Monoclonal antibodies against diagnostic Anisakis simplex antigens

Five monoclonal antibodies (UA2, UA3, UA5, UA6, and UA8) specific for Anisakis simplex are described. All are IgG1/κ monoclonal antibodies, except for UA2, which is an antibody IgM/κ. The molecular weights of the major components recognized in immunoblotting are 48 and 67 kDa (UA2); 139 kDa (UA3 and UA5; same epitope); 35, 38, and 139 kDa (UA6); and 205 kDa (UA8). UA2 was the only monoclonal antibody to recognize both components of an excretion-secretion antigen preparation and antigens in the excretory cell and esophageal glands of third-stage A.␣simplex larvae; antigens in the excretory cell were also recognized by UA3 and UA6. Cross-reactivity studies using a hyperimmune polyclonal rabbit serum reacting with various ascaridoid nematodes indicated that the antigens captured by our monoclonal antibodies were specific for A. simplex. Finally, comparative studies of our monoclonal antibodies and An2 (the only monoclonal antibody currently available for serodiagnosis of human anisakiasis), based on the calculation of multiples of normal activity for human anisakiasis sera, indicated that our monoclonal antibodies (and particularly UA3) recognized antigens that are good candidates for serodiagnostic purposes. Received: 13 February 1997 / Accepted: 16 March 1997     

Cellular immune responses to salivary antigens in autoimmune liver disease with sicca syndrome.

Twenty-six patients with primary biliary cirrhosis (PBC) and twenty-two with active chronic hepatitis (ACH) were examined for evidence of the sicca syndrome (keratoconjunctivitis sicca, xerostomia). Measurements of tear flow and total saliva flow showed that at least one sicca feature was present in twenty (77%) of the patients with PBC and ten (45%) of those with ACH. Examination of cellular immune responses to a protein fraction of normal human saliva using the leucocyte migration test showed sensitization to the saliva protein in twenty-three of the thirty cases with sicca syndrome but in only two of the eighteen in whom sicca features were not detected. Antisera raised in guinea-pigs against the saliva protein gave specific immunofluorescent staining of bile duct epithelial cells in sections of normal human liver. These findings suggest that damage to structures in the liver may lead to sensitization to various self-antigens which cross-react with other tissues in which a similar disease process may be consequently be initiated.     

Cellular immune responses to recombinant antigens in pregnant women chronically infected with Toxoplasma gondii

The parasite Toxoplasma gondii can infect most mammals and birds, sometimes causing severe pathology. Primary infection during pregnancy can result in abortion or fetal defects. Host immunity, particularly cellular immunity towards antigenic peptides, can control infection, but an efficient vaccine is not yet available. We have evaluated T-cell responses to a crude soluble toxoplasma antigen (ST-Ag) and to five recombinant peptide antigens of cells in whole-blood cultures from 22 pregnant women with preexisting infections and from 7 pregnant negative controls. Cells from all infected patients but from none of the controls responded specifically to ST-Ag by expressing surface CD25 on culture. Responses to the recombinant antigens showed considerable variation between individuals. rGRA1 elicited a response in 16 of the 22 samples (73%), rSAG1 in 13, rGRA7 in 9, rGRA6-CT in 4, and rGRA6-NT in only 1. Most responding cells were CD4(+). Cells from infected subjects cultured with ST-Ag all released high levels of gamma interferon (IFN-gamma) into the culture supernatant (4,343 +/- 2,536 pg/ml). Cells from 12 patients released IFN-gamma after culture with rGRA1 (130 +/- 98 pg/ml), those from 10 patients released it after culture with rSAG1 (183 +/- 128 pg/ml), and those from 4 patients released it after culture with rGRA7 (324 +/- 374 pg/ml). Intensity of IFN-gamma production in response to the latter two recombinant antigens correlated with responses to ST-Ag (r = 0.61 and 0.53, respectively; P < 0.01). Interleukin-4 was always absent from supernatants of cells stimulated with toxoplasma antigens. The heterogeneity of human responses to individual recombinant toxoplasma antigens should be considered in the design of potential vaccines.     

