Isolierung von Bordetella-Phagen aus Bakterienstämen der Impfstoffproduktion

It has been demonstrated that strains of Bordetella pertussis used for vaccine production contain temperate phages. It can be conducted from many experiments performed in our laboratory. that 10–100 phages per 10¹⁰ bacteria are released. However, the production of bacterial mass is not markedly influenced by lysogeny. Strains of Bordetella bronchiseptica used for production of vaccine against Rhinitis atrophicans of pigs have temperate phages too. These phages may cause a complete lysis during a submerse cultivation. The phages of Bordetella pertussis and Bordetella bronchiseptica can be propagted on Bordetella parapertussis.     

Increased incidence of pertussis and parapertussis in HIV-1-positive adolescents vaccinated previously with whole-cell pertussis vaccine

We investigated 296 adolescents (11–18 years), who had been immunized previously with the three doses of DPT vaccines. 48 were diagnosed positive for HIV-1. Nasopharyngeal swabs were obtained from 296 adolescents who presented with persistent cough and nasopharyngeal secretions. Nasopharyngeal swabs (calcium alginate) specimens were collected by passing the swabs through the nares into the posterior nasopharynx and rotating the swabs for a few seconds. The swabs were plated for culture of Bordetella organisms in charcoal cephalexin blood agar (CCBA). The CCBA plates were incubated for 2–6 days at 35 °C in a humid aerobic atmosphere. The suspected, shiny (mercury-like) colonies were tested by slide agglutination with antisera to B. pertussis and B. parapertussis, and urease, oxidase activities were performed. Results indicate that out of 48 HIV-1-positive adolescents, 18 had positive cultures for Bordetella organisms (14, Bordetella pertussis, and 4, Bordetella parapertussis). Of 248 HIV-1-negative subjects, 3 had Bordetella organisms (2, Bordetella pertussis, 1, Bordetella bronchiseptica). One of the subjects, a boy, aged 14 years, with Bordetella bronchiseptica had a dog as pet, which was found to be infected. The results indicate that adolescents with HIV-1 infection, despite being vaccinated against pertussis have a higher rate of infection when exposed to pertussis bacteria than HIV-1-negative adolescents. This revised version was published online in August 2006 with corrections to the Cover Date.     

Passive immunization of piglets against enterotoxigenic colibacillosis by vaccinating dams with K88ac pili bearing bacterins

Day-old-piglets were passively immunized by vaccinating the pregnant sows with K88ac enterotoxigenic Escherichia coli (ETEC) vaccine. High level of ETEC specific antibodies was excreted in colostrum (3733.33 +/- 1152.13) and maintained a detectable level (100.00 +/- 0.00) up to 21 day post partum (DPP). The IgG was the predominant immunoglobulin followed by IgA and IgM. Piglets born of vaccinated dam (group A) and unvaccinated dam (group B) were challenged in 7 day of age. Clinical and faecal scores were significantly (P < 0.01) low in group A than that of group B. Piglets of group A developed mild diarrhoea (33.33%), while all the control piglets developed profuse diarrhoea and 3 of these died before 14 day of challenge infection.     

Development of vaccines against pertussis caused by Bordetella holmesii using a mouse intranasal challenge model

Bordetella holmesii is recognized as the third causative agent of pertussis (whooping cough) in addition to Bordetella pertussis and Bordetella parapertussis. Pertussis caused by B. holmesii is not rare around the world. However, to date, there is no effective vaccine against B. holmesii. We examined the protective potency of pertussis vaccines available in Japan and vaccines prepared from B. holmesii. A murine model of respiratory infection was exploited to evaluate protective potency. No Japanese commercial pertussis vaccines were effective against B. holmesii. In contrast, a wBH vaccine and an aBH vaccine prepared from B. holmesii were both protective. Passive immunization with sera from mice immunized with aBH vaccine established protection against B. holmesii, indicating that B. holmesii‐specific serum antibodies might play an important role in protection. Immuno‐proteomic analysis with sera from mice immunized with aBH vaccine revealed that the sera recognized a BipA‐like protein of B. holmesii. An aBH vaccine prepared from a BipA‐like protein‐deficient mutant strain did not have a protective effect against B. holmesii. Taken together, our results suggest that the BipA‐like protein plays an important role in the protective efficacy of aBH vaccine.     

