肺炎链球菌毒力蛋白在侵入性感染中的免疫原性评价

目的观察肺炎链球菌表面蛋白A(PspA)、肺炎链球菌表面黏附素A(PsaA)及肺炎链球菌溶菌素(Ply)激发自然途径感染肺炎链球菌机体保护性体液免疫应答的情况,评价这三种毒力蛋白作为治疗性肺炎链球菌疫苗候选抗原的免疫原性。方法采集侵入性肺炎链球菌感染患者(实验组)及同期确诊为侵入性其它细菌感染患者(对照第一组)和同期确诊为非感染性疾病患者(对照第二组)各36例急性期(第0+3天)及恢复期(第21±3天)血清样本,酶联免疫吸附法(ELISA)检测患者血清中肺炎链球菌3种毒力蛋白抗原特异性抗体IgG水平变化。结果实验组与两组对照组间第(0+3)天血清的3种肺炎链球菌毒力蛋白特异性抗体水平无显著性差异(P>0.05)。第一组对照组恢复期较急性期血清中3种毒力蛋白特异性抗体水平无显著变化(P>0.05),而实验组恢复期血清3种肺炎链球菌毒力蛋白特异性抗体水平明显高于急性期血清抗体水平8～30倍(P<0.01),其中PspA和Ply蛋白抗原特异性抗体水平升高更明显,血清中针对PspA家族1(PspA-Faml)特异性抗体水平高于针对PspA家族2(PspA-Fam2)的特异性抗体水平(P<0.01)。结论 PsaA,P...     

ClpP免疫小鼠可抵抗TIGR4型肺炎链球菌的侵袭性感染

目的 原核表达肺炎链球菌毒力因子ClpP,探讨其作为肺炎链球菌候选蛋白疫苗的价值.方法 分离培养TIGR4型肺炎链球菌,获取其染色体DNA,采用基因体外重组的方法将完整的ClpP开放读码框架克隆到pET-32a原核表达载体内,经测序鉴定、原核表达及纯化,ClpP主动和阳性抗体血清被动免疫小鼠,TIGR4型肺炎链球菌攻击后,监测其生存时间.结果 获得了高表达的重组抗原蛋白,表达的抗原蛋白用Western blot鉴定,镍柱纯化并透析复性可得到纯度达90%以上的重组蛋白.主动和被动免疫BALB/c小鼠,与未免疫组相比,产生的保护性作用具有统计学意义.结论 ClpP蛋白免疫小鼠可抵抗肺炎链球菌侵袭性感染,ClpP蛋白可作为肺炎链球菌的候选蛋白疫苗.     

肺炎链球菌菌体蛋白LytA和CbpA对感染小鼠的诊断价值

目的 通过基因工程技术获取肺炎链球菌自溶素(autolysin,LytA)和胆碱结合蛋白A(choline binding protein A,CbpA),探讨其对肺炎链球菌感染小鼠的血清学诊断价值.方法 根据GenBank中lytA和cbpA基因保守序列设计合成特异性引物,采用PCR技术从肺炎链球菌基因组中扩增lytA和cbpA保守序列片段;经由IPTG诱导并通过等电点洗脱方法获取纯化的重组蛋白LytA和CbpA;Western blot测定表达蛋白的抗体结合活性.以LytA和CbpA为抗原,建市ELISA反应模式测定感染小鼠血清中相应的IgG和IgM抗体,评价诊断价值.结果 成功构建了原核表达载体pET-32a(+)/lytA和pET-32a(+)/cbpA,表达的重组蛋白LytA和CbpA具有较好的抗体结合活性.实验组小鼠(肺炎链球菌感染组)IgM和IgG类抗LytA抗体滴度高于对照组(止常对照组和乙型链球菌感染对照组),差异有统计学意义(P<0.05),CbpA抗体与正常对照组差异有统计学意义,与乙型链球菌感染组差异无统计学意义(P>0.05).CbpA蛋白对感染小鼠诊断的敏感性较高(IgG:83.3%;lgM:75.0%),而LytA特异性较高(IgG:100%;IgM:100%).结论 协同利用重组蛋白CbpA诊断敏感性及LytA的特异性,对肺炎链球菌感染小鼠的血清学诊断具有一定价值,为进一步用于临床检验奠定基础.

