7S Antibody Research Articles

In Vitro Immune Responses of Rabbits with Persistent Adenovirus Type 5 Infection

Abstract Antibody responses of rabbits to adenovirus type 5 were studied. Serum antibodies were still present 12 months after the animals were given a single injection of infectious virus. Isolated lymphoid tissues from these animals were capable of synthesizing high antibody titers in vitro if exposed to viral antigen prior to cultivation. Antibody-producing cultures were maintained up to 120 days. Serum antibody titers of animals immunized with the soluble antigens declined rapidly. Synthesis of antibody could be inhibited by the addition of chloramphenicol and puromycin to the culture media. Phytohemagglutinin enhanced antibody synthesis of established antibody-producing cultures. The antibody species synthesized in vitro was primarily 7S although some 19S antibody was produced. Evidence for a persistent infection in the lymphoid tissues of rabbits given infectious virus was obtained by recovery of the virus from the tissues of the animals up to 12 months after the initial injection. The virus was also isolated from spleen and lymph node fragments maintained in vitro. This study indicates that persistent infection of the rabbit with adenovirus type 5 was responsible for the prolonged immune response.

The secondary antibody response in vitro. I. The conditions of contact with antigen, cultivation and detection of antibody formation to soluble protein antigens.

The present publication deals with the conditions under which thein vitro secondary reaction to diphtheria toxoid (DT) and BSA antigens can be elicited. Using DT the reaction could be obtained as much as 30 months after antigen prestimulationin vivo; using BSA the period was shorter. The doses of antigen used for the secondary stimulusin vivo, which besides the intradermal route, were given intravenously and intraperitoneally at a certain time interval before thein vitro challenge, produced a state of unresponsiveness. This paralysis is explained as an exhaustion of cells which are ready for the secondary response. The period in which the antibody response could not be obtained following this kind of anin vivo prestimulation, lasted as Long as 3 months. On the basis of these results it is impossible to conclude how soon the restoration of the ability to react to the challenging dose of antigenin vitro takes place. The induction, duration and intensity of the secondary reaction afterin vitro contact was compared with the reaction obtained after intravital short-lasting antigen stimulation with subsequent cultivation. In both cases both 7S and 19S antibodies were found. Other parameters of the course of the reaction including the possibilities of influencing thein vitro secondary response, are discussed.

The development of clones of antibody-forming cells in the spleens of irradiated mice. II. Some properties of plaque-forming cell colonies, and evidence for their clonal nature.

SummaryLocalised concentrations of 19S antibody plaque‐forming cells developed in the spleens of lethally irradiated mice which had been injected with syngeneic spleen cells und sheep erythrocytes. Numbers of plaque‐forming cells in such colonies ranged from 28.000 down to less than 40, the largest aggregations appearing after 6–8 days of culture.Evidence that these colonies were clones came from the finding that, in most cases, all the cells in any one colony released antibody of apparently identical specificity. In addition, the appearance of the plaques themselves was often conspicuously uniform within a colony.Individual plaque‐forming cells were obtained from 9 colonies by micromanipulation and examined morphologically. In 4 cases all the cells examined from a single colony were closely similar in appearance.Localized concentrations of cells producing 7S antibody were also found, although these were relatively uncommon. Nearly half of the 7S colonies appeared in the same spleen segments as 19S colonies, statistically a highly significant association which supports the idea of a 19S to 7S antibody switch in the differentiation of some antibody‐forming cell clones.

Protection antisera afford against death by endotoxin.

Whole antisera were shown to afford substantial protection against death from endotoxin in mice in an environment held constant at 22 C, but 19S or 7S antibodies acting alone did not afford significant protection.

Passive administration of antibodies during the primary immunization. The influence on the secondary response.

Groups of mice were injected with different quantities of sheep red blood cells (SRBC: 108, 109 and 1010) and simultaneously with different quantities of antibodies against SRBC. The response was tested 15 and 30 days after the primary dose and 4 days after the secondary response. The action of antiserum regulates the quantity of antigen available for the immunization process. With a large dose of antiserum it is possible to inhibit the primary, as well as the secondary response. A smaller dose of antiserum suppresses primary antibody formation but the process of preparing the secondary response is not inhibited. An inhibitory dose of antiserum injected 24 and 48 hours after antigen significantly depresses the primary response but is followed by a pronounced secondary response. When the antigen is bound 24 and 48 hours after the primary stimulus, the secondary response is only of the 19S type. If the antigen is present at least 72 hours after the primary dose of antigen cells forming 7S antibodies appear also in the secondary response. Experimental data support the hypothesis that there is a common precursor cell for the 19S and 7S Ab-forming cell; during limited proliferation the cellular basis for the 19S secondary response and with intensive proliferation (only after 6 or 7 generations) the precursors for 7S antibody forming cells, appear.

