Subcutaneous immunization with a novel immunogenic candidate (urease) confers protection against Brucella abortus and Brucella melitensis infections

Brucellosis is a world prevalent endemic illness that is transmitted from domestic animals to humans. Brucella spp. exploits urease for survival in the harsh conditions of stomach during the gastrointestinal infection. In this study, we examined the immune response and the protection elicited by using recombinant Brucella urease (rUrease) vaccination in BALB/c mice. The urease gene was cloned in pET28a and the resulting recombinant protein was employed as subunit vaccine. Recombinant protein was administered subcutaneously and intraperitoneally. Dosage reduction was observed with subcutaneous (SC) vaccination when compared with intraperitoneal (IP) vaccination. rUrease induced mixed Th1–Th2 immune responses with high titers of specific IgG1 and IgG2a. In lymphocyte proliferation assay, splenocytes from IP and SC‐vaccinated mice displayed a strong recall proliferative response with high amounts of IL‐4, IL‐12 and IFN‐γ production. Vaccinated mice were challenged with virulent Brucella melitensis, B. abortus and B. suis. The SC vaccination route exhibited a higher degree of protection than IP vaccination (p value ≤ 0.05). Altogether, our results indicated that rUrease could be a useful antigen candidate for the development of subunit vaccines against brucellosis.     

Protective Live Oral Brucellosis Vaccines Stimulate Th1 and Th17 Cell Responses

Zoonotic transmission of brucellosis often results from exposure to Brucella-infected livestock, feral animals, or wildlife or frequently via consumption of unpasteurized milk products or raw meat. Since natural infection of humans often occurs by the oral route, mucosal vaccination may offer a means to confer protection for both mucosal and systemic tissues. Significant efforts have focused on developing a live brucellosis vaccine, and deletion of the znuA gene involved in zinc transport has been found to attenuate Brucella abortus. A similar mutation has been adapted for Brucella melitensis and tested to determine whether oral administration of ΔznuA B. melitensis can confer protection against nasal B. melitensis challenge. A single oral vaccination with ΔznuA B. melitensis rapidly cleared from mice within 2 weeks and effectively protected mice upon nasal challenge with wild-type B. melitensis 16M. In 83% of the vaccinated mice, no detectable brucellae were found in their spleens, unlike with phosphate-buffered saline (PBS)-dosed mice, and vaccination also enhanced the clearance of brucellae from the lungs. Moreover, vaccinated gamma interferon-deficient (IFN-γ^−/−) mice also showed protection in both spleens and lungs, albeit protection that was not as effective as in immunocompetent mice. Although IFN-γ, interleukin 17 (IL-17), and IL-22 were stimulated by these live vaccines, only RB51-mediated protection was codependent upon IL-17 in BALB/c mice. These data suggest that oral immunization with the live, attenuated ΔznuA B. melitensis vaccine provides an attractive strategy to protect against inhalational infection with virulent B. melitensis.     

Characteristics of Schistosoma japonicum infection induced IFN‐γ and IL‐4 co‐expressing plasticity Th cells

Schistosoma japonicum infection can induce granulomatous inflammation and cause tissue damage in the mouse liver. The cytokine secretion profile of T helper (Th) cells depends on both the nature of the activating stimulus and the local microenvironment (e.g. cytokines and other soluble factors). In the present study, we found an accumulation of large numbers of IFN‐γ⁺ IL‐4⁺ CD4⁺ T cells in mouse livers. This IFN‐γ⁺ IL‐4⁺ cell population increased from 0·68 ± 0·57% in uninfected mice to 7·05 ± 3·0% by week 4 following infection and to 9·6 ± 5·28% by week 6, before decreasing to 6·3 ± 5·9% by week 8 in CD4 T cells. Moreover, IFN‐γ⁺ IL‐4⁺ Th cells were also found in mouse spleen and mesenteric lymph nodes 6 weeks after infection. The majority of the IFN‐γ⁺ IL‐4⁺ Th cells were thought to be related to a state of immune activation, and some were memory T cells. Moreover, we found that these S. japonicum infection‐induced IFN‐γ⁺ IL‐4⁺ cells could express interleukin‐2 (IL‐2), IL‐9, IL‐17 and high IL‐10 levels at 6 weeks after S. japonicum infection. Taken together, our data suggest the existence of a population of IFN‐γ⁺ IL‐4⁺ plasticity effector/memory Th cells following S. japonicum infection in C57BL/6 mice.     

