Evaluation of humoral and cellular immune responses to BP26 and OMP31 epitopes in the attenuated Brucella melitensis vaccinated sheep

In recent years, the number of cases of human brucellosis has been increasing by approximately 10% per year in China. Most cases were caused by Brucella melitensis through contacts with infected sheep, goats or their products. An attenuated B. melitensis vaccine M5-90 is currently used to vaccinate both animals in China. This vaccine has not been investigated for critical parameters such as immune response and its association with protective efficacy. In this study, humoral and cellular immune response to the periplasmic protein BP26 and the outer membrane protein OMP31 were evaluated in M5-90 vaccinated Chinese merino and Kazak sheep. Antibodies to BP26 or OMP31 were detected at low levels, and specific IFN-γ response was quantified. Strongly reactive peptides derived from BP26 and OMP31 identified five T-cell epitopes (BP26-6, -8, -11, -12 and OMP31-23) common to both sheep species, five species-specific epitopes (BP26-10, -18, -21 and -22 and OMP31-12) and four animal-specific epitopes (BP26-15, -23, OMP31-6 and -21), which stimulated specific IFN-γ response in vaccinated sheep. Among those T-cell epitopes, reactivity to BP26-18 and -21 epitopes was significantly associated with MHC-I B allele (P = 0.024). However, a specific T-cell response induced by the M5-90 vaccine was relatively week and did not sustain long enough, which might be suppressed by rapid activation of T-regulatory (Treg) cells following vaccination. These findings provide an insight in designing a safer and more effective vaccine for use in animals and in humans.     

Characterization of possible correlates of protective response against Brucella ovis infection in rams immunized with the B. melitensis Rev 1 vaccine

Vaccination with the live attenuated Brucella melitensis Rev 1 vaccine is used to control ovine brucellosis caused by Brucella ovis in sheep. The objective of this study was to identify possible correlates of protective response to B. ovis infection through the characterization by microarray hybridization and real-time RT-PCR of inflammatory and immune response genes differentially expressed in rams previously immunized with B. melitensis Rev 1 and experimentally challenged with B. ovis. Gene expression profiles were compared before and after challenge with B. ovis between rams protected and those vaccinated but found infected after challenge. The TLR10, Bak and ANXI genes were expressed at higher levels in vaccinated and protected rams. These genes provide possible correlates of protective response to B. ovis infection in rams immunized with the B. melitensis Rev 1 vaccine.     

Inactivation of formyltransferase (wbkC) gene generates a Brucella abortus rough strain that is attenuated in macrophages and in mice

Rough mutants of Brucella abortus were generated by disruption of wbkC gene which encodes the formyltransferase enzyme involved in LPS biosynthesis. In bone marrow-derived macrophages the B. abortusΔwbkC mutants were attenuated, could not reach a replicative niche and induced higher levels of IL-12 and TNF-α when compared to parental smooth strains. Additionally, mutants exhibited attenuation in vivo in C57BL/6 and interferon regulatory factor-1 knockout mice. ΔwbkC mutant strains induced lower protective immunity in C56BL/6 than smooth vaccine S19 but similar to rough vaccine RB51. Finally, we demonstrated that Brucella wbkC is critical for LPS biosynthesis and full bacterial virulence.     

Evaluation of immunogenicity and protective efficacy of a plasmid DNA vaccine encoding ribosomal protein L9 of Brucella abortus in BALB/c mice

Brucellosis is a worldwide zoonotic disease. No Brucella vaccine is available for use in humans and existing animal vaccines have limitations. We have previously described the ribosomal protein L9 to have the vaccine potential. In this study, L9 based DNA vaccine (pVaxL9) was generated and evaluated in mouse model. Intramuscular immunisation of pVaxL9 was able to elicit the anti-L9 IgG antibody response of both IgG1 and IgG2a isotypes when compared with PBS and pVax immunised control animals. Heightened antibody response was observed in mice groups immunised with pVaxL9 priming and recombinant L9 boosting (PB) and where pDNA immunisation was carried out by in vivo electroporation (EP). The vaccine groups proliferated splenocytes and released Th1 type cytokines e.g. IFN-γ, TNF-α, IL-2. Further, flow cytometric analysis revealed that IFN-γ was released by both by CD4+ and CD8+ T cells particularly in PB and EP groups when compared with mice immunised with empty control vector. The L9 based pDNA vaccine was able to confer significant protection in mice against challenge with virulent B. abortus with PB and EP groups offering better protection. Taken together, it can be concluded that L9 based DNA vaccine is immunogenic and confer protection in mouse model.     

