Induction of Immune Response in BALB/c Mice with a DNA Vaccine Encoding Bacterioferritin or P39 of Brucella spp.

In this study, we evaluated the ability of DNA vaccines encoding the bacterioferritin (BFR) or P39 proteins of Brucella spp. to induce cellular and humoral immune responses and to protect BALB/c mice against a challenge with B. abortus 544. We constructed eukaryotic expression vectors called pCIBFR and pCIP39, encoding BFR or P39 antigens, respectively, and we verified that these proteins were produced after transfection of COS-7 cells. PCIBFR or pCIP39 was injected intramuscularly three times, at 3-week intervals. pCIP39 induced higher antibody responses than did the DNA vector encoding BFR. Both vectors elicited a T-cell-proliferative response and also induced a strong gamma interferon production upon restimulation with either the specific antigens or Brucella extract. In this report, we also demonstrate that animals immunized with these plasmids elicited a strong and long-lived memory immune response which persisted at least 3 months after the third vaccination. Furthermore, pCIBFR and pCIP39 induced a typical T-helper 1-dominated immune response in mice, as determined by cytokine or immunoglobulin G isotype analysis. The pCIP39 delivered by intramuscular injection (but not the pCIBFR or control vectors) induced a moderate protection in BALB/c mice challenged with B. abortus 544 compared to that observed in positive control mice vaccinated with S19.     

Vaccination with Brucella abortus rough mutant RB51 protects BALB/c mice against virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis.

Vaccination of BALB/c mice with live Brucella abortus RB51, a stable rough mutant, produced protection against challenge with virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis. Passive-transfer experiments indicated that vaccinated mice were protected against B. abortus 2308 through cell-mediated immunity, against B. ovis PA through humoral immunity, and against B. melitensis 16M through both forms of immunity. Live bacteria were required for the induction of protective cell-mediated immunity; vaccination with whole killed cells of strain RB51 failed to protect mice against B. abortus 2308 despite development of good delayed-type hypersensitivity reactions. Protective antibodies against the heterologous species were generated in vaccinated mice primarily through anamnestic responses following challenge infections. Growth of the antigenically unrelated bacterium Listeria monocytogenes in the spleens of vaccinated mice indicated that nonspecific killing by residual activated macrophages contributed minimally to protection. These results encourage the continued investigation of strain RB51 as an alternative vaccine against heterologous Brucella species. However, its usefulness against B. ovis would be limited if, as suggested here, epitopes critical for protective cell-mediated immunity are not shared between B. abortus and B. ovis.     

Efficacy of Dart or Booster Vaccination with Strain RB51 in Protecting Bison against Experimental Brucella abortus Challenge

This study characterized the efficacy of the Brucella abortus strain RB51 vaccine in bison when delivered by single intramuscular vaccination (hand RB51), by single pneumatic dart delivery (dart RB51), or as two vaccinations approximately 13 months apart (booster RB51) in comparison to control bison. All bison were challenged intraconjunctivally in midgestation with 10⁷ CFU of B. abortus strain 2308 (S2308). Bison were necropsied and sampled within 72 h of abortion or delivery of a live calf. Compared to nonvaccinated bison, bison in the booster RB51 treatment had a reduced (P < 0.05) incidence of abortion, uterine infection, or infection in maternal tissues other than the mammary gland at necropsy. Bison in single-vaccination treatment groups (hand RB51 and dart RB51) did not differ (P > 0.05) from the control group in the incidence of abortion or recovery of S2308 from uterine, mammary, fetal, or maternal tissues at necropsy. Compared to nonvaccinated animals, all RB51 vaccination groups had reduced (P < 0.05) mean colonization or incidence of infection in at least 2 of 4 target tissues, with the booster RB51 group having reduced (P < 0.05) colonization and incidence of infection in all target tissues. Our data suggest that booster vaccination of bison with RB51 enhances protective immunity against Brucella challenge compared to single vaccination with RB51 by hand or by pneumatic dart. Our study also suggests that an initial vaccination of calves followed by booster vaccination as yearlings should be an effective strategy for brucellosis control in bison.     