Impact of the probiotic bacteria Enterococcus faecium NCIMB 10415 (SF68) and Bacillus cereus var. toyoi NCIMB 40112 on the development of serum IgG and faecal IgA of sows and their piglets

Abstract

To examine the influence of two different probiotic bacteria on the humoral immune system of swine, two animal studies were carried out with sows and their litters. The sows' feed was supplemented with either Enterococcus faecium NCIMB 10415 (SF68) or Bacillus cereus var. toyoi NCIMB 40112 beginning early in pregnancy. The total IgA content in the faeces as well as the total IgG concentration in the blood of the sows was recorded before and after weaning. The same parameters were determined in the blood and faeces of the piglets. In sows, only feed supplementation with B. cereus led to a clear increase in faecal IgA. Serum IgG levels were not significantly affected by any probiotic feeding in sows. In piglets, the group that was fed B. cereus showed significantly higher faecal IgA levels shortly before weaning, whereas in the E. faecium group, a significant decrease in IgA levels was observed one week after weaning. In both probiotic fed groups the post-weaning IgG levels were significantly decreased compared to the respective control groups. We conclude that B. cereus var. toyoi feed supplementation led to an increased intestinal IgA secretion both in sows and piglets. This effect could be related to a more successful mucosal defence which in turn led to a lower level in systemic IgG production in piglets after weaning.     

Field trials on transmissible gastroenteritis live virus vaccine in newborn piglets.

Field trials were conducted on attenuated live virus vaccine of transmissible gastroenteritis to confer active immunity to newborn piglets. To examine innocuity and efficacy of the vaccine, a total of 714 newborn piglets were subjected to these trials. Of them, 357 piglets were administered orally with 10(7.0) TCID50 within 3 days after birth, and the other 357 piglets served as nonvaccinated controls. No undesirable postvaccinal reaction was observed in any vaccinated piglet. Suckling piglets born from nonimmune sows showed a good antibody response after vaccination. They were different, however, in antibody titer from one experimental place to another. Antibody levels were high in piglets raised in the northern experimental places. On the contrary, the antibody response of suckling piglets born from immune sows was influenced by vaccination. In most of these piglets, antibody titers declined markedly and disappeared finally 3 months after vaccination. About 25% of the non-vaccinated piglets showed an antibody response by pen contact with vaccinated ones.     

一株支气管炎鲍特杆菌国家标准菌种的再鉴定

通过生化试验、质谱鉴定、16S rRNA基因序列分析、全基因组序列测定及生物信息学分析等方法对国家标准菌种CMCC(B)40001进行再鉴定。对菌株的毒力、耐药和MLST进行分析，并对鲍特杆菌（Bordetella spp.）的群体进化进行分析。结合不同方法，CMCC(B)40001再鉴定为支气管炎鲍特杆菌（Bordetella bronchiseptica），且其作为标准菌种能够发挥国家标准描述的质控作用。同时获取该菌株的新ST型，在进化关系上，其与百日咳鲍特杆菌(Bordetella pertussis)和副百日咳鲍特杆菌(Bordetella parapertussis)遗传距离近。本研究再鉴定了国家标准菌种CMCC(B)40001为支气管炎鲍特杆菌，并分析了其遗传特征，为国家标准菌种的应用提供资源描述支持。     

Comparison of real-time PCR and culture isolation in colostrum-deprived pigs immunized and challenged with Haemophilus parasuis

Aims: A real‐time PCR (RT‐PCR) based on the detection of the infB gene of Haemophilus parasuis is compared with culture isolation (Frandoloso et al., (2011) Clin Vaccine Immunol 18 , 50–58.), evaluating different subunit or commercial vaccines. Methods and Results: Samples from different tissues of 24 experimentally infected and challenged colostrum‐deprived piglets were tested. The RT‐PCR gave globally a 23·3% more of positive results than culture, and all samples being positive by culture were positive by RT‐PCR also. H. parasuis could not be cultured from any of the samples of the piglets included in the three vaccinated groups resulting in a strong protection, but it could be detected by RT‐PCR in six samples in the group immunized with the commercial vaccine, in three in that vaccinated with native proteins with affinity to porcine transferrin (NPAPT) administered intramuscularly and in only two in that immunized with NPAPT intratracheally. Conclusions: The RT‐PCR was more sensitive than culture for H. parasuis detection in the organs compared. Significance and Impact of the Study: The RT‐PCR evidenced that NPAPT vaccines were those yielding the best protection results in terms of H. parasuis clearance.     

