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The hemolytic plaque formation of cells producing antibody against heterologous albumins was tested for sensitivity to specific inhibition by free antigen. The inhibition characteristics of plaques in this system were found to be a measure for the avidity of the antibody produced by the plaque-forming cells (PFC:s). High avidity-producing PFC:s were more sensitive to inhibition than low avidity PFC:s. Immunization with a high dose of antigen induced PFC:s that produced antibody with a lower avidity as compared to PFC:s from animals immunized with a low dose. The avidity was increased with time. Determinations of avidity at the serum level were also made, and the results were in agreement with the findings at the cellular level.
The present method made it possible to demonstrate differences in avidity of antibody at the level of the single antibody-forming cell. It may also constitute a useful tool for the analysis of the cellular events leading to the production of antibodies with varying affinities during the immune response.
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... Affinity of antibody was determined after transfer to sublethally irradiated host animals and stimulation with antigen. The antibody affinity was studied at the cellular level, using the sensitivity of hemolytic plaque-forming cells to hapten inhibition as a measure of affinity (10,11). ...
... In the DNP40BSA-SRBC test 1 mg of free BSA was added to the agar together with the erythrocytes. This is a concentration of BSA that will completely inhibit BSA-specific plaques (11). Antibody-forming cell suspension was added in 0.1 ml of EME. ...
... Detection of A~nity at the Cellular Level.--The sensitivity of the hemolytic plaques to inhibition with free hapten was used as a measure for affinity (10,11). Immediately before plating, 0.I ml of PBS containing 0.001-100 pg of NIP-e-aminocaproic acid (NIP-EACA) or DNP-e-aminocaproic acid (DNP-EACA) was added to the top layer of agar. ...
Heterogeneity with regard to affinity of anti-hapten antibody was demonstrated at the cellular level in mice. The heterogeneity was shown at the level of single antibody-forming cells using hapten inhibition of hemolytic antibody plaque formation as a measure of affinity. The affinity increased with time after immunization. A high antigen dose initially resulted in relatively low affinity antibody production as compared to the affinity of the antibody production in animals immunized with a low dose. Affinity specialization of immunological memory cells was demonstrated, since it was possible to specifically fractionate such cells with regard to affinity on hapten-protein-coated plastic bead columns. High affinity memory cells showed a higher tendency to become retained in the columns than did low affinity memory cells. The data in a direct way demonstrate that memory cells carrying membrane-associated receptors of a certain affinity for the antigen are determined to release antibody of a similar affinity after stimulation with antigen.
... It has been found that changes in the affinity of serum antibody with time after immunization reflect changes in the population of antibody-secreting cells. By determining the concentration of antigen required to inhibit individual plaque-forming cells (PFC) assayed at various times after immunization, a time-dependent avidity increase at the antibody-secreting cell level is seen and is comparable to the increase in affinity found in serum antibody (3). Thus, a good correlation between the (intrinsic) affinity of serum antibody and the avidity of the antibody-secreting cell products has been established. ...
... The precursor activity of the purified DNP-binding cells was tested by transferring these cells together with DNP-keyhole limpet hemocyanin (KLH) and a source of KLH carrier-primed cooperator cells into irradiated recipients and subsequently assaying the recipient spleens for anti-DNP-plaque-forming cells (PFC). In addition to quantitating the precursor activity of the purified DNP-binding cells, the hemolytic plaque assay was used to study the avidity of single antibody-secreting cells (3). By determining the avidity of anti-DNP-PFC resulting from the transfer of purified DNP-binding cells with varying avidities for ~DNP-MGG, we have found a direct correlation between the avidity of the DNP antigen-binding cells and the anti-DNP antibody secreted by the PFC derived from them. ...
Cells binding DNP groups conjugated to fluoresceinated mouse gamma globulin (FDNP-MGG) were isolated from spleens of unprimed mice using a fluorescence-activated cell sorter (FACS). The isolated cells were specifically enriched at least 100-fold for anti-DNP precursor activity in an adoptive transfer assay as compared to unfractionated spleen. The fraction depleted of binding cells, although depleted of anti-DNP precursor activity, responded as well as unfractionated spleen when assayed for anticarrier (keyhole limpet hemocyanin [KLH]) precursor activity. High avidity binding cells were stained using low concentrations of FDNP-MGG. Medium and low avidity binding cells were stained using high concentrations of FDNP-MGG in the presence of free hapten which selectively blocked staining of the high avidity binding cells. Cells were supplemented with an excess of carrier-primed (KLH), nylon-purified splenic T cells and transferred to irradiated recipients. DNP-KLH was given at transfer and 5 days later. The anti-DNP plaque-forming cell (DNP-PFC) response and the avidities of the DNP-PFC in the irradiated recipients were measured by hapten inhibition of direct PFC plaque formation 12 days after transfer. At this time, very few indirect PFC were found. There was a positive correlation between the avidity of the DNP-binding cells and the avidity of the anti-DNP antibody secreted by their progeny. High avidity DNP-binding cells gave rise to predominantly high avidity anti-DNP-PFC. Medium and low avidity binding cells gave rise to medium and low avidity DNP-PFC.
... However, a higher antigen dose may not necessarily be better . For example, unlike with a low antigen dose, when primed with a high antigen dose, the antibody response is higher and quicker, but the antibody titer levels off earlier . Additionally, in contrast to low-dose-induced antibodies, the antibodies due to a high antigen dose are of higher affinity/avidity, yet such antibodies impair the antibody formation mechanism after immunization . ...
... For example, unlike with a low antigen dose, when primed with a high antigen dose, the antibody response is higher and quicker, but the antibody titer levels off earlier . Additionally, in contrast to low-dose-induced antibodies, the antibodies due to a high antigen dose are of higher affinity/avidity, yet such antibodies impair the antibody formation mechanism after immunization . In other words, low-affinity antibodies enhance the initial immune response and B-cell activation. ...
Ticks are ubiquitous blood-sucking ectoparasites capable of transmitting a wide range of pathogens such as bacteria, viruses, protozoa, and fungi to animals and humans. Although the use of chemicals (acaricides) is the predominant method of tick-control, there are increasing incidents of acaricide tick resistance. Furthermore, there are concerns over accumulation of acaricide residues in meat, milk and in the environment. Therefore, alternative methods of tick-control have been proposed, of which anti-tick cattle vaccination is regarded as sustainable and user-friendly. Over the years, tremendous progress has been made in identifying and evaluating novel candidate tick vaccines, yet none of them have reached the global market. Until now, Bm86-based vaccines (Gavac™ in Cuba and TickGARDPLUS™ Australia-ceased in 2010) are still the only globally commercialized anti-tick vaccines. In contrast to Bm86, often, the novel candidate anti-tick vaccines show a lower protection efficacy. Why is this so? In response, herein, the potential bottlenecks to formulating efficacious anti-tick vaccines are examined. Aside from Bm86, the effectiveness of other anti-tick vaccines is rarely assessed. So, how can the researchers assess anti-tick vaccine effectiveness before field application? The approaches that are currently used to determine anti-tick vaccine efficacy are re-examined in this review. In addition, a model is proposed to aid in assessing anti-tick vaccine effectiveness. Finally, based on the principles for the development of general veterinary vaccines, a pipeline is proposed to guide in the development of anti-tick vaccines.
... In the present article, we have attempted to clarify the effect of LPS on anti-2,4,6-trinitrophenyl (TNP) 19S antibody-producing cell (APC) responses in the spleen of mice, on the regulation of affinity of anti-TNP 19S antibody synthesized at the cellular level, and on the correlation between its adjuvant action on the APC response and the alteration of the affinity of antibodies synthesized by these cells. the affinity of the antibody produced by the PFC [1,7] ; i.e. high affinity-producing PFC was more sensitive to inhibition than low affinity PFC. Inhibition of plaque formation was conducted by adding 0.1 ml of PBS containing TNP-BSA to the liquid agar which was used as the PFC assay immediately before plating. ...
... In addition, adding some concentrations of fluid antigens to the agar results in an apparent reduction of PFC numbers in the plates. The affinity (avidity) of the antibody produced by the PFC is reflected by the relative sensitivity of the plaques to inhibition by soluble antigens added to agar gel [1,7]. Plaques formed by low affinity require higher antigen concentrations for inhibition than high affinity plaques. ...
The effect of lipopolysaccharide (LPS) on anti-trinitrophenyl (TNP) direct plaque-forming cells (PFC) in the spleen of mice and the affinity of antibodies produced by these PFC were examined. Simultaneous injection of bacterial LPS and TNP-coupled sheep red blood cells(SRBC) induced an obvious increase in anti-TNP PFC numbers and heightened the antibody affinity at cellular levels. The higher the doses of LPS, the greater the effects. Concomitant injection of LPS in TNP-coupled homologous mouse red blood cells (MRBC) also elicited good anti-TNP PFC response and slightly heightened the affinity. Priming with LPS and SRBC together 7 days prior to immunization did not enhance the anti-TNP PFC response and it was difficult to alter the affinity. Preinjection with small amounts of TNP-MRBC or -rabbit red blood cells and LPS simultaneously did not induce any significant increase in anti-TNP PFC secondary response after reimmunization with TNP-SRBC, but obviously heightened the antibody affinity. Injection of LPS simultaneously with the secondary immunization was effective for both the anti-TNP PFC response and the alteration of antibody affinity. These results suggest that LPS affects the control mechanisms of anti-TNP antibody affinity via the non-thymus-derived helper cell function, and the adjuvant action and alteration of antibody affinity induced by LPS are regulated by different mechanisms.
