Characterization and specificity of antibodies to protein I of Neisseria gonorrhoeae produced by injection with various protein I-adjuvant preparations.

A major goal of gonococcal research is the development of a gonorrheal vaccine. A vaccine candidate is the major outer membrane protein (PI) of the gonococcus, which has limited antigenic variability. Two main subtypes, PIA and PIB, and nine main serotypes have been described. To avoid raising anti-protein III (PIII)-blocking antibodies and limit potential lipooligosaccharide toxicity, PI was chromatographically isolated with minimal PIII contamination (less than 1%) from Pgh 3-2 (PIB), a serum-sensitive gonococcal strain and UU1 (PIA), a serum-resistant gonococcal strain. Alum was used as an adjuvant and the antibodies raised in rabbits did not agglutinate the organisms, were not opsonic, and bactericidal titers were not increased. To present PI in a form mimicking its in vivo disposition, it was inserted into liposomes. The resulting antisera did agglutinate the organism and contained opsonic and bactericidal activity greater than the preimmune sera or alum-generated sera. The PIB liposome antisera also had higher ELISA titers to a synthetic peptide equivalent to an exposed portion of PIB and a higher percentage of antibodies absorbed by whole organisms than the PIB alum antisera. We speculate that when PI is presented in liposomes, the antibodies raised are mainly to surface-exposed epitopes of the protein as opposed to when PI is presented absorbed to alum, where the antibodies are produced mainly to buried epitopes.

A surface receptor specific for human IgA on group B streptococci possessing the Ibc protein antigen.

A number of group B streptococcal strains of various serotypes, Ia, Ib, Ic, II, and III were examined for their ability to bind human IgG and IgA. No strains of group B streptococci were found to bind IgG, but many strains possessing the Ibc protein antigen(s) were found to bind a significant amount of IgA. The extent of IgA binding correlated with the amount of a 130,000 mol wt, detergent-extractable protein, and reactivity with the Ic typing sera. Using nitrocellulose blots, it was found that the 130,000 mol wt protein bound human IgA. A method was developed to purify the protein while retaining its ability to bind human IgA. Using solid phase radioimmunoassays, it was determined that the protein bound to the Fc region of monomeric or polymeric IgA and that it failed to bind IgM or any IgG isotype.

Successful recovery of the normal electrophysiological properties of PorB (class 3) porin from Neisseria meningitidis after expression in Escherichia coli and renaturation.

Neisseria meningitidis PorB class 3 porins obtained either from native membranes (wild-type) or recovered from inclusion bodies following expression in Escherichia coli (recombinant), have been reconstituted into solvent-free planar phospholipid membranes. The wild-type and recombinant porins exhibited the same single-trimer conductance (1-1.3 nS in 200 mM NaCl), tri-level closure pattern, characteristic of functional channel trimers, and pattern of insertion into planar membranes. Both proteins were open at low voltages and displayed two voltage-dependent closure processes, one at positive and the other at negative potentials. Both showed asymmetric voltage dependence such that one gating process occurred at lower voltages (Vo=15 mV) than the other (Vo=25 mV). The sign of the potential that resulted in closure at low voltages varied from membrane to membrane indicating that they may have the property of auto-directed insertion (in analogy to the mitochondrial channel, VDAC). In the case of the recombinant porin, the steepness of the voltage dependence of one gating process was slightly less (n=1.3) than that observed for the other process or for the wild-type channel (n=1.5-1.7). Both channels have a high (40%) probability of closure even at 0 mV. While both channels show a slight selectivity for Cl- over Na+, the selectivity of the recombinant porin is a bit higher (permeability ratio of 2.8 vs. 1.6) as measured using a 2-fold salt gradient. Thus, the method employed to refold the recombinant porin was successful in not only restoring wild-type structure [H.L. Qi, J.Y. Tai, M.S. Blake, Expression of large amounts of Neisserial porin proteins in Escherichia coli and refolding of the proteins into native trimers, Infect. Immun. 62 (1994) 2432-2439; C.A.S.A. Minetti, J.Y. Tai, M.S. Blake, J.K. Pullen, S.M. Liang, D.P. Remeta, Structural and functional characterization of a recombinant PorB class 2 protein from Neisseria meningitidis. Conformational stability and porin activity, J. Biol. Chem. 272 (1997) 10710-10720] but also the overall electrophysiological function.

