Biomedical subjects
Publications and source records attributed to C R Young.
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Recently, in investigating the responses of T-cells from high responder mice that were primed with myoglobin (Mb) or with synthetic peptides containing antigenic site 5 and increasing in length stepwise by increments of two residues, we observed that T-cell recognition was highly dependent on conformation. In the present studies, tolerization experiments were carried out to further investigate this finding. Neonatal mice (BALB/cByJ) were either tolerized with Mb or with synthetic peptides of Mb containing antigenic site 5. Tolerization with Mb and subsequent immunization with Mb gave T-cells that did not proliferate in vitro to Mb or any of the peptides. T-cells from mice that were tolerized with a truncated peptide 139-153 (having deletions at Tyr-151 and Ala-144) and subsequently immunized with Mb proliferated in vitro to Mb and to peptides 132-153, 135-153 and 143-153. T-cells from mice that were tolerized with native Mb and subsequently immunized with peptides (which are unfolded in solution) did not proliferate in vitro to Mb, but responded well to the peptides. Conversely, tolerization with peptides had no effect on the recognition of, and the response to, native Mb by the T-cells, whereas the response to the peptides was completely removed. It was thus concluded that the recognition of protein antigens (or at least of Mb) by T-cells is (like the recognition by antibody) dependent on the conformation of the antigen.
Previous studies in this laboratory have resulted in the determination of the antigenic structure of myoglobin. The present work was carried out to investigate the genetic control of the murine antibody response to myoglobin following immunization with free (i.e., not coupled to a carrier) synthetic antigenic sites or other peptides corresponding to surface regions of myoglobin that are not immunogenic when the native molecule is the immunizing antigen. Synthetic peptides corresponding to antigenic site 1 (peptide 15-22), site 2 (peptide 56-62), site 3 (peptide 94-100), site 4 (peptide 113-120), site 5 (peptide 145-151) and two surface regions, peptide 1-6 and peptide 121-127, were injected in complete Freund's adjuvant in different strains of mice. Serum antibodies specific for myoglobin were subsequently obtained and were measured by means of a radioimmune plate binding assay in which Mb was used as the solid phase antigen. It was found that the genetic control of the antibody response to myoglobin following immunization with the free synthetic peptides was different from the genetic control obtained following immunization with native myoglobin. The significance of this finding is discussed.
Previously described GM1 ganglioside enzyme-linked immunosorbent assays (GM1-ELISA) for the detection of Escherichia coli heat-labile enterotoxin (LT) showed sensitivity equal to the Y-1 adrenal cell assay when anti-LT (a reagent not commercially available) was used. However, when antitoxin to immunologically related (commercially available) cholera toxin was substituted, a marked loss in sensitivity occurred. We modified the GM1-ELISA that employed anti-cholera toxin to make it comparable in sensitivity to the Y-1 adrenal cell assay. When five media commonly used for LT production were compared, Mundell's Casamino Acids medium was shown to be significantly superior. Lincomycin (45 micrograms/ml) added to E. coli cultures significantly increased net optical densities in the GM1-ELISA, a direct measure of the amount of LT. Treatment of broth cultures or bacterial cell pellets with polymyxin B or extension of culture time to 48 h also significantly increased net optical density by allowing enhanced release of periplasmic LT. A major innovation involved the direct culture of E. coli strains in GM1-coated wells of microtiter plates followed by ELISA. This direct culture method GM1-ELISA (DCM-GM1-ELISA) saved not only assay time, but also materials and reagents. The net optical densities that result from this assay allow the test to be read visually without a spectrophotometer. Three independent observers read plates with E. coli tested by DCM-GM1-ELISA. Thirty-four of 35 adrenal cell-positive strains (97% sensitivity) and 30 of 30 LT-negative control E. coli strains (100% specificity) were identified by all three observers reading coded plates. The DCM-GM1-ELISA provides a simple, practical and efficient assay for LT for less sophisticated laboratories.
Previously, we have shown by radioimmune antibody binding studies that immunization with each of the five synthetic antigenic sites of sperm-whale myoglobin (Mb), in their free form (i.e. not coupled to a carrier), resulted in the formation of antibodies that bound specifically to Mb and exclusively to the peptide that was used in immunization. In the present series of studies we have immunized separate groups of Balb/cByJ mice with different amounts of each of the free synthetic antigenic sites so as to determine the optimum immunizing dose for eliciting serum antibodies that bind specifically to Mb. The synthetic peptides corresponded to: Site 1 (peptide 15-22), Site 2 (peptide 56-62), Site 3 (peptide 94-100), Site 4 (peptide 113-120, and Site 5 (peptide 145-151). The dose range of each of the antigenic sites used in immunization was from 6 micrograms to 100 micrograms. Radioimmune antibody binding studies indicated that there is an optimal immunizing dose for each of the five antigenic sites which was smaller than anticipated. The significance of these findings is discussed.
