Enhancement of immune responses by co-administration of bacterial ghosts-mediated Neisseria gonorrhoeae DNA vaccines.

AIM: Gonorrhoea remains a leading public health burden and the development of vaccine against gonorrhoea becomes more urgent. Here, a novel Neisseria gonorrhoeae DNA vaccine delivered by Salmonella enteritidis ghosts was developed and the immune responses of the vaccine candidate were evaluated. METHODS AND RESULTS: Neisseria gonorrhoeae nspA gene was cloned into the pVAX1 vector. The constructed recombinant plasmid pVAX1-nspA was loaded into the lyophilized SE ghosts to produce SE ghosts (pVAX1-nspA). Then, the immune responses induced by SE ghosts (pVAX1-nspA) alone and co-administrated with SE ghosts (pVAX1-porB) were evaluated in mouse model. Co-administered SE ghosts (pVAX1-nspA) and SE ghosts (pVAX1-porB) could elicited significantly higher levels of specific IgG antibody responses and lymphocyte proliferative responses than the control groups. Furthermore, the group co-administered SE ghosts (pVAX1-nspA) and SE ghosts (pVAX1-porB) had the highest bactericidal antibody titres. CONCLUSIONS: Co-administration of SE ghosts (pVAX1-nspA) and SE ghosts (pVAX1-porB) elicited significant specific humoral and cellular immune responses. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the potential of co-administration of SE ghosts (pVAX1-nspA) and SE ghosts (pVAX1-porB) as an attractive vaccination regimen for gonorrhoea.

Genetic diversity of Leptospira interrogans circulating isolates and vaccine strains in China from 1954-2014.

Leptospirosis is one of the most important but neglected, infectious tropical diseases worldwide. Leptospira interrogans is now recognized as a leading cause of the disease. Little is known of the genetic diversity and phylogenetic characteristics of L. interrogans within China. To better understand the transmission and genetic diversity of L. interrogans populations, we characterized 271 isolates and seven vaccine strains from China during 1954-2014 using multilocus variable-number tandem repeat analysis (MLVA). 110 different L. interrogans MLVA profiles (MTs) were identified, of which five were predominant, reflecting a high level of genetic diversity in L. interrogans population in China. Different from that of circulating isolates, seven vaccine strains have different MT, of which some are phylogenetically away from the circulating isolates. The results showed that Icterohaemorrhagiae, Hebdomadis, and Canicola ranked as the top three serogroups among L. interrogans strains tested. The cluster analysis demonstrate the clonal links between rodent and human isolates, suggesting the rodent species played a key role in the transmission of leptospirosis to humans, and contributed to the circulation of the pathogen in humans. Taken together, these findings should provide insight into a better knowledge of the epidemiology and molecular evolution of L. interrogans in China. Furthermore, the results should facilitate the selection of candidate vaccine strains in the future.

Development of a highly sensitive PCR/DNA chip method to detect mycoplasmas in a veterinary modified live vaccine.

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.

Leptospira spp. vaccinal antibodies do not react with Borrelia burgdorferi peptides used in the AccuPlex 4.

We attempted to determine if Leptospira spp. antibodies induced by vaccination would cross-react with Borrelia burgdorferi antigens used in a commercial automated immunofluorescent assay (AccuPlex 4 BioCD; Antech). Staff- and student-owned dogs ( n = 31) were recruited at a veterinary teaching hospital in a B. burgdorferi nonendemic area. The dogs were randomized and administered 1 of 4 commercial Leptospira spp. vaccines that contained serovars Canicola, Grippotyphosa, Icterohaemorrhagiae, and Pomona, then booster vaccinated 3 wk later. Blood was collected on weeks 0, 3, 4, 8, and 12. After confirming that maximal Leptospira spp. titers occurred on week 4, aliquots of sera from week 4 were shipped frozen for analysis of B. burgdorferi antibodies against OspA, OspC, OspF, P39, and SLP with the AccuPlex system. Week 4 sera from all 31 dogs had a titer of 1:100 for at least 1 Leptospira spp. serovar. Titers of 1:800 or greater were detected against multiple serovars in 27 dogs. None of the samples contained antibodies against the B. burgdorferi OspA, OspC, OspF, P39, and SLP peptides used in the commercial assay. The B. burgdorferi peptides used in the AccuPlex system do not recognize naturally occurring Leptospira spp. antibodies or those induced by the commercial Leptospira spp. vaccines administered in our study.

Development of a duplex PCR for rapid detection and differentiation of Erysipelothrix rhusiopathiae vaccine strains and wild type strains.