STUDIES ON INTERACTION OF BACTERIA, SERUM FACTORS AND POLYMORPHONUCLEAR LEUKOCYTES IN MOTHERS AND NEWBORNS

The bactericidal capacity of newborn infants' whole blood for E. coli was deficient compared to the mothers, and attempts were made to identify cellular or humoral factors responsible for this deficiency. Separated polymorphonuclear leukocytes from newborn infants were found to be similar to polymorphs from mothers in capacity to engulf and kill E. coli and other bacteria so that cellular deficiency was not evident. Comparison of the serum opsonic capacity of newborn infants' and mothers' sera revealed deficient opsonic capacity for E. coli in newborn sera. The mean opsonic titer for E. coli was 46.7 in mothers and 4.3 in neonates. Serum opsonic titers for Staph. aureus and group B streptococcus were similar. The opsonic capacity for all bacterial species was decreased when the sera were heated or decomplemented with immune complexes indicating the phagocytosis amplifying role of complement. The newborn-maternal difference in opsonic capacity for E. coli was presumably a result of deficient 19S antibodies, the primary opsonic antibodies for this organism. Maternal 19S serum fractions alone, however, showed no opsonic capacity for E. coli. Addition of a complement source (newborn serum absorbed with E. coli) revealed the opsonic capacity of these 19S maternal serum fractions for E. coli. Antibodies in 19S serum fractions therefore are efficient opsonins for E. coli; however, complement is necessary to demonstrate their opsonic potential.

Cellular electrophysiology — XVII. Effect of specific antibody on the coulokinetic behavior of Escherichia coli

Results indicate that prior treatment of E. coli with either 19S or 7S antibody fractions had little or no effect on the ability of organisms to metabolize glucose as indicated by the same coulombic outputs. It appears, however, that the rate of availability of glucose to the sides of enzymatic activity in the micro-organism may be lessened.

Regulation of the immune response. I. Reduction in ability of specific antibody to inhibit long-lasting IgG immunological priming after removal of the Fc fragment.

The ability of 7S and F(ab')(2) antibody fragments to suppress priming with low doses of antigen was compared. The 7S preparation was approximately 100-1000 times more potent than the F(ab')(2) preparation when the agglutinin titers of the two preparations were the same. The presence of any ability to suppress priming in the F(ab')(2) preparation may reflect an inherent capacity of the F(ab')(2) antibody or contamination with small amounts of 7S antibody. The difference between 7S and F(ab')(2) antibody in ability to suppress priming is attributed to the lack of the Fc portion on the F(ab')(2) antibody. The Fc portion may be needed to prevent rapid excretion of antibody from the body, to induce rapid phagocytosis of antigen-antibody complexes with consequent breakdown and elimination of antigen, or to inactivate or suppress the antigen-sensitive cells from reacting to antigenic determinants. More detailed studies will permit a better assessment of the importance of these three possible regulatory roles of the Fc portion of the immunoglobulin in the immune response.

Requirement for continuous antigenic stimulation in the development and differentiation of antibody-forming cells. The effect of passive antibody on the primary and secondary response.