Abstract：

Objective To obtain the pneumococcal autolysin(LytA)and choline binding protein A(CbpA)by prokaryotic expression system and investigate their diagnosis for infection caused by Streptococcus pneumoniae.Methods The specific primers were designed according to lytA and cbpA of Streptococcus pneumoniae gene sequence.lytA and cbpA were amplified by PCR form the pneumococcus genome.After IPTG inducing,the recombinant proteins were purified by electroeluting of bag filter,detected by SDS-PAGE and Western blot.Serum lgG and IgM antibodies accordingly of BALB/c mice infected with Streptococcus pneurnoniae were detected by ELISA.Results The recombinant plasmid pET-32a(+)/lytA and pET-32a (+)/cbpA were constructed successfully.Fusion proteins LytA and CbpA were expressed and displayed expected antigenicity.IgM and IgG antibodies level anti LytA were significantly higher than the control group (infections with B Streptococcus group and healthy mice),(P<0.05),but antibodies level anti CbpA did not increase as compared with group infected with B Streptococcus(P>0.05).Diagnostic sensitivity of CbpA was 83.3%(IgG)and 75.0%(IgM).Diagnostic specificity of LytA was 100%(IgG and IgM).Conclusion The synergistic use of specificity of LytA and sensitivity of CbpA may be worthy of serological diagnosis for Streptococcus pneumoniae infection,and may be used for further clinical test.     

肺炎链球菌抗原试验快速检测阳性血培养瓶肺炎链球菌

目的探讨肺炎链球菌抗原用于辅助诊断阳性血培养瓶肺炎链球菌的临床意义。方法采用双盲法检测人工模拟血培养链球菌阳性标本34例和人工模拟血培养链球菌阴性标本180例，血培养仪报警后，移出培养瓶用注射器抽取部分血培养液，部分用于革兰染色，部分用于肺炎链球菌抗原试验和标准培养鉴定法试验。结果34例链球菌阳性标本中，肺炎链球菌24例，肺炎链球菌抗原试验均为阳性．缓症链球菌2例和血链球菌1例，肺炎链球菌抗原试验亦为阳性，7例其它链球菌肺炎链球菌抗原试验阴性。结论肺炎链球菌抗原试验可应用于阳性血培养瓶肺炎链球菌的快速鉴定。     

ClpP融合蛋白诱导的免疫反应可抵抗不同型别肺炎链球菌的侵袭性感染

目的 ClpP融合蛋白在调节肺炎链球菌其他毒力因子表达和入血过程中起重要作用,评价Trx-ClpP融合蛋白对不同型别肺炎链球菌的侵袭性感染的免疫保护效果,为后续蛋白疫苗的开发提供依据.方法 含有重组质粒pET-32a(+)/ClpP工程菌BL21(DE3)经IPTG诱导后表达Trx-ClpP融合蛋白,纯化后与铝佐剂混合经腹腔注射免疫小鼠,而对照小鼠则用PBS与铝佐剂混合免疫.免疫程序:初次免疫5周后用ELISA法测小鼠针对ClpP融合蛋白的抗体免疫应答水平,末次免疫2周后用12个型别肺炎链球菌攻击,监测其生存时间.结果 重组ClpP融合蛋白主动免疫BALB/c小鼠后,产生了高滴度的anti-ClpP融合蛋白抗体,对多个血清型的肺炎链球菌菌血症小鼠模型的保护效果较对照组具有统计学意义.结论 ClpP融合蛋白免疫小鼠后可抵抗1、2、3、4、5、6B、7F、9V、14、18C、19F、23F等12个型别的肺炎链球菌侵袭性感染,提示以ClpP融合蛋白为活性成分的疫苗具有较高的开发价值.     

肺炎链球菌毒力相关基因licC的活性研究

目的:克隆、表达肺炎链球菌LicC及LicC3′端25个氨基酸缺失片段(记作△LicC)基因;分析、比较目标蛋白活性,证实LicC部分缺失对其活性的影响。方法:通过基因重组技术构建重组质粒pQE80-licC、pQE80-△licC,转化大肠杆菌BL21,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,自建生物荧光技术测定酶活性。结果:成功构建重组表达载体,获得可溶性LicC和LicC截短体蛋白,活性测定表明LicC截短体活性显著低于LicC(P0.05)。结论:自建生物荧光技术测定LicC活性准确、可靠;证实LicC3′端25个氨基酸对其活性有重要影响;提示抑制LicC活性有可能成为一种有效的杀灭耐药肺炎链球菌的措施。     

肺炎链球菌抗生素敏感性研究

肺炎链球菌抗生素敏感性研究解静平，曹玉璞，薛冰，袁林，陈树生，廖冰肺炎链球菌是小儿细菌性肺炎的第一位致病菌〔１〕。１９８８年秋至１９８９年冬本室及广州市儿童医院检验科对肺炎患儿鼻咽分泌物进行了细菌分离鉴定及肺炎链球菌抗生素敏感试验－应用纸片扩散法及最...     

