Human anisakiasis: Diversity in antibody response profiles to the changing antigens in larval excretions/secretions

Anisakiasis is an infectious parasitic disease contracted by eating third stage larvae of Anisakis simplex (L3) carried by marine fishes. Human anisakiasis was researched for specific IgG with L3 excretory secretory products (ESP). L3ESP were prepared by daily harvesting of culture supernatant from day 2 to day 5, using two kinds culture media of RPMI-1640 and phosphate buffered saline (PBS). When the sera from persons diagnosed with anisakiasis by means of endoscopy were analyzed using indirect ELISA and Western blot, the sera was classified into four groups depending on specific antigen recognition patterns. In addition, the presence of a new antigen for L3, located at less than 13 kDa (AgLT13) was demonstrated in L3ESP with a modified Western blot condition. The production of AgLT13 was mainly found in L3ESP harvested both on day 2 and day 3, and that in PBS was predominant over that in RPMI-1640. Among those sera, the high reactive sera to L3ESP-day two prepared with phosphate buffer in indirect ELISA recognized AgLT13, and 33% of the low reactive sera did so. These results indicate that the binding pattern of human L3 specific antibody is diverse depending on L3ESP preparations and that AgLT13 demonstrated with a Western blot condition is a specific antigen for L3.     

Age-dependency of serum isotype responses and antigen recognition in human whipworm (Trichuris trichiura) infection

The present study examines the age-dependency of parasite-specific isotype responses and antigen recognition profiles of individuals within a Trichuris trichiura endemic community, in order to evaluate the significance of serum antibodies as determinants of observed age-related patterns of infection intensity. A high degree of individual heterogeneity is observed in isotype responses to separated T. trichiura antigens by Western blot. Recognition by IgG₁ antibodies exhibits marked age-dependency. The age-profiles of IgG₁ responses to selected antigens of 16–17 kDa and 90 kDa molecular weight reflect the age-related changes in current infection intensity at the population level. Similarly, mean age patterns of IgG₂ responses to a 90 kDa antigen, and mean IgG₄ responses to a 16–17 kDa antigen reflect mean infection levels. IgG₃ responses are negligible, and for methodological reasons, both IgE and IgM specificities are not presented. IgA responses to separated antigens of 16–17 kDa and 90 kDa, exhibit age-profiles which may suggest the development of an IgA-mediated acquired resistance to T. trichiura with age. IgA levels remain elevated throughout early adulthood, when infection intensity levels markedly decrease, supporting the hypothesis that IgA antibodies may be significant in generating the convex nature of the age-infection profile of T. trichiura.     

Interrelationship of eosinophilia and IgE antibody production to larval ES antigen in Toxocara canis infected mice

Summary In order to elucidate the relationship between eosinophilia and IgE antibody production in parasitic infection, the degree of eosinophilia and IgE antibody levels to larval ES antigen of Toxocara canis were investigated in eight inbred strains of mice after infection with T. canis. Eosinophilia was far higher in degree in SJL mice than in the other inbred strains. However, IgE antibody levels to larval ES antigen in SJL mice were the lowest among those inbred strains. Similarly, the degree of eosinophilia in AKR mice was slightly higher than those in the other strains except SJL mice, but IgE antibody levels to larval ES antigen in AKR mice was as low as that in SJL mice. Consequently, high responders for eosinophilia were not always high responders for IgE antibody production to larval ES antigen. Inheritance of the two traits, marked eosinophilia and poor IgE antibody production to larval ES antigen in T. canis infected SJL mice, were investigated using Fi hybrid and back-cross mice. The former was regulated by more than one gene, while the latter was a recessive trait. The degree of eosinophilia after infection showed no relationship with the total numbers of larvae recovered from T. canis infected inbred mice.     

