A DNA vaccine encoding Cu,Zn superoxide dismutase of Brucella abortus induces protective immunity in BALB/c mice

This study was conducted to evaluate the immunogenicity and protective efficacy of a DNA vaccine encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). Intramuscular injection of plasmid DNA carrying the SOD gene (pcDNA-SOD) into BALB/c mice elicited both humoral and cellular immune responses. Animals injected with pcDNA-SOD developed SOD-specific antibodies which exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. In addition, the DNA vaccine elicited a T-cell-proliferative response and also induced the production of gamma interferon, but not interleukin-10 (IL-10) or IL-4, upon restimulation with either recombinant SOD or crude Brucella protein, suggesting the induction of a typical T-helper-1-dominated immune response in mice. The pcDNA-SOD (but not the control vector) induced a strong, significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308; the level of protection was similar to the one induced by B. abortus vaccine strain RB51. Altogether, these data suggest that pcDNA-SOD is a good candidate for use in future studies of vaccination against brucellosis.     

A DNA vaccine encoding lumazine synthase from Brucella abortus induces protective immunity in BALB/c mice

This study was conducted to evaluate the immunogenicity of the Brucella abortus lumazine synthase (BLS) gene cloned into the pcDNA3 plasmid, which is driven by the cytomegalovirus promoter. Injection of plasmid DNA carrying the BLS gene (pcDNA-BLS) into BALB/c mice elicited both humoral and cellular immune responses. Antibodies to the encoded BLS included immunoglobulin G1 (IgG1) IgG2a, IgG2b, IgG3, and IgM isotypes. Animals injected with pcDNA-BLS exhibited a dominance of IgG2a over IgG1. In addition, spleen cells from vaccinated animals produced interleukin-2 and gamma interferon but not IL-10 or IL-4 after in vitro stimulation with recombinant BLS (rBLS), suggesting the induction of a Th1 response. Protection was evaluated by comparing the levels of infection in the spleens of vaccinated mice challenged with B. abortus 544. Immunization with pcDNA-BLS- reduced the bacterial burden relative to those in the control groups. Mice immunized with rBLS produced a significant humoral response but did not show a specific cellular response or any protection from challenge. Altogether, these data suggest that pcDNA-BLS is a good immunogen for the production of humoral and cell-mediated responses in mice and is a candidate for use in future studies of vaccination against brucellosis.     

Brucella abortus deficient in copper/zinc superoxide dismutase is virulent in BALB/c mice.

The gene encoding the Cu/Zn superoxide dismutase (SOD) of Brucella abortus strain 2308 was identified in a Brucella genomic library utilizing a combination of Western blotting and native gel electrophoresis. The Cu/Zn SOD gene was inactivated in vitro by ligation of a kanamycin resistance gene into the open reading frame encoding SOD. The plasmid born construct was introduced back into B. abortus by electroporation. Replacement of the wild-type Cu/Zn SOD by recombination was demonstrated by showing that both the KnR gene and the Cu/Zn SOD gene hybridized to the same band in a Southern analysis of genomic DNA. In addition, KnR strains were deficient in Cu/Zn SOD activity as assessed by lack of Cu/Zn SOD activity on a native gel and by lack of reactivity with specific serum in a Western analysis. Either strain 2308 or the Cu/Zn SOD deficient mutant injected intraperitoneally into BALB/c mice, exhibited no differences in their ability to colonize the spleen at 7 and 28 days post-inoculation. Thus, the inability to produce Cu/Zn SOD by B. abortus does not significantly impair its virulence in mice.     

