Anti-Plasmodium falciparum antibodies acquired by residents in a holoendemic area of Liberia during development of clinical immunity

Sera from 48 children and adolescents (2-15 years of age), residing in a malaria holoendemic area of Liberia were investigated for specificities and isotypes of anti-P. falciparum antibodies. No clear-cut relationship to the development of clinical immunity was found when the overall antibody activities to total parasite antigens were determined by enzyme-linked immunosorbent assay (ELISA). Although there was a certain rise of IgM, total IgG- and IgG2 antibody activities, this was most pronounced at ages when a clinical but nonsterile immunity is already present. When the sera were investigated by immunoprecipitation of 35S-methionine labeled parasite polypeptides, the total number of parasite antigens precipitated was similar at all ages. Analysis by indirect immunofluorescence (IFA), registering antibodies to intracellular parasite antigens, revealed no age-dependent changes in antibody titers. In contrast, when the sera were assayed by a novel IFA, specific for a restricted number of parasite antigens in the membrane of infected erythrocytes, the frequency of positive sera as well as the anti-P. falciparum titers rose in parallel with the development of clinical immunity. Thus, these antigens appeared to be important inducers of protective immune responses and may be suitable candidates for a vaccine against the asexual blood stages of P. falciparum.     

Cultured Plasmodium falciparum used as antigen in a malaria indirect fluorescent antibody test.

Human sera obtained from persons infected with Plasmodium falciparum were tested by a standard indirect fluorescent antibody (IFA) technique using antigen obtained from long term in vitro cultures of two strains of P. falciparum, and antibody in high titer was reproducibly detected. Sera from uninfected persons had undetectable or very low titers of antibody. The use of cultured parasites offers a convenient, stable source of antigens from different P. falciparum strains without requiring their adaptation to primates. Differences observed in IFA titers obtained by reacting immune serum with two different P. falciparum strains suggests the need for further evaluation of strain specificity.     

大肠埃希菌表达的恶性疟原虫裂殖子表面蛋白MSP1-42的免疫原性

目的纯化大肠埃希菌Rosettagami（DE3）表达的可溶性裂殖子表面蛋白1C末端蛋白（MSP1-42,3D7株）,检测其体外抑制恶性疟原虫生长的效果。方法利用大肠埃希菌Rosettagami（DE3）为宿主菌诱导表达MSP1-42,用带组氨酸标签的镍柱纯化可溶性的MSP1-42。6只新西兰白兔随机分为免疫组和对照组,每组3只。用MSP142与弗氏佐剂乳化后,皮下注射,抗原免疫剂量每次为200“g／只,共免疫4次,每次间隔2周。佐剂对照组以PBS代替抗原同法免疫。免疫前及末次免疫2周后取血,用酶联免疫吸附试验和间接荧光抗体试验检测血清中特异性抗体及其与天然抗原的反应,用含10％和20％免疫血清的培养基体外培养恶性疟原虫（海南分离株,Fcc1／HN）,检测免疫血清体外抑制恶性疟原虫生长的效果。结果经镍柱纯化后获得纯度为95％以上的MSP1-42蛋白;免疫组个体在第4次免疫后血清特异性抗体的滴度依次为1：640000、1：640000、1：160000,间接荧光抗体试验检测表明MSP1-42免疫兔血清与恶性疟原虫表面蛋白有阳性反应;免疫血清能抑制恶性疟原虫在体外生长,在10％浓度时,3只免疫兔血清的抑制率依次为（51．94-24．2）％、（29．4±8．6）％和（86．7±7．4）％,在20％浓度时,抑制率分别为（93．3±7．5）％、（65．3±10．6）％和（96．4±1．0）％。结论大肠埃希菌Rosettagami（DE3）表达的MSP142蛋白免疫血清能识别恶性疟原虫天然抗原,体外培养能抑制恶性疟原虫的生长。     