肺炎链球菌外膜蛋白PspA和PsaA的免疫原性比较

目的 比较肺炎链球菌(Streptococcus pneumoniae)表面黏附素A(PsaA)和表面蛋白A(PspA)的免疫原性.方法 电泳分析Sp6B亚型菌株的两种外膜蛋白基因及所编码蛋白相对分子质量的可变性,采用Western blot方法比较两种重组外膜蛋白对5种亚型菌株相应蛋白抗血清的交叉反应,采用酶联免疫吸附法(ELISA)分析两种外膜蛋白的抗体类型和抗体水平以及抗血清对5种亚型菌株的亲和性,以小鼠保护实验比较两种蛋白对5种亚型菌株的交叉保护作用.结果 两种蛋白产生的抗体亚型水平相当;PspA蛋白与其他亚型蛋白的交叉反应广泛性不如PsaA蛋白,只限于同一支系的蛋白之间;PspA抗体与病原菌具有可接近性,并具备交叉免疫保护作用.可作为一种更有效的抗原成分.结论 PspA对同家族同支系菌株攻击具有交叉免疫保护作用,可作为一种更有效的抗原成分.     

重组白细胞介素-17A鼻黏膜免疫抗肺炎链球菌感染的实验研究

目的 观察小鼠重组白细胞介素-17A (rIL-17A)鼻腔黏膜免疫对感染肺炎链球菌(Streptococcus pneumoniae,Sp)小鼠防御素β-2(Defb2)、巨噬细胞炎症蛋白(MIP)等表达的影响,探讨rIL-17A抗肺炎链球菌感染的机制.方法 SPF级BALB/c小鼠随机分为3组:肺炎组、干预组、对照组,以鼻腔接种的方法建立肺炎模型,采用鼻黏膜免疫的方法进行rIL-17A干预.以real-time荧光定量PCR法检测肺组织Defb2、MIP-1α、MIP-2β mRNA的表达,ELISA法检测支气管肺泡灌洗液(BALF)、脾细胞以及纵膈淋巴结细胞培养上清中MIP-1α、MIP-2β、IFN-γ、IL-4的浓度,并对BALF进行白细胞分类计数和Sp菌落计数,对肺组织进行病理分析.结果 rIL-17A干预组BALF中Sp菌落数较肺炎组明显降低(2.18±0.94 vs 4.37+0.57,P＜0.01),中性粒细胞及巨噬细胞数量显著高于肺炎组(P＜0.01);干预组肺组织Defb2、MIP-1α mRNA表达上调,其中Defb2 mRNA表达量为对照组的53.93倍,与肺炎组比较差异有统计学意义(53.93±4.80 vs 14.49±5.84,P＜0.01);rILL-17A干预组与肺炎组比较,淋巴结细胞培养上清中MIP-1α浓度增高明显(431.80±31.57 vs 291.10±5.62,P＜0.01);MIP-2β浓度在脾细胞和淋巴结细胞培养上清中较肺炎组显著增高(246.20±11.50 vs 183.70±10.64,508.50+20.26 vs 290.90+15.20,P＜0.01),而肺泡灌洗液中浓度变化不显著(P＞0.05);IFN-γ浓度在BALF和细胞培养上清中均较肺炎组显著升高;ILM除淋巴结细胞培养上清中外,BALF和脾细胞培养上清中均较肺炎组显著升高(92.42±3.82 vs 80.68±4.83,106.80±8.07 vs 73.57+7.43,P＜0.01);干预组与肺炎组小鼠支气管及血管周围炎症细胞浸润无显著差异,但干预组组织损伤较轻.结论rIL-17A可通过促进Ddb2以及MIP、IFN-y、IL-4等炎症因子的表达,增加炎症部位白细胞募集,提高Sp清除率来增强肺炎小鼠的抗感染能力.     