The essential role of continuous antigenic stimulation in the development and differentiation of antibody-forming cells as defined in the X-Y-Z immune cell maturation scheme was examined in these studies. Mice were primed with sheep erythrocytes (SRBC) in an attempt to induce maximum immune progenitor cell conversion (X --> Y). Subsequently antigen was depleted at 1 or 4 days after priming with isologous specific antibody in order to interrupt further immune cell differentiation (Y --> Z). It was reasoned that this condition would result in depression of the functional antibody-producing cell compartment as measured in the intact mice and subsequently in enhancement of the sensitized (Y cell) compartment as measured in the spleen cell transfer system. These data were also correlated with systematic studies of the hyperplasia of the spleen germinal centers. The effect of passive antibody on the primary response to SRBC was a marked decrease indirect and indirect hemolysin-producing cells (DPFC and IPFC). However, there was a lack of correlation in the degree of antibody-mediated 19S and 7S immune cell suppression during the primary response, the DPFC being much less depressed than the IPFC. As measured in the transfer system there was an enhanced 19S sensitized cell compartment and a depressed 7S sensitized cell compartment in 1 day passively immunized mice. This was true whether or not transfers were performed 1, 2, or 4 wk after priming. Similarly, there was an enhanced 19S-sensitized cell compartment with little or no effect on the 7S-sensitized. cell compartment in 4 day passively immunized mice. These data suggest that progeny of the antigen-stimulated progenitor cells (X cell), as a consequence of lack of further antigenic stimulation, were forced into maturation arrest. These studies further demonstrate that isologous passive antibody suppresses germinal center growth regardless of whether the antibody is infused 1, 2, or 4 days after priming. In terms of formation of sensitized cells, the marked depression of 7S sensitized cell compartment after passive immunization at 24 hr in contrast to the enhancement of the 19S sensitized cell compartment corresponds to the suppressed growth of germinal centers during the primary response. Thus, if the germinal center is, as suggested, the site of proliferative expansion of immunocompetent cells, these data indicate that the germinal center growth is related to the 7S antibody response and in the formation of "7S memory."

Decreased 19S antibody response to bacterial antigens in systemic lupus erythematosus.

The antibody response to immunization with Brucella and the levels of natural antibody to Escherichia coli and Shigella were compared in patients with systemic lupus erythematosus and control groups. After Brucella immunization, SLE patients showed a significantly lower antibody response in whole serum and in the macroglobulin antibody fraction separated by sucrose density gradient centrifugation. Sucrose gradient fractionation of natural antibodies to E. coli and a polyvalent Shigella antigen showed a significant decrease in macroglobulin antibody against four of the five E. coli antigens tested and the Shigella polyvalent antigen in SLE patients when compared with a group of normal individuals and a matched control group with pulmonary tuberculosis. Whole serum natural antibody titers against 5 of 13 Shigella antigens were significantly lower in the SLE patients when compared with the normal group, and against 7 of 13 when compared with the matched tuberculosis controls. Whole serum titers against 8 of 13 E. coli antigens were significantly lower in the SLE patients when compared with normal subjects. The observed decreased antibody response to bacterial antigens in SLE patients, occurring mainly in the macroglobulin fraction, is discussed in relation to the increased incidence of infection commonly observed in these patients.

Immunoglobulin classes of serum antibodies formed in response to natural infections with myxo- and pseudomyxoviruses among infants and children.

ABSTRACTThe occurrence of immunoglobulin classes of serum antibody in response to natural infections with myxo‐ and pseudomyxoviruses among infants and children was investigated. Two types of the 19S antibody response have been demonstrated. In the first type, in which are included the measles and mumps virus infections, the 19S antibody appeared dominantly as a primary response to the virus infection. In the second type, which includes the influenza, parainfluenza and RS virus infections, the 19S antibody was either not detectable or produced in a limiting amount in infants even at an early stage of the illnesses. A possibility was discussed that production of the 19S antibody in response to the infection with myxo‐ and pseudomyxoviruses may be primarily determined by the nature of virus infection itself, particularly the presence or absence of a viremia.

Anti-trinitrophenyl (TNP) antibody detection by neutralization of TNP-coliphage T4

Anti-trinitrophenyl antibodies are detected by the specific neutralization of bacteriophage T4 haptenated with the picryl radical (TNP). TNP-phage neutralization by anti-TNP is compared to neutralization by anti-phage. Anti-TNP neutralized haptenated phage by two separable first order processes. The initial reaction was operative until the surviving fraction approached 0·1 at which point a second, slower, apparent first order process was detectable. Neutralization of haptenated phage by anti-phage antibody appeared to proceed by a single first order reaction and thus differed from neutralization by anti-TNP antibody. Neutralization by anti-TNP is hapten specific since anti-hapten antibody was unable to neutralize native phage and since neutralization of TNP-phage by anti-TNP was inhibited by TNP-ϵ-aminocaproic acid which does not affect anti-phage antibodies. Neutralization of TNP-phage by anti-hapten also differs from neutralization of TNP-phage in the rat the former is more dependent on antigen concentration. This assay was used to detect primary 19S and 7S anti-hapten antibodies separated by sucrose gradient centrifugation and provides a means of detecting the two classes of anti-TNP antibodies at very low concentration.

Immunochemical studies of foot-and-mouth disease. V. Antigenic variants of virus demonstrated by immunodiffusion analyses with 19S but not 7S antibodies.