The differentiation of vaccine strains from wild type strains is important for disease control. A duplex PCR for rapid detection and differentiation of Erysipelothrix rhusiopathiae vaccine strains and wild type strains was developed based on the DNA polymerase IV gene. This duplex PCR was sensitive and specific. The detection results were coincident with that of a single nucleotide polymorphisms based PCR but the detection process was more rapid. In conclusion, this duplex PCR was a useful tool for Erysipelothrix rhusiopathiae infections' differential diagnosis in China.

Small ruminant pasteurellosis in Tigray region, Ethiopia: marked serotype diversity may affect vaccine efficacy.

The aim of this study was to investigate the prevalent Bibersteinia, Mannheimia and Pasteurella serotypes, risk factors and degree of serotype co-infections in sheep and goats in the Tigray region of Ethiopia. Serum was collected from 384 sheep and goats from the Tanqua-Abergelle district of Tigray region using cross-sectional random sampling. An indirect haemagglutination test was used for serotyping. Risk factors for infections were evaluated by logistic regression. Potential clustering of multiple serotypes within individual animals due to common risk factors was evaluated by redundancy analysis. Eight serotypes were identified: all studied animals were serologically positive for at least one serotype. Overall, 355 (92·45%) of the animals were infected by four or more serotypes. Of the five risk factors studied, peasant association (PA), animal species, age (serotype A1), and bodyweight (serotype T15) were significantly associated with infection, but sex was not significant. Only PA explained a significant proportion of the variation (adjusted R 2 = 0·16) in the serological responses. After the effect of PA was accounted for, T3 and T4; A7 and Pasteurella multocida A; and A7 and T10 were positively correlated for co-infection, while T4 and T10 were less likely to be found within the same animal. Diverse serotypes were circulating in the Tigray region and could be a challenge in selecting serotypes for vaccine.

The Riemerella anatipestifer AS87_01735 Gene Encodes Nicotinamidase PncA, an Important Virulence Factor.

UNLABELLED: Riemerella anatipestifer is a major bacterial pathogen that causes septicemic and exudative diseases in domestic ducks. In our previous study, we found that deletion of the AS87_01735 gene significantly decreased the bacterial virulence of R. anatipestifer strain Yb2 (mutant RA625). The AS87_01735 gene was predicted to encode a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD(+) salvage pathway. In this study, the AS87_01735 gene was expressed and identified as the PncA-encoding gene, using an enzymatic assay. Western blot analysis demonstrated that R. anatipestifer PncA was localized to the cytoplasm. The mutant strain RA625 (named Yb2ΔpncA in this study) showed a similar growth rate but decreased NAD(+) quantities in both the exponential and stationary phases in tryptic soy broth culture, compared with the wild-type strain Yb2. In addition, Yb2ΔpncA-infected ducks showed much lower bacterial loads in their blood, and no visible histological changes were observed in the heart, liver, and spleen. Furthermore, Yb2ΔpncA immunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Our results suggest that the R. anatipestifer AS87_01735 gene encodes PncA, which is an important virulence factor, and that the Yb2ΔpncA mutant can be used as a novel live vaccine candidate. IMPORTANCE: Riemerella anatipestifer is reported worldwide as a cause of septicemic and exudative diseases of domestic ducks. The pncA gene encodes a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD(+) salvage pathway. In this study, we identified and characterized the pncA-homologous gene AS87_01735 in R. anatipestifer strain Yb2. R. anatipestifer PncA is a cytoplasmic protein that possesses similar PncA activity, compared with other organisms. Generation of the pncA mutant Yb2ΔpncA led to a decrease in the NAD(+) content, which was associated with decreased capacity for invasion and attenuated virulence in ducks. Furthermore, Yb2ΔpncA immunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Altogether, these results suggest that PncA contributes to the virulence of R. anatipestifer and that the Yb2ΔpncA mutant can be used as a novel live vaccine candidate.

Detection and differentiation of wild-type and a vaccine strain of Streptococcus equi ssp. equi using pyrosequencing.

Streptococcus equi subspecies equi (S. equi), the causative agent of strangles, is an important equine pathogen. Strangles is a highly contagious disease and a commercial modified live vaccine (MLV) is used for protection, which although effective, may also result in clinical signs of the disease. A rapid means to differentiate between the MLV and wild-type infection is crucial for quarantine release and limiting the disease spread. This study describes the use of a pyrosequencing assay targeting a single nucleotide deletion upstream of the SzPSe gene to distinguish between the wild-type and vaccine strains. A set of 96 characterized clinical specimens and isolates were tested using the assay. The assay was successful in differentiating between wild-type S. equi and the vaccine strains and in discriminating S. equi from other Streptococci. The vaccine strain was identified in 61.7% (29/47) of the strangles cases in horses with a history of MLV vaccination.