Comparison of inducers of crisis forms in Plasmodium falciparum in vitro

A variety of known or suspected inducers of crisis form parasites in cultivated Plasmodium falciparum were examined. Sera from Sudanese residents of malaria-endemic areas, sera from American tuberculosis patients, and rabbit sera containing tumor necrosis factor were assayed in vitro for cytotoxic activities against P. falciparum and mouse L-M cell cultures. Inhibition was determined by measurement of incorporation of radiolabeled nucleic acid precursors. When compared to normal serum, parasites grown in the presence of a 1:4 dilution of rabbit sera containing tumor necrosis factor, TB patient sera, or Sudanese sera were metabolically inhibited 73%, 75%, and 95%, respectively. However, only the rabbit sera containing tumor necrosis factor were cytotoxic to L-M cells, inhibiting radiolabel incorporation by 80% at a 1:1,000 serum dilution. These findings suggest that tumor necrosis factor is apparently not responsible for the induction of parasite crisis forms by the inhibitory human sera tested. In addition, human gamma-interferon had no effect on parasite growth.     

Tumour necrosis factor and interleukin-6 production induced by components associated with merozoite proteins of Plasmodium falciparum

P. falciparum merozoite antigens, merozoite surface protein-l (MSP-1) and rhoptry associated protein-1 (RAP-1), were shown to be liberated into the supernatant of in vitro parasite cultures and to be included in the endotoxin-like exoantigen complex, previously designated Ag7. Material affinity purified from culture supernatants, using immobilized monoclonal antibodies specific for RAP-1 or MSP-1, stimulated normal human mononuclear cells to produce TNF and IL-6 in vitro. However, stimulation of TNF was absent, and that of IL-6 was reduced, when the antigens were purified from detergent extracts of infected erythro-cytes. These results indicate that the RAP-1 and MSP-1 proteins themselves do not stimulate the production of TNF. Instead, other components associating with these exoantigens may be responsible for the TNF production. Mouse antisera blocking TNF production stimulated by P. yoelii exoantigens also blocked TNF production stimulated by material affinity purified from P. falciparum culture supernatants using RAP-1 specific monoclonal antibody, indicating the conserved structure of the TNF inducing component.     

The presence of liver auto-antibodies induced by Entamoeba histolytica in the sera from both naturally infected humans and immunized rabbits.

Auto-antibodies against normal human liver have been detected in the sera of humans with highly positive indirect hemagglutination (IHA) amebiasis titers and with clinically-proven amebic liver abscess. Sera of amebiasis patients and rabbits immunized with killed Entamoeba histolytica were tested for anti-amebic antibodies by the IHA test and for auto-antibodies by the complement fixation test, using the antigens prepared from extracts of human liver and rabbit liver. A direct correlation was found to exist between high anti-Entamoeba antibody titers and the presence of anti-liver antibody in the serum. It is proposed that, in addition to direct parasite damage to host tissue, immunological damage could result from the attachment of circulating antigen to the cell surfaces of host tissues such as the liver.     

抗恶性疟重组复合抗原的免疫血清对恶性疟原虫体外生长的抑制作用

本文初步观察了兔抗两种重组恶性疟复合抗原（C和CAC）的免疫血清对恶性疟原虫的体外抑制作用。实验结果显示：抗C和抗CAC两种免疫血清对疟原虫体外生长均有明显抑制作用，但抗CAC血清的抑制效果更明显（P＜0．05）。抑制程度随培养物中免疫血清浓度的增高及作用时间的延长而增强，其中抗CAC免疫血清在1％、10％和20％浓度时对疟原虫第2增殖周期（72h）的抑制率分别可达15％、54％和82％。免疫血清作用后较多原虫呈现发育不良及裂殖子凝集和成熟原虫退变现象，提示可能与复合抗原中的多个保护性抗原位点所产生的多功能抗体有关。     

Comparison of some methods for the detection of antigen-antibody reactions in amoebiasis.

The indirect hemagglutination test (IHA) and counterimmuno-electrophoresis (CIE) were used to test for antibodies to Entamoeba histolytica in human sera using antigens from axenic cultures of the HK9 strain. Correlation between the test was excellent with most sera from patients with amoebiasis demonstrating precipitin lines by CIE at IHA titers considered diagnostically positive, larger than or equal to 1:128. Precipitin reactions with positive sera were also compared by CIE, immunoelectrophoresis (IEP) and two-dimensional electrophoresis is (2D-IEP). The greatest number of antigen-antibody components were demonstrated by 2D-IEP. Further studies utilizing 2D-IEP should be of value in the antigenic analysis of E. histolytica.     

