Antibodies to meningococcal H.8 (Lip) antigen fail to show bactericidal activity

Purified H.8 (Lip) antigen was coupled to tresyl-activated Sepharose 4B and used in affinity columns to purify anti-Lip antibodies from convalescent patient sera and from immune rabbit sera. Affinity-purified anti-Lip antibodies isolated from two convalescent patient sera contained 1000 and 1280 ELISA units of antibody and included antibodies of IgG, IgA, and IgM isotypes. An anti-Lip mouse monoclonal ascites (2-1-CA2) had 28,400 ELISA units of antibody. Bactericidal assays were performed using three different case strains of Neisseria meningitidis group B, namely 44/76, 8532, and 8047. Neither preparation of purified human anti-Lip antibodies had detectable bactericidal activity against strains 44/76 and 8532, but one of the two had a titer of 1:4 against strain 8047. Anti-Lip antibodies that were purified from immune rabbit serum and contained 1600 ELISA units of anti-Lip antibodies also failed to show detectable bactericidal activity. The rabbits were immunized with purified Lip antigen and showed specific antibody levels of 2000-2200 units by ELISA, but even the unfractionated sera had little or no bactericidal activity against the test strains. The high titer mouse monoclonal ascites had no bactericidal activity against the test strains. The poor bactericidal activity associated with monoclonal and polyclonal antibodies to the Lip antigen suggest that in spite of other attractive properties it may not be useful as a meningococcal vaccine.     

Development of a piezoelectric immunosensor for the detection of enterobacteria.

A piezoelectric crystal immunosensor has been developed for the detection of enterobacteria in drinking water using antibodies against the enterobacterial common antigen. Applying an anti-enterobacterial antibody layer via protein A immobilization onto a 10-MHz crystal, a response is observed for 10(6) to 10(9) cells ml-1 of Escherichia coli K12, and for various other antigens of the Enterobacteriaceae family.     

A fluorescent antibody method to distinguish vegetative cells from spheroplasts of anEscherichia coli strain

An immunological method for distinguishing bacteria from spheroplasts of anE. coli strain is presented. Antisera prepared against fractions of a lysate of spheroplasts are able, after absorption of the O antibodies, to label spheroplasts but not normal bacteria by the fluorescent antibody method. The immunologic bases of the specificity are studied. By diffusion precipitation tests it was shown that spheroplasts contain at least 7 antigens, 2 of which are part of the O antigen complex of the bacterium.This investigation was supported by the Fonds National de la Recherche Scientifique. The author wishes to thand Doctor J. Salmon for useful suggestions and help in immunological methods. The technical assistance of Miss E. Ligot is gratefully acknowledged.     

Purification of cross-reacting protein antigen shared by Yersinia enterocolitica and other gram-negative bacteria with monoclonal antibody

A monoclonal antibody against the Yersinia enterocolitica 60-kilodalton (kDa) antigen, designated cross-reacting protein antigen (CRPA), was obtained by cell fusion. The CRPA common to gram-negative bacteria was purified from Y. enterocolitica by the affinity chromatography with the monoclonal antibody (IgG1) thus obtained. The purified CRPA showed a single band of 60 kDa in SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and reacted with rabbit antisera against Y. enterocolitica, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, and Shigella sonnei in Western blot analysis. The monoclonal antibody, however, reacted with a 60 kDa peptide from Y. enterocolitica, but not with the antigens from other gram-negative bacteria such as V. cholerae, E. coli, S. sonnei, Salmonella enteritidis, Serratia marcescens, Klebsiella pneumoniae, Proteus mirabilis, and P. aeruginosa. The results suggested that both species-specific and cross-reactive epitopes were present on a CRPA molecule.     

Comparison of serological specificity of anti-endotoxin sera directed against whole bacterial cells and core oligosaccharide of Escherichia coli J5-tetanus toxoid conjugate

The rough mutants of Gram-negative bacteria are widely used to induce protective antisera but the nature of the target epitope for such antibodies is not precisely defined. Endotoxin is one of several antigens present on the surface of bacterial cells, which are able to elicit specific antibodies. We studied the specificity of antibodies produced against a conjugate of E. coli J5 endotoxin core oligosaccharide with tetanus toxoid. The use of chemically defined antigen for immunisation excludes the possibility of production of antibodies against other cell surface antigens. A comparison of this monospecific anti-endotoxin serum with antiserum against E. coli J5 whole cells was performed in order to distinguish the role that endotoxin core oligosaccharide plays in the interaction with humoral host defences from that of other potentially important Gram-negative bacterial surface antigens. The reactivity of both sera with smooth and rough lipopolysaccharides was determined in ELISA, immunoblotting and by flow cytometry. Both antisera reacted with similar specificity with most lipopolysaccharides of identical or related core type. Less distinct reactions with endotoxins of the antibacterial serum in comparison with the anti-conjugate serum were found in all serological tests. LPS of E. coli O100 that showed the strongest reactions with both sera was used to stimulate IL-6, TNFalpha and nitric oxide production by the J-774A.1 cell line. Both sera were used to inhibit that stimulation and no inhibitory effects of the examined sera in comparison with non-immune serum were observed.     

