ribosomal-protein-l7-l12 and Brucellosis

The present study employs the Brucella abortus L7/L12 antigen in a Salmonella secretion platform and investigates its ability to induce protective immune responses against wild type challenge in BALB/c mice. The highly conserved L7/L12 open reading frame was PCR amplified from B. abortus and cloned into a prokaryotic expression vector, pJHL65, directly under the beta-lactamase secretory signal. The plasmid constructs pJHL65::L7/L12 was then transformed into an attenuated Salmonella Typhimurium strain, JOL1800 (∆lon, ∆cpxR, ∆asd, and ∆rfaL), and protein secretion was verified by Western blot. Three mice groups were inoculated with either phosphate-buffered saline (PBS), vector-only control, or the vaccine strain secreting L7/L12 antigen. Assessment of humoral and cell-mediated immune responses revealed successful elicitation of Brucella antigen-specific Th1 and Th2 immune responses that were significantly higher than PBS and vector control groups. The immune responses were confirmed by splenocyte proliferation assay, flow cytometry analysis for CD4+ and CD8+ markers, and RT-PCR based cytokine profiling upon restimulation with L7/L12 purified antigen. Results indicate that immunization with Salmonella secreting L7/L12 antigen demonstrated significant enhancement of cell-mediated immune (CMI) responses in immunized mice. The overall effectiveness of the immunization was evaluated by challenging with virulent B. abortus that revealed significant reduction in Brucella colonization in spleen and liver tissues in Salmonella L7/L12 immunized mice. Delivery of Brucella protective antigen L7/L12 using the Salmonella secretion system can effectively accomplish immunogenic advantages of both Salmonella and L7/L12 to derive robust CMI responses and induce humoral immunity to protect against Brucella infection in the mouse model.

Topics: Animals; Antigens, Bacterial; Brucella abortus; Brucella Vaccine; Brucellosis; Female; Immunity, Cellular; Immunity, Humoral; Mice; Mice, Inbred BALB C; Ribosomal Proteins; Salmonella typhimurium; Specific Pathogen-Free Organisms; Vaccines, Attenuated

Brucella abortus is the etiologic agent of Brucellosis, a zoonotic infection affecting a wide range of animals. It is a highly infectious disease of pandemic potential reporting over 500,000 new human cases annually. Till date, there is no reported vaccine for humans and the available animal vaccines are unsafe, therefore a safe and effective subunit vaccine is highly sought for. In this study, we have evaluated rL7/L12 protein encapsulated in microparticles of PLGA (85:15), a biocompatible and biodegradable polymer approved by FDA for human use. In this work, BALB/c mice have been immunized with rL7/L12 entrapped in microparticles in a prime-boost regimen. Further, evaluation of the immunogenicity of the formulation showed that the IgG antibody titre reached a maxima of 2.2×10(5) (p value 0.0001 v/s control) after the injection of the booster dose. A mixed IgG isotype profile (IgG1/IgG2a) indicated the stimulation of both the cellular as well as humoral immunity which has increased parallely and gradually since the first immunization. High levels of IFN-γ, 815±55pg/ml were recorded depicting an optimal elicitation of the cellular wing of immunity leading to clearance of splenic bacteria upto 1.69 log units.

Topics: Animals; Antibodies, Bacterial; Brucella abortus; Brucella Vaccine; Brucellosis; Enzyme-Linked Immunospot Assay; Female; Humans; Immunity, Cellular; Interferon-gamma; Lactic Acid; Mice, Inbred BALB C; Microspheres; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Recombinant Proteins; Ribosomal Proteins; Spleen; Vaccines, Synthetic

