Immunological studies of the disulfide bridge region of Pseudomonas aeruginosa PAK and PAO pilins, using anti-PAK pilus and antipeptide antibodies.

Pseudomonas aeruginosa is an opportunistic pathogen that attaches to host cells via their pili. The pilus of P. aeruginosa PAK consists of a polymer of a single subunit, pilin, which is a 144-residue polypeptide. The C-terminal end of this protein is semiconserved in a number of strains and contains a disulfide bridge. We have synthesized the C-terminal peptide PAK (128-144)-OH in both its reduced and oxidized forms and the analog PAK(A-129) (128-144)-OH, in which cysteine-129 was substituted by alanine. These three peptides were used to immunize rabbits and prepare antipeptide antisera. It was found that antipeptide antisera to reduced peptide (17-R) and to oxidized peptide (17-O) bound to native PAK pili and cross-reacted with strain PAO pili in direct enzyme-linked immunosorbent assay (ELISA) and immunoblot experiments. However, the antiserum to the peptide immunogen PAK(A-129)(128-144)-OH, which does not have the ability to form the disulfide bridge, did not bind to either PAK or PAO pili. Competitive ELISA experiments with reduced and oxidized peptides of Ac-PAK(128-144)-OH showed that there was no difference in binding between the two peptides for 17-R or 17-O immunoglobulin G. When immunoglobulin G from native PAK antipilus antiserum was used in competitive or direct ELISA experiments, there was also no preference in binding to reduced or oxidized Ac-PAK(128-144)-OH or to PAK(A-129)(128-144)-OH. This result showed that the disulfide bridge in Pseudomonas pili is not critical to the immunogenicity of this region. However, the disulfide bridge is important in the immunogenicity of the C-terminal peptide when preparing antipeptide antisera that are cross-reactive with pili from different strains, since only the disulfide bridge peptide antisera cross-reacted well with the PAO pili as shown by competitive ELISA, suggesting that this region could be an important candidate for development of a synthetic vaccine.     

Adherence of Pseudomonas aeruginosa and Candida albicans to glycosphingolipid (Asialo-GM1) receptors is achieved by a conserved receptor-binding domain present on their adhesins.

Pseudomonas aeruginosa, a gram-negative bacterium, and Candida albicans, a dimorphic yeast, are evolutionarily distant microorganisms which can utilize filamentous structures termed pili and fimbriae, respectively, to mediate adherence to glycosphingolipids (asialoganglioside-GM1) receptors. The mechanism of adherence to glycosphingolipid receptors was investigated in these studies. By using monoclonal antibodies (MAbs) against purified pili of P. aeruginosa PAK (PK99H) and monospecific anti-peptide antibodies against the PAK pilin peptides [anti-PAK(128-144) and anti-PAK(134-140)], we demonstrated that these antibodies agglutinated C. albicans whole cells and cross-reacted with C. albicans fimbriae in immunoblots. A control MAb, PKL1, and anti-PAK(75-84) peptide antibodies failed to agglutinate C. albicans whole cells or cross-react with the fimbrial proteins. Conversely, the anti-C. albicans fimbrial MAb Fm16, but not Fm34, agglutinated P. aeruginosa PAK whole cells and Western blots (immunoblots). The interactions between PK99H and Fm16 and their respective homologous antigens were competitively inhibited by heterologous antigens; this demonstrated that the interactions between the antibodies and the heterologous antigens, i.e., PK99H with C. albicans fimbriae and Fm16 with P. aeruginosa pili, were highly specific and suggested that both adhesins share a common antigenic determinant. The immunological cross-reactivity between Fm16 and P. aeruginosa PAK pilin is localized onto the PAK(134-140) region as shown by a competitive enzyme-linked immunosorbent assay. The PAK(134-140) region of PAK pilin contains the epitope recognized by PK99H and also constitutes part of the receptor-binding domain of the pilus adhesin. Thus, the results from these studies suggest that common cell surface receptors are recognized by the P. aeruginosa and C. albicans adhesins because of a conserved receptor-binding domain on the adhesins.     

