Monoclonal antibodies to gonococcal outer membrane protein IB: use in investigation of the potential protective effect of antibodies directed against conserved and type-specific epitopes

Several monoclonal antibodies directed against gonococcal outer membrane protein IB have been used in in vitro assays to investigate their potential efficacy in protection against gonococcal infection. In a cytotoxicity assay, virulence of the variant P9-17 for epithelial cells in tissue culture was reduced in the presence of three of the four antibodies which recognized type-specific epitopes. Similarly, virulence of P9-17 as well as a recent isolate was reduced in the presence of the one antibody, SM24, which reacted with a conserved epitope. This antibody was also bactericidal in the presence of complement, and in addition was opsonic for several protein IB-expressing strains as determined by polymorphonuclear leucocyte chemiluminescence measurements. Similarly, all the type-specific antibodies were opsonic for P9 variants. However, only two of these antibodies mediated complement-dependent killing although those which were ineffective were nevertheless complement-fixing antibodies. These results indicate that antibodies to closely positioned epitopes on protein I vary in their biological activities and that the conserved epitope recognized by the antibody SM24 is potentially an effective target on the gonococcal surface for immunoprophylaxis.     

Immunobiology of gonococcal outer membrane protein I

Monoclonal antibodies have been obtained which react with gonococcal outer membrane protein I. One antibody recognised the majority of strains expressing P.IA and another recognised the majority of strains expressing P.IB. In in vitro tests both antibodies were bactericidal in the presence of complement, opsonic for phagocytosis by human PMN and protected epithelial cells against gonococcal invasion. Thus conserved epitopes on P.I. are potentially effective targets for immunoprophylaxis.     

Identification of epitopes recognized by monoclonal antibodies SM1 and SM2 which react with all pili of Neisseria gonorrhoeae but which differentiate between two structural classes of pili expressed by Neisseria meningitidis and the distribution of their encoding sequences in the genomes of Neisseria spp

The pili expressed by all isolates of Neisseria gonorrhoeae react with two monoclonal antibodies, SM1 and SM2. In contrast, although many isolates of Neisseria meningitidis also express pili (class I) which react with antibodies SM1 and SM2, a proportion express pili (class II) which fail to react. In order to define the epitopes recognized by these antibodies, a series of overlapping peptides corresponding to the amino acid sequence of conserved regions of gonococcal pili have been synthesized. The minimum epitope recognized by antibody SM1 was found to comprise a linear peptide EYYLN, corresponding to residues 49-53 of mature pilin. In contrast, antibody SM2 reacted with a number of peptides from around the cysteine residue (Cys 1) at position 120, suggesting that an extended region may contribute to a conformational epitope recognized by this antibody in the native protein. The identification of the two epitopes defines structural differences between the classes of pili expressed by meningococci. In order to determine the distribution of pilin gene sequences in Neisseria we used as hybridization probes an oligonucleotide (PS1) with the sequence 5'-GAGTATTACCTGAATCA-3' which spans the coding region for the SM1 epitope, and a fragment of the 3' end of the gonococcal pilE gene which contains conserved sequences flanking the two Cys codons and encodes the SM2 epitope. All strains of N. gonorrhoeae and N. meningitidis tested, regardless of piliation phenotype, harboured DNA sequences homologous to those encoding the carboxy-terminus of meningococcal class I pilin. Furthermore, all gonococci and all meningococci producing class I pili hybridized with oligonucleotide probe PS1. Non-reverting non-piliated derivatives of previously class I pilus-producing strains showed reduced hybridization signals with this probe, but nevertheless retained sequences homologous to the coding sequence for the SM1 epitope. However, meningococci producing class II pili could be divided into two groups on the basis of their reaction with the PS1 probe: half the strains tested failed to react, which is consistent with our previous analysis of silent class I pilin sequences; the remainder reacted (relatively weakly) with the probe, suggesting that the silent pil sequences in these strains extend further towards the 5' end of the pilin gene than in strains studied previously. Some strains of Neisseria lactamica reacted weakly with both types of probe but failed to produce SM1-reactive pili. In contrast, isolates of Neisseria flava, Neisseria pharyngis, Neisseria sicca and a series of unrelated bacteria failed to react with both SM1 antibody and the DNA probes. This confirms that possession of 'gonococcal' pilin sequences is limited to the pathogenic neisseriae.     

