphocytes. Furthermore, the protective immunity of SLW07 was not affected after ArcA mutation. Immunization with SLW07 could provide a complete protection following virulent A. pleuropneumoniae challenge in mice. Our results suggest that SLW07 is a promising live vaccine candidate, which is further attenuated from and shares similar protective efficacy with its quadruple deletion parent SLW06., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Reverse vaccinology is a widely used approach that has facilitated the rapid identification of vaccine candidates suitable in vaccine development for pathogens. Vibrio anguillarum is a major pathogen responsible for vibriosis in fish and shellfish leading to huge economic losses to the aquaculture industry. Although commercial vaccines are available for fish against this bacterium they have their own limitations. In this study, we used the reverse vaccinology strategy to screen and identify V. anguillarum outer membrane proteins (OMPs) that could serve as vaccine candidates. Our analysis identified 23 antigenic outer membrane proteins which were highly conserved (>98% identity) across serovars of this bacterium. Of the 23, two were identified as outer membrane lipoproteins. Among the OMPs identified 18 were novel to this study and conserved across several Vibrio spp. with an identity of 21-93%. While the least (>48%) identity was observed for V. anguillarum ferrichrome-iron transporter protein, the highest identity (>80%) was seen for outer membrane proteins OmpK, BamA, OmpU, Fatty acid transporter, and two hypothetical proteins. These potential vaccine targets identified could contribute to the development of effective vaccine not only against V. anguillarum but also across other Vibrio spp. In addition, several B-cell and T-cell epitopes were predicted for the novel OMPs in this study which could aid in narrowing down peptide selection in designing a suitable epitope-based vaccine.

Objective: To develop a safe and effective oral vaccine against Helicobacter pylori using its HpaA protein expressed in Lactococcus lactis., Results: The gene encoding HpaA was obtained by PCR and ligated to pNZ8110-lysM following digestion with NaeI + SphI. The recombinant plasmid was transferred into E. coli for multiplication, and then into L. lactis. The recombinant L. lactis was induced to express HpaA, resulting in two products of 29 and 25 kDa, both of which yielded positive immunoreaction with mouse antisera against H. pylori, as confirmed by immunoblot assays. The 29 kDa product constituted 12% of the cell lysates. Oral inoculation with the engineered L. lactis evoked significantly elevated serum IgG level in mice (P < 0.05)., Conclusions: A novel engineered L. lactis strain was developed that efficiently produces whole HpaA protein with desired antigenicity and potent immunogenicity. It provides a basis for approaches to L. lactis-delivered anti-H. pylori vaccination.

A vaccine against Moraxella catarrhalis would reduce tremendous morbidity, mortality, and financial burden by preventing otitis media in children and exacerbations of chronic obstructive pulmonary disease (COPD) in adults. Oligopeptide permease A (OppA) is a candidate vaccine antigen that is (i) a nutritional virulence factor expressed on the bacterial cell surface during infection, (ii) widely conserved among strains, (iii) highly immunogenic, and (iv) a protective antigen based on its capacity to induce protective responses in immunized animals. In the present study, we show that the antibodies to OppA following vaccination mediate accelerated clearance in animals after pulmonary challenge. To identify regions of OppA that bind protective antibodies, truncated constructs of OppA were engineered and studied to map regions of OppA with surface-accessible epitopes that bind high-avidity antibodies following vaccination. Protective epitopes were located in the N and C termini of the protein. Immunization of mice with constructs corresponding to these regions (T5 and T8) induced protective responses. Studies of overlapping peptide libraries of constructs T5 and T8 with OppA immune serum identified two discrete regions on each construct. These potentially protective regions were mapped on a three-dimensional computational model of OppA, where regions with solvent-accessible amino acids were identified as three potentially protective epitopes. In all, these studies revealed two regions with three specific epitopes in OppA that induce potentially protective antibody responses following vaccination. Detection of antibodies to these regions could serve to guide vaccine formulation and as a diagnostic tool for monitoring development of protective responses during clinical trials., (Copyright © 2018 American Society for Microbiology.)