Characterization of the Pseudomonas aeruginosa pilus adhesin: confirmation that the pilin structural protein subunit contains a human epithelial cell-binding domain.

Previous studies have suggested that the Pseudomonas aeruginosa PAK pilus adhesin moiety resides in an epithelial cell-binding domain located in the C-terminal region of the PAK pilin structural protein. Synthetic peptides Ac17red (a synthetic peptide with a sequence identical to that of PAK pilin residues 128 to 144, with the Cys-129 and Cys-142 residues being in the reduced state) and Ac17ox (a synthetic peptide with a sequence identical to that of PAK pilin residues 128 to 144, with a formed disulfide bridge between the amino acid residues Cys-129 and Cys-142), which should contain the epithelial cell-binding domain, were synthesized. Ac17red and Ac17ox both bound to buccal epithelial cells (BECs) and to ciliated tracheal epithelial cells (TECs). Ac17ox had a Km of 6.40 microM for binding to BECs, while Ac17red had a Km of 9.87 microM. Ac17red bound to the same receptor sites that purified pili did and competitively inhibited the binding of purified PAK pili to BECs. BEC glycoproteins with molecular masses of 82, 55 to 51, and 40 kilodaltons immobilized on nitrocellulose exhibited periodate-sensitive receptor activity for Ac17red; similar activity has been found for PAK pili. Ac17red, Ac17ox, and PAK pili bound to the cilia and luminal portions of the cytoplasmic membrane of human TECs, the same regions to which P. aeruginosa whole cells bind. PAK pilin has an epithelial cell-binding domain that resides in the C-terminal region of the protein.     

The antigenicity of synthetic peptide fragments of lactate dehydrogenase C4

Lactate dehydrogenase C4 (LDH-C4) is an antigenic protein found only in spermatozoa and the mature testis. Synthetic peptides containing the amino acid sequences of the C-subunit, designated MC5-15, MC97-110 and MC211-220, were each conjugated to the carrier proteins diphtheria toxoid or bovine serum albumin. Rabbits immunized with these peptide-carrier conjugates produced antibodies to the peptide that cross-reacted with native LDH-C4. These data support our map that identifies antigenic domains of LDH-C4. Such synthetic peptides will be useful in the design of a contraceptive vaccine.     

Serial isolates of Pseudomonas aeruginosa from a cystic fibrosis patient have identical pilin sequences.

Five isolates of Pseudomonas aeruginosa (CD2, CD3, CD4, CD5, and CD10) from a patient with cystic fibrosis were examined with regard to several genotypic and phenotypic characteristics to determine whether the patient was colonized with one or several distinct strains. Isolates CD2, CD3, and CD4 were obtained from a single sputum sample, and CD5 and CD10 were obtained 1 and 2 years later, respectively. On the basis of colonial morphology, serotyping, and antibiograms, the five isolates appeared to be different strains. However, Southern blot analysis with a 1.2-kilobase DNA probe containing the P. aeruginosa PAK pilin gene indicated that all five strains were identical at that genetic locus. The pilin genes of the five isolates were cloned and sequenced at the nucleotide level and found to be identical. Southern blot analysis with a probe from a separate region of the P. aeruginosa chromosome, a 741-base-pair PstI-NruI DNA fragment adjacent to the exotoxin A gene, also revealed genetic identity among these five clinical isolates. On this basis, it was concluded that this patient was colonized with a single strain of P. aeruginosa and that the strain had remained genetically stable over a period of 2 years. The predicted pilin sequence of the CD isolates was almost identical to that of strain PA103 (97% homology) and serologically related to PAO pilin, with which it shared 80% homology. No immunological cross-reactivity was detected between the CD and PAK pilins, which shared the least homology (62%) among the four pilins considered in this study. Although all five CD isolates contained identical pilin genes, three had acquired mutations which prevented normal expression of the pilus system. CD3 was a putative regulatory mutant which was unable to produce normal amounts of pilin, and CD4 and CD10 were putative assembly mutants which produced normal amounts of pilin but were unable to assemble the pilin subunit into intact pili.     

