Vaccination with meningococcal outer membrane vesicles carrying Borrelia OspA protects against experimental Lyme borreliosis.

Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines, as such a vaccine has been proven to be successful in the past. The expression of recombinant antigens in meningococcal Outer Membrane Vesicles (OMVs), with the OMV functioning both as adjuvant and delivery vehicle, greatly enhances their potential. Immunization studies in mice have shown that OMV-based vaccines can protect against various pathogens and an OMV-based meningococcal vaccine is approved and available for human use. Because of its surface localization in Borrelia and the detailed knowledge regarding its immunogenicity and structure, OspA was chosen as a suitable lipoprotein to be tested as an OMV-based vaccine against Lyme borreliosis. We have previously shown that the OMV-OspA vaccine was immunogenic in mice and here we assessed the efficacy of OMV-OspA. We generated a second-generation OMV-OspA vaccine and vaccinated C3H/HeN mice with (EDTA extracted) meningococcal OMVs expressing OspA from B. burgdorferi strain B31. The adjuvant effect of empty OMVs on recombinant OspA was tested as well. We subsequently challenged mice with a subcutaneous injection of B. burgdorferi. Average antibody end-point titers against the OspA-OMV construct were high, although lower compared to the antibodies raised against recombinant OspA. Interestingly, antibody titers between recombinant OspA adjuvanted with aluminum hydroxide and recombinant OspA with OMV as adjuvant were comparable. Finally, qPCR and culture data show that both the OspA-OMV and the vaccine based on recombinant OspA with OMV as adjuvant provided significant, yet partial protection, against Borrelia infection. OMV-based vaccines using Borrelia (lipo)proteins are an easy and feasible vaccination method protecting against B. burgdorferi infection and could be a promising strategy in humans.

Molecular and cellular signatures underlying superior immunity against Bordetella pertussis upon pulmonary vaccination.

This corrects the article DOI: 10.1038/mi.2017.81.

Molecular and cellular signatures underlying superior immunity against Bordetella pertussis upon pulmonary vaccination.

Mucosal immunity is often required for protection against respiratory pathogens but the underlying cellular and molecular mechanisms of induction remain poorly understood. Here, systems vaccinology was used to identify immune signatures after pulmonary or subcutaneous immunization of mice with pertussis outer membrane vesicles. Pulmonary immunization led to improved protection, exclusively induced mucosal immunoglobulin A (IgA) and T helper type 17 (Th17) responses, and in addition evoked elevated systemic immunoglobulin G (IgG) antibody levels, IgG-producing plasma cells, memory B cells, and Th17 cells. These adaptive responses were preceded by unique local expression of genes of the innate immune response related to Th17 (e.g., Rorc) and IgA responses (e.g., Pigr) in addition to local and systemic secretion of Th1/Th17-promoting cytokines. This comprehensive systems approach identifies the effect of the administration route on the development of mucosal immunity, its importance in protection against Bordetella pertussis, and reveals potential molecular correlates of vaccine immunity to this reemerging pathogen.

Trichinella spiralis-secreted products modulate DC functionality and expand regulatory T cells in vitro.

Helminths and their products can suppress the host immune response which may benefit parasite survival. Trichinella spiralis can establish chronic infections in a wide range of mammalian hosts including humans and mice. Here, we aim at studying the effect of T. spiralis muscle larvae excretory/secretory products (TspES) on the functionality of DC and T cell activation. We found that TspES suppress in vitro DC maturation induced by both S- and R-form lipopolysaccharide(LPS) from enterobacteria. Using different toll-like receptor (TLR) agonists, we show that the suppressive effect of TspES on DC maturation is restricted to TLR4. These helminth products also interfere with the expression of several genes related to the TLR-mediated signal transduction pathways. To investigate the effect of TspES on T cell activation, we used splenocytes derived from OVA-TCR transgenic D011.10 that were incubated with OVA and TspES-pulsed DC. Results indicate that the presence of TspES resulted in the expansion of CD4(+) CD25(+) Foxp3+ T cells. These regulatory T (Treg) cells were shown to have suppressive activity and to produce TGF-ß. Together these results suggest that T. spiralis secretion products can suppress DC maturation and induce the expansion of functional Treg cells in vitro.

Suppression of dendritic cell maturation by Trichinella spiralis excretory/secretory products.