Texas Star-SR, a laboratory-derived mutant of Vibrio cholerae El Tor Ogawa 3083, which produces B but not A subunit of cholera toxin was given to 68 healthy adult volunteers in doses of 10(5) to 5 X 10(10) organisms. 16 of 68 exhibited loose stools but in only one individual was stool volume notable. Vomiting occurred in 1 and abdominal cramps in 3 vaccines; malaise and fever were not seen. Texas star was recovered from stools of 22% who received low doses (10(5) or 10(6) organisms) and from 63% who received high doses (10(8), 10(10), 2 X 10(10) or 5 X 10(10)). The attenuated strain was also recovered from jejunal fluid of 76% in the high dose group; cultures revealed 10(2)-10(5) organisms/ml. Seroconversions of vibriocidal antibody occurred in 93% and peak organisms/ml. Seroconversions of vibriocidal antibody occurred in 93% and peak titers were resembling those seen following clinical cholera. In contrast, serum IgG ELISA antitoxin rose significantly in only 29% and levels were below those encountered after clinical cholera. Only 5 of 18 vaccinees tested so far had significant rises in intestinal SIgA antitoxin; these also manifested rises in serum antitoxin. The occurrence of loose stools did not correlate with dose ingested, excretion of vibrios or rise in serum antitoxin. 503 clones recovered from coprocultures and jejunal fluids were negative when tested for enterotoxin. One month following a single 5 X 10(10) organism dose of Texas Star, 7 vaccinees and 6 controls were challenged with 10(6) pathogenic El Tor Ogawa vibrios. Diarrhea occurred in 7 of 10 controls but in only 1 of 7 vaccinees (p = 0.05). Despite clinical protection, excretion of pathogenic V. cholerae was similar in vaccinated and control groups. Twelve vaccinees who received two 10(9) or two 2 X 10(10) organisms doses of Texas Star 1 week apart were challenged with 10(6) pathogenic V. cholerae El Tor of the heterologous serotype. Diarrhea occurred in 11 of 15 controls but in only 3 of 12 vaccinees (p. 0.01). The 3 vaccinees with diarrhea all had mild illness. Texas Star-SR is a prototype that commonly causes mild diarrheal responses but is genetically stable in vivo and stimulates protective immunity against challenge with either the homologous or heterologous serotype.
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Previous studies from this laboratory have resulted in the determination of the antigenic structure of sperm-whale myoglobin (Mb). In the present work, we have investigated the fine specificity requirements for T-cell recognition of one of the Mb antigenic sites (antigenic site 5). The antigenic site (peptide 145-153) and seven progressively longer peptides, increasing in length stepwise by two residues at a time up to 22 residues in length (peptide 132-153), were synthesized. In addition, four truncated peptides were synthesized with intentional deletions at Tyr-151 and Ala-144. The T-cell recognition of these purified synthetic peptides was examined here in detail in three strains of mice (BALB/cByJ, B10.D2/n and SJL/J). Mb-primed mice afforded T-cells which proliferated to smaller peptides (two or four residues longer than the site; i.e. peptides 145-153 and 143-153) and more so to the longer peptides 135-153 and 132-153 and to Mb. No response was obtained to the truncated peptides, thus underscoring the fine specificity of T-cells. No response was obtained also to intermediate-sized peptides. The latter result, due to an unfavorable mode of folding, suggested a conformational dependency in T-lymphocyte recognition and was confirmed by additional studies employing peptide priming. Thus priming with either the 11- or 22-residue peptides afforded T-cells which proliferated to both of these two peptides, but not to intermediate-sized peptides. More importantly, peptide-primed cells did not proliferate on challenge in vitro with Mb. This indicated that the in vivo recognition (priming) was to the unfolded peptide and these cells will not therefore recognize native Mb in vitro. Tolerization of neonatal mice with native Mb, which was total for the native protein at the T-cell level, had no effect on in vivo recognition (priming) and in vitro proliferation due to the unfolded peptide. Conversely, tolerization by any unfolded peptide had little or no effect on in vivo recognition of, and proliferation to, native Mb. It was concluded that T-cell recognition of native proteins (or at least of Mb) is dependent on protein conformation. A peptide may not cause proliferation of native protein-primed T-cells even though it contains an antigenic site. Finally, macrophage presentation of protein antigens is not related to processing via fragmentation of antigen by macrophage.