内蒙古自治区乌兰察布市羊种布鲁氏菌HOOF基因分型特征

目的 调查2012-2015年内蒙古自治区乌兰察布市83株布鲁氏菌HOOF分型特征。方法 对临床分离的83株布鲁氏菌采用常规方法和AMOS-PCR进行鉴定;利用8个数目可变串联重复序列 位点的HOOF分型方法对菌株进行基因分型,并用Hunter-Gaston 分辨指数评估各VNTR 位点的多态性和对菌株的综合辨别能力,采用BioNumerics 5.0软件聚类分析和构建聚类图。结果 83株试验菌全部为羊种布鲁氏菌,全部8个分型位点具有极高的多态性,多态性指数为0.998;其中6个位点（1、2、4~7）的多态性指数 ≥ 0.678,其分辨力主要来自多态性指数较高的位点。83株羊种布鲁氏菌聚为8大类76个基因型。6个共享基因型包括13株布鲁氏菌,提示感染患者具有流行病学相关性;另70株菌呈现独特的基因型,提示病原分离自无流行病学相关的散发患者。结论 乌兰察布市人布鲁氏菌病流行以散发和局部暴发共存,且散发为主,而交叉感染患者与传染源转移有关。     

Immunization of mice with a novel recombinant molecular chaperon confers protection against Brucella melitensis infection

Brucella spp. are zoonotic Gram-negative intracellular pathogens with the ability to survive and replicate in phagocytes. It has been shown that bacterial proteins expressed abundantly in this niche are stress-related proteins capable of triggering effective immune responses. BMEI1549 is a molecular chaperone designated DnaK that is expressed under stress conditions and helps to prevent formation of protein aggregates. In order to study the potential of DnaK as a prospective Brucella subunit vaccine, immunogenicity and protective efficacy of recombinant DnaK from Brucella melitensis was evaluated in BALB/c mice. The dnak gene was cloned, expressed in Escherichia coli, and the resulting recombinant protein used as subunit vaccine. DnaK-immunized mice showed a strong lymphocyte proliferative response to in vitro antigen stimulation. Although comparable levels of antigen-specific IgG2a and IgG1 were observed in immunized mice, high amounts of IFN-γ, IL-12 and IL-6, no detectable level of IL-4 and very low levels of IL-10 and IL-5 were produced by splenocytes of vaccinated mice suggesting induction of a Th1 dominant immune response by DnaK. Compared to control animals, mice vaccinated with DnaK exhibited a significant degree of protection against subsequent Brucella infection (p < 0.001), albeit this protection was less than the protection conferred by Rev.1 (p < 0.05). A further increase in protection was observed, when DnaK was combined with recombinant Omp31. Notably, this combination, as opposed to each component alone, induced statistically similar level of protection as induced by Rev.1 suggesting that DnaK could be viewed as a promising candidate for the development of a subunit vaccine against brucellosis.     

Novel vector vaccine against Brucella abortus based on influenza A viruses expressing Brucella L7/L12 or Omp16 proteins: Evaluation of protection in pregnant heifers

The present study provides the first information about the protection of a novel influenza viral vector vaccine expressing the Brucella proteins ribosomal L7/L12 or Omp16 containing the adjuvant Montanide Gel01 in pregnant heifers. Immunization of pregnant heifers was conducted via the conjunctival (n = 10) or subcutaneous (n = 10) route using cross prime and booster vaccination schedules at an interval of 28 days. The vector vaccine was evaluated in comparison with positive control groups vaccinated with Brucella abortus S19 (n = 10) or B. abortus RB51 (n = 10) and a negative (PBS + Montanide Gel01; n = 10) control group. Via both the conjunctival or subcutaneous route, evaluation of protectiveness against abortion, effectiveness of vaccination and index of infection (in heifers and their fetuses or calves) demonstrated the vector vaccine provided good protection against B. abortus 544 infection compared to the negative control group (PBS + Montanide Gel01) and comparable protection to commercial vaccines B. abortus S19 or B. abortus RB51.     