Cellular immune responses of hydatid patients to Echinococcus granulosus antigens.

The reactivity of peripheral blood mononuclear cells (PBMC) from 40 hydatid patients to hydatid fluid (HF) and to two hydatid fractions (pH5PPT and pH5SUP) was evaluated by the incorporation of 3H-methylthymidine into DNA. Maximal responses were detected using 200 micrograms/ml protein after 7-9 days incubation. The three antigen preparations were inducers of PBMC proliferation, with a good correlation (r2 = 0.87) of the responses induced by HF and by the fraction pH5PPT, which contains the two major hydatid antigens (5 and B). Lymphocytes from healthy donors and non-hydatid patients showed no response to these antigens. Neither direct nor inverse correlation was found between the results of the serological tests and of the PBMC proliferation assays. The majority of the patients (75%) responded in serological and in cellular tests. Of the remaining patients, six showed high antibody response associated with a negative PBMC proliferation assay and conversely four seronegative patients were found to respond positively in the PBMC proliferation assay. No relationship of the pattern of immune responsiveness to the patient's clinical forms could be established. Use of the PBMC proliferation assay with hydatid antigens appears rational in those patients which are low antibody producers, but the test is still not to be considered applicable for routine diagnosis.     

Cellular immune response detected in blast transformation test with lymphocytes from rabbits immunized with nucleocapsid of herpes simplex type 2 virus

A preparation of herpes simplex virus type 2 (HSV-2) nucleocapsid (NC) was obtained after treatment of intracellular virus with detergents followed by centrifugation in linear sucrose density gradient. Electron microscopic analysis revealed polymorphism of HSV-2 NC population. Three main types of capsid structures were found: empty capsids, NC containing various amounts of nucleoid material, and NC with complete nucleoid. Morphological heterogeneity correlated with heterogenous sedimentation profile of the total NC preparation of labeled virus represented by light, intermediate, and heavy structures. Specific blasttranformation tests with lymphocytes from rabbits immunized with HSV-2 NC demonstrated differences in the primary and secondary cellular immune responses. The secondary cellular immune response was characterized by a more rapid and strong increase in BTT values than the primary immune response.     

Delayed-type hypersensitivity in mice immunized with Trypanosoma rhodesiense antigens.

When mice were immunized intravenously, subcutaneously, or by the footpad route with formaldehyde-killed Trypanosoma rhodesiense, delayed-type hypersensitivity was elicited by the use of frozen-thawed trypanosomal antigen. The delayed footpad swelling technique was used to measure delayed hypersensitivity. Hypersensitivity induction was dose dependent (greater than or equal to 10(6) formaldehyde-treated T. rhodesiense) and was affected by the route of immunization. The footpad route induced higher levels of hypersensitivity than other routes of immunization. Mice immunized with a single dose of formaldehyde-treated antigen and challenged with live T. rhodesiense did not survive. Yet, mice immunized subcutaneously with formaldehyde-treated antigen and then injected with frozen-thawed antigen and challenged 28 days after immunization survived. The results suggest that T-cell activation, manifested by delayed hypersensitivity responses, was a necessary component in the protective response, perhaps functioning in a helper cell capacity.     

Cellular immune responses of leprosy contacts to fractionated Mycobacterium leprae antigens.

Antigens of armadillo-derived Mycobacterium leprae sonic extract were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane, and the unstained blot was converted into 20 fractions of antigen-bearing particles. These were tested in cellular proliferation assays, and reproducible results were obtained between batches of fractions. Peripheral blood mononuclear cells from healthy contacts of leprosy patients (presumed to have protective immunity) were tested with the fractions to investigate which antigens they recognized. A small group of tuberculoid leprosy patients were also tested. Both groups showed a wide range of responses. Almost every fraction stimulated proliferation with at least one donor, yet none was clearly immunodominant or inhibitory in either group. Thus, protective immunity did not appear to be associated with proliferation caused by any single fraction.     