Aerosol infection of BALB/c mice with Brucella melitensis and Brucella abortus and protective efficacy against aerosol challenge

Brucellosis is a zoonotic disease with a worldwide distribution that can be transmitted via intentional or accidental aerosol exposure. In order to engineer superior vaccine strains against Brucella species for use in animals as well as in humans, the possibility of challenge infection via aerosol needs to be considered to properly evaluate vaccine efficacy. In this study, we assessed the use of an aerosol chamber to infect deep lung tissue of mice to elicit systemic infections with either Brucella abortus or B. melitensis at various doses. The results reveal that B. abortus causes a chronic infection of lung tissue in BALB/c mice and peripheral organs at low doses. In contrast, B. melitensis infection diminishes more rapidly, and higher infectious doses are required to obtain infection rates in animals similar to those of B. abortus. Whether this difference translates to severity of human infection remains to be elucidated. Despite these differences, unmarked deletion mutants BAΔasp24 and BMΔasp24 consistently confer superior protection to mice against homologous and heterologous aerosol challenge infection and should be considered viable candidates as vaccine strains against brucellosis.     

Vaccination with Live Escherichia coli Expressing Brucella abortus Cu/Zn Superoxide Dismutase Protects Mice against Virulent B. abortus

Vaccination of mice with Escherichia coli expressing Brucella Cu/Zn superoxide dismutase (SOD) [E. coli(pBSSOD)] induced a significant level of protection against virulent Brucella abortus challenge, although this level was not as high as the one reached with B. abortus vaccine strain RB51. In addition, vaccination with E. coli(pBSSOD) induced antibodies to Cu/Zn SOD and a strong proliferative response of splenocytes when stimulated in vitro with a thioredoxin-Cu/Zn SOD fusion protein.     

单纯疱疹病毒糖蛋白单克隆抗体对致死性腹腔感染BALB/c幼鼠的被动保护作用

用单纯疤疹病毒(HSV)Ⅰ型SM44株和Ⅱ型SaV株分别腹腔感染BALB/c小鼠,于感染前后不同时间经腹腔注射HSV单克隆抗体(McAb)观察6株McAbs对致死性腹腔感染小鼠的被动保护作用。结果4株McAbs(2C5、1A12、Mad-2、1D10)对HSV-Ⅰ感染的小鼠有保护作用,5株McAbs(2C5、1A12、2A8、1D10、CH-A9)对HSV-Ⅱ感染的小鼠有保护作用。体内保护作用与体外的中和活性相关;并分析了McAbs在中和试验中有无补体参与条件下的保护能力。还证实了HSV糖蛋白在急性感染病程中其型特异性和型共同性抗原决定簇在体内的表达。     

Variation of Brucella abortus 2308 infection in BALB/c mice induced by prior vaccination with salt-extractable periplasmic proteins from Brucella abortus 19.

The study compared the immune and protective responses induced in BALB/c mice vaccinated with six salt-extractable periplasmic protein fractions (Brucella cell surface proteins [BCSP]) of Brucella abortus 19 and later challenge exposed with B. abortus 2308. BCSP70 was precipitated with ammonium sulfate at 70% saturation, and BCSP100 was precipitated with ammonium sulfate at 100% saturation by use of supernatant fluid of BCSP70 that had been precipitated with 70% ammonium sulfate. Four subfractions were separated from BCSP100 by anion-exchange high-performance liquid chromatography (HPLC). Monophosphoryl lipid A (MPL) from Salmonella typhimurium Re mutant strain was used as a potential immune response modifier in some vaccines. Reduced or increased numbers of CFU and increased spleen size in the principal groups of mice relative to that of the nonvaccinated control group were considered protectiveness or virulence (survival) criteria. Results indicated that vaccines prepared from BCSP70 and BCSP100 were moderately protective and immunogenic. The subfractions designated BCSP100-A through BCSP100-D purified by anion-exchange HPLC were not protective when MPL was not used as an immune response modifier. However, two subfractions were associated with significant (P < 0.05) increases in CFU per spleen and splenomegaly in vaccinated mice compared with those in nonvaccinated challenge-exposed mice. MPL enhanced protection or was neutral when used with BCSP70, BCSP100, BCSP100-C, and BCSP100-D. Serologic results of an enzyme-linked immunosorbent assay indicated that MPL modulated the immunoglobulin G responses induced by BCSP70, BCSP100, and subfraction BCSP100-B vaccines only. The overall results suggest that certain proteinaceous periplasmic fractions might serve as virulence or survival factors in B. abortus infections.     

