Immunization with recombinant Opc outer membrane protein from Neisseria meningitidis: influence of sequence variation and levels of expression on the bactericidal immune response against meningococci.

The opc gene from Neisseria meningitidis was cloned into the pRSETA vector, and recombinant protein was expressed at high levels in Escherichia coli. The protein was readily purified by affinity chromatography and used for immunization with conventional Al(OH)3 adjuvant or after incorporation into liposomes and Zwittergent micelles. The resulting sera were analyzed for their ability to recognize purified recombinant protein and "native" protein in an enzyme immunoassay with outer membranes and by whole-cell immunofluorescence. Immunization with Al(OH)3 induced high levels of antibodies which reacted with the purified protein but did not recognize whole cells. In contrast, liposomes and micelles induced antibodies which reacted with the native protein in whole cells. The addition of monophosphoryl lipid A (MPLA) to either liposomes or micelle preparations increased the magnitude of the immune response and induced a wider range of immunoglobulin subclasses. This was associated with the ability of the sera to induce complement-mediated killing of the homologous strain. The most effective bactericidal activity was observed with Opc protein incorporated into liposomes containing MPLA. The magnitude of the bactericidal effect was strongly influenced by the level of expression of the Opc protein and was abolished by limited variation in the sequence of the protein expressed by heterologous strains.

Interactions of Neisseria gonorrhoeae with mature human macrophage opacity proteins influence production of proinflammatory cytokines.

The pathological features of ascending gonococcal infection suggest that proinflammatory mediators secreted by tissue-resident macrophages are important components of the host response. Challenge of fully differentiated, mature macrophages with variants of Neisseria gonorrhoeae strain P9 or purified bacterial surface components (pili, lipooligosaccharide, and outer membrane vesicles) induced the secretion of interleukin 6 (IL-6), tumor necrosis factor alpha, growth-related protein alpha, macrophage inflammatory protein 1alpha (MIP-1alpha), and RANTES cytokines but had no effect on IL-8 production. No secretion of IL-1beta, epithelial-derived neutrophil attractant 78, granulocyte-macrophage colony-stimulating factor, IL-10, or IL-12 cytokines was observed. Notably, the P9-Opa(b) protein, in comparison to P9-Opa(a), increased the association of gonococci with macrophages and elevated the secretion of cytokines. Thus, variation in Opa protein expression by the gonococcus may be a determining factor in the severity of pelvic inflammatory disease.

Recombinant proteins in vaccine development.

The outer membrane of Neisseria meningitidis contains a variety of proteins with the potential for inclusion in new meningococcal vaccines (1). Studies on the vaccine potential of these proteins would be facilitated by the production of pure recombinant protein, free from other components of the Neisseria outer membrane. At present, the class 1 outer-membrane protein (OMP) is generally regarded as the most promising candidate. In this chapter, we describe four protocols involved in the preparation of recombinant class 1 OMP for vaccine development. This integrated set of methods can also be readily used to study the potential of other meningococcal OMP as vaccine candidates, and moreover, their utilities make them attractive for vaccine studies relating to many other human pathogens.

Epitope mapping.

An epitope is defined as the site on an antigen at which an antibody binds. In the case of proteins the epitopes can be classified as continuous (or sequential) and discontinuous according to whether or not the amino acids recognized are close together in the primary sequence or are well-separated but brought together by the folding of the protein. The methods described have permitted the localization or "mapping" of continuous epitopes on meningococcal outer-membrane proteins (OMPs), which are recognized by both monoclonal and polyclonal antibodies (MAbs/PAbs).

Interactions of Neisseria meningitidis with cells of the human meninges.

The interaction of Neisseria meningitidis with the meninges that surround and protect the brain is a pivotal event in the progression of bacterial meningitis. Two models of the human meninges were established in vitro, using (i) sections of fresh human brain and (ii) cultures of viable cells grown from human meningiomas. Neisseria meningitidis showed a specific predilection for binding to the leptomeninges and meningeal blood vessels in human brain and not to the cerebral cortex. There was a close correlation between the adherence of different Neisseria species to leptomeninges and cultured cells. The major ligand that mediated adherence was the pilus, and pilin variation modulated the interactions. The presence of Opa protein increased the association of Cap+ meningococci that expressed low-adhesive pili, but did not influence the association of high-adhesive pili. In contrast, Opc did not influence the adherence of Cap+ meningococci, whereas loss of capsule was associated with a more intimate interaction between the bacteria and the meningioma cell that was not apparent with Cap+ meningococci. There was no evidence of internalization of meningococci by meningioma cells in vitro, an observation that is consistent with the barrier properties of the leptomeninges to N. meningitidis observed in vivo.