Protection against Streptococcus pneumoniae Elicited by Immunization with Pneumolysin and CbpA

The need for the development of cheap and effective vaccines against pneumococcal disease has necessitated the evaluation of common virulence-associated proteins of Streptococcus pneumoniae as potential vaccine antigens. In this study, we examined the capacity of active immunization with a genetic toxoid derivative of pneumolysin (PdB) and/or a fragment of choline binding protein A (CbpA; also known as PspC, Hic, and SpsA) to protect mice from intraperitoneal challenge with medium to very high doses of a highly virulent capsular type 2 pneumococcal strain, D39. The median survival times for mice immunized with the individual protein antigens in different adjuvant combinations were significantly longer than those for mice that received the respective adjuvants alone. Mice immunized with CbpA alone were significantly better protected than mice immunized with PdB alone. Correspondingly, the median survival times for mice that were immunized with a combination of PdB and CbpA were significantly longer than those for mice that received PdB alone but not significantly different from those that received CbpA alone. Mice immunized with the protein antigens in a mixture of monophospholipid A (MPL) and aluminium phosphate (AlPO4) adjuvants had higher antibody titers than mice that received the antigens in AlPO4 alone. Mice immunized with PdB in MPL plus AlPO4 were also significantly better protected than mice that received PdB in AlPO4 alone.     

Analysis of serum cross-reactivity and cross-protection elicited by immunization with DNA vaccines against Streptococcus pneumoniae expressing PspA fragments from different clades

Streptococcus pneumoniae is a major cause of disease, especially in developing countries, and cost-effective alternatives to the currently licensed vaccines are needed. We constructed DNA vaccines based on pneumococcal surface protein A (PspA), an antigen shown to induce protection against pneumococcal bacteremia. PspA fragments can be divided into three families, which can be subdivided into six clades, on the basis of PspA amino acid sequence divergence (S. K. Hollingshead, R. Becker, and D. E. Briles, Infect. Immun. 68:5889-5900, 2000). Since most clinical isolates belong to family 1 or family 2, PspA fragments from members of both of these families were analyzed. Vectors encoding the complete N-terminal regions of PspAs elicited significant humoral responses, and cross-reactivity was mainly restricted to the same family. DNA vaccines encoding fusions between PspA fragments from family 1 and family 2 were also constructed and were able to broaden the cross-reactivity, with induction of antibodies that showed reactions with members of both families. At least for the pneumococcal strains tested, the cross-reactivity of antibodies was not reflected in cross-protection. Animals immunized with DNA vaccines expressing the complete N-terminal regions of PspA fragments were protected only against intraperitoneal challenge with a strain expressing PspA from the same clade.     

Protection against Shiga toxin-producing Escherichia coli infection by transcutaneous immunization with Shiga toxin subunit B

Enterohemorrhagic Escherichia coli (EHEC) strains are important human food-borne pathogens. EHEC strains elaborate potent Shiga toxins (Stx1, and/or Stx2) implicated in the development of hemorrhagic colitis (HC) or hemolytic-uremic syndrome (HUS). In this report, we evaluated the immunogenicity and protective efficacy of Stx1 subunit B (StxB1) administered by transcutaneous immunization (TCI). Three groups of Dutch Belted rabbits received patches containing StxB1, StxB1 in combination with Escherichia coli heat-labile enterotoxin (LT), or LT alone. An additional group of naïve rabbits served as controls. The protective efficacy following TCI with StxB1 was assessed by challenging rabbits with a virulent Stx1-producing strain, RDEC-H19A, capable of inducing HC and HUS in rabbits. Antibodies specific to StxB1 from serum and bile samples were determined by enzyme-linked immunosorbent assay and toxin neutralization test. Rabbits immunized with StxB1 demonstrated improved weight gain and reduced Stx-induced histopathology. Rabbits receiving StxB or StxB1/LT showed a significant increase in serum immunoglobulin G titers specific to StxB1 as well as toxin neutralization titers. These data demonstrated that the StxB delivered by TCI could induce significant systemic immune responses. Thus, Stx subunit B vaccine delivered by a patch for a high-risk population may be a practical approach to prevent (and/or reduce) Stx-induced pathology.     

Protection against experimental Bordetella bronchiseptica infection in mice by active immunization with killed vaccine.