The Brucella abortus S19 ΔvjbR Live Vaccine Candidate Is Safer than S19 and Confers Protection against Wild-Type Challenge in BALB/c Mice When Delivered in a Sustained-Release Vehicle

Brucellosis is an important zoonotic disease of nearly worldwide distribution. Despite the availability of live vaccine strains for bovine (S19, RB51) and small ruminants (Rev-1), these vaccines have several drawbacks, including residual virulence for animals and humans. Safe and efficacious immunization systems are therefore needed to overcome these disadvantages. A vjbR knockout was generated in the S19 vaccine and investigated for its potential use as an improved vaccine candidate. Vaccination with a sustained-release vehicle to enhance vaccination efficacy was evaluated utilizing the live S19 ΔvjbR::Kan in encapsulated alginate microspheres containing a nonimmunogenic eggshell precursor protein of the parasite Fasciola hepatica (vitelline protein B). BALB/c mice were immunized intraperitoneally with either encapsulated or nonencapsulated S19 ΔvjbR::Kan at a dose of 1 × 10⁵ CFU per animal to evaluate immunogenicity, safety, and protective efficacy. Humoral responses postvaccination indicate that the vaccine candidate was able to elicit an anti-Brucella-specific immunoglobulin G response even when the vaccine was administered in an encapsulated format. The safety was revealed by the absence of splenomegaly in mice that were inoculated with the mutant. Finally, a single dose with the encapsulated mutant conferred higher levels of protection compared to the nonencapsulated vaccine. These results suggest that S19 ΔvjbR::Kan is safer than S19, induces protection in mice, and should be considered as a vaccine candidate when administered in a sustained-release manner.     

Evaluation of the Efficacy of Outer Membrane Protein 31 Vaccine Formulations for Protection against Brucella canis in BALB/c Mice

Canine brucellosis is an infectious disease caused by the Gram-negative bacterium Brucella canis. Unlike conventional control programs for other species of the genus Brucella, currently there is no vaccine available against canine brucellosis, and preventive measures are simply diagnosis and isolation of infected dogs. New approaches are therefore needed to develop an effective and safe immunization strategy against this zoonotic pathogen. In this study, BALB/c mice were subcutaneously immunized with the following: (i) the recombinant Brucella Omp31 antigen formulated in different adjuvants (incomplete Freund adjuvant, aluminum hydroxide, Quil A, and Montanide IMS 3012 VGPR), (ii) plasmid pCIOmp31, or (iii) pCIOmp31 plasmid followed by boosting with recombinant Omp31 (rOmp31). The immune response and the protective efficacy against B. canis infection were characterized. The different strategies induced a strong immunoglobulin G (IgG) response. Furthermore, spleen cells from rOmp31-immunized mice produced gamma interferon and interleukin-4 (IL-4) after in vitro stimulation with rOmp31, indicating the induction of a mixed Th1-Th2 response. Recombinant Omp31 administered with different adjuvants as well as the prime-boost strategy conferred protection against B. canis. In conclusion, our results suggest that Omp31 could be a useful candidate for the development of a subcellular vaccine against B. canis infection.     

Vaccination with recombinant L7/L12-truncated Omp31 protein induces protection against Brucella infection in BALB/c mice

Brucellosis is the most common bacterial zoonotic disease worldwide and no vaccine is available for the prevention of human brucellosis. In humans, brucellosis is mostly caused by Brucella melitensis and Brucella abortus. The Outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved in human Brucella pathogens. In the present study, we evaluated the humoral and cellular immune responses induced by a fusion protein designed based on the Truncated form of Omp31 (TOmp31) and L7-L12 antigens. Vaccination of BALB/c mice with the recombinant fusion protein (rL7/L12-TOmp31) provided the significant protection level against B. melitensis and B. abortus challenge. Moreover, rL7/L12-TOmp31 elicited a strong specific IgG response (higher IgG2a titers) and significant IFN-γ/IL2 production and T-cell proliferation was also observed. The T helper1 (Th1) oriented response persisted for 12 weeks after the first immunization. The rL7/L12-TOmp31 could be a new potential antigen candidate for the development of a subunit vaccine against B. melitensis and B. abortus.     

