Polarity-variable birefringence on hyperlens structure.

A variable birefringence effect has been observed with 1D PMMA surface gratings on a gold film substrate. By changing the operation wavelength on the Au film, the birefringence value Δn(eff) changes from positive to negative. The result verified that this uniaxial crystal-like plasmonic surface gratings showed good superlensing effect at 515 nm when PMMA width:Air width=1:1 where the absolute value of Δn(eff) is relatively large.

Structural and functional characterization of a recombinant PorB class 2 protein from Neisseria meningitidis. Conformational stability and porin activity.

An outer membrane PorB class 2 protein from Neisseria meningitidis has been overexpressed in Escherichia coli, isolated from inclusion bodies, and refolded in the presence of zwitterionic detergent. The purified recombinant and native (strain M986) counterpart exhibit most of the typical functional and structural properties that are characteristic of bacterial porins. Channel forming activity has been monitored by incorporating class 2 into reconstituted liposomes and measuring the permeation rates of various oligosaccharides through the proteoliposomes to derive a pore diameter of approximately 1.6 nm. Structural studies employing a combination of spectroscopic and electrophoretic techniques reveal that recombinant and native class 2 are identical in terms of overall conformational stability. Both proteins form stable trimers in zwitterionic detergent and retain significant secondary and tertiary structure in the presence of SDS. The thermal unfolding of zwittergen-solubilized class 2 trimers (Tm = 88 degrees C) is reversible and characterized by solvent exposure of aromatic residues with concomitant disruption of tertiary and partial loss of secondary structures. SDS-induced destabilization and irreversible unfolding of the native trimeric assembly occurs at temperatures above 60 degrees C. Our physicochemical studies of PorB class 2 protein furnish significant insight regarding the structural and functional properties of this meningococcal outer membrane protein within the porin superfamily.

Preclinical evaluation of group B Neisseria meningitidis and Escherichia coli K92 capsular polysaccharide-protein conjugate vaccines in juvenile rhesus monkeys.

We reported the first use of group B meningococcal conjugate vaccines in a nonhuman primate model (S. J. N. Devi, C. E. Frasch, W. Zollinger, and P. J. Snoy, p. 427-429, in J. S. Evans, S. E. Yost, M. C. J. Maiden, and I. M. Feavers, ed., Proceedings of the Ninth International Pathogenic Neisseria Conference, 1994). Three different group B Neisseria meningitidis capsular polysaccharide (B PS)-protein conjugate vaccines and an Escherichia coli K92 capsular polysaccharide-tetanus toxoid (K92-TT) conjugate vaccine are here evaluated for safety and relative immunogenicities in juvenile rhesus monkeys with or without adjuvants. Monkeys were immunized intramuscularly with either B PS-cross-reactive material 197 conjugate, B PS-outer membrane vesicle (B-OMV) conjugate, or N-propionylated B PS-outer membrane protein 3 (N-pr. B-OMP3) conjugate vaccine with or without adjuvants at weeks 0, 6, and 14. A control group of monkeys received one injection of the purified B PS alone, and another group received three injections of B PS noncovalently complexed with OMV. Antibody responses as measured by enzyme-linked immunosorbent assay varied among individual monkeys. All vaccines except B PS and the K92-TT conjugate elicited a twofold or greater increase in total B PS antibodies after one immunization. All vaccines, including the K92-TT conjugate, elicited a rise in geometric mean B PS antibody levels of ninefold or more over the preimmune levels following the third immunization. Antibodies elicited by N-pr. B-OMP3 and B-OMV conjugates were directed to the N-propionylated or to the spacer-containing B PS antigens as well as to the native B PS complexed with methylated human serum albumin. None of the vaccines caused discernible safety-related symptoms.

Standardization and a multilaboratory comparison of Neisseria meningitidis serogroup A and C serum bactericidal assays. The Multilaboratory Study Group.

