Antibody responses of experimentally infected lambs to antigens collected during in vitro maintenance of the adult, metacestode or oncosphere stages of Taenia hydatigena and Taenia ovis with further observations on anti-oncospheral antibodies

The antigenicity and specificity of crude antigens collected during the in vitro maintenance of Taenia hydatigena and T. ovis, excretory/secretory (ES) antigens, were assessed in a peroxidase microenzyme-linked immunosorbent assay (ELISA), using sera from lambs given experimental monospecific infections with T. hydatigena, T. ovis, Echinococcus granulosus or Fasciola hepatica. ES antigens of larval cysts of T. ovis and T. hydatigena were less reactive than those of adult or oncosphere stages. Strong interspecific cros-reactions occurred between all antigen preparations, and these antigens offered no better specificity than crude somatic extracts. IgG1 was the major immunoglobulin detected in sera from lambs experimentally infected with T. ovis or T. hydatigena using antigens prepared from sonicated oncospheres. Discrete peaks of anti-oncospheral antibodies were detected following initial and challenge infections with eggs (whether the homologous or heterologous species), when sera were assayed with a PBS sonicate or an ES antigen from oncospheres. However, when oncospheres solubilised with sodium deoxycholate were used, the antibody response was prolonged and resembled that reported previously when somatic extracts of adult and metacestode stages were used as antigen. The results showed that oncospheres share antigens in common with other life-cycle stages, but also support the notion that they may possess some unique stage-specific antigenic determinants.     

Echinococcus granulosus: use of an intermediate host mouse model to evaluate sources of protective antigens and a role for antibody in the immune response.

A Balb/cJ mouse model was used to determine which stage of the E. granulosus life cycle possessed the most potent protective antigens. Mice were immunized with crude extracts of protoscoleces, brood capsules, cyst fluid, adult worm tissue, eggs or oncospheres and then challenged intraperitoneally with 600 activated oncospheres. Sonically disrupted oncospheres induced the highest levels of protection (greater than 90%) at doses greater than or equal to 10(3) oncosphere equivalents per mouse. High levels of protection were maintained when these preparations were solubilized in SDS. Immunization with Taenia ovis or T. hydatigena oncosphere preparations induced a maximum of 62 and 40% cross-protection, respectively. In passive transfer experiments, serum from triple-infected immune donors that were completely resistant to subsequent challenge induced 69% protection in naive recipients (P less than 0.01). Serum from mice that had been immunized with oncosphere sonicates that were shown to be highly immune, failed to induce statistically significant protection in recipients. A sheep trial confirmed the protective ability of prior infections. Immunization of sheep with a SDS solubilized oncosphere preparation produced 91% protection (P less than 0.01).     

Antigenic affinity of cestodes in the genus Taenia

Antigens of four species of the genus Taenia (T. ovis ovis, T. ovis krabbei, T. hydatigena and T. parenchimatosa) were studied by means of immunodiffusion reaction in agar gel with the use of hyperimmune sera. It has been established that extracts of the studied cestodes contain a great number of antigens, which during parenteral administration cause a synthesis of antibodies in rabbits. In homologous systems the number of recorded antigen-antibody complexes varied from 5 to 10. The most close antigenic affinity was found between T. ovis ovis and T. ovis krabbei, T. ovis ovis and T. hydatigena, as far as the main mass of precipitation bands in the immunodiffusion reaction fused together that suggests the identity of corresponding antigenic components. In all cases when analysing antiserum to T. parenchimatosa extract no differences of species-specific character in heterologous systems were traced.     

Killing of Taenia hydatigena oncospheres by sheep neutrophils

Neutrophils collected from the mammary glands of uninfected sheep or from sheep infected with Taenia hydatigena, attached to and killed T. hydatigena oncospheres in vitro in the presence of serum from infected sheep. Infected sheep serum alone was not deleterious to the parasite in vitro. Fc receptors for antibody were detected on both normal and immune neutrophils; they were present at a greater density on the latter. Immune neutrophils were more reactive towards oncospheres than normal neutrophils and formed extensive capsules around the parasite. Fc receptors were not detected on oncospheres. It is hypothesised that neutrophils may kill the parasite by producing hydrogen peroxide and the superoxide anion, both of which are toxic to a variety of cell types and protozoa. The function of antibody may be to facilitate attachment of neutrophils to oncospheres by way of their Fc receptors.     