Nonbactericidal antibodies against Neisseria gonorrhoeae: evaluation of their blocking effect on bactericidal antibodies directed against outer membrane antigens

Nonbactericidal monoclonal antibodies (MAbs) directed against gonococcal surface antigens were examined for their effect on complement-mediated bactericidal killing by other MAbs and normal human serum. One MAb, SM73, directed against the H.8 antigen activated complement only moderately well and had little influence on bactericidal antibodies. Two antibodies directed against an epitope on protein III had very different effects. Antibody SM51 activated complement poorly and had no effect on bactericidal killing, whereas antibody SM50, although itself nonbactericidal, activated complement and blocked the bactericidal effect of other antibodies. The extent of the blocking ability of MAb SM50 was studied using MAbs of different specificities as well as polyclonal antisera raised against gonococcal surface antigens. Antibody SM50 blocked IgG MAbs of all specificities, but several MAbs of the IgM class retained their bactericidal effect. Each of these IgM MAbs reacted with lipopolysaccharide, but with different epitopes.     

Gonococcal outer-membrane protein PIB: comparative sequence analysis and localization of epitopes which are recognized by type-specific and cross-reacting monoclonal antibodies.

Comparison of the inferred amino acid sequence of outer-membrane protein PIB from gonococcal strain P9 with those from other serovars reveals that sequence variations occur in two discrete regions of the molecule centred on residues 196 (Var1) and 237 (Var2). A series of peptides spanning the amino acid sequence of the protein were synthesized on solid-phase supports and reacted with a panel of monoclonal antibodies (mAbs) which recognize either type-specific or conserved antigenic determinants on PIB. Four type-specific mAbs reacted with overlapping peptides in Var1 between residues 192-198. Analysis of the effect of amino acid substitutions revealed that the mAb specificity is generated by differences in the effect of single amino acid changes on mAb binding, so that antigenic differences between strains are revealed by different patterns of reactivity within a panel of antibodies. The variable epitopes in Var1 recognized by the type-specific mAbs lie in a hydrophilic region of the protein exposed on the gonococcal surface, and are accessible to complement-mediated bactericidal lysis. In contrast, the epitope recognized by mAb SM198 is highly conserved but is not exposed in the native protein and the antibody is non-bactericidal. However, the conserved epitope recognized by mAb SM24 is centred on residues 198-199, close to Var1 , and is exposed for bactericidal killing.     

Variability of the lytic susceptibility of Neisseria gonorrhoeae to human sera.

The bactericidal activity of human sera for Neisseria gonorhoeae was studied. Sera were obtained from a group of patients with gonococcal infections who had acute urethritis, acute pelvic inflammatory disease, disseminated gonococcal infection, or who were asymptomatic carriers. The homologous and heterologous strains were tested with these sera. The development of serum bactericidal antibodies was not a universal event. With few exceptions, the susceptibility of a particular strain to human antibody and complement appeared to be largely independent of the particular person from whom the serum was obtained and was due instead to antigenic properties intrinsic to each individual strain. Lipopolysaccharide appeared to be the predominant antigen against which bactericidal antibodies were directed. The principal bactericidal antibody class was IgM. Blocking antibodies were not found to account for the lack of lytic activity. A correlation of bactericidal antibodies with protection from developing gonococcal infection could not be demonstrated in three pateints.     