A standardized serum bactericidal assay (SBA) is required to evaluate the functional activity of antibody produced in response to Neisseria meningitidis serogroup A and C vaccines. We evaluated assay parameters (assay buffer, target strains, growth of target cells, target cell number, complement source and concentration, and methods for growth of surviving bacteria) which may affect the reproducibility of SBA titers. The various assay parameters and specificity of anticapsular antibody to five serogroup A strains (A1, ATCC 13077, F8238, F9205, and F7485) and four serogroup C strains (C11, G7880, G8050, and 1002-90) were evaluated with Centers for Disease Control and Prevention meningococcal quality control sera. The critical assay parameters for the reproducible measurement of SBA titers were found to include the target strain, assay incubation time, and complement. The resulting standardized SBA was used by 10 laboratories to measure functional anticapsular antibody against serogroup A strains F8238 and serogroup C strain C11. In the multilaboratory study, SBA titers were measured in duplicate for 14 pairs of sera (seven adults and seven children) before and after immunization with a quadrivalent polysaccharide (A, C, Y, and W-135) vaccine. The standardized SBA was reliable in all laboratories regardless of experience in performing SBAs. For most sera, intralaboratory reproducibility was +/- 1 dilution; interlaboratory reproducibility was +/- 2 dilutions. The correlation between median titers (interlaboratory) and enzyme-linked immunosorbent assay total antibody concentrations was high for both serogroup A (r = 0.86; P < 0.001; slope = 0.5) and serogroup C (n = 0.86; P < 0.001; slope = 0.7). The specified assay, which includes the critical parameters of target strain, incubation time, and complement source, will facilitate interlaboratory comparisons of the functional antibody produced in response to current or developing serogroup A and C meningococcal vaccines.

Preclinical evaluation of a novel group B meningococcal conjugate vaccine that elicits bactericidal activity in both mice and nonhuman primates.

Group B meningococcal (GBM) conjugate vaccines were prepared using chemically modified N-propionylated polysialic acid, from Escherichia coli K1 polysaccharide capsule, coupled by reductive amination to tetanus toxoid and purified recombinant GBM porin (rPorB). All conjugates elicited high antibody levels in mice with good booster responses. However, only rPorB conjugates elicited bactericidal activity specific against a broad spectrum of five different GBM serotypes. Bactericial activity was completely inhibited by free N-propionylated polysaccharide. In baboons and rhesus monkeys, rPorB conjugates elicited high antibody titers, with IgG booster responses 9- to 15-fold higher than primary responses. Bactericial activity increased 19- to 39-fold over preimmune values, using rabbit complement; increased bactericial activity was also confirmed with human and monkey complement. IgG cross-reactivity for unmodified N-acetyl polysaccharide was <5% for 79% of mice and <10% for 80% of primates. These studies strongly suggest that the N-propionylated polysialic acid-rPorB conjugate is an excellent vaccine candidate for human use.

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.

Production of Haemophilus influenzae type-b porin in Escherichia coli and its folding into the trimeric form.

The P2 protein from pathogenic Haemophilus influenzae type b (Hib) functions as a bacterial porin and is one of several immunogenic outer membrane proteins. The P2 gene was expressed in Escherichia coli and the recombinant P2 protein (re-P2) purified to facilitate functional and immunologic studies. P2 was obtained from Hib strain Eagan using PCR and the pET vectors (17b and 11a) were used to produce re-P2 at levels exceeding 30% of the total E. coli proteins. Since previous reports had indicated that P2 was toxic to E. coli, steps were taken to control the toxicity. The plasmid was stabilized by tightly controlling the synthesis of re-P2 prior to induction. Subsequent to induction, re-P2 was sequestered into inclusion bodies rather than to membrane compartments. The refolding of the denatured re-P2 into the trimeric form involved high salt and calcium ions. re-P2 was then purified to homogeneity using gel-filtration and ion-exchange chromatography.

Maternal immunization of mice with group B streptococcal type III polysaccharide-beta C protein conjugate elicits protective antibody to multiple serotypes.

Group B streptococcal infection is a major cause of neonatal mortality. Antibody to the capsular polysaccharide protects against invasive neonatal disease, but immunization with capsular polysaccharides fails to elicit protective antibody in many recipients. Conjugation of the polysaccharide to tetanus toxoid has been shown to increase immune response to the polysaccharide. In animal models, C proteins of group B streptococci are also protective determinants. We examined the ability of the beta C protein to serve in the dual role of carrier for the polysaccharide and protective immunogen. Type III polysaccharide was covalently coupled to beta C protein by reductive amination. Immunization of rabbits with the polysaccharide-protein conjugate elicited high titers of antibody to both components, and the serum induced opsonophagocytic killing of type III, Ia/C, and Ib/C strains of group B streptococci. Female mice were immunized with the conjugate vaccine and then bred; 93% of neonatal pups born to these dams vaccinated with conjugate survived type III group B streptococcal challenge and 76% survived type Ia/C challenge, compared with 3% and 8% survival, respectively, in controls (P < 0.001). The beta C protein acted as an effective carrier for the type III polysaccharide while simultaneously induced protective immunity against beta C protein--containing strains of group B streptococci.

Expression of large amounts of neisserial porin proteins in Escherichia coli and refolding of the proteins into native trimers.