Studies on recombinant glucokinase (r-glk) protein of Brucella abortus as a candidate vaccine molecule for brucellosis

Brucellosis is one of the most prevalent zoonotic diseases of worldwide distribution caused by the infection of genus Brucella. Live attenuated vaccines such as B. abortus S19, B. abortus RB51 and B. melitensis Rev1 are found most effective against brucellosis infection in animals, contriving a number of serious side effects and having chances to revert back into their active pathogenic form. In order to engineer a safe and effective vaccine candidate to be used in both animals and human, a recombinant subunit vaccine molecule comprising the truncated region of glucokinase (r-glk) gene from B. abortus S19 was cloned and expressed in Escherichia coli BL21DE3 host. Female BALB/c mice immunized with purified recombinant protein developed specific antibody titer of 1:64,000. The predominant IgG2a and IgG2b isotypes signified development of Th1 directed immune responses. In vitro cell cytotoxicity assay using anti-r-glk antibodies incubated with HeLa cells showed 81.20% and 78.5% cell viability against lethal challenge of B. abortus 544 and B. melitensis 16M, respectively. The lymphocyte proliferative assay indicated a higher splenic lymphocyte responses at 25 μg/ml concentration of protein which implies the elevated development of memory immune responses. In contrast to control, the immunized group of mice intra-peritoneal (I.P.) challenged with B. abortus 544 were significantly protected with no signs of necrosis and vacuolization in their liver and spleen tissue. The elevated B-cell response associated with Th1 adopted immunity, significant in vitro cell viability as well as protection afforded in experimental animals after challenge, supplemented with histopathological analysis are suggestive of r-glk protein as a prospective candidate vaccine molecule against brucellosis.     

江西省羊种布鲁氏菌分子流行病学特征分析

目的 掌握江西省羊种布鲁氏菌分子特征,了解分离菌株流行病学间相关性。方法 采用多位点可变数目串联重复序列分析方法（MLVA）对江西省17个县（区） 25株人分离羊种布鲁氏菌进行分析。结果 25株羊种布鲁氏菌聚类可分为24个独立基因型,相似度为67.00%～100.00%,辛普森指数在0.000～0.773之间,MLVA8型有3个基因型,60.00%（15/25）为42型,32.00%（8/25）为43型,8.00%（2/25）为63型。MLVA11型有7个基因型,116、125型为主要基因型,两型菌株占56.00%（14/25）。结论 25株人分离羊种布鲁氏菌基因具有高度遗传多样性,菌株基因型繁多,各菌株间无明显流行病学相关性,表明江西省羊种布鲁氏菌传染来源复杂。     

IL4R and IL13 polymorphic variants and development of antibodies to surface antigen of hepatitis B virus in hemodialysis patients in response to HBV vaccination or infection

We searched for an association between the interleukin 4 receptor gene (IL4R) rs1805015 and interleukin 13 gene (IL13) rs20541 polymorphisms and the development of antibodies to hepatitis B surface antigen (anti-HBs) in the case of hepatitis B virus (HBV) vaccination or infection in hemodialysis (HD) patients. HD patients who failed to respond to HBV vaccination did not differ in genotype frequencies of IL4R (TT 72.7%, CT 22.6%, CC 4.7%) and IL13 (CC 59.0%, CT 34.2%, TT 6.8%) from vaccine responders (IL4R TT 68.0%, CT 27.3%, CC 4.7%; IL13 CC 55.0%, CT 38.5%, TT 6.5%). HD patients who did not develop anti-HBs despite HBV infection also did not differ in genotype frequencies of IL4R (TT 67.8%, CT 26.8%, CC 5.4%) and IL13 (CC 60.7%, CT 33.9%, TT 5.4%) from HD patients who developed an anti-HBs response (IL4R TT 65.4%, CT 30.8%, CC 3.8%; IL13 CC 60.5%, CT 34.6%, TT 4.9%). In HD patients, neither the IL4R nor IL13 polymorphism is associated with anti-HBs development irrespective of whether an immunization is provoked by HBV vaccination or HBV infection.     