Localization and characterization of a specific linear epitope of the Brucella DnaK protein

We have previously produced and characterized four monoclonal antibodies to the Brucella DnaK protein which were derived from mice infected with B. melitensis or immunized with the B. melitensis cell wall fraction. By use of a recombinant DNA technique, we have localized a linear epitope, recognized by two of these monoclonal antibodies (V78/07B01/G11 and V78/09D04/D08), in the last 21 amino acids of the C-terminal region of the Brucella DnaK protein. The C-terminal region has been reported to be the most variable region among DnaK proteins. The two other monoclonal antibodies (A53/09G03/D02 and A53/01C10/A10) failed to react with the recombinant clones and might recognize discontinuous epitopes of the Brucella DnaK protein. The four monoclonal antibodies reacted with all recognized Brucella species and biovars in immunoblotting after SDS-PAGE. Monoclonal antibodies V78/07B01/G11 and V78/09D04/D08 did not react with reported cross-reacting bacteria nor with bacteria of the α-2 subdivision of the class Proteobacteria for which a close genetic relationship with Brucella spp. has been reported. However, monoclonal antibodies A53/09G03/D02 and A53/01C10/A10 reacted with Phyllobacterium rubiacearum and/or Ochrobactrum anthropi, both bacteria of the α-2 subdivision of the class Proteobacteria. The Brucella genus DnaK specific epitopes could be of importance for diagnostic purposes.     

Vaccination against Taenia solium cysticercosis in pigs using native and recombinant oncosphere antigens.

Pigs were immunised with antigens derived from Taenia solium oncospheres or with a pool of three recombinant antigens from Taenia ovis, and subsequently challenged with T. solium eggs. The native oncosphere antigens induced 83% protection against viable, and 89% protection against the total number of cysticerci established following the challenge infection. Immunisation with the recombinant T. ovis antigens induced 93% protection against the establishment of viable cysticerci, and 74% protection against the total number of cysticerci. These results, and those achieved elsewhere with Taenia saginata and T. ovis, support the possibility of developing a practical vaccine to assist in the control of transmission of T. solium through pigs.     

The location of Taenia pisiformis, Taenia ovis and Taenia hydatigena in the gut of the dog and its effect on net environmental contamination with ova.

Autopsy of dogs 56 days after infection with either T. pisiformis, T. ovis or T. hydatigena showed that these worms could be found attached at any point along the length of the small intestine, but were most commonly in the anterior half. The mean relaxed lengths of T. pisiformis, T. ovis and T. hydatigena were 107 cm, 156 cm and 177 cm respectively. Attached gravid proglottides contained a mean of 41 000 eggs each in T. pisiformis, 31 000 eggs in T. hydatigena and 95000 eggs in T. ovis, whereas proglottides free in the gut contained means of only 1370, 500 and 1400 eggs respectively; therefore, the majority of eggs were released into the gut before segments passed out into the faeces. It was shown that eggs of all 3 species of worms hatched and activated in the small intestine of the dog, especially in the anterior half. Eggs of T. pisiformis which had been passaged through the intestine of the dog and stored in the faeces for 5 days were poorly infective for rabbits compared with eggs only stored in faeces. It was concluded, therefore, that during taeniid infections of dogs the point of apolysis in the gut plays a significant role in determining environmental contamination with eggs. Puppies which had been fed 10000 T. ovis eggs daily for 6 weeks prior to infection with T. ovis cysticerci showed no difference in susceptibility to the infection when compared with untreated puppies.     