Effect of P39 Gene Deletion in Live Brucella Vaccine Strains on Residual Virulence and Protective Activity in Mice

The 39-kilodalton protein (P39) has previously been shown to be an immunodominant protein in Brucella infections. P39 gene deletion mutants of vaccine strains Brucella abortus S19 and Brucella melitensis Rev.1 were constructed by gene replacement. This deletion did not significantly modify the residual virulence of both vaccine strains in CD-1 mice. CD-1 mice vaccinated with the parent or mutant strains were protected against a virulent challenge. Mutant vaccine strains devoid of P39 could provide a means for differentiating vaccinated from infected animals.     

Immunogenicity and efficacy of a bivalent DNA vaccine containing LeIF and TSA genes against murine cutaneous leishmaniasis

There is no effective vaccine for the prevention and elimination of leishmaniasis. For this reason, we assessed the protective effects of DNA vaccines containing LeIF, TSA genes alone, or LeIF–TSA fusion against cutaneous leishmaniasis pEGFP‐N1 plasmid (empty vector) and phosphate buffer saline (PBS) were used as control groups. Therefore, cellular and humoral immune responses were evaluated before and after the challenge with Leishmania major. Lesion diameter was also measured 3–12 weeks after challenge. All immunized mice with plasmid DNA encoding Leishmania antigens induced the partial immunity characterized by increased IFN‐γ and IgG2a levels compared with control groups (p < 0.001). Furthermore, the immunized mice showed significant reduction in mean lesion sizes compared with mice in empty vector and PBS groups (p < 0.05). The reduction in lesion diameter was 29.3%, 34.1%, and 46.2% less in groups vaccinated with LeIF, TSA, and LeIF‐TSA, respectively, than in PBS group at 12th week post infection. IFN/IL‐4 and IgG2a/IgG1 ratios indicated that group receiving LeIF–TSA fusion had the highest IFN‐γ and IgG2a levels. In this study, DNA immunization promoted Th1 immune response characterized by higher IFN‐γ and IgG2a levels and also reduction in lesion size. These results showed that a bivalent vaccine containing two distinct antigens may induce more potent immune responses against leishmaniasis.     

Limitations of the BP26 Protein-Based Indirect Enzyme-Linked Immunosorbent Assay for Diagnosis of Brucellosis

Brucellosis is a serious zoonosis that occurs worldwide, and its diagnosis is typically based on the detection of antibodies against Brucella lipopolysaccharide (LPS). However, the specificity of the LPS-based test is compromised by cross-reactivity with Escherichia coli O157:H7 and Yersinia enterocolitica O:9. Also, diagnosis based on the LPS test cannot differentiate between vaccinated and infected individuals. The detection of the 26-kDa cytosoluble protein (BP26) antibody is considered an alternative that circumvents these drawbacks because it is exclusively expressed by infectious Brucella. A BP26-based enzyme-linked immunosorbent assay (ELISA) has been tried for the diagnosis of Brucella-infected animals and humans, but a few results showed that BP26 couldn''t react with all Brucella-positive sera. In order to explore whether different animals could produce antibodies against BP26 after being infected with various Brucella species, we infected sheep, goats, and beef cattle with common virulent reference Brucella species. All sera were collected from the experimental animals and tested using both LPS-based ELISAs and BP26-based ELISAs. The results showed that all Brucella-infected individuals could produce high levels of antibodies against LPS, but only B. melitensis 16M- and B. melitensis M28-infected sheep and B. melitensis 16M- and B. abortus 2308-infected goats could produce antibodies against BP26. Therefore, we concluded that the BP26-based indirect ELISA (i-ELISA) showed both Brucella species and host specificity, which obviously limits its reliability as a substitute for the traditional LPS-based ELISA for the detection of brucellosis.     