Brucellosis is one of the most common zoonotic diseases caused by species of Brucella. At present, there is no commercially available vaccine for the human brucellosis. Brucella melitensis and Brucella abortus are the main causes of human brucellosis, worldwide. The outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved among human Brucella pathogens. The purpose of the current study was to evaluate and compare the immunogenicity and protective efficacy of the L7/L12-TOmp31 construct administered as DNA/DNA and DNA/Pro vaccine regimens. Vaccination of BALB/c mice with the DNA/Pro regimen provided more protection levels against B. melitenisis and B. abortus challenge than did the DNA/DNA regimen. IgG1 and IgG2a titers were higher in the sera from DNA/Pro-immunized mice than in those from mice immunized with DNA alone. Moreover, splenocytes from DNA/Pro-immunized mice produced significantly higher levels of IFN-γ than did those from mice given DNA alone. The pcDNA-L7/L12-TOmp31 priming followed by rL7/L12-TOmp31 boosting led to improved protection against B. abortus or B. melitensis infection.

Topics: Animals; Antibodies, Bacterial; Bacterial Outer Membrane Proteins; Brucella abortus; Brucella melitensis; Brucella Vaccine; Brucellosis; DNA, Bacterial; Female; Humans; Immunity, Cellular; Immunity, Humoral; Immunization, Secondary; Immunoglobulin G; Interferon-gamma; Mice; Mice, Inbred BALB C; Plasmids; Recombinant Fusion Proteins; Ribosomal Proteins; Spleen; Vaccines, DNA

There is a compelling need for the development of suitable adjuvants for human use to enhance the efficacy of the upcoming vaccines for the prevention of life threatening infections. In the current study, we have tried to explore the immunogenic potential of nanoparticles (NPs) made of PLGA (poly lactic-co-glycolic acid), a biodegradable and biocompatible polymer approved by FDA for human use after entrapping rL7/L12 protein, an immunodominant antigen of Brucella. Adjuvant properties were exhibited by the formulation as it elicited high IgG antibody titers just after first immunization which increased significantly after the booster administration. A good elicitation of the Th1 cytokines especially IFN-γ was recorded. Amongst the IgG antibody subclasses, IgG1 remained the predominant subclass to be elicited in mice serum after immunization; however IgG1/2a ratio showed a mixed profile of Th1/Th2 response. Lymphocyte proliferation assay as a marker of amplification in cellular immunity demonstrated that the splenocytes of the immunized mice had a high proliferation index with reference to the control, revealing that L7/L12 entrapping PLGA nanoparticles are potent inducer of inflammatory cell response indispensable to combat Brucella infection. Enumeration of splenic CFU after 14 days of infection with Brucella abortus 544 showed a significant reduction in log CFU of splenic bacteria in the vaccinated mice as compared to the control group. Therefore it is evident that PLGA nano formulations delivering the entrapped vaccine candidate in mice elicit specific humoral as well as cellular responses specific to the entrapped Brucella antigen. So there is much promise in this approach and this work by highlighting the adjuvant properties of the PLGA nanospheres will accelerate the development of improved vaccines safe for human as well as veterinary use.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Antigens, Bacterial; Brucella abortus; Brucella Vaccine; Brucellosis; Colony Count, Microbial; Drug Delivery Systems; Escherichia coli; Female; Gene Expression; Immunity, Cellular; Immunity, Humoral; Immunization, Secondary; Immunoglobulin G; Lactic Acid; Mice; Nanoparticles; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Recombinant Proteins; Ribosomal Proteins; Spleen