... Avidity maturation is thought to take place when B cells compete for a limited supply of antigens to receive signals to survive. It is tempting to speculate that, if sufficient antigen is available, there is no advantage for higher-avidity clones to overgrow the low-avidity populations (24). Nonetheless, there are other possible explanations for these findings, as it has been shown that somatic hypermutation still takes place in the bone marrow of mice vaccinated with NP-Ficoll, supporting the notion that antigens have independent effects as well (25,26). ...
Anti-citrullinated protein antibodies (ACPAs) are highly specific for rheumatoid arthritis (RA) and are present years before the onset of symptoms. The avidity of autoantibodies can have a strong impact on their effector potency. This study was undertaken to analyze the avidity of ACPAs in serum samples obtained from ACPA-positive healthy individuals (predisease), patients with early disease, and patients with established RA as well as the avidity maturation over time in samples from healthy subjects who later developed RA.
We measured ACPA avidity in serum samples from ACPA-positive healthy individuals, symptomatic individuals, and patients with established RA in 5 collections from The Netherlands, Canada, and Austria. We determined the dynamics of avidity maturation of ACPAs from the predisease stage to established disease in 1 case from the native North American population and in 10 cases from a Dutch blood donor cohort.
The overall ACPA response was characterized by low-avidity antibodies. Higher-avidity ACPAs were observed in symptomatic patients only, while low-avidity ACPAs were observed in both healthy subjects and patients. In longitudinal samples obtained from subjects prior to disease onset, ACPA avidity increased over time until disease onset. No further avidity maturation was observed after disease onset.
Our findings indicate that avidity maturation of the ACPA response takes place prior to disease onset.
... conceivable that the inhibitory effect seen in these experiments was due to antigen that had been carried over from the preincubation stage into the assay and which diffused out into the assay mixture, thus inhibiting plaque formation by competing for secreted antibody with the DNP-SRC (17). Therefore, aliquots pretreated with DNP-POL were mixed with aliquots preincubated with medium alone, and the mixture assayed for anti-DNP-AFC. ...
This study describes the effects of incubating antibody-forming cells (AFC), either as mass cell suspensions, or as single
AFC in microdroplets, with antigens against which the cells display specificity. Most of the work was done with hapten-specific
anti-DNP-AFC, but AFC with specificity against flagellar antigens or fowl gamma globulin (FGG) were also included. It was
noted that 30-min incubation of AFC with highly multivalent forms of antigen caused a substantial partial suppression of the
antibody-forming performance of the AFC as measured by a hemolytic plaque test. Thus, when cell suspensions containing anti-DNP
plaque-forming cells (PFC), were incubated for 30 min at 37°C with 100 µg of DNP-polymerized flagellin (DNP-POL), the number
of plaques appearing after washing of the cells and placing them in plaque-revealing erythrocyte monolayers was reduced to
50% or less compared with the number of plaques observed with control portions preincubated with medium alone. Preincubation
with DNP-lysine, with oligovalent DNP-protein conjugates, or with irrelevant antigens produced no such inhibition. Studies
where preinhibited PFC suspensions were mixed with control suspensions before assay showed that a nonspecific carryover of
antigen into the assay system was not involved. The inhibitory effect could also be initiated by holding cells at 0°C with
DNP-POL, but in that case, inhibition only became manifest after cells were incubated for 30 min at 37°C before being placed
in plaque-revealing monolayers. This suggested that inhibition was initiated by adsorption of multivalent antigen onto PFC-surface
Ig, but required some active process before secretion actually slowed down. The effect was dose- and time-dependent, antigen-specific,
and generalized for all antigens studied.
As well as yielding reduced plaque numbers, the preinhibited cells also gave smaller, more turbid plaques, suggesting a reduction
in antibody-forming rate by each PFC rather than the elimination of PFC. Consistent with this suggestion was the observation
that the degree of inhibition of plaque formation could be increased by decreasing the sensitivity of the assay so that only
AFC secreting at high rates were detected. A micromanipulation study, where single PFC were subjected to inhibition, and were
then tested for the rate at which they could cause hemolysis, showed a 68% inhibition of mean secretory rate.
Micromanipulation studies were performed to test the amount of cell surface-associated Ig on control and preinhibited PFC.
For this, single PFC were held with [125I]antiglobulin and quantitative radioautography was performed. No significant difference emerged, suggesting that retention
of secreted Ig on cell-attached antigen was not the cause of inhibition.
The results are discussed in the framework of tolerance models and blocking effects at the T-cell level by antigen-antibody
complexes. The name effector cell blockade is suggested in the belief that the phenomenon may be a general one applying to
both T and B cells.
... While other authors have measured the affinity or avidity of antibodies released in vitro (2,13,14) the maturation of the immune response in tissue culture has not been described except for our previous communication (9). According to the present results obtained with optimal challenge, the maturation in vitro is similar to what happens during the primary response in the intact animal (1, 2, 14-16), or in the secondary adoptive response with a small number of transferred cells (6), in terms of range and kinetics of the excursion of affinity measurements in "early" and "late" antibodies. ...
We have cultivated lymph node microfragments from ß-D-galactosidase (Escherichia coli) primed rabbits and have measured their secondary response directed towards the whole molecule (precipitating antibodies)
and to a single determinant (activating antibodies) of the antigen. By decreasing the size of the fragments to 105 cells, we began to observe heterogeneity among identical cultures in terms of positivity of response, antibody specificity,
and titers. The affinity of "early" activating antibodies was inversely proportional to the dose of challenge. While no maturation
was seen in low and excessive challenge, in all cultures receiving intermediate doses the association constant was raised
several orders of magnitude within periods of 20 days. The relevance of these data to the mechanism of affinity selection
of antigen-sensitive cells is discussed.
... (c) They were inhibited 50% by DNP-lys in relative low concentrations (10-6-10 -9 M) (24). (d) They displayed the expected increase in affinity with time after immunization (40,41). As 10 -4 M DNP-lys inhibit almost completely the DNP-RFCs (Table I), this concentration of DNP-lys was used in the column separation experiments to be described. ...
... (c) They were inhibited 50% by DNP-lys in relative low concentrations (10-6-10 -9 M) (24). (d) They displayed the expected increase in affinity with time after immunization (40,41). As 10 -4 M DNP-lys inhibit almost completely the DNP-RFCs (Table I), this concentration of DNP-lys was used in the column separation experiments to be described. ...
Mice immunized with hapten-autologous serum albumin conjugates (DNP-mouse serum albumin) were shown to contain immune B and T cells with specificity for the conjugate. Fractionation on antigen-coated Sepharose beads showed that B cells could be subdivided in two major groups: those reacting against the haptenic group (DNP) and those reactive against the new antigenic determinants (NADs) introduced into the protein carrier by the hapten coupling. It was shown previously that humoral antibodies formed against hapten-mouse serum albumin conjugates also were directed against these two groups of antigenic determinants and that the immune response to the NADs does not follow the genetic rules of high or low response against the hapten used. All together these findings support the distinct nature of the NADs over the haptenic groups, as recognized both at the humoral and cellular level.
Absorption of mouse cells immune to hapten-autologous serum albumin conjugates on antigen-coated Sepharose beads using a variety of incubation conditions resulted in no specific retention of T cells. Therefore we had to resort to specificity studies of T cells in relation to T cell function. Relatively pure immune T cell suspensions were obtained using fractionation on anti-immunoglobulin-coated columns. DNP-MSA-specific T cells were shown to be very specific for the DNP-MSA conjugate with only one exception: they cross-reacted with antigenic determinants on DNP-rat serum albumin. As DNP-specific help was excluded in the present transfer system (as shown by the inability of cells from DNP-skin-painted mice and DNP-heterologous protein conjugate specific T cells [anti-immunoglobulin column purified] to help a DNP-MSA response), these results demonstrate the NAD specificity of the DNP-MSA-reactive T cells. The cross-reactivity pattern of DNP-MSA-specific T cells was similar to that found for humoral anti-NAD antibodies produced against the same immunogen.
Whether B and T cells are activated by the same antigenic determinants is discussed.
... Avidity of DNP-PFC. Avidity was estimated by including ~-DNP-lysine in the plaquing chambers at 10-fold concentration steps from 10 -~° to 10 -~ M as previously described (16). Developing Antisera. ...
