Comparison of protective efficacy of subcutaneous versus intranasal immunization of mice with a Brucella melitensis lipopolysaccharide subunit vaccine

Groups of mice were immunized either subcutaneously or intranasally with purified Brucella melitensis lipopolysaccharide (LPS) or with LPS as a noncovalent complex with Neisseria meningitidis group B outer membrane protein (LPS-GBOMP). Control mice were inoculated with sterile saline. Two doses of vaccine were given 4 weeks apart. Mice were challenged intranasally with virulent B. melitensis strain 16M 4 weeks after the second dose of vaccine. Sera, spleens, lungs, and livers of mice were harvested 8 weeks after challenge. The bacterial loads in the organs were determined by culture on brucella agar plates. Protective efficacy was determined by comparing the clearance of bacteria from organs of immunized mice with the clearance of bacteria from organs of control mice. At 8 weeks postchallenge there was significant protection from disseminated infection of spleens and livers of mice intranasally immunized with either vaccine compared to infection of control mice (P < 0.01). There was no significant difference in clearance of bacteria from the lungs of immunized mice and control mice. However, mice immunized subcutaneously with either LPS or LPS-GBOMP vaccine showed significant protection against infection of the spleen (P < 0.001), liver (P < 0.001), and lungs (P < 0.05). These results show that intranasal immunization of mice with either vaccine provided significant protection against disseminated infection of the spleen and liver but subcutaneous immunization of mice with the vaccines conferred significant protection against infection of the spleen, liver, and lungs.     

Paucibacillary tuberculosis in mice after prior aerosol immunization with Mycobacterium bovis BCG

To develop a murine model of paucibacillary tuberculosis for experimental chemotherapy of latent tuberculosis infection, mice were immunized with viable Mycobacterium bovis BCG by the aerosol or intravenous route and then challenged six weeks later with virulent Mycobacterium tuberculosis. The day after immunization, the counts were 3.71 +/- 0.10 log(10) CFU in the lungs of aerosol-immunized mice and 3.65 +/- 0.11 and 4.93 +/- 0.07 log(10) CFU in the lungs and spleens of intravenously immunized mice, respectively. Six weeks later, the lungs of all BCG-immunized mice had many gross lung lesions and splenomegaly; the counts were 5.97 +/- 0.14 and 3.54 +/- 0.07 log(10) CFU in the lungs and spleens of aerosol-immunized mice, respectively, and 4.36 +/- 0.28 and 5.12 +/- 0.23 log(10) CFU in the lungs and spleens of intravenously immunized mice, respectively. Mice were then aerosol challenged with M. tuberculosis by implanting 2.37 +/- 0.13 log(10) CFU in the lungs. Six weeks after challenge, M. tuberculosis had multiplied so that the counts were 6.41 +/- 0.27 and 4.44 +/- 0.14 log(10) CFU in the lungs and spleens of control mice, respectively. Multiplication of M. tuberculosis was greatly limited in BCG-immunized mice. Six weeks after challenge, the counts were 4.76 +/- 0.24 and 3.73 +/- 0.34 log(10) CFU in the lungs of intravenously immunized and aerosol-immunized mice, respectively. In contrast to intravenously immunized mice, there was no detectable dissemination to the spleen in aerosol-immunized mice. Therefore, immunization of mice with BCG by the aerosol route prior to challenge with a low dose of M. tuberculosis resulted in improved containment of infection and a stable paucibacillary infection. This model may prove to be useful for evaluation of new treatments for latent tuberculosis infection in humans.     