Ascaris suum infection negatively affects the response to a Mycoplasma hyopneumoniae vaccination and subsequent challenge infection in pigs

Since their first introduction more than a century ago, vaccines have become one of the most cost-effective tools to prevent and manage infectious diseases in human and animal populations. It is vital to understand the possible mechanisms that may impair optimal vaccine efficacy. The hypothesis posed in this study was that a concurrent Ascaris suum infection of pigs vaccinated with a Mycoplasma hyopneumoniae (Mh) vaccine would modulate the protective immune response to a subsequent challenge infection. Four groups of pigs were either (1) untreated (group C), (2) vaccinated against Mh 3 weeks after the start of the study (group V), (3) given a trickle infection with A. suum throughout the study (group A), or (4) given a trickle infection with A. suum and vaccinated against Mh (group AV). All pigs were subsequently inoculated with live Mh bacteria 4 weeks after the Mh vaccination and necropsied after another 4 weeks. All pigs in group V sero-converted 3 weeks after vaccination (100%), as opposed to only 33% of group AV pigs that were Mh-vaccinated and given A. suum. At the end of the study, only 78% of pigs in group AV had sero-converted. Pigs in group AV had a higher mean percentage of lung pathology and the variation was significantly higher in these pigs compared to pigs in group V. The pattern of gene expression in the lungs and draining lymph nodes indicated a local Th2-skewed response induced by A. suum. Our study indicated that A. suum significantly compromised the effect of Mh vaccination. The impact of reduced vaccine efficacy caused by a common gastrointestinal helminth emphasises the importance of parasite control. More focus should be put into this area of research to outline the practical consequences of this interaction, and to be able to predict, prevent and correct negative interactions.     

Attenuation of Theileria lestoquardi infected cells and immunization of sheep against malignant ovine theileriosis

Malignant ovine theileriosis caused by Theileria lestoquardi is an economically important disease infecting small ruminants in the Sudan. The disease causes massive losses among sheep in many regions of Northern Sudan. The present studies were done to isolate lymphoblastiod cells infected with malignant ovine theileriosis and attenuate them by passage using culture media to develop and produce schizonts candidate vaccine, then test its efficacy and safety by exposing immunized lambs to field challenge in an area endemic with T. lestoquardi. In the present experiments we isolated and established an in vitro culture of T. lestoquardi infected lymphoblast cell line. Long-term culture of T. lestoquardi infected lymphoplastoid cells was shown to result in attenuation of their virulence and lambs inoculated with different doses of such cells at passage 105 exhibited very mild reactions with fever that lasted for 1–5 days and parasitaemia of <0.2%. The experimental lambs immunized with this candidate vaccine were immune and protected when exposed to field challenge in an area endemic of ovine theileriosis, while morbidity and mortality among non-immunized animals reached 76.9% and 46.15%, respectively, and they exhibited the clinical signs of malignant ovine theileriosis that included, high fever, loss of appetite, enlargement of lymph nodes, jaundice, loss of weight and death. The present study demonstrates the efficacy and the safety of this attenuated cell line as a live attenuated candidate vaccine.     