A conditionally lethal mutant of Salmonella Typhimurium induces a protective response in mice.

Here we present the design of a conditionally lethal mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium) which growth depends on tetracycline (Tet). Four mutants of S. Typhimurium, with Tet-conditional growth, were created by inserting the tetRA cassette. Three of the mutants presented a conditional-lethal phenotype in vitro. One mutant in the yabB gene remained conditional inside cells and did not persisted after 24 h in cell cultures. The capacity of S. Typhimurium yabB::tetRA to invade deep organs was investigated in intraperitoneally (IP) infected mice fed with or without chlortetracycline (CTet), a Tet analog with lower antibiotic activity. The yabB::tetRA mutant was undetectable in liver or spleen of animals under normal diet, while in mice under diet including CTet, yabB::tetRA invaded at a level comparable to the WT in mice under normal diet. Moreover, yabB::tetRA produced a strong humoral-immunoresponse after one IP immunization with 10(6) bacteria, measured as serum reactivity against S. Typhimurium whole cell extract. By contrast, oral immunization with 10(6) bacteria was weaker and variable on inducing antibodies. Consistently, IP infected mice were fully protected in a challenge with 10(4) oral S. Typhimurium, while protection was partial in orally immunized mice. Our data indicate that S. Typhimurium yabB::tetRA is a conditionally attenuated strain capable of inducing a protective response in mice in non-permissive conditions.

Vaccination with Klebsiella pneumoniae-derived extracellular vesicles protects against bacteria-induced lethality via both humoral and cellular immunity.

The emergence of multidrug-resistant Klebsiella pneumoniae highlights the need to develop preventive measures to ameliorate Klebsiella infections. Bacteria-derived extracellular vesicles (EVs) are spherical nanometer-sized proteolipids enriched with outer membrane proteins. Gram-negative bacteria-derived EVs have gained interest for use as nonliving complex vaccines. In the present study, we evaluated whether K. pneumoniae-derived EVs confer protection against bacteria-induced lethality. K. pneumoniae-derived EVs isolated from in vitro bacterial culture supernatants induced innate immunity, including the upregulation of co-stimulatory molecule expression and proinflammatory mediator production. EV vaccination via the intraperitoneal route elicited EV-reactive antibodies and interferon-gamma-producing T-cell responses. Three vaccinations with the EVs prevented bacteria-induced lethality. As verified by sera and splenocytes adoptive transfer, the protective effect of EV vaccination was dependent on both humoral and cellular immunity. Taken together, these findings suggest that K. pneumoniae-derived EVs are a novel vaccine candidate against K. pneumoniae infections.

Identification of the 1B vaccine strain of Chlamydia abortus in aborted placentas during the investigation of toxaemic and systemic disease in sheep.

CASE HISTORY: One hundred and forty Cheviot and 100 Suffolk cross Mule primiparous 1-2-year-old ewes, from a flock of about 700 ewes, were vaccinated with an attenuated live 1B strain Chlamydia abortus vaccine about 4 weeks before ram introduction (September 2011). Between 08 March and 01 April 2012, 50 2-year-old ewes aborted and 29 of these died, despite antimicrobial and anti-inflammatory treatment and supportive care. PATHOLOGICAL FINDINGS: Seven fetuses and three placentae from five 2-year-old ewes were submitted for pathological investigation. The aborted fetuses showed stages of autolysis ranging from being moderately fresh to putrefaction. Unusual, large multifocal regions of thickened membranes, with a dull red granular surface and moderate amounts of grey-white surface exudate were seen on each of the placentae. Intracellular, magenta-staining, acid fast inclusions were identified in Ziehl Neelsen-stained placental smears. Immunohistochemistry for Chlamydia-specific lipopolysaccharide showed extensive positive labelling of the placental epithelia. LABORATORY FINDINGS: Molecular analyses of the aborted placentae demonstrated the presence of the 1B vaccine-type strain of C. abortus and absence of any wild-type field strain. The vaccine strain bacterial load of the placental tissue samples was consistent with there being an association between vaccination and abortion. DIAGNOSIS: Initial laboratory investigations resulted in a diagnosis of chlamydial abortion. Further investigations led to the identification of the 1B vaccine strain of C. abortus in material from all three of the submitted aborted placentae. CLINICAL RELEVANCE: Timely knowledge and understanding of any potential problems caused by vaccination against C. abortus are prerequisites for sustainable control of chlamydial abortion. This report describes the investigation of an atypical abortion storm in sheep, and describes the identification of the 1B vaccine strain of C. abortus in products of abortion. The significance of this novel putative association between the vaccine strain of C. abortus and severe clinical disease is unknown. Aspects of the approach that is described are relevant to the investigation of all outbreaks of ovine abortion, irrespective of the diagnosis. Awareness of the changing role of C. abortus as a major global cause of abortion ought to reinforce the importance of monitoring of adequate biosecurity in those countries which are currently free from chlamydial abortion.