Trichuris muris: antigen recognition and transfer of immunity in mice by IgA monoclonal antibodies

Mesenteric node lymphocytes from mice that had been infected with the nematode Trichuris muris, and then boosted with adult worm excretory-secretory antigens were fused with myeloma cells to produce a panel of 9 monoclonal antibodies (MoAbs). Five of the MoAbs were of the IgA isotype. The antigen recognition profiles of these MoAbs were studied using SDS-PAGE and immunoblotting; three major profile patterns were identified. Five MoAbs recognized a major band in the MW range 43-48 kD; all recognized a range of antigens. Three MoAbs were used to localize antigens in the bodies of adult worms. Granules within the anterior stichocytes were recognized strongly, as was material within the eggs and pseudocoelom. Two MoAbs stained the cuticle. Although the phosphorylcholine (PC) determinant was widely distributed within worm tissues none of the MoAbs tested recognized PC. Passive transfer of immunity was achieved using two of the IgA monoclonals; no immunity was transferred by the IgM and IgG MoAbs used. The limited recognition profiles of these IgA MoAbs, and the ability to stain stichocyte granules, suggest that their protective activity results from an interaction with ES antigens.     

IgE antibody during infection with the ovine abomasal nematode, Teladorsagia circumcincta: primary and secondary responses in serum and gastric lymph of sheep

A monoclonal antibody to ovine IgE was employed in an ELISA to investigate the IgE antibody responses in serum and gastric lymph to a primary infection of Teladorsagia circumcincta, and following challenge in previously infected sheep. During a primary response, IgE antibody to antigens derived from the infective third stage (L3) and adult (L5) worms were negligable, with low levels of IgE antibody detected in serum and lymph. In contrast, there was a pronounced IgE antibody response in 2/4 sheep to L3 antigens during 2–8 days after challenge of previously infected animals but low levels of IgE antibody to L5 antigens. This response was confirmed in a second but similar experiment, where relatively high levels of IgE antibody was detected to antigens from L3. Antibody levels were higher in lymph than in serum from the same animals, and Western blots of L3 antigen following SDS-PAGE under reducing conditions revealed several bands of MW26–96KD which reacted with the IgE antibody from gastric lymph. Immunohistochemical staining indicated that these IgE antibodies may be reacting with allergens associated with the surface cuticle of the worms.     

戊型肝炎病毒抗体双抗原夹心法ELISA的建立与初步应用

目的　建立抗戊型肝炎病毒 (HEV)抗体检测的双抗原夹心ELISA(DS -ELISA) ,并应用于多种动物血清的检测。方法　将大肠杆菌表达的一段HEVORF2区重组抗原分别包被微孔板和进行辣根过氧化物酶 (HRP)标记 ,利用 5份阳性血清和 4 0份阴性血清建立双抗原夹心ELISA ;用 4 0 0份义务献血员血清比较双抗原夹心ELISA与间接法IgG抗体ELISA的符合情况 ;用 3只HEV感染猴系列血清比较双抗原夹心ELISA试剂和Genelabs公司HEVIgG试剂 ;用双抗原夹心ELISA试剂检测新疆地区的部分牛、绵羊、山羊、猪血清和上海地区的部分鸡血清中的HEV抗体。结果　建立了检测HEV抗体的双抗原夹心ELISA方法 ,对 4 0 0份义务献血员血清的检测表明其与间接法IgG抗体ELISA试剂的符合情况良好 ,并且有更高的s/co比值 ;与Genelabs公司HEVIgG试剂的比较表明双抗原夹心ELISA试剂的检出更早 ,尤其是持续时间及强度明显优于Genelabs试剂 ;在所检测的各种动物中均发现了HEV抗体 ,其中猪抗体的阳性率最高 ,表明双抗原夹心ELISA试剂可同时用于不同动物的抗HEV抗体检测。结论　利用大肠杆菌表达的HEV重组抗原建立了双抗原夹心法ELISA ,并可同时用于不同动物的抗HEV抗体检测。     

Combination hepatitis C virus antigen and antibody immunoassay as a new tool for early diagnosis of infection