Evidence from experimental studies indicates that during chronic infections with certain helminth species a regulatory network is induced that can down-modulate not only parasite-induced inflammation but also reduce other immunopathologies such as allergies and autoimmune diseases. The mechanisms however, and the molecules involved in this immunomodulation are unknown. Here, we focus on the effect of Trichinella spiralis excretory/secretory antigens (TspES) on the innate immune response by studying the effect of TspES on DC maturation in vitro. Bone marrow-derived DC from BALB/c mice were incubated with TspES either alone or in combination with LPS derived from two different bacteria. As indicators of DC maturation, the cytokine production (IL-1alpha, IL-6, IL-10, IL-12p70 and TNF-alpha) and the expression of various surface molecules (MHC-II, CD40, CD80 and CD86) were measured. Results indicate that while TspES alone did not change the expression of the different surface molecules or the cytokine production, it completely inhibited DC maturation induced by Escherichia coli LPS (E. coli LPS). In contrast, DC maturation induced by LPS from another bacterium, Neisseria meningitidis, was not affected by TspES. These results were confirmed using TLR4/MD2/CD14 transfected HEK 293 cells. In conclusion, T. spiralis ES antigens lead to suppression of DC maturation but this effect depends on the type of LPS used to activate these cells.

Incorporation of LpxL1, a detoxified lipopolysaccharide adjuvant, in influenza H5N1 virosomes increases vaccine immunogenicity.

The increasing number of human influenza H5N1 infections accentuates the need for the development of H5N1 vaccine candidates to prevent a potential influenza pandemic. The use of adjuvants in such vaccines can contribute significantly to antigen dose-sparing. In this study, we evaluated the capacity of the non-toxic Neisseria meningitidis lipopolysaccharide analog LpxL1 to function as an adjuvant for an influenza H5N1 virosomal vaccine. Inactivated influenza H5N1 virus (NIBRG-14) was used to construct virosomes (reconstituted virus envelopes) with LpxL1 incorporated in the virosomal membrane thus combining the influenza hemagglutinin (HA) antigen and the adjuvant in the same particle. Mice were immunized in a one- or two-dose immunization regimen with H5N1 virosomes with or without incorporated LpxL1. After a single immunization, H5N1 virosomes with incorporated LpxL1 induced significantly enhanced H5N1-specific total IgG titers as compared to non-adjuvanted virosomes but hemagglutination inhibition (HI) titers remained low. In the two-dose immunization regimen, LpxL1-modified H5N1 virosomes induced HI titers above 40 which were significantly higher than those obtained with non-adjuvanted virosomes. Incorporation of LpxL1 had little effect on virosome-induced IgG1 levels, but significantly increased IgG2a levels in both the one- and two-dose immunization regimen. Compared to non-adjuvanted virosomes, LpxL1-modified virosomes induced similar numbers of IFNgamma-producing T cells but decreased numbers of IL-4-producing T cells irrespective of the number of immunizations. We conclude that LpxL1 incorporated in H5N1 influenza virosomes has the capacity to function as a potent adjuvant particularly stimulating Th1-type immune reactions.

The mannose cap of mycobacterial lipoarabinomannan does not dominate the Mycobacterium-host interaction.

Pathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL-10 production via interaction with the mannose receptor or DC-SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M. bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose-dependent mannosyltransferase. Compared with parent strain, capless M. marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M. bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC-SIGN-transfected Raji cells, but no differences in IL-10 induction were observed. In mice, capless M. bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose-capped LAM does not dominate the Mycobacterium-host interaction.

High-level endothelial E-selectin (CD62E) cell adhesion molecule expression by a lipopolysaccharide-deficient strain of Neisseria meningitidis despite poor activation of NF-kappaB transcription factor.