Intranasal immunization with multivalent group A streptococcal vaccines protects mice against intranasal challenge infections

We have previously shown that a hexavalent group A streptococcal M protein-based vaccine evoked bactericidal antibodies after intramuscular injection. In the present study, we show that the hexavalent vaccine formulated with several different mucosal adjuvants and delivered intranasally induced serum and salivary antibodies that protected mice from intranasal challenge infections with virulent group A streptococci. The hexavalent vaccine was formulated with liposomes with or without monophosphorylated lipid A (MPL), cholera toxin B subunit with or without holotoxin, or proteosomes from Neisseria meningitidis outer membrane proteins complexed with lipopolysaccharide from Shigella flexneri. Intranasal immunization with the hexavalent vaccine mixed with these adjuvants resulted in significant levels of antibodies in serum 2 weeks after the final dose. Mean serum antibody titers were equivalent in all groups of mice except those that were immunized with hexavalent protein plus liposomes without MPL, which were significantly lower. Salivary antibodies were also detected in mice that received the vaccine formulated with the four strongest adjuvants. T-cell proliferative assays and cytokine assays using lymphocytes from cervical lymph nodes and spleens from mice immunized with the hexavalent vaccine formulated with proteosomes indicated the presence of hexavalent protein-specific T cells and a Th1-weighted mixed Th1-Th2 cytokine profile. Intranasal immunization with adjuvanted formulations of the hexavalent vaccine resulted in significant levels of protection (80 to 100%) following intranasal challenge infections with type 24 group A streptococci. Our results indicate that intranasal delivery of adjuvanted multivalent M protein vaccines induces protective antibody responses and may provide an alternative to parenteral vaccine formulations.     

Comparison of host resistance to primary and secondary Listeria monocytogenes infections in mice by intranasal and intravenous routes

There have been no studies on the susceptibility and host immune responses to an intranasal infection with Listeria monocytogenes. In this study, we compared the susceptibilities and cytokine responses between intranasal and intravenous infections with L. monocytogenes in mice. Moreover, we compared efficiency of acquisition of host resistance to L. monocytogenes infection between intranasally and intravenously immunized mice because an intranasal immunization of vaccines is reportedly available for induction of adaptive immunity against various infectious pathogens. The susceptibility to an intranasal infection with L. monocytogenes was markedly lower than that to the intravenous infection. The bacterial growth in the lungs, spleens, and livers was substantially similar between intranasally and intravenously infected mice. Titers of endogenous gamma interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in the spleens, livers, and lungs were parallel to bacterial numbers in each organ of mice during intranasal infection and intravenous infection. IFN-gamma-deficient mice and TNF-alpha-deficient mice were highly susceptible to intranasal infection as well as intravenous infection. Susceptibilities to intranasal and intravenous L. monocytogenes infection were the same in these cytokine-deficient mice. These results suggest that both IFN-gamma and TNF-alpha play critical roles in host resistance to intranasal L. monocytogenes infection as well as the intravenous infection. Acquisition of host resistance to intravenous and intranasal L. monocytogenes infection was induced in intranasally immunized mice as well as intravenously immunized mice, suggesting that intranasal immunization is effective for prevention of a systemic infection with L. monocytogenes.     

结核分枝杆菌Ag85B基因疫苗免疫保护作用的初步研究

目的:研究编码结核分枝杆菌分泌蛋白Ag85B的基因疫苗pTB30m和pTB30s对免疫动物的保护作用。方法:以基因疫苗pTB30m和pTB30s肌注免疫BALB/c小鼠。免疫完成6 wk后,用5×105 CFU的MTB H37Rv毒株经小鼠尾静脉攻击感染。同时用尼龙毛柱分离基因免疫BALB/c小鼠的T细胞,并以5×106 T细胞/只小鼠过继免疫正常BALB/c小鼠,立即用105 CFU的MTB毒株经小鼠尾静脉攻击感染。4 wk后分别计数脾脏中的细菌负荷。结果:与生理盐水对照组相比较,pTB30m及pTB30s质粒免疫组BALB/c小鼠脾脏中的细菌负荷均减少,分别为0.645(log10 CFU,P<0.01)和0.839(log10CFU,P<0.001);而空质粒对照组小鼠脾脏中的细菌负荷减少较少。经质粒pTB30m和pTB30s免疫的BALB/c小鼠的T细胞,过继免疫的正常BALB/c小鼠,对攻击感染的MTB H37Rv毒株在脾脏中的增殖具有部分抑制作用。结论:pTB30s免疫的BALB/c小鼠,对MTB H37 Rv毒株攻击的保护作用优于pTB30m质粒免疫,有望进一步用于结核病的防治研究。     