The potential protective effect of immunization with outer-membrane protein I from Neisseria gonorrhoeae

Immunization of rabbits with outer membranes (OM) of Neisseria gonorrhoeae produced antibodies directed against outer-membrane proteins PI and PIII. The antibodies directed against PIII reacted equally well on Western blots with all strains tested, but antibodies directed against PI reacted only with the homologous strain. When purified PIB was used for immunization the immune response was quite different: the sera obtained reacted with both homologous and heterologous PIB types and also reacted with strains expressing PIA. Western blotting of peptides produced by sequential cleavage of PIB revealed that the antigenic determinants recognized by anti-OM sera were predominantly located in the central surface-exposed region of PIB, as is the epitope recognized by the protective anti-PIB monoclonal antibody SM24. In contrast antibodies produced by immunization with purified PI reacted with antigenic determinants in the N-terminal portion of PIB. Nevertheless these determinants are accessible to immune attack on the native protein since the anti-PI sera were opsonic and were strongly bactericidal for both PIA- and PIB-expressing strains.     

Outer membrane protein III of Neisseria gonorrhoeae: variations in biological properties of antibodies directed against different epitopes

Monoclonal antibodies (mAbs) have been raised to gonococcal outer membranes. A panel of six mAbs was identified by several criteria as reacting with outer membrane protein III (P.III). Competitive radioimmunoassays showed that the mAbs could be grouped into three pairs recognizing different epitopes on P.III. These epitopes are equally present on all pathogenic Neisseria. The mAbs demonstrated differing protective effects in model systems. Those directed against one epitope were particularly effective in protecting Chang conjunctiva epithelial cells against gonococcal challenge. mAbs against this epitope and another promoted complement-mediated bactericidal activity, while those directed against the third epitope were ineffective. Thus the biological effects of mAbs directed against P.III vary according to the epitope recognized.     

Specific cross-protective antigonococcal immunity in the murine genital tract

Specific acquired immunity to gonococci was studied in systemically immunized mice, challenged with 10(7) gonococci by intrauterine inoculation. Protection after intraperitoneal immunization was monitored by vaginal cultures taken 24 h post-challenge, since events during the first 24 h postexposure to gonococci are crucial in determining the outcome of infection. Mice were protected against gonococcal challenge by two inoculations with either live or boiled gonococci given 4 weeks apart, whereas immunization with one inoculation did not protect against challenge 1 week later. Protection was correlated with high titers of IgG antibody in serum after two immunizations, but not with the high titers of serum IgM antibody found after the one immunization. IgG antibodies, but not IgM antibodies, were shown to pass into genital secretions. Protection could be passively transferred by serum with high titers of antibody. Of most practical importance was the finding that not only were heat-stable antigens protective, but also heterologous protection resulted after immunization with three strains differing in source (disseminated gonococcal infection versus gonorrhea), opacity-transparency characteristics, and serum sensitivity. The data indicate that IgG antibodies resulting from systemic immunization with heat-stable antigens may be able to provide cross-protection immunity against gonorrhea.     

Synthetic peptide antigens elicit monoclonal and polyclonal antibodies to cytochrome P450 IA2

Two peptide sequences from cytochrome P450 IA2 were synthesized, coupled to ovalbumin and used as antigens to generate anti-peptide monoclonal and polyclonal antibodies. Antisera to both peptides reacted with rat IA2 but not the structurally similar IA1 form as determined by enzyme-linked immunosorbent assay. However, antisera to both peptides detected both rat IA2 and IA1 on immunoblots. In addition immunoblots of human liver microsomes revealed that both antisera recognized human IA2, but not IA1. Monoclonal antibodies generated against one of the peptides recognized rat IA2 and IA1 but did not detect human IA2. These results demonstrate the utility of anti-peptide antisera as a practical approach for the generation of P450 specific antibodies.     