We generated novel, effective candidate vaccine against Brucella abortus based on recombinant influenza viruses expressing the Brucella ribosomal protein L7/L12 or outer membrane protein (Omp)-16 from the NS1 open reading frame. The main purpose of this work was to evaluate the safety, immunogenicity and protectiveness of vaccine candidate in laboratory animals.. Four recombinant influenza A viral constructs of the subtypes Н5N1 or H1N1 expressing the Brucella proteins L7/L12 or Omp16 were obtained by a reverse genetics method: Flu-NS1-124-L7/L12-H5N1, Flu-NS1-124-Omp16-H5N1, Flu-NS1-124-L7/L12-H1N1 and Flu-NS1-124-Omp16-H1N1. Despite of substantial modification of NS1 gene, all constructs replicated well and were retain their Brucella inserts over five passages in embryonated chicken eggs (CE). Administration of the mono- or bivalent vaccine formulation via prime-boost intranasal (i.n.), conjunctival (c.) or subcutaneous (s.c.) immunization was safe in mice; no deaths, body weight loss or pathomorphological changes were observed over 56 days. Moreover, guinea pigs vaccinated i.n. with vaccine vectors did not shed the vaccine viruses through their upper respiratory tract after the prime and booster vaccination. These findings confirmed the replication-deficient phenotype of viral vectors. The highest antibody response to Brucella antigen was obtained with constructs expressing L7/L12 (ELISA, GMT 242.5-735.0); whereas the highest T-cell immune response- with construct expressing Omp16 (ELISPOT, 337 ± 52-651 ± 45 spots/4×105cells), which was comparable (P > 0.05) to the response induced by the commercial vaccine B. abortus 19. Interestingly, c. immunization appeared to be optimal for eliciting T-cell immune response. In guinea pigs, the highest protective efficacy after challenge with B. abortus 544 was achieved with Omp16 expressing constructs in both monovalent or bivalent vaccine formulations; protective efficacy was comparable to those induced by a commercial live B. abortus 19 vaccine.. Thus, influenza vectors expressing Brucella protective antigens can be developed as novel influenza vectored vaccine against B. abortus infection.

Topics: Animals; Antibodies, Bacterial; Antigens, Bacterial; Bacterial Outer Membrane Proteins; Brucella abortus; Brucella Vaccine; Brucellosis; Disease Models, Animal; Drug Carriers; Genetic Vectors; Genomic Instability; Guinea Pigs; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H5N1 Subtype; Mice; Ribosomal Proteins; Survival Analysis; T-Lymphocytes; Vaccination; Vaccines, Synthetic; Virus Replication

Because of high morbidity of the brucellosis in humans and the potential use of the microorganism as an agent of biologic warfare, protection of effective vaccines and specific diagnostic reagents become necessary to eradicate brucellosis.. In this study we aimed to investigate the cytokine responses and changes in peripheral blood lymphocyte subgroups of acute brucellosis patients in response to L7/L12 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) recombinant proteins derived from Brucella abortus.. levels of IFN-γ, IL-4 and IL-10 secreted from PBMCs of 25 acute brucellosis patients and 15 healthy controls, stimulated with Phytohemagglutinin (PHA), L7/L12 or GAPDH were measured by ELISA. Furthermore alterations in lymphocyte subgroups in response to these Brucella antigens were determined by flow cytometry.. Extracellular IFN-γ levels were found to be elevated after stimulation with L7/L12 in patients with acute brucellosis, whereas no significant changes were found in IL-4 and IL-10 levels. Similar data was also obtained with GAPDH, but the stimulation of IFN-γ production was not observed in all patients and was not as strong as that observed for L7/L12. Moreover, when the distribution of lymphocytes subgroups (CD3+, CD3+ CD4+, CD3+ CD8+, CD4+ CD25+, CD3+ CD69+ and CD3+ CD152+) was evaluated, it was found that the stimulation with L7/L12 and GAPDH only led to an increase in the percentage of CD3+ CD69+ lymphocytes.. These data indicate that Brucella abortus L7/L12 or GAPDH induce a Th1 type immune response in acute brucellosis patients. Additionally, these recombinant proteins, especially L7/L12, may be used in new vaccine preparations and diagnostic tests.

