Immunology 1990 71 127-132
Effects of major histocompatibility genes and antigen delivery on induction of protective mucosal immunity to E. acervulina following immunization with a recombinant merozoite antigen H. S. LILLEHOJ, M. C. JENKINS & L. D. BACON* Protozoan Diseases Laboratory, Livestock and Poultry Sciences Institute, Agricultural Research Service, U.S. Department of Agriculture, BARC-East, Beltsville, Maryland and *Regional Poultry Research Laboratory, U.S. Department of Agriculture, East Lansing, Michigan, U.S.A. Acceptedfor publication 9 May, 1990
SUMMARY Intramuscular immunization with the recombinant p250 surface antigen of Eimeria acervulina merozoite (rEAMZp250) or oral inoculation with live recombinant Escherichia coli expressing the rEAMZp250 protein resulted in antigen-specific T-cell and humoral responses and conferred a significant reduction in mucosal parasitism compared to immunization with the negative control antigen preparation. Among the major histocompatibility complex (MHC) (B)-congenic chickens receiving intramuscular immunization, strain .6-2 (B2B2) showed significant (P < 0-05) protection to live E. acervulina challenge compared to the other strains examined. In contrast, strains .C-12 (B'2B'2) and .P-13 (B'3B'3) showed significant protection among the groups given live recombinant E. coli. In general, strains showing enhanced T-cell responses to the rEAMZp250 protein were better protected compared to those showing minimal or no T-cell responses. Thus the results suggest that the B haplotypes of the host and the mode of antigen presentation influence the outcome of protection following an immunization of chickens with recombinant coccidial antigen.
INTRODUCTION Eimeria acervulina is an intestinal protozoa that causes intense damage to the duodenum in chickens, resulting in poor feed utilization and severe weight loss. Since it is not practical or safe to use live attenuated vaccines for preventing coccidiosis, the subunit vaccine approach is being explored using recombinant DNA techniques to create bacterial and viral vectors to direct immunity toward the mucosal immune system (Jenkins, Lillehoj & Dame 1988; Kim, Jenkins & Lillehoj, 1989; Turner, 1989; Wallach et al., 1989). Although both humoral and cell-mediated immunity against coccidial antigens are detected following Eimeria infection, resistance to coccidiosis is ascribed to the development of cell-mediated immunity (Lillehoj, 1987a; Rose, 1987; Lillehoj et al., 1988). Recent studies indicate that the development of protective immunity is influenced by both major histocompatibility complex (MHC) (Clare et al., 1985) and nonMHC genes (Bumstead & Millard, 1986; Lillehoj et al., 1988: Lillehoj et al., 1989b). As a step towards developing a protective anti-coccidial vaccine, a lambda bacteriophage vector was constructed that expressed an immunodominant epitope of the 250,000 molecular weight (MW) merozoite surface protein of E. acertulina (rEAMZp250), composed of tandemly repeated
amino acid sequences (Kim et al., 1989). The recombinant antigen was recognized by immune lymphocytes and serum from E. acervulina-immune chickens (Jenkins et al., 1988; Lillehoj et al., 1988). Chickens receiving an oral inoculation with live Escherichia coli expressing the rEAMZp250 recombinant protein developed antigen-specific humoral and T-cell responses and were partially protected against oral coccidial challenge (Kim et al., 1989). This study was carried out to investigate: (i) the vaccine potential of the rEAMZp250 recombinant merozoite protein using two different antigen delivery systems: (ii) the protective effects of rEAMZp250 antigen-specific humoral and cellular immunity in the immunized chickens; and (iii) the role of MHC genes in the induction of protective intestinal immune response following immunization with the rEAMZp250 recombinant coccidial antigen. MATERIALS AND METHODS
Chickens and parasites Fertile 15I5-B congenic chicken eggs were obtained from the Regional Poultry Research Laboratory (RPRL) and hatched at the Livestock and Poultry Science Institute. The development and histocompatibility status of these B congenic strains have been described elsewhere (Bacon, Ismail & Motta, 1986). Inbred SC and FP chicken eggs were purchased from Hy-line International Production Center, Dallas Center, IA. All chickens
Correspondence: Dr H. Lillehoj, Protozoan Diseases Laboratory, Livestock and Poultry Sciences Institute, U.S. Dept. of Agriculture, BARC-East, Building 1040, Beltsville, MD 20705, U.S.A.
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were housed in clean wire-floored cages and provided feed and water ad libitum. The wild-type strain of E. acerrulina developed and maintained at the Protozoan Diseases Laboratory, LPSI, was used for challenge experiments.