Three antigenic variants of foot-and-mouth disease virus, type A, strain 119, were demonstrated in Ouchterlony analyses utilizing serum collected from guinea pigs 7 days postinfection (DPI). Such antisera contain antibodies of the 19S class. Guinea pig antisera that contained antibodies of the 7S class were unable to distinguish between the antigenic variants. Similarly, 19S antibody was able to demonstrate antigenic differences in trypsin- and chymotrypsin-treated viruses that were not detected by 7S antibody-containing antisera. One of the antigenic variants of virus is apparently the wild type and is tentatively considered to have two antigenic determinant groupings termed the a- and b-sites (140S-ab). The 140S-ab variant was the sole or predominant antigenic type produced in guinea pigs and in large plaque-forming- and tissue culture-low passage sources of the virus. Another antigenic variant appears to possess only the b-site (140S-b) and was the major constituent in tissue culture-high passage virus preparations. The third variant, a small plaque former, was also devoid of the a-site and contains an antigenic determinant that is related to, but not identical with, the b-site. This variant appears to be a minor constituent of tissue culture-high passage virus. 7-DPI serum could be absorbed with a suitable concentration of tissue culture-high passage virus to remove antibody reactive with the b-determinant site. This absorbed serum still precipitated 140S-ab virus by virtue of still containing antibody reactive with the a-determinant site; however, the neutralizing activity was eliminated. This suggests that the b-site is critical with respect to neutralization while the a-site is noncritical.

Protection Antisera Afford Against Death by Endotoxin1

Whole antisera were shown to afford substantial protection against death from endotoxin in mice in an environment held constant at 22 C, but 19S or 7S antibodies acting alone did not afford significant protection.

Role of the Fc fragment in the regulation of the primary immune response.

SPECIFIC antibody, passively transferred, has the capacity to inhibit the production of the specific immune response (reviewed in ref. 1), and may even terminate a response2. There have been several reports dealing with the ability of F(ab′)2 antibody (the antibody fragment which has the two antigen-binding sites but lacks the Fc fragment) to suppress the primary immune response (refs. 3–5, and F. J. Dixon, quoted in ref. 1). The specificity of this type of immunosuppression is best explained in terms of antigen–antibody binding so that the antigenic site is masked, and so it was not surprising that F(ab′)2 was found to induce antibody-mediated suppression of the immune response. In an initial attempt at preparing F(ab′)2 antibody, however, we found that all the suppressive activity in the F(ab′)2 preparation could be explained on the basis of contaminating whole 7S antibody. We report here the results of experiments in which the 5S F(ab′)2 antibody digested by pepsin and remaining undigested 7S antibody were separated on sucrose gradients and their activities in suppressing the primary antibody response tested separately.

The effect of antigenic competition on various manifestations of humoral antibody formation and cellular immunity.

The effect of antigenic competition on various parameters of humoral antibody formation and cellular immunity was studied in mice. Several pairs of antigens were employed in the investigation of the competitive aspects of induction of humoral antibody formation. Induction of a primary immune response to hemocyanin in Swiss white female mice moderately suppressed the induction of both 19S and 7S antibody formation to goose or rat erythrocytes. Suppression of 7S antibody formation was maximal when a time interval of 1-3 days separated the sequence of injections, although suppression was noted for intervals of up to 14 days. The induction of a primary immune response to rat RBC, the second of the two antigens in sequence, also suppressed The induction of both 19S and 7S antibody formation to goose RBC when appropriate intervals of 1-3 days were employed between injections. The induction of a secondary immune response to rat RBC totally suppressed the primary induction of both 19S and 7S antibody formation to goose RBC administered in the appropriate time sequence. Subsequently, it was shown that the secondary immune response to the suppressed antigen (goose RBC) elicited 30 days after induction of a primary immune response (5 days after secondary immunization with rat RBC) was characterized by deficient 19S and 7S antibody production. These levels were suppressed even in comparison with a normal primary immune response to this antigen. The results were interpreted in part on the basis of a deficiency of formation of primed cells associated with immunological memory. Alternatively, evidence was obtained for the development of a split type of immunological tolerance in 6 of 10 animals studied, since a total suppression of 7S antibody production was obtained in association with deficient 19S antibody synthesis (titers < 1/10). The induction of a primary immune response to several antigens in A/J female mice suppressed the processes of cellular immunity as manifested by prolonged survival of skin grafts from C57 BL/6J female donors. These results were interpreted as evidence for the existence and utilization of processing cells by the initial immune stimulus yielding a deficiency of cells available for processing the second antigen administered in sequence.

