Analysis of heavy-chain antibody responses and resistance to Parelaphostrongylus tenuis in experimentally infected alpacas.

The parasitic nematode Parelaphostrongylus tenuis is an important cause of neurologic disease of camelids in central and eastern North America. The aim of this study was to determine whether alpacas develop resistance to disease caused by P. tenuis in response to a previous infection or a combination of controlled infection and immunization. Alpacas were immunized with a homogenate of third-stage larvae (L3) and simultaneously implanted subcutaneously with diffusion chambers containing 20 live L3. Sham-treated animals received adjuvant alone and empty chambers. The protocol was not effective in inducing resistance to oral challenge with 10 L3, and disease developed between 60 and 71 days following infection. Immediately following the onset of neurologic disease, affected animals were treated with a regimen of anthelmintic and anti-inflammatory drugs, and all recovered. One year later, a subset of alpacas from this experiment was challenged with 20 L3 and the results showed that prior infection induced resistance to disease. Primary and secondary infections induced production of conventional and heavy-chain IgGs that reacted with soluble antigens in L3 homogenates but did not consistently recognize a recombinant form of a parasite-derived aspartyl protease inhibitor. Thus, the latter antigen may not be a good candidate for serology-based diagnostic tests. Antibody responses to parasite antigens occurred in the absence of overt disease, demonstrating that P. tenuis infection can be subclinical in a host that has been considered to be highly susceptible to disease. The potential for immunoprophylaxis to be effective in preventing disease caused by P. tenuis was supported by evidence of resistance to reinfection.

Contributions of conventional and heavy-chain IgG to immunity in fetal, neonatal, and adult alpacas.

In addition to conventional immunoglobulins, camelids produce antibodies that do not incorporate light chains into their structures. These so-called heavy-chain (HC) antibodies have incited great interest in the biomedical community, as they have considerable potential for biotechnological and therapeutic application. Recently, we have begun to elucidate the immunological functions of HC antibodies, yet little is known about their significance in maternal immunity or about the B lymphocytes that produce them. This study describes the application of isotype-specific reagents toward physiological assessments of camelid IgGs and the B cells that produce them. We document the specificities of monoclonal antibodies that distinguish two conventional IgG1 isotypes and two HC IgG3 variants produced by alpacas. Next, we report that the relative concentrations of five isotypes are similar in serum, milk, and colostrum; however, following passive transfer, the concentrations of HC IgG2 and IgG3 declined more rapidly than the concentration of conventional IgG1 in the sera of neonates. Finally, we assessed the distribution of B cells of distinct isotypes within lymphoid tissues during fetal and adult life. We detected IgG1, IgG2, and IgG3 in lymphocytes located in lymph node follicles, suggesting that HC B cells affinity mature and/or class switch. One IgG3 isotype was present in B cells located in ileal Peyer's patches, and one conventional IgG1 isotype was detected in splenic marginal zone B cells. Our findings contribute to the growing body of knowledge pertaining to HC antibodies and are compatible with functional specialization among conventional and HC IgGs in the alpaca.

Application of monoclonal antibodies in functional and comparative investigations of heavy-chain immunoglobulins in new world camelids.

Of the three immunoglobulin G (IgG) isotypes described to occur in camelids, IgG2 and IgG3 are distinct in that they do not incorporate light chains. These heavy-chain antibodies (HCAbs) constitute approximately 50% of the IgG in llama serum and as much as 75% of the IgG in camel serum. We have produced isotype-specific mouse monoclonal antibodies (MAbs) in order to investigate the roles of HCAbs in camelid immunity. Seventeen stable hybridomas were cloned, and three MAbs that were specific for epitopes on the gamma chains of llama IgG1, IgG2, or IgG3 were characterized in detail. Affinity chromatography revealed that each MAb bound its isotype in solution in llama serum. The antibodies bound to the corresponding alpaca IgGs, to guanaco IgG1 and IgG2, and to camel IgG1. Interestingly, anti-IgG2 MAbs bound three heavy-chain species in llama serum, confirming the presence of three IgG2 subisotypes. Two IgG2 subisotypes were detected in alpaca and guanaco sera. The MAbs detected llama serum IgGs when they were bound to antigen in enzyme-linked immunosorbent assays and were used to discern among isotypes induced during infection with a parasitic nematode. Diseased animals, infected with Parelaphostrongylus tenuis, did not produce antigen-specific HCAbs; rather, they produced the conventional isotype, IgG1, exclusively. Our data document the utility of these MAbs in functional and physiologic investigations of the immune systems of New World camelids.