Cellular immune responses in neonates

Reduced numbers of lymphocytes and antigen presenting cells have been described as some of the main factors responsible for antigenic tolerance or low responsiveness in neonates. However, by changing the parameters of immunization, such as dose of antigen and frequency of antigen presenting cells we and others have shown that neonates have the option of developing the same variety of immune responses seen in adults. Several aspects of the development of cellular immunity in human and murine neonates are reviewed in this article, with a special focus on the development of T cell mediated responses, from ontogeny to effector function.     

重组BCG-Sj26GST疫苗诱导小鼠体液免疫应答的动态观察

目的　检测日本血吸虫重组BCG Sj2 6GST疫苗免疫小鼠不同时间后的血清IgG及其亚类和循环抗原 (circulat ingantigen ,CAg)反应。方法　 1× 10 6克隆形成单位 (colony formingunit,CFU)疫苗皮下和静脉注射免疫BALB/c鼠 ,分别于免疫后 0、4、8、10、14和 16周各剖杀 4只 ,收集血清 ,常规ELISA法检测IgG及其亚类和CAg ,同时设PBS皮下注射对照组。结果皮下注射组IgG在免疫后 10周 ,IgG1在免疫后 14～ 16周 ,IgG2a在免疫后 4周和IgG2b在免疫后 16周达最高水平 ,未能测到CAg ;静脉注射组IgG在免疫后 10周 ,IgG1和IgG2a在免疫后 8周以及IgG2b在免疫后 16周达最高水平 ,CAg在免疫后 14周升高。结论　日本血吸虫重组BCG Sj2 6GST疫苗能诱导宿主产生高水平的IgG、IgG2a和IgG2b ,静脉注射的免疫效果优于皮下注射     

Cellular immune responses in aspergillosis

Diminished platelet aggregation responses to one or more aggregating agents were found in 25 of 32 patients with nasal allergy studied at the peak of the allergy season. Abnormal in-season platelet aggregation induced by epinephrine and collagen was significantly improved when repeated out-of-season, while incomplete platelet aggregation induced by adenosine diphosphate (ADP) and thrombin was unchanged. Recombination of an in-season serum factor with autologous, out-of-season normally aggregating platelets caused marked inhibition of platelet aggregation. Mean bleeding times of 20 symptomatic patients were also prolonged during the height of the pollination season. These data suggest that the allergic diathesis is a model for the study of cyclic, nondrug induction of platelet dysfunction.     

Cellular immune responses in mice challenged with an amyocarditic variant of Coxsackievirus B3

An amyocarditic variant of a temperature-sensitive (ts) mutant derived from the parent myocarditic variant Coxsackievirus B3 (CVB3m) was studied in a murine model of CVB3m-induced myocarditis to assess virus-induced antigens and their possible role in the disease process. Amyocarditic variant ts5R induced a heart tissue antigen(s), extractable by hypertonic KC1, which inhibited migration of peritoneal exudate cells from CVB3-inoculated myocarditic mice in an agarose droplet cell-migration-inhibition assay. The ts5R variant was amyocarditic at inoculum doses of 10(3) to 10(8) plaque-forming units per mouse, but in cyclophosphamide-immunosuppressed mice, ts5R induced myocarditis. Viable ts5R served as a vaccine and protected mice against CVB3m-induced myocarditis. Murine neonatal skin fibroblasts (MNSF) infected with either virus served as in vitro targets and were lysed by splenic cytotoxic T lymphocytes from mice inoculated with either virus variant. ts5R and CVB3m replicated to similar titers in murine neonatal skin fibroblasts (MNSF) at 24 hr postinoculation (pi), but differences in titers were found by 72 hr pi. Levels of natural killer cell activities in spleens of ts5R-inoculated mice were slightly lower than in spleens of CVB3m-inoculated mice at 7 days pi. The data suggest that viral induction of new antigens on target cells and viral induction of specific cytotoxic T lymphocytes that recognize these antigenic changes do not always result in induction of myocarditis.     