Reduction of the window period of hepatitis C virus (HCV) infection represents an important goal in the transfusional and diagnostic setting. A prototype assay designed to simultaneously detect circulating HCV antigen and anti-HCV, has been developed. Aim of this study was to evaluate the performance of this new assay in terms of specificity and sensitivity and to compare its efficacy with commercial assays. To evaluate the specificity of the assay, 400 samples from the general population and 100 'difficult' sera, negative for anti-HCV, were tested. To assess sensitivity, the new test was used on 76 PCR-positive and anti-HCV negative sera, seven natural or commercial seroconversion panels that included 17 RNA-positive and anti-HCV negative sera and 31 anti-HCV positive sera, 20 weak anti-HCV positive sera, 80 viraemic and anti-HCV-positive sera from patients infected with different subtypes and 10 sera from patients with HBV-HCV or HIV-HCV co-infections. Of 500 anti-HCV negative samples, 499 (99.8%) were negative with a cut-off index <0.5, while one sample was within the grey zone. Of the 93 HCV-RNA positive and anti-HCV negative sera from patients and panels, 85 (91.4%) resulted positive, and one had the cut-off index in the grey zone. The reduction in the diagnostic window period observed with the new test and HCV-RNA assays were equal, on average, to 24 and 34.4 days respectively. All anti-HCV positive sera were positive. The new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or HCV-RNA detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible because of costs, organization, emergency and/or logistic difficulties.     

Immunological relationships during primary infection with Heligmosomoides polygyrus (Nematospiroides dubius): parasite specific IgG1 antibody responses and primary response phenotype

IgG1 antibody responses to Heligmosomoides polygyrus were measured in eight mouse strains supporting acute (< 8 weeks, SJL, SWR), intermediate (10–20 weeks, NIH, BALB/c) or chronic (> 25 weeks, C57BL/0, CBA, C3H, AKR) primary infections. Mice supporting acute or intermediate infections produced more intense antibody responses and total serum IgG1 concentrations were higher than in mice tolerating chronic infections. Positive correlations across mouse strains between the intensity of the antibody response and the percentage loss of worms in weeks 6 and 10 were established. No correlation was found between the response within mouse strains and loss of worms by individual mice. Heavy infections gave marginally higher antibody titres than low intensity infections, but few significant differences were detected and it was concluded that infection intensity did not markedly influence the magnitude of the antibody response. Male and female mice responded similarly despite the earlier loss of worms from females. No association was found between the primary response phenotype and recognition of particular antigens in Western blot analysis, nor did intensity of infection or host gender affect recognition. The possibility that immunomodulatory properties of adult worms may have had a differential influence on ability of strains of contrasting response phenotype to mount IgG1 responses was discussed.     

Characterization of excretory-secretory products of adult Dictyocaulus viviparus and the antibody response to them in infection and vaccination

In vitro released products of the adult stage of the bovine lungworm, Dictyocaulus viviparus, were characterized according to their SDS-PAGE profile, glycosylation pattern, in vitro synthesis and antigenicity in the context of infection and vaccination with irradiated larvae. Biosynthetic labelling experiments with ³⁵S-methionine indicated active synthesis of ES throughout this time. There was, however, little incorporation of ³H-glucosamine into ES products, and lectin affinity chromatography and glycopeptidase F digestion identified only one glycosylated component. Immunoprecipitation of ¹²⁵l-labelled ES products with sera from calves patently infected with D. viviparus demonstrated that all of these, with the exception of two components, are antigenic to the bovine host. One of those not immunoprecipitated was shown to be host serum albumin carried over into culture. A limited degree of cross-reactivity between nematode species was observed, with a D. viviparus female-specific antigen of 290 kDa being recognized by serum antibody from calves infected with the gastrointestinal nematodes Cooperia oncophora and Ostertagia ostertagi. Calves vaccinated with irradiated larvae of D. viviparus, despite not being exposed to the adult stage of the parasite, also showed some recognition of adult ES products. This might suggest that vaccination with irradiated larvae operates against both pre-pulmonary and pulmonary stages of the infection.     