Natural antibodies against three distinct and defined antigens of Plasmodium falciparum in residents of a mesoendemic area in Gabon

The magnitude of the antibody response to three distinct and defined antigens of Plasmodium falciparum was assessed in 144 inhabitants of the Kassa district (Haut Ogooué Province, Gabon), a region where malaria is mesoendemic. Antibodies against a polypeptide consisting of 40 (Asn-Ala-Asn-Pro) repeats of P. falciparum circumsporozoite protein [(NANP)40] were detected by ELISA. Antibodies against the fusion peptide 31.1, which consists of the N-terminal portion of the 190-200 kDa glycoprotein, were also detected by ELISA. Antibodies against ring-infected erythrocyte surface antigens (RESA), mainly the P. falciparum 155 kDa antigen (Pf 155), were detected by IFA on glutaraldehyde-fixed P. falciparum infected red blood cells (IRBC). In addition, a standard IFA employing air-dried P. falciparum IRBC was used to detect antibodies against intraerythrocytic asexual forms. Parasitemia and spleen enlargement were also recorded. The frequency of sera positive for specific antibodies increased with age for all the antigens tested. Plateau antibody levels were reached at different ages for the different antigens. Individual antibody responses varied in titer to the different antigens. Subjects with parasite-negative thick smears showed higher titers of anti-31.1 as well as an increased frequency of anti-RESA antibodies compared to subjects having positive smears. No differences in the titer and in the prevalence of anti-(NANP)40 antibodies were found between these groups. The results suggest that the antibody response against asexual blood stage antigens, especially anti-RESA and anti-31.1, may play a role in controlling parasitemia.     

Use of antigen preparations of the amastigote stage of Trypanosoma cruzi in the serology of Chagas' disease

Antigens derived from the amastigote or the epimastigote stages of Trypanosoma cruzi and prepared by sonication or formalinization were examined and compared in the immunofluorescent antibody (IFA) test and enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to the parasite in sera of infected humans. The results revealed that antigens derived of amastigotes from cell cultures can be used for the detection of antibodies to T. cruzi in both tests. In the IFA test, 46.3% of the sera had higher titers with amastigote antigens, 41.5% had equal titers with antigens from both stages, and 12.2% had higher titers with epimastigote antigens. In the ELISA, 43.9% had higher titers with amastigote antigens, 43.9% had equal titers with antigens of both stages and 12.2% had higher titers with the epimastigote antigens. The differences in titers, however, were not of a magnitude sufficient to indicate a higher sensitivity for the amastigote antigens. The ELISA was performed successfully with formalinized or sonicated organisms of both stages. The use of formalinized parasites introduces a new advantage over previously reported ELISA methods. Formalinized antigens are easier to prepare and can be stored for prolonged periods of time at 4 degrees C. The reason for the higher titers with amastigote antigens was examined by using 125I-labeled antigens to determine the binding of antigens to the plastic solid-phase used in the ELISA and to compare the antigens of each stage that could be immunoprecipitated by antibodies to T. cruzi. The ability of amastigote antigens to bind to the plastic solid phase appeared to be slightly higher than that of epimastigote antigens although the differences were not statistically significant. On the other hand, the amount of antigens in the amastigote preparation immunoprecipitated by antibodies in 6 of 10 sera examined was higher than the amount of antigens in the epimastigote preparation immunoprecipitated by antibodies in the same sera. In two sera the amount was similar, and in the remainder more antigens in the epimastigote antigen preparation were immunoprecipitated. These results are of interest and may suggest clinical implications.     

Anti-sporozoite antibodies induced by natural infection

Serum samples from 120 individuals living in a malaria-endemic area, 31 patients with Plasmodium falciparum infection, and 58 healthy blood donors were tested for antibodies against P. falciparum and P. vivax sporozoites. Specific antibodies were determined by the circumsporozoite precipitation (CSP) reaction and indirect immunofluorescent (IFA) tests for IgG and IgM antibodies. It was found that a high proportion of adults living in the endemic area had IFA anti-sporozoite antibodies, usually IgG. Children and healthy donors were either negative or had low antibody titers. A positive correlation was found between IgG antibody titers against P. falciparum sporozoites and those against P. vivax sporozoites. CSP reactivity was demonstrated in 5 of 31 sera from patients with falciparum malaria, and was always associated with a high level of IFA antibodies. The anti-sporozoite antibodies were found to be stage- and species-specific.     