Role of pili in adhesion of Pseudomonas aeruginosa to human respiratory epithelial cells.

The ability of pili from Pseudomonas aeruginosa K (PAK) to act as an adhesin to human respiratory epithelial cells was examined using an in vitro adhesion assay. Equilibrium analysis of PAK binding to human buccal epithelial cells (BECs) and tracheal epithelial cells (TECs) by means of a Langmuir adsorption isotherm revealed that the maximum numbers of binding sites per epithelial cell (N) were 255 for BECs and 236 for TECs, with apparent association constants (Ka) of 2.8 x 10(-9) and 5.8 x 10(-9) ml/CFU, respectively. Trypsinization of the BECs before the binding assay increased N to 605 and decreased the Ka to 1.7 x 10(-9) ml/CFU. Addition of homologous pili to the binding assay with BECs or TECs or the addition of anti-pilus Fab fragments inhibited PAK adherence. Binding of purified pili to BECs was shown to reach saturation. Purified pili and PAK competed for the same receptor on the BEC surface. Further, by using peptide fragments of PAK pilin (derived from the native pili or produced synthetically) in the binding assay for PAK to BECs, we have presumptively identified the pilus binding domain in the C-terminal region of the pilin and shown that the C-terminal disulfide bridge is important in maintaining the functionality of the binding domain.     

Mapping of the antigenic determinants of Pseudomonas aeruginosa PAK polar pili.

The polar pili of Pseudomonas aeruginosa are flexible filaments 5.2 nm in diameter and 2.5 microns in average length. They consist of a single subunit, pilin, which is a 144-residue polypeptide containing a hydrophobic N-terminal region (residues 1 to 30) and eight hydrophilic regions distributed throughout the remainder of the molecule. To delineate the antigenic regions of pilin, we cleaved the protein at Arg30, Arg53, and Arg120 to produce peptides TCI (residues 1 to 30), TCII (31 to 53), TCIII (54 to 120), and TCIV (121 to 144). TCIII and TCIV were further cleaved into several subfragments. The purified peptides were coupled to bovine serum albumin by using the N-hydroxysuccinimide ester of 4-azidobenzoic acid and were then subjected to immunological analysis, using the enzyme-linked immunosorbent assay and immunoblot procedures with polyclonal antiserum. Four antigenic regions were identified; one in TCI was found to be common to both PAK and PAO pilin. The remaining three were found to be specific to PAK pilin. Two of these were subfragments of TCIII, whereas the third was located close to the C-terminus of the molecule, most likely between Cys129 and Cys142. Modification of these cysteines by reduction and carboxymethylation of the disulfide linkage did not abolish the antigenicity of the C-terminal type-specific antigenic determinant.     

Identification of the mucin-binding adhesin of Pseudomonas cepacia isolated from patients with cystic fibrosis.

In previous experiments, we have shown that isolates of Pseudomonas cepacia from sputa of patients with cystic fibrosis (CF), particularly those with severe lung infection, exhibited specific binding to purified respiratory or intestinal mucins (U. Sajjan, M. Corey, M. Karmali, and J. Forstner, J. Clin. Invest. 89:648-656, 1992). The present report describes the identification of the adhesin as a protein located on fimbriae of mucin-binding P. cepacia. From a total of 53 isolates available (from 22 patients with CF), we used three mucin-binding and three non-mucin-binding isolates for our experiments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude P. cepacia homogenates was performed, the separated proteins were blotted onto nitrocellulose and overlaid with purified mucin, and mucin-binding components were detected with an antimucin antibody and then a second-antibody-alkaline phosphatase conjugate system. Only mucin-binding isolates exhibited a positively stained band at an Mr of 22,000. The 22-kDa protein was purified, and a polyclonal antibody specific for it was developed in rabbits. By electron microscopy and immunogold labelling, both the antibody and mucin (separately) were localized to pili present over the entire surface of the bacterial cells. Non-mucin-binding isolates did not have (or had very few) pili and did not stain with either mucin or the antibody to the 22-kDa protein. The purified 22-kDa protein and its antibody were each able to inhibit piliated P. cepacia binding to mucin. The amino acid composition of the 22-kDa protein was dissimilar to those of the major pilin proteins of Escherichia coli (type 1 pilus) and P. aeruginosa (PAK and PAO1 strains). Both the pili of P. aeruginosa PAK and PAO1 and antibodies to these pili failed to inhibit P. cepacia binding to mucin. Thus, P. cepacia adhesion to mucin is mediated by a pilin-associated 22-kDa protein which differs from epithelial-cell-binding pilin proteins of P. aeruginosa. We postulate that the 22-kDa adhesin may play a role in the virulence of P. cepacia lung infections of patients with CF.     