Background: The bacterial pathogen Mycoplasma synoviae can cause subclinical respiratory disease, synovitis, airsacculitis and reproductive tract disease in poultry and is a major cause of economic loss worldwide. The M. synoviae strain MS-H was developed by chemical mutagenesis of an Australian isolate and has been used as a live attenuated vaccine in many countries over the past two decades. As a result it may now be the most prevalent strain of M. synoviae globally. Differentiation of the MS-H vaccine from local field strains is important for epidemiological investigations and is often required for registration of the vaccine., Results: The complete genomic sequence of the MS-H strain was determined using a combination of Illumina and Nanopore methods and compared to WVU-1853, the M. synoviae type strain isolated in the USA 30 years before the parent strain of MS-H, and MS53, a more recent isolate from Brazil. The vaccine strain genome had a slightly larger number of pseudogenes than the two other strains and contained a unique 55 kb chromosomal inversion partially affecting a putative genomic island. Variations in gene content were also noted, including a deoxyribose-phosphate aldolase (deoC) fragment and an ATP-dependent DNA helicase gene found only in MS-H. Some of these sequences may have been acquired horizontally from other avian mycoplasma species., Conclusions: MS-H was somewhat more similar to WVU-1853 than to MS53. The genome sequence of MS-H will enable identification of vaccine-specific genetic markers for use as diagnostic and epidemiological tools to better control M. synoviae.

Objective To investigate the effect of aluminum phosphate (AP) and aluminum hydroxide (AH) as adjuvants on Brucella outer membrane protein 31 (Omp31) in inducing humoral and cellular immune responses and immune protection. Methods AP and AH adjuvants were prepared and separately mixed with Brucella Omp31 protein to measure the adsorption rates. The AP- and AH-absorbed Omp31 protein were intraperitoneally injected into BLAB/c mice at 0, 2, and 4 weeks, and meanwhile, unabsorbed Omp31 protein and PBS were used as controls. The levels of serum IgG, IgG1, IgG2a and genital tract secretion sIgA were determined by ELISA at 0, 2, 4 and 6 weeks. Spleen cells were collected for culture at 6 weeks, and the cells were stimulated by Omp31 for 48 hours followed by the analysis of IFN-γ and IL-10 levels in the supernatants by ELISA, and the determination of lymphocyte proliferation by CCK-8 assay. The mice were challenged with Brucella at 6 weeks, and bacterial content in spleen tissue was determined 1 and 2 weeks later. Results AP and AH could absorb over 70% and 85% of the Omp31 protein, respectively, for solutions at all the tested concentrations. ELISA suggested that serum IgG, IgG1, IgG2a and genital tract sIgA levels peaked 2 weeks after the last immunization for both AP and AH groups, and antibody level was higher in the AP and AH groups than the control groups, and higher in the AH group than in the AP group. CCK-8 assay showed that the proliferating rate of lymphocytes induced by the AH group was significantly higher than that by the AP group, and the AH group also showed significantly higher IFN-γ level in the supernatant than the AP group, but no significant difference in IL-10 level. The AH group had remarkably lower bacterial load in the spleen than the AP group 2 weeks after challenged by Brucella 16M strain. Conclusion Both AP and AH adjuvants effectively enhanced immunogenicity and immune protection of the Brucella Omp31 protein, and AH was superior to AP in this respect.