Biophysical and antigenic characterization of gonococcal protein I incorporated into liposomes.

The major gonococcal outer membrane protein, protein I (Por), was reconstituted into liposomes composed of either 1-palmitoyl, 2-oleoyl phosphatidylcholine (POPC) or POPC:1-palmitoyl, 2-oleoyl phosphatidylethanolamine (POPE) (1:1 weight ratio) and the resulting proteoliposomes characterized with respect to their biophysical and antigenic properties. Isopycnic density gradient centrifugation studies established that essentially all of the protein was reconstituted into the lipid bilayer with no significant differences in incorporation seen as a function of lipid composition. Examination of Por orientation in these proteoliposomes revealed that over 80% of the protein was oriented facing outwards in the same 'hairpin loop' fashion found in the native bacterial membrane. Reconstituted Por proteoliposomes exhibited a mean vesicle diameter of > 0.5 micron but could be reduced by extrusion without significant loss of protein or lipid. These extruded systems were suitable for sterilization by terminal filtration. The antibody binding activities of various Por liposome formulations were determined using both anti-Por monoclonal antibodies and an immunized rabbit sera. No significant differences in antibody binding were observed as a function of proteoliposome lipid composition. However, consistently higher levels of antibody binding were obtained for Por liposomes prepared in this way compared with reconstituted systems prepared as described in earlier publications.

Characterization of the structure, function, and conformational stability of PorB class 3 protein from Neisseria meningitidis. A porin with unusual physicochemical properties.

PorB proteins constitute the vast majority of channels in neisserial outer membranes and can be subdivided within meningococcal strains into two distinct and mutually exclusive families that are designated as class 2 and class 3 proteins. We recently characterized the functional activity and conformational stability of a PorB class 2 protein from Neisseria meningitidis (Minetti, C. A. S. A., Tai, J. Y., Blake, M. S., Pullen, J. K., Liang, S. M., and Remeta, D. P. (1997) J. Biol. Chem. 272, 10710-10720). To evaluate the structure-function relatedness among the PorB proteins, we have employed a combination of electrophoretic and spectroscopic techniques to assess the conformational stability of zwittergent-solubilized class 3 trimers. The functional, physicochemical, and structural properties of the meningococcal class 2 and class 3 proteins are comparable with the notable exception that the latter exhibits a significantly higher susceptibility to SDS. The SDS-induced dissociation and partial unfolding of PorB class 3 is characterized by a single two-state transition with a midpoint at 0.35% SDS. The native trimeric assembly dissociates reversibly, forming partially folded monomers that retain the characteristic beta-sheet content of the transmembrane domain with a concomitant increase in random coil structure arising from unfolding the rigid surface loops. These results provide new insight into the elucidation of porin folding pathways and the factors that govern the overall structural stability of meningococcal proteins.

A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots.

A rapid, sensitive method has been developed to detect antibody-antigen complexes on "Western blots." The methods of H. Towbin, T. Staehlin, and J. Gordon were used to separate and blot the antigens onto nitrocellulose. The remaining sites of attachment were blocked and the nitrocellulose was washed with polyoxyethylenesorbitan monolaurate (Tween 20). The blot was then reacted with the antiserum or hybridoma supernate to be tested. After the antigen-antibody reaction was completed, the blot was washed and treated with anti-antibody which had been conjugated to alkaline phosphatase. The alkaline phosphatase was detected by the reduction of the tetrazolium salt to diformazan by the hydrogen ions released in the formation of indigo by the reaction of the phosphatase on the indoxyl phosphate. The advantages of this method over previously described techniques are (1) use of Tween 20 allows the blot to be stained with Coomassie blue, (2) the substrates of the alkaline phosphatase reaction are stable for long periods of time, (3) the reaction products form an intense blue color which does not fade, (4) the resolution is extremely good with little to no band broadening, (5) the reaction is sensitive to picogram quantities of antigen, and (6) the reaction is quantitative.

Meningococcal PorA/C1, a channel that combines high conductance and high selectivity.