An antigenic recombinant serine protease from Trichinella spiralis induces protective immunity in BALB/c mice

In this study, we report the cloning and characterization of a cDNA encoding a Trichinella serine protease gene (TspSP-1.3) from GenBank. The recombinant TspSP-1.3 protein (rTspSP-1.3) was expressed in an Escherichia coli expression system and purified with Ni-affinity chromatography. Real-time quantitative PCR analysis revealed that TspSP-1.3 was expressed at significantly higher levels in muscle larvae and adult worms than in newborn larvae. TspSP-1.3 was detected in excretory–secretory proteins of Trichinella spiralis with western blotting. Immunization with the rTspSP-1.3 antigen induced humoral immune responses, which manifested as elevated specific anti-rTspSP-1.3 IgG and IgE antibodies and a mixed Th1/Th2 response. To determine whether purified rTspSP-1.3 had good antigenicity and could be a vaccine candidate for the control of T. spiralis infection, we immunized BALB/c mice with rTspSP-1.3 and subsequently challenged the mice with T. spiralis larvae. The results showed that mice vaccinated with rTspSP-1.3 exhibited an average reduction in the muscle larvae burden of 39 % relative to the control group. These results suggest that TspSP-1.3 could be a novel vaccine candidate for controlling Trichinella infection.     

Recombinant Ochrobactrum anthropi expressing Brucella abortus Cu,Zn superoxide dismutase protects mice against B. abortus infection only after switching of immune responses to Th1 type

The members of the genus Brucella are gram-negative, facultatively intracellular bacterial pathogens that cause brucellosis in many animal species and humans. Although live, attenuated vaccines are available to protect several animal species from the disease, there is no safe and effective vaccine for human use. Here we report that a bacterium that is closely related to Brucella species, Ochrobactrum anthropi, can be used as a vaccine vector for the delivery of Brucella antigens to mice, leading to the elicitation of protective immunity against brucellosis. Brucella abortus Cu,Zn superoxide dismutase (SOD), a protective Brucella antigen, was expressed in large amounts in O. anthropi strain 49237 by use of the broad-host-range plasmid pBBR1MCS. Neither O. anthropi strain 49237 nor the recombinant O. anthropi strain 49237SOD, expressing B. abortus Cu,Zn SOD, provided protection against virulent Brucella infection in mice. Analysis of immune responses indicated that strains 49237 and 49237SOD stimulated a mix of Th1 and Th2 type responses in the mice. After the immune response was switched to a Th1-biased response by addition of oligonucleotides containing unmethylated CpG motifs, both O. anthropi strain 49237 and the recombinant O. anthropi strain 49237SOD induced protection in mice. However, the protection conferred by strain 49237SOD was significantly better than that induced by the parental strain, 49237.     

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 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.     

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.     

The Brucella abortus Cu,Zn superoxide dismutase is required for optimal resistance to oxidative killing by murine macrophages and wild-type virulence in experimentally infected mice

Two-dimensional gel electrophoretic analysis of cell lysates from Brucella abortus 2308 and the isogenic hfq mutant Hfq3 revealed that the RNA binding protein Hfq (also known as host factor I or HF-I) is required for the optimal stationary phase production of the periplasmic Cu,Zn superoxide dismutase SodC. An isogenic sodC mutant, designated MEK2, was constructed from B. abortus 2308 by gene replacement, and the sodC mutant exhibited much greater susceptibility to killing by O(2)(-) generated by pyrogallol and the xanthine oxidase reaction than the parental 2308 strain supporting a role for SodC in protecting this bacterium from O(2)(-) of exogenous origin. The B. abortus sodC mutant was also found to be much more sensitive to killing by cultured resident peritoneal macrophages from C57BL6J mice than 2308, and the attenuation displayed by MEK2 in cultured murine macrophages was enhanced when these phagocytes were treated with gamma interferon (IFN-gamma). The attenuation displayed by the B. abortus sodC mutant in both resting and IFN-gamma-activated macrophages was alleviated, however, when these host cells were treated with the NADPH oxidase inhibitor apocynin. Consistent with its increased susceptibility to killing by cultured murine macrophages, the B. abortus sodC mutant also displayed significant attenuation in experimentally infected C57BL6J mice compared to the parental strain. These experimental findings indicate that SodC protects B. abortus 2308 from the respiratory burst of host macrophages. They also suggest that reduced SodC levels may contribute to the attenuation displayed by the B. abortus hfq mutant Hfq3 in the mouse model.     