Oral vaccination with Brucella melitensis WR201 protects mice against intranasal challenge with virulent Brucella melitensis 16M

Human brucellosis can be acquired from infected animal tissues by ingestion, inhalation, or contamination of conjunctiva or traumatized skin by infected animal products. In addition, Brucella is recognized as a biowarfare threat agent. Although a vaccine to protect humans from natural or deliberate infection could be useful, vaccines presently used in animals are unsuitable for human use. We tested orally administered live, attenuated, purine auxotrophic B. melitensis WR201 bacteria for their ability to elicit cellular and humoral immune responses and to protect mice against intranasal challenge with B. melitensis 16M bacteria. Immunized mice made serum antibody to lipopolysaccharide and non-O-polysaccharide antigens. Splenocytes from immunized animals released interleukin-2 and gamma interferon when grown in cultures with Brucella antigens. Immunization led to protection from disseminated infection and enhanced clearance of the challenge inoculum from the lungs. Optimal protection required administration of live bacteria, was related to immunizing dose, and was enhanced by booster immunization. These results establish the usefulness of oral vaccination against respiratory challenge with virulent Brucella and suggest that WR201 should be further investigated as a vaccine to prevent human brucellosis.     

Protection of BALB/c Mice against Brucella abortus 544 Challenge by Vaccination with Bacterioferritin or P39 Recombinant Proteins with CpG Oligodeoxynucleotides as Adjuvant

The P39 and the bacterioferrin (BFR) antigens of Brucella melitensis 16M were previously identified as T dominant antigens able to induce both delayed-type hypersensivity in sensitized guinea pigs and in vitro gamma interferon (IFN-gamma) production by peripheral blood mononuclear cells from infected cattle. Here, we analyzed the potential for these antigens to function as a subunitary vaccine against Brucella abortus infection in BALB/c mice, and we characterized the humoral and cellular immune responses induced. Mice were injected with each of the recombinant proteins alone or adjuvanted with either CpG oligodeoxynucleotides (CpG ODN) or non-CpG ODN. Mice immunized with the recombinant antigens with CpG ODN were the only group demonstrating both significant IFN-gamma production and T-cell proliferation in response to either Brucella extract or to the respective antigen. The same conclusion holds true for the antibody response, which was only demonstrated in mice immunized with recombinant antigens mixed with CpG ODN. The antibody titers (both immunoglobulin G1 [IgG1] and IgG2a) induced by P39 immunization were higher than the titers induced by BFR (only IgG2a). Using a B. abortus 544 challenge, the level of protection was analyzed and compared to the protection conferred by one immunization with the vaccine strain B19. Immunization with P39 and CpG ODN gave a level of protection comparable to the one conferred by B19 at 4 weeks postchallenge, and the mice were still significantly protected at 8 weeks postchallenge, although to a lesser extent than the B19-vaccinated group. Intriguingly, no protection was detected after BFR vaccination. All other groups did not demonstrate any protection.     