Topics: Adult; Brucella abortus; Brucellosis; Case-Control Studies; Cytokines; Female; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); Humans; Leukocytes, Mononuclear; Lymphocyte Subsets; Male; Middle Aged; Recombinant Proteins; Ribosomal Proteins; Th1 Cells

The immunogenic Brucella abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of subunit vaccines against brucellosis.. This study was aimed to evaluate the protection of recombinant Human Serum Albumin (HAS)-L7/L12 fusion protein in Balb/c mice.. The amplified L7/L12 gene was cloned in pYHSA5 vector, pYHSA5-L7/L12 construct was transformed in Saccharomyces cerevisiae and the expressed protein from supernatant was purified by affinity chromatography. Balb/c mice were immunized in five groups by tHSA-L7/L12 fusion protein (group 1), Brucella abortus S19 (group 2), HSA (group 3), recombinant L7/L12 (group 4), PBS (group 5). ELISA to detect antibody production, LTT test to assess antigen specific lymphocyte response were conducted prior to virulent B. abortus strain 544 challenge two weeks after the last injection. Bacterial counts from spleens of immunized mice were done four weeks after challenge.. In ELISA tests, the specific antibodies exhibited a dominance of immunoglobulin IgG1 over IgG2a. In addition, the tHSA-L7/L12 fusion protein and L7/L12 elicited a strong T-cell proliferative response upon restimulation in vitro with recombinant tHSA-L7/L12 and L7/L12, suggesting the induction of a cellular immunity response in vivo. However, there was no significant difference in proliferative response of L7/L12 and tHSA-L7/L12 fusion protein (p>0.05). The L7/L12 and tHSA-L7/L12 fusion protein vaccines could also induce significant protection against challenge with the virulent strain B. abortus 544 in Balb/c mice (p< or =0.05).. The tHSA-L7/L12 fusion protein, similar to L7/L12 has the ability to induce antigen specific lymphocyte proliferation, stimulate humoral immunity and engender protection.

Topics: Animals; Antibody Formation; Bacterial Proteins; Brucella abortus; Brucellosis; Female; Genetic Vectors; Humans; Immunity, Cellular; Immunoglobulin G; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Models, Animal; Recombinant Fusion Proteins; Ribosomal Proteins; Saccharomyces cerevisiae; Serum Albumin; Spleen; T-Lymphocytes; Vaccination

Brucella abortus, a facultative intracellular pathogen, is of tremendous zoonotic importance because of its ability to induce spontaneous abortion in cattle and other livestock. It is also known to cause persistent undulant fever, endocarditis, arthritis, osteomyelitis and meningitis in humans. The available vaccines against this dreadful infection suffer from limitations like short-term immunity, increased risk of hypersensitivity and low prophylactic index in the recipients. In the present study, we have demonstrated that liposomal form of a recombinant ribosomal L7/L12 protein, a B-T cell antigen of B. abortus, activates strong immune response in the host. In contrast, free antigen generates moderate immune response in the immunised animals. The liposomisation of rL7/L12 protein causes tremendous increase in cell-mediated immune response in terms of delayed type hypersensitivity, T-cell proliferation and up-regulation in type I cytokine expression, etc. Moreover, the liposome encapsulated antigen elicited stronger humoral immune response as compared to standard vaccine (S-19) or IFA-L7/L12 combination in the immunised animals. The effectiveness of liposome-based vaccine was also substantiated by better systemic clearance of bacterial load after challenging the animals with B. abortus 544 pathogen. The results of the present study suggest the potential of liposome-based rL7/L12 antigen as prospective and efficient candidate vaccine capable of eliciting both cell mediated as well as humoral immune responses against experimental murine brucellosis.

Topics: Animals; Antibodies, Bacterial; Brucella abortus; Brucella Vaccine; Brucellosis; CD4-Positive T-Lymphocytes; Female; Immunization; Interferon-gamma; Liposomes; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Recombinant Proteins; Ribosomal Proteins