Effect of AcHERV-GmCSF as an Influenza Virus Vaccine Adjuvant

Introduction

The first identification of swine-originated influenza A/CA/04/2009 (pH1N1) as the cause of an outbreak of human influenza accelerated efforts to develop vaccines to prevent and control influenza viruses. The current norm in many countries is to prepare influenza vaccines using cell-based or egg-based killed vaccines, but it is difficult to elicit a sufficient immune response using this approach. To improve immune responses, researchers have examined the use of cytokines as vaccine adjuvants, and extensively investigated their functions as chemoattractants of immune cells and boosters of vaccine-mediated protection. Here, we evaluated the effect of Granulocyte-macrophage Colony-Stimulating Factor (GmCSF) as an influenza vaccine adjuvant in BALB/c mice.

Method and Results

Female BALB/c mice were immunized with killed vaccine together with a murine GmCSF gene delivered by human endogenous retrovirus (HERV) envelope coated baculovirus (1×10⁷ FFU AcHERV-GmCSF, i.m.) and were compared with mice immunized with the killed vaccine alone. On day 14, immunized mice were challenged with 10 median lethal dose of mouse adapted pH1N1 virus. The vaccination together with GmCSF treatment exerted a strong adjuvant effect on humoral and cellular immune responses. In addition, the vaccinated mice together with GmCSF were fully protected against infection by the lethal influenza pH1N1 virus.

Conclusion

Thus, these results indicate that AcHERV-GmCSF is an effective molecular adjuvant that augments immune responses against influenza virus.     

In-vitro and in-vivo analysis of the production of the Bordetella type three secretion system effector A in Bordetella pertussis,Bordetella parapertussis and Bordetella bronchiseptica

Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica are three closely related pathogens.They all possess the gene coding for the Bordetella type three secretion system effector A (bteA) toxin that became a focus of interest since it was demonstrated that B. pertussis Japanese non-vaccine-type isolates produce BteA unlike vaccine-type isolates. We thus explored the in-vitro production of BteA in B. pertussis isolates collected in France during periods of different vaccine policy as well as in B. parapertussis and B. bronchiseptica isolates. We also analyzed the in-vivo induction of anti-BteA antibodies after infection with different isolates of the three species.We produced a recombinant His6-tagged BteA (rBteA) protein. Specific rBteA polyclonal serum was prepared which enabled us to screen Bordetella isolates for in-vitro BteA production: 99.0% (293/296) of tested B. pertussis isolates, including French vaccine strains, and 97.5% (79/81) of B. bronchiseptica isolates produced BteA in-vitro but only the latter was capable of inducing an in-vivo immune response. No in-vitro or in-vivo production of BteA was detected by any of the B. parapertussis isolates tested.     

Analysis of humoral immune responses in rhesus macaques vaccinated with attenuated SIVmac239Δnef and challenged with pathogenic SIVmac251

Background To determine the correlation between protection and humoral immune response against simian immunodeficiency virus (SIVmac251), 11 macaques were immunized with live‐attenuated SIVmac239Δnef either intravenously or via the tonsils and exposed to SIVmac251 after either 6 or 15 months along with unvaccinated controls. Results Independent of the route of vaccine application, viremia was significantly reduced in vaccinees compared with controls 2 weeks post‐challenge. Concomitantly, viremia correlated inversely with SIV‐specific IgG, complement‐mediated lysis and neutralizing antibodies and these parameters seemed to contribute to reduced viremia. During chronic infection, six monkeys controlled viremia in the circulation (two or fewer infectious units per 10⁶ PBMCs) and showed no signs of trapping in lymphatic tissues (Appendix S1). Conclusions As no significant differences were observed throughout the study, with respect to the humoral immune response and viremia control, between the two vaccinated cohorts, mucosal immunization strategies are recommended due to more simplified application.     

大豆黄酮对母猪免疫功能和血清及初乳中GH、PRL、SS水平的影响

研究口服大豆黄酮对妊娠母猪及其仔猪的免疫功能和血清、初轧中生长激素（ＧＨ）、催乳素（ＰＲＬ）、生长抑素（ＳＳ）水平的影响，结果表明：１）大豆黄酮能明显提高母猪血清和初乳中特异性抗体（猪瘟抗体或溶血素）的水平，表明母猪整体和乳腺器官的体液免疫功能明显增强。２）通过对初乳的吸收，其新生仔猪血清中母源抗体水平明显高于对照组。３）母猪血清和初乳中ＧＨ、ＰＲＬ含量明显高于对照组，而血清ＳＳ含量显著低于对照组。本实验结果提示：垂体ＰＲＬ、ＧＨ和体内ＳＳ可能参与了大豆黄酮对猪免疫功能的调节过程。     

Epitope specificity of protective lactogenic immunity against swine transmissible gastroenteritis virus.