Antibody responses against natural Taenia hydatigena infection in dogs in Kenya

Antibody responses (IgG) against Taenia hydatigena infection in dogs in Kenya were analysed in ELISA using excretory/secretory products of T. hydatigena scoleces derived from goat cysticercus cysts. Helminth infections of individual dogs were confirmed at autopsy. T. hydatigena worms were found in 89.5% of 143 dogs, and positive anti-T. hydatigena antibody levels were detected in 58.7% of infected dogs. Positive antiscolex antibody levels were detected in 40.0% of Turkana dogs uninfected with T. hydatigena, suggesting previous infection. Antibody was not detected in 34.4% of infected dogs. There was no relationship between individual T. hydatigena worm burdens and absorbance values for sera in ELISA. It was not possible to distinguish between sera from T. hydatigena-infected and uninfected dogs.     

Intestinal cestodes of stray dogs in Jordan

Five species of cestodes namely Echinococcus granulosus, Taenia hydatigena, Taenia pisiformis, Taenia ovis and Dipylidium caninum were recovered post mortem from 120 out of 173 stray dogs collected from the 5 governorates of Jordan during the period June 1979 to November 1980. Twenty-five of the examined dogs (14%) were found to be infected with E. granulosus, 79 (46%) with T. hydatigena, 14 (8%) with T. pisiformis and 5 (3%) with T. ovis. Dipylidium caninum was encountered in 33 (19%) of the examined dogs and infection with this parasite was significantly higher in males than in females. The parasites, except for D. caninum which was encountered in the ileum, were almost exclusively recovered from the duodenum and the jejunum. Single, double and triple infections with those cestodes were recorded.     

Schistosoma mansoni: cloning of antigen gene sequences in Escherichia coli

Fischer rat protective antiserum (F-2x) prepared from Schistosoma mansoni-infected rats was used to screen an adult worm cDNA library constructed in a lambda gt11 bacteriophage expression vector. This led to the isolation of several clones yielding proteins reactive with antibodies in the infection serum. Counter-screening of these clones with Wistar-Furth rat nonprotective antiserum (W-2x) enabled identification of clones either uniquely or preferentially reacting with F-2x, in addition to clones of nearly equal reactivity with both antisera. Six clones were further characterized. Five expressed beta-galactosidase/S. mansoni fusion proteins which migrated more slowly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than beta-galactosidase and all were reactive in a Western immunoblot assay. The cDNA insert sizes in the clones ranged from 150 to 900 base pairs. Rabbit antibodies prepared against fusion proteins from three of the clones recognized biosynthetically radio-labeled 4-week worm proteins of sizes 20, 38, and 70 kDa, respectively. The 20- and 38-kDa proteins were among the protein antigens uniquely recognized by the F-2x protective antiserum. These proteins are therefore candidates for protective vaccine antigens and the recombinant lambda clones are now serving as useful reagents for obtaining the corresponding nucleotide gene sequences.     

Trichinella spiralis: characterization of phage-displayed specific epitopes and their protective immunity in BALB/c mice

Trichinellosis is a global zoonosis mainly caused by Trichinella spiralis. We have previously reported that a novel Ts87 gene from the cDNA library of adult T. spiralis was cloned and expressed in a prokaryotic expression system. Vaccination with recombinant Ts87 protein (rTs87) induced a muscle larvae burden reduction in BALB/c mice by 29% in response to T. spiralis infection. In the present study, we screened a random phage-displayed peptide library using monoclonal antibody 5A3 which recognized Ts87 protein. Four positive phage clones were selected to subcutaneously immunize BALB/c mice without adjuvant. Two phage clones could effectively stimulate specific antibodies against rTs87. Mice vaccinated with these two combined phage clones showed a 28.7% worm burden reduction as compared to the control group. Therefore, the identified phage clones displayed peptides representing specific epitopes of Ts87 protein and could be considered as potential vaccine candidates for T. spiralis.     