Cloning and Sequencing of yajC and secD Homologs of Brucella abortus and Demonstration of Immune Responses to YajC in Mice Vaccinated with B. abortus RB51

To identify Brucella antigens that are potentially involved in stimulating a protective cell-mediated immune response, a gene library of Brucella abortus 2308 was screened for the expression of antigens reacting with immunoglobulin G2a antibodies from BALB/c mice vaccinated with B. abortus RB51. One selected positive clone (clone MCB68) contained an insert of 2.6 kb; nucleotide sequence analysis of this insert revealed two open reading frames (ORFs). The deduced amino acid sequences of the first and second ORFs had significant similarities with the YajC and SecD proteins, respectively, of several bacterial species. Both the YajC and SecD proteins were expressed in Escherichia coli as fusion proteins with maltose binding protein (MBP). In Western blots, sera from mice vaccinated with B. abortus RB51 recognized YajC but not SecD. Further Western blot analysis with purified recombinant YajC protein indicated that mice inoculated with B. abortus 19 or 2308 or B. melitensis RM1 also produced antibodies to YajC. In response to in vitro stimulation with recombinant MBP-YajC fusion protein, splenocytes from mice vaccinated with B. abortus RB51 were able to proliferate and produce gamma interferon but not interleukin-4. This study demonstrates, for the first time, the involvement of YajC protein in an immune response to an infectious agent.     

Influence of Brucella endotoxins on the initiation of antibody-forming spleen cells in mice immunized with sheep red blood cells

Changes in the formation of antibodies to sheep red blood cells (sRBC) in the presence of Brucella extracts was studied in mice whose spleen cells were assayed by the Jerne procedure. Two strains of female mice were employed. Brucella extracts were prepared: (i) by trichloroacetic acid extraction (LPSN), (ii) by phenol extraction (LPS), and (iii) by hot acetic acid hydrolysis (Ps). B. abortus LPSN and B. melitensis LPSN or LPS, administered with sRBC, stimulated the specific response to sRBC, but only at high doses of endotoxins. B. abortus LPSN and B. melitensis LPSN suppressed nonspecific responses against horse red blood cells (hRBC), in contrast to the typical events following administration of Serratia marcescens endotoxin (or endotoxins from other ubiquitous organisms). In CD-1 mice, B. abortus Ps depressed the specific anti-sRBC response. Attempts to presensitize mice with abortus LPSN resulted in a stimulation of the response to sRBC, but pretreatment with B. melitensis LPSN had an inhibitory effect. When injected alone, Brucella endotoxins activated anti-sRBC antibody-forming cells but not anti-hRBC cells. B. abortus Ps was unable to modify the background number of anti-sRBC cells and inhibited the hRBC response. These data suggest (i) that there exists a “common antigen” between Brucella cells and sRBC and (ii) that the so-called primary response to endotoxins from ubiquitous organisms represents a secondary response to already naturally sensitized animals.     

Improved immunogenicity and protective efficacy of a divalent DNA vaccine encoding Brucella L7/L12-truncated Omp31 fusion protein by a DNA priming and protein boosting regimen

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.     

Identification of a new immunogenic candidate conferring protection against Brucella melitensis infection in Mice

Identification of bacterial proteins that contribute to the replication and survival of the engulfed bacteria within phagolysosome is critical in the pathogenesis of intracellular bacteria. Heat shock proteins (HSPs) are molecular chaperones that prevent unwanted protein aggregation and protect the bacteria against cell stress. In order to study the potential of HspA for development of a Brucella subunit vaccine, immunogenicity and protective efficacy of recombinant HspA (rHspA) from Brucella melitensis was evaluated in BALB/c mice. The hspA gene was cloned in pDEST42 and the resulting recombinant protein was used as subunit vaccine. rHspA elicited mixed TH1/TH2 immune responses with higher titers of specific IgG1 than IgG2a. In lymphocyte transformation assay, splenocytes of immunized mice exhibited a strong recall proliferative response with high amounts of IFN-γ, IL-12, IL-10 and IL-6 and very low levels of IL-5 and IL-4 production. The protective effect of rHspA was evaluated by administering rHspA to mice that resulted in a significant reduction in bacterial load and high degree of protection against B. melitensis challenge compared to control mice (p < 0.001). These results suggest that rHspA may be a useful candidate for the development of subunit vaccine against brucellosis.     