Cellular and humoral immune responses of mice subclinically infected with Pneumocystis carinii.

Cellular and humoral immune responses to Pneumocystis carinii were investigated. ICR and DDD mice were intranasally infected with 10(4) mouse lung-derived P. carinii, and the delayed-type hypersensitivity (DTH) reaction and antibody titers to P. carinii were measured along with the number of P. carinii cysts in the lungs after infection. The number of P. carinii cysts in the lungs peaked at 2 weeks after infection and then decreased to barely detectable levels by 4 weeks. Serum antibody (immunoglobulin G) titers measured by indirect immunofluorescence increased up to 4 weeks. The DTH footpad reaction was most prominent at 2 weeks postinfection and declined thereafter. Thus, the decline in the number of P. carinii cysts in the lungs corresponded well with the time of the peak of the DTH reaction but not with the serum antibody response. Spleen T cells from infected mice mediated the DTH reaction when transferred intravenously into normal recipients and reduced the number of P. carinii cysts in the lungs when transferred intravenously into P. carinii-infected mice. The results indicated that cellular immunity is important for protection from subclinical P. carinii infections.     

A phage display system for the identification of novel Anisakis simplex antigens

Anisakis simplex has been recognized as an important cause of disease in man and as a foodborne allergen source. Actually, this food-borne was recently identified as an emerging food safety risk including allergenic symptoms. This parasite contains a large variety of allergenic proteins enforcing the necessity to detect new allergens. Commonly, these efforts have been focused on the developing of cDNA libraries, where virtually all expressed mRNAs are present, by using immunoreactive patient serum or polyclonal antibodies. Phage display system is an alternative strategy which permits the physical binding of the genotype with the phenotype, since the products are expressed by the phage on its surface, thereby allowing more efficient selection. In this work we have constructed two libraries in the pJuFo phage, obtaining a primary titer of around 103 cfu/ml and an amplified titer of the order of 1013 cfu/ml whereas the insert sizes varied from 0.35 to 1.2kb. Both libraries were subsequently analyzed by enrichment with polyclonal antibodies to an A. simplex extract and immunoreactive sera from patients with a clinical history of allergy to this parasite. Finally, 30 clones were scrutinized detecting several Anisakis candidate antigens. Actually, one protein, belongs to the fructose-1,6-bisphosphatase family, was found in 34% of scrutinized clones revealing as a promising novel A. simplex allergen. Phage display technology has to date not yet been applied to the identification of new A. simplex allergens, and the present work opens up new avenues to the understanding of the Anisakis allergenic process.     

Comparison of immune responses of mice immunized with five different Mycobacterium bovis BCG vaccine strains.

Among the various parameters which may contribute to Mycobacterium bovis BCG vaccination efficiency, the choice of the vaccine strain may play an important role. In the present study, we therefore compared the immunogenicity of five different BCG strains that are commonly used for BCG vaccine production (Glaxo 1077, Japanese 172, Pasteur 1173P2, Prague, and Russian strains). The comparison of the growth capacity of these BCG strains in BALB/c and C3H mice demonstrated that a great difference exists between the capacity of various BCG strains to multiply and persist in target organs. A much lower recovery of BCG could be shown in mice immunized with Prague and Japanese BCG strains. T-cell responses of BCG-immunized mice were also examined by analyzing T-cell proliferative responses, cytokine production, delayed-type hypersensitivity responses, and cytotoxic activity. All these assays demonstrated that BCG immunization induced strong CD4+ T-cell responses, mostly of the Th1 type, as demonstrated by interleukin-2 and gamma interferon production. These studies also demonstrated that there are differences between BCG strains in stimulating these T-cell responses. A lack of induction of cytotoxic activity was observed following immunization with the Japanese strain. Lower anti-purified protein derivative antibody responses were also observed after intravenous or oral immunization with this BCG strain. Finally, the protective activity of these BCG strains was tested by measuring the capacity of immunized mice to eliminate recombinant Pasteur and Japanese BCG strains which expressed beta-galactosidase. The results of these experiments clearly demonstrated that the Prague and Japanese strains were unable to protect mice against a second mycobacterial challenge whereas mice immunized with the Glaxo, Pasteur, or Russian strain eliminated the recombinant BCG very efficiently. Altogether, the results of the present study strongly support the view that there are considerable differences in the immunogenicity of various BCG vaccine strains and that these differences may play a major role in BCG vaccination efficiency.     