Nutrition, immunity and helminth infection: effect of dietary protein on the dynamics of the primary antibody response to Trichuris muris (Nematoda) in CBA/Ca mice

The effect of dietary protein on the specific antibody responses (total immunoglobulins, IgG1 and IgA) to the intestinal nematode Trichuris muris was studied in CBA/Ca mice fed isocaloric diets containing 16% or 4% protein. Mice fed the 16% diet and given a high infection dose of 650 eggs expelled almost their entire primary infection by day 21 post infection. In similarly infected animals fed the 4% protein diet, there was prolonged survival of adult worms. At a low infection dose of 10 eggs, there was no evidence of an expulsion response in either dietary group. The primary antibody response to parasite excretory/secretory (E/S) antigen was time-dependent, regardless of dietary protein or infection dose, and was predominantly an IgG1 response. Within each dietary group, antibody production and antigen recognition occurred earlier and the antibody responses were more intense in mice given the higher infection dose. The principal finding was that the specific antibody response was more vigorous, both quantitatively (serum titres) and qualitatively (antigen recognition by IgG1), in mice on a low protein diet, even though worm expulsion did not occur in these hosts. This result suggests that serum antibody level or antigen recognition is not related simply to protective immunity against T. muris in CBA/Ca mice.     

恶性疟原虫复合抗原重组真核表达质粒的构建与表达

目的　构建编码恶性疟原虫不同发育期抗原表位的复合抗原基因HGFSP的重组真核表达质粒。　方法　将HGFSP亚克隆于真核表达载体pcDNA3构建重组真核表达质粒 pc HGFSP ;琼脂糖凝胶电泳和限制性内切酶分析鉴定 ;脂质体介导转染HepG2细胞。取经G418筛选的阳性细胞克隆进行免疫学鉴定。 　结果　酶切及电泳鉴定证实pc HGFSP含有HGFSP。间接免疫荧光试验 (IFAT)、SDS PAGE及Westernblotting结果显示 ,pc HGFSP转染细胞含有可与HGFSP原核表达蛋白免疫血清产生特异性免疫反应的蛋白 (包含恶性疟原虫MSA1、MSA2、RESA和CSP中的抗原表位 ) ,其分子量与按HGFSP长度推算的蛋白分子量接近。　结论　用HGFSP成功构建恶性疟原虫复合抗原真核表达质粒 pc HGFSP ,其表达产物具免疫反应性。     

Identification of a Theileria mutans-specific antigen for use in an antibody and antigen detection ELISA

Summary Purified piroplasms of Theileria mutans were used to immunize BALB/c mice to generate monoclonal antibodies (MoAbs). The MoAbs recognized an antigen of a relative molecular mass of 32 kDa in Western blots. This antigen was also recognized by sera from cattle which had recovered naturally from experimental tick-transmission or infections induced by the blood stages of T. mutans. The MoAbs did not react, in indirect immunofluorescence or enzyme-linked immunosorbent assays (ELISA), with the common haemoparasites of cattle, namely, T. parva, T. annulata, Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma congolense, T. vivax or T. brucei. An antigen capture ELISA was established with two of the MoAbs which recognized different epitopes on the 32 kDa molecule. Using this test it was possible to detect circulating antigens or immune complexes in sera collected from cattle during the acute or chronic phases of infection. When the purified 32 kDa protein was used as antigen in a micro-ELISA to detect circulating antibodies in both experimental and field cattle sera, it was found that the titres of antibodies ranged between 1:20 and 1:10 240. Results of this study indicate that the antigen and immune complex capture assays and the antibody detection ELISA can be complementary in the immunodiagnosis of acute and chronic T. mutans infections. Moreover, the tests are useful in the differential diagnosis of the disease and for epidemiological studies.     