Porins from different neisserial strains and species have been shown to have differences in both primary amino acid sequence and biophysical characteristics as observed by functional assays. A closer examination of how the changes in the primary amino acid sequence of Neisseria porin molecules correlate with these observed biophysical changes has been impeded by the inability to easily manipulate the cloned porin genes by modern molecular techniques and then obtain enough of the expressed modified porin protein to purify and use in these biophysical functional assays. In this report, we describe a method by which the genes encoding three different porin proteins, lacking their neisserial promoter and signal sequences, were cloned into an expression plasmid and transformed into Escherichia coli. Upon induction, large amounts of the porin proteins were produced. The expressed porin proteins were then manipulated to regenerate their native trimer structure and purified by standard protein chemistry. Sufficient purified recombinant porin protein was obtained for further antigenic as well as biophysical characterization. This sets the stage for the biophysical characterization of these neisserial porin proteins in more detail.

Substitution of lysine for arginine at position 42 of human transforming growth factor-alpha eliminates biological activity without changing internal disulfide bonds.

Transforming growth factor-alpha (TGF-alpha) is a growth-promoting protein that binds to the epidermal growth factor (EGF) receptor. To identify critical residues that govern TGF-alpha-EGF receptor binding, we prepared site-specific substitution mutants of TGF-alpha. Mutant proteins were tested in receptor-binding and mitogenesis assays. Semiconservative substitutions at positions 4, 12, 18, and 45 decreased biological activity 2.1- to 14-fold. The conservative substitution of lysine for arginine at position 42 completely eliminated biological activity. Amino acid composition analysis of proteolytic fragments from TGF-alpha and the Lys-42 mutant indicated that these proteins contained the same disulfide bonds. These studies suggest that arginine 42 may be a contact point for TGF-alpha-EGF receptor interaction.

Epidermal growth factor receptor binding is affected by structural determinants in the toxin domain of transforming growth factor-alpha-Pseudomonas exotoxin fusion proteins.

TGF-alpha-PE40 is a hybrid protein composed of transforming growth factor-alpha (TGF-alpha) fused to a 40,000-dalton segment of Pseudomonas exotoxin A (PE40). This hybrid protein possesses the receptor-binding activity of TGF-alpha and the cell-killing properties of PE40. These properties enable TGF-alpha-PE40 to bind to and kill tumor cells that possess epidermal growth factor (EGF) receptors. Unexpectedly, TGF-alpha-PE40 binds approximately 100-fold less effectively to EGF receptors than does native TGF-alpha (receptor-binding inhibition IC50 = 540 and 5.5 nM, respectively). To understand the factors governing receptor binding, deletions and site-specific substitutions were introduced into the PE40 domain of TGF-alpha-PE40. Removal of the N-terminal 59 or 130 amino acids from the PE40 domain of TGF-alpha-PE40 improved receptor binding (IC50 = 340 and 180 nM, respectively) but decreased cell-killing activity. Substitution of alanines for cysteines at positions 265 and 287 within the PE40 domain dramatically improved receptor binding (IC50 = 37 nM) but also decreased cell-killing activity. Similar substitutions of alanines for cysteines at positions 372 and 379 within the PE40 domain did not significantly affect receptor-binding or cell-killing activities. These studies indicate that the PE40 domain of TGF-alpha-PE40 interferes with EGF receptor binding. The cysteine residues at positions 265 and 287 of PE40 are responsible for a major part of this interference.

Structure-function analysis of synthetic and recombinant derivatives of transforming growth factor alpha.

Transforming growth factor alpha (TGF-alpha) is a 50-amino-acid peptide that stimulates cell proliferation via binding to cell surface receptors. To identify the structural features of TGF-alpha that govern receptor-ligand interactions, we prepared synthetic peptide fragments and recombinant mutant proteins of TGF-alpha. These TGF-alpha derivatives were tested in receptor binding and mitogenesis assays. Synthetic peptides representing the N terminus, the C terminus, or the individual disulfide constrained rings of TGF-alpha did not exhibit receptor-binding or mitogenic activity. Replacement of the cysteines with alanines at positions 8 and 21, 16 and 32, and 34 and 43 or at positions 8 and 21 and 34 and 43 yielded inactive mutant proteins. However, mutant proteins containing substitutions or deletions in the N-terminal region retained significant biologic activity. Conservative amino acid changes at residue 29 or 38 or both and a nonconservative amino acid change at residue 12 had little effect on binding or mitogenesis. However, nonconservative amino acid changes at residues 15, 38, and 47 produced dramatic decreases in receptor binding (23- to 71-fold) and mitogenic activity (38- to 125-fold). These studies indicate that at least three distinct regions of TGF-alpha contribute to biologic activity.