Cellular immune responses in human neurocysticercosis

Studies on the role of cell-mediated immune responses in human neurocysticercosis (NCC) are lacking. Various cell-mediated immune responses such as lymphocyte subpopulation, lymphocyte transformation to cysticercus antigens and cytokine profile were carried out in NCC patients. Lymphocyte transformation assays using larval antigens showed significantly higher ³H-thymidine uptake. Immunophenotyping analysis showed an insignificant increase in B cells and a decrease in total T cells. However, there was a significant decrease (P < 0.05) in CD8+ T cells whereas there was no change in other cells like CD4+, HLA-DR+ and CD16+/CD56+. Cytokine profile revealed significantly higher (P < 0.01) production of Th1 cytokines (γ-IFN and IL-2) using cysticercal antigens as stimulants for peripheral blood mononuclear cells, while there was no difference in IL-4 levels between NCC patients and healthy controls. The cytokine profile indicated the involvement of Th-1-like responses in NCC patients. Received: 28 October 1999 / Accepted: 9 December 1999     

Cellular immune responses of patients with juvenile chronic arthritis to retinal antigens and their synthetic peptides

The objective of this study was to determine the proliferative responses of peripheral blood lymphocytes of ocular antigens like retinal S-antigen, peptides M and G of S-antigen, yeast histone H3 peptide 106–121 homologous to peptide M and peptide R16 of interphotoreceptor retinoid binding protein (IRBP) in children with juvenile chronic arthritis (JCA). We have studied the in vitro proliferative response of peripheral blood lymphocytes from 41 patients with JCA (10 with and 31 without uveitis) and 23 healthy controls against the above antigens. The responders were retested after 1 or 6 months. Fifty (5/10) and 9.7% (3/31) of JCA patients with and without uveitis, respectively, responded (stimulation index >3) to S-antigen or one of its peptide listed above or yeast histone H3 peptide or R16 of IRBP. None of the healthy controls responded to any of these antigens. The difference in the frequency of responders (SI>3) between JCA associated with uveitis and healthy controls was statistically significant (p=0.001). Similarly, the difference between JCA with and without uveitis was also significant (p=0.013). Our findings suggest that these antigens may have a role in the pathogenesis of uveitis in a subset of patients with JCA.     

Anisakis simplex: from obscure infectious worm to inducer of immune hypersensitivity

Infection of humans with the nematode worm parasite Anisakis simplex was first described in the 1960s in association with the consumption of raw or undercooked fish. During the 1990s it was realized that even the ingestion of dead worms in food fish can cause severe hypersensitivity reactions, that these may be more prevalent than infection itself, and that this outcome could be associated with food preparations previously considered safe. Not only may allergic symptoms arise from infection by the parasites ("gastroallergic anisakiasis"), but true anaphylactic reactions can also occur following exposure to allergens from dead worms by food-borne, airborne, or skin contact routes. This review discusses A. simplex pathogenesis in humans, covering immune hypersensitivity reactions both in the context of a living infection and in terms of exposure to its allergens by other routes. Over the last 20 years, several studies have concentrated on A. simplex antigen characterization and innate as well as adaptive immune response to this parasite. Molecular characterization of Anisakis allergens and isolation of their encoding cDNAs is now an active field of research that should provide improved diagnostic tools in addition to tools with which to enhance our understanding of pathogenesis and controversial aspects of A. simplex allergy. We also discuss the potential relevance of parasite products such as allergens, proteinases, and proteinase inhibitors and the activation of basophils, eosinophils, and mast cells in the induction of A. simplex-related immune hypersensitivity states induced by exposure to the parasite, dead or alive.     