Investigation of a Providencia rettgeri strain that has enterobacterial common antigen in the immunogenic state

Antigenic material obtained by phenol-water extraction from Providencia rettgeri strains, Escherichia coli O:14 strains, and mutants of the E. coli O:14 strain were examined by the passive (indirect) hemagglutination technique, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and by immune blotting (lipopolysaccharide (LPS) blotting). Providencia rettgeri 965, like E. coli O:14, was demonstrated to have an enterobacterial common antigen (ECA) in the immunogenic form but, unlike E. coli O:14, it possessed characteristics of a smooth strain. Two populations of molecules were observed to occur in P. rettgeri 965 phenol-water extracts: one consisting of LPS identifiable with specific O antisera and the other of ECA molecules identifiable with E. coli O:14 antiserum or with a monoclonal antibody against ECA.     

Normal serum cytotoxicity for P32-labeled smooth Enterobacteriaceae. 3. Isolation of a gammag normal antibody and characterization of other serum factors causing P32 loss

Spitznagel, John K. (University of North Carolina School of Medicine, Chapel Hill). Normal serum cytotoxicity for P(32)-labeled smooth Enterobacteriaceae. III. Isolation of a gammaG normal antibody and characterization of other serum factors causing P(32) loss. J. Bacteriol. 91:401-408. 1966.-Gram-negative bacteria lost metabolically incorporated P(32) when suspended in serum only if the serum contained heat-labile in addition to heat-stable factors. Gram-positive bacteria labeled with P(32) and included for comparison lost P(32) in heat-inactivated as well as in fresh normal serum. Further investigation of gram-negative bacteria showed that a smooth Escherichia coli (O117:H27) lost P(32) only if suspended in serum containing complement fractions C'1, C'2, C'3, and C'4 "normal" antibody and lysozyme. The normal antibody was recovered by absorption on and subsequent elution from E. coli O117:H27 cell walls. Immunoelectrophoresis showed that it was a gammaG-globulin. Its P(32)-releasing activity was destroyed by 2-mercaptoethanol. Lysozyme was found to potentiate the P(32)-releasing action of normal antibody plus complement. Evidence was obtained suggesting that beta(1C) globulin was the component absorbed to zymosan during serum absorption at 15 C. Reduction of the beta(1C) level evidently upsets an important balance that exists in normal serum among complement, antibody, and lysozyme. This balance is essential for maximal P(32) release from labeled bacteria, or possibly for a maximal antibacterial effect from normal serum. The possible relationships of bactericidal, bacteriolytic, and opsonic action of normal serum are discussed.     

Analysis of a common-antigen lipopolysaccharide from Pseudomonas aeruginosa.

Lipopolysaccharide isolated from Pseudomonas aeruginosa PAO1 (O5 serotype) was separated into two antigenically distinct fractions. A minor fraction, containing shorter polysaccharide chains, reacted with a monoclonal antibody to a P. aeruginosa common antigen but did not react with antibodies specific to O5-serotype lipopolysaccharide. In contrast, fractions containing long polysaccharide chains reacted only with the O5-specific monoclonal antibodies. The shorter, common-antigen fraction lacked phosphate and contained stoichiometric amounts of sulfate, and the fatty acid composition of this fraction was similar to that of the O-antigen-specific fraction. The lipid A derived from the serotype-specific lipopolysaccharide cross-reacted with monoclonal antibodies against lipid A from Escherichia coli, while the lipid A derived from the common antigen did not react. We propose that many serotypes of P. aeruginosa produce two chemically and antigenically distinct lipopolysaccharide molecules, one of which is a common antigen with a short polysaccharide and a unique core-lipid A structure.     