Impaired control of Brucella melitensis infection in Rag1-deficient mice

After intranasal inoculation, Brucella melitensis chronically infects the mononuclear phagocyte system in BALB/c mice, but it causes no apparent illness. Adaptive immunity, which can be transferred by either T cells or antibody from immune to naive animals, confers resistance to challenge infection. The role of innate, non-B-, non-T-cell-mediated immunity in control of murine brucellosis, however, is unknown. In the present study, we documented that BALB/c and C57BL/6 mice had a similar course of infection after intranasal administration of 16M, validating the usefulness of the model in the latter mouse strain. We then compared the course of infection in Rag1 knockout mice (C57BL/6 background) (referred to here as RAG-1 mice) which have no B or T cells as a consequence of deletion of Rag1 (recombination-activating gene 1), with infection in normal C57BL/6 animals after intranasal administration of B. melitensis 16M. C57BL/6 mice cleared brucellae from their lungs by 8 to 12 weeks and controlled infection in the liver and spleen at a low level. In contrast, RAG-1 mice failed to reduce the number of bacteria in any of these organs. From 1 to 4 weeks after inoculation, the number of splenic bacteria increased from 2 to 4.5 logs and remained at that level. In contrast to the consistently high numbers of brucellae observed in the spleens, the number of bacteria rose in the livers sampled for up to 20 weeks. Immunohistologic examination at 8 weeks after infection disclosed foci of persistent pneumonia and large amounts of Brucella antigen in macrophages in lung, liver, and spleen in RAG-1, but not C57BL/6, mice. These studies indicate that T- and B-cell-independent immunity can control Brucella infection at a high level in the murine spleen, but not in the liver. Immunity mediated by T and/or B cells is required for clearance of bacteria from spleen and lung and for control of bacterial replication in the liver.     

双价志贺菌苗滴鼻免疫对小鼠NALT、GALT和脾淋巴细胞的影响

本文是探讨滴鼻免疫对NALT、GALT和脾淋巴细胞中特异性抗体分泌细胞及淋巴细胞表型变化的影响。将Balb/c小鼠随机分为 3组 ,每组 2 0只。FSM 2 117或FS 5 416 4× 10 7CFU/只经滴鼻途径免疫小鼠 ,间隔 2周 ,3次免疫后 7d活杀 ,分离NALT、鼻通道、脾、小肠PP及MLN淋巴细胞 ,采用BA ELISPOT法检测其中特异性抗福氏、宋内LPSIgA和IgG分泌细胞 ;采用流式细胞术检测其淋巴细胞表型的变化。结果是两株菌苗经鼻内免疫都诱发了NALT、NP、GALT内 (PP、MLN、LPL )以及代表系统免疫反应的脾淋巴细胞中特异性抗福氏、宋内LPSIgA、IgG ASC的显著增加 (P <0 0 1) ;NALT、NP和PP淋巴细胞中CD3+T细胞显著增加 ,其中以CD4+T细胞增加为主 ;FSM 2 117免疫组的脾细胞中B2 2 0 +细胞显著增加 (P <0 0 1) ,而FS 5 416免疫组的脾细胞中CD3+T细胞显著增加 (P <0 0 1)。说明两株菌苗经鼻粘膜免疫均可诱导鼻粘膜局部、GALT及系统免疫反应的发生 ,是一个安全有效的免疫途径     

Intranasal vaccination with chitosan-DNA nanoparticles expressing pneumococcal surface antigen a protects mice against nasopharyngeal colonization by Streptococcus pneumoniae

Streptococcus pneumoniae is a respiratory pathogen, and mucosal immune response plays a significant role in the defense against pneumococcal infections. Thus, intranasal vaccination may be an alternative approach to current immunization strategies, and effective delivery systems to mucosal organism are necessary. In this study, BALB/c mice were immunized intranasally with chitosan-DNA nanoparticles expressing pneumococcal surface antigen A (PsaA). Compared to levels in mice immunized with naked DNA or chitosan-pVAX1, anti-PsaA IgG antibody in serum and anti-IgA antibody in mucosal lavages were elevated significantly in mice immunized with chitosan-psaA. The balanced IgG1/IgG2a antibody ratio in serum, enhanced gamma interferon (IFN-γ) and IL-17A levels in spleen lymphocytes, and mucosal washes of mice immunized with chitosan-psaA suggested that cellular immune responses were induced. Furthermore, significantly fewer pneumococci were recovered from the nasopharynx of mice immunized with chitosan-psaA than for the control group following intranasal challenge with ATCC 6303 (serotype 3). These results demonstrated that mucosal immunization with chitosan-psaA may successfully generate mucosal and systemic immune responses and prevent pneumococcal nasopharyngeal colonization. Hence, a chitosan-DNA nanoparticle vaccine expressing pneumococcal major immunodominant antigens after intranasal administration could be developed to prevent pneumococcal infections.     