The duration of passive protection against Taenia ovis larvae in lambs

In an attempt to induce passive protection in lambs against Taenia ovis larvae that would last for the 15-20 weeks from birth to slaughter as fat lambs, one group of ewes was immunized by a series of injections of 2000, 4000, 8000, 16 000 and 32 000 activated oncospheres of Taenia ovis prior to parturition. Another group of ewes was not immunized. All ewes had previously grazed pasture lightly infected with T. ovis eggs. Most lambs from non-immunized ewes developed cysts after oral infection with T. ovis eggs. However, no lambs from immunized ewes developed cysts up to and including 6 weeks after birth. Between 8 and 16 weeks after birth a proportion of lambs were found to be susceptible to infection. By 18 weeks after birth all lambs were apparently susceptible. The 99% confidence band for the mean duration of demonstrable complement-fixing antibody titres was 6.2-7.8 weeks for lambs from immunized ewes. The persistence of maternal protective antibody in some lambs could possibly preclude successful active immunization of all lambs against T. ovis larvae before 18 weeks of age.     

Immunoprevalence and Immunodominance of HLA-Cw?0801-Restricted T Cell Response Targeting the Hepatitis B Virus Envelope Transmembrane Region

HLA-C-restricted T cells have been shown to play an important role in HIV control, but their impact on protection or pathogenesis in other viral infections remains elusive. Here, we characterized the hierarchy of HLA class I-restricted hepatitis B virus (HBV) epitopes targeted by CD8 T cells in HBV-infected subjects. The frequency of CD8 T cells specific for a panel of 18 HBV epitopes (restricted by HLA-A∗0201/03/07 [hereinafter HLA-A0201/03/07], -A1101, -A2402/07, -B5801, -B4001, -B1301, and -Cw0801) was quantified in a total of 59 subjects who resolved HBV infection. We found that the HLA-Cw0801-restricted epitope comprised of Env residues 171 to 180 (Env_171–180) is immunoprevalent in the Southeast Asian subjects (10/17 HLA-Cw0801-positive subjects) and immunodominant in the majority of HLA-Cw0801-positive subjects able to control HBV infection. HLA-Cw0801-restricted Env_171–180-specific CD8 T cells recognized endogenously produced HBV surface antigen (HBsAg) and tolerated amino acid variations within the epitope detected in HBV genotypes B and C. In conclusion, we demonstrate that the HLA-Cw0801-restricted Env_171–180 T cell response is an important component of the HBV-specific adaptive T cell immunity in Asians infected with HBV. Thus, HLA-C restricted T cells might play an important role in various viral infections.     

The use of excretory and secretory antigens of the scolex of Taenia ovis for the serodiagnosis of infection in dogs

The excretory and secretory antigens from the evaginated scoleces of Taenia ovis were collected for 3 days in vitro, and used in an ELISA test to detect antibodies to T. ovis in the serum of dogs. When tested on sequentially collected sera, diagnostic ELISA values could be detected in many dogs 4 wk after infection, and remained for an average of a further 4 wk after worms were removed from dogs with an anthelmintic. Using an ELISA discriminant value that eliminated all false positives from 70 uninfected laboratory dog sera and from 57 uninfected farm dog sera, 54/62 true positives were found in sera from dogs infected with various numbers of T. ovis for various intervals. Sera from dogs infected with T. hydatigena gave 11/15 false positive reactions, whereas sera from 15 dogs infected with Echinococcus granulosus or 7 dogs infected with T. pisiformis were all negative. For T. ovis the test had a high repeatability, was not greatly influenced by the number of worms carried by the dog and higher titres were correlated with long-standing infections. Approximately 1,000 scoleces could be recovered from each experimentally infected sheep. Using the ELISA test with undiluted antigen and serum diluted 1:40, approximately 10 sera could be tested in duplicate with the excretions and secretions from each T. ovis scolex.     