Protection of Mice against Brucellosis by Vaccination with Brucella melitensis WR201(16MΔpurEK)

Human brucellosis can be acquired from infected animal tissues by ingestion, inhalation, or contamination of the conjunctiva or traumatized skin by infected animal products. A vaccine to protect humans from occupational exposure or from zoonotic infection in areas where the disease is endemic would reduce an important cause of morbidity worldwide. Vaccines currently used in animals are unsuitable for human use. We tested a live, attenuated, purine-auxotrophic mutant strain of Brucella melitensis, WR201, for its ability to elicit cellular and humoral immune responses and to protect mice against intranasal challenge with B. melitensis 16M. Mice inoculated intraperitoneally with WR201 made serum antibody to lipopolysaccharide and non-O-polysaccharide antigens. Splenocytes from immunized animals released interleukin-2 (IL-2), gamma interferon, and IL-10 when cultured with Brucella antigens. Immunization led to protection from disseminated infection but had only a slight effect on clearance of the challenge inoculum from the lungs. These studies suggest that WR201 should be further investigated as a vaccine to prevent human brucellosis.     

Evaluation of protection afforded by Brucella abortus and Brucella melitensis unmarked deletion mutants exhibiting different rates of clearance in BALB/c mice

Research for novel Brucella vaccines has focused upon the development of live vaccine strains, which have proven more efficacious than killed or subunit vaccines. In an effort to develop improved vaccines, signature-tagged mutant banks were screened to identify mutants attenuated for survival. Mutants selected from these screens exhibited various degrees of attenuation characterized by the rate of clearance, ranging from a failure to grow in macrophages after 24 h of infection to a failure to persist in the mouse model beyond 8 weeks. Ideal vaccine candidates should be safe to the host, while evoking protective immunity. In the present work, we constructed unmarked deletion mutants of three gene candidates, manBA, virB2, and asp24, in both Brucella abortus and Brucella melitensis. The Deltaasp24 mutants, which persist for extended periods in vivo, are superior to current vaccine strains and to other deletion strains tested in the mouse model against homologous challenge infection after 12, 16, and 20 weeks postvaccination. The Deltaasp24 mutants also display superior protection compared to DeltamanBA and DeltavirB2 mutants against heterologous challenge in mice. From this study, a direct association between protection against infection and cytokine response was not apparent between all vaccine groups and, therefore, correlates of protective immunity will need to be considered further. A distinct correlation between persistence of the vaccine strain and protection against infection was corroborated.     

HBV和HIV的复合型DNA免疫可诱发BALB/c小鼠的细胞免疫应答

本研究是通过细胞免疫检测来研究含有两种不同病毒基因的复合型DNA免疫的效果 ,同时和单一病毒基因的DNA免疫作比较。在本实验中 ,将HBV的S基因和 (或 )HIV 1的gp12 0基因插入到真核表达载体pcDNA3中 ,得到能表达HBsAg和gp12 0融合蛋白的重组体pcDNA S 12 0 ,和能表达HBsAg的重组体pcDNA S。在实验中 ,将重组质粒DNA直接注射到BALB/c小鼠股四头肌内 ,按每只小鼠 10 0 μg/ 10 0 μl的剂量经 3次免疫后 ,在T细胞增殖实验中 ,用HBsAg蛋白刺激体外培养的被免疫小鼠T细胞后 ,出现了明显的T细胞增殖。采用51Cr释放法检测特异性CTL杀伤作用时 ,发现致敏的CTL对HIVgp12 0多肽孵育后的靶细胞P815有明显的杀伤作用。     

Identification and Characterization of a 14-Kilodalton Brucella abortus Protein Reactive with Antibodies from Naturally and Experimentally Infected Hosts and T Lymphocytes from Experimentally Infected BALB/c Mice

A low-molecular-weight recombinant Brucella abortus protein reactive with antibodies from a variety of naturally and experimentally infected hosts and T lymphocytes from experimentally infected mice was identified and given the designation BA14K. The gene encoding BA14K was cloned and characterized, and the predicted amino acid sequence of this immunoreactive protein showed no significant homology with previously described proteins. Sequences homologous to the cloned fragment encoding BA14K were identified by Southern blot analysis of genomic DNAs from representatives of all of the currently recognized Brucella species. Studies employing BA14K should contribute to our efforts to better understand the antigenic specificity of protective immunity to brucellosis.     

Alteration of protective and serologic responses in BALB/c mice vaccinated with chemically modified versus nonmodified proteins of Brucella abortus 19.