Quantitation of virus-specific classes of antibodies following immunization of mice with attenuated equine herpesvirus 1 and viral glycoprotein D

The antibody responses of CBA/J mice infected intranasally (i.n.) with either the attenuated KyA strain or the pathogenic RacL11 strain of equine herpesvirus 1 (EHV-1) or immunized with recombinant glycoprotein D (rgD) were investigated using the ELISPOT assay to measure EHV-1-specific antibody-secreting cells (ASC) in the regional lymphoid tissue of the respiratory tract. IgG, IgA, and IgM ASC specific for EHV-1 were detected in the mediastinal lymph nodes (MLN) and lungs 2 weeks after i.n. infection with EHV-1 strain KyA or RacL11, or immunization with heat-killed KyA or rgD. EHV-1-specific ASC were present in the MLN and lungs at 4 and 8 weeks, but declined in frequency by fivefold in the lung at 8 weeks. However, i.n. immunized (2 x 10(6) pfu KyA or 50 microgram rgD/mouse) mice infected at 8 weeks with pathogenic EHV-1 RacL11 resisted challenge and showed eight- and tenfold increases in MLN ASC and lung ASC, respectively, by 3 days after challenge. In contrast to the intranasal route of immunization, intraperitoneal immunization yielded ASC frequencies in the MLN and lungs that were only slightly above those of nonimmunized control mice. These data indicate that immunization with infectious or heat-killed EHV-1 KyA, or rgD, induces significant levels of virus-specific ASC both in the MLN and lungs, a specific memory B-cell response, and long-term protective immunity. The finding that the numbers of ASC induced by the pathogenic strain versus the attenuated strain of EHV-1, which were virtually identical, indicated that the ability to generate a B-cell response is independent of and does not contribute to EHV-1 virulence.     

pHSP65疫苗增强结核杆菌特异性T细胞产生IFN-γ

为提高BCG的免疫效果,以BCG进行初次免疫,比较pHSP65基因疫苗和BCG疫苗加强免疫的效果,寻找新型结核疫苗的初免-加强免疫策略。于第0周皮下接种BCG,第6、8周给予pHSP65滴鼻免疫或BCG加强免疫,末次免疫后2周检测全身及肺脏局部IFN-γ的产生。与BCG初免/BCG加强免疫相比,经pHSP65基因疫苗滴鼻加强免疫后,ELISPOT结果显示脾脏和肺局部分泌IFN-γ的淋巴细胞数量显著增加,流式检测显示IFN-γ+CD4+T细胞数量显著增加,ELISA结果证实淋巴细胞IFN-γ的分泌显著增高,提示经BCG初次免疫后,以pHSP65 DNA滴鼻加强免疫可显著增强全身和肺局部T淋巴细胞IFN-γ的产生,具有良好的抗结核感染潜能。     