Production and high-pressure liquid chromatographY (HPLC) purification of recombinant fusion proteins Detailed molecular and biochemical characterizations of the EAMZp250 recombinant antigen have been described elsewhere (Jenkins et al., 1988; Jenkins, 1988). Partial purification of this beta-galactosidase fusion protein was performed by HPLC size exclusion chromatography, as described elsewhere (Lillehoj et al., 1988). HPLC fractions representing recombinant rEAMZp250 antigen, free of bacterial protein as assessed by SDS-PAGE, were pooled and used for immunization and in ritro studies.
Production of E. coli trans/ormnant carrying the cMZ8 cDNA E. coliN6405 transformants were produced as described previously (Androphy, Lowry & Schiller, 1987; Kim et al., 1989). Transformants were grown in culture at 32 , induced for production of fusion protein at 42 for 1-2 hr. and harvested by centrifugation. The recombinant E. coli transformant pCO,MZ8 encodes the 42,000-46,000 MW protein (p 42-46) immunoreactive with serum obtained from chickens inoculated
with E. acerrulina (Kim et al., 1989).
Inimuni-ation o/ chickens and assessment of antibody and antigen-specific T-cell responses 1 51 -B congenic and inbred chickens were immunized intramuscularly with various doses of the HPLC-purified rEAMZp250 merozoite surface antigen or control extracts emulsified in complete Freund's adjuvant (CFA), followed by a second immunization I week later with antigen in incomplete Freund's adjuvant (IFA). Immunization with live E. coli expressing the rEAMZp250 protein or control E. coli harbouring a nonrecombinant plasmid was with 109 temperature-induced E. coli. Protection against E. aceruldina challenge was assessed by oocyst output in faecal samples collected 5-9 days after challenge with 104 E. acerrulina. expressed as oocyst reduction
between means were evaluated using Duncan's multiple range test. RESULTS Kinetics of antigen-specific serum antibody and T-cell responses after intramuscular immunization with the rEAMZp250 recombinant antigen Three-week-old inbred SC and FP chickens (n = 6) were given an intramuscular injection of HPLC-purified rEAMZp250 recombinant antigen twice at I -week intervals and the antigen-specific humoral responses were assessed by ELISA. The level of antibody responses depended upon the chicken strain and the dose of the antigen used (Fig. 1). At 7 days post-immunization, antibody levels of FP chickens immunized with 12 ticg rEAMZp250 were significantly higher (P
Effects of major histocompatibility genes and antigen delivery on induction of protective mucosal immunity to E. acervulina following immunization with a recombinant merozoite antigen H. S. LILLEHOJ, M. C. JENKINS & L. D. BACON* Protozoan Diseases Laboratory, Livestock and Poultry Sciences Institute, Agricultural Research Service, U.S. Department of Agriculture, BARC-East, Beltsville, Maryland and *Regional Poultry Research Laboratory, U.S. Department of Agriculture, East Lansing, Michigan, U.S.A. Acceptedfor publication 9 May, 1990
SUMMARY Intramuscular immunization with the recombinant p250 surface antigen of Eimeria acervulina merozoite (rEAMZp250) or oral inoculation with live recombinant Escherichia coli expressing the rEAMZp250 protein resulted in antigen-specific T-cell and humoral responses and conferred a significant reduction in mucosal parasitism compared to immunization with the negative control antigen preparation. Among the major histocompatibility complex (MHC) (B)-congenic chickens receiving intramuscular immunization, strain .6-2 (B2B2) showed significant (P < 0-05) protection to live E. acervulina challenge compared to the other strains examined. In contrast, strains .C-12 (B'2B'2) and .P-13 (B'3B'3) showed significant protection among the groups given live recombinant E. coli. In general, strains showing enhanced T-cell responses to the rEAMZp250 protein were better protected compared to those showing minimal or no T-cell responses. Thus the results suggest that the B haplotypes of the host and the mode of antigen presentation influence the outcome of protection following an immunization of chickens with recombinant coccidial antigen.