Recombinant Mycobacterium bovis BCG is a promising platform to develop vaccines against Trypansoma cruzi infection

Chagas disease, caused by the hemoflagelate parasite Trypanosoma cruzi, is one of the most prevalent endemic parasitoses, affecting 7–8 million people. Due to the complexity of the infection, no vaccines are available at present. The extraordinary adjuvant capacity of bacille Calmette–Guérin (BCG) was explored in this work to develop a vaccine candidate to protect against T. cruzi infection using the recombinant BCG (rBCG) vaccine platform. Three antigens of the parasite corresponding to the N and C terminal fragments of the enzyme trans-sialidase (NT-TS and CT-TS, respectively) and a fragment of the cruzipain enzyme (CZf) were cloned into the vectors pUS997 and pUS2000 and transformed into the BCG Pasteur strain. In vaccinated mice, rBCG expressing NT-TS in pUS2000 plasmid provided the highest protection and the lowest parasitemia after challenging BALB/c mice with a 50% lethal dose of parasites. When mice vaccinated with pUS2000-NT-TS were challenged with a 100% lethal dose of parasite, high levels of protection were also obtained, together with a low degree of cardiac lesions 120 days after infection. In immunized mice with pUS2000-NT-TS/rBCG clone, the proliferation of CD4⁺ cells from splenocytes stimulated with the TS antigen was significant; this stimulation increased interferon (IFN)-γ and interleukin (IL)-17 within CD4⁺ T lymphocytes (LTCD4⁺) cells and IFN-γ and CD107 expression within LTCD8⁺ cells. Therefore, pUS2000-NT-TS/rBCG conferred high levels of protection, which correlated with an immune response orientated towards a T helper type 1 (Th1)/Th17 profile, together with an LTC-specific response, indicating that rBCG is a promising platform to develop vaccines against T. cruzi.     

A vaccine formulated with the major outer membrane protein can protect C3H/HeN,a highly susceptible strain of mice,from a Chlamydia muridarum genital challenge

C3H/HeN female mice were vaccinated with native Chlamydia muridarum major outer membrane protein (MOMP), using Montanide+CpG or Alum+CpG as adjuvants. Negative control groups were immunized with ovalbumin (OVA) and the same adjuvants. As positive control, mice were inoculated intranasally with live Chlamydia. Mice were challenged in the ovarian bursa with 10⁵ C. muridarum inclusion forming units. Six weeks after the genital challenge the animals were caged with male mice and monitored for pregnancy. Mice vaccinated with MOMP+Montanide+CpG developed high levels of C. muridarum‐specific antibodies, with a high IgG2a/IgG1 ratio and neutralizing titres. Animals immunized using Alum+CpG had low antibody levels. Cellular immune responses were significantly higher in mice vaccinated with MOMP and Montanide+CpG, but not with Alum+CpG, when compared with negative controls. Following the genital challenge, only 20% (4/20) of mice vaccinated with MOMP+CpG+Montanide had positive vaginal cultures whereas 100% (9/9) of mice immunized with MOMP+CpG+Alum had positive cultures. Of the positive control animals inoculated with live Chlamydia only 15% (3/20) had positive vaginal cultures. In contrast, 100% (20/20) of mice immunized with OVA+CpG+Montanide, or minimal essential medium, had positive cultures. Following mating, 80% (16/20) of mice vaccinated with MOMP+CpG+Montanide, and 85% (17/20) of animals inoculated intranasally with live C. muridarum carried embryos in both uterine horns. No protection against infertility was observed in mice immunized with MOMP and CpG+Alum or OVA. In conclusion, this is the first time that a subunit vaccine has been shown to elicit a protective immune response in the highly susceptible C3H/HeN strain of mice against an upper genital challenge.     

Type-III interferons and rheumatoid arthritis: Correlation between interferon lambda 1 (interleukin 29) and antimutated citrullinated vimentin antibody levels

Aim: To assess serum type III or lambda (λ) interferons (IFN) levels and its clinical and laboratory associations in rheumatoid arthritis (RA). Methods: A cross-sectional study including 43 patients with RA (86% females; age 45.3?±?10.3 years) and 43 healthy individuals was performed. Clinical data including disease activity, acute-phase reactants, rheumatoid factor and anticyclic citrullinated peptide (anti-CCP) antibodies were collected. Serum IFNλ1, IFNλ2, IFNλ3, CXCL8 and anti-mutated citrullinated vimentin (anti-MCV) antibody levels were measured. Results: Patients with RA had higher IFNλ1 (113.5?±?118.6?pg/mL versus 55.9?±?122.3?pg/mL; p?<?0.0001) and IFNλ2 (245.4?±?327.7?pg/mL versus 5.1?±?11.0?pg/mL; p?=?0.009) levels than controls, but not IFNλ3 levels. Notably, IFNλ1 levels were found to be higher in both patients with active disease (124.9?±?135.9?pg/mL; p?<?0.001) and quiescent disease (99.0?±?93.7?pg/mL; p?<?0.01), while IFNλ2 levels were higher only in patients with active disease (264.0?±?356.1?pg/mL; p?=?0.02). A noteworthy association between serum IFNλ1 levels and anti-MCV antibody titers (Spearman's rho coefficient 0.36, 95% CI 0.36 to 0.61; p?=?0.02) was observed. Conclusion: Serum IFNλ1 and IFNλ2 levels are abnormally elevated in patients with RA and the former are linearly associated with circulating anti-MCV antibody levels. These results may place type-III IFN as an attractive new therapeutic target in RA.     