Inter-strain homology of pilin gene sequences in Neisseria meningitidis isolates that express markedly different antigenic pilus types

Neisseria meningitidis isolates examined in this study elaborated one of two pilus types that were antigenically markedly different. Each pilus type reacted either with SM1, a monoclonal antibody that recognizes an epitope common to all gonococcal pili, or with a polyclonal antiserum raised against meningococcal pili that did not react with SM1, but not both. Total genomic DNA from all N. meningitidis isolates analysed, irrespective of pilus type, contained at least one region with extensive homology to a gonococcal pilE probe. Different N. meningitidis strains possessed one of several configurations of genomic pilE-homologous segments. Chromosomal rearrangement of pilE-homologous sequences was associated with P+ to P- pilus phase transition in the strains examined. The arrangement of pilE-homologous segments in total genomic DNA from N. meningitidis isolated from the blood and cerebro-spinal fluid of the same patient was apparently identical.     

Monoclonal, but not polyclonal, antibodies protect against Plasmodium yoelii sporozoites

One of the primary strategies for malaria vaccine development has been to design subunit vaccines that induce protective levels of antibodies against the circumsporozoite (CS) protein of malaria sporozoites. In the Plasmodium yoelii mouse model system such vaccines have been uniformly unsuccessful in protecting against sporozoite-induced malaria. To demonstrate that antibodies to P. yoelii CS protein could provide protection we established a passive transfer model. Passive transfer of Navy yoelii sporozoite 1 (NYS1), an IgG3 mAb against the P. yoelii CS protein, protected 100% of mice against challenge with 5000 P. yoelii sporozoites. Binding of NYS1 to sporozoites was inhibited by incubation with (QGPGAP)2, a synthetic peptide derived from the repeat region of the P. yoelii CS protein, indicating that the epitope on sporozoites recognized by this mAb was included within this peptide. The levels of antibodies to (QGPGAP)2 by ELISA, and to sporozoites by indirect fluorescent antibody test and CS precipitation reaction were similar in sera from mice that received NYS1 in passive transfer and were protected against challenge with 5000 sporozoites, and from mice that had been immunized with subunit vaccines containing (QGPGAP)2 but were not protected against challenge with 40-200 sporozoites. To determine if antibody avidity, not absolute concentration could explain the striking differences in protection, we established a thiocyanate elution assay. The results suggest that NYS1, the protective mAb, has a lower avidity for (QGPGAP)2 and for sporozoites than do the vaccine-induced antibodies. Although the results of the conventional antibody assays did not correlate with protection, sera from the protected animals inhibited sporozoite development in mouse hepatocyte cultures significantly more than did the sera from the unprotected, subunit vaccine-immunized animals, correlating with protection. The data clearly demonstrate that antibodies to the CS protein can protect against intense sporozoite infection. Improved understanding of the differences between protective mAb and nonprotective polyclonal antibodies will be important in the further development of malaria vaccines.     

A protective monoclonal antibody with dual specificity for Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins.

An IgM monoclonal antibody (Mab 36) which reacts with the circumsporozoite (CS) proteins of both P. falciparum and P. berghei was isolated from Plasmodium falciparum sporozoite-immunized mice. In assays of biological activity, Mab 36 induces the CS precipitation reaction with live sporozoites and blocks the invasion of hepatoma cells by sporozoites in vitro at concentrations much lower than those observed for previously reported CS protein-specific monoclonal antibodies. Mab 36 also provided complete protection against P. berghei sporozoite challenge in mice at low doses. Linear epitope mapping revealed that the epitope specificities recognized by Mab 36 are completely encompassed by other monoclonals previously shown to be associated in vivo with protection against P. falciparum or P. berghei sporozoite infection. These results suggest that the ability to make high-affinity IgM antibody to specific CS protein repeat epitopes may be important for eliciting protection against malarial infection.     