The life cycle of antibody-forming cells. I. The generation time of 19S hemolytic plaque-forming cells during the primary and secondary responses.

The generation, time of cells synthesizing 19S antibody was measured during the exponential phase of the primary and secondary immune responses. Using a plaque-forming cell assay together with a double label isotope method, the duration of the generation time was found to be about 13 hr, that of the S period was about 8 hr, and that of G-1 and G-2 were about 3 and 2 hr, respectively, in both immune responses. Previous work was confirmed showing that all plaque-forming cells observed during the exponential phase of the immune responses incorporate labeled thymidine, and therefore take part in proliferation. These results were obtained at points during both responses when the doubling time of the PFC was observed to be 6-7 hr. This finding makes untenable the hypothesis that the rate of appearance of PFC equals the rate of proliferation of these cells. A hypothesis is presented to account for these findings and to serve as a basis for further experiments.

Receptors for complement of leukocytes.

Sheep red blood cells sensitized by 7S, but not by 19S rabbit anti-Forssman antibodies, adhere and form rosettes on mouse macrophages and on a few monocytes and polymorphonuclear cells (PMN). When, however, C' factors from mouse serum are added to the antigen-19S antibody complex (EAC'), rosettes are formed on most mouse peritoneal macrophages and PMN and on a few monocytes. In addition EAC' also adheres to 10-25% of lymph node lymphocytes but not to thymus lymphocytes. EAC' prepared with 7S anti-Forssman antibodies has identical properties. The adherence of red cells induces an increase in the membrane activity of the leukocytes and causes injury to the red cells which rapidly become deformed and fragmented. Adherence of EAC' occurs at 37 degrees C and is minimal at 4 degrees C. Probably only the first four C' components are involved in this phenomenon as mouse serum deficient in C'5 or rabbit serum, deficient in C'6 can be used as a source of C' components. Treatment of EAC' with EDTA does not modify its leukocyte-adherence properties. The adherence of EAC' to the leukocytes is not inhibited in the presence of serum. The receptors for C' on macrophages, PMN, and monocytes differ from those found on lymphocytes. Rosette formation by EAC' on macrophages, PMN, and monocytes depends on divalent cations (Mg(++)) and can be reversed by Na(3)H EDTA, while adherence to lymphocytes is independent of these ions and occurs in the presence of 0.01 M Na(3)H EDTA. Both types of receptors for C' components are destroyed by trypsin treatment of the leukocytes, in contrast with the receptors for 7S antibodies on the same cells which persist after enzyme treatment.

RECOVERY IN THE MOUSE SPLEEN AFTER SUBLETHAL IRRADIATION

ABSTRACT1. CBA and C57B1 mice were exposed to LD50/30 doses of γ‐radiation and at intervals after irradiation the numbers of haemopoietic colony‐forming units (CFU) and of antibody‐producing cells in the spleen were determined.2. CFU repression was observed after transfer of non‐irradiated cells to hybrid recipients. However, this repression did not appear until 60 days after irradiation of the donor.3. The recovery after irradiation of the capacity to inactivate CFUs in spleen cell mixtures was studied. The results suggested that cells which produce 19S antibody are different from those which are reactive against foreign cells.

Greater specificity of 19S than 7S antibodies on haemagglutination-inhibition tests with closely related group B arboviruses.

ATTEMPTS to demonstrate differences in specificity of 19S and 7S antibodies have produced conflicting results. For example, recent reports refer to (i) greater specificity of 7S than 19S rabbit antibodies in agglutination tests with strains of Shigella flexneri1; (ii) similar specificities with these two antibodies in complement-fixation tests with rabbit antibody fractions to Southern bean mosaic virus2; (iii) specific 19S antibody, in haemagglutination-inhibition (HI) tests, against mumps virus in monkeys previously immune to the antigenically related DA virus, in the presence of 7S antibody equally reactive with both viruses3; and (iv) 19S rabbit antibodies to SW influenza virus which showed high cross-reactivity with related viruses compared with relatively specific early 7S antibody, both in HI and neutralization tests4. Because of the strong serological cross-reactivity of group B arboviruses, especially in HI tests, efforts to improve group B antibody specificity have employed rather tedious antigen absorptions5,6, an indirect HI test using erythrocytes presensitized with each antigen of interest7, or Casals's8 kinetic HI test9,10 which uses a checker-board arrangement for each antigen–antibody reaction and hence becomes rather cumbersome.