Some structural and biological properties of Brucella endotoxin

Hot phenol-water extraction of smooth Brucella abortus and B. melitensis cells yielded a toxic fraction which was recovered from the phenol phase (fraction 5). Chemically, fractions 5 from both Brucella species were lipid-carbohydrate-protein-2 keto-3-deoxyoctulosonic acid complexes which were stable to heat and resistant to Pronase digestion. Electron micrographs of the Brucella toxins were morphologically indistinguishable from those of enterobacterial endotoxins. Biologically, Brucella toxins were lethal for mice and immunogenic for rabbits. An intravenous injection of Brucella toxin induced severe leukopenia with subsequent leukocytosis in mice. Cross-tolerance experiments with mice demonstrated that pretreatment with B. abortus toxin lessened the hypoferremia produced by challenge with Escherichia coli endotoxin. Furthermore, fractions 5 from B. abortus and B. melitensis were able to form hybrids with E. coli and Salmonella enteritidis endotoxins and also with each other. Although Brucella toxins possess many structural and biological properties in common with endotoxins from the Enterobacteriaceae, some quantitative differences in their biological potencies were observed. Brucella toxins were relatively innocuous in tests for pyrogenicity in rabbits and lethality for chick embryos. In nonspecific protection tests, Brucella toxin had only 1/75 the potency of E. coli endotoxin in protecting mice against challenge with virulent S. typhi. However, on the basis of the data presented and on the work done previously, we concluded that the heat-stable toxins of B. abortus and B. melitensis were endotoxins.     

CD4+ Foxp3+ regulatory T‐cell number increases in the gastric tissue of C57BL/6 mice infected with Helicobacter pylori

Helicobacter pylori (H. pylori), one of the most common infections, is associated with various clinical outcomes. In addition to inducing inflammation, immunological clearance of the pathogen is often incomplete. Regulatory T cells (Treg cells) have been recently demonstrated to play an important role in H. pylori infection and the final clinical outcome. The aim of this study was to investigate the number and localization of CD4⁺Foxp3⁺ Treg cells in stomachs and spleens of H. pylori‐infected mice. The expression levels of Foxp3 as well as anti‐ and pro‐inflammatory cytokines before and after H. pylori triple eradication therapy were examined. We found that the percentages of CD4⁺Foxp3⁺ Treg cells out of the lamina propria lymphocytes (LPLs) and spleen lymphocytes in the infection group were higher than the PBS negative control group and the treatment group. H. pylori antigen stimulation was associated with an increased number of Treg cells in vitro. Furthermore, compared with the PBS and treatment groups, a higher mRNA expression level of Foxp3 in the gastric tissue was detected in the infection group. IL‐10 and TGF‐β1 contents were increased significantly in the culture supernatant of spleen lymphocyte stimulated with H. pylori antigen. A marked elevation in serum IFN‐γ level was observed in H. pylori‐infected mice. In addition, gastric tissues of the infection group contained more Foxp3⁺ cells. These results indicate that the percentage of CD4⁺Foxp3⁺ Treg cells are increased in H. pylori‐infected mice, suggesting a role of Treg cells in H. pylori‐induced pathologies, even at the early stages of chronic gastritis and gastric tumorigenesis.     