Haemophilus influenzae type b polysaccharide-protein conjugate vaccine.

An Haemophilus influenzae type b capsular polysaccharide-protein conjugate has been prepared. The polysaccharide was coupled to the serotype II protein of group B meningococcus through the spacer 6-aminocaproic acid using cyanogen bromide and water soluble carbodiimide. The conjugate can be shown to be reproducible and is stable and highly immunogenic in mice and African green monkeys. Clinical evaluation of this conjugate in children 3 months to 4 years of age showed that it elicited an antibody titer to the polysaccharide moiety greater than 1000 ng/ml in children 8 months of age or older.

Molecular cloning of the temperature-sensitive 371 Kirsten murine sarcoma virus and expression in Escherichia coli of the mutant and wild-type viral Kirsten ras p21 proteins.

Rodent fibroblasts infected with the ts371 Kirsten murine sarcoma virus (KiMuSV) are temperature sensitive for the maintenance of transformation because of the production of an abnormal p21 protein. We cloned the ts371 KiMuSV provirus from the genome of a conditionally transformed nonproducer cell line, ts371 KiMuSV NRK clone 5 (T. Y. Shih, M. O. Weeks, H. A. Young, and E. M. Scolnick, J. Virol. 31:546-556, 1979). The molecularly cloned virus had 1,000-fold lower transformed focus-forming activity at 39 degrees C than at 34 degrees C. The ts371-v-Ki-ras gene differed from the wild type (wt) by a single point mutation, resulting in the substitution of arginine for glutamine at amino acid residue 43 of the encoded p21. A second difference from the published sequence for wt v-Ki-ras (N. Tsuchida, T. Ryder, and E. Ohtsubo, Science 217:937-939, 1982) at amino acid residue 37 was found. However, on sequencing the wt v-Ki-ras in this region, we found that it also contained a glutamate at residue 37. Preliminary characterization of bacterially expressed wt and ts371-v-Ki-ras p21 proteins is discussed.

Gonococcal pili. Primary structure and receptor binding domain.

The complete amino acid sequence of pilin from gonococcal strain MS11 and the sequence of constant and variable regions from strain R10 pilin have been determined in order to elucidate the structural basis for adherence function, antigenic diversity, and polymeric structure. The MS11 pilin sequence consists of 159 amino acids in a single polypeptide chain with two cysteines in disulfide linkage and serine-bonded phosphate residues. TC-2 (31-111), a soluble monomeric pilus peptide prepared by arginine-specific digestion, bound human endocervical, but not buccal or HeLa cells and therefore is postulated to encompass the receptor binding domain. Variable regions of CNBr-3 appear to confer antigenic diversity and comprise segments in which changes in the position of charged residues occur in hydrophilic, beta-turns. Residues 2-21 and 202-221 of gonococcal pilins and lower eucaryotic actins, respectively, exhibit 50% homology. When these residues are arranged at intervals of 100 degrees of arc on "helical wheels," the identical amino acids comprise a hydrophobic face on one side of the helix. This observation, the hydrophobic character of this region and the tendency for TC-1 (residues 1-30) to aggregate in water, suggest that this stretch interacts with other subunits to stabilize polymeric structure.

Isolation of type-specific polysaccharide antigen from group B type Ib streptococci.

Group B streptococcus type Ib (strain H36B) was subjected to digestion with extracellular muralytic enzymes prepared from Streptomyces albus. Type Ib-specific polysaccharide antigen was isolated from the lysate by alcohol precipitation and Sepharose 6B chromatography. The purified type Ib antigen has a Kd value of 0.31 on a Sepharose 4B column and contains four sugars, galactose, glucose, N-acetyl glucosamine, and sialic acid in a molar ratio of 2.05:0.86:1.00:0.90. Acid treatment (pH 2.0) of this polysaccharide results in partial degradation of the antigen (Kd = 0.41 on Sepharose 4B) with the loss of 93% of the sialic acid. The molar ratio of the remaining sugars in the polysaccharide remains identical to that in the native one. This suggests that the sialic acid is at the terminal position in the molecule. Both intact and acid-treated antigen cross-react with some type Ia and type Ic antisera as a result of the common Iabc determinant, but not with type II and type III antisera. Absorption studies indicate that Ib-specific determinant and Iabc determinant are on the same molecule and that sialic acid is not the cross-reactive determinant.