Lack of immunity in university students before an outbreak of serogroup C meningococcal infection.

Immunity to meningococci was determined in infected and uninfected students before and during an outbreak of serogroup C meningococcal infection at a university in the United Kingdom. No immunity against the outbreak strain was detected in serum taken from infected students prior to the outbreak or at the time of admission; bactericidal activity developed during convalescence. Carriage of all strains of serogroup C meningococci in asymptomatic students was low (0.9%), and no carriage of the outbreak strain could be detected. Immunity in the at-risk student population before the outbreak was low: 90% of students had no significant bactericidal activity against the outbreak strain. A low prevalence of carriage of the outbreak strain, together with a low prevalence of protective immunity within the student population, was associated with a high incidence of invasive disease in those who acquired the outbreak strain.

Interaction of primary human endometrial cells with Neisseria gonorrhoeae expressing green fluorescent protein.

Infection of the endometrium by Neisseria gonorrhoeae is a pivotal stage in the development of pelvic inflammatory disease in women. An ex vivo model of cultures of primary human endometrial cells was developed to study gonococcal-host cell interactions. To facilitate these studies, gonococci were transformed with a hybrid shuttle vector containing the gfp gene from Aequoria victoria, encoding the green fluorescent protein (GFP), to produce intrinsically fluorescent bacteria. The model demonstrated that both pili and Opa proteins were important for both mediating gonococcal interactions with endometrial cells and inducing the secretion of pro-inflammatory cytokines and chemokines. Pil+ gonococci showed high levels of adherence and invasion, regardless of Opa expression, which was associated with increased secretion of IL-8 chemokine and reduced secretion of IL-6 cytokine. Gonococcal challenge also caused increased secretion of TNF-alpha cytokine, but this did not correlate with expression of pili or Opa, suggesting that release of components from non-adherent bacteria may be involved in TNF-alpha induction. Thus, the use of cultured primary endometrial cells, together with gonococci expressing green fluorescent protein, has the potential to extend significantly our knowledge, at the molecular level, of the role of this important human pathogen in the immunobiology of pelvic inflammatory disease.

Effect of adjuvant composition on immune response to a multiple antigen peptide (MAP) containing a protective epitope from Neisseria meningitidis class 1 porin.

A variety of adjuvants with the potential for use with experimental human vaccines were used for immunisation of mice, in an attempt to augment the humoral immune response to a multiple antigen peptide (MAP) containing a protective epitope from the sero-subtype specific class 1 porin protein of Neisseria meningitidis, in tandem with a Th-cell epitope. Surface plasmon resonance showed that combinations of the immunomodulators pluronic block co-polymer, muramyl dipeptide and monophosphoryl lipid A (MPL), increased the magnitude and avidity of the immune response in comparison with both Al(OH)3 and Freund-type adjuvants. In addition, the incorporation of MPL was essential for the induction of a broad distribution of antibody isotypes. The antibodies induced recognised the native protein in meningococcal outer membranes in a subtype-specific manner. The formulations containing these multiple immunomodulators which have already been used in human phase I/II trials with experimental vaccines, are candidates for inclusion in future human vaccines based on synthetic peptides containing defined, protective epitopes.

Dynamics of carriage of Neisseria meningitidis in a group of military recruits: subtype stability and specificity of the immune response following colonization.

Meningococcal carriage and the immune response to colonization were studied in a group of military recruits undergoing basic training. Subtyping by determination of the class 1 protein sequence clearly differentiated between strains and demonstrated the dynamics of carriage and transmission. Expression of class 1 protein by each strain remained stable during prolonged carriage by different subjects. Following colonization, a marked increase in serum bactericidal response occurred, which was specific for the subtype of the acquired strain and was associated with an increase in reactivity by Western blot to the homologous class 1 protein. Subjects colonized by multiple strains showed evidence of a specific immune response to the class 1 protein of each strain acquired. The subtype specificity of the bactericidal response to meningococci and the stability of expression of the class 1 protein have important implications for the design of vaccines for prevention of serogroup B meningococcal disease.