重组幽门螺杆菌尿素酶B亚单位疫苗鼻腔免疫的实验研究

目的:探讨基因工程疫苗Hp重组尿素酶B亚单位(rUreB)鼻腔接种的免疫效果。方法:以rUreB不同剂量或加不同佐剂滴鼻免疫BALB/c小鼠。末次免疫7 d后,收集血清及胃黏膜、小肠黏膜、鼻黏膜及气管黏膜冲洗液,用ELISA法检测抗rUreB特异性抗体。结果:rUreB鼻腔免疫后各实验组血清特异性IgG及各黏膜冲洗液中特异性IgA的水平均明显增高,与对照组相比较差异显著(P<0.01)。20μg剂量组与10μg剂量组相比较,仅血清特异性IgG水平增高,其它黏膜特异性IgA的水平未见增高。大肠杆菌不耐热肠毒素B亚单位(LTB)的佐剂效果较霍乱毒素B亚单位(CTB)强,卡泊波可增强鼻腔接种疫苗在胃黏膜洗液中的抗体应答水平。结论:CTB、LTB、卡泊波均可作为rUreB鼻腔黏膜接种的佐剂。HprUreB鼻黏膜接种,不仅可诱导血清特异性抗体反应,而且能引起多个黏膜部位的免疫应答,是一种方便、有效、廉价的免疫途径。     

重组BCG—Sj26GST疫苗诱导小鼠sIL—2R和IFN—γ的变化

目的研究日本血吸虫重组 BCG- Sj2 6 GST疫苗对小鼠脾细胞 s IL- 2 R和 IFN- γ的影响。方法第 1次实验采用 1× 10 6 和 1× 10 8CFU疫苗皮下免疫 BAL B/ C鼠 ,免疫后 8周用日本血吸虫尾蚴进行攻击感染 ,感染后 6周剖杀小鼠 ,同时设有 PBS对照组。第 2次实验用 1× 10 6 CFU疫苗皮下和静脉注射分别免疫小鼠 ,于免疫后 0、4、8、10、14和 16周各剖杀 4只 ,分离脾脏 ,用 Sj2 6或 Con A刺激脾细胞 ,用 EL ISA法检测脾细胞上清液中 s IL 2 - R和 IFN -γ含量。结果疫苗免疫 ,尾蚴攻击后 ,s IL- 2 R和 IFN- γ水平显著升高 ;动态观察发现 s IL- 2 R和 IFN- γ分别于免疫后 8～ 10周和 4～ 8周达最高水平。结论日本血吸虫重组 BCG- Sj2 6 GST疫苗可加强宿主 Th1反应 ,促进 Th1细胞分泌 IL- 2和 IFN- γ,它们与免疫细胞相互作用 ,提高宿主抗血吸虫感染的保护力     

Bartonella henselae-Specific Cell-Mediated Immune Responses Display a Predominantly Th1 Phenotype in Experimentally Infected C57BL/6 Mice

Immune responses of the immunocompetent host to Bartonella henselae infection were investigated in the murine infection model using C57BL/6 mice. Following intraperitoneal infection with human-derived B. henselae strain Berlin-1, viable bacteria could be recovered from livers and spleens during the first week postinfection, while Bartonella DNA remained detectable by PCR in the liver for up to 12 weeks after infection. Granulomatous lesions developed in livers of infected mice, reached maximal density at 12 weeks after infection, and persisted for up to 20 weeks, indicating that B. henselae induced a chronic granulomatous hepatitis in the immunocompetent murine host. T-cell-mediated immune responses were analyzed in vitro by means of spleen cell proliferation and cytokine release assays as well as analysis of immunoglobulin G (IgG) isotypes. Spleen cells from infected mice proliferated specifically upon stimulation with heat-killed Bartonella antigen. Proliferative responses were mainly mediated by CD4+ T cells, increased during the course of infection, peaked at 8 weeks postinfection, and decreased thereafter. Gamma interferon, but not interleukin-4, was produced in vitro by spleen cells from infected animals upon stimulation with Bartonella antigens. Bartonella-specific IgG was detectable in serum of infected mice by 2 weeks, and the antibody concentration peaked at 12 weeks postinfection. IgG2b was the prominent isotype among the Bartonella-specific serum IgG antibodies. These data indicate that B. henselae induces cell-mediated immune responses with a Th1 phenotype in immunocompetent C57BL/6 mice.     