The Brucella abortus L7/L12 gene encoding ribosomal protein L7/L12 and the Listeria monocytogenes partial hly gene encoding the protective region of the hemolysin (partial listeriolysin, pLLO) were cloned into vaccinia virus by homologous recombination to produce recombinants WRL7/L12 and WRpLLO, respectively. The ability of these recombinants to induce humoral, cell mediated and protective immune response in mice was assessed. Although mice inoculated with WRL7/L12 recombinant produced antibodies specific to vaccinia virus and L7/L12 antigens, they were not protected against a virulent challenge with B. abortus 2308 strain. In contrast, mice inoculated with WRpLLO were protected against a challenge with virulent L. monocytogenes. Stimulation with purified fusion listeriolysin protein (MBP-LLO), but not with unrelated control protein (MBP), induced splenocytes from WRpLLO-inoculated mice to secrete significantly higher amounts of IFN-gamma than saline inoculated mice. Mice inoculated with either WRpLLO or WRL7/L12 recombinants produced predominantly IgG2a isotype antibody responses, indicative of a Th1 type of immune response. The protective potential of the WRpLLO recombinant correlated with the level of IFN-gamma produced in these mice.

Topics: Animals; Antibodies, Bacterial; Bacterial Toxins; Bacterial Vaccines; Brucella abortus; Brucellosis; Heat-Shock Proteins; Hemolysin Proteins; Immunization; Interferon-gamma; Listeria monocytogenes; Listeriosis; Mice; Mice, Inbred BALB C; Recombinant Proteins; Ribosomal Proteins; Vaccines, Synthetic; Vaccinia virus

The Brucella abortus ribosomal protein L7/L12 is an immunodominant antigen and an interesting candidate for the development of oral live vaccines against brucellosis. Here, a recombinant Lactococcus lactis strain producing L7/L12 under the control of nisin inducible promoter was orally administered to BALB/c mice. Significant levels of anti-L7/L12 specific IgA detected in feces revealed an induced local humoral immune response. However, serum analysis did not reveal any anti-L7/L12 antibodies suggesting the absence of a systemic response. Nevertheless, the vaccinated mice showed a partial protective immunity against B. abortus virulent strain (S2308) challenged by intraperitoneal inoculation.

Topics: Administration, Oral; Animals; Antigens, Bacterial; Brucella abortus; Brucella Vaccine; Brucellosis; Enzyme-Linked Immunosorbent Assay; Lactococcus lactis; Male; Mice; Mice, Inbred BALB C; Ribosomal Proteins

Brucella abortus is a facultative intracellular gram-negative bacterial pathogen that infects humans and animals by entry mainly through the digestive tract. B. abortus causes abortion in pregnant cattle and undulant fever in humans. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of oral live vaccines against brucellosis, using food-grade lactic acid bacteria (LAB) as a carrier. The L7/L12 gene was expressed in Lactococcus lactis, the model LAB, under the nisin-inducible promoter. Using different signals, L7/L12 was produced in cytoplasmic, cell-wall-anchored, and secreted forms. Cytoplasmic production of L7/L12 gave a low yield, estimated at 0.5 mg/liter. Interestingly, a secretable form of this normally cytoplasmic protein via fusion with a signal peptide resulted in increased yield of L7/L12 to 3 mg/liter; secretion efficiency (SE) was 35%. A fusion between the mature moiety of the staphylococcal nuclease (Nuc) and L7/L12 further increased yield to 8 mg/liter. Fusion with a synthetic propeptide (LEISSTCDA) previously described as an enhancer for heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895-1903, 1998) raised the yield to 8 mg/liter and SE to 50%. A surface-anchored L7/L12 form in L. lactis was obtained by fusing the cell wall anchor of Streptococcus pyogenes M6 protein to the C-terminal end of L7/L12. The fusions described allow the production and targeting of L7/L12 in three different locations in L. lactis. This is the first example of a B. abortus antigen produced in a food-grade bacterium and opens new perspectives for alternative vaccine strategies against brucellosis.