Comparison of Dot-ELISA with Sandwich-ELISA for the detection of circulating antigens in patients with bancroftian filariasis

We compared the performance of a newly developed Dot-ELISA with that of a previously described Sandwich-ELISA to detect parasite antigens in sera from patients with bancroftian filariasis. The same monoclonal antibody and the same sera were used in both tests. In the Dot-ELISA, 67 of 70 sera from microfilaremic donors were deemed to contain filarial antigens when screened at a dilution of 1:50. End titers were 1:80-1:1280. With the Sandwich-ELISA, 64 of the same sera were positive at a dilution of 1:10 and 42 were positive at a dilution of 1:50. End titers were 1:10-1:320. The specificity of both assays was greater than 95%, but their sensitivity was remarkably different. The Dot-ELISA could detect as little as 0.055 ng/ml microfilarial antigen added to normal human sera, whereas the lower limit with the Sandwich-ELISA was 10 ng/ml parasite antigen. Additionally, the Dot-ELISA does not require radioactivity or sophisticated equipment and, therefore, can be performed in virtually all filariasis-endemic areas.     

华支睾吸虫富甘氨酸2a类抗原Cs4重组蛋白的免疫学特性与定位

目的 对华支睾吸虫富甘氨酸2a(glycine rich antigen 2a,GRA2a)类抗原Cs4重组蛋白(rCs4)进行免疫学特性分析,并对GKA2a类抗原进行组织定位.方法 使用蛋白质印迹(Western印迹)分析rCs4与华支睾吸虫病患者混合血清的免疫反应性,并分析GRA2a类抗原的分泌性.应用ELISA法检测rCs4免疫小鼠血清中抗rCs4特异性IgG水平,并动态检测华支睾吸虫感染兔0～44周的血清中抗rCs4特异性IgG水平.通过免疫荧光实验(immunofluorescence assay,IFA)对华支睾吸虫GRA2a类抗原进行组织定位.结果 纯化的rCsd可被华支睾吸虫病患者混合血清识别.rCs4单抗(mAb Cs4-31)与华支睾吸虫排泄分泌抗原和可溶性抗原在相对分子质量(Mr)26 000与55 000之间分别有13、15个反应条带,均呈阶梯状分布.ELISA检测rCs4免疫小鼠抗rCs4和ESA的IgG效价均达1:64 000,感染兔的抗rCs4特异性抗体平均水平自感染第4周开始上升,于第6周达到高峰,此后逐步递减,至第20周已呈较低水平.IFA检测发现GRA2a类抗原定位于华支睾吸虫成虫的表皮.结论 Cs4蛋白是华支睾吸虫在感染早期的分泌型蛋白,具较好的抗原性.GRA2a类抗原定位于华支睾吸虫成虫表皮.

Abstract：

Objective To study immunological characteristics of Cs4 recombinant protein(rCs4)of Clonorchis sinensis,one of the glycine rich antigen 2a(GRA2a)gene family member,and the histolocalization of GRA2a antigens. Methods The immunological reactivity of Clonorcihs sinensis Cs4 protein to pooled sera from clonorchiasis patients was investigated by using purified rCs4 in Western blotting.The secretory property of GRA2a antigens was also analyzed by Western blotting.The level of specific antibodies against rCs4 in the sera from mice immunized with rCs4,and the dynamic change of antibodies against rCs4 in the sera of C. sinensisinfected New Zealand rabbits(O-44 w post infection)were examined by ELISA.Immunofluorescence assay (IFA)was performed using the anti-rCs4 monoclonal antibody Cs4-31(mAb Cs4-31)to determine the localization of GRA2a antigens in C. sinensis adult worm.Results The purified rCs4 was recognized by pooled sera from clonorchiasis patients.13 and 15 bands appeared at the sites between M,26 000 and 55 000 after mAb Cs4-31 reacting with excretory-secretory antigen(ESA)and soluble adult worm antigen(AWA),respectively.The anti-rCs4 and anti-ESA serum antibody titers in mice immunized with rCs4 were 1:64 000.In C. sinensis infected rabbits,antibody level began to elevate at 4 w after infection,peaked at 6 w,and declined rapidly to a lower level at 20 w.IFA revealed that GRA2a antigens were localized in the tegument of C.sinensis adult worm.Conclusion The Cs4 protein was a secretory antigen appeared in the early stage of infection,and exhibited high antigenieity.GRA2a antigens were localized in tIle tegument of C. sinensis adult worm.     