We present evidence here for two stages in B-memory cell development, the first of which is T independent and the second T dependent. For these studies, we use a new type of T-deficient mouse (allotype suppressed) which specifically lacks T-helper activity (Th) for a subset of memory B cells responsible for approximately 10% of the overall IgG antibody response. We have shown elsewhere that these mice (SJL X BALB/c hybrids suppressed for Ig-1b) lack Th capable of helping Ig-1b memory cells, although they have normal Th activity for all other IgG memory B cells. This selective Th deficiency allows study of the effects of T depletion on memory development and avidity maturation of one population of B cells under conditions where the bulk of the immune response in the animal is proceeding normally, thus obviating environmental problems due to secondary effects of T depletion. With this sytem, we show that after a single priming dose of 2,4-dinitrophenyl-keyhole limpet hemocyanin, the memory B-cell pool in suppressed and nonsuppressed donors is indistinguishable with respect to magnitude and avidity of the response for all IgG antibodies produced, including Ig-1b antibody, despite the fact that expression of Ig-1b memory cells is prevented in intact Ig-1b-suppressed mice by the absence of Th capbale of cooperating with these memory cells. We have shown elsewhere that virtually all of the Ig-1b memory is carried by Ig-1b bearing cells. In contrast with the lack of suppressor T-cell effect on initial Ig-1b memory cell development, our data show that continued Ig-1b memory development is selectively impaired in suppressed mice. When primed mice are boosted repeatedly with the priming antigen, the average avidity of most of the IgG memory cells increases over 100-fold while there is no avidity increase in the Ig-1b component. To explain these data, we suggest that the development of high avidity memory occurs in two stages. The first stage, which occurs as a result of primary antigenic exposure, is the creation of a pool of IgG-bearing memory cells with a relatively low average avidity for the antigen. The appearance of these first stage memory cells does not require help from (post-thymic) Th, although Th are required for the expression of these memory cells (antibody production). The second stage of B-memory development requires both further antigenic stimulation and B-memory cell interaction with competent Th. This is a continuing process in which the number of memory cells in the pool remains relatively constant but the average avidity of these cells increases with continued antigenic exposure.
... Assay of Avidity of Anti-DNP PFC. The avidity of the indirect anti-DNP PFC was assayed by inhibition of plaque formation by various concentrations of DNP-EACA essentially according to the method described by Andersson (34). Nine concentrations of DNP-EACA ranging from 1 × 10 9 to 1 x 10 5 M in half-log increments were used. ...
The ontogeny of the ability of B lymphocytes to produce an antihapten response which is heterogeneous with respect to affinity for the antigenic determinant was studied in a cell transfer system. The heterogeneity of affinity of the immune response of lethally irradiated mice reconstituted with syngeneic, adult thymus cells and fetal or neonatal tissues as a source of B lymphocytes was studied. It was found that B cells from 17 day fetal liver or neonatal liver are highly restricted with respect to heterogeneity of affinity as compared with adult spleen or bone marrow. The B-cell population achieves an adult character with respect to heterogeneity of affinity by 2 wk of age. The peripheral lymphoid tissues (spleen) appear to mature in this respect more rapidly than do central lymphoid tissues (bone marrow). Spleens from 10-day old donors behave in an adult, heterogeneous manner while bone marrow from the same donors exhibit a marked restriction in heterogeneity of affinity. Germfree mice produce an immune response which is indistinguishable from conventionally reared adult animals with respect to heterogeneity of affinity. The earlier appearance of the ability to transfer a heterogeneous immune response in spleen as compared with bone marrow suggests that the increasing heterogeneity of the B-lymphocyte population which occurs between birth and 2 wk of age is the result of a differentiation event and not of a somatic mutation or recombination event.
... (c) They were inhibited 50% by DNP-lys in relative low concentrations (10-6-10 -9 M) (24). (d) They displayed the expected increase in affinity with time after immunization (40,41). As 10 -4 M DNP-lys inhibit almost completely the DNP-RFCs (Table I), this concentration of DNP-lys was used in the column separation experiments to be described. ...
The carrier specificity of secondary anti-hapten responses using different haptenic densities on autologous or heterologous carrier proteins was studied. In addition, the T cell requirements for anti-hapten antibody synthesis were investigated. The results demonstrated a clearcut decrease in carrier specificity as hapten density goes up. Also, memory against autologous hapten carrier conjugates at the T cell level waned with time in contrast to the memory against heterologous hapten-carrier conjugates. Specific helper cells against autologous conjugates were demonstrated by filtration through anti-immunoglobulin coated columns.
... (Received for publication 12 July 1971) The average intrinsic association constant of anti-hapten antibody increases with time after immunization with hapten-protein complexes, and the rate of change is influenced by the size of the initial dose of antigen (1)(2)(3)(4)(5)(6)(7). Steiner and Eisen (3) suggested that as the concentration of antigen falls after immunization, new populations of cells are selected which produce antibody of progressively increasing affinity. ...
Secondary immune responsiveness to dinitrophenylated bovine gamma globulin (DNP-BGG) was transferred to heavily irradiated rats by means of small lymphocytes from the thoracic duct of immunized syngeneic donors. Affinity of the antibody produced by the adoptive recipients when challenged immediately was the same as that seen in immunized controls and cell donors, suggesting that the performance of lymphocytes in the thoracic duct of immunized rats provide an accurate measure of the immunologic history of memory cells both in the intact animal and the lymphocyte-depleted donor. The relevance of this to cell cooperation in the production of antibody to hapten-protein complexes is discussed. Direct evidence on antigen modulation of the immune response was also obtained. When immune thoracic duct lymphocytes were transferred to adoptive recipients and challenged either immediately or after a delay of 6 wk, it was found that, although the amount of antibody decreased with this delay in challenge, the affinity remained constant. Thus it would seem that in control rats, the increase in the affinity of antibody with time after immunization is, in fact, due to the selection of new populations of cells by the progressively waning concentration of antigen.
... It is important to note that, since antigenic essence comprises about 1% of the total cell protein content, its use allows up to 100 times more cells and, therefore, target antigens to be used for vaccination as compared to whole cells. However, vaccination with very low amounts of antigenic essence may be also important because high doses of antigens have been shown to yield lower-quality antibodies, especially in terms of affinity . High avidity T cells are capable of being stimulated by extremely low concentrations of antigen , and it has been shown that T cells with higher functional avidity are more effective at treating tumors . ...
The creation of cancer vaccines is a constant priority for research and biotechnology. Therefore, the emergence of any new technology in this field is a significant event, especially because previous technologies have not yielded results. Recently, the development of a cancer vaccine has been complemented by a new proteomics technology platform that allows the creation of antigen compositions known as antigenic essences. Antigenic essence comprises a target fraction of cellular antigens, the composition of which is precisely controlled by peptide mass spectrometry and compared to the proteomic footprint of the target cells to ensure similarity. This proteomics platform offers potential for a massive upgrade of conventional cellular cancer vaccines. Antigenic essences have the same mechanism of action, but without the disadvantages, and with notable advantages such as precise targeting of the immune response, safety, controlled composition, improved immunogenicity, addressed MHC restriction, and extended range of vaccination doses. The present paper calls attention to this novel platform, stimulates discussion of the role of antigenic essence in vaccine development, and consolidates academic science with biotech capabilities. A brief description of the platform, list of cellular cancer vaccines suitable for the upgrade, main recommendations, limitations, and legal and ethical aspects of vaccine upgrade are reported here.
... The avidity distribution of the anti-DNP PFC was assayed by the inhibition of plaque formation using various concentrations of DNP-EACA, essentially according to the method of Andersson (13) as validated by previous work (14,15). Concentrations of DNP-EACA ranging from 1 x 10 -9 to Table I is illustrated the anti-DNP PFC response 13 and 20 days after DNP-BGG immunization of lethally irradiated, thymectomized mice reconstituted with syngeneic bone marrow which had been treated with anti-brain 0-antiserum and C. Half of the animals also received 1 × 10 s syngeneic thymus cells. ...
Tolerance can be induced in adult mice by a single intravenous injection of 0.5 mg dinitrophenylated bovine gamma globulin. The cellular mechanism of the unresponsive state is different depending upon whether the tolerance is induced in normal intact adult mice or in reconstituted, irradiated mice. The tolerant state induced in intact mice is characterized by a high avidity of the residual antibody-forming cells in partially tolerant animals and a prompt reversibility on cell transfer. The overall properties of this unresponsive state are consistent with the hypothesis that it is mediated by the production of small amounts of high affinity antibody in response to the tolerance-inducing injection of antigen. In contrast, the unresponsiveness induced in reconstituted, irradiated mice by the same procedure was characterized by a low avidity of the residual antibody-forming cells in partially tolerant animals and stability on transfer of spleen cells from unresponsive into irradiated recipients. No suppressor cell activity was detected and mixed cell transfer studies were consitent with the view that this unresponsive state represented a B-lymphocyte clonal deletion. The presence or absence of T lymphocytes in the population of cells used for reconstituting the irradiated recipients did not effect the ease of tolernace induction or the cellular mechanism of the tolerant state which was produced. If irradiated mice reconstituted with B and T lymphocytes were rested for 2 wk before tolerance induction then a reversible "high affinity"-type tolerance is obtained such as is typical of normal intact animals. Restorationof a "normal" response to the tolerance-inducing injection of antigen is dependent upon the presence of thymus cells in the population of cells used for reconstitution. It is suggested that the structural integrity of the lymphoid tissue is critical in determining whether B cell will be rendered tolerant after exposure to antigen in vivo.
... The distribution of affinity of the antibody response was determined by the PFC inhibition procedure of Andersson (16). E-TNP-L-iysine (ICN Pharmaceuticals, Cleveland, OH) or NP-caproate (Biosearch, San Rafael, CA) was used to inhibit anti-TNP and anti-NP plaques, respectively and was added to both the agarose and the complement where indicated. ...