Background & Aims: Helicobacter pylori is remarkable for its genetic variation; yet, little is known about its genetic changes during early stages of human infection, as the bacteria adapt to their new environment. We analyzed genome and methylome variations in a fully virulent strain of H pylori during experimental infection., Methods: We performed a randomized Phase I/II, observer-blind, placebo-controlled study of 12 healthy, H pylori-negative adults in Germany from October 2008 through March 2010. The volunteers were given a prophylactic vaccine candidate (n = 7) or placebo (n = 5) and then challenged with H pylori strain BCM-300. Biopsy samples were collected and H pylori were isolated. Genomes of the challenge strain and 12 reisolates, obtained 12 weeks after (or in 1 case, 62 weeks after) infection were sequenced by single-molecule, real-time technology, which, in parallel, permitted determination of genome-wide methylation patterns for all strains. Functional effects of genetic changes observed in H pylori strains during human infection were assessed by measuring release of interleukin 8 from AGS cells (to detect cag pathogenicity island function), neutral red uptake (to detect vacuolating cytotoxin activity), and adhesion assays., Results: The observed mutation rate was in agreement with rates previously determined from patients with chronic H pylori infections, without evidence of a mutation burst. A loss of cag pathogenicity island function was observed in 3 reisolates. In addition, 3 reisolates from the vaccine group acquired mutations in the vacuolating cytotoxin gene vacA, resulting in loss of vacuolization activity. We observed interstrain variation in methylomes due to phase variation in genes encoding methyltransferases., Conclusions: We analyzed adaptation of a fully virulent strain of H pylori to 12 different volunteers to obtain a robust estimate of the frequency of genetic and epigenetic changes in the absence of interstrain recombination. Our findings indicate that the large amount of genetic variation in H pylori poses a challenge to vaccine development. ClinicalTrials.gov no: NCT00736476., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Clostridium perfringens alpha toxin, encoded by plc gene, has been implicated in gas gangrene, a life threatening infection. Vaccination is considered one of the best solutions against Clostridium infections. Although studies have identified many low quality clostridial vaccines, the use of recombinant proteins has been considered a promising alternative. Previously, a naturally occurring alpha toxin isoform (αAV1b) was identified with a mutation at residue 11 (His/Tyr), which can affect its enzymatic activity. The aim of the present study was to evaluate whether the mutation in the αAV1b isoform could result in an inactive toxin and was able to induce protection against the native alpha toxin. We used recombinant protein techniques to determine whether this mutation in αAV1b could result in an inactive toxin compared to the active isoform, αZ23. Rabbits were immunized with the recombinant toxins (αAV1b and αZ23) and with native alpha toxin. αAV1b showed no enzymatic and hemolytic activities. ELISA titration assays showed a high titer of both anti-recombinant toxin (anti-rec-αAV1b and anti-rec-αZ23) antibodies against the native alpha toxin. The alpha antitoxin titer detected in the rabbits' serum pool was 24.0 IU/mL for both recombinant toxins. These results demonstrate that the inactive naturally mutated αAV1b is able to induce an immune response, and suggest it can be considered as a target for the development of a commercial vaccine against C. perfringens alpha toxin., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Human respiratory syncytial virus (RSV) can cause severe acute lower respiratory tract disease leading to numerous hospitalizations and deaths in the infant and elderly populations worldwide, while no vaccine or effective drug is available for RSV infections. In the present study, truncated G protein was successfully expressed both in prokaryotic and eukaryotic system, and high levels of serum IgG in response to truncated G protein were observed both in GD-protein group (intramuscularly with purified GD protein) and GD-VNP20009 group (challenged via the oral route with 1 × 10 9  CFU of pLIVE-RSV-GD-VNP20009 strains) since 21th day, and GD-VNP20009 significantly reduced the productions of IL-1β, IL-6, and TNF-α, histamine and pathological features caused by the RSV Long strain (P < .01). Our data indicated that Salmonella typhimurium can be used to deliver truncated G DNA vaccine and represents a promising effect to protect host against RSV., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Actinobacillus pleuropneumoniae (APP) causes serious economic losses in the swine industry, and is the etiologic agent of porcine pleuropneumonia. In this study we have engineered a trivalent Apx fusion protein enclosed in outer membrane vesicles (Apxr-OMV) and studied its immunoprotective efficacy against APP serotypes 1 and 7 challenge in mice. The results showed that the IgG levels in the Apxr-OMVs immune group were significantly higher than those of the negative control (P < 0.05). Up-regulation of both Th1 (IFN-γ, IL-2) and Th2 (IL-4) cytokines were detected in splenocytes of Apxr-OMVs immune group. The survival rates 87.5% and 62.5% were observed against APP strain 1516 of serotype 7 and APP strain 2701 of serotype 1 in the groups of Apxr-OMVs immune group, respectively. Histopathological lesions of the pulmonary structure alveoli were found to be minimal in APX-OMV group challenged with APP serotypes 1 and 7. These results strongly indicated that engineered OMVs could effectively induce specific humoral or cellular immune responses. Moreover, Apxr-OMVs used as novel vaccine provides cross-protective immunity against different serotype 1 and 7 of APP infection in a mouse model. In contrast, the OMV-empty and PBS as negative controls or inactivated strain of APP-2701 and APP-1516 as positive controls for the animal study cannot provide protection or cross-protection.