Protective immunity elicited by a divalent DNA vaccine encoding both the L7/L12 and Omp16 genes of Brucella abortus in BALB/c mice

This study was designed to evaluate the immunogenicity and the protective efficacy of a divalent fusion DNA vaccine encoding both the Brucella abortus L7/L12 protein (ribosomal protein) and Omp16 protein (outer membrane lipoprotein), designated pcDNA3.1-L7/L12-Omp16. Intramuscular injection of this divalent DNA vaccine into BALB/c mice elicited markedly both humoral and cellular immune responses. The specific antibodies exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. In addition, the dual-gene DNA vaccine elicited a strong T-cell proliferative response and induced a large amount of gamma interferon-producing T cells upon restimulation in vitro with recombinant fusion protein L7/L12-Omp16, suggesting the induction of a typical T-helper-1-dominated immune response in vivo. This divalent DNA vaccine could also induce a significant level of protection against challenge with the virulent strain B. abortus 544 in BALB/c mice. Furthermore, the protection level induced by the divalent DNA vaccine was significantly higher than that induced by the univalent DNA vaccines pcDNA3.1-L7/L12 or pcDNA3.1-Omp16. Taken together, the results of this study verify for the first time that the Omp16 gene can be a candidate target for a DNA vaccine against brucellosis. Additionally, a divalent genetic vaccine based on the L7/L12 and Omp16 genes can elicit a stronger cellular immune response and better immunoprotection than the relevant univalent vaccines can.     

乙脑病毒prME蛋白与BALB/c鼠IgG Fc段编码基因联合构建DNA免疫研究

目的 研究IgG Fc编码基因对流行性乙型脑炎(Japanese encephalitis,JE)DNA疫苗免疫增强效应的影响.方法 巢式RT-PCR法从BALB/c鼠脾组织获取IgG Fc段编码基因,用限制性内切酶从含流行性乙型脑炎病毒(Japanese encephalitis virus,JEV)prME蛋白基因重组子获取prME蛋白基因,分别插入同-真核表达载体pcDNA3.1(+)不同酶切位点,构建蘑组子pJME/IgG Fc并经酶切及DNA测序分析.脂质体法将pJMrY/IgG Fc转染CHO细胞.免疫荧光、Western blot法检测转染的CHO细胞中融合蛋白分布与表达.将pJME/IgG Fc肌注免疫BALB/c鼠,检测小鼠脾特异性细胞毒T细胞(CTL)杀伤活性和中和抗体滴度.结果 pJME/IgG Fc经BamH Ⅰ/EcoR Ⅰ和BamH Ⅰ/Not Ⅰ酶切释出的插入子大小,(2001 bp,2730 bp)分别与预期结果相符合.所编码的融合蛋白相对分子质量(Mr)为101×103,主要分布于胞浆,少最分布于胞膜,pJME/IgG Fc转染CHO细胞经32次传代仍可表达融合蛋白.pJME/IgGFc免疫组中和抗体滴度与CTL活性较pJME及灭活疫苗组均升高(P<0.05).结论 pJME/IgG Fc成功构建,转染的CHO细胞可稳定表达融合蛋白,IgG Fc段编码基因能够增强JEV DNA疫苗的细胞和体液免疫应答.     

Characterization of specific immune responses of mice inoculated with recombinant vaccinia virus expressing an 18-kilodalton outer membrane protein of Brucella abortus

Using the shuttle vector pMCO2 and the vaccinia virus wild-type WR strain, we constructed a recombinant virus expressing an 18-kDa outer membrane protein of Brucella abortus. BALB/c mice inoculated with this virus produced 18-kDa protein-specific antibodies, mostly of immunoglobulin G2a isotype, and in vitro stimulation of splenocytes from these mice with purified maltose binding protein-18-kDa protein fusion resulted in lymphocyte proliferation and gamma interferon production. However, these mice were not protected against a challenge with the virulent strain B. abortus 2308. Disruption of the 18-kDa protein's gene in vaccine strain B. abortus RB51 did not affect either the strain's protective capabilities or its in vivo attenuation characteristics. These observations suggest that the 18-kDa protein plays no role in protective immunity.     

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.     