Nonabsorbable rabbit anti-Salmonella typhimurium antibody as detected by the complement-mediated bactericidal reaction

Herzberg, Mendel (University of Florida, Gainesville), Kathryn V. Kenny, and John B. Robbins. Nonabsorbable rabbit anti-Salmonella typhimurium antibody as detected by the complement-mediated bactericidal reaction. J. Bacteriol. 91:1548-1555. 1966.-A portion of antibody active in the complement-mediated bactericidal reaction against Salmonella typhimurium from hyperimmune rabbit serum has been shown to be nonabsorbable by repeated serial absorptions with whole heat-killed or living bacteria. The first two absorptions remove 90 to 95% of the activity, but 1 to 5% cannot be removed by subsequent absorptions. The nonabsorbable antibody appears to be a macroglobulin by density-gradient centrifugation and by comparison of activity and absorbability of purified gamma-M and gamma-G immune globulins. Alternative hypotheses involving low avidity antibody or antibody to minor cell antigenic components are offered in explanation of the phenomenon.     

Antibody to an artificial disaccharide antigen cross-reactive with Neisseria gonorrhoeae lipopolysaccharide.

An artificial antigen was prepared from 4-O-beta-I-galactopyranosyl-D-glucose (lactose) and 8-ethoxycarbonyloctanol. Covalent attachment to bovine serum albumin provided an antigen that elicited antilactose antibody in rabbits and goat. These antibodies were active against Neisseria gonorrhoeae lipopolysaccharide in passive hemagglutination tests. The same antibody agglutinated cells of Streptococcus faecalis, strain N, and precipitated the lactose-containing cell wall diheteroglycan of this organism. Fractionation of rabbit and goat antibody raised against the synthetic antigen of S. faecalis vaccine provided two antibody fractions only one of which, eluted from the immunoadsorbent by galactose, was active against N. gonorrhoeae lipopolysaccharide.     

Major heat-modifiable outer membrane protein in gram-negative bacteria: comparison with the ompA protein of Escherichia coli.

The outer membranes of several strains of Escherichia coli, other enteric bacteria, and a variety of nonenteric gram-negative bacteria all contain a major heat-modifiable protein similar to the OmpA protein of E. coli K-12. The heat-modifiable proteins from these bacteria resemble the K-12 protein in molecular weight, in preferential release from the outer membrane by sodium dodecyl sulfate in the presence of Mg2+, and in characteristic cleavage by proteases to yield a smaller fragment which remains membrane bound. Antiserum directed against the K-12 protein precipitated the heat-modifiable protein from all strains of Enterobacteriaceae, and chemical comparison by isoelectric focusing, cyanogen bromide cleavage profiles, and proteolytic peptide analysis indicated that the proteins from the various enteric bacteria were nearly identical in primary structure. The heat-modifiable proteins from bacteria phylogenically distant from E. coli shared many of the properties of the E. coli protein but were chemically distinct. Thus, it appears that the structure (and, presumably, the function) of the heat-modifiable protein of gram-negative bacteria is strongly conserved during evolution.     

dGTP triphosphohydrolase, a unique enzyme confined to members of the family Enterobacteriaceae.

The enzyme dGTP triphosphohydrolase (dGTPase; EC 3.1.5.1) was assayed in partially purified extracts of several genera of bacteria, and it was found to be strictly confined to members of the family Enterobacteriaceae. Whereas 11 of 12 enteric bacteria had comparable activity for this enzyme, 8 of 8 nonenteric bacteria, including species in the very closely related genera Vibrio and Aeromonas, did not assay positively for this enzyme. When challenged with Escherichia coli anti-dGTPase antiserum, the active enzymes fell into three groups, retaining 0, approximately 50, or 100% of their original activity. A computer search has revealed an amino acid sequence in the E. coli enzyme which matches well with the single-stranded-DNA binding motif of Prasad and Chiu (J. Mol. Biol. 193:579-584, 1987) and may account for the enzyme's observed interaction with DNA. As far as we are aware, this is the only enzymatic activity so far reported to be present solely in the enteric bacteria.     

Development of oligonucleotide primers for the specific PCR-based detection of the most frequent Enterobacteriaceae species DNA using wec gene templates

Oligonucleotide primers were designed for the PCR-based detection of the wec gene cluster involved in the biosynthetic pathway leading to the production of enterobacterial common antigen (ECA). Escherichia coli DNA was detected using wec A, wec E, and wec F gene primers. The wec A primers were specific for E. coli. The wec E and wec F primers enabled the detection of the most frequent species of the Enterobacteriaceae found in blood and urine specimens as well as in water. The sensitivity of the assay was approximately 1.2 x 102 bacteria/mL of water. Thus, these primers represent an important step in the molecular diagnosis of major Enterobacteriaceae infections. Their role in the routine testing of contamination in drinking water and food may prove to be very useful. The DNA of Enterobacteriaceae species is detected in a first step PCR, followed by specific identification of important pathogens like E. coli O157, Shigella spp., Salmonella spp., and Yersinia spp.     