Nonenterotoxigenic strains no. 1196-78 and no. 1074-78 of Vibrio cholerae serogroup O1 (biotype El Tor, serotype Ogawa) were isolated from sewage water in Brazil and fed to 20 volunteers. Neither strain caused diarrhea. None of the seven volunteers who ingested Ogawa strain no. 1074-78 (10(6) organisms) excreted the organism whereas eight of the 13 volunteers who ingested Ogawa strain no. 1196-78 (10(6) or 10(8) organisms) did excrete the organism in their stools. None of 114 stool-culture isolates yielded cholera enterotoxin, and none of the 20 volunteers had significant increases in serum titers of antitoxin as measured by enzyme-linked immunosorbent assay although six of the volunteers had slightly elevated vibriocidal antibody levels. Six volunteers used as controls and four volunteers who had stool cultures positive for strain no. 1196-78 of V. cholerae were challenged with pathogenic El Tor Ogawa strain no. E7946 (10(6) organisms) to determine if previous ingestion of the Brazilian strain would induce protective immunity. All 10 of the volunteers developed diarrhea, and the severity of the illness was similar in both groups.
Sera from 147 volunteers were examined for vibriocidal antibody by the tube-dilution method before and after they ingested 10(3)-10(6) Vibrio cholerae organisms. Titers increased significantly after challenge in 97% of 110 persons who excreted V. cholerae. In 12% of the bacteriologically confirmed infections, the titer increased significantly only to the homologous (Inaba or Ogawa) serotype. Levels of vibriocidal antibody decreased substantially between one and six months after challenge, but they usually remained elevated over base-line values. Levels of antitoxin to V. cholerae were measured by IgG enzyme-linked immunosorbent assay (ELISA), rabbit skin permeability tests, and adrenal cell techniques in 73 pairs of prechallenge and postchallenge sera. Results of the vibriocidal assay agreed most closely with those of the ELISA, the most sensitive antitoxin assay, in serologically detecting clinical and subclinical infections. Recent infection could be accurately serodiagnosed by levels of vibriocidal antibody and antitoxin (by IgG ELISA) in a convalescent-phase serum.
Previously, it was reported that the immune response to myoglobin (Mg) was under genetic control, with the response to each site being under separate Ir-gene control. Here we have investigated the effect of antigen dose on the control of the antibody response to the five antigenic sites of sperm-whale Mb to determine whether or not the overcoming of genetic control by antigen dose has a uniform effect on all five antigenic sites. The antibody response to sperm whale myoglobin (Mb) and its five antigenic was measured in the following inbred strains of mice, C57BL/6J, AKR and SWR/J. These strains of mice are low responders to Mb following immunization with 50 micrograms, responding only to site 4. After immunization with 200 micrograms Mb: C57BL/6J mice are high responders to Mb and respond to antigenic sites 1, 3, 4 and 5; AKR mice are high responders to Mb and respond to antigenic sites 1 and 4; SWR/J mice are high responders to Mb and respond to all five antigenic sites. It was concluded that the genetic control of the immune response to Mb and its synthetic antigenic sites is dependent on antigen dose. Also, these studies have enabled us for the first time to separate the response to site 1 from the response to site 2 and thus have conclusively established that sites 1 and 2 are controlled by separate Ir-genes.
Previous studies from this laboratory have resulted in the determination of the entire antigenic structure of myoglobin (Mb). The present work was carried out to investigate the antigenicity of the synthetic antigenic sites of Mb in their free form (i.e. without coupling to a carrier) and the effect of peptide size on antigenicity. Site 5 (peptide 145-151) and the longer peptides 145-153, 143-153 and 132-153, each of which carries within it the region of site 5, were synthesized. Immunization of three different mouse strains with each of these peptides, in their free form, resulted in the formation of antibodies that bound specifically to intact Mb. The advantages and significance of these findings are discussed.