Cloning,characterization and construction of htrA and htrA-like mutants of Brucella abortus and their survival in BALB/c mice

A genomic library of Brucella abortus S2308 was screened for expression of recombinant proteins recognized by sera from mice and from cattle infected with B. abortus. A positive clone, BA1, expressing a 50 kDa peptide was recognized by both sera. Plasmid pBA1, isolated from BA1, was shown by restriction enzyme digestion to possess a 1.9 kb insert. The nucleotide sequence of the pBA1 insert revealed an open reading frame with of 1539 bases with a coding capacity of 513 amino acids and a predicted molecular weight of 50 992. The predicted amino acid sequence showed 37% identity to E. coli HtrA, a temperature inducible serine protease. A second B. abortus htrA gene, designated htrA-like, was identified on a different cloned frag ment that also encoded B. abortus recA. The nucleotide sequence of the htrA -like gene revealed an open reading frame of 1422 nucleotides with a coding capacity of 474 amino acids and a predicted molecular weight of 50 155. The deduced amino acid sequence of the htrA-like gene showed 42% and 36% identity with B. abortus and E. coli HtrAs respectively. Western blotting of E. coli lysate containing the htrA-like gene was not recognized by sera from B. abortus-infected cattle or mice. B. abortus htrA but not htrA-like relieved the temperature sensitive phenotype and permitted growth of an E. coli htrA mutant at 42°C. B. abortus htrA and htrA-like mutants were constructed and their survival and growth in BALB/c mice was compared to the parental strain S2308. Splenic levels of htrA or htrA-like mutants were initially lower but after 60 days post-infection both were higher than the parental strain. Histologic analysis of hepatic inflammatory responses suggested that an initial intense granuloma formation in the htrA group was the basis for early low splenic titers of bacteria, but that failure to maintain granulomas, as did mice given the parental strain, resulted in a marked secondary rise in splenic bacterial titers.     

The Leishmania infantum acidic ribosomal protein P0 administered as a DNA vaccine confers protective immunity to Leishmania major infection in BALB/c mice

In this study, we examined the immunogenic properties of the Leishmania infantum acidic ribosomal protein P0 (LiP0) in the BALB/c mouse model. The humoral and cellular responses induced by the administration of the LiP0 antigen, either as soluble recombinant LiP0 (rLiP0) or as a plasmid DNA formulation (pcDNA3-LiP0), were determined. Also, the immunological response associated with a prime-boost strategy, consisting of immunization with pcDNA3-LiP0 followed by a boost with rLiP0, was assayed. Immunization with rLiP0 induced a predominant Th2-like humoral response, but no anti-LiP0 antibodies were induced after immunization with pcDNA3-LiP0, whereas a strong humoral response consisting of a mixed immunoglobulin G2a (IgG2a)-IgG1 isotype profile was induced in mice immunized with the prime-boost regime. For all three immunization protocols, rLiP0-stimulated production of gamma interferon (IFN-γ) in both splenocytes and lymph node cells from immunized mice was observed. However, it was only when mice were immunized with pcDNA3-LiP0 that noticeable protection against L. major infection was achieved, as determined by both lesion development and parasite burden. Immunization of mice with LiP0-DNA primes both CD4⁺ and CD8⁺ T cells, which, with the L. major challenge, were boosted to produce significant levels of IL-12-dependent, antigen-specific IFN-γ. Taken together, these data indicate that genetic vaccination with LiP0 induces protective immunological effector mechanisms, yet the immunological response elicited by LiP0 is not sufficient to keep the infection from progressing.     

Whole influenza virus vaccine is more immunogenic than split influenza virus vaccine and induces primarily an IgG2a response in BALB/c mice

The aim of this study was to compare the kinetics and the magnitude of the humoral immune response to two different influenza vaccine formulations, whole and split virus vaccines. BALB/c mice were immunized intramuscularly with one or two doses (3 weeks apart) of 7.5, 15 or 30 µg of haemagglutinin of monovalent A/Panama/2007/99 (H3N2) split or whole virus vaccine. The two vaccine formulations induced similar kinetics of the antibody-secreting cells response; however, differences in the magnitude were observed in the spleen and bone marrow. Vaccination with whole virus vaccine generally elicited a quicker and higher neutralizing antibody response, particularly after the first dose of vaccine. The two vaccine formulations gave different immunoglobulin G (IgG) subclass profiles. Split virus vaccine stimulated both IgG1 and IgG2a antibodies suggestive of mixed T-helper 1 (Th1) and Th2 response, whereas whole virus vaccine induced mainly an IgG2a antibody response, which is indicative of a dominant Th1 response. The increased immunogenicity of whole virus vaccine in a naïve population could reduce the vaccine concentration needed to provide protective immunity.     