Binding of host inflammatory cells to the endothelium is a critical contributor to the vascular damage characteristic of severe meningococcal disease and is regulated by endothelial cell adhesion molecules such as ICAM-1, VCAM-1 and CD62E. Intact meningococci induce far higher levels of CD62E than lipopolysaccharide (LPS) alone, whereas LPS is at least as potent as meningococci at inducing both VCAM-1 and ICAM-1 expression. This suggests that meningococci possess additional factors other than LPS present in whole bacteria that result in differential adhesion molecule expression. To investigate this possibility, we studied the capacity of an LPS-deficient isogenic strain of serogroup B Neisseria meningitidis H44/76 (lpxA-) to induce endothelial cell adhesion molecule expression and translocation of the transcription factor NF-kappaB, and compared it to both parent and unencapsulated strains of both B1940 and H44/76 and purified LPS. Although the LPS-deficient isogenic mutant of strain H44/76 was found to be a poor inducer of NF-kappaB, it induced higher levels of CD62E expression than LPS alone. These data provide evidence that intact meningococci induce a range of signals in the endothelium that are distinct from those seen with purified LPS alone and that they occur in a LPS-dependent and LPS-independent manner. These signals may explain the potent effects of N. meningitidis on CD62E expression on vascular endothelium and provide a basis for the complex endothelial dysregulation seen in meningococcal sepsis.

Functional activity of antibodies against the recombinant OpaJ protein from Neisseria meningitidis.

The opacity proteins belong to the major outer membrane proteins of the pathogenic Neisseria and are involved in adhesion and invasion. We studied the functional activity of antibodies raised against the OpaJ protein from strain H44/76. Recombinant OpaJ protein was obtained from Escherichia coli in two different ways: cytoplasmic expression in the form of inclusion bodies followed by purification and refolding and cell surface expression followed by isolation of outer membrane complexes (OMCs). Immunization with purified protein and Quillaja saponin A (QuilA) induced high levels of Opa-specific antibodies, whereas the E. coli OMC preparations generally induced lower levels of antibodies. Two chimeric Opa proteins, hybrids between OpaB and OpaJ, were generated to demonstrate that the hypervariable region 2 is immunodominant. Denatured OpaJ with QuilA induced high levels of immunoglobulin G2a (IgG2a) in addition to IgG1, whereas refolded OpaJ with QuilA induced IgG1 exclusively. These sera did not induce significant complement-mediated killing. However, all sera blocked the interaction of OpaJ-expressing bacteria to CEACAM1-transfected cells. In addition, cross-reactive blocking of OpaB-expressing bacteria to both CEACAM1- and CEA-transfected cells was found for all sera. Sera raised against purified OpaJ and against OpaJ-containing meningococcal OMCs also blocked the nonopsonic interaction of Opa-expressing meningococci with human polymorphonuclear leukocytes.

Phase variation in meningococcal lipooligosaccharide biosynthesis genes.

Neisseria meningitidis expresses a range of lipooligosaccharide (LOS) structures, comprising of at least 13 immunotypes (ITs). Meningococcal LOS is subject to phase variation of its terminal structures allowing switching between ITs, which is proposed to have functional significance in disease. The objectives of this study were to investigate the repertoire of structures that can be expressed in clinical isolates, and to examine the role of phase-variable expression of LOS genes during invasive disease. Southern blotting was used to detect the presence of LOS biosynthetic genes in two collections of meningococci, a global set of strains previously assigned to lineages of greater or lesser virulence, and a collection of local clinical isolates which included paired throat and blood isolates from individual patients. Where the phase-variable genes lgtA, lgtC or lgtG were identified, they were amplified by PCR and the homopolymeric tracts, responsible for their phase-variable expression, were sequenced. The results revealed great potential for variation between alternate LOS structures in the isolates studied, with most strains capable of expressing several alternative terminal structures. The structures predicted to be currently expressed by the genotype of the strains agreed well with conventional immunotyping. No correlation was observed between the structural repertoire and virulence of the isolate. Based on the potential for LOS phase variation in the clinical collection and observations with the paired patient isolates, our data suggest that phase variation of LOS structures is not required for translocation between distinct compartments in the host.

Outer membrane composition of a lipopolysaccharide-deficient Neisseria meningitidis mutant.

In the pathogen Neisseria meningitidis, a completely lipopolysaccharide (LPS)-deficient but viable mutant can be obtained by insertional inactivation of the lpxA gene, encoding UDP-GlcNAc acyltransferase required for the first step of lipid A biosynthesis. To study how outer membrane structure and biogenesis are affected by the absence of this normally major component, inner and outer membranes were separated and their composition analysed. The expression and assembly of integral outer membrane proteins appeared largely unaffected. However, the expression of iron limitation-inducible, cell surface-exposed lipoproteins was greatly reduced. Major changes were seen in the phospholipid composition, with a shift towards phosphatidylethanolamine and phosphatidylglycerol species containing mostly shorter chain, saturated fatty acids, one of which was unique to the LPS-deficient outer membrane. The presence of the capsular polysaccharide turned out to be essential for viability without LPS, as demonstrated by using a strain in which LPS biosynthesis could be switched on or off through a tac promoter-controlled lpxA gene. Taken together, these results can help to explain why meningococci have the unique ability to survive without LPS.