A novel intranasal virus-like particle (VLP) vaccine designed to protect against the pandemic 1918 influenza A virus (H1N1)

We have prepared a virus-like particle (VLP) vaccine bearing the surface glycoproteins HA and NA of the 1918 influenza A virus by infecting Sf9 cells with a quadruple recombinant baculovirus that expresses the four influenza proteins (HA, NA, M1, and M2) required for the assembly and budding of the VLPs. The presence of HA and M1 in the purified VLPs was confirmed by Western blot, and that of NA by a neuraminidase enzymatic assay. For in vivo studies, the 1918 VLP vaccine was formulated with or without an oligonucleotide containing two CpG motifs and administered in two doses 2 wk apart via the intranasal route. The antibody titers in mice immunized with VLP vaccines were higher than in mice vaccinated with an inactivated swine virus (H1N1) control, when CHO cells expressing 1918 HA were used as antigen. The opposite result was obtained when disrupted swine virus was the antigen for the ELISA test. Vaccine efficacy was evaluated by challenging immunized mice with the 1918 antigenically related influenza virus A/swine/Iowa/15/30 (H1N1) and measuring viral titers in the upper and lower respiratory tract. Mice immunized with VLP vaccine plus CpG demonstrated significantly lower viral titers in the nose and lungs than did the control on days 2 and 4 postchallenge and completely cleared the virus by day 6. Furthermore, they did not show symptoms of disease although there was a minor decrease in body weight. Mice vaccinated with VLP alone also demonstrated significantly lower viral titers in the nose and lungs than did the placebo group as well as the inactivated virus group on days 4 and 6 postchallenge. These results suggest that it is feasible to make a safe and immunogenic vaccine to protect against the extremely virulent 1918 virus, using a novel and safe cell-based technology.     

Protection against nasal colonization with Streptococcus pneumoniae by parenteral immunization with a DNA vaccine encoding PspA (Pneumococcal surface protein A)

Pneumococcal surface protein A (PspA) is an important candidate for a cost-effective vaccine with broad coverage against Streptococcus pneumoniae. We have previously shown that intramuscular immunization with PspA as a DNA vaccine induces an immune response characterized by the induction of a balanced IgG1/IgG2a antibody response in BALB/c mice, which was able to efficiently mediate complement deposition onto intact bacteria and to induce protection against an intraperitoneal challenge. We now confirm the results in C57BL/6 mice and further show that the response induced by the DNA vaccine expressing PspA is able to mediate protection against colonization of the nasopharyngeal mucosa even though immunization was given parenterally. Moreover, a positive correlation was observed between IgG1 and the numbers of CFU recovered, whereas an inverse correlation was observed between nasal CFU levels and IgG2a. A positive correlation was also found for IgG1/IgG2a antibody ratios with CFU recovered from the nasopharynx. Therefore, reduction of nasal colonization was strongly associated with increased levels of serum IgG2a complement fixing antibody and low levels of IgG1 antibody which has much less complement fixing activity. Passive transfer of serum from animals immunized with the DNA vaccine expressing PspA was also able to reduce the fraction of mice with high density of colonization of the nasopharynx. Secretion of IFN-γ, but not IL-17, was observed in splenocytes from mice immunized with the DNA vaccine.     