The epitope specificity of the protective immune response against swine transmissible gastroenteritis (TGE) has been investigated by using circulating and secretory antibodies. This study was carried out with sows vaccinated with TGEV or the antigenically related porcine respiratory coronavirus (PRCV). TGEV vaccination of sows resulted in greater lactogenic protection of suckling piglets against TGEV challenge and a higher secretory immune response than PRCV vaccination did. These differences in the immune response were conditioned by the route of antigen presentation as a result of the different tropism of each virus. Epitopes on S protein, and in particular those contained in its antigenic site. A, were more immunogenic than epitopes on N and M proteins in both groups of vaccinated sows, as determined by a competitive radioimmunoassay. Minor differences in antibody response against the previously defined antigenic subsites Aa, Ab, and Ac were also detected, with subsite Ab being the most antigenic in both TGEV- and PRCV-immune sows. These findings suggest that antigenic site A on S protein, involved in virus neutralization, is the immunodominant site in pregnant sows that confer lactogenic protection. They also validate, in experiments with secretory antibodies, the antigenic maps made with murine monoclonal antibodies. Therefore, this antigenic site should be considered for vaccine or diagnostic development.     

Pertactin antigens extracted from Bordetella pertussis and Bordetella bronchiseptica differ in the isoelectric point

Pertactin, which is a membrane-associated antigen of Bordetella pertussis and which is present in many acellular vaccines against whooping cough, has been reported to be similar to the homologous protein in Bordetella bronchiseptica. By running parallel experiments using proteins derived from the two species, we show that the isoelectric point of pertactin from B. pertussis is lower than reported and clearly distinguishable from the homologous protein of B. bronchiseptica. Received: 9 April 1997 / Accepted: 20 May 1997     

Immunity Elicited by an Experimental Vaccine Based on Recombinant Flagellin-Porcine Circovirus Type 2 Cap Fusion Protein in Piglets

In a recent study, we reported that a recombinant protein from fusion expression of flagellin to porcine circovirus type 2 (PCV2) Cap induced robust humoral and cell-mediated immunity that afforded full protection for PCV2 infection using BALB/c mice. Here, we further evaluated the immunogenicity and protection of the recombinant protein using specific pathogen free (SPF) pigs. Twenty-five 3-week-old piglets without passively acquired immunity were divided into 5 groups. All piglets except negative controls were challenged with a virulent PCV2 at 21 days after booster vaccination and necropsied at 21 days post-challenge. Vaccination of piglets with the recombinant protein without adjuvant induced strong humoral and cellular immune responses as observed by high levels of PCV2-specific IgG antibodies and neutralizing antibodies, as well as frequencies of PCV2-specific IFN-γ-secreting cells that conferred good protection against PCV2 challenge, with significant reduced PCV2 viremia, mild lesions, low PCV2 antigen-positive cells, as well as improved body weight gain, comparable to piglets vaccinated with a commercial PCV2 subunit vaccine. These results further demonstrated that the recombinant flagellin-Cap fusion protein is capable of inducing solid protective humoral and cellular immunity when administered to pigs, thereby becoming an effective PCV2 vaccine candidate for control of PCV2 infection.     

Immunogenicity and protective efficacy of recombinant iron superoxide dismutase protein from Bordetella pertussis in mice models

Whooping cough (pertussis) is a highly contagious respiratory infection caused by Bordetella pertussis. Although availability of effective pertussis vaccines reportedly decreases the incidence of the disease, B. pertussis circulation in populations has not been eliminated. Thus, it is necessary to find new protein candidates with greater immune protective capacities than the currently available acellular pertussis vaccines. In this study, iron superoxide dismutase (FeSOD) gene (sodB) was cloned, expressed in Escherichia coli and recombinant FeSOD protein thence purified. The recombinant protein (rFeSOD) was formulated with aluminum hydroxide (Alum) or monophosphoryl lipid A (MPLA) and injected intraperitoneally to immunize mice, after which IgG1, IgG2a and IFN‐γ titers were measured to assess humoral and cellular responses, respectively, to these immunizations. The extent of bacterial colonization in lungs of intranasally challenged mice was determined 5, 8 and 14 days post‐challenge. IgG1 and IgG2a responses were significantly stronger in mice that had been immunized with rFeSOD–MPLA than in those that had received rFeSOD‐Alum (P < 0.05). Additionally, IgG2a titers were higher in mice vaccinated with recombinant protein FeSOD (rFeSOD) formulated with MPLA, especially after the second immunization. Immunization with rFeSOD–MPLA also provided a modest, but significant decrease in bacterial counts in lungs of mice (P < 0.05). Antigen specific‐IFN‐γ responses were significantly stronger in the group vaccinated with rFeSOD–MPLA, which could account for the lower bacterial counts. These findings suggest that rFeSOD protein formulated with MPLA has potential as an acellular pertussis vaccine candidate component.     