A single immunization with a recombinant canine adenovirus expressing the rabies virus G protein confers protective immunity against rabies in mice

Rabies vaccines based on live attenuated rabies viruses or recombinant pox viruses expressing the rabies virus (RV) glycoprotein (G) hold the greatest promise of safety and efficacy, particularly for oral immunization of wildlife. However, while these vaccines induce protective immunity in foxes, they are less effective in other animals, and safety concerns have been raised for some of these vaccines. Because canine adenovirus 2 (CAV2) is licensed for use as a live vaccine for dogs and has an excellent efficacy and safety record, we used this virus as an expression vector for the RVG. The recombinant CAV2-RV G produces virus titers similar to those produced by wild-type CAV2, indicating that the RVG gene does not affect virus replication. Comparison of RVG expressed by CAV2-RV G with that of vaccinia-RV G recombinant virus (V-RG) revealed similar amounts of RV G on the cell surface. A single intramuscular or intranasal immunization of mice with CAV2-RVG induced protective immunity in a dose-dependent manner, with no clinical signs or discomfort from the virus infection regardless of the route of administration or the amount of virus.     

Immune and mitogenic responses by BALB/c, C3H/HeJ, and nude mice to Brucella abortus bacterin and lipopolysaccharide.

The immunogenic and mitogenic properties of Brucella abortus 1119-3 bacterin (BA) and biologically active B. abortus lipopolysaccharide (BA-LPS) were studied using normal and athymic (nude) BALB/c and C3H/HeJ mice. Although BA stimulated 2-mercaptoethanol-sensitive (2-ME-S) primary and secondary antibody responses in all mice, nude mice, in contrast to normal BALB/c and C3H/HeJ mice, did not make substantial 2-mercaptoethanol-resistant (2-ME-R) antibody responses. Similarly, all mice injected with BA-LPS made 2-ME-S primary responses, and the secondary response of thymus-bearing mice contained a substantial 2-ME-R component. Collectively, these observations suggest that although both BA and BA-LPS can stimulate thymus-independent 2-ME-S antibody synthesis, thymus-derived cells are required for optimal immune responses containing a 2-ME-R component. The antibody responses of normal BALB/c and C3H/HeJ mice to BA and BA-LPS were qualitatively and quantitatively similar. Both BA and BA-LPS were mitogenic for spleen cells from normal and nude BALB/c and C3H/HeJ mice but not for thymus cells from normal BALB/c or C3H/HeJ mice, suggesting that both preparations are B-cell mitogens.     

嗜肺军团菌免疫原蛋白核酸疫苗诱导的小鼠免疫应答及其保护力

目的:研究嗜肺军团菌免疫原蛋白核酸疫苗诱导的小鼠免疫原性以及对LP感染小鼠的保护能力。方法:用嗜肺军团菌免疫原蛋白基因真核表达重组质粒pcDNA3.1-ip作为DNA疫苗免疫BALB/c小鼠,检测免疫小鼠体内抗原特异性抗体水平、脾淋巴细胞增殖活性、IFNγ-产生水平和CTL特异杀伤活性等指标,以评价疫苗的免疫原性。真核表达重组质粒pcDNA3.1-ipDNA疫苗重复免疫BALB/c小鼠2次,末次免疫2周后,用10倍LD50剂量攻击小鼠,计数小鼠的存活数及小鼠肺中的细菌数,观察感染鼠的肺部病理变化。结果:pcDNA3.1-ip免疫小鼠后诱导产生了特异的体液免疫应答和细胞免疫应答,免疫组的免疫原性和免疫保护性均高于对照组pcDNA3.1( )组(P<0.01)。结论:免疫原蛋白基因可作为嗜肺军团菌核酸疫苗的侯选基因。