A laminated polymer film formulation for enteric delivery of live vaccine and probiotic bacteria.

Live bacterial cells (LBCs) are administered orally as attenuated vaccines to deliver biopharmaceutical agents and as probiotics to improve gastrointestinal (GI) health. However, LBCs present unique formulation challenges and must survive GI antimicrobial defenses including gastric acid after administration. We present a simple new formulation concept, termed polymer film laminate (PFL). LBCs are ambient dried onto cast acid-resistant enteric polymer films that are then laminated together to produce a solid oral dosage form. LBC of a model live bacterial vaccine and a probiotic were dried directly onto a cast film of enteric polymer. The effectiveness at protecting dried cells in a simulated gastric fluid (SGF, pH 2.0) depended on the composition of enteric polymer film used, with a blend of ethylcellulose plus Eudragit L100 55 providing greater protection from acid than Eudragit alone. However, although PFL made from blended polymer films completely released low-molecular-weight dye into intestinal conditions (pH 7.0), they failed to release LBCs. In contrast, PFL made from Eudragit alone successfully protected dried probiotic or vaccine LBC from SGF for 2 h, and subsequently released all viable cells within 60 min of transfer into simulated intestinal fluid. Release kinetics could be controlled by modifying the lamination method.

Mucosal adjuvant activity of IL-2 presenting spores of bacillus subtilis in a murine model of Helicobacter pylori vaccination.

The endospores of Bacillus subtilis are now widely used as a platform for presentation of heterologous proteins and due to their safety record and high resistance to harsh environmental conditions can be considered as potential vehicles for oral vaccination. In this research we show that recombinant B. subtilis spores presenting a fragment of the Helicobacter acinonychis UreB protein and expressing the ureB gene under vegetative promoter elicit a strong cellular immune response in orally immunized mice when co-administered with spores presenting IL-2. We show for the first time the successful application of two types of recombinant spores, one carrying an antigen and the other an adjuvant, in a single oral immunization.

Immunization against a saccharide epitope accelerates clearance of experimental gonococcal infection.

The emergence of ceftriaxone-resistant strains of Neisseria gonorrhoeae may herald an era of untreatable gonorrhea. Vaccines against this infection are urgently needed. The 2C7 epitope is a conserved oligosaccharide (OS) structure, a part of lipooligosaccharide (LOS) on N gonorrhoeae. The epitope is expressed by 94% of gonococci that reside in the human genital tract (in vivo) and by 95% of first passaged isolates. Absence of the 2C7 epitope shortens the time of gonococcal carriage in a mouse model of genital infection. To circumvent the limitations of saccharide immunogens in producing long lived immune responses, previously we developed a peptide mimic (called PEP1) as an immunologic surrogate of the 2C7-OS epitope and reconfigured it into a multi-antigenic peptide, (MAP1). To test vaccine efficacy of MAP1, female BALB/c mice were passively immunized with a complement-dependent bactericidal monoclonal antibody specific for the 2C7 epitope or were actively immunized with MAP1. Mice immunized with MAP1 developed a TH1-biased anti-LOS IgG antibody response that was also bactericidal. Length of carriage was shortened in immune mice; clearance occurred in 4 days in mice passively administered 2C7 antibody vs. 6 days in mice administered control IgG3λ mAb in one experiment (p = 0.03) and 6 vs. 9 days in a replicate experiment (p = 0.008). Mice vaccinated with MAP1 cleared infection in 5 days vs. 9 days in mice immunized with control peptide (p = 0.0001 and p = 0.0002, respectively in two replicate experiments). Bacterial burden was lower over the course of infection in passively immunized vs. control mice in both experiments (p = 0.008 and p = 0.0005); burdens were also lower in MAP1 immunized mice vs. controls (p<0.0001) and were inversely related to vaccine antibodies induced in the vagina (p = 0.043). The OS epitope defined by mAb 2C7 may represent an effective vaccine target against gonorrhea, which is rapidly becoming incurable with currently available antibiotics.

Construction and immunogenicity of a ∆apxIC/ompP2 mutant of Actinobacillus pleuropneumoniae and Haemophilus parasuis.