The biologic activity of molecules synthesized and secreted by hapten-specific inducer T cells was examined. After activation, a single inducer clone secretes both antigen-specific inducer peptides as well as nonspecific factors. The nonspecific factors augment the in vitro response of B cells to sheep erythrocytes (SRBC) and Type 2 T-independent antigens. The antigen-specific molecules (ABM) induce plaque-forming cell (PFC) responses in cultures containing ABM, B cells, and antigen that links the epitope recognized by ABM with the B cell epitope. Induction of B cells by ABM is limited to B cells expressing the same I-A allele as the source of the ABM and this reflects binding by ABM to I-A products on B lymphocytes. The data reported here strongly support the view that inducer cells can activate at least some B cells by secretion of a modified form of the T cell surface receptor.
... Experiments with inhibition of NNP-PFC by free NNP6sHGG (16) were done by adding to the assay mixture 0.05 ml of dilutions from 10°-10 -7 of a NNP65HGG solution 3.8 mg/mL Thus, the 10 ° dilution represents a NNPasHGG concentration in the gel of 190/~g/ml. (Table I). ...
Purified protein derivative (PPD) tuberculin induced immunoglobulin production in cultures of nonimmune mouse spleen cells, as measured by plaque-forming cells (PFC) against sheep erythrocytes (SRBC), horse erythrocytes, and 4-hydroxy-3,5-dinitrophenacetyl-SRBC. The increase started between 15 and 20 h of culture and reached a peak at 48–72 h. Higher PPD concentrations resulted in earlier peak responses than low concentrations. The Ig produced was mainly 19S and of very low avidity. The response elicited by PPD was of the same type as that caused by lipopolysaccharide of bacterial origin. Mitomycin treatment of cells before culture did not change the numbers of PFC/10⁶ recovered cells but the cell recovery was considerably lower.
Also injection of PPD in vivo resulted in increased numbers of PFC. On the basis of these results it is suggested that PPD nonspecifically activates a majority of the B cell population to proliferation and immunoglobulin synthesis.
... Heterogeneity Indices of PFC during the Response to TNP-F. An attempt was made to relate the above findings on blocked antibody-forming cells to the decrease in apparent heterogeneity of antibody as measured by the conventional plaque inhibition assay (39). In these calculations, any hapten-augmentable PFC were purposely ignored. ...
Attempts were made to elucidate the cause of the downward regulation of the splenic plaque-forming cell (PFC) response in AKR/J and BALB/c mice between days 4 and 7 after a single intravenous injection of 2,4,6,trinitrophenyl- lys-Ficoll(TNP-F). AKR/J spleen cells, taken 7 d after injection of TNP-F, were transferred, together with TNP-F, into normal AKR/J mice. The day-3 or -4 PFC response of the recipients was much lower than that of recipients of normal cells. However, the suppression was only apparent because the presence of 10(-8)-10(-7) M 2,4,6-trinitrophenyl-ε-amino-n-caproic acid (TNP- EACA) (or 10(-7)-10(-6) M 2,4,-dinitrophenyl-ε-amino-n-caproic acid) in the PFC assay caused a dramatic increase in observed PFC, averaging 298 percent on day 3 and 122 percent on day 4. Recipients of normal cells showed no such hapten-augmentable PFC. T-depleted immune spleen cells did not cause any apparent suppression of the response to TNP-F, but hapten-augmentable PFC in recipient spleens were again prevalent. Suppression of the PFC response, as well as hapten-augmentable PFC, were seen after transfer of immune serum. It was postulated that hapten augmentation of PFC was caused by displacement of auto-anti-idiotypic antibody from the surface of blocked antibody- synthesizing cells.
Further studies showed that such hapten-augmentable PFC occurred in the spleens of a large percentage of both AKR/J and BALB/c mice examined after day 4 of the primary response to TNP-F. Thus, it was hypothesized that the downward regulation of the magnitude and, possibly, also of the heterogeneity of the splenic-PFC response was due to an auto-antibody response to one or more major idiotypes of the anti-TNP response.
... Affinity of indirect anti-DNP PFC was assayed by the inhibition of PFC by DNP-EACA according to the method of Andersson (10), as modified by Goidl et al. (11). Nine concentrations of DNP-EACA phosphate saline ranging from 1 × 10 -5 M to 1 X 10 -9 M in half-log increments were added to both the TNP-SRBC agarose suspension and the complement source. ...
Aged mice preferentially lose the capacity to make IgG and high affinity PFC after immunization with the T-dependent antigen DNP-BGG. We have found that thymectomy accelerates the appearances of these immune deficiencies associated with aging. When splenocytes from old mice are transferred to young lethally irradiated, syngeneic mice and the recipients immunized 7 wk later, the number of IgG and high affinity PFC was increased compared to the response of old splenocytes transferred to young thymectomized mice. These immune deficiencies of aged mice were also reversed when old mice were treated with thymopoietin in vivo or splenocytes from old mice were incubated with thymopoietin before adoptive transfer to young irradiated, thymectomized syngeneic mice. The T-cell independent response to DNP-Ficoll was less impaired than the T-cell dependent response to DNP-BGG in old animals. These data suggest that a decline in thymic function that occurs during aging may contribute to the immunological deficiencies of old animals.
... High peptide antigen doses have been shown experimentally to result in low avidity and T cell responses (88,89). However dosing timing strategy has been shown to have a significant effect on the average avidity of a T cell population (90,91). ...
Therapeutic vaccines can elicit tumor-specific cytotoxic T lymphocytes (CTLs), but durable reductions in tumor burden require vaccines that stimulate high-avidity CTLs. Recent advances in immunotherapy responses have led to renewed interest in vaccine approaches, including dendritic cell vaccine strategies. However, dendritic cell requirements for vaccines that generate potent anti-tumor T-cell responses are unclear. Here we use mathematical modeling to show that, counterintuitively, increasing levels of immature dendritic cells may lead to selective expansion of high-avidity CTLs. This finding is in contrast with traditional dendritic cell vaccine approaches that have sought to harness ex vivo generated mature dendritic cells. We show that the injection of vaccine antigens in the context of increased numbers of immature dendritic cells results in a decreased overall peptide:MHC complex load that favors high-avidity CTL activation and expansion. Overall, our results provide a firm basis for further development of this approach, both alone and in combination with other immunotherapies such as checkpoint blockade.
... Interestingly, we observed no increased potency in responses to higher ID2 immunogen, indicating that once an optimal level of B cell receptor saturation has occurred, no added benefit to antibody affinity will occur. This is in agreement with previously published data studying the effect of antigen amount which show that a higher antigen load leads to increased plasma cell differentiation but lower antibody affinity, while a lower dose of antigen leads to an increased B cell memory pool as well as antibodies of higher affinity . . Ability of ID-2 immunized sera to compete with A32 and N5-i5 for ID2 binding. ...
Fc-mediated effector functions of antibodies, including antibody-dependent cytotoxicity (ADCC), have been shown to contribute to vaccine-induced protection from HIV-1 infection, especially those directed against non-neutralizing, CD4 inducible (CD4i) epitopes within the gp120 constant 1 and 2 regions (C1/C2 or Cluster A epitopes). However, recent passive immunization studies have not been able to definitively confirm roles for these antibodies in HIV-1 prevention mostly due to the complications of cross-species Fc-FcR interactions and suboptimal dosing strategies. Here, we use our stabilized gp120 Inner domain (ID2) immunogen that displays the Cluster A epitopes within a minimal structural unit of HIV-1 Env to investigate an immunization protocol that induces a fine-tuned antibody repertoire capable of an effective Fc-effector response. This includes the generation of isotypes and the enhanced antibody specificity known to be vital for maximal Fc-effector activities, while minimizing the induction of isotypes know to be detrimental for these functions. Although our studies were done in in BALB/c mice we conclude that when optimally titrated for the species of interest, ID2 with GLA-SE adjuvant will elicit high titers of antibodies targeting the Cluster A region with potent Fc-mediated effector functions, making it a valuable immunogen candidate for testing an exclusive role of non-neutralizing antibody response in HIV-1 protection in vaccine settings.
The increase in affinity and heterogeneity of antibody with respect to time after immunization to the 2,4,6-trinitrophenyl (TNP) determinant was studied using TNP-brucella (BA) and TNP-type III pneumococcal polysaccharide (SIII). Experimental evidence is presented in support of the maturation of 19S antibody affinity. The difficulties which have been encountered in some previous investigations in detecting such a maturation appear to be the tendency of the cells to switch from IgM to IgG synthesis early after the peak of the primary response. Data are presented indicating that this switch occurs in a non-antibody-secreting memory cell population prior to, or more likely very shortly after, boosting. We also present evidence that the use of an antigen that does not induce a massive switch from IgM to IgG antibody synthesis offers a way of unmasking maturation of the 19S response. Thus, with the T-independent antigen TNP-SIII, a definite increase in heterogeneity could be detected in the 19S response upon secondary boosting. A greater increase in heterogeneity was noted in nude mice and was possibly due to the absence of suppressor T cells.