Acellular and whole cell pertussis vaccines protect against the lethal effects of intracerebral challenge by two different T-cell dependent humoral routes

Athymic (nu/nu) and euthymic (+/nu) BALB/c mice were immunized with a whole cell pertussis vaccine or with an acellular vaccine which contained detoxified pertussis toxin (PT) and filamentous hemagglutinin (FHA). Only the euthymic mice were protected against intracerebral challenge with virulent Bordetella pertussis which implies involvement of T-cells. As a cell transfer from mice immunized with whole cell or acellular vaccine prior to the challenge did not protect naive euthymic recipients, cellular immunity seems to be non-protective as an effector mechanism. Mice could be protected passively against a challenge by administration of immune sera. Therefore, T-cell dependent humoral immune responses to B. pertussis appear to be crucial for protection. The humoral response was further studied with athymic and euthymic mice. In euthymic mice the whole cell vaccine induced antibodies to FHA, pililipopolysaccharides (LPS) and an outer membrane protein (OMP) preparation, whereas the acellular vaccine induced antibodies to PT, FHA and OMP. Both IgM and IgG could be detected. From the nude mice only those immunized with the whole cell vaccine showed an antibody response which consisted of low titres of IgM directed to LPS. Sera from both +/nu and nu/nu mice immunized with the whole cell vaccine were bactericidal in vitro. These data demonstrate that in the mouse model protection to intracerebral challenge with B. pertussis is T-cell dependent as is the humoral response to PT, FHA, OMP and pili. The T-independent B-cell activation by the whole cell preparation is due to the presence of LPS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative analysis of the immune responses induced by native versus recombinant versions of the ASP-based vaccine against the bovine intestinal parasite Cooperia oncophora

The protective capacities of a native double-domain activation-associated secreted protein (ndd-ASP)-based vaccine against the cattle intestinal nematode Cooperia oncophora has previously been demonstrated. However, protection analysis upon vaccination with a recombinantly produced antigen has never been performed. Therefore, the aim of the current study was to test the protective potential of a Pichia-produced double-domain ASP (pdd-ASP)-based vaccine against C. oncophora. Additionally, we aimed to compare the cellular and humoral mechanisms underlying the vaccine-induced responses by the native (ndd-ASP) and recombinant vaccines. Immunisation of cattle with the native C. oncophora vaccine conferred significant levels of protection after an experimental challenge infection, whereas the recombinant vaccine did not. Moreover, vaccination with ndd-ASP resulted in a higher proliferation of CD4-T cells both systemically and in the small intestinal mucosa when compared with animals vaccinated with the recombinant antigen. In terms of humoral response, although both native and recombinant vaccines induced similar levels of antibodies, animals vaccinated with the native vaccine were able to raise antibodies with greater specificity towards ndd-ASP in comparison with antibodies raised by vaccination with the recombinant vaccine, suggesting a differential immune recognition towards the ndd-ASP and pdd-ASP. Finally, the observation that animals displaying antibodies with higher percentages of recognition towards ndd-ASP also exhibited the lowest egg counts suggests a potential relationship between antibody specificity and protection.     

Isotypic characteristics of serum and secretory O antibodies and local immunological memory in the parenteral immunization of guinea pigs with a ribosomal Shigella vaccine

Guinea pigs were immunized subcutaneously with ribosomal vaccine prepared from S. sonnei and their systemic and local humoral response was studied by means of ELISA techniques with the use of monospecific antisera to guinea pig IgA and IgG. Injection of the ribosomal vaccine leads to a significant rise in the levels of IgA O-antibodies in tears, IgG and IgA O-antibodies in the serum. The presence of IgA O-antibodies in tears was seemingly the result of their local synthesis rather than the seepage of serum IgA. The stimulation of the local and systemic anti-O response was more pronounced after parenteral immunization with the ribosomal vaccine than after immunization with the corresponding dose of lipopolysaccharide (LPS). Parenteral immunization with the ribosomal vaccine induced the development of both systemic and local memory. The priming effect produced by relatively small doses of this vaccine (40 micrograms), administered parenterally, was similar to the effect of prolonged and intensive stimulation ensured by 10-day feeding with LPS (the total dose being 5,000 micrograms).