Modification of lipid A biosynthesis in Neisseria meningitidis lpxL mutants: influence on lipopolysaccharide structure, toxicity, and adjuvant activity.

Two genes homologous to lpxL and lpxM from Escherichia coli and other gram-negative bacteria, which are involved in lipid A acyloxyacylation, were identified in Neisseria meningitidis strain H44/76 and insertionally inactivated. Analysis by tandem mass spectrometry showed that one of the resulting mutants, termed lpxL1, makes lipopolysaccharide (LPS) with penta- instead of hexa-acylated lipid A, in which the secondary lauroyl chain is specifically missing from the nonreducing end of the GlcN disaccharide. Insertional inactivation of the other (lpxL2) gene was not possible in wild-type strain H44/76 expressing full-length immunotype L3 lipopolysaccharide (LPS) but could be readily achieved in a galE mutant expressing a truncated oligosaccharide chain. Structural analysis of lpxL2 mutant lipid A showed a major tetra-acylated species lacking both secondary lauroyl chains and a minor penta-acylated species. The lpxL1 mutant LPS has retained adjuvant activity similar to wild-type meningococcal LPS when used for immunization of mice in combination with LPS-deficient outer membrane complexes from N. meningitidis but has reduced toxicity as measured in a tumor necrosis factor alpha induction assay with whole bacteria. In contrast, both adjuvant activity and toxicity of the lpxL2 mutant LPS are strongly reduced. As the combination of reduced toxicity and retained adjuvant activity has not been reported before for either lpxL or lpxM mutants from other bacterial species, our results demonstrate that modification of meningococcal lipid A biosynthesis can lead to novel LPS species more suitable for inclusion in human vaccines.

Contributions of Neisseria meningitidis LPS and non-LPS to proinflammatory cytokine response.

To determine the relative contribution of lipopolysaccharide (LPS) and non-LPS components of Neisseria meningitidis to the pathogenesis of meningococcal sepsis, this study quantitatively compared cytokine induction by isolated LPS, wild-type serogroup B meningococci (strain H44/76), and LPS-deficient mutant meningococci (strain H44/76[pLAK33]). Stimulation of human peripheral-blood mononuclear cells with wild-type and LPS-deficient meningococci showed that non-LPS components of meningococci are responsible for a substantial part of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta production and virtually all interferon (IFN)-gamma production. Based on tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of LPS in proteinase K-treated lysates of N. meningitidis H44/76, a quantitative comparison was made between the cytokine-inducing capacity of isolated and purified LPS and LPS-containing meningococci. At concentrations of >10(7) bacteria/mL, intact bacteria were more potent cytokine inductors than equivalent amounts of isolated LPS, and cytokine induction by non-LPS components was additive to that by LPS. Experiments with mice showed that non-LPS components of meningococci were able to induce cytokine production and mortality. The principal conclusion is that non-LPS parts of N. meningitidis may play a role in the pathogenesis of meningococcal sepsis by inducing substantial TNF-alpha, IL-1beta, and IFN-gamma production.

Dendritic cell activation and cytokine production induced by group B Neisseria meningitidis: interleukin-12 production depends on lipopolysaccharide expression in intact bacteria.

Interactions between dendritic cells (DCs) and microbial pathogens are fundamental to the generation of innate and adaptive immune responses. Upon stimulation with bacteria or bacterial components such as lipopolysaccharide (LPS), immature DCs undergo a maturation process that involves expression of costimulatory molecules, HLA molecules, and cytokines and chemokines, thus providing critical signals for lymphocyte development and differentiation. In this study, we investigated the response of in vitro-generated human DCs to a serogroup B strain of Neisseria meningitidis compared to an isogenic mutant lpxA strain totally deficient in LPS and purified LPS from the same strain. We show that the parent strain, lpxA mutant, and meningococcal LPS all induce DC maturation as measured by increased surface expression of costimulatory molecules and HLA class I and II molecules. Both the parent and lpxA strains induced production of tumor necrosis factor alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), and IL-6 in DCs, although the parent was the more potent stimulus. In contrast, high-level IL-12 production was only seen with the parent strain. Compared to intact bacteria, purified LPS was a very poor inducer of IL-1alpha, IL-6, and TNF-alpha production and induced no detectable IL-12. Addition of exogenous LPS to the lpxA strain only partially restored cytokine production and did not restore IL-12 production. These data show that non-LPS components of N. meningitidis induce DC maturation, but that LPS in the context of the intact bacterium is required for high-level cytokine production, especially that of IL-12. These findings may be useful in assessing components of N. meningitidis as potential vaccine candidates.