Spleen cells from normal unimmunized adult mice were subjected to a 2-stage fractionation procedure designed to prepare a subpopulation highly enriched for the capacity to form antibody to the hapten fluorescein (FLU). First, the spleen cell suspension was rocked in a petri dish coated with a thin layer of FLU-gelatin (15 min at 4 °C), the nonadherent cells washed away and the adherent cells recovered by melting the gelatin and treated with collagenase. The cells were relabeled with a fluorescent protein, usually FLU-gelatin at 100 μg/ml, (25 °C, 15 min). After washing, the cells were sorted into cohorts of relatively homogeneous fluorescence intensity in the fluorescence-activated cell sorter (FACS). Finally, the sorted cells were again collagenase-treated and stimulated with FLU-coupled polymerized flagellin in a limit dilution microculture system capable of generating single clones of anti-hapten plaque-forming cells (PFC).It was found that the cohort of cells representing the 10 % most intensely fluorescent cells, yielded PFC clones with a frequency of 1 in 8, about 5 times higher than the hapten-gelatin-enriched cells. Less fluorescent cohorts gave progressively lower frequencies of clonable PFC precursors. Some evidence was obtained, suggesting that the most highly fluorescent cells produced antibody of higher median avidity than the FLU-gelatin-fractionated cells, which in turn showed higher avidity than unfractionated spleen cells, findings consistent with the clonal selection hypothesis.Somewhat surprisingly, cells with high fluorescence intensity prepared by FACS sorting of FLU-gelatin-labeled unfractionated spleen cells, yielded much lower PFC precursor frequencies than cells of equivalent fluorescence intensity derived through the 2-stage fractionation procedure.Spleen cells from newborn mice were subjected to 2-stage fractionation. The method proved equally applicable to them, and preparations with a 1 in 11 PFC precursor frequency could be readily generated. However, the neonatal cells differed from the adult cells in responding significantly better to the polyclonal B cell activator, lipopolysaccharide, than to antigen.The theoretical implications of these findings and the potential uses of pure populations of hapten-binding cells are briefly discussed.
19S IgM rheumatoid factors (RF) may play an important role in sustaining inflammation in rheumatoid arthritis (RA). As yet, no unique antigenic specificity for RF in RA has been identified. Because the synovium is central to the pathogenic changes in RA, RF produced therein might be pathogenically more important than serum RF. Therefore, we examined the reactivity and relative avidity of 19S IgM-RF in serum and rheumatoid synovial cells (RSC) from 20 patients with seropositive RA. Reactivities were determined by competitive inhibition of serum RF hemolytic activity and RSC RF-plaque-forming cells (PFC) by added soluble antigen, i.e., monomeric human IgG subclasses. Estimation of relative avidities of RSC RF for human IgG subclasses was done by calculation of fractional RF expression in the RSC RF-PFC assay following inhibition by IgG subclasses. RSC RF had greatest reactivity with IgG3 and IgG1, some reactivity with IgG2, and the least reactivity with IgG4. Serum RF reacted most with IgG1 and IgG2, reacted some with IgG4, but reacted poorly with IgG3. The antigenic determinants with which RSC RF reacted were common to many IgG3 molecules. The highest relative avidity of RSC RF was for IgG3. These observations indicate a selective deficiency of serum RF compared with RSC RF and suggest an important pathogenic role for these qualitatively different RSC RF molecules for in situ RF immune complex-mediated inflammation in RA synovial tissue.
The plaque inhibition method of measuring the affinity of single antibody-forming cells (AFC) developed by Andersson has been modified for use with haptens. Using this method, it was possible to make simultaneous determinations of the relative affinities of IgG and IgM plaques during the primary and secondary responses.
It was shown that there was no maturation of the affinity of antibody-released IgM AFC in the primary response in mice. There was an increase in affinity of the antibody released from IgG AFC, which was antigen dose- and time-dependent, confirming results obtained by others. Furthermore, at any time during the course of the primary response, the affinity of the IgM antibody released by AFC was always lower than that of IgG plaque-forming cells. This difference averaged about 10-fold.
The same factors were shown to influence the affinity changes of IgG AFC observed during the secondary response. In addition the booster, or secondary dose of antigen used, was important. Highest affinity antibody was produced in response to intermediate antigen doses. The maturation of affinity affects the generation of memory cells to the same degree as the antibody-secreting cells during the primary response, since similar affinity changes occurred in the absence of detectable primary responses.
Finally, when the period between primary stimulation and challenge is 5 months, there is a decrease of between 2 and 2.6 log10 in the concentrations of DNP lysine needed to inhibit 50 % of the IgG antibody-forming cells depending on the doses used for priming. The variation of affinity of IgM antibody was much less obvious, with a 6-fold difference at the maximum, which was independent of the doses of antigen used for priming or boosting, and of the time elapsed between the two antigenic stimuli.
Anti-citrullinated protein antibodies (ACPA) are highly specific for rheumatoid arthritis (RA) and have been implicated in disease pathogenesis. Recent ongoing evidence indicates that the ACPA response broadens before precipitation of full-blown RA, as indicated by a more extensive isotype usage and an increased citrullinated epitope recognition profile. Nonetheless, the evolution of the ACPA response is still poorly understood and might intrinsically differ from the protective responses against pathogens.
The avidity and the avidity maturation of ACPA in relation to the avidity of antibodies against recall antigens were analysed.
The avidity of ACPA was significantly lower than the avidity of antibodies to the recall antigens tetanus toxoid and diptheria toxoid. Moreover, ACPA did not show avidity maturation during longitudinal follow-up and ACPA avidity was also relatively low in patients who displayed extensive isotype switching.
These observations indicate that the natural evolution of ACPA differs from the development of antibodies against recall antigens. These data also indicate that ACPA avidity maturation and isotype switching are disconnected, whereby extensive isotype switching occurs in the setting of restricted avidity maturation. Intrinsic differences between the RA-specific autoantibody system and protective antibody responses against pathogens could be of relevance for designing novel B cell-targeted therapies for RA.
Spleen cells of mice from eight inbred strains and three F1 hybrids, undergoing a secondary immune response to dinitrophenylated keyhole limpet hemocyanin (DNP-KLH), were examined for numbers of indirect DNP-specific plaque forming cells (PFC) as well as avidity of anti-DNP antibodies. The results indicated that the magnitude of the immune response is under genetic control. Differences in average avidity and heterogeneity of avidity were found among different mouse strains, suggesting genetic control of these parameters. However, no simple pattern of inheritance for these characteristics emerged from the study.
Mice primed with a thymus-dependent form of Type 3 pneumococcal polysaccharide (S3), i.e. S3 coupled to erythrocytes (S3-RBC) produces S3-specific IgG antibody after secondary challenge with S3-RBC. When mice are depleted of T cells by treatment with anti-lymphocyte serum (ALS) at the time of priming, no IgG antibody is produced after secondary challenge. In order to determine the cellular basis for this phenomenon, various combinations of T and/or B cells from ALS-treated or normal primed mice were transferred to irradiated recipients prior to secondary challenge with S3-RBC. The results indicated that T cells were required at the time of priming with S3-RBC in order to (a) prevent the induction of tolerance in S3-specific B cells in mice primed with high doses of S3-RBC, and (b) induced differentiation of IgG-producing B cell precursors to Bgamma memory cells in mice primed with low doses of antigen.
The humoral immune response to a unique haptenic determinant has been characterized at the single cell level. We report here that the hapten, phthalate, when conjugated to keyhole limpet hemocyanin (4-azophthalate-KLH) is highly immunogenic in adult BALB/c mice, and that the immunogenicity of the hapten is largely restricted to the two charged carboxylate groups on phthalate. Based upon plaque-forming cell (PFC) inhibition with increasing concentrations of free phthalate, relative affinity distribution profiles of antibody-forming cells have been established for individual mice. The antibody-forming cells in the spleens of these mice consist of five or six individual relative affinity sets. PFC inhibition studies with hybridomas secreting phthalate specific monoclonal antibodies reveal, as expected, a more restricted inhibition profile. The majority of PFC produced by the hybridomas could be assigned to one of the relative affinity sets. PFC inhibition studies employing a panel of cross-reactive phthalate analogs as inhibitors, have made it possible to segregate the splenic antibody-forming cell population further into distinct fine specificity sets. The recognized immunodominance of the two negatively charged carboxylate groups of phthalate and the rigid positioning of substituted charged groups on the phthalate analogs are unique to this hapten system and they are fundamental to the meaningful interpretation of the fine specificity data generated by the inhibition studies reported here. The ability to compartmentalize the phthalate response into relative affinity and fine specificity sets at the single cell level has made it possible for us to restrict our studies of immune regulation to a defined and manageable portion of a conventional thymic dependent B cell repertoire.