Mice immunized with a killed vaccine of phase I Bordetella bronchiseptica were challenged with various numbers of virulent B. bronchiseptica by intraperitoneal, intracerebral, or intranasal routes. The course of infection was compared among these routes, and the protective effect of vaccination was quantitatively analyzed. In ddN mice infected intraperitoneally with 1.8 X 10(8) cells (ca. 80 times the 50% lethal dose [LD50]) the organisms rapidly increased in the intraperitoneal fluid, spleen, and liver within few days and caused splenic atrophy, septicemia, and death. However, immunizations with 5 X 10(9) cells gave the mice a high agglutinin titer and suppressed the increase in the number of organisms. With four immunizations, the lungs and livers were clear within 3 days, and with one or two immunizations, they were clear within 7 days. These immunizations effectively protected the mice from death but did not protect them from splenic atrophy. In the intracerebral infection with 1.4 X 10(6) cells (ca. 1.2 X 10(5) LD50), the number of organisms rapidly increased in the brain and caused encephalitis, splenic atrophy, and death. However, four or five immunizations completely suppressed the increase in the brain and protected the mice from death and splenic atrophy. After intranasal infection with 4 X 10(6) cells (ca. 25 LD50), the organisms rapidly increased in the nasal cavity and lungs and caused pneumonia and death. Immunization with 5 X 10(9) cells was effective in clearing the organisms from the lungs and in protecting against death and splenic atrophy. However, the organisms were not cleared from the nasal cavity for 60 to 150 days after the challenge with as little as 10(2) cells, even in the mice with an agglutinin titer as high as 1:10,000.     

Protective DNA immunization against Chlamydia pneumoniae

We have investigated the efficacy of the DNA vaccination using the heat shock protein 60 (HSP-60) gene of C. pneumoniae, for protection of mice against infection with the bacteria. C57Bl/6 mice had a 5-20-fold reduction of C. pneumoniae numbers in lungs when immunized intranasally (i.n.) with plasmids (p) encoding pHSP-60. The reduction of the bacterial load coincided with a decreased severity of disease. No specific antibodies were detected after protective i. n. immunization. In contrast, mice immunized intradermally (i.d.) were not protected against challenge with C. pneumoniae, although specific humoral Immunoglobulin (Ig)G responses were generated. Co-inoculation i.n. of pHSP-60 with pIL-12 but not with pGM-CSF further increased protection of mice against infection with C. pneumoniae. Lungs from pHSP-60 i.n. immunized and infected mice showed higher levels of interferon (IFN)-gamma mRNA, and spleen cells from these mice co-cultured with r-HSP-60 released higher levels of IFN-gamma and displayed higher proliferative responses than nonimmunized and infected controls. pHSP-60 immunized IFN-gamma receptor (R)-/- mice were not protected against infection with C. pneumoniae. Likewise, i.n. administration of pIFN-gamma alone induced significant protection. DNA vaccine-induced protection was CD4+ and CD8+ T-cell dependent, as shown by DNA-vaccination of MHC class II-/-, CD4-/-, CD8-/- and CD4-/-CD8-/-mice. Interestingly, DNA vaccine induced CD4+ T cells, in the absence of CD8+ T cells, were involved in worsening the outcome of infection. This worsening was linked with a shift towards a Th2 cytokine pattern.     

Protection against experimental Pseudomonas aeruginosa infection in mice by active immunization with exotoxin A toxoids.

The immunoprophylactic effect of chemically inactivated Pseudomonas aeruginosa exotoxin A in experimental pseudomonas infections was studied. Exotoxin A toxoids were prepared by Formalin (f-TXD) or glutaraldehyde (g-TXD) treatment. Immunization of mice with three or four doses (10 micrograms each) of f-TXD and the synthetic adjuvant N-acetylmuramyl-L-alanyl-D-isoglutamine (50 micrograms) induced high levels of antiexotoxin A antibodies as measured by passive hemagglutination assay and enzyme-linked immunosorbent assay. Immunization with toxoid alone did not elicit antitoxin. A significant increase in survival time and survival rate (P less than 0.01) was seen in immunized (f-TXD) and in burned and infected mice (50 to 85%) as compared with control mice immunized with formalinized bovine serum albumin (6 to 20%). Virtually 100% survival was obtained when preinfection immunization weas combined with single-dose gentamicin treatment within 24 h of infection. Immunization with g-TXD increased survival time (P less than 0.01) but did not consistently increase survival rate, and the results were not as satisfactory as those with formalinized exotoxin. The data presented indicate that active immunization with formalinized exotoxin A toxoid and adjuvant induced protective immunity to various degrees against infections in mice and could be potentially useful in prophylaxis of P. aeruginosa infections.     