INTRODUCTION Eimeria acervulina is an intestinal protozoa that causes intense damage to the duodenum in chickens, resulting in poor feed utilization and severe weight loss. Since it is not practical or safe to use live attenuated vaccines for preventing coccidiosis, the subunit vaccine approach is being explored using recombinant DNA techniques to create bacterial and viral vectors to direct immunity toward the mucosal immune system (Jenkins, Lillehoj & Dame 1988; Kim, Jenkins & Lillehoj, 1989; Turner, 1989; Wallach et al., 1989). Although both humoral and cell-mediated immunity against coccidial antigens are detected following Eimeria infection, resistance to coccidiosis is ascribed to the development of cell-mediated immunity (Lillehoj, 1987a; Rose, 1987; Lillehoj et al., 1988). Recent studies indicate that the development of protective immunity is influenced by both major histocompatibility complex (MHC) (Clare et al., 1985) and nonMHC genes (Bumstead & Millard, 1986; Lillehoj et al., 1988: Lillehoj et al., 1989b). As a step towards developing a protective anti-coccidial vaccine, a lambda bacteriophage vector was constructed that expressed an immunodominant epitope of the 250,000 molecular weight (MW) merozoite surface protein of E. acertulina (rEAMZp250), composed of tandemly repeated
amino acid sequences (Kim et al., 1989). The recombinant antigen was recognized by immune lymphocytes and serum from E. acervulina-immune chickens (Jenkins et al., 1988; Lillehoj et al., 1988). Chickens receiving an oral inoculation with live Escherichia coli expressing the rEAMZp250 recombinant protein developed antigen-specific humoral and T-cell responses and were partially protected against oral coccidial challenge (Kim et al., 1989). This study was carried out to investigate: (i) the vaccine potential of the rEAMZp250 recombinant merozoite protein using two different antigen delivery systems: (ii) the protective effects of rEAMZp250 antigen-specific humoral and cellular immunity in the immunized chickens; and (iii) the role of MHC genes in the induction of protective intestinal immune response following immunization with the rEAMZp250 recombinant coccidial antigen. MATERIALS AND METHODS
Chickens and parasites Fertile 15I5-B congenic chicken eggs were obtained from the Regional Poultry Research Laboratory (RPRL) and hatched at the Livestock and Poultry Science Institute. The development and histocompatibility status of these B congenic strains have been described elsewhere (Bacon, Ismail & Motta, 1986). Inbred SC and FP chicken eggs were purchased from Hy-line International Production Center, Dallas Center, IA. All chickens
Correspondence: Dr H. Lillehoj, Protozoan Diseases Laboratory, Livestock and Poultry Sciences Institute, U.S. Dept. of Agriculture, BARC-East, Building 1040, Beltsville, MD 20705, U.S.A.
127
128
H. S. Lillehoj, M. C. Jenkins & L. D. Bacon
were housed in clean wire-floored cages and provided feed and water ad libitum. The wild-type strain of E. acerrulina developed and maintained at the Protozoan Diseases Laboratory, LPSI, was used for challenge experiments.
Production and high-pressure liquid chromatographY (HPLC) purification of recombinant fusion proteins Detailed molecular and biochemical characterizations of the EAMZp250 recombinant antigen have been described elsewhere (Jenkins et al., 1988; Jenkins, 1988). Partial purification of this beta-galactosidase fusion protein was performed by HPLC size exclusion chromatography, as described elsewhere (Lillehoj et al., 1988). HPLC fractions representing recombinant rEAMZp250 antigen, free of bacterial protein as assessed by SDS-PAGE, were pooled and used for immunization and in ritro studies.
Production of E. coli trans/ormnant carrying the cMZ8 cDNA E. coliN6405 transformants were produced as described previously (Androphy, Lowry & Schiller, 1987; Kim et al., 1989). Transformants were grown in culture at 32 , induced for production of fusion protein at 42 for 1-2 hr. and harvested by centrifugation. The recombinant E. coli transformant pCO,MZ8 encodes the 42,000-46,000 MW protein (p 42-46) immunoreactive with serum obtained from chickens inoculated
with E. acerrulina (Kim et al., 1989).
Inimuni-ation o/ chickens and assessment of antibody and antigen-specific T-cell responses 1 51 -B congenic and inbred chickens were immunized intramuscularly with various doses of the HPLC-purified rEAMZp250 merozoite surface antigen or control extracts emulsified in complete Freund's adjuvant (CFA), followed by a second immunization I week later with antigen in incomplete Freund's adjuvant (IFA). Immunization with live E. coli expressing the rEAMZp250 protein or control E. coli harbouring a nonrecombinant plasmid was with 109 temperature-induced E. coli. Protection against E. aceruldina challenge was assessed by oocyst output in faecal samples collected 5-9 days after challenge with 104 E. acerrulina. expressed as oocyst reduction
between means were evaluated using Duncan's multiple range test. RESULTS Kinetics of antigen-specific serum antibody and T-cell responses after intramuscular immunization with the rEAMZp250 recombinant antigen Three-week-old inbred SC and FP chickens (n = 6) were given an intramuscular injection of HPLC-purified rEAMZp250 recombinant antigen twice at I -week intervals and the antigen-specific humoral responses were assessed by ELISA. The level of antibody responses depended upon the chicken strain and the dose of the antigen used (Fig. 1). At 7 days post-immunization, antibody levels of FP chickens immunized with 12 ticg rEAMZp250 were significantly higher (P