Molting, ecdysis, and reproduction of Trichinella spiralis are supported in vitro by intestinal epithelial cells.

Trichinella spiralis is an obligate parasite of animals that has an unusual intracellular life cycle. Investigation of parasitism at the cellular and molecular levels has been challenging because of a shortage of tools for in vitro cultivation of T. spiralis. We have found that T. spiralis larvae molt, ecdyse, develop to adulthood, and reproduce when they are inoculated onto cultured intestinal epithelial cells. Initially, larvae invade and migrate through cells in a monolayer (T. ManWarren, L. Gagliardo, J. Geyer, C. McVay, S. Pearce-Kelling, and J. Appleton, Infect. Immun. 65:4806-4812, 1997). During prolonged culture in Caco-2 epithelial cells, L1 larvae molted and ecdysed with efficiencies as high as 50%. Molting and ecdysis in vitro required entry of the parasite into cells; conditions that prevented entry into cells also prevented ecdysis. When larvae were inoculated at a low density and cultured for 5 to 9 days, as many as 50% of the larvae developed to adult stages. Low numbers of mature male worms with copulatory appendages were observed in these cultures. The majority of worms that survived for five or more days were unfertilized females. Low-density cultures supported development of female worms with embryos at rates of 4 to 5%. These results show that the intestinal life cycle of T. spiralis can be supported entirely by host epithelial cells. Our model should allow more detailed investigation of intracellular parasitism by T. spiralis.

Larvae-induced plasma membrane wounds and glycoprotein deposition are insufficient for Trichinella spiralis invasion of epithelial cells.

Trichinella spiralis L1 larvae infect susceptible hosts by invading epithelial cells that line the small intestine. Invasion also occurs in vitro when larvae are inoculated into cultures of epithelial cells from several different animal species. To further investigate the mechanism of invasion, we studied the interaction of larvae with the rat epithelial cell line IEC-6. Larvae did not invade IEC-6 cells, but did cause the cells to take up parasite glycoproteins. Glycoprotein bearing cells remained viable and were detectable in monolayers for as long as 24 h, suggesting that the glycoproteins were not toxic for cells. Immunofluorescence revealed that parasite glycoproteins localized in the nuclei, mitochondria and cytoplasm and we found evidence for selection of certain molecules between nuclear and cytoplasmic compartments. Using fluorescent dextrans as fluid phase markers we found 17-38% of the cells in inoculated monolayers were engorged with dextran and that dextran was free in the cytoplasm. Increased dextran uptake was not lethal, required the presence of activated larvae, and was often associated with uptake of parasite glycoproteins. These observations suggest that larvae caused plasma membrane wounds. Our results showed that neither delivery of glycoproteins nor mechanical wounding is sufficient to allow entry of the parasite into resistant epithelial cells. Because both invasion-resistant and susceptible epithelial cells undergo non-lethal wounding, we propose that larvae-induced injury to epithelial cells may result in release of cell-specific mediators that signal larva to invade a particular cell line or, alternatively, to ignore it.

Antibodies to tyvelose exhibit multiple modes of interference with the epithelial niche of Trichinella spiralis.

Infection with the parasitic nematode Trichinella spiralis is initiated when the L1 larva invades host intestinal epithelial cells. Monoclonal antibodies specific for glycans on the larval surface and secreted glycoproteins protect the intestine against infection. Protective antibodies recognize tyvelose which caps the target glycan. In this study, we used an in vitro model of invasion to further examine the mechanism(s) by which tyvelose-specific antibodies protect epithelial cells against T. spiralis. Using cell lines that vary in susceptibility to invasion, we confirmed and clarified the results of our in vivo studies by documenting three modes of interference: exclusion of larvae from cells, encumbrance of larvae as they migrated within epithelial monolayers, and inhibition of parasite development. Excluded larvae bear cephalic caps (C. S. McVay et al., Infect. Immun. 66:1941-1945, 1998) of immune complexes that may physically block invasion or may interfere with sensory reception. Monovalent Fab fragments prepared from a tyvelose-specific antibody also excluded larvae from cells, demonstrating that antibody binding can inhibit the parasite in the absence of antigen aggregation and cap formation. In contrast, encumbered larvae caused extensive damage to the monolayer yet were not successful in establishing a niche, as reflected by their failure to molt. These results show that antibodies to tyvelose exhibit multiple modes of inhibitory activity, further implicating tyvelose-bearing glycoproteins as mediators of invasion and niche establishment by T. spiralis.