Murine monoclonal antibodies to PorA of Neisseria meningitidis show reduced protective activity in vivo against B:15:P1.7,16 subtype variants in an infant rat infection model.

The major outer membrane protein PorA of Neisseria meningitidis is the target for bactericidal serosubtyping antibodies and is currently considered as a potential vaccine candidate against group B meningococcal disease. Although the minor antigenic variability of the PorA has been increasingly recognized and described, its implication for vaccine design remains unclear. In this study, the protective activity of murine monoclonal PorA specific antibodies against four isogenic meningococcal P1.7,16 target strains, the prototype P1.7,16a and three loop 4 point mutation variants (designated P1.7,16b to d) constructed from reference strain H44/76 (B:15:P1.7,16a), was evaluated in the infant rat infection model. All monoclonal antibodies had been obtained by immunization of mice with outer membrane protein preparations from meningococcal serosubtype P1.7,16 reference strain H44/76. A challenge dose of 10(5)cfu/pup was given i.p. 1-2 h after the i.p. injection of 1:100 diluted antibodies, and the development of bacteremia was assessed by culturing blood samples taken 6 h after challenge. MN14C11.6, a reference monoclonal antibody for serosubtype P1.7 epitope located in predicted loop 1 (VR1) identical in all the variants, was equally protective against all loop 4 variants. The three P1.16 specific monoclonal antibodies tested (MN5C11G, MN12H2 and 62D12-8) all completely protected animals against the prototype P1.7,16a, variably against the P1.7,16b and P1.7,16c, but not against the P1.7,16d variant. Our findings therefore suggest that certain subtype variants may escape protection in vivo conferred by PorA specific antibodies.

Construction of porA Mutants.

The PorA or class 1 protein is one of the major meningococcal outermembrane proteins (OMPs). It is one of the two porins found in this organism, the other one being the PorB or class 2/3 protein. It folds into a 16-stranded ß-barrel structure, which is now well-established for bacterial porins, in which seven loops are exposed at the cell surface and the remaining one forms the constriction of the pore (1,2). There are approx 20 different serosubtypes of PorA (3), based on sequence variability in the longest surface-exposed loops 1 and 4 (see Fig. 1). In addition, minor sequence variations within individual subtypes have been observed. As a result, some subtypes such as P1.10 actually constitute a family of variants differing by single amino acid substitutions, which may affect antibody recognition; for other subtypes such as P1.4 the number of variants is more limited (4,5). Several studies with experimental outer membrane-derived vaccines have shown that PorA is a major inducer of bactericidal antibodies (6-8), making it a crucial component of any meningococcal vaccine. These antibodies are highly subtype-specific. Epidemic strains tend to be clonal and mainly express a single PorA subtype that changes only slowly over time (9). In hyperendemic situations, more variation is found but it is generally still possible to select a limited number of PorA subtypes that will cover most of the strains (10). However, PorA variation in both time and geography means that it is unlikely that a universal once-and-for-all meningococcal vaccine based on this protein alone can ever be made. This necessitates the use of vaccine strains with flexible PorA composition, in which new variants can be inserted into established production strains when required by new epidemiological circumstances. This chapter will describe methods to construct isogenic meningococcal strains with altered porA genes, which can be used both for vaccine production and as test strains to determine the precise epitope specificity of bactericidal antibodies directed against the various loops of PorA. Fig. 1. Topology model for PorA protein. Residues shown in boldface represent the surface-exposed P1.5 and P1.2 epitopes in loop 1 and 4. Residues marked with an asterisk represent the points of insertion into the KpnI site in loop 5 or 6.

Construction of LPS mutants.