Location of a blocking epitope on outer-membrane protein III of Neisseria gonorrhoeae by synthetic peptide analysis

A series of overlapping peptides spanning the deduced amino acid sequence of outer-membrane protein PIII of Neisseria gonorrhoeae have been synthesized on solid-phase supports. The peptides were used in an attempt to locate the epitopes recognized by anti-PIII monoclonal antibodies with defined biological properties. Four bactericidal and two nonbactericidal antibodies were reacted with the synthetic peptides. None of the bactericidal antibodies reacted with the linear peptides. However, the two nonbactericidal antibodies were found to react within the disulphide loop thought to be exposed on the bacterial surface. Monoclonal antibody SM51 recognized a decapeptide corresponding to amino acid residues 24-33, while monoclonal antibody SM50 recognized an octapeptide contained within the decapeptide. The difference in the ability of the two antibodies to block the bactericidal effect of antibodies directed against other surface antigens therefore appears to be related to a difference in their ability to activate complement rather than to the location of the epitope recognized.     

Localization of antibody-binding sites by sequence analysis of cloned pilin genes from Neisseria gonorrhoeae

Immunological analysis of gonococcal pilin (the protein structural subunit of pili) has demonstrated the existence of cross-reacting and type-specific epitopes. The role in adhesion of the domains represented by these epitopes remains unclear. DNA sequencing of a series of pilin-expressing (pilE) genes from a number of otherwise isogenic pilus antigenic variants combined with previous immunological analysis of the corresponding encoded pilins has allowed us to correlate certain predicted amino acid sequences with monoclonal antibody reactivities. The putative epitopes for type-specific antibodies lie predominantly in hydrophilic domains that also contain beta turns. The epitopes for type-specific monoclonal antibodies were shown to depend on amino acid changes either in three separated blocks of amino acid sequence in the semi-variable (SV) region of pilin, or in discrete regions that lie in the disulphide loop in the hypervariable (HV) region of the polypeptide. In contrast, antibody SM1, which reacts with all gonococcal pili, recognizes a poorly immunogenic region of moderate hydrophilicity but low turn potential lying in a conserved portion of the pilin molecule. Our results confirm that antibodies directed against epitopes in both the SV and HV regions are able to inhibit adhesion.     

Dependence of the surface expression of the glycolipid cerebroside sulfate on its lipid environment: comparison of sphingomyelin and phosphatidylcholine

The influence of the membrane lipid environment on the reactivity with antibody of the acidic glycolipid cerebroside sulfate (CBS) was examined by using a spin membrane immunoassay. Fewer antibodies in a polyclonal anti-CBS antiserum recognized the antigen in a bovine brain sphingomyelin/cholesterol (SM/CHOL) environment than in dipalmitoylphosphatidylcholine/cholesterol (DPPC/CHOL). Changes in the CBS ceramide group appeared to have less influence on antibody recognition of CBS in SM/CHOL than in DPPC/CHOL [Crook et al. (1986) Biochemistry 25, 7488-7494]. Although the fatty acid chain length of phosphatidylcholine strongly influences CBS recognition, the fatty acid chain length of sphingomyelin had only a moderate effect on CBS recognition and did not account for the decreased recognition in SM compared to in DPPC. Inhibition studies revealed that the antibodies which recognize CBS in SM/CHOL (S antibodies) form a population distinct from those which recognize CBS in DPPC/CHOL (P antibodies). The specificity of the P and S antibodies was examined further by comparing the efficacy of various substances, which share chemical features with the components of CBS in a SM/CHOL or DPPC/CHOL environment, to inhibit lysis of liposomes containing CBS. Intact CBS, cholesterol, and a phosphocholine lipid, at certain antigen densities, were required for optimal recognition of the antigen, especially by the P antibodies, suggesting that a complex of all three lipids in a multivalent array may be recognized by these antibodies. The S antibodies may recognize a smaller complex or monomers of CBS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microbial carbohydrate specific antibodies distinguish between different stages of differentiating mouse cerebellum

