Role of immunoglobulin A in protection against reovirus entry into Murine Peyer's patches.

Reovirus type 1 Lang (T1L) infects the mouse intestinal mucosa by adhering specifically to epithelial M cells and exploiting M-cell transport to enter the Peyer's patches. Oral inoculation of adult mice has been shown to elicit cellular and humoral immune responses that clear the infection within 10 days. This study was designed to determine whether adult mice that have cleared a primary infection are protected against viral entry upon oral rechallenge and, if so, whether antireovirus secretory immunoglobulin A (S-IgA) is a necessary component of protection. Adult BALB/c mice that were orally inoculated on day 0 with reovirus T1L produced antiviral S-IgA in feces and IgG in serum directed primarily against the reovirus sigma1 attachment protein. Eight hours after oral reovirus challenge on day 21, the Peyer's patches of previously exposed mice contained no detectable virus whereas Peyer's patches of naive controls contained up to 2,300 PFU of reovirus/mg of tissue. Orally inoculated IgA knockout (IgA(-/-)) mice cleared the initial infection as effectively as wild-type mice and produced higher levels of reovirus-specific serum IgG and secretory IgM than C57BL/6 wild-type mice. When IgA(-/-) mice were rechallenged on day 21, however, their Peyer's patches became infected. These results indicate that intestinal S-IgA is an essential component of immune protection against reovirus entry into Peyer's patch mucosa.

Schistosoma mansoni gene GP22 encodes the tegumental antigen sm25: (1) antibodies to a predicted B-cell epitope of Sm25 cross-react with other candidate vaccine worm antigens; (2) characterization of a recombinant product containing tandem-repeats of this peptide as a vaccine.

Monospecific antibodies against two putative epitopes of schistosome protein encoded by gene GP22 (182 codons, no introns) were used to probe worm extracts fractionated by lentil-lectin affinity chromatography or by electrophoresis. Anti-peptide-alpha (codons 70-84) exclusively identifies the N-glycanase-sensitive, 25 kDa tegumental glycoprotein Sm25 in the lectin-bound fraction of detergent-solubilized adult worm extract S3. In contrast, antipeptide-delta (codons 151-162) does not react with Sm25 but cross-reacts with other schistosome proteins, including candidate vaccine antigens paramyosin (Sm97) and glutathione-S-transferases (Sm26, Sm28, Sj26). Recombinant protein r4 x 47, constructed to express multiple copies of codon sequence 117-163 (containing delta), reacts with anti-delta and is uniquely recognized by protective Fischer twice-infected (F-2x) rat antiserum. Immunization with r4 x 47 induces antibodies with cross reactivities similar to anti-delta, but which also recognize Sm25. Despite these cross-reactivities with protective antigens, rodents vaccinated with r4 x 47 were not protected against cercarial infection. On the basis of these data, two hypotheses are proposed: (1) antigenic epitopes other than delta are present within the r4 x 47 sequence which induce antibodies reactive with Sm25 and/or (2) peptide-delta assumes alternative antigenic conformations, dependent upon the context of neighbouring sequences, some of which mimic epitopes of proteins encoded by other schistosome genes. These mimotopes are not targets of protective antibodies.

Identification and molecular cloning of a 67-kilodalton protein in Schistosoma japonicum homologous to a family of actin-binding proteins.

A monoclonal antibody to Schistosoma japonicum which conferred significant protection against cercarial challenge in mice was produced. The predicted translation product of the cDNA corresponding to the antigen recognized by this antibody was homologous to a newly identified family of actin-binding proteins. The expressed protein bound polymerized actin and was recognized by serum from patients infected with S. japonicum.

Evaluation of recombinant protein r140, a polypeptide segment of tegumental glycoprotein Sm25, as a defined antigen vaccine against Schistosoma mansoni.

To investigate the role of tegumental glycoprotein Sm25 in protective immunity against schistosomiasis, codons 43-182 of its gene (GP22) were amplified by PCR and cloned in the pET 15b bacterial expression system. Recombinant protein r140 was inducibly expressed in the presence of rifampicin and purified by Ni-affinity chromatography. In different vaccination trials, Balb/c mice and Fischer rats repeatedly immunized with r140 in combination with one of several adjuvants (alum, cholera toxin or complexed into proteosomes) produced high titre anti-r140 responses. These antibodies detected an N-glycanase sensitive. 25 kDa antigen in a detergent solubilized worm fraction using Western immunoblotting. The choice of adjuvant affected the isotype distribution of the specific anti-r140 antibodies. Despite the presence of high antibody titres and isotypes which have been shown to correlate with protective immunity, protection against subsequent cercarial challenge was not observed. In addition, no appreciable effects on worm sex ratios or liver egg yields were detected in mice. Studies involving biotin labelling of membrane proteins in live worms showed that the majority of anti-r140 reactive molecules present in adult schistosomes are biotinylated after permeabilization of the parasite surface. Several possibilities to account for the lack of protective immunity are analysed.

Immobilization of Schistosoma mansoni miracidia by activation of the alternate complement pathway at unusually high serum dilution.

Free-swimming Schistosoma mansoni miracidia were immobilized by adding normal mammalian serum to the water. Miracidial immobilizing activity (MIA) was shown to result from activating the alternate pathway of complement (APC). MIA in normal sera was heat-sensitive and antibody independent; it was greatly reduced in factor B-depleted or C6-depleted, but not in C1-depleted, human serum. Addition of purified factor B to B-depleted serum totally restored MIA. Half-maximal MIA in normal human, rabbit, and guinea pig sera was detectable at final dilutions exceeding 1/100, 1/200, and 1/500, respectively, and normal rat serum was particularly potent, with MIA at dilutions exceeding 1/2000. Detection of APC activity at such high dilutions is quite extraordinary and attributed to the hypotonic conditions. We confirmed and extended previous findings that heat-inactivated infection sera also display MIA. Immobilizing activity in irradiated-cercarial vaccine rat serum cofractionated with rat IgG and anti-SWAP antibody activity. Antibody-dependent MIA titres were much lower than for APC-dependent MIA. Based upon light microscope and transmission EM studies, immobilization of miracidia by APC activation was attributed to severe tegumental damage. Miracidia within egg shells were insensitive to MIA.