The antibody response of plasmacytoma-bearing mice (PC-mice) is severely reduced. In order to understand the nature of the effect of the tumor on the cells making antibody, quantitative and qualitative studies of the humoral response of PC-mice were undertaken. In these studies, the affinity of the antibody produced by tumor-bearing and normal mice was compared to determine whether the small amount of antibody produced by PC-mice is the product of a normal or an altered population of B cells. Antibody to TNP-Ficoll made by PC-mice 3 days after immunization was less heterogeneous and of an affinity lower than that of antibody made by normal mice. However, at 7 days, the antibody made by PC- and normal mice did not differ significantly. These data suggest that, prior to antigenic stimulation, the B cells of PC-mice are relatively immature, reflecting a possible retardation in the generation and turnover of B lymphocytes. The process of antigen-driven selection of high-affinity antibody-producing cells, however, appears to function normally in PC-mice. These studies, then, reveal a qualitative as well as quantitative defect in the primary humoral response of PC-mice which may reflect an abnormality in the development and differentiation of B cells in these mice.
The average affinity of rabbit antibodies directed against the capsular polysaccharide of Type III pneumococci and harvesed at various times during the immunization procedure was determined by equilibrium dialysis. The results show that during prolonged immunization with pneumococci, the immune response matures, that is, there is a progressive increase in the average affinity of the elicited antibodies. This is attributed to a selective process occurring in the system based on a competition for antigen between antigen-sensitive cells and serum antibody. On occasions, antibody components of restricted structural heterogeneity are produced. These are assumed to be the product of clones of cells that have overgrown the immune system. It is proposed that the selective advantage possessed by these clones is that their product is of markedly higher affinity than that of prior antibody in the system.
The in vivo effects of histamine injection in LAF1 male mice on the immune reactivity to trinitrophenylated bovine γ-globulin was studied using plaque-forming cell (PFC) responses and their avidity distributions. Splenic anti-trinitrophenyl (anti-TNP) PFC responses of mice treated with histamine (5 × 10−6 mol or 1 mg, intravenously) were significantly reduced in number and restricted in heterogeneity and characterized by a preferential loss of high-avidity IgG PFCs. The reduced PFC response in histamine-treated mice was dose and time dependent. No evidence of suppressor cell activity in the spleens from histamine-treated mice was demonstrable. Only histamine-treated mice produced a significantly high percentage of anti-idiotype-blocked, hapten-augmentable IgG PFCs, suggesting the presence of auto-anti-idiotypic activity. Immune sera taken from histamine-treated mice caused an inhibition of anti-TNP PFC in vitro. This PFC-inhibiting factor in immune sera of histamine-treated mice was an antibody of the IgG1 and IgG2a class, lacked anti-TNP antibody activity, but reacted with anti-TNP antibody of LAF1 origin. Passive hemagglutination study of this sera showed anti-(anti-TNP F(ab′)2-IgG) titer. Thus, the results of this study suggest that histamine in combination with antigen induces auto-anti-idiotypic antibody which, in turn, is involved in the normal regulation of the immune response to trinitrophenylated bovine γ-globulin in vivo.
We have studied the effects of lipopolysaccharide (LPS) on the primary in vivo immune response to the hapten (4-hydroxy-3,5-dinitrophenyl)acetyl (NNP), with special reference to the avidity and affinity of the early appearing 19S and antibodies Comparisons were made of the immune response to NNP in groups of mice given either antigen alone. LPS alone, or antigen plus LPS The avidity of antibodies induced by LPS plus antigen was similar to that found after injection of antigen alone, in spite of the fact that the antibodies were more numerous However, when comparing the avidity of antibodies produced in animals given only LPS with those given LPS plus antigen, the latter group was often found to have fewer low-avidity 19S-antibody-producing cells The affinity of 7S antibodies was also similar in the two groups given antigen or antigen plus LPS Kinetic studies of the effect of LPS on the primary immune response to NNP showed that synergy was observed only before or after the peak response in groups given antigen alone It is concluded that LPS under synergy conditions acts preferentially on specific antigen-sensitive cells which are distinct from those that are activated to polyclonal antibody synthesis by LPS alone. Possible mechanisms for the adjuvant effect of LPS are discussed
Mice of six strains were immunized with (4-hydroxy-3-nitrophenyl)acetyl (NP) groups coupled to chicken globulin. The fine specificities of these antibodies were characterized by estimating relative affinities for related compounds, (4-hydroxy-3, 5-dinitro-phenyl)acetyl (NNP), (5-bromo-4-hydroxy-3-nitrophenyl)acetyl (NBrP) and (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP) groups. These measurements were carried out on both serum and single plaque-forming cell antibodies. At least three patterns of fine specificity could be recognized among these strains.C57BL/6 produced 7 S (IgG) anti-NP that consistently had 3–10 times higher affinity for NIP than for NP, and higher affinity for NNP and NBrP than for NP (heteroclitic). By doing affinity measurements on serum and plaque-forming cell antibodies, we could demonstrate that the majority of the 7 S (IgG) anti-NP produced by C57BL/6 mice was heteroclitic at both early and later stages of immunization. Moreover, the antibody from this strain exhibited only a slight change in affinity during the immune response, while the other strains underwent clearcut maturation (progressively increased affinity for NP and increased discrimination between NP and the related compound).Nine F1 hybrid mice between C57BL/6 and CBA produced anti-NP antibody that was very similar to C57BL/6 anti-NP.
The primary and secondary immune response was studied in mice in which tolerance was induced by injection of sheep's erythrocytes and cyclophosphamide (CP). In the early stages (from 1 to 8 weeks) after induction the mice were immunized with sheep's erythrocytes either in a single dose of 5·108 cells or in two doses each of 1·106 cells. Both methods of immunization gave equal results in the control animals. In the experimental animals the process of formation and (or) realization of immunological memory was impaired to a greater degree and recovered more slowly than ability to give a primary response.
A radial diffusion agar plaque technique is presented for estimation of the average avidity of bactericidal antibodies, based upon quantitative inhibition of the antibodies with free lipopolysaccharide. With this technique and with Vibrio cholerae as model organisms the avidities of vibriocidal rabbit antisera were determined. It appeared that primary response antisera had lower avidity than secondary response antisera, which showed an inverse relation between avidity and booster antigen dose. However, the avidity differences observed in these systems were only 10- to 20-fold, i. e. considerably less than reported for hapten-protein conjugate antigen. Avidity estimations by the Fair ammonium-sulphate precipitation method were in agreement with the results obtained with the described technique. The separated IgG and IgM fractions showed the described relation to immunization schedule; however, the IgM avidity appeared lower than the IgG avidity.
Adult mice were immunologically paralyzed by repeated injections of bovine serum albumin. Partial paralysis was achieved both with a high dose regimen of 10–100 mg and with a low dose regimen of 1–10 μg. The avidity of the antibody produced in response to an immunizing dose of antigen in Freund's complete adjuvant was determined both at the serum level using an antigen-binding test and at the level of single antibody-forming cells using inhibition of antibody plaque-forming cells by free antigens as a test system. The antibody was of relatively low avidity in high dose paralyzed animals, whereas low dose paralysis did not result in a change in the avidity. These data indicate that the B lymphocytes are target cells in high dose paralysis, since a cellular selection was shown to occur. In the low dose paralysis the B lymphocytes seem not to be the immediate target cells, since no cellular selection was demonstrated. Instead, it was shown that the main cellular defect in low dose paralysis was in the T lymphocytes, since carrier-specific helper cells in an anti-hapten response were shown to be depleted. Further, studying column purified B and T lymphocytes, it was shown that the cellular site of unresponsiveness in high dose paralysis was both at the B cell and at the T cell level. Low dose paralyzed animals had B cells of normal reactivity, whereas their T cells were paralyzed.
The affinity of anti-hapten antibodies, produced by single cells, was studied by hapten inhibition of plaque-forming cells using the local hemolysis in gel assay. The shift in affinity of anti-hapten antibodies, that occurs with time after immunization, was parallelled by an analogous shift, at the cellular level, with regard to indirect plaque-forming cells (PFC). Thus, there was a gradual decrease of the hapten concentration which was needed to suppress indirect plaque formation with time after immunization, indicating a gradual increase in the number of high affinity cells. No such shift was observed with cells causing direct plaque formation.
Analogous studies were performed with hapten-specific antigen-binding cells of T and B origin by using hapten-inhibition of rosette-forming cells. Hapten-specific antigen-binding cells were present in both cell populations. Lymphocyte binding of haptenated red cells could be specifically inhibited by free hapten. It was found that antigen-binding B lymphoid cells became gradually more susceptible to hapten inhibition with time after immunization, whereas no such change was observed in antigen-binding T lymphoid cells. Possible reasons for this discrepancy are discussed, as well as the implications for these findings regarding the specificity and structure of the T cell receptors.
The number and avidity curves of IgM-positive B lymphocytes forming rosettes with trinitrophenyl-treated sheep's erythrocytes (TNP-RFC) were compared in BALB/c, C57BL/6, (C57BL/6×BALB/c)F1, F1×BALB/c, and F1×C57BL/6 mice. Trinitrophenyl-bovine serum albumin (TNP24-BSA), dinitrophenyl-BSA (DNP23-BSA), and sulfanyl-BSA (Sulf17-BSA), in various dilutions, were used as inhibitors. The number of TNP-RFC was 60% greater in the spleen of the BALB/c mice than in that of the C57BL/6 mice. The F1 hybrids occupied an intermediate position, whereas the number of TNP-RFC was 35% greater in F1×BALB/c hybrids than in F1×C57BL/6 hybrids. Inhibition (avidity) curves differed in the two strains and the F1 hybrids tested. It is concluded that the number and avidity of TNP-RFC are under genetic control. Together with the postulated random (stochastic) expression of the V genes (genes of idiotypes?) in lymphocytes this suggests that the simplest mechanism of genetic control may be the ratio between the corresponding groups of V genes.