Antigen-specific antibody responses in B-1a and their relationship to natural immunity

B-1a cells are primarily thought of as natural antibody-producing cells. However, we now show that appropriate antigenic stimulation induces IgM and IgG B-1a antibody responses and long-lived T-independent antigen-specific B-1a memory that differs markedly from canonical B-2 humoral immunity. Thus, we show here that in the absence of inflammation, priming with glycolipid (FtL) from Francisella tularensis live vaccine strain induces splenic FtL-specific B-1a to mount dominant IgM and activation-induced cytidine deaminase-dependent IgG anti-FtL responses that occur within 3-5 d of FtL priming and fade within 1 wk to natural antibody levels that persist indefinitely in the absence of secondary FtL immunization. Equally surprising, FtL priming elicits long-term FtL-specific B-1a memory cells (IgM>IgG) that migrate rapidly to the peritoneal cavity and persist there indefinitely, ready to respond to appropriately administrated secondary antigenic stimulation. Unlike B-2 responses, primary FtL-specific B-1a responses and establishment of persistent FtL-specific B-1a memory occur readily in the absence of adjuvants, IL-7, T cells, or germinal center support. However, in another marked departure from the mechanisms controlling B-2 memory responses, rechallenge with FtL in an inflammatory context is required to induce B-1a secondary antibody responses. These findings introduce previously unexplored vaccination strategies for pathogens that target the B-1a repertoire.     

A comparison of cellular and humoral immune responses to trichuroid derived antigens in human trichuriasis

Individuals, residing in a region highly endemic for Trichuris trichiura, were examined for cytokine and proliferative responses to T. trichiura worm homogenate (TtAg), T. trichiura excretory/secretory products (TtES) and the equivalent antigenic preparations from the murine whipworm, Trichuris muris. Serum antibody levels against TtAg, T. muris worm homogenate and T. muris ES products were also studied. Measurable levels of immunoglobulin (Ig)G1, IgG4, IgA and IgE against T. muris antigens were detected, indicating a degree of conservation of epitopes between antigens derived from both species. Although levels of interleukin (IL)-4, IL-10, IL-13, tumour necrosis factor (TNF)-alpha and proliferative responses produced were comparable between homogenate antigens of either species and ES antigens of either species, a markedly different cellular response was observed in cultures stimulated with homogenate antigens compared to ES antigens. ES antigens preferentially induced IL-10 (P > 0.001) and TNF-alpha (P > 0.001) production, whereas levels of IL-4 (P > 0.001), IL-13 (P > 0.001) and proliferative responses (P > 0.001) were greater in cultures stimulated with whole worm extracts. Our findings suggest that T. muris preparations could be used as an alternative to T. trichiura proteins as a source of antigens in ex vivo cultures and that ES products stimulate a distinct immune response compared to somatic antigens.     

Mapping the T cell epitopes of the Babesia bovis antigen 12D3: implications for vaccine design

The Babesia bovis antigen 12D3 was analysed to identify potential T-cell epitopes. Two predictive algorithms identified 13 possible sites but there was minimal agreement between the different predictive methods. Experimental determination of the T-cell epitopes recognized by nine cattle was achieved using a panel of overlapping peptides which identified seven different epitopes, five of which were clustered together around residues 210–320 of the molecule. No T cell epitopes were located within the tightly disulphide bonded core of 12D3. Using a series of truncated peptides, the location of two of the epitopes was mapped to residues 35–43 and 266–275. The sequences of these two epitopes was compared with a database of previously described binding motifs for MHC II alleles and each epitope was found to contain three sequence motifs recognized by HLA-DR alleles. The BoLA-DRB3 alleles occurring in these cattle were determined by a sequence specific oligonucleotide hybridization assay. Within those cattle whose T cells proliferated in response to 12D3, there was a consistent pattern of epitope recognition and presence of particular DRB3 alleles. The implications for effective vaccine design are discussed.     

Primary cytomegalovirus infection in pregnancy: comparison of antibody responses to virus-encoded proteins between women with and without intrauterine infection

Using radioimmunoassay followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we compared serial IgG precipitin antibody responses to cytomegalovirus (CMV) proteins in three groups of 29 pregnant women who had primary CMV infection. Five women had CMV mononucleosis, and four of them infected their fetuses. Twenty-four women had subclinical infection, and eight infected their fetuses. There were no qualitative differences in the precipitin responses against the virus-encoded proteins in three different infected cellular antigens (cytoplasmic, nuclear, and high-speed pellet) between these three groups of women. There was also no qualitative difference in responses whether the infection was clinically apparent or subclinical. Quantitation by densitometer, however, revealed that women with mononucleosis and those with subclinical infection who infected their fetuses had a more intense and prolonged antibody response than did women with subclinical infection who failed to transmit CMV in utero.     