Protection of mice against Schistosoma mansoni infection by passive transfer of sera from infected rabbits

Sera from rabbits infected with unattenuated Schistosoma mansoni cercariae conferred significant levels of protection against S. mansoni challenge ( P  < 0.001) after passive transfer to mice. Infected rabbit sera were only effective in conferring protection when transferred during the first week of infection, and were not effective when administered against liver-stage worms. Immunoglobulins isolated from the infected rabbit sera with Protein A-Sepharose were shown to be responsible for the transfer of protection to mice. Immunofluorescence studies demonstrated that the sera were more reactive against the surface of three hour-old mechanically transformed schistosomula than against the surfaces of lung-stage schistosomula. The sera from infected rabbits reacted polyspecifically against antigens in cercaria, schistosomula, and the worm and egg stages of the S. mansoni life-cycle. The host parasite relationship of S. mansoni in the rabbit is discussed.     

Reactivity of serum from patients with suspected legionellosis against 29 antigens of legionellaceae and Legionella-like organisms by indirect immunofluorescence assay

Sets of sera (444) submitted for diagnostic testing for legionellosis were tested against 29 indirect immunofluorescence assay (IFA) antigens prepared from the characterized Legionella species and Legionella-like organisms to determine the prevalence of antibodies to Legionella organisms. Reciprocal titers of 15% of the serum sets rose fourfold or more to greater than or equal to 128 (indicating seroconversion) against one or more Legionella antigens. The specificity of the test was 96% when evaluated in patients with pneumonia due to non-Legionella organisms. Antibodies were of the IgG, IgM, and (infrequently) IgA classes and were either specific for a single species (as defined by a difference in titer of fourfold or more) or reacted with common Legionella antigens (30 [45%] vs. 36 [55%] of 66 seroconversions, respectively). No single antigen detected half of the positive sera. Elevated IFA titers (of greater than or equal to 256) against single or multiple Legionella antigens occurred in 12% of 184 normal control sera. Therefore, only seroconversions to titers of greater than or equal to 128 should be considered indicative of recent Legionella infection.     

The seroepidemiology of malaria in Middle America. II. Studies on the Pacific coast of Costa Rica.

Serologic studies for malaria using the indirect fluorescent antibody technique suggest that active transmission is either absent or very low in 6 villages on the Pacific side of Costa Rica. Positive titers (1:20 or higher) were seen in the under-15-year age group in three of the study localities, but only 5 such responses were encountered among 249 people examined in this age range. In the adults (15 years and over) from the same 3 villages there were 68 positive titers among 161 examined. There were 43 positive responses in 189 adults from the remaining 3 villages where none of 307 persons under 15 years of age showed a titer of 1:20 or higher to any of the 3 malaria antigens tested (Plasmodium falciparum, P. vivax and P. malariae). These data suggest that the positive responses in the latter villages are more likely to be associated with old or imported cases than with current local transmission. Serologic responses of 1:80 or higher to the P. falciparum antigen suggested the continued presence of this parasite in the population in spite of the paucity of positive blood smears with this species in recent years. Positive titers with the P. malariae antigen suggest that this parasite is probably still present in the area. Such serologic studies help to indicate areas where malaria transmission is active and provide information on parasite reservoirs in particular populations.     