Irradiated rats reconstituted with immune spleen cells demonstrated a decline in their ability to mount a secondary response when challenged at various times after transfer. This decline was biphasic in nature, suggesting the existence of at least two memory cell populations: one with a half-life of 4.5 days and the other with a half-life of 40 days.When antigen (DNP-BGG) was given immediately after reconstitution and the rats rechallenged 1 mo later, a significant potentiation of immunological memory occurred. This effect of antigen appeared to be independent of its ability to stimulate the memory cells into becoming antibody-forming cells. This suggested that antigen may have at least two distinct effects on the memory cell population: (1) it can stimulate the cells to become antibody forming cells or (2) it can somehow act on the cells to potentiate their functional survival.
We present a mathematical theory of hapten inhibition of hemolytic plaque formation. The treatment is based upon the mathematical model for plaque growth presented by DeLisi & Bell (1974). The lymphocyte under consideration is embedded in an infinite three-dimensional medium, and is secreting antibodies isotropically at a constant rate. As the antibodies diffuse from the source they can bind reversibly to hapten, and in the most general case reversibly to red blood cell (RBC) epitope. The model leads to a non-linear diffusion equation coupled to a set of first order differential equations. The system must, in general, be solved numerically. However, in many cases of experimental interest simplifications arise which permit closed form solutions to be obtained. In this paper we have developed solutions for three special cases.
Guinea pigs immunized with 2,4-dinitrophenyl-guinea pig albumin (DNP-GPA) possess lymphocytes which specifically bind sufficient DNP-GPA-125I to their surface to be detected by radioautography. These lymphocytes are present in the draining lymph nodes in a frequency of ∼50/1000 lymphocytes in animals immunized 2–4 wk earlier with DNP-GPA in complete Freund's adjuvant. Nonimmunized animals have ∼0.4 DNP-GPA antigen-binding cells (ABC) per 1000 lymphocytes. An increase in the frequency of DNP-GPA ABC in peripheral blood is detectable by 5 days after immunization, which is before the time that serum anti-DNP antibody is measurable. The receptors of these ABC are hapten specific in that free ε-DNP-L-lysine, at low concentration, inhibits the binding of DNP-GPA-125I; DNP bovine serum alumbin (DNP-BSA) is equivalent to DNP-GPA in the inhibition of binding of DNP-GPA-125I to ABC; and both DNP-GPA agarose beads and DNP-BSA agarose beads specifically adsorb DNP-GPA-125I ABC. Anti-immunoglobulin antisera, particularly anti-γ2 sera, inhibit the binding of DNP-GPA-125I to these cells implying that the receptors are immunoglobulin, primarily of the γ2 heavy chain class. DNP-GPA-125I ABC appear to represent precursors of antibody-secreting cells and have specificity characteristics which are very different from cells, of similarly immunized guinea pigs, which mediate a cellular immune response to DNP-GPA.
During the past decade, the concept of antibody affinity has been increasingly employed in the analysis of immunological phenomena, and it is now generally understood to be an essential element in developing molecular interpretations of immunological processes. Consequently, there has been increased attention to a number of distinctive features of antibody affinity, particularly its variability and regulation, the role of antibody multivalence, and the biological concomitants of these properties. In an earlier discussion of the affinity of antibody (Karush, 1962), the main emphasis was placed on the nature and quantitative significance of the intermolecular forces involved and on some thermodynamic aspects of the reversible reaction of antibody and ligand. This analysis differs in its focus because it is based on experimental studies concerned with the variability of affinity (“the maturation process”) as well as the range of affinity, the kinetic features of the rapid antibody-ligand interaction, and the contribution of multivalence to the effective affinity of the antibody molecule. The information now available in these areas stems from the introduction of new experimental methods into immunological research. These methods include (1) the techniques of stop-flow and temperature jump for evaluating the kinetic parameters of the formation of antibody-ligand complexes; (2) the inhibition of hemolytic plaques, formed by suspensions of antibody-secreting cells, by soluble ligands to estimate relative affinities; and (3) the neutralization of hapten-conjugated bacteriophage with antihapten antibody to measure rates of binding of antibody to large ligands and to evaluate the quantitative effects of multivalent interaction.
Perhaps the most important function of antibody molecules is to combine with the corresponding antigen to form an antibody—antigen complex which is then eliminated from the circulation by the cells of the mononuclear phagocytic system. The specific interaction of antibody with the antigen involves the interaction of the antigen binding site, (which is comprised of the hypervariable regions of both heavy and light chains), with the antigenic determinant.
Variations of antibody affinity with antigen dose and time after immunization, such as a more rapid increase of affinity after lower doses of antigen (1), have been explained by the maturation theory (2). According to this theory, there are specific B lymphocytes with different receptor affinity prior to antigenic stimulation. Upon interaction of cell receptors with antigen the lymphocyte will proliferate and secrete antibodies with the same affinity as that of the cell receptors. As the antigen concentration decreases with time after immunization, the higher affinity cells will be selected out and predominate in the population. After lower doses of antigen this selection proceeds more rapidly than after higher doses. Hence, during the immune response there are parallel changes in average affinity at the serum antibody and cell receptor level (3, 4).
During the past 3–4 decades, an increasing amount of evidence has pointed to the complex role of the antigen dose or T cell receptor (TCR) stimulation strength on the subsequent type, duration and “flavor” or quality of the response. Antigen dose was initially shown to impact Th1/Th2 bias, and later also shown to differentially affect development and induction of Tregs, Th17, T-follicular helper (Tfh), cells, and others.
In recent years the quality of both CD4/8 T cells during infections, cancer and/or autoimmunity has turned out to be critical for subsequent disease outcome. Importantly, different vaccination strategies also lead to different types of T cell responses, and the role of the antigen dose is emerging as an important factor as well as a tool for investigators to utilize in fine-tuning vaccine efficacy. This commentary will highlight essential background of how antigen dose can impact and affect the quality of T cell responses, and discuss how this translates in different vaccine settings.
Neonatal liver or adult spleen was used as a source of B-lymphocytes in reconstituting lethally irradiated, syngeneic mice. Recipients were all given excess adult, syngeneic thymus cells and were immunized with dinitrophenylated bovine gamma globulin. The distribution of avidities of plaque-forming cells produced by immunized recipients of neonatal liver was highly restricted in comparison with animals reconstituted with adult spleen indicating a restriction of B-lymphocyte heterogeneity in the neonatal mouse.
Rabbit sera were found to possess neutralizing activity (normal antibody) to polioviruses and Coxsackie B viruses. This normal antibody showed high specificity in cross-neutralization and absorption tests. It was associated with heat-stable, mercaptan-sensitive, 19S γ1-β-macroglobulins, which formed weak complexes with the viral antigen. In rare instances, sera with normal macroglobulin antibody, also contained very low activity which was due to 7S γ2-globulins. The neutralization of poliovirus by normal 19S γ1-β-antibody appeared to follow first order kinetics, and the thermodynamic parameters of this reaction were the same as those of serological reactions employing immune antibody. The electrophoretic mobility, sedimentation properties, sensitivity to mercaptan, thermostability, and avidity of normal and early (up to day 3) immune antibodies to poliovirus were similar, but differed in several respects from those of late immune antibodies. Thus, the available evidence suggested, that earlier reported differences between normal and immune antibodies reflected differences between antibodies of diverse physicochemical properties rather than between normal and immune antibodies per se. It is proposed that the normal macroglobulin antibody is associated with an immunological response to repeated stimulation with minute amounts of antigen.
Sheep erythrocytes sensitized with the immunoglobulin G (IgG) fraction of commercial rabbit anti-sheep hemolysin were not hemolyzed directly by complement, but were hemolyzed after treatment with anti-rabbit globulin and complement. Erythrocytes so sensitized were plated in nonanticomplementary agar, which also contained spleen cells from mice immunized with rabbit IgG. After incubation and addition of complement, hemolytic plaques were found around single spleen cells producing antibodies specific for rabbit IgG. The primary and secondary responses of mice to both aggregate-free and heat-aggregated rabbit IgG are described.
Enumeration of cells producing antibody against arsanilic acid was made feasible by the use of hapten-conjugated sheep erythrocytes for the localized hemolysis-in-gel technique. The time of appearance and numbers of these hapten-specific plaque-forming cells correlated closely with the development of arsanilic acid-specific serum antibodies.
The effect of antigen dose on the kinetics of circulating antibody synthesis and on antibody affinity was studied in a haptenic system. High doses of antigen resulted, early in immunization, in higher concentrations of antibody followed later in the immune response by decreased serum levels of antibody as compared with lower doses of antigen. The affinity of the initial antibody synthesized was very similar over a wide antigen dose range. Subsequently, however, a rapid rise in affinity was seen in animals immunized with low doses of antigen, while relatively little change in affinity was seen in animals immunized with higher antigen doses. Suppression of active antibody formation by passive antiserum led to an increase in antibody affinity.