Naturally occurring isolates of Neisseria gonorrhoeae, which display anomalous serovar properties, express PIA/PIB hybrid porins, deletions in PIB or novel PIA molecules.

The por gene of Neisseria gonorrhoeae encodes the Protein I porin responsible for serovar specificity. In this study the por genes have been sequenced from clinical isolates which exhibited anomalous serovar reactivity. One group of 'intermediate' strains differed significantly from both Protein IA and IB strains, were more closely related to IA but appeared to represent a distinct class of Protein I. Another strain was closely related to Protein IB of serovar IB-6 but contained a deletion of six amino acids in surface exposed loop 6 which removed epitopes recognized by IB specific monoclonal antibodies. The third group of strains, which reacted with both IA and IB specific monoclonal antibodies, expressed hybrid Protein I molecules containing both IA and IB epitopes. These strains appeared to originate from a double crossover between Proteins IA and IB with the amino and carboxy terminal residues homologous to IB while the surface exposed loop 6 demonstrated close homology to IA. This is the first demonstration of naturally occurring gonococci expressing a hybrid Protein IA/IB.

Construction of an epitope vector utilising the diphtheria toxin B-subunit.

An immunogenic loop within the diphtheria toxin has been deleted from the B-subunit by a modification of the inverse polymerase chain reaction (IPCR) and replaced by a unique restriction endonuclease site. An oligonucleotide encoding an identified epitope sequence from the major outer membrane protein of Neisseria meningitidis of similar size and structure to that deleted has been introduced into the restriction site. Expression of the resulting chimeric B-subunit from Escherichia coli yielded a protein that was recognised by a panel of antibodies specific for the meningococcal epitope. Initial immunisation data suggest that this protein could elicit an antibody response against both diphtheria toxin and meningococcal proteins.

Expression of Neisseria meningitidis class 1 porin as a fusion protein in Escherichia coli: the influence of liposomes and adjuvants on the production of a bactericidal immune response.

High level expression of meningococcal class 1 protein was achieved in Escherichia coli using the p-GEMEX-1 vector, in which the protein was expressed in inclusion bodies (IB), as a fusion with the bacteriophage T7 gene 10 capsid protein. The fusion protein (FP) was engineered with a factor Xa protease site between the gene 10 and class 1 protein, but treatment with the enzyme resulted in cleavage at additional sites within the class 1 protein. Since it was not possible to remove the leader protein, the intact FP provided an alternative antigen for immunization. Antisera raised to FP, solubilized from IB and incorporated into liposomes, generated a subtype-specific response which was weakly bactericidal for meningococci. In order to remove any possible effect of E. coli LPS present in IB, the FP was further purified by SDS-PAGE and incorporated into liposomes, either alone or in combination with the adjuvants monophosphoryl lipid A or muramyl dipeptide. The incorporation of adjuvants in liposomes resulted in stimulation of the overall immune response to FP, but the resulting antisera were not bactericidal. However an effective bactericidal response was obtained with the purest preparation of FP in liposomes, without any additional adjuvants, revealing that attempts to increase further the immunogenicity of such antigens must not be at the expense of interfering with optimal protein folding.

Sequence variation in class 1 outer membrane protein in Neisseria meningitidis isolated from patients with meningococcal infection and close household contacts.

The meningococcal porA gene encodes the class 1 outer membrane protein which contains the VR1 and VR2 regions responsible for sero-subtype specificity. However, sequence variations may occur within these regions which are not recognised by the currently available subtype antibodies. Since this "silent" microheterogeneity represents a potential hidden source of information, in the current study we have used porA gene sequence analysis to study strains isolated from cases of meningococcal infection and close household contacts. With each of the three subtypes studied, the index cases could be differentiated from each other by sequence variations within at least one of the VR1, VR2 and SV1 regions. In addition, although isolates from close household contacts showed a high degree of homology significant differences could be detected within some family groups. These data demonstrate that it is possible to use sequence information to differentiate between potential sources of infection which appear identical using conventional serological methods.

Effect of sialylation of lipopolysaccharide of Neisseria gonorrhoeae on recognition and complement-mediated killing by monoclonal antibodies directed against different outer-membrane antigens.

