Characterization of the antibody response to Type III pneumococcal polysaccharide at the cellular level: I. Dose-response studies and the effect of prior immunization on the magnitude of the antibody response

The cytokinetics of the antibody to Type III pneumococcal polysaccharide (SSS-III) were characterized by an immuno-plaque procedure using erythrocytes sensitized with SSS-III. Prior immunization, irrespective of the doses employed, did not result in the development of immunological memory; instead, low-dose paralysis was produced in mice previously immunized with all doses of SSS-III.

Dose-response studies revealed that within a given dose range, there was a direct relationship between the immunizing dose and the magnitude of the antibody response obtained. The dose-response curve for SSS-III showed a single optimal dose for immunization; doses only slightly in excess of the optimal dose produced a significant reduction in the magnitude of the antibody response. The implications of these findings, with respect to the development of paralysis to SSS-III, are discussed.

     

Correlation of Circulating Capsular Polysaccharide with Bacteremia in Pneumococcal Pneumonia

Immunoelectroosmophoresis with rabbit anticapsular antibody was used to detect type-specific pneumococcal polysaccharide in serum from bacteremic patients with pneumococcal pneumonia. The test could detect as little as 0.1 to 1.0 mug per ml or 0.2 to 2 ng per test of polysaccharide from types 1, 2, 3, 4, 5, 8, 12, and 18, and its sensitivity was 10 times that of double immunodiffusion. Although antigen could be detected by double immunodiffusion with types 7 and 14, no antigen could be detected by immunoelectroosmophoresis. Types 7 and 14 polysaccharides were found to be positively charged, whereas the other polysaccharides were negatively charged. Forty-six patients with pneumonia were selected for study because pneumococci corresponding to those types where the test was known to work had been isolated from blood or the respiratory tract. Antigenemia correlated strongly with bacteremia: 12 of 20 bacteremic patients with pneumonia showed antigenemia, whereas 26 patients negative for bacteremia did not show circulating antigen detectable with antisera against the pneumococcal type isolated from the respiratory tract. The apparent concentration of circulating polysaccharide ranged from 0.1 to 100 mug per ml of serum, and the concentration did not appear to diminish appreciably in 10 to 15 days. Three of 12 patients with antigenemia died, and two of these had the highest levels of circulating antigen observed.     

Inactivation of suppressor T-cell activity by nontoxic monophosphoryl lipid A.

Treatment with nontoxic monophosphoryl lipid A (MPL), which was derived from a polysaccharide-deficient, heptoseless Re mutant of Salmonella typhimurium, was found to inactivate suppressor T-cell activity, as evidenced by a decrease in the degree of low-dose immunological paralysis expressed and an increase in the magnitude of the antibody response to type III pneumococcal polysaccharide. The effects produced, which could not be attributed to the polyclonal activation of immune B cells by MPL, were dependent upon the dose of MPL used, as well as the time when MPL was given relative to low-dose priming or immunization with type III pneumococcal polysaccharide. Neither amplifier nor helper T-cell activity was decreased by treatment with the same, or larger, doses of MPL. The significance of these findings to the use of MPL as an immunological adjuvant or an immunomodulating agent is discussed.     

Studies on immunological paralysis: IX. The immunogenicity and tolerogenicity of levan (polyfructose) in mice

The immunological response to native levan (a fructose homopolymer with molecular weight 2×10⁷) has been studied in (DBA/1×CBA-T6T6)F₁ mice by passive haemagglutination and plaque-forming cell (PFC) assays. It resembles type-specific pneumococcal polysaccharide (SSS) in eliciting a prolonged humoral antibody response over a wide dose range (0.0001–100 μg) and in inducing longlasting `high zone' tolerance with a single injection (1 mg or more). Other similarities include an exclusively IgM response, independence of synergy with thymus-derived lymphocytes and absence of immunological memory. On the other hand, parallelism between serum haemagglutinin and PFC levels following all doses of antigen implies that higher immunizing doses of levan, unlike SSS, do not engage in peripheral neutralization of antibody. It was concluded from studying the fate of ¹⁴C-labelled levan that this was attributable to more rapid elimination from the circulation and subsequent slow metabolism of this polysaccharide. Levan also differs from SSS in inducing tolerance directly, without a detectable prior immune phase.     

On the mechanism of immunological tolerance in cyclophosphamide-treated mice

The mechanism of immunological tolerance of sheep red blood cells (SRBC) in mice treated with a single dose of cyclophosphamide was studied. Specific tolerance lasted for as long as nine months in some animals, and its maintenance required the repeated administration of SRBC. Anti-SRBC antibody-forming cells were significantly reduced in the tolerant mice, and X-irradiated recipients of their spleens were specifically tolerant of SRBC. The smallest number of SRBC required for the induction of tolerance was 10⁷; this was the smallest number of SRBC that could elicit antibody synthesis within 5 days in normal mice. Since the effects of cyclophosphamide on antibody-forming cells last only 5 days, it was concluded that the mechanism of tolerance induction involved destruction of antigen-stimulated cells. In support of this is the finding that mice treated with small doses of cyclophosphamide were rendered tolerant only when SRBC were given before the drug. Drug-induced immunological tolerance thus appears to differ significantly from both pneumococcal polysaccharide paralysis and classical, acquired tolerance. A central loss of immunocompetence does not occur in the former, while the latter requires for its induction the administration of antigen in a dose or form that does not stimulate antibody synthesis.     

Effect of the MER tubercle bacillus fraction on the responsiveness of mice to T-independent antigens.

The effect of treatment with the methanol extraction residue (MER) mycobacterial fraction on the immunological responsiveness of BALB/c mice to the T-independent antigens pneumococcal polysaccharide type III (SIIII) and trinitrophenyl-lipopolysaccharide conjugate (TNP-LPS) was ascertained. Pretreatment with MER prevented the establishment of immunological paralysis by threshold doses (10 or 15 microgram) of SIII and by a paralyzing dose of 100 microgram TNP-LPS. The induction of immunological paralysis by SIII was unaffected by treatment with the bacterial adjuvant Corynebacterium parvum and with the B cell mitogens PPD, LPS (Escherichia coli lipopolysaccharide), and dextran sulfate.     

Characterization of the antibody response to Type III pneumococcal polysaccharide at the cellular level: II. Studies on the relative rate of antibody synthesis and release by antibody-producing cells

A procedure based on the rate of appearance of plaque-forming cells (PFC) in agarose was used to measure the relative rates of antibody synthesis and release by cells making antibody specific for Type III pneumococcal polysaccharide (SSS-III). The rate of antibody synthesis and release by SSS-III-specific PFC was directly related to the immunizing dose employed; maximal values were obtained with mice given an optimally immunogenic dose (0.5 μg) of SSS-III. However, dose-dependent reductions, not only in the magnitude of the antibody response, but also in the rate of antibody synthesis and release by specific PFC, were noted in mice receiving doses greater than 0.5 μg. The latter suggests that a decrease in the rate of antibody synthesis and release by antibody-forming cells may be an initial step in the induction of immunological paralysis by high doses of SSS-III.

Increases in the magnitude of the serum antibody and the PFC response were associated with corresponding increases in the rate of antibody synthesis and release by SSS-III-specific PFC following immunization; this suggests that cell differentiation—rather than proliferation—plays a major rôle in the development of the antibody response to SSS-III. In contrast, the results obtained in similar studies with sheep erythrocytes indicate that cell proliferation influences to a greater degree the magnitude of the antibody response elicited to this antigen.

     

Humoral immune response in chinchillas to the capsular polysaccharides of Streptococcus pneumoniae.

Vaccines made from the capsular polysaccharides of Streptococcus pneumoniae have been shown to reduce the incidence of pneumococcal disease in certain populations and have recently been evaluated for their ability to elicit protection against experimental pneumococcal otitis media in a chinchilla model. In this study, chinchillas were vaccinated with a dodecavalent preparation of pneumococcal capsular polysaccharides (PCP) to obtain more information on the immunogenicity of these polysaccharide antigens. All 12 PCP types elicited an antibody response, but the optimum PCP dose and the kinetics of the antibody response varied among types. Immunological paralysis was demonstrated with an immunogenic dose of PCP after primary immunization with a large PCP dose (25 micrograms or more). Pertussis vaccine acted as neither an immunoadjuvant nor an immunosuppressant in the serum antibody response to type 7F PCP in chinchillas.     

Immunological Unresponsiveness Induced by Cryptococcal Capsular Polysaccharide Assayed by the Hemolytic Plaque Technique

Numerous studies have suggested that cryptococcal capsular polysaccharide could induce an immunological paralysis. To investigate this possibility, mice were given various concentrations of purified cryptococcal polysaccharide and then 14 days later were challenge-immunized with the same material in Freund's incomplete adjuvant. Anticryptococcal agglutinin titers were determined at various periods after polysaccharide treatment and after challenge immunization. At the same periods the hemolytic plaque technique was used to determine the number of spleen cells capable of producing antibody against cryptococcal polysaccharide. The data indicated that there was a transitory immune response which preceded tolerance induction. In animals given the largest doses of polysaccharide, “in vivo” neutralization was responsible for low serum agglutinin titers during the transitory response. The capsular polysaccharide was considered to have induced immunological unresponsiveness at the highest concentration, because challenge immunization did not stimulate an increase in the number of plaque-forming cells (PFC). A sixfold increase in numbers of PFC was found in animals injected initially with the lowest concentration of polysaccharide. These results support the idea that tolerance was due to terminal differentiation without proliferation of the immunocompetent cells. The central failure of the immune mechanism which was apparent in the paralyzed mice was temporary under the conditions of this experiment.     

Non-specific stimulation of immunoglobulin synthesis in mice by capsular polysaccharide of Klebsiella pneumoniae

Intramuscular injection of a non-immunogenic dose (1 mg) of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) into mice caused a marked and prolonged increase in the amount of the serum IgM and IgG. The increase of IgM after this injection was over 100 times greater than that of antigen-specific IgM antibodies produced by injection of an immunogenic dose of CPS-K (5 μg) or sheep red blood cells (SRBC) (2 X 10⁸). The increase in the amount of immunoglobulins was not related to induction of immunological paralysis to the major subcomponent of CPS-K (acidic CPS-K) by a large dose of antigen, but was induced by non-antigenic stimulation by the minor subcomponent of CPS-K (neutral CPS-K), which was non-immunogenic at any dose. In mice injected with a non-immunogenic dose of CPS-K, antibody levels in the serum to three kinds of non-cross-reacting antigens, SRBC, bovine serum albumin (BSA) and a possibly autochthonous IgG (IgM—IgG mixed type cryoglobulin) were also increased. The number of plaque-forming cells (PFC) for SRBC in the spleens of mice was also markedly increased after CPS-K injection. These antibodies to SRBC and BSA and PFC for SRBC were exclusively of IgM type and increased to as high levels as those found after specific antigenic stimulation. This marked increase in IgM antibody was neither followed by an increase in IgG antibody nor by immunological memory for a secondary IgG response.     

Effect of Fluosol DA 20% on antibody response to type 3 pneumococcal polysaccharide in rats

Exchange transfusion with the oxygen-carrying resuscitation fluid, Fluosol DA 20% (FDA), interferes with the efficacy of penicillin therapy of pneumococcal infection in rats. Because this effect could not be attributed to an interaction between FDA and penicillin, the effect of FDA on the ability of rats to mount an antibody response to type 3 pneumococcal polysaccharide was tested. FDA (25 ml) was administered by isovolumetric exchange transfusion. Rats were immunized intravenously with 0.2 microgram of type 3 pneumococcal polysaccharide 3 days before, 1 day before, 1 day after, or 3 days after transfusion with FDA. IgM and IgG antibody responses were determined by ELISA 0, 3, 7, 10, 14, 21, and 28 days after immunization. When rats were immunized 3 days before or 1 day before transfusion with FDA, antibody levels were increased above control levels and remained relatively high through Day 28. When the animals were immunized 1 day after transfusion, antibody levels were approximately the same as in the control group. When the rats were immunized 3 days after transfusion, antibody levels were suppressed. These data suggest that FDA does not inhibit the humoral immune response when administered after or within 1 day before immunization, but does inhibit the response when immunization is given 3 days after transfusion.     

Maternal-foetal interaction, antibody formation, and metabolic response in mice immunized with pneumococcal polysacharides.

The maternal transfer of pneumococcal polysaccharides to foetus, as well as the antibody formation and metabolic response were studied in mice exposed to pneumococcal polysaccharides during pregnancy. Type 19 and type 57 pneumococcal polysaccharides display cross-placental transfer to foetus. These polysaccharides also transfer through mother's milk to neonates. Maternal immunization of type 19 polysaccharide during pregnancy induced higher antibody formation in the offspring than the group from non-immunized mothers. Young mice, which received a second dose of polysaccharide at 2 weeks of age, showed a higher antibody response than those which did not receive polysacharide. Treatment of mothers with anti-lymphocyte serum, following by administration of polysaccharide, significantly increased the neonatal immune response to the polysaccharide. Treatment of the mother with a high dose of type 19 or type 57 polysaccharide did not cause significant changes in neonatal growth and organ weights. The offspring from mothers treated with high doses of these polysaccharides did not exhibit abnormalities in chemical contents of their tissues.     

Examination of the differential characteristics of amplifier and contrasuppressor T cells

Vicia villosa lectin-adherent Lyt-1+ spleen cells, obtained 4 days after immunization with an optimally immunogenic dose (0.5 micrograms) of Type III pneumococcal polysaccharide (SSS-III), increased the magnitude of the antibody response of mice to SSS-III upon transfer to recipients also immunized with the same antigen; however, the ability to demonstrate such enhancement depended greatly upon when such cells were transferred relative to immunization of recipients. Lectin-adherent cells augmented the antibody response of athymic nude (nu/nu) mice to SSS-III, and abrogated the expression - but not the induction - of low-dose immunological paralysis, a form of unresponsiveness mediated by suppressor T cells. These findings are consistent with effects usually attributed to the action of amplifier, rather than contrasuppressor, T cells.     

Pneumococcal conjugate vaccines.

We have prepared conjugates of pneumococcal type 4 polysaccharides (PS4) or oligosaccharides to tetanus toxoid using the carbodiimide method. The use of a spacer, 6-aminohexanoic acid, resulted in higher incorporation of carrier protein. Conjugates contained up to 10% free polysaccharide, but no free protein. In general, polysaccharide conjugates induced higher anti-PS4 IgG antibody titers than oligosaccharide conjugates. Conjugates with the highest amount of incorporated protein were the most immunogenic. The response to conjugated PS4 does show characteristics of a T cell-dependent antibody response, in terms of both isotype distribution and induction of immunological memory. Repeated immunization with high doses of PS4TT conjugate resulted in a virtually negative anti-PS4 IgG response, suggestive of the induction of high dose tolerance.     

Influence of preimmunization antibody levels on the specificity of the immune response to related polysaccharide antigens

We studied the influence of preimmunization antibody level on the immune response of adults to one of two structurally related yet immunologically distinct type-specific polysaccharides from Type III Group B streptococcus and Type 14 pneumonococcus. Four weeks after immunization with multivalent pneumococcal vaccine, 20 subjects with low levels of antibody to Type III Group B streptococcus antigen had no significant increase in antibody to this antigen (P greater than 0.05), but all volunteers with moderate to high preimmunization antibody levels who were immunized with Pneumovax had significant increases (P less than 0.01). However, the streptococcal antibody response to pneumococcal Type 14 antigen was weaker and briefer than that in 10 adults given Type III Group B streptococcus vaccine(P less than 0.05). Preimmunization antibody levels influenced the immune response to a structurally similar polysaccharide antigen, but specific Type III polysaccharide antigen appeared necessary to induce a primary antibody response in "nonimmune" adults. We conclude that immunization of mothers with pneumococcal vaccine is not likely to prevent neonatal Type III Group B streptococcal infection, despite immunologic similarities between the two organisms.     

Inactivation of suppressor T cell activity by the nontoxic lipopolysaccharide of Rhodopseudomonas sphaeroides.

Antibody responses of mice immunized with type III pneumococcal polysaccharide were examined with and without treatment with nontoxic lipopolysaccharide from Rhodopseudomonas sphaeroides (Rs-LPS). The results obtained were similar to those described previously for mice treated with monophosphoryl lipid A (MPL) except that lower amounts of Rs-LPS were needed. Both were without effect when given at the time of immunization with type III pneumococcal polysaccharide but elicited significant enhancement when given 2 to 3 days later. Such enhancement was T cell dependent and not due to polyclonal activation of immunoglobulin M synthesis by B cells. Treatment with either Rs-LPS or MPL abolished the expression but not induction of low-dose paralysis, a form of immunological unresponsiveness known to be mediated by suppressor T cells (Ts). The in vitro treatment of cell suspensions containing Ts with extremely small amounts of Rs-LPS or MPI completely eliminated the capacity of such cells to transfer suppression to other mice. These findings indicate that the immunomodulatory effects of both MPL and Rs-LPS are mainly the result of eliminating the inhibitors effects of Ts; this permits the positive effects of amplifier T cells to be more fully expressed, thereby resulting in an increased antibody response. The significance of these and other findings to the use of Rs-LPS as a pharmacotherapeutic agent for gram-negative bacterial sepsis is discussed.     

A peptide mimotope of type 8 pneumococcal capsular polysaccharide induces a protective immune response in mice

Increasing antibiotic resistance and a rising patient population at risk for infection due to impaired immunity underscore the importance of vaccination against pneumococci. However, available capsular polysaccharide vaccines are often poorly immunogenic in patients at risk for pneumococcal disease. The goal of this study was to explore the potential of peptide mimotopes to function as alternative vaccine antigens to elicit a type-specific antibody response to pneumococci. We used a human monoclonal immunoglobulin A (IgA) antibody (NAD) to type 8 Streptococcus pneumoniae capsular polysaccharide (type 8 PS) to screen a phage display library, and the phage PUB1 displaying the peptide FHLPYNHNWFAL was selected after three rounds of biopanning. Inhibition studies with phage-displayed peptide or the peptide PUB1 and type 8 PS showed that PUB1 is a mimetic of type 8 PS. PUB1 conjugated to tetanus toxoid (PUB1-TT) induced a type 8 PS-specific antibody response in BALB/c mice, further defining it as a mimotope of type 8 PS. The administration of immune sera obtained from PUB1-TT-immunized mice earlier (days 14 and 21) and later (days 87 and 100) after primary and reimmunization resulted in a highly significant prolongation of the survival of naive mice after pneumococcal challenge compared to controls. The survival of PUB1-TT-immunized mice was also prolonged after pneumococcal challenge nearly 4 months after primary immunization. The efficacy of PUB1-TT-induced immune sera provides proof of principle that a mimotope-induced antibody response can protect against pneumococci and suggests that peptide mimotopes selected by type-specific human antibodies could hold promise as immunogens for pneumococci.     

Primary murine immunoglobulin M responses to certain pneumococcal capsular polysaccharides consist primarily of anti-pneumococcal cell wall carbohydrate antibodies.

The antibody induced in mice immunized with a vaccine preparation of type 6 (Danish type 6A) pneumococcal capsular polysaccharide (S6) reacted with several chemically disparate pneumococcal capsular polysaccharides. Equivalent numbers of plaque-forming cells were observed when sheep erythrocytes coated with either S6, type 19 pneumococcal capsular polysaccharide (S19), or the pneumococcal cell wall carbohydrate (PnC) were used to detect the response to S6 or to S19. The addition of exogenous PnC to the plaquing mixtures of spleen cells from S6-, S19-, or PnC-immunized mice inhibited the appearance of most (greater than or equal to 85%) of the plaque-forming cells. Furthermore, the addition of monoclonal antibody specific for the dominant (TEPC 15) idiotype of anti-phosphorylcholine (a component of PnC) antibodies also inhibited the appearance of most of the plaque-forming cells. A suppressed S19 response was induced by priming mice with a low dose of S19 or PnC 3 days before immunization with an optimal dose of S19 (low-dose paralysis). These results demonstrated that most, if not all, of the antibody stimulated by these preparations of S6 and S19 was actually induced by and was specific for PnC.     

Effect of cocaine on the immune response and host resistance in BALB/c mice

This study focuses on the effect of varying regimens of cocaine administration on three parameters of the immune response: antibody production, resistance to infection by Streptococcus pneumoniae following immunization, and resistance to tumors. The effect of cocaine on antibody production of female and male BALB/c mice was investigated to both a T-independent (pneumococcal polysaccharide type III [SSS-III]) and a T-dependent antigen (the 2,4-dinitrophenyl ligand [DNP]). It was found that high doses of cocaine injected 3 times/day prior to SSS-III resulted in a small rise in antibody levels in male mice. Low doses given for 4 days prior to or subsequent to SSS-III injection had no effect on the antibody response nor on the susceptibility to infection by live S. pneumoniae. High dosages of cocaine administered 3-5 times/day had no effect on the anti-DNP immune response of male mice but resulted in an almost 2-fold increase of anti-DNP plaque-forming cells in female mice.     

Cellular events in protein tolerant inbred rats. II. Antibody avidity maturation during induction and loss of tolerance

Complete and partial states of tolerance were induced in (AS2 x AS)F₁ hybrid rats after single or repeated injections of high, medium and low doses of fluid HSA (100 mg to 10 μg). Changes in antibody avidity, following challenge with HSA plus adjuvant, were used to detect those affinity subclasses of HSA-specific cells which were not eliminated by tolerogenic antigen. Rats undergoing prolonged low dose treatments of fluid HSA induced an appreciable quantity of high avidity antibody before challenge. The avidity decreased significantly following challenge, indicating a progressive loss of high affinity cells, but only after the synthesis of antibody. Rats made partially tolerant against large and medium doses of fluid HSA when challenged, synthesized antibody which failed to mature to a high level, indicating again that high affinity cells were among the first to be deleted. The higher affinity cells in primed rats (including partially unresponsive rats) were the first to disappear during the memory response which was also triggered by fluid HSA. It is proposed that protein antigens induce a state of tolerance by eliminating B cells, but only after driving them through a stage of antibody synthesis. Avidity of antibody was measured following restoration of HSA responsiveness. Antibody failed to mature to a high level in previously tolerant rats that were allowed to recover naturally (stem cell replacement) or were given normal thoracic duct lymphocytes after the tolerance inducing treatment.