Purified type 1 somatic pili from enterotoxigenic Escherichia coli (ETEC) strain H10407 (O78:H11) was evaluated as a parenteral immunizing agent in the hope that this antigen might enhance a contemplated polyvalent pilus vaccine. Intramuscular inoculation with 45, 90, 900 or 1 800 mcg of pili vaccine was tolerated without incident in 82 volunteers. Six of 15 persons who received a 28 day booster of 1 800 mcg developed local reactions while none of 52 persons receiving 180 or 450 mcg boosters evinced such reactions. Pili vaccine did not significantly alter intestinal transit time, absorptive capacity or the prevalence of colonic E. coli bearing type 1 somatic pili of the H10407 antigenic variety. All vaccinees developed significant rises in circulating IgG antibody to type 1 somatic pili, the magnitude of the response being directly proportioned to the vaccine dose. None of the vaccinees had significant rises to CFA I or II pili nor to heat-labile enterotoxin. However, many had rises in O antibody, particularly among those inoculated with 1 800 mcg. Three challenge studies were carried out with E. coli H10407 to assess vaccine efficacy. In the initial study the vaccinees were either protected against diarrhea (2 of 6 vaccinees versus 7/7 of controls) or had milder disease than the controls. In two subsequent challenges with H10407 significant protection was not seen. It was not clear whether protection exhibited by the vaccinee group in the first challenge was due to O antibody, pili antibody, or both acting synergistically. To clarify this, a group of the immunized volunteers were challenged with ETEC strain B7A which is a different serotype (O148:H28) lacks CFA/I or II pili, but possesses type 1 somatic pili antigenically distantly related to those of H10407. Attack rates and severity of illness were similar in both vaccinee and control groups. While most volunteers challenged with E. coli H10407 developed significant rises in circulating antibody to CFA/I, LT and O antigen, none had rises to type 1 somatic pili. It is unclear if this is due to immune tolerance to this antigen when encountered enterally or whether these pili are not present in vivo in ETEC initiating diarrhea in the proximal small intestine. In summary, parenterally inoculated type 1 somatic pili were safe and highly immunogenic in man but did not consistently induce protection. Further studies are planned to clarify the role of antibody to type 1 somatic pili in mediating protection.
The antibody response to the synthetic polypeptide poly (L Tyr, L Glu)-poly (DL Ala)--poly (L Lys) designated (T,G)-A--L, was investigated in inbred, congenic, F1 and F2 hybrid strains of mice. The antibody response was analysed at both low (10 microgram) and high (50 microgram) immunizing doses of (T,G)-A--L. Antibodies were measured using both a modified Farr assay and a plate binding assay. At low immunizing doses it was found that all of the congenic and non-congenic (low responder x low responder) F1 hybrids were low responders. However, the quantitative antibody response of one non-congenic (low responder x low responder) F2 hybrid segregated in a 1:1 ratio of high responders to low responders, suggesting some form of complementation of (T,G)-A--L Ir genes. At high immunizing doses it was found that congenic and non-congenic (low responder x low responder) F1 hybrids were all high responders, indicating a complementation of Ir genes to (T,G)-A--L. This complementation was confirmed using two different routes of immunization, namely footpad and intraperitoneal. Furthermore the quantitative antibody responses of (low responder x low responder) F2 hybrids segregate in a 1:1 ratio of high responders to low responders. The class of antibodies produced to (T,G)-A--L in (low responder x low responder) F1 hybrids was determined by gel filtration on Sephadex G-200, and found to be predominantly IgG, with lesser amounts of IgM.
Four volunteers were rechallenged with Vibrio cholerae (10(6) classical Ogawa 395 organisms) 33-36 months after their initial induced cholera infection; none of the four veterans and four of five control volunteers developed diarrhea (P = 0.04). All control subjects, but only one veteran, had positive coprocultures. Three of the four veterans had significant levels of serum IgG antitoxin before challenge, but none had measurable intestinal levels of secretory IgA antitoxin. Significant rises in levels of serum vibriocidal and antitoxic antibody occurred in all control subjects and in two veterans, who also manifested rises in levels of intestinal secretory IgA antitoxin. The impressive duration of infection-derived immunity suggests that the most promising approach to development of cholera vaccines may be to mimic natural immunity with orally administered, attenuated strains of V. cholerae.
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We have previously shown that the antibody response to mammalian myoglobins is under genetic control. In the present study we examined antibodies to sperm-whale. Atlantic bottlenosed dolphin, Black Sea dolphin, horse and badger myoglobins, raised in high responder strains of mice, to ascertain whether there is genetic control of antibody affinity to mammalian myoglobins. Using antisera of varying dilutions, the binding to 125I-labelled homologous myoglobins was studied by inhibition with homologous myoglobin over a wide range of inhibitor concentration in a modified Farr essay. The results indicated that there are no large differences between high responder strains of mice in the affinity of antibodies to mammalian myoglobins.
Previously it has been shown that the immune responses to sperm-whale myoglobin are under H-2-linked Ir-gene control. More importantly the responses to the synthetic antigenic sites are each under separate genetic control. In the present studies we investigated the genetic control of the antibody response to four different myoglobins of known structure, to determine whether this genetic control is influenced by changes in the properties of the sites. The results suggest that genetic control of the responses to individual antigenic sites on a protein antigen is not only determined by the genetic constitution of the host species but also by the chemical properties of the individual sites. It appears that the H-2 subregions mapping the responses to given antigenic sites can also recognize other sites, which were previously unrecognizable in a homologous protein, if their chemical properties are suitably altered.