High titered anticarbohydrate antibodies were used to identify cell surface carbohydrates during different stages in histogenesis of mouse cerebellum in a micro tissueculture system which mimics selected features of in vivo cerebellum development. Blockage of fiber formation within the first few days in vitro and inhibition of cell migrations by carbohydrate-specific antibodies served as an assay system for possible contributions of surface carbohydrates to the behavior of developing cerebellar cells. Microbial strains were selected on the basis of carbohydrate structures of their cell wall antigens, and anticarbohydrate antibodies were raised against treated whole bacteria and yeast in rabbits. We found that antibodies to mannan were active at all stages of development tested (embryonic day 13, E13; the day of birth, P0; and postnatal day 7, P7). Antibodies to sialic acids prepared against strains B and C of Neisseria meningitidis distinguish different subterminal structures: anti-B reacted with E 13 and P0 cerebellar cells, and anti-C mostly with cells older than P7. Antifetuin antibody recognized E 13 and P0 but not P7 cell populations. Pneumococcus C strain R36A-specific antibodies were effective only after coating cells to C type carbohydrate before application of the antibody. The results demonstrate that antimicrobial carbohydrate antibodies cross-react with mammalian cell surface carbohydrate structures and therefore can be used as a powerful tool in tissue culture to analyze those structures which might control cell behaviors pertinent to cerebellar development.     

Generation of Anti-Idiotype scFv for Pharmacokinetic Measurement in Lymphoma Patients Treated with Chimera Anti-CD22 Antibody SM03

Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen.     

Defining the protective antibody response for HIV-1

The development of an effective HIV-1 vaccine would be greatly facilitated by knowledge regarding the type and quantity of antibodies that are protective. Since definitive immune correlates are established only after a vaccine has been shown to be effective in humans, animal models are often used to guide vaccine development. Experimental lentivirus infection of non-human primates has shown that neutralizing antibodies can protect against infection. Most specifically, studies of passive antibody transfer in the chimeric SIV/HIV-1 immunodeficiency virus (SHIV) model have provided quantitative data on the level of protective antibody required. While direct extrapolation to humans is difficult, these data likely provide important insights into the protection afforded by antibodies against HIV-1. When used alone, high levels of neutralizing antibodies are required to completely block infection. However, even modest levels of antibody can provide partial protection and affect disease course. Understanding the exact level of protective antibody becomes even more complex in the setting of active immunization and coexisting cellular immunity. Despite this uncertainty, recent findings from lentiviral animal models strongly suggest that neutralizing antibodies will contribute to protection against HIV-1. Based on these data, a major goal of HIV-1 vaccine strategies is the induction of neutralizing antibodies against circulating primary HIV-1 strains.     

Antibodies directed at a conserved motif in loop 6 of outer membrane protein P2 of nontypeable Haemophilus influenzae recognize multiple strains in immunoassays

The P2 porin is the most abundant protein in the outer membrane of nontypeable Haemophilus influenzae. Analysis of P2 sequences from a limited number of strains reveals the presence of both heterogeneous and conserved surface-exposed loops of the P2 molecule among strains. We have previously shown that antibodies raised against the loop 6 sequence of P2 from strain 5657 are bactericidal against multiple isolates. In this study, we determined the nucleotide sequence of the loop 6 region of the P2 molecule from 108 strains of nontypeable H. influenzae in order to assess more rigorously the degree of conservation of loop 6. Based on this analysis, we identified a conserved sequence, different from that of strain 5657, that occurs in approximately one-third of the strains sequenced. To assess the potential of this peptide as a vaccine antigen, antibodies raised to a multiple antigenic peptide corresponding to this sequence were characterized with respect to specificity for the P2 molecule and reactivity with heterologous strains in immunoblot assay, flow cytometry and bactericidal assays. Antibodies were reactive to the P2 molecule of 16 of 20 strains tested by immunoblot assay. Antibodies recognized nine of the 20 strains in a flow cytometry assay, and 13 of 20 demonstrated complement-mediated killing in bactericidal assays. These results support the concept of using conserved regions of the P2 protein as a vaccine antigen.