Growth of gonococci in the presence of CMP-N-acetylneuraminic acid (CMP-NANA) has previously been shown to induce resistance to the bactericidal effect of normal human serum and is accompanied by sialylation of the gonococcal lipopolysaccharide (LPS). We have used monoclonal antibodies (mAbs) to compare the effect of LPS sialylation on recognition of gonococci and complement-mediated killing by antibodies directed either against LPS or against defined epitopes on outer-membrane protein PI. Despite differences in binding to sialylated LPS on Western blots, all three mAbs directed against LPS showed considerably reduced binding to gonococci grown in the presence of CMP-NANA and a concomitant reduction in ability to promote complement-mediated killing. In contrast, mAbs directed against previously defined epitopes on a surface exposed loop of PI showed little difference in binding between sialylated and non-sialylated gonococci and promoted killing of the sialylated gonococci. Similarly a mAb directed against an epitope on a loop of the outer-membrane Rmp protein, which had previously been shown to block killing by antibodies directed against other surface antigens, also exerted a blocking effect with sialylated gonococci. Thus in the present study the continued biological effect of mAbs was correlated with the ability of the antibody to recognize surface-exposed epitopes on sialylated gonococci. Despite the presence of the sialylation which is likely to occur in vivo, it should be possible to induce complement-mediated killing by focusing the immune response to those surface-exposed epitopes which are least susceptible to the potential inhibitory effect of LPS sialylation.

Comparison of the class-1 outer membrane protein from B:15:P1.16 Neisseria meningitidis strains isolated from patients previously immunized with a serogroup B outer membrane protein vaccine in Norway.

A previous report of a large, double blind, efficacy trial of an experimental Group B meningococcal outer membrane protein vaccine carried out in Norwegian Teenagers, showed a protection rate of 57%. Previous studies had demonstrated the occurrence of mutations in the class-1 outer membrane protein which alter its immunological properties. The occurrence of new mutations might compromise the efficacy of a vaccine and explain the occurrence of any vaccine failures. The porA gene, which encodes expression of the class 1 protein, was sequenced in all isolates from vaccine failures and compared to that of the vaccinating strain H44/76 (B:15:P1.7,16). The porA DNA and deduced amino acid sequences were all identical to that of the vaccinating strain except for that of one isolate which had a sequence identical to strains previously reported in Norway and England with a 'masked P1.7' epitope. The absence of new mutations in the trial was encouraging for the further development of outer membrane protein vaccines.

Immunization with a multiple antigen peptide containing defined B- and T-cell epitopes: production of bactericidal antibodies against group B Neisseria meningitidis.

Previous analysis of the class 1 outer-membrane (OM) protein of Neisseria meningitidis has identified discrete epitopes to be potential targets for immune attack. The conformation of these epitopes is important for inducing antibodies which can react with the native protein and promote complement-mediated lysis of the meningococcus. The multiple antigen peptide (MAP) system, which consists of an oligomeric branching lysine core to which are attached dendritic arms of defined peptide antigens, confers some conformational stability and also allows for the preparation of immunogens containing both B-cell and T helper (Th)-cell epitopes. In this study, MAPs were synthesized to contain (i) the subtype P1.16b meningococcal class 1 protein B-cell epitope (B-MAP), and (ii) the P1.16b epitope in tandem with a defined Th-cell epitope, chosen from tetanus toxin (BT-MAP). The B-MAP was nonimmunogenic in animals. In contrast, incorporation of the Th-cell epitope into BT-MAP induced a strong humoral response towards the class 1 protein B-cell epitope. Antisera from immunized mice and rabbits reacted in ELISA with synthetic peptides containing the B-cell epitope, and also cross-reacted with meningococcal OMs from strains of subtype P1.16b and P1.16a. Murine and rabbit antisera showed similar reactivity and epitope specificity, but did not react with denatured class 1 protein in Western blotting, indicating the predominance of antibodies directed towards conformational epitopes. The antisera from rabbits immunized with BT-MAP promoted complement-mediated bactericidal killing not only of the homologous meningococcal subtype P1.16b strain but also of subtype P1.16a.

A comparison of human and murine monoclonal IgGs specific for the P1.7 PorA protein of Neisseria meningitidis.