T Cell Repertoire Diversity Is Decreased in Type 1 Diabetes Patients

Type 1 diabetes mellitus (T1D) is an immune-mediated disease. The autoreactive T cells in T1D patients attack and destroy their own pancreatic cells. In order to systematically investigate the potential autoreactive T cell receptors (TCRs), we used a high-throughput immune repertoire sequencing technique to profile the spectrum of TCRs in individual T1D patients and controls. We sequenced the T cell repertoire of nine T1D patients, four type 2 diabetes (T2D) patients and six nondiabetic controls. The diversity of the T cell repertoire in T1D patients was significantly decreased in comparison with T2D patients (P = 7.0E
08 for CD4+ T cells, P = 1.4E
04 for CD8+ T cells) and nondiabetic controls (P = 2.7E
09 for CD4+ T cells, P = 7.6E
06 for CD8+ T cells). Moreover, T1D patients had significantly more highly-expanded T cell clones than T2D patients (P = 5.2E
06 for CD4+ T cells, P = 1.9E
07 for CD8+ T cells) and nondiabetic controls (P =1.7E
07 for CD4+ T cells, P= 3.3E
03 for CD8+ T cells). Furthermore, we identified a group of highly-expanded T cell receptor clones that are shared by more than two T1D patients. Although further validation in larger cohorts is needed, our data suggest that T cell receptor diversity measurements may become a valuable tool in investigating diabetes, such as using the diversity as an index to distinguish different types of diabetes.     

Dictyocaulus viviparus: isolation and characterization of a recombinant antigen with potential for immunodiagnosis.

Crude adult worm antigen of Dictyocaulus viviparus was examined for specific antigens by SDS-PAGE and immunoblotting using sera from cattle experimentally infected with D. viviparus, vaccinated with a normal or a reduced dosage of the commercial lungworm vaccine, and helminth-free cattle. A D. viviparus-specific region M(r) 18,000 was identified and isolated. A lambda ZAP II cDNA expression library consisting of 4.4 x 10(5) recombinant clones (88% of the total number of clones) was constructed from D. viviparus adult worm mRNA. Rabbit antiserum to the M(r) 18,000 antigen was used to screen the cDNA library and eight positive clones were picked and allocated to the same antigenic family by sibling analysis. All clones were subcloned into the plasmid pGEX-2T, and the clone with highest expression yields was expressed as a glutathione S-transferase fusion protein (DvGST3-14) or, after cleavage with thrombin, as pure recombinant parasite protein (Dv3-14). The native parasite antigen encoded by the clone was identified. The immunodiagnostic potential of the recombinant proteins was assessed by immunoblotting.     

Prior exposure to infection with Chlamydia pneumoniae can influence the T-cell-mediated response to Chlamydia trachomatis

Using T-cell clones derived from patients with Chlamydia trachomatis (CT)-induced reactive arthritis, we identified target antigens and mapped the peptide epitopes that were recognized. Several epitopes were conserved in homologous proteins of Chlamydia pneumoniae (CPN), and it was shown that these epitopes were generated following processing of the CPN proteins or CPN elementary bodies, i.e. the T-cell clones were indeed CT and CPN cross-reactive. Given that CPN infection is frequent, we wished to determine whether prior infection with CPN could have an effect on the response to subsequent infection with CT. First, we showed that the CPN antigen, OmcB, was recognized by polyclonal peripheral blood T cells from additional subjects with CT-induced reactive arthritis; they were chosen to be HLA-DR-matched with the T-cell clones used to map epitopes in OmcB. Responses to a peptide previously shown to be conserved in CT and CPN OmcB were also seen, but only in CPN-seropositive individuals. These subjects also produced interferon-gamma (IFN-gamma) in response to CPN OmcB, and did not recognize a nonconserved epitope in OmcB. Secondly, OmcB-responsive clones from CPN-seropositive subjects were dominated by those recognizing the cross-reactive epitope, despite the recent exposure of these subjects to CT. Lastly, healthy CPN-seropositive subjects, without evidence of exposure to CT, showed greater responses, measured as IFN-gamma secretion, to CT proteins in vitro than those shown by seronegative subjects. This is consistent with the idea that prior CPN infection primes a Th1 T-cell response to CT antigens. This finding is relevant to the pathogenesis of the sequelae of CT infection (trachoma, infertility and arthritis), which may be influenced by prior exposure to CPN, and to the choice of CT antigens as vaccine candidates.