Protection to respiratory challenge of Brucella abortus strain 2308 in the lung

Brucella is amongst the top 5 causes of zoonotic disease worldwide. Infection is through ingestion, inhalation or contact exposure. Brucella is characterized as a class B pathogen by Centers of Disease Control and Prevention (CDC). Currently, there are no efficacious vaccines available in people. Currently available USDA approved vaccines for animals include B. abortus strain RB51 and B. melitensis Rev1. Protection is mediated by a strong innate and CD4 Th1, CD8 Tc1 immune response. If protective vaccines can be developed, disease in people and animals can be controlled. While strain RB51 protects in cattle, and against intraperitoneal challenge in mice, it does not protect against respiratory challenge. Therefore, we assessed the efficacy of strain RB51 combined with different TLR agonists, and O-side chain from LPS, to enhance protection against respiratory challenge with strain 2308. We hypothesized that TLR agonists and O-side chain would enhance protection. Strains RB51 with TLR2 agonist, RB51 with TLR4 agonist and strain 19 provided significant protection in the lung. Protection using strain RB51 with TLR agonists was associated with increased IgG2a and IgG1 in the (bronchoalveolar lavage) BAL and serum, and increased IgA (serum). Splenocytes from strain RB51 with TLR2 vaccinated mice up-regulated antigen specific interferon-gamma and TNF-alpha production. Vaccination and challenge resulted in significant increases in activated dendritic cells (DCs), and increased CD4 and CD8 cells in the BAL. Overall, this study demonstrates the ability of TLR agonists 2 and 4 to up-regulate strain RB51 mediated protection in the lung to respiratory challenge against strain 2308.     

Brucellosis as a cause of acute febrile illness in Egypt

To develop better estimates of brucellosis incidence, we conducted population-based surveillance for acute febrile illness (AFI) in Fayoum governorate (population 2347249), Egypt during two summer periods (2002 and 2003). All hospitals and a representative sample of community healthcare providers were included. AFI patients without obvious etiology were tested for brucellosis by culture and serology. Incidence estimates were calculated adjusting for sampling methodology and study period. Of 4490 AFI patients enrolled, 321 (7%) met the brucellosis case definition. The estimated annual incidence of brucellosis per 100000 population was 64 and 70 in 2002 and 2003, respectively. The median age of brucellosis patients was 26 years and 70% were male; 53% were initially diagnosed as typhoid fever. Close contact with animals and consumption of unpasteurized milk products were associated with brucellosis. The high incidence of brucellosis in Fayoum highlights its public health importance, and the need to implement prevention strategies in humans and animals.     

The polymeric antigen BLSOmp31 confers protection against Brucella ovis infection in rams

We have engineered the polymeric vaccine BLSOmp31 by decorating the highly immunogenic and decameric Brucella lumazine synthase with an exposed loop of the Brucella outer membrane protein Omp31. In the present study, we have immunized different groups of rams with the recombinant chimera rBLSOmp31 in two different adjuvants (Incomplete Freund Adjuvant—IFA and QUIL A) and with the plasmid pCIBLSOmp31 administered either by i.m. injection alone or by using electroporation. In addition, we have used a heterologous prime-boost strategy consisting of repeated pCIBLSOmp31 electroporation priming followed by a single protein boost. Both, chimera rBLSOmp31 in IFA and the prime-boost strategy induced the highest IgG specific antibodies with bacteriolytic activity. While electroporation-enhanced humoral immune responses as compared to pCIBLSOmp31 injection alone, the highest levels of specific IFN-γ and protection against bacterial challenge were achieved with prime-boost (76%) and chimera rBLSOmp31 in IFA (63%). Taken together these results strongly support the usefulness of the chimera BLSOmp31 as a vaccine against Brucella ovis in ovine brucellosis.     

Modulation of inter-vaccination interval to avoid antigenic competition in multivalent footrot (Dichelobacter nodosus) vaccines in sheep