Monoclonal human IgG SS269 reacts with Neisseria meningitidis expressing the P1.7 PorA protein and with linear peptides containing NGGAS, which accounts for the P1.7 specificity. Murine monoclonal antibody to P1.7 reacts with peptides containing the overlapping epitope, ASGQ. The human and murine antibodies have similar affinities. The low avidity human antibody is very inefficient at stimulating complement-mediated bactericidal killing while the high avidity murine antibody efficiently kills bacteria. However, efficient opsonophagocytosis was mediated even at low concentrations of the human antibody and in the absence of complement, suggesting that low avidity antibodies might be protective against disease.

Structural comparison and epitope analysis of outer-membrane protein PIA from strains of Neisseria gonorrhoeae with differing serovar specificities.

The sequences of the por genes, encoding outer-membrane protein PI, have been obtained from a number of strains of Neisseria gonorrhoeae that express PIA molecules with differing serovar specificities. The inferred amino acid sequences of the mature proteins each comprise 308 residues and show considerable homology, with the degree of sequence variation between PIA molecules being considerably less than seen previously with PIB, but more evenly distributed throughout the molecule. The positions of sequence variation are largely confined to the regions predicted to form one of eight surface-exposed loops, suggesting a more widespread distribution of potential antigenic diversity. The deduced amino acid sequences were used to synthesize peptides for epitope mapping experiments. Some epitopes responsible for serovar specificity or recognized by bactericidal monoclonal antibodies could be identified on the basis of their reactivity with simple linear peptides, whilst others recognized conformational epitopes. By comparison of sequence differences with mAb reactivity it was possible to identify regions that appear to contribute to such determinants, including separated regions of the molecule which together were required for the formation of the conformational epitopes. All the epitopes identified lie at or close to the apices of the predicted surface-exposed loops 1, 3, 6, or 8, focusing attention on these regions as accessible targets for immune attack.

A new variant of serosubtype P1.16 in Neisseria meningitidis from Norway, associated with increased resistance to bactericidal antibodies induced by a serogroup B outer membrane protein vaccine.

Based on differences in reaction pattern with monoclonal antibodies against the P1.16 epitope, a new variant of the class 1 protein in Neisseria meningitidis serogroup B was identified in Norway. A single amino acid deletion was revealed when the part of the gene region encoding the second variable region of the protein was sequenced. This new variant was designated P1.16c. About 5% of the B:15:P1.7,16 strains in Norway from the time period 1987-1991 were P1.16c. In a localized area in Southern Norway, 5/8 (62%) of the P1.7,16 strains were P1.16c. The P1.16b mutant, recently described in England, was not found among the Norwegian meningococcal isolates. Strains carrying the P1.16c mutation showed increased resistance to bactericidal killing, not only by P1.16-specific monoclonal antibodies, but also by the sera from individuals immunized with a vaccine based on outer membranes from a B:15:P1.7,16 strain.

Immunization with synthetic peptides containing epitopes of the class 1 outer-membrane protein of Neisseria meningitidis: production of bactericidal antibodies on immunization with a cyclic peptide.

The class 1 outer-membrane protein of Neisseria meningitidis is the target for subtype-specific, bactericidal monoclonal antibodies (mAbs). The epitopes recognized by these antibodies have been mapped previously to linear peptides corresponding to the sequences thought to be exposed at the apices of surface-exposed loops of the protein. In this work several synthetic peptides containing the subtype Pl.16b epitope have been synthetized with the aim of inducing a polyclonal immune response resembling the reactivity of the mAbs. Initially, peptides of 9 and 15 amino acid residues were synthesized and used for immunization after coupling to a carrier protein. The reactivity of the resulting antisera, with synthetic linear decapeptides, resembled that seen in previous epitope mapping experiments with the protective mAbs. However, despite the induction of antibodies having the desired specificity, the antisera reacted poorly with the native protein in outer membranes, and were non-bactericidal. A 36mer peptide, consisting of the entire surface-exposed loop 4 of the class 1 protein was then synthesized and used for immunization as (i) free peptide, (ii) peptide coupled to carrier and (iii) peptide subjected to cyclization, in an attempt to restrict it to conformations that might more closely resemble the native loop structure. In contrast to antisera raised against linear peptides, antibodies raised by immunization with the 36mer cyclic peptide, did not react with linear peptides recognized by the mAbs, but instead appeared to recognize conformational determinants. This antiserum promoted complement-mediated bactericidal killing of the homologous meningococcal strain, demonstrating the potential of synthetic peptide immunogens for inducing a protective immune response against group B meningococci.