Cellular basis of decreased immune responses to pneumococcal vaccines in aged mice.

Previously, model systems were developed in our laboratory to study murine immune responses to the 23-valent pneumococcal polysaccharide vaccine Pnu-Imune, both in vivo and in vitro (M. Garg and B. Subbarao, Infect. Immun. 60:2329-2336, 1992; M. Garg, A. M. Kaplan, and S. Bondada, J. Immunol. 152: 1589-1596, 1994). Using these systems, we found that aged mice did not respond to the vaccine in vivo or in vitro. Cell separation studies showed that the unresponsiveness of the aged spleen cells to the vaccine was not due to an intrinsic B-cell defect or to T-cell-mediated immunosuppression but resulted from an accessory cell deficiency. Irradiated spleen cells from young mice enabled the old mouse spleen cells to respond to the vaccine. Interestingly, irradiated spleen cells from old mice also restored the vaccine responsiveness in old mice but were required in greater numbers than the young mouse spleen cells to induce similar levels of response. The accessory cell was an adherent cell that could be removed by passage through Sephadex G-10 and thus may be a macrophage. Accessory function could also be provided by the cytokine interleukin-1 (IL-1), IL-4, or IL-5 but not IL-2 or IL-6. Thus, one reason for the deficient immune response to pneumococcal vaccine in aged mice is a quantitative defect in adherent accessory cells.     

Presence of IL-4-like molecules in larval excretory-secretory products and crude extracts from Anisakis simplex

Acute symptoms of anisakidosis are caused by a type I allergic reaction in the gastrointestinal wall with elevated specific immunoglobulin (Ig)E. The purpose of this study was to investigate the presence of interleukin (IL)-4 in the larval antigens of Anisakis simplex. We detected concentrations of IL-4 in pg/ml when larval excretory--secretory products and crude extract from A. simplex were investigated by ELISA. Specific antibodies were obtained by immunization of rabbits with mouse IL-4 and tested in ELISA against A. simplex antigens obtaining higher values of optical density, that were confirmed by western blot analysis. The absorption of these sera with A. simplex antigen resulted in a 70--80% inhibition of antigen binding when retested in ELISA. We demonstrated that A. simplex antigens react with antibodies raised against vertebrate IL-4. The results obtained by us support the hypothesis that A. simplex shares several epitopes with IL-4, important for the Th2 response development in human anisakiasis, where the parasite may modulate the Th1--Th2 dichotomy for its own benefit by mucosal inflammation control in an attempt to avoid the larval expelling.     

Immune responses and resistance to Toxoplasma gondii in mice immunized with antigens of the parasite incorporated into immunostimulating complexes.

Immunostimulating complexes were prepared with antigens extracted from tachyzoites of Toxoplasma gondii and were used to immunize mice. The major antigens incorporated into the immunostimulating complexes were the P30 and P22 antigens and an antigen with an approximate molecular weight of 6,000. Other antigens of molecular weights above 30,000 were also present. High antibody titers to T. gondii antigens and a delayed-type hypersensitivity reaction were noted for the immunized mice. Challenge of these mice with tachyzoites injected interperitoneally or with oocysts administered orally resulted in a statistically significant (P < 0.001) conditional probability of survival compared with that of controls. In contrast, the differences between immunized mice and controls challenged with tissue cysts did not attain statistical significance.