Therapeutic effects of lysophosphatidylcholine in experimental sepsis

Sepsis represents a major cause of death in intensive care units. Here we show that administration of lysophosphatidylcholine (LPC), an endogenous lysophospholipid, protected mice against lethality after cecal ligation and puncture (CLP) or intraperitoneal injection of Escherichia coli. In vivo treatment with LPC markedly enhanced clearance of intraperitoneal bacteria and blocked CLP-induced deactivation of neutrophils. In vitro, LPC increased bactericidal activity of neutrophils, but not macrophages, by enhancing H(2)O(2) production in neutrophils that ingested E. coli. Incubation with an antibody to the LPC receptor, G2A, inhibited LPC-induced protection from CLP lethality and inhibited the effects of LPC in neutrophils. G2A-specific antibody also blocked the inhibitory effects of LPC on certain actions of lipopolysaccharides (LPS), including lethality and the release of tumor necrosis factor-alpha (TNF-alpha) from neutrophils. These results suggest that LPC can effectively prevent and treat sepsis and microbial infections.     

Growth at sub-optimal temperatures allows the production of functional, antigen-binding Fab fragments in Escherichia coli

Expression in Escherichia coli of recombinant genes coding for the kappa-chain and the Fd fragment of an antibody directed against carcinoembryonic antigen gives rise to Fab dimers. These Fab fragments possess antibody activity, as demonstrated by enzyme-linked immunosorbent assay as well as by ligand competition assay. Effective production of soluble Fab in Escherichia coli was achieved by a decrease in the growth temperature. Following a one step purification by anion exchange chromatography, the bacterially-produced Fab retains its activity at 4 degrees C for at least two months. The relatively simple methodology described in this study should be useful for the design and production of antibodies in bacteria.     

Induction of intestinal IgA and IgG antibodies preventing adhesion of verotoxin-producing Escherichia coli to Caco-2 cells by oral immunization with liposomes

AIMS: To examine the efficacy of liposome oral administration to induce systemic and mucosal immune responses against verotoxin-producing Escherichia coli (VTEC) and the effect of the induced antibodies on the binding of the bacteria to Caco-2 cells. METHODS AND RESULTS: Mice were immunized orally with VTEC antigen and monophosphoryl lipid A (MPL)-containing liposomes composed of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylserine and cholesterol (1 : 1 : 2, molar ratio) (PS-liposome). After immunization, significant IgA and IgG responses to VTEC were induced in both serum and the intestinal lavage fluid in all mice tested. Furthermore, anti-VTEC IgA and IgG antibodies in the lavage fluid effectively inhibited the adhesion of VTEC to Caco-2 cells. CONCLUSIONS: Oral immunization with liposome-associated E. coli O157:H7 antigen can induce significant systemic and mucosal antibody responses against the bacterial antigen and antibodies produced in the intestinal tract, thus functioning as inhibitors for preventing VTEC infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Oral PS-liposome vaccines containing MPL have the potential usefulness for the induction of a protective mucosal immune response against intestinal diseases.     

The action of human and rabbit serum phospholipase A2 on Escherichia coli phospholipids

We have compared the properties of phospholipase A (E.C. 3.1.1.4) activity in whole human and rabbit serum toward the phospholipids of Escherichia coli. Using as substrate E. coli labeled during growth with either [1-(14)C]-palmitic acid or [1-(14)C]oleic acid, and then autoclaved to inactivate E. coli phospholipases and to render the labeled phospholipids accessible to exogenous phospholipases, we show that the deacylating activity in both human and rabbit serum is almost exclusively of the A(2) type. Rabbit serum is at least 20-fold more active than human serum. Activity in both sera is maximal at physiological Ca(2+) concentrations (2 mM) and is abolished by ethylenediaminetetraacetic acid. To examine hydrolysis of intact (unautoclaved) E. coli treated with 25% serum, use was made of a phospholipase A-deficient E. coli strain (E. coli S17), thereby eliminating the possible contribution of bacterial phospholipases to degradation. Human and rabbit serum are about equally bactericidal toward E. coli and cause comparable structural damage. However, only rabbit serum produces substantial hydrolysis of the phospholipids of intact E. coli S17. Heated (56 degrees C, 30 min) rabbit serum is non-bactericidal and retains phospholipase A(2) activity toward autoclaved, but not intact E. coli. The ability of heated serum to degrade phospholipids of intact E. coli S17 is restored, however, by adding 25% normal human serum, which is bactericidal. In this combination, doses of heated rabbit serum containing as much phospholipase A(2) activity (toward autoclaved E. coli) as is present in 25% unheated rabbit serum, produce roughly the same extent of hydrolysis of intact E. coli as does normal rabbit serum alone. Low doses with a phospholipase A(2) activity comparable to that of normal human serum elicit little or no hydrolysis. These findings indicate that hydrolysis of the phospholipids of intact E. coli S17 by serum occurs when: 1) the serum is bactericidal, and 2) when sufficient phospholipase A(2) is present. The difference in phospholipid hydrolysis that accompanies killing of E. coli by human or rabbit serum appears to reflect, therefore, the different amounts of phospholipase A(2) activity in the two sera. Phospholipid degradation is not required for the bactericidal action of serum. Bacterial phospholipid breakdown may be important, however, in the overall destruction and digestion of invading bacteria by the host.-Kaplan-Harris, L., J. Weiss, C. Mooney, S. Beckerdite-Quagliata, and P. Elsbach. The action of human and rabbit serum phospholipase A(2) on Escherichia coli phospholipids.     