Isotypic variation in antibody responses in a community in Papua New Guinea to larval and adult antigens during infection, and following reinfection, with the hookworm Necator americanus

The natural infection of a community with the hookworm Necator americanus induces a vigorous humoral response to both larval and adult parasite antigens. This response occurs in all five human antibody isotypes, and data are presented to show that, at the population level, isotypes respond differently, following chemotherapy and during reinfection, to changes in antigen stimulation. The differential response probably reflects the fact that the parasite, during the course of its life cycle, presents different amounts of antigens at different anatomical locations. It is suggested that IgG and IgM responses against adult excretory-secretory (ES) products most accurately reflect the efficacy of chemotherapy, and the load of resident adult infection, while IgG responses against larval somatic antigens reflect continuous exposure to infection. These hypotheses should now be tested, at the level of the individual, in a longitudinal manner using more closely spaced sampling intervals. This repetitive sampling, and the inclusion of a measure of the exposure of the population to infective stages, will allow more definitive conclusions to be made about the role of the immune response in controlling worm burdens.     

Immuno-epidemiology of Ascaris lumbricoides infection in a high transmission community: antibody responses and their impact on current and future infection intensity

The role of the humoral immune system in human infection with Ascaris lumbricoides remains unclear. This study documents an epidemiological investigation in a highly endemic community in Vietnam, whereby serum antibody levels were assessed before treatment and after a 6-month reinfection period. These data were examined by correlation with infection status using an age-structured approach in an attempt to help shed light on the role of the humoral immune response. The first part of this study characterized levels of all serum antibody isotypes from the community in response to antigens of both adult and larval A. lumbricoides. Data were assessed in terms of their relation to host age and infection intensity with the aim to provide a broadly detailed account of immune responses to the parasite. In the second part, antibody responses to both life-stages of A. lumbricoides in serum samples collected before anthelmintic chemotherapy were analysed in relation to intensity of re-infection with the parasite 6 months following treatment. The results suggest that antibody responses may not confer protection from current infection or re-infection with A. lumbricoides and may not serve as reliable indicators of future infection intensity. Our results thereby lend support to the theory that immunity to A. lumbricoides may not be based on the humoral immune system.     

Investigation of a pertussis outbreak and comparison of two acellular booster pertussis vaccines in a junior school in South East England, 2019

In March 2019, a pertussis outbreak occurred in children in a junior school (7–11 years) in England who had been offered pertussis-containing booster vaccine at 40 months of age. In a case–control investigation, we assessed the extent of transmission and any difference in protection afforded to those who had previously received a booster 3- or 5-component acellular pertussis vaccine (aP). We took oral fluid specimens from the students to determine IgG antibodies against pertussis toxin (anti-PT). Parents of students attending the school were sent a questionnaire on pertussis symptoms and vaccination status was retrieved from general practitioner records for all students. Of 381 students, 134 (35.2%) were classified as pertussis cases, 133 by demonstration of significant anti-PT IgG titres and one clinically. There was no significant difference in the risk of pertussis between students receiving 3-component (33.7%) or 5-component (32.3%) aP boosters. However, pertussis infection differed significantly in school year 4, with 22.9%, 50.0%, 23.7% and 38.1% pertussis cases in years 3, 4, 5 and 6, respectively. The proportion of students with incomplete vaccinations recorded was higher than the proportion of those not covered according to the national reported coverage, possibly contributing to sustained transmission within the school.     

弓形虫主要抗原及核酸疫苗研究进展

弓形虫分布广泛,且人畜感染弓形虫会带来很严重的后果,所以弓形虫病的防治一直困扰着各国学者。近年来,随着对弓形虫研究的深入以及分子生物学的发展,弓形虫核酸疫苗的研究也取得了较大的进展。本文综述了弓形虫主要抗原以及弓形虫核酸疫苗的研究现状,探索弓形虫疫苗的发展前景,并对核酸疫苗的优缺点进行讨论。