Class 1 porins (PorA/C1) from Neisseria meningitidis achieve both high selectivity and high conductance. The channel is highly selective (24:1 Na+ over Cl-), suggesting a highly negatively charged selectivity filter. The trimeric nature of PorA/C1 accounts for part of the enormous conductance in 200 mM NaCl (0.97nS). However, the currents that can be achieved exceed the simple infinite-sink calculation for a pore 0.7 nm in radius (estimated from nonelectrolyte permeability). The conductance is linear with salt activity from 20 mM to 2.0 M NaCl with no sign of saturation at low salt. Impermeant polymers reduce the conductance in a manner consistent with their ability to reduce bulk conductivity. Extrapolating from the known structure of homologous porins, the selectivity filter is likely to be small and localized. If small and highly negatively charged ( approximately 9 charges), the predicted conductance would be an order of magnitude higher than that observed. The rate at which ions reach the selectivity filter seems to limit overall ionic flux. PorA/C1 rectifies strongly, and this rectification can be accounted for by calculated differences in the voltage and concentration profiles in the access regions. Thus, it appears that the conductance of this channel is determined by the access resistance and the selectivity by a highly-conductive filter.

Group A streptococcus-liposome ELISA antibody titers to group A polysaccharide and opsonophagocytic capabilities of the antibodies.

Antibodies reactive with group A streptococci (GAS) carbohydrate were studied by ELISA and in an indirect bactericidal assay. The ELISA used GAS carbohydrate covalently bound to phosphatidylethanolamine incorporated into liposomes so that both precipitating and nonprecipitating antibodies were measured. Sera from children from different geographic areas exhibited marked differences in levels of anti-GAS carbohydrate antibody, which increased with age. The antibodies were predominantly of IgG. In bactericidal assays, most of these sera promoted phagocytosis of several type-specific M-positive strains. Opsonization was also related to serum levels of anti-GAS carbohydrate antibodies. These opsonizing antibodies were depleted from the serum by absorption of the sera on an N-acetyl-D-glucosamine affinity column. Antibody eluted from this column could partially restore opsonization of GAS. Anti-GAS carbohydrate antibodies play a major role in these opsonophagocytosis assays.

Gonococcal porin vaccine evaluation: comparison of Por proteosomes, liposomes, and blebs isolated from rmp deletion mutants.

The gonococcal major outer membrane protein, Por (protein I), is a potential vaccine candidate. A large-scale animal trial compared the immunogenicity of Por inserted in liposomes (mimicking its in vivo structure) with previously used vaccines containing Por. Por was purified from Rmp (protein III)-negative gonococcal mutants and made into five different formulations: proteosomes, proteosomes absorbed to alum, liposomes, gonococcal membrane blebs, and gonococcal membrane blebs absorbed to alum. Por liposomes induced the greatest amount of Por antibodies; proteosomes and both preparations of blebs induced minimal amounts of Por antibodies. Proteosomes absorbed to alum induced slightly lower amounts of Por antibodies than did liposomes, especially when protein IA was used. Whole-organism absorption studies revealed that a significantly greater percentage of liposome-induced Por antibodies recognized exposed portions of the protein than did proteosome- or proteosome/alum-induced antibodies.

GD3/proteosome vaccines induce consistent IgM antibodies against the ganglioside GD3.

The gangliosides of melanoma and other tumours of neuroectodermal origin are suitable targets for immune intervention with tumour vaccines. The optimal vaccines in current use contain ganglioside plus bacillus Calmette-Guérin and induce considerable morbidity. We have screened a variety of new adjuvants in the mouse, and describe one antigen-delivery system, proteosomes, which is especially effective. Highly hydrophobic Neisserial outer membrane proteins (OMP) form multimolecular liposome-like vesicular structures termed proteosomes which can readily incorporate amphiphilic molecules such as GD3 ganglioside. The optimal GD3/proteosome vaccine formulation for induction of GD3 antibodies in the mouse is determined. Interestingly, the use of potent immunological adjuvants in addition to proteosomes augments the IgM and IgG antibody titres against OMP in these vaccines but GD3 antibody titres are unaffected. The application of proteosomes to enhance the immune response to GD3 extends the concept of the proteosome immunopotentiating system from lipopeptides to amphipathic carbohydrate epitopes such as cell-surface gangliosides. The demonstrated safety of meningococcal OMP in humans and the data in mice presented here suggest that proteosome vaccines have potential for augmenting the immunogenicity of amphipathic tumour antigens in humans.