Virulent footrot is a significant disease of sheep in most sheep farming countries; a strain/serogroup of the anaerobic bacterium Dichelobacter nodosus is the essential transmitting agent. Commercial multivalent footrot vaccines containing nine fimbrial serogroups (A through I) of D. nodosus produce relatively low and short term antibody responses due to antigenic competition, in contrast to higher and longer responses provided by monovalent or bivalent vaccines. The latter were important components of successful eradication programs for endemic footrot caused by either one or two serogroups of D. nodosus in Nepal, Bhutan, and several flocks in Australia. However, the presence of up to six serogroups in some Australian flocks and the use of an annual bivalent vaccination regime to progressively eradicate serogroups would require a long term program. In this study we report the results of a sequential vaccination trial testing different time intervals between different bivalent vaccinations. Intervals of 12, 9, 6, 3 and 0 months were tested. The 1st vaccination was with recombinant fimbrial antigens for serogroups A and B while the 2nd vaccination was with D and E. There were no significant differences between the antibody responses for time intervals of 3, 6, 9 and 12 months whereas there was a reduced response when sheep were vaccinated with two bivalent vaccines (four antigens) concurrently, indicating antigenic competition. Therefore an inter-vaccination interval of 3 months can be applied between two different bivalent vaccines without detrimental impact on the humoral immune responses to the various fimbrial antigens of D. nodosus. These results could have wider applications in vaccination against diseases caused by multivalent or multistrain microbes.     

Evaluation of protection in a mouse model after vaccination with Mycobacterium avium subsp. paratuberculois protein cocktails

Whole-cell vaccines successfully reduce signs of clinical disease and fecal shedding of Mycobacterium avium subsp. paratuberculosis (MAP), however, these vaccines have some limitations. The present study was conducted to identify MAP proteins that might be candidates for the development of an improved vaccine. MAP proteins were screened for immunogenicity in naturally infected cattle and selected based upon reactivity in the interferon-γ (IFN-γ) and Western blot assays. Proteins (MAP1087, MAP1204, MAP1272c, and MAP2077c) were arrayed into 4 overlapping cocktails containing 3 proteins each. The efficacy of the proteins within these cocktails as vaccine candidates was evaluated by subcutaneous immunization of mice, followed by challenge with live, virulent MAP. All MAP protein cocktails significantly reduced the recovery of live MAP from the ileum, while cocktails 1 and 3 reduced colonization in the liver. No significant differences were seen in the mesenteric lymph node or spleen, however, cocktail 1 reduced viable MAP in the mesenteric lymph node compared to other treatments. Stimulation of splenocytes upregulated antigen-specific IFN-γ and IL-23 secretion in all treatment groups, regardless of vaccination. Interestingly, IL-4 was moderately downregulated for vaccinates compared to control infected mice. An increase in total CD25 expression was noted for 3 of the 4 vaccinate groups upon stimulation of splenocytes with a whole cell sonicate of MAP, with this effect becoming more significant within CD4CD25+ and CD8CD25+ subpopulations. The present study demonstrated that MAP proteins are useful as vaccine candidates to reduce MAP tissue burden.     

Evaluation of efficacy,biodistribution and safety of antibiotic-free plasmid encoding somatostatin genes delivered by attenuated Salmonella enterica serovar Choleraesuis

We describe here a balanced-lethal system using an Asd⁺ expression plasmid pVGS/2SS-asd encoding two copies of somatostatin (SS) genes carried by Δasd/Δcrp double mutant Salmonella enterica serovar Choleraesuis (named C501). The advantage of this novel system is the use of asd (aspartate-semialdehyde dehydrogenase) gene as selection marker to replace the antibiotic resistance markers, thus eliminating the industrial cultivation and environmental problems. We then evaluated the efficacy, biodistribution and safety of antibiotic-free plasmid delivered by strains C501. Mice orally immunized with C501 (pVGS/2SS-asd) had significantly higher levels of anti-SS total IgG and IgA antibodies than control mice and demonstrated a bias toward Th2-associated responses (IgG1/IgG2a ratio > 1). Safety evaluation indicated that vaccinated mice displayed no abnormal clinical signs and histological changes. Biodistribution result revealed that the GS/2SS message was detected in several examined tissues with the exception of ovary and brain, but was rapidly cleared from the body (approximately 10 days). Furthermore, the risk of integration of plasmid pVGS/2SS-asd into the host cellular genome was considered to be negligible. These results may have important implications for the use of vaccine strain C501 (pVGS/2SS-asd) in domestic animals and prompt new perspectives on the safety of DNA vaccines delivered by attenuated bacteria.