A study was conducted to determine whether the covalent chemical modification of Brucella abortus 19 salt-extractable proteins (BCSP) and BCSP derivatives would modulate the immune responses in BALB/c mice. Salt-extractable proteins BCSP 0-70 and BCSP 70-100 were modified with acetoacetic anhydride, and recombinant proteins rBCSP20 (20 kDa), rBCSP31 (31 kDa), and rBCSP45 (45 kDa) were modified with succinic and dodecanoyl anhydrides. Four weeks after mice were vaccinated with the different preparations, principal and control mice were challenge exposed with a virulent culture of B. abortus 2308, and mice were necropsied 2 weeks later. Serum samples were obtained immediately before mice were challenge exposed and at necropsy. Sera were tested for specific immunoglobulin M (IgM) and G (IgG) antibodies by using an enzyme-linked immunosorbent assay. Acylation decreased the immune responses (increased IgG antibodies and reduced spleen CFU and splenomegaly) induced by both BCSP 0-70 and BCSP 70-100. Modification of the recombinant proteins by dodecanoyl and succinic anhydrides had no effect on the protection induced; however, the IgG serologic responses to the homologous and heterologous proteins were altered. Monophosphoryl lipid A markedly enhanced the immunogenicity of BCSP 0-70.     

Maternal Vaccination with a Fimbrial Tip Adhesin and Passive Protection of Neonatal Mice against Lethal Human Enterotoxigenic Escherichia coli Challenge

Globally, enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood and travelers'' diarrhea, for which an effective vaccine is needed. Prevalent intestinal colonization factors (CFs) such as CFA/I fimbriae and heat-labile enterotoxin (LT) are important virulence factors and protective antigens. We tested the hypothesis that donor strand-complemented CfaE (dscCfaE), a stabilized form of the CFA/I fimbrial tip adhesin, is a protective antigen, using a lethal neonatal mouse ETEC challenge model and passive dam vaccination. For CFA/I-ETEC strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407, which has been extensively studied in volunteers, an inoculum of 2 × 10⁷ bacteria resulted in 50% lethal doses (LD₅₀) in neonatal DBA/2 mice. Vaccination of female DBA/2 mice with CFA/I fimbriae or dscCfaE, each given with a genetically attenuated LT adjuvant (LTK63) by intranasal or orogastric delivery, induced high antigen-specific serum IgG and fecal IgA titers and detectable milk IgA responses. Neonates born to and suckled by dams antenatally vaccinated with each of these four regimens showed 78 to 93% survival after a 20× LD₅₀ challenge with {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407, compared to 100% mortality in pups from dams vaccinated with sham vaccine or LTK63 only. Crossover experiments showed that high pup survival rates after ETEC challenge were associated with suckling but not birthing from vaccinated dams, suggesting that vaccine-specific milk antibodies are protective. In corroboration, preincubation of the ETEC inoculum with antiadhesin and antifimbrial bovine colostral antibodies conferred a dose-dependent increase in pup survival after challenge. These findings indicate that the dscCfaE fimbrial tip adhesin serves as a protective passive vaccine antigen in this small animal model and merits further evaluation.     

日本血吸虫TCTP编码基因DNA免疫的效果评价

目的评价pEGFP—TCTP真核重组质粒联合免疫佐剂CpG诱导小鼠保护性免疫的效果。方法构建日本血吸虫TCTP真核重组质粒．将pEGFP—Tc口注射入BABL／c小鼠，采用ELISA法检测免疫小鼠血清中IFN-γ水平；尾蚴攻击感染后45d处死小鼠，收集成虫和虫卵，计算减虫率和减卵率；小鼠肝脏送病理切片，计算虫卵肉芽肿的数量。结果TCTP DNA免疫保护性效果显示：实验组与阴性对照组相比，显示出一定的保护性效果．TCTP组的减虫率和减卵率分别为43．91％和53．48％．与GST组效果相似。DNA免疫后及感染后期，TCTP组与PBS对照组血清IFN-γ水平有显著性差异（P〈0．05）；攻击感染前后组间比较发现，GST组和GST＋CpG组有显著性差异（P〈0．05），提示CpG显著提高了DNA免疫的效果。结论SjTCTP DNA免疫能诱导较明显的保护性Thl免疫应答。CpG基序可以有效的诱导保护性Th1免疫应答，是一种有效的DNA免疫佐剂。     

Protection of Mice against Plasmodium yoelii Sporozoite Challenge with P. yoelii Merozoite Surface Protein 1 DNA Vaccines

Immunization of mice with DNA vaccines encoding the full-length form and C and N termini of Plasmodium yoelii merozoite surface protein 1 provided partial protection against sporozoite challenge and resulted in boosting of antibody titers after challenge. In C57BL/6 mice, two DNA vaccines provided protection comparable to that of recombinant protein consisting of the C terminus in Freund’s adjuvant.