In vitro growth inhibition of Plasmodium falciparum by sera from different regions of the Philippines

Sera from different malaria endemic regions of the Republic of the Philippines were compared for their ability to inhibit growth of Plasmodium falciparum in vitro. Dialyzed serum was added to synchronous cultures containing schizonts for either the total 48 hr test period or only the last 24 hr in order to analyze the effects on erythrocytic invasion and intraerythrocytic growth, respectively. Reduction in 3H-hypoxanthine uptake was used to determine the percent of inhibition compared to nonimmune serum. One hundred seventy sera from Mindanao and Palawan in the South, the centrally located island of Mindoro, and Luzon in the North, were tested against 4 P. falciparum strains from the Philippines and 1 from Africa. Indirect fluorescent antibody titers were not predictive of inhibition. Inhibition of merozoite invasion rather than intraerythrocytic parasite growth is suggested by this study. Generally, sera were more inhibitory to parasite strains from the same geographical area than to those from more remote areas.     

Sharing of antigens between Plasmodium falciparum and Anopheles albimanus

The presence of common antigens between Plasmodium falciparum and Anopheles albimanus was demonstrated. Different groups of rabbits were immunized with: crude extract from female An. albimanus (EAaF), red blood cells infected with Plasmodium falciparum (EPfs), and the SPf66 synthetic malaria vaccine. The rabbit's polyclonal antibodies were evaluated by ELISA, Multiple Antigen Blot Assay (MABA), and immunoblotting. All extracts were immunogenic in rabbits according to these three techniques, when they were evaluated against the homologous antigens. Ten molecules were identified in female mosquitoes and also in P. falciparum antigens by the autologous sera. The electrophoretic pattern by SDS-PAGE was different for the three antigens evaluated. Cross-reactions between An. albimanus and P. falciparum were found by ELISA, MABA, and immunoblotting. Anti-P. falciparum and anti-SPf66 antibodies recognized ten and five components in the EAaF crude extract, respectively. Likewise, immune sera against female An. albimanus identified four molecules in the P. falciparum extract antigen. As far as we know, this is the first work that demonstrates shared antigens between anophelines and malaria parasites. This finding could be useful for diagnosis, vaccines, and the study of physiology of the immune response to malaria.     

Circulating staphylococcal antigen in humans and immune rabbits with endocarditis due to Staphylococcus aureus: inhibition of detection by preexisting antibodies.

A radioimmunoassay for staphylococcal antigen that had detected antigenemia in each of 12 nonimmune rabbits with staphylococcal endocarditis detected antigen in sera from one of nine humans and two of eight immune rabbits with Staphylococcus aureus endocarditis. Staphylococcal antigens could be detected at concentrations as low as 0.78 microgram/ml when diluted in normal rabbit serum, compared with 6.25 microgram/ml when diluted in normal human serum and greater than 25 microgram/ml when diluted in immune rabbit or human serum. Low titers of staphylococcal antibody were found in normal rabbit serum compared with immune rabbit and normal or immune human sera. Detection of staphylococcal antigen was inhibited when the antigen was diluted in the IgG and IgM fractions, but not in the albumin fraction, of normal human serum. This study demonstrated that antigenemia can be detected infrequently in patients and immune rabbits with staphylococcal endocarditis; staphylococcal antibodies inhibit detection of antigen, presumably through formation of immune complexes.     

Expression of Plasmodium falciparum surface antigens in Escherichia coli.

The asexual blood stages of the human malarial parasite Plasmodium falciparum produce many antigens, only some of which are important for protective immunity. Most of the putative protective antigens are believed to be expressed in schizonts and merozoites, the late stages of the asexual cycle. With the aim of cloning and characterizing genes for important parasite antigens, we used late-stage P. falciparum mRNA to construct a library of cDNA sequences inserted in the Escherichia coli expression vector pUC8. Nine thousand clones from the expression library were immunologically screened in situ with serum from Aotus monkeys immune to P. falciparum, and 95 clones expressing parasite antigens were identified. Mice were immunized with lysates from 49 of the bacterial clones that reacted with Aotus sera, and the mouse sera were tested for their reactivity with parasite antigens by indirect immunofluorescence, immunoprecipitation, and immunoblotting assays. Several different P. falciparum antigens were identified by these assays. Indirect immunofluorescence studies of extracellular merozoites showed that three of these antigens appear to be located on the merozoite surface. Thus, we have identified cDNA clones to three different P. falciparum antigens that may be important in protective immunity.