Protection against Schistosoma mansoni achieved by immunization with sonicated parasite

Partial protection of (C57BL/6 X DBA/2)F1 mice against infection with Schistosoma mansoni has been achieved by immunization with small amounts (0.2 to 2.0 microgram) of crude cercarial sonicate adsorbed on aluminium hydroxide gel adjuvant (alum). A decrease of 34-90% in the adult worm burden, of the immunized mice, as compared to that of untreated mice or those injected with adjuvant alone, has been found in five experiments by liver perfusion six weeks after percutaneous challenge infection. High titers of anti-cercarial IgE antibodies have been found in the sera of the immunized mice by two independent techniques, radioimmunoassay and degranulation of rat basophilic leukemia cells (RBL-2H3), determined by 3H-serotonin release. By counting the live worms in the lungs of immunized and uniummized mice on days 4-7 after infection it was observed that the schistosomula were killed before they reached the lungs, probably at the skin. Mice immunized with the same amounts of cercarial antigen in Freund's complete adjuvant were not protected against infection with S. mansoni. These animals developed high titer of total anti-cercaria antibodies (determined by radioimmunoassay) but had low levels of antiparasite IgE. The possible role of IgE in protective immunity is discussed.     

Protection against infection with Neisseria meningitidis group B serotype 2b by passive immunization with serotype-specific monoclonal antibody

Hybridomas derived from mice immunized with Neisseria meningitidis serogroup B serotype 2b (B,2b) outer membrane preparations produced monoclonal antibodies (MAbs) specific for major outer membrane proteins of classes 1, 2, and 5. The MAbs were examined by enzyme-linked immunosorbent assay against a selected panel of seven strains of N. meningitidis (B,2b) of different sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns, a serotype 2a, and a nontypable strain. The five MAbs selected were all bactericidal and of different immunoglobulin subclasses. None of the MAbs reacted with other bacterial strains in a dot-enzyme immunoassay. The corresponding antigenic determinant for each MAb was localized on a specific outer membrane protein by immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of major outer membrane proteins. MAbs M5-11 and M5-30 bound to the class 2 protein and were serotype 2b specific. MAb M2-20 bound to the class 1 protein, and MAbs M5-16 and M5-19 bound to the class 5 protein. A mouse model of infection was established whereby a local infection progressed to lethal bacteremia over 3 days, and 50% of the animals were killed with an intraperitoneal injection of 10 meningococci plus 4% mucin and 1.6% hemoglobin. The ability of the MAbs to provide passive protection against experimental infection with N. meningitidis (B,2b) was examined. Both serotype-specific MAbs M5-11 and M5-30 were highly protective even though they were of different immunoglobulin subclasses. The class 5-specific MAb offered no protection, while the class 1-specific MAb gave limited protection. It may therefore be possible to provide protection against serotype 2b infection by using as vaccine the class 2 serotype-specific surface-exposed outer membrane protein epitopes defined by MAb M5-11 or M5-30.     

Protection against influenza A virus infection in mice by oral immunization with a polyvalent bacterial lysate.

A study is presented which investigated whether oral immunization with a polyvalent bacterial lysate (Paspat oral) can sufficiently enhance cell-mediated defense mechanisms to protect mice against influenza A virus infection. It was found that oral immunization reduced mortality due to influenza A infection with 15-70%, depending on the quantity of virus administered and and the moment of infection. Cyclosporin A severely reduced the protective effect of oral immunization, suggesting that a major effect of oral immunization in these studies is T-cell activation. The effect of oral immunization on macrophageal activity was evaluated by measuring cyclic-AMP in alveolar macrophages (AMs) obtained by bronchoalveolar lavage. Before infection, basal activity levels of AMs in immunized mice were significantly lower than in controls. Five days after infection, however, basal activity level of AMs in immunized mice was significantly higher than AM activity in controls. Stimulation of AMs with PGE2 significantly reduced cellular activity in both groups, before and after infection. However, cellular activity of AMs from immunized animals was less reduced than cellular activity of control macrophages. Activity of AMs of immunized animals was significantly more reduced by histamine than activity of control macrophages. It is concluded that oral immunization with Paspat oral stimulates T-cell-dependent immune mechanisms, resulting in protection against influenza A virus infection in mice.