Evolution in primates by “Catastrophic-selection” interplay between enveloped virus epidemics,mutated genes of enzymes synthesizing carbohydrate antigens,and natural anti-carbohydrate antibodies

“Catastrophic-selection” is an evolutionary mechanism, by which entire parental-populations are eliminated but very few mutated offspring survive and replace extinct parental-populations. The human natural anti-carbohydrate antibodies, anti-Gal and anti-Neu5Gc suggest the occurrence of catastrophic-selection events in primate evolution. Parental-populations synthesizing corresponding carbohydrate-antigens underwent extinction in viral epidemics, and few offspring survived. These offspring carried accidental mutations that inactivated carbohydrate-antigen synthesis and produced natural-antibody against the lost antigen. Such natural anti-carbohydrate antibody was produced against environmental carbohydrate-antigens (e.g., gastrointestinal bacteria). The carbohydrate-antigen in infected parental-populations was also synthesized on viruses by the host glycosylation-machinery. The natural-antibody in the offspring bound to the carbohydrate-antigen on infecting viruses produced in parental-populations, destroyed the viruses and protected these offspring from extinction. This process occurred in ancestral Old-World monkeys and apes synthesizing α-gal epitopes, which were replaced 20–30 million-years-ago by offspring lacking α-gal epitopes and producing natural anti-Gal antibody against this antigen, and later in hominins synthesizing the sialic-acid antigen Neu5Gc, which were replaced by offspring lacking Neu5Gc and producing anti-Neu5Gc antibody. A present-day example for accidental mutations in very few humans that lost a common carbohydrate-antigen and produce a natural antibody against it is the rare blood-group “Bombay” individuals. These individuals lack the H-antigen (blood-group O) which is synthesized in all other humans, and produce the natural anti-H antibody against blood-group O. Overall, it is suggested that natural anti-carbohydrate antibodies played a critical role in preventing complete extinction of mammalian species in epidemics of highly virulent viruses and may have similar role in future events.     

Conservation of Chi cutting activity in terrestrial and marine enteric bacteria

Homologous recombination in Escherichia coli occurs at increased frequency near Chi sites, 5'G-C-T-G-G-T-G-G3'. Cutting of DNA close to the Chi sequence by the E. coli RecBC enzyme is essential to Chi's stimulation of recombination. We have detected Chi-dependent cutting activity in extracts of several genera of terrestrial enteric bacteria (family Enterobacteriaceae) and of two genera of marine enteric bacteria (family Vibrionaceae). More distantly related bacteria had no detectable Chi-dependent cutting activity. These results support the view that recognition of this specific nucleotide sequence as a signal activating recombination has been maintained during the evolution of certain groups of bacteria. We discuss the possibility that other sequences play a similar role in other groups of bacteria.     

Mouse protection induced by Pseudomonas aeruginosa PAC1R and its defective mutants, Salmonella minnesota Re-mutant and Escherichia coli O14

Abstract Pseudomonas aeruginosa PAC1R and its defective mutants (acetone-killed bacteria), Salmonella minnesota Re mutant (acetone-killed bacteria and Re-LPS) and Escherichia coli O14 (acetone-killed bacteria and enterobacterial common antigen, ECA) were studied in a mouse active protection test. Immunized mice were challenged with wild-type P. aeruginosa strains. It was established that P. aeruginosa LPS-defective mutants induced cross-immunity against different Fisher immunotypes of P. aeruginosa. S. minnesota Re-LPS and ECA gave mice protection against P. aeruginosa .