Lipopolysaccharide (LPS) is a major component of the meningococcal outer membrane. It consists of a hexa-acylated glucosamine disaccharide substituted at both ends with diphosphoethanolamine, to which an oligosaccharide chain of up to 10 sugar residues is attached (1,2). It lacks a long repeating O-antigen side chain, as is typically found in many Enterobacteriaceae, and is therefore also sometimes referred to as lipooligosaccharide or LOS. The oligosaccharide part shows structural variation among strains, which forms the basis for division into the different immunotypes L1 to L12 (3). In addition, individual strains can vary their LPS structure through high-frequency phase variation of several genes encoding glycosyltransferases (4). This can affect virulence-related properties such as invasion of host cells and serum resistance (5). In the context of vaccine development, meningococcal LPS is relevant in several ways. First, the cell surface-exposed oligosaccharide part may contain epitopes recognized by bactericidal or otherwise protective antibodies; however, the presence of host-identical structures such as the terminal lacto-N-neotetraose means that the possibility of inducing autoimmune pathology should also be considered (6). Second, the membrane-anchoring lipid A part has strong endotoxin activity, by inducing the synthesis of proinflammatory cytokines in a variety of host cells (7). This plays a major role in the pathological manifestations of meningococcal sepsis, and is also responsible for most of the reactogenicity found with outer membrane vesicle (OMV)-based vaccines.

Gram-negative bacteria induce proinflammatory cytokine production by monocytes in the absence of lipopolysaccharide (LPS).

Tumour necrosis factor-alpha (TNF-alpha), IL-1alpha and IL-6 production by human monocytes in response to a clinical strain of the Gram-negative encapsulated bacteria Neisseria meningitidis and an isogenic lpxA- strain deficient in LPS was investigated. Wild-type N. meningitidis at concentrations between 105 and 108 organisms/ml and purified LPS induced proinflammatory cytokine production. High levels of these cytokines were also produced in response to the lpxA- strain at 107 and 108 organisms/ml. The specific LPS antagonist bactericidal/permeability-increasing protein (rBPI21) inhibited cytokine production induced by LPS and wild-type bacteria at 105 organisms/ml but not at higher concentrations, and not by LPS-deficient bacteria at any concentration. These data show that proinflammatory cytokine production by monocytes in response to N. meningitidis does not require the presence of LPS. Therapeutic strategies designed to block LPS alone may not therefore be sufficient for interrupting the inflammatory response in severe meningococcal disease.

Effect of sequence variation in meningococcal PorA outer membrane protein on the effectiveness of a hexavalent PorA outer membrane vesicle vaccine.

Though meningococcal serogroup C conjugate vaccines have been introduced into the UK infant immunisation schedule, there is currently no vaccine solution for serogroup B disease. PorA outer membrane protein (OMP) is a potential serogroup B vaccine candidate. A hexavalent PorA outer membrane vesicle (OMV) vaccine has been evaluated in phase I and II trials with promising results. This vaccine contains six different PorA OMPs each representing a different serosubtype. However, considerable sequence variation occurs in the variable regions (VRs) encoding these serosubtypes. By using recombinant P1.5,10 PorA variants we have demonstrated that the killing of this particular serosubtype combination was due mainly to the induction of antibody to the VR2 (P1.10) epitope region, and that after three or four doses of vaccine there was a significant reduction in the killing of variants P1.10a (three doses, p<0.0001; four doses, p = 0.003) and P1.10f (three doses, p<0.0001; four doses, p = 0.002), as compared to responses to the P1.10 strain, when the P1.10 serosubtype was used as the immunogen. Since large numbers of serosubtype variants are known to exist, this finding may have implications for the use of PorA as a meningococcal serogroup B vaccine.

Immunogenicity of in vitro folded outer membrane protein PorA of Neisseria meningitidis.

In vitro folded and the denatured form of PorA P1.6 from Neisseria meningitidis strain M990 were used for immunization studies in mice. Previously, the antigen was isolated from cytoplasmic inclusion bodies, folded and purified. Its immunogenicity without adjuvant appeared to be low. The addition of the adjuvant QuilA, but not of galE lipooligosaccharide, considerably enhanced the immunogenicity. Moreover, when immunized with folded PorA P1.6 plus QuilA, a clear switch towards the IgG2a subclass of antibodies and concomitantly, the appearance of serum bactericidal activity, which is believed to be important for protective immunity, was observed. Hence, a tool for preparing vaccines against serogroup B meningococci devoid of endotoxin is available.