LOS诱导的特异性抗体分泌细胞的ELISPOT法检测

目的 :动态测定卡他性莫拉氏菌 (Moraxellacatarrhalis,M .cat)脱毒脂寡糖 (dLOS)蛋白质结合疫苗诱导的抗体分泌细胞的应答状态。方法 :以M .catdLOS蛋白质结合疫苗滴鼻免疫BALB/c小鼠。应用酶联免疫斑点试验 (ELISPOT)检测免疫小鼠不同免疫诱导部位和免疫效应部位 ,包括 :鼻相关淋巴组织 (NALT)、脾脏、颈部淋巴结、鼻内容物、肺脏和派伊尔氏结的特异性抗体分泌细胞 ,并同时测定血清、鼻冲洗液、肺泡灌洗液、唾液及粪便提取液中特异性IgA、IgG和IgM的水平。结果 :M .catdLOS蛋白质结合疫苗免疫小鼠的NALT、脾脏、颈部淋巴结、鼻内容物、肺脏和派伊尔氏结中 ,均测出分泌LOS特异抗体的抗体分泌细胞 ,以鼻内容物中IgA分泌细胞的数目最多 ,其次是在NALT和肺脏中 ,这与特异性抗体测定的结果相一致。结论 :M .catdLOS蛋白质结合疫苗经滴鼻免疫 ,能刺激产生LOS特异的黏膜和全身抗体分泌细胞的应答。ELISPOT试验具有快速、灵敏、特异的优点 ,为动态分析单个抗体分泌细胞应答规律提供了新方法。     

Effects of diet and genetics on Mycobacterium bovis BCG vaccine efficacy in inbred guinea pigs.

Strain 2 and strain 13 guinea pigs were vaccinated with Mycobacterium bovis BCG and placed on low-protein or protein-adequate diets. Five weeks later all animals were infected by the respiratory route with virulent Mycobacterium tuberculosis H37Rv organisms. Four weeks postchallenge, guinea pigs were skin tested with purified protein derivative and sacrificed. Protein deficiency resulted in significant reductions in body weight and thymus weight and in an impairment in the ability to control the M. bovis BCG vaccine organisms and to mount delayed hypersensitivity reactions. Protein deficiency also adversely affected the efficacy of the BCG vaccine as demonstrated by the numbers of virulent organisms recovered in spleens and lungs. Strain differences were observed in the number of leukocytes, thymus weight, and the responsiveness of blood lymphocytes to purified protein derivative stimulation. In general, strain 13 guinea pigs responded more dramatically to dietary insult than did their strain 2 counterparts. Protein deprivation completely abolished BCG vaccine protection in the lungs and spleens of strain 13 animals and significantly reduced the protection afforded to strain 2 animals. In both strains, the BCG vaccine protected normally nourished guinea pigs. There was no significant difference between strains with respect to susceptibility to pulmonary infection with virulent mycobacteria. Thus, diet and genetic pedigree each had a significant influence on BCG vaccine efficacy.     

Comparison of antibody- and cell-mediated immune responses after intramuscular hepatitis C immunizations of BALB/c mice

Current treatments for hepatitis C infection have limited efficacy, and there is no vaccine available. The goal of this study was to compare the immune response to several immunization combinations against hepatitis C virus (HCV). Six groups of mice were immunized at weeks 0, 4, and 8 with different combinations of a candidate HCV vaccine consisting of 100 microg recombinant HCV core/E1/E2 (rHCV) DNA plasmid and/or 25 microg rHCV polyprotein and 50 microL Montanide ISA- 51. Four weeks after the last injection, all groups of mice were sacrificed and blood samples and spleens were collected for measuring the levels of specific HCV antibodies (total IgG, IgG1, and IgG2a). Cell proliferation and intracellular interferon-gamma were also measured. Among the groups of immunized mice, only the mice immunized with rHCV DNA plasmid, rHCV polyprotein, and montanide (group D) and mice immunized with rHCV polyprotein and montanide (group F) demonstrated a significant increase in the total IgG titer after immunization. IgG1 was the predominant antibody detected in both groups D and F. No IgG2a was detected in any of the groups. Proliferation assays demonstrated that splenocytes from group D and group C (rHCV DNA primed/rHCV polyprotein boost) developed significant anti-HCV proliferative responses. The combination of an rHCV DNA plasmid, rHCV polyprotein, and montanide induced a high antibody titer with a predominance of IgG1 antibodies and recognized the major neutralization epitopes in HVR1. In contrast, group C did not show an increase in anti-HCV antibodies, but did show a proliferative response.