Induction and treatment of anergy in murine leprosy

Leprosy is a disease consisting of a spectrum of clinical, bacteriological, histopathological and immunological manifestations. Tuberculoid leprosy is frequently recognized as the benign polar form of the disease, while lepromatous leprosy is regarded as the malignant form. The different forms of leprosy depend on the genetic and immunological characteristics of the patient and on the characteristics of the leprosy bacillus. The malignant manifestations of lepromatous leprosy result from the mycobacterial‐specific anergy that develops in this form of the disease. Using murine leprosy as a model of anergy in this study, we first induced the development of anergy to Mycobacterium lepraemurium (MLM) in mice and then attempted to reverse it by the administration of dialysable leucocyte extracts (DLE) prepared from healthy (HLT), BCG‐inoculated and MLM‐inoculated mice. Mice inoculated with either MLM or BCG developed a robust cell‐mediated immune response (CMI) that was temporary in the MLM‐inoculated group and long‐lasting in the BCG‐inoculated group. DLE were prepared from the spleens of MLM‐ and BCG‐inoculated mice at the peak of CMI. Independent MLM intradermally‐inoculated groups were treated every other day with HLT‐DLE, BCG‐DLE or MLM‐DLE, and the effect was documented for 98 days. DLE administered at a dose of 1.0 U (1 × 10⁶ splenocytes) did not affect the evolution of leprosy, while DLE given at a dose of 0.1 U showed beneficial effects regardless of the DLE source. The dose but not the specificity of DLE was the determining factor for reversing anergy.     

Enhanced protective efficacy against Mycobacterium tuberculosis afforded by BCG prime‐DNA boost regimen in an early challenge mouse model is associated with increased splenic interleukin‐2‐producing CD4 T‐cell frequency post‐vaccination

The development of improved vaccines and vaccination strategies against Mycobacterium tuberculosis has been hindered by a limited understanding of the immune correlates of anti‐tuberculosis protective immunity. Simple measurement of interferon‐γ frequency or production per se does not provide adequate prediction of immune protection. In this study, we examined the relationship between T‐cell immune responses and protective efficacy conferred by the heterologous vaccination strategy, bacillus Calmette–Guérin (BCG) prime‐Ag85A DNA boost (B/D), in an early challenge mouse model of pulmonary tuberculosis. The results demonstrated that mice vaccinated with the B/D regimen had a significantly reduced bacillary load compared with BCG‐vaccinated mice, and the reduction in colony‐forming units was associated with decreased pathology and lower levels of inflammatory cytokines in the infected lungs. Further analysis of immunogenicity showed that the superior protection afforded by the B/D regimen was associated with significantly increased frequency of splenic interleukin‐2 (IL‐2) ‐producing CD4 T cells and increased IL‐2 production when measured as integrated mean fluorescence intensity post‐vaccination as well. These data suggest that measurement of elevated frequency of IL‐2‐producing CD4 T cells or IL‐2 production in the spleens of vaccinated mice can predict vaccine efficacy, at least in the B/D strategy, and add to the accumulating body of evidence suggesting that BCG prime‐boost strategies may be a useful approach to the control of M. tuberclosis infection.     

Impact of heat shock protein 60KD in combination with outer membrane proteins on immune response against Brucella melitensis

Brucellosis caused by the bacterium Brucella affects various domestic and wild species. The outer membrane proteins 25 and 31 play key roles on stimulation of cell‐mediated immune response against Brucella. GroEL as one of the major Brucella antigens stimulates the immune system and increases intracellular survival of bacteria. In the present study, we assumed injection of GroEL in combination with OMP25 and OMP31 would offer higher immunity levels. So, the impact of GroEL with different concentrations of recombinant outer membrane proteins emulsified in Chitosan Nanoparticles on immune responses was evaluated in mice model. Results showed both univalent (except rGroEL) and divalent immunized groups induced higher IFN‐γ, TNF‐α, and IL‐4 titers in comparison to negative control groups. While GroEL showed negative effect on TNF‐α titer, there were positive increase trends in IFN‐γ in some treatments. Analysis of humoral antibody response revealed both univalent and divalent immunized groups induced higher IgG2a titer than IgG1 titer, indicating strong bent of Th1 immune response. Also, results showed GroEL can have positive impact on lymphocyte proliferation response. Overall, mice immunization using individual OMP25 or OMP31 demonstrated more effective cell‐mediated immunity, although some combinations of rGroEL and rOMP31 vaccines were more efficient than other divalent ones.     

Thermostable Cross-Protective Subunit Vaccine against Brucella Species

A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 10⁵ CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation.