Studies on antibody avidity at the cellular level. Effects of immunological paralysis and administered antibody

The avidity of antibodies produced by single cells has been studied using inhibition of hemolytic plaque formation by free antigen as a test system. The effect of a paralyzing injection of antigen into mice immunized with heterologous albumins was studied. After a lag period of approximately two days the number of plaque-forming cells (PFC) decreased in the paralyzed mice. The PFC that remained in the paralyzed animals released antibodies with a low avidity for the antigen as compared to control PFC. The impact of an injection of antibody into immune mice was then studied. A reduction in the number of PFC was found in the antiserum suppressed animals. The remaining PFC released antibody of a relatively high avidity for the antigen. The data support the concept of a similar binding constant for the cell-associated antibody-like receptor operating in induction of immunity and paralysis and the antibody released from the cell after stimulation.     

Experimental Escherichia coli O6 pyelonephritis in rabbits. Effect on O6 antibody quantity and avidity of prior immunization with E. coli O2 bacteria.

Haematogenous pyelonephritis was induced in rabbits using Escherichia coli 06:K13:H1 bacteria and the amounts and avidities of antibodies to the 06 antigen were analysed by the ammonium sulphate precipitation technique of Farr. In a group of six animals preimmunized with E. coli 02:K2ab:H1, five developed pyelonephritis and one pyelitis, as determined by histological examination. All aminals showed a considerable antibody response to E. coli 06 antigen during the infection. The animal with pyelitis gave a slightly smaller response than the others. The antibody avidity showed a pronounced variation. In a second group of six rabbits not preimmunized, five animals developed pyelonephritis. The titres of antibodies against E. coli 06 antigen increased during the infection inall of the six animals. However, the increase was significantly smaller than for the animals preimmunized with E. coli 02:K2ab:H1 (P smaller than 0.01). The pattern of the antibody avidities in this group was also heterogenous. The results are consistent with previous findings that exposure to serologically heterologous E. coli bacteria can enhance the development of the homologous antibody titres. This could be of relevance for serological diagnostic work as well as in the determination of the protective capacity of the antibody.     

The influence of amount and avidity of persisting primary antibodies on the secondary response to human serum albumin in rabbits

The primary antibody response in rabbits to human serum albumin (HSA) has been analysed. Doses of 0.04–25 mg were given subcutaneously with Freund's complete adjuvant. Maximum antibody response about 60 days after primary stimulation was independent of the antigen dose given.

After 175 days the amount of persisting antibody was lowest in the animals stimulated with the lowest dose of antigen. The avidity of the antibody was inversely proportional to the antibody titre. Secondary stimulation of all rabbits with 1 mg of HSA intravenously caused secondary antibody responses which were insignificantly different from each other.

It is suggested that the magnitude of the secondary antibody response is determined by the regulatory activity of the circulating antibodies following primary stimulation.

     

Mucosal and systemic antibody responses to the lipopolysaccharide of Escherichia coli O157 in health and disease

Mucosal immunity in the gastrointestinal (GI) tract is a primary defence against GI pathogens. We hypothesise that a mucosal response to lipopolysaccharide (LPS), especially to the common (core) determinants of GI pathogenic Escherichia coli strains, is protective. The aims of this study were to investigate the specificities, levels and development of humoral responses in health and GI disease to the R3 LPS core and O-polysaccharide of E. coli O157. The purpose was to try to predict whether vaccination or passive immunisation might induce protection. Wherever possible, paired whole gut lavage fluid (WGLF) and serum samples were collected for comparison of the mucosal and systemic responses. Matched saliva samples were also collected from some study groups. The patient groups included those with acute E. coli O157 disease (serum only), patients convalescing after E. coli O157 infections, and patients undergoing routine investigation for GI conditions but subsequently shown to be immunologically normal. Some samples of WGLF from patients with Crohn's disease (CRO) and ulcerative colitis (UC) were included to allow comparisons with patients with inflammatory conditions known to alter antibody secretion in the GI tract. The healthy groups from whom serum and saliva only were taken included blood donors, healthy volunteers and a group of slaughterhouse workers. This latter group was likely to have been exposed regularly to faecal bacteria from animals and antibody specificities might have been expected to be different from other healthy individuals. Levels and classes of antibodies were determined by ELISA with microtitration plates coated with polymyxin complexes of whole LPS extracted from E. coli O157 and LPS from the E. coli R3 rough mutant. Antibodies of IgG and IgM classes were measured in serum and IgA was measured in WGLF and saliva. IgG antibodies to the O157 LPS and the R3 core oligosaccharide were detected in the serum of healthy blood donors. Patients with acute E. coli O157 disease showed elevated levels of serum IgM to O157 LPS and R3, with IgG levels raised only to R3. In serum from convalescent patients, IgG to O157 LPS was significantly above the control groups only in the period 6-16 weeks after infection. Total IgA levels were similar in WGLF specimens from all groups, except the patients with UC, whose levels were much higher. Specific IgA levels were higher in the E. coli O157 convalescent group, but there were no significant correlations overall. UC patients had significantly lower levels of IgA to O157 and CRO patients had higher O157 IgA levels than UC patients and healthy volunteers. In serum, inhibition of ELISA showed that the response to the O157 LPS was due in part to a response to the R3 oligosaccharide component. This response was much more pronounced in the healthy and non-O157 groups than in convalescent patients. There was no correlation between specific IgA antibody levels in saliva and matched specimens of WGLF, and levels in sequential saliva specimens fluctuated widely. The significant IgG and IgA responses to the R3 core suggest that there is immunological memory to this oligosaccharide LPS component which may have a role in protection against E. coli LPS both systemically and locally in the GI tract. Boosting of this mucosal response to the LPS core, either naturally through exposure or by active or passive immunisation, may confer protection. Finally, antibody responses to E. coli O157 must be interpreted with caution, as the response detected is a sum of responses to the O-specific polysaccharide and the R3 core.     

Maternal antibody inhibits both cellular and humoral immunity in response to measles vaccination at birth

Maternal antibody prevents the use of live, attenuated measles vaccine (LAV) before 6-9 months of age, but vaccinated 6-month-old infants can mount a T cell response. An infant macaque model was used to study the immune response to LAV in the newborn in the presence or absence of maternal antibody. Four newborn monkeys without detectable maternal antibody and 9 newborns with passive measles antibody were vaccinated with LAV. Only the infants without passive antibody seroconverted after vaccination and 3 of 4 of these infants also developed measles-specific interferon gamma+ T cells. The monkeys were challenged with wild-type measles virus at 5 months of age, and 7 of 9 infants vaccinated in the presence of passive antibody had systemic infection and skin rash, while 3 of the 4 infants vaccinated in the absence of passive antibody were protected from viremia and rash. This suggests that the newborn can respond to LAV but that maternal antibody suppresses the priming of both humoral and cellular immunity at birth.     

Human response to Escherichia coli O157:H7 infection: antibodies to secreted virulence factors

Vaccination has been proposed for the prevention of disease due to enterohemorrhagic Escherichia coli (EHEC), but the immune response following human infection, including the choice of potential antigens, has not been well characterized. To study this, sera were obtained from five pediatric patients with acute diarrhea caused by E. coli O157:H7 0, 8, and 60 days after hospitalization. These sera were used to examine the immune response to four different EHEC virulence factors: Tir (translocated intimin receptor, which is inserted into the host cell membrane), intimin (bacterial outer membrane protein which binds to Tir), EspA (secreted protein which forms filamentous structures on EHEC surface), and EspB (inserted into the host membrane and cytoplasm). The response to O157:H7 lipopolysaccharide was also examined. Sera were assayed against purified recombinant proteins using immunoblot analysis and by enzyme-linked immunosorbent assay to determine the sera's titers to each of the antigens in all patients. We found that there was little reaction to EspA, EspB, and intimin in the acute-phase sera, although there was some reactivity to Tir. By day 8, titers of antibody to all four virulence factors were present in all patients, with a very strong response against Tir (up to a titer of 1:256,000), especially in hemolytic-uremic syndrome patients, and lesser strong responses to the other three antigens. The titer to the antigens 60 days after hospitalization was decreased but was still highest for Tir. These results suggest that there is a strong immune response to Tir, and to a lesser extent to the other three virulence factors, following EHEC disease, indicating that these bacterial molecules are potential vaccine candidates for preventing EHEC disease. They also suggest that bacterial virulence factors that are inserted into host cells during infection by type III secretion systems (Tir or EspB) are still recognized by the host immune response.     

Production and characterization of monoclonal antibodies specific for the lipopolysaccharide of Escherichia coli O157.

Identification of the O157 antigen is an essential part of the detection of Escherichia coli O157:H7, which is recognized as a major etiologic agent of hemorrhagic colitis. However, polyclonal antibodies produced against E. coli O157:H7 lipopolysaccharide (LPS) may react with several other bacteria including Brucella abortus, Brucella melitensis, Yersinia enterocolitica O9, Escherichia hermannii, and Stenotrophomonas maltophilia. We produced eight monoclonal antibodies (MAbs) specific for the LPS of E. coli O157. Western blots (immunoblots) of both the phenol phase (smooth) and the aqueous phase (rough) of hot phenol-water-purified LPS indicated that three of the MAbs were specific for the O antigen and five were reactive with the LPS core. The eight MAbs could be further differentiated by their reactivities to Salmonella O30 LPS (group N), which is reported to be identical to the E. coli O157 antigen. All eight MAbs reacted strongly to all of the 64 strains of E. coli O157 tested, which included 47 isolates of O157:H7 and 17 other O157 strains. None of the eight MAbs cross-reacted with any of the 38 other E. coli serotypes tested, which consisted of 29 different O-antigen serotypes, or with 38 strains (22 genera) of non-E. coli gram-negative enteric bacteria.     

Neutralizing antibodies to Escherichia coli Vero cytotoxin 1 and antibodies to O157 lipopolysaccharide in healthy farm family members and urban residents.

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Escherichia coli O157 lipopolysaccharide (LPS) was developed with sera from 63 children with confirmed recent E. coli O157 infection and from 256 age-stratified urban controls. The median ELISA values for control and case sera were 0.05 (interquartile range, 0 to 0.20; mean +/- standard deviation [SD], 0.15 +/- 0.22) and 1.41 (interquartile range, 1.11 to 1.59; mean +/- SD, 1.41 +/- 0.53), respectively (P < 0.001). With a breakpoint of 0.59 (mean ELISA value of the control sera + 2 SDs), the assay had a sensitivity, specificity, and positive and negative predictive values of 95, 94, 80, and 98%, respectively, for recent E. coli O157 infection. The O157 LPS assay and Vero cytotoxin (VT) 1-neutralizing-antibody (NAb) assay were used to compare the relative frequencies of O157 LPS antibodies and VT1-NAbs in an age-stratified urban population from Toronto, Ontario, Canada, and in 216 healthy family members from dairy farm in southern Ontario. The frequency of O157 LPS antibodies was about threefold higher in dairy farm residents (12.5%) than in urban residents (4.7%) (P < 0.01). Similarly, the frequency of VT1-NAbs was about sixfold higher in dairy farm residents (42.0%) than in urban residents (7.7%) (P < 0.001). These findings are consistent with a greater level of exposure of dairy farm residents to VT-producing E. coli (VTEC) strains. The high rate of seropositivity to VT1 in farm residents probably reflects the booster effect of repeated VTEC exposures and argues against a sustained generalized immunosuppressive effect of VT1. Seroepidemiological studies may help in assessing the level of exposure of different populations to VTEC strains.     

Determination of antibody avidity at the cellular level by the plaque inhibition technique: effect of valence of the inhibitor.

Inhibition of plaque formation by multivalent and univalent ligands was compared as an assay of avidity of antibody produced by PFC. Multivalent ligands are much more effective as inhibitors and their use tends to impart an appearance of lack of heterogeneity and high avidity to the PFC populations being studied. It is thus probably generally advisable to employ univalent ligands in such studies.     

Systemic antibody response to diarrheagenic Escherichia coli and LPS O111, O157 and O55 in healthy Brazilian adults

Enterohaemorrhagic Escherichia coli (EHEC) can cause a variety of human illnesses ranging from uncomplicated diarrhoea to haemorrhagic colitis and haemolytic uremic syndrome. The serotype O157:H7 has been associated with numerous outbreaks worldwide, but in Brazil the infection is rare. Brazilian adults present antibodies reactive with the principal virulence factors of enteropathogenic E. coli (EPEC) that have many genetic and antigenic similarities with EHEC. Lipopolysaccharides (LPS) are components of outer membranes and important virulence factors of Gram‐negative bacteria. LPS O111 is present in EPEC and EHEC strains. LPS O157 is found only in EHEC strains, but it has some structural similarities with LPS O55 present in EPEC strains. This study investigates the levels of IgG and IgM seric antibodies reactive with EHEC O157:H7, EHEC O111:H–, EPEC O111:H– and the levels of anti‐LPS O111, LPS O157 and LPS O55 antibodies in healthy adults living in São Paulo, Brazil. The antibody levels were determined by an enzyme‐linked immunosorbent assay for 100 individual serum samples, and the presence of anti‐bacterial and anti‐LPS seric antibodies was confirmed. Positive correlations were found among the three kinds of antibodies. The concentrations of IgM anti‐LPS were significantly higher than those of IgG, and surprisingly, the concentrations of anti‐LPS O157 were high in view of the infrequent isolation of O157 bacteria in Brazil. Our results suggest that there is a cross‐reacting immunity to EHEC in the Brazilian population, which may be a result of the immunity to EPEC antigens. Alternatively, Brazilians may be exposed to EHEC more frequently than has previously been thought.     

The interaction of Escherichia coli with normal human serum: factors affecting the capacity of serum to mediate lipopolysaccharide release

We previously demonstrated that incubation of E. coli in normal human serum (NHS) resulted in the release of a finite fraction (approximately 30%) of LPS from the bacterial outer membrane. In experiments reported here, we examined factors which may enhance or diminish the capacity of NHS to mediate this limited LPS release. Both the susceptibility to serum killing and LPS release were dependent on growth phase. Optimal killing and release coincided with the midlogarithmic growth phase. The composition of LPS subunits in the outer membrane appeared to influence serum-mediated LPS release. Serum treated E. coli enriched for Rc-chemotype LPS released less LPS from their outer membrane than the wildtype 'smooth' bacteria during exponential growth. LPS fractions released by NHS or EDTA appeared to a large degree to overlap, suggesting that NHS-mediated LPS release may involve the action of a serum chelator. A serum-resistant mutant failed to release LPS in either NHS or EDTA. This latter observation suggests that LPS release may be a relevant event in serum killing. We did not detect any modulation of LPS release when E. coli were pre-incubated with a series of antibiotics prior to treatment with NHS.     

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and monoclonal antibodies as tools for the subgrouping of Escherichia coli lipopolysaccharide O18 and O23 antigens

The lipopolysaccharide (LPS) of Escherichia coli O18 isolated from a wide variety of sources was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Four different LPS types, designated O18A, O18A1, O18B, and O18B1, were identified. Most O18 strains possess O18A, O18A1, or O18B LPS types, and these types are clonally associated. A reference test strain with the classical O18ab designation possessed O18B LPS, while two reference O18ac strains possessed O18A and O18A1 LPS, respectively. A panel of 15 anti-O18A B-cell hybridomas was isolated. Enzyme-linked immunosorbent assays revealed that some of the monoclonal antibodies produced by these cells recognize different epitopes. Four of these antibodies suffice to distinguish the four O18 types. Numerous strains whose LPS had been typed by SDS-PAGE were tested by agglutination with seven monoclonal antibodies whose specificities had been determined by enzyme-linked immunosorbent assays. The results indicated a perfect correlation between the two methods. Rabbit antisera raised against O18A bacteria agglutinated boiled bacteria of each of the O18 LPS types efficiently. The antisera were adsorbed with bacteria possessing each of the LPS types. The adsorbed sera only distinguished between two groups: O18A and O18A1 versus O18B and O18B1, as shown by agglutination assays and Western blotting. E. coli O4 and O23 and Serratia marcescens O8 antigens, which are reputed to cross-react with O18, were also analyzed. One O4, one O8, and four O23 strains were tested. All made an LPS which was distinguishable from O18 LPS types by SDS-PAGE. Each O23 strain synthesized a different LPS, and three of them synthesized only few short chains. Some of the monoclonal antibodies reacted with O4, O8, and O23A LPSs. The results are interpreted as indicating that numerous E. coli O serogroups will prove to be chemically heterogeneous and that future analyses of subgroup heterogeneity should be guided by results from SDS-PAGE and rely preferentially on monoclonal antibodies as opposed to rabbit hyperimmune sera.     

The natural antibody response to E. coli includes antibodies of the IgD class.

Antibodies to E. coli of the IgM, IgG and IgA class are readily demonstrable in normal human serum. Using the sensitive red cell-linked antigen-antiglobulin system, it has been demonstrated that antibodies of the IgD class are also part of this normal response. The IgD antibody titre is low and often could only be demonstrated in partially purified IgD preparations. The availability of purified IgD paraproteins and their Fabdelta and Fcdelta fragments, as well as antisera specific for these fragments, allowed the necessary critical specificity controls to be performed.     

The early cellular and humoral immune response to primary and booster oral immunization with cholera toxin B subunit

The immune response to cholera toxin B subunit given orally was studied in 13 human volunteers. A serum IgG and IgA antitoxin response was observed, which was boosted by a second immunization. Using an immunospot assay, cells spontaneously secreting anti-toxin IgG and IgA, but not IgM appeared transiently in the blood after immunization. There were 105 IgG- and 87 IgA-secreting cells per 2 x 10(6) mononuclear cells 7 days after the first immunization, and 282 IgG- and 413 IgA-secreting cells 5 days after the second immunization. A polyclonal increase in total IgM-secreting cells was observed. Few anti-toxin-secreting cells were observed in the bone marrow at the peak of the circulating cell response, which could be accounted for by contamination of the sample with peripheral blood, suggesting that the bone marrow is not a significant site of anti-toxin-secreting cells after oral immunization.     

Alteration of the antibody response to Escherichia coli O antigen in mice by prior exposure to various somatic antigens.

In the present study in mice we used the Jerne plaque assay to compare the immunity enhancing potential of different Gram-negative bacteria with special regard to their endotoxin. The results confirm the recent finding that injection of Escherichia coli bacteria of various serotypes may enhance the IgG antibody response to the O antigen of a serologically unrelated E. coli strain injected subsequently, but may suppress the IgM antibody formation. The O antibodies formed were of low avidity but were antigen specific. Smaller amounts of antibodies were formed to a serologically unrelated antigen, E. coli O76, which had not been injected. Of the strains tested as primary stimuli E. coli O4 gave considerably greater enhancement than any other serotype including the homologous E. coli O6, when a short interval between the injections was used. The influence of O4 on the serologically unrelated anti-O6 response was stronger than on the response to the cross-reactive E. coli O18 antigen, suggesting that O antigen cross-reactivity is not the basis for the immunomodulation. Formalin-killed bacteria were more effective in this respect than boiled bacteria or purified lipopolysaccharide and rough mutants (E. coli R1--R4) and E. coli O4 were less effective than many of the other smooth E. coli. These findings suggest that shared determinants in the lipid, basic carbohydrate core or Kunin common antigen portions of the endotoxin do not play the major immunomodulating role in this system. Salmonella reading but not Pseudomonas aeruginosa affected the anti-E. coli O6 response in a similar manner. One explanation for the alterations in the immune response observed implies the presence of an antigen determinant shared by many Enterobacteriaceae in such a position in relation to the O antigen that it can be utilized for cellular co-operative events in the O antibody response. The protein portion of the endotoxin protein--lipid--carbohydrate complex is a possible location.     

Serum response of Escherichia coli strains causing dyspepsia and urinary tract infection: relation to alpha-hemolysin production and O type.

A total of 109 alpha-hemolytic and 104 nonhemolytic Escherichia coli isolates from children with dyspepsia and urinary tract infections were investigated for resistance to the bactericidal activity of human serum. A significantly higher proportion of serum resistance was found in alpha-hemolytic E. coli isolates than in nonhemolytic isolates (P < 0.01). An association between the titer of alpha-hemolysin produced and serum resistance was found.     

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.

     

Antibody class and complement requirement on neutralizing antibodies in the primary and secondary antibody response of cattle to infectious bovine rhinotracheitis virus vaccine

Summary Calves responded to a single intramuscular injection of an attenuated strain of infectious bovine rhinotracheitis virus by producing IgM followed by IgG antibody. Both IgM and IgG antibody produced during the first month were primarily complement-requiring neutralizing antibody (CRNAb), especially IgM antibody. After a month, IgG had replaced IgM as the predominant immunoglobulin, and titers with and without complement (C') decreased in both IgG and IgM fractions. The largest decrease was in the IgM CRNAb fraction.Seven days after a second injection given on day 196, calves responded with an anamnestic IgG response in which CRNAb titers were 1 or 2 two-fold dilutions higher than non-CRNAb titers. One calf developed an IgM response similar to its primary response, whereas inhibition of the IgM response occurred in the other 3 calves which had much lower IgM antibody titers than those attained in the primary response. Twenty-eight days after the second injection the titers of IgG were the same or only a 2-fold dilution less than their 7-day secondary titers, whereas IgM titers generally decreased considerably more than this. Guinea pig and rabbit sera were equally effective as C' sources in potentiating CRNAb, whereas bovine serum was a poor C' source.