Dominance of immunoglobulin G2c in the antiphosphorylcholine response of rats infected with Trichinella spiralis.

The antibody response to the L1 stage of Trichinella spiralis has been described as biphasic. Worms resident in the intestine during the first week of infection stimulate an antibody response against a subset of larval proteins. L1 larvae in the muscle at the end stage of infection stimulate a second antibody response against tyvelose-bearing glycoproteins. Antityvelose antibodies protect rats against challenge infection with larvae. The aim of this study was to characterize the rat B-cell response against larval antigens during the intestinal phase of T. spiralis infection and to test the antiparasitic effects of such antibodies. Strain PVG rats were infected orally with 500 larvae. Antibodies specific for phosphorylcholine-bearing proteins of L1 larvae first appeared in serum 9 days postinfection. Absorption experiments showed that the majority of antilarval antibodies produced in rats 16 days after infection with T. spiralis were specific for phosphorylcholine-bearing proteins. A fraction of these antibodies bound to free phosphorylcholine. Immunoglobulin G2c (IgG2c) producing cells in the mesenteric lymph node dominated this early antibody response. IgG2c is associated with T-independent immune responses in the rat; however, a comparison of athymic rats with euthymic controls suggested that only a small fraction of the phosphorylcholine-related antibody response against T. spiralis was T independent. Phosphorylcholine is a common epitope in antigens of bacteria and nematode parasites and has been shown to be a target of protective immunity in certain bacteria. A monoclonal IgG2c antibody was prepared from infected rats and shown to be specific for phosphorylcholine. Monoclonal phosphorylcholine-specific IgG2c failed to protect rats against intestinal infection with T. spiralis. Therefore, our findings do not support a role for phosphorylcholine-bearing antigens in immune defense against T. spiralis; however, the potency of the immune response induced suggests an immunomodulatory role for the lymphocytes involved.

Diversity of the antibody responses produced in ponies and mice against the equine influenza A virus H7 haemagglutinin.

A large panel of mouse monoclonal antibodies was produced and tested against field isolates of the equine H7N7 influenza A virus subtype. Only a limited degree of H7 haemagglutinin variation was detected. At least four antigenic sites were identified by selecting variant viruses in eggs. The limited variation in the field did not correlate with the frequency of variant viruses detected in eggs; this frequency was similar to those reported for other influenza viruses. We sought to determine whether the limited amount of variation could be correlated with an epitope-restricted antibody response in vaccinated horses. To this end, limiting dilution cultures were established with peripheral blood leukocytes from vaccinated ponies and the antibodies released into culture supernatants were assayed for binding to variant H7 viruses in ELISA. Three neutralizable antigenic sites mapped by mouse antibodies were also recognized by antibodies in pony limiting dilution culture supernatants, indicating that the equine antibody response against the influenza virus H7 haemagglutinin is diverse, and should be effective in selecting variant viruses.

Production of an equine monoclonal antibody specific for the H7 hemagglutinin of equine influenza virus.

Peripheral blood leucocytes from a pony previously exposed to equine influenza virus (H3, N8) and vaccinated with killed virus (H3, N8 and H7, N7 subtypes) were cultured in vitro with live A/equine/Prague/56 (H7, N7). On the sixth day of culture, cells were harvested and fused with mouse myeloma cells (X63-Ag8.653). From this fusion, one hemagglutinin specific, equine IgG monoclonal antibody secreting hybridoma was identified and cloned twice by limiting dilution. The antibody inhibited hemagglutination by nine H7 equine influenza virus isolates obtained over a 21-year period, but did not inhibit A/equine/Miami/63 (H3, N8), or A/PR/8/34 (H1, N1). The neutralizing titer of hybridoma induced, nude mouse ascitic fluid was 10(-4.5) when tested in eggs against 100 egg infective doses (EID50) A/equine/Prague/1/56. The hybridoma continued to synthesize antibody during more than 4 months in continuous culture.