The apxIC genes of the Actinobacillus pleuropneumoniae serovar 5 (SC-1), encoding the ApxIactivating proteins, was deleted by a method involving sucrose counter-selection. In this study, a mutant strain of A. pleuropneumoniae (SC-1) was constructed and named DapxIC/ ompP2. The mutant strain contained foreign DNA in the deletion site of ompP2 gene of Haemophilus parasuis. It showed no haemolytic activity and lower virulence of cytotoxicity in mice compared with the parent strain, and its safety and immunogenicity were also evaluated in mice. The LD50 data shown that the mutant strain was attenuated 30-fold, compared with the parent strain (LD50 of the mutant strain and parent strain in mice were determined to be 1.0 × 10(7) CFU and 3.5 × 10(5) CFU respectively). The mutant strain that was attenuated could secrete inactivated ApxIA RTX toxins with complete antigenicity and could be used as a candidate live vaccine strain against infections of A. pleuropneumoniae and H. parasuis.

Prevalence of the Leptospira serovars bratislava, grippotyphosa, mozdok and pomona in French dogs.

Although most French dogs are correctly vaccinated against leptospirosis with inactivated strains of canicola and icterohaemorrhagiae, the disease is still very prevalent in France raising the question of whether the vaccines used require updating. The aim of the present study was to provide serological data regarding circulation of the Leptospira serovars: grippotyphosa, bratislava, pomona and mozdok, which are contained in vaccines available in other parts of the world and which could be rapidly adapted for France. Results indicated that the epidemiology was consistent with the circulation of Leptospira belonging to the serogroups Australis and Grippotyphosa and that the case to support the inclusion of either pomona or mozdok in a dog vaccine for France was weak.

Attenuation of a virulent Aeromonas hydrophila with novobiocin and pathogenic characterization of the novobiocin-resistant strain.

AIM: To determine whether novobiocin resistance strategy could be used to attenuate a virulent Aeromonas hydrophila AH11P strain and to characterize the growth and pathogenic differences between the novobiocin-resistant strain and its virulent parent strain AH11P. METHODS AND RESULTS: A novobiocin-resistant strain AH11NOVO was obtained from a virulent Aer. hydrophila strain AH11P through selection of resistance to novobiocin. AH11NOVO was found to be avirulent to channel catfish (Ictalurus punctatus), whereas AH11P was virulent. When AH11NOVO vaccinated channel catfish were challenged with AH11P at 14 days postvaccination, relative per cent of survival of vaccinated fish was 100%. The cell proliferation rate of AH11NOVO was found to be significantly (P < 0.05) less than that of AH11P. In vitro motility assay revealed that AH11NOVO was nonmotile, whereas AH11P was motile. AH11NOVO had significantly (P < 0.05) lower in vitro chemotactic response to catfish mucus than that of AH11P. Although the ability of AH11NOVO to attach catfish gill cells was similar to that of AH11P, the ability of AH11NOVO to invade catfish gill cells was significantly (P < 0.05) lower than that of AH11P. CONCLUSIONS: The novobiocin-resistant AH11NOVO is attenuated and different from its parent AH11P in pathogenicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The significantly lower chemotactic response and invasion ability of AH11NOVO compared with that of its virulent parent strain AH11P might shed light on the pathogenesis of Aer. hydrophila.

Mice vaccinated with enteropathogenic Escherichia coli ghosts show significant protection against lethal challenges.

AIM: To prepare enteropathogenic Escherichia coli (EPEC) E2348/69 ghosts and investigate whether immunization with EPEC bacterial ghosts can elicit protective immune responses. METHODS AND RESULTS: A recombinant plasmid with double λPL/PR-cI857 temperature-sensitive regulatory cassettes was constructed. The lysis gene E and/or the staphylococcal nuclease A (SNA) gene were separately inserted downstream of the two regulatory cassettes to construct the lysis plasmids pBV220::E and pBV220::E::CI-P-SNA. An EPEC reference strain E2348/69 (serotype O127:H6) was transformed with the lysis plasmids to produce EPEC ghosts. Mice injected with bacterial ghosts EGE (EPEC ghosts produced using lysis protein E) or EGES (EPEC ghosts produced using a combination of lysis protein E and SNA) gained weight normally and showed no clinical signs of disease. Vaccination trials showed that mice immunized with EGE or EGES were significantly protected against subsequent challenge with the wild-type virulent parent strain, EPEC E2348/69 (42/50 and 45/50 survival, respectively); in contrast, none of the 30 control mice survived. CONCLUSIONS: Immunization with EPEC ghosts can elicit protective immune responses in BALB/c mice. SIGNIFICANCE AND IMPACT OF THE STUDY: EPEC ghosts may represent a promising new approach for vaccination against EPEC infection.