Topics: Animals; Antigens, Bacterial; Bacterial Proteins; Bacterial Vaccines; Brucella abortus; Brucellosis; Cattle; Cell Wall; Cytoplasm; Humans; Lactococcus lactis; Micrococcal Nuclease; Protein Sorting Signals; Recombinant Fusion Proteins; Ribosomal Proteins

Nucleic acid vaccines provide an exciting approach for antigen presentation to the immune system. As a test of this new methodology, the immune response to the in vivo-expressed Brucella abortus ribosomal L7/12 gene in the muscle cells of mice was examined. To accomplish this goal the eukaryotic expression systems pcDNA3 and p6 were used. Single intramuscular injection of the L7/L12 gene driven by the human cytomegalovirus (CMV) promoter (pcDNA3) or bovine MHC 1 promoter (p6) resulted in intracellular expression of the B. abortus L7/L12 immunodominant protein encoded by this gene. This application facilitated directed antigen presentation to the immune system and established specific antibody and T-cell responses compared with vector only (pcDNA3) negative controls and B. abortus S19 injected positive controls. Although pcDNA3-encoded L7/L12 gene-inoculated mice possessed significant protection, p6-L7/L12 did not engender significant protection against B. abortus S2308 infection compared to positive control mice. These data suggest a promising antigen-specific response, and L7/L12 nucleic acid vaccination may be an initial step in the development of genetically engineered candidate vaccines against brucellosis. This study for the first time focuses on DNA immunization of a gene from B. abortus.

Topics: Animals; Antibodies, Bacterial; Antibody Formation; Antibody Specificity; Brucella abortus; Brucella Vaccine; Brucellosis; Cattle; DNA, Ribosomal; Escherichia coli; Female; Genes, Bacterial; Genetic Vectors; Humans; Lymphocyte Activation; Male; Mice; Mice, Inbred BALB C; Ribosomal Proteins; T-Lymphocytes; Vaccines, DNA

BALB/c mice were immunized with the recombinant Brucella abortus L7/L12 ribosomal protein fused to maltose binding protein (MBP). Vaccinated animals mounted a specific immune response to the recombinant fusion protein as demonstrated by immunoblot analyses. Additionally, B. abortus L7/L12 ribosomal protein conferred a significant degree of protection when compared to mice vaccinated with adjuvant alone, adjuvant plus MBP or B. abortus. These results indicate that a recombinant B. abortus protein, previously identified as T-cell-reactive, engendered protective immunity to mice against brucellosis.

Topics: Animals; Antibodies, Bacterial; Bacterial Vaccines; Brucella abortus; Brucellosis; Female; Immunity, Cellular; Mice; Mice, Inbred BALB C; Ribosomal Proteins; Vaccines, Synthetic

A delayed-type hypersensitivity (DTH) reaction in the course of brucellosis in humans and animals can be revealed by the brucellin INRA (Brucellergen) skin test. Brucellergen is composed of more than 20 proteins of different molecular weights. A 12-kDa protein eliciting DTH in Brucella melitensis Rev1-sensitized guinea pigs was found to be a significant component for the allergenic properties of Brucellergen. Sequencing of the gene encoding this protein identified it as the L7/L12 ribosomal protein. The L7/L12 gene of B. melitensis was amplified by PCR and subcloned in the Escherichia coli pQE30 plasmid. The resulting recombinant protein did not produce a DTH reaction in sensitized animals. It was used to raise specific antibodies in a rabbit. Affinity chromatography with these antibodies was used to isolate a single protein from Brucellergen and from B. melitensis cytosol preparations which produced a DTH reaction in guinea pigs sensitized with B. melitensis Rev1. N-terminal amino acid sequencing of the protein confirmed that it was the L7/L12 ribosomal protein. This is the first complete report on the involvement of a defined bacterial ribosomal protein in the DTH response of animals infected with intracellularly multiplying bacteria.

Topics: Allergens; Animals; Antigens, Bacterial; Base Sequence; Brucella melitensis; Brucellosis; Escherichia coli Proteins; Genes, Bacterial; Guinea Pigs; Hypersensitivity, Delayed; Male; Molecular Sequence Data; Ribosomal Proteins; Skin Tests