The results are discussed in terms of the mechanisms involved in the selection of a population of cells to participate in the immune response and the mechanisms whereby antigen dose and circulating antibody function to control antibody synthesis.
A method is presented which allows the in vitro enumeration of cells producing antibody against a variety of protein antigens. The proteins are covalently linked to red blood cells by means of a carbodiimide reagent. The concentrations of protein required for the plaque assay are greater than those required for passive agglutination.
The method is simple and sensitive and the results mimic the kinetics of the response that is seen in in vivo assays of serum antibody. Both direct (probably 19 S) and indirect (probably 7 S) plaque-producing cells are detected.
Serum samples taken from rabbits 5 days after vaccination with SW influenza virus by the intravenous route contained high levels of IgM antibodies. IgG antibodies were either not detected or were present at very low levels. By the 10th day after vaccination both IgM and IgG antibodies were present in the serum. The early IgM antibodies were of high avidity while the early IgG antibodies were of very low avidity. The presence of low avidity IgG antibodies in whole serum caused a decrease in the average avidity of the antibodies in whole serum from the 5th to the 10th day post vaccination. The avidity of the IgM antibodies remained fairly constant for the first 20 days of the immune response but a slight increase was detected after secondary vaccination. The avidity of the early IgG antibodies increased during the test period of 20 days. The early IgM and IgG antibodies were heterogeneous with respect to avidity.The highly avid IgM antibodies showed high cross-reactivity with related influenza viruses, i.e. they were of low specificity. The early IgG antibodies that were of low avidity cross-reacted with only one other influenza virus out of the four tested, i.e. they were more specific; as the avidity of the IgG antibodies increased so did their cross-reactivity.
Suppression ofanti-DNP antibody formation by passively administered anti-DNP antibody was studied quantitatively. The ability of an antiserum to sup-press antibody formation was found to be related to the affinity of the antibody for the homologous antigenic determinant (e-DNP-L-lysine), high affinity antibody being capable of causing suppression at far lower concentrations than low affinity antibody. In addition, very low concentrations of high affinity antibody were found to bring about enhancement of antibody formation. The results are discussed with respect to the significance of circulating antibody in the control of antibody synthesis. Partial suppression of antibody formation by a single injection ofpassive antibody slightly lowered the affinity ofthe antibody synthesized. The affinity ofthe antibody synthesized was independent of the affinity of the passive antibody used to bring about partial suppression.
Following immunization of Swiss Webster mice with sheep erythrocytes or Escherichia coli lipopolysaccharide antigens, γA antibody-releasing cells were detected by a modification of the localized hemolysis-in-gel technique. When purified rabbit anti-mouse γA immunoglobulin was added to spleen cell suspensions before plating and enumerating, augmented numbers of antibody-releasing cells were detected. These “γAPFC” were found following primary and secondary injections of both antigens and were specifically inhibited by γA myeloma protein.
By the use of antiglobulin sera of known specificity mouse cells releasing γM, γG1, γG2a, γG2b or γA antibody to sheep erythrocytes can be identified in the LHG assay. The properties of these sera are described. Cells releasing γG1 or γG2b antibody appear as soon after immunization as cells releasing γM antibody. High doses of sheep RBC (1×1010) were needed to stimulate the appearance of γA releasing cells in the spleen after intraperitoneal immunization.
A method was described for the sensitization of erythrocytes with purified type-specific pneumococcal polysaccharide antigens using chromium chloride as a coupling agent. Erythrocytes so sensitized can be used in routine passive hemagglutination and hemolysis tests as well as in the technique of localized hemolysis-in-gel for the detection of specific antibody and specific antibody-producing cells, respectively.
In vitro antigen stimulation of DNA synthesis in lymph node cultures from immunized guinea pigs can be obtained with very low (10–4 µg/ml) antigen concentrations in the culture fluid. Immunization with low doses of DNP-GPA leads to a cell population capable of being stimulated, on the average, by low concentration of antigen whereas immunization with large antigen doses results in a sensitive cell population requiring, on the average, high antigen concentrations for stimulation. These findings correlate well with the affinity for hapten of the serum antibodies produced by these guinea pigs.
Both delayed reactions in vivo and DNA synthesis in vitro can be stimulated by hapten conjugated to proteins different from that used in primary immunization. However the immunizing conjugate is much more effective in terms of antigen concentration required for a given response.
These results can be understood in terms of a thermodynamically driven interaction of antigen (or "processed" antigen) with cell-associated antibody.
Induction of partial tolerance to a haptenic determinant, by the neonatal injection of antigen, resulted in a decrease in the affinity of the antibody formed. Thus, tolerance was more readily induced in those cells capable of synthesizing high affinity as opposed to low affinity antibody. The results are interpreted in terms of a thermodynamically driven selection of cell populations participating in immunologic reactions.
Lymph node tissue from rabbits sensitized with the synthetic terpolymer, G42L28A30, was found to contain antibody-forming cells capable of reacting with GLA-coated erythrocytes in the agar plate method originally devised by Jerne and Nordin. Sheep anti-rabbit globulin completely inhibited the anti-GLA activity in the plaque-forming cells obtained during the early stages following both primary and secondary immunization. Soluble GLA30 (2.4 mg) incorporated into the upper layer also inhibited the response; in contrast, lower concentrations of GLA30 (0.02 mg) had no effect.
Passive hemagglutination titers of the sera against GLA-coated sheep cells indicated no correlation between these titers and the magnitude of the plaque-forming cell response in the lymph node tissue, with the possible exception of sera obtained in the early phases following primary immunization. Differences in the reactions against untreated sheep cells and the GLA-coated cells were discussed, including the appearance of the hemolytic zones and the effect of sheep anti-rabbit globulin sera.
Examination of the effects of anti-allotype sera on hemolytic plaque formation by rabbit spleen cells has revealed the following effects:
1. Plaque formation is usually inhibited by the anti-allotype sera if the cells are obtained from rabbits undergoing the initial response to sheep red blood cells.
2. Plaque formation is usually enhanced by the anti-allotype sera if the cells are obtained from rabbits undergoing the secondary response to sheep red blood cells.
3. A single anti-allotype serum directed against an allotypic specificity present on either the H-chain or the L-chain may give either inhibition or enhancement depending upon the source of the PFC as given in 1 and 2 above.
4. Both the inhibition and the enhancement appear to be mediated through the action of the anti-allotype sera on the hemolysin produced rather than directly on the cells producing it.
A technique is described which makes it possible to detect individual antibody-forming cells using a localized hemolysis reaction in a thickened culture medium containing sheep erythrocytes and guinea pig complement. This technique has the advantage over single cell isolation in that it is technically feasible to survey large populations in order to detect a very small active fraction. The cells can be observed continuously during the time of antibody release, and it appears that an estimate of the relative antibody-forming activity can be made from the size of the areas of lysis. Experiments with metabolic inhibitors indicate that active synthesis is occurring rather than release of preformed antibody. Some experiments on the detection of antibody other than anti-red cell antibodies are reported.
This technique has been applied to a study of the induction period of the primary response of rabbits to sheep red blood cells. The results of this experiment are consistent with an induction period of 2 to 3 days during which there is no increase in the number of active cells in spleen and lymph node reflecting the lag in appearance of detectable serum antibody followed by an abrupt rise of 50- to 100-fold between the 3rd and 5th day. However, the present data are not sufficient to exclude various other mechanisms.
Rabbit sera were found to possess neutralizing activity (normal antibody) to polioviruses and Coxsackie B viruses. This normal antibody showed high specificity in cross-neutralization and absorption tests. It was associated with heat-stable, mercaptan-sensitive, 19S γ1-ß-macroglobulins, which formed weak complexes with the viral antigen. In rare instances, sera with normal macroglobulin antibody, also contained very low activity which was due to 7S γ2-globulins. The neutralization of poliovirus by normal 19S γ1-ß-antibody appeared to follow first order kinetics, and the thermodynamic parameters of this reaction were the same as those of serological reactions employing immune antibody.
The electrophoretic mobility, sedimentation properties, sensitivity to mercaptan, thermostability, and avidity of normal and early (up to day 3) immune antibodies to poliovirus were similar, but differed in several respects from those of late immune antibodies. Thus, the available evidence suggested, that earlier reported differences between normal and immune antibodies reflected differences between antibodies of diverse physicochemical properties rather than between normal and immune antibodies per se.
It is proposed that the normal macroglobulin antibody is associated with an immunological response to repeated stimulation with minute amounts of antigen.
RECENT procedures for the immunological assay of protein hormones in human plasma1-3 require the routine preparation of hormones labelled with iodine-131 of high specific activity.
Localized haelnolysis in gel
W D Dresser
H H Worfis
Dresser, W. D., and H. H. Worfis. 1967. Localized haelnolysis in gel. In Handbook of Experimental Immunology. D. M. Weir, editor. Blackwell Scientific Publications, Ltd., Oxford, England. 1054.
The agar plaque technique for recognizing antibody producing cells
N K Jerne
A A Nordin
Jerne, N. K., A. A. Nordin, and C. Henry. 1963. The agar plaque technique for
recognizing antibody producing cells. In Cell Bound Antibodies. B. Amos and
H. Koprowski, editors. Wistar Institute Press, Philadelphia, Pa.