Differentiation of Pseudomonas aeruginosa pili based on sequence and B-cell epitope analyses.

The nucleotide sequences of three previously undescribed Pseudomonas aeruginosa pilin structural genes are presented. Comparisons of deduced pilin primary structure and flanking DNA sequence allowed placement of these and six previously published sequences into one of two groups. Epitope mapping, using overlapping immobilized peptides representing the pilin primary structure, with antipilin monoclonal antibodies revealed several B-cell determinants grouped near the carboxyl terminus of P. aeruginosa 1244 pilin. One determinant was found to reside near the pilin constant region. These determinants were found associated with the pili of 31 of 95 P. aeruginosa clinical isolates.     

Application and limitations of the multiple antigen peptide (MAP) system in the production and evaluation of anti-peptide and anti-protein antibodies.

The multiple antigen peptide (MAP) system has been proposed as a novel and valuable approach for eliciting antibodies to peptides and developing synthetic vaccines. The MAP system consists of a small immunogenically inert core matrix of lysine residues with alpha- and epsilon-amino groups for anchoring multiple copies of the same or different synthetic peptides. Several MAP systems, each containing eight copies of 6-15 residue-long peptides derived from the terminal and central regions of various proteins were analyzed in this study. The immunogenicity of MAPs was compared to that of the same peptides linked to carrier protein by means of conventional conjugation procedures. The various peptide antisera were tested in ELISA with homologous peptides conjugated to a carrier protein via their C terminal (as in the MAP system) or their N terminal end, or with their parent proteins. The antigenic properties of MAPs were studied with anti-peptide sera obtained by classical methods and with anti-protein sera. The results showed that the MAP system was an efficient antigen in ELISA except when the peptide corresponded to a C terminal epitope. However, the value of MAPs for raising anti-peptide antibodies cross-reactive with the cognate protein appeared much more limited. In the case of one N terminal peptide, the MAP construction was not immunogenic while the conventionally conjugated peptide induced antibodies that reacted strongly with the corresponding protein. In the case of the two C terminal peptides tested, the antibodies raised against MAP constructs reacted well with homologous MAPs but did not cross-react with the whole protein. Only in the case of a peptide from an internal domain of histone H2A did immunization with a MAP generate antibodies that cross-reacted with the protein.     

DNA sequences of three papA genes from uropathogenic Escherichia coli strains: evidence of structural and serological conservation.

Pyelonephritis-associated pili (Pap) are important in the pathogenesis of ascending, unobstructive Escherichia coli-caused renal infections because these surface bacterial organelles mediate digalactoside-specific binding to host uroepithelial cells. Pap are composed of many different polypeptides, of which only the tip proteins mediate specific binding. The PapA moiety polymerizes to form the bulk of the pilus structure and has been employed in vaccines despite its lack of Gal alpha(1-4)Gal receptor specificity. Animal recipients of PapA pilus-based vaccines are protected against experimental pyelonephritis caused by homologous and heterologous Gal-Gal-binding uropathogenic E. coli strains. Specific PapA immunoglobulin G antibodies in urine are correlated with protection in these infection models. The nucleotide sequences of the gene encoding PapA were determined for three E. coli clones expressing F7(1), F7(2), and F9 pili and were compared with corresponding sequences for other F serotypes. Specific rabbit antisera were employed in enzyme-linked immunosorbent assays to study the cross-reactivity between Gal-Gal pili purified from recombinant strains expressing F7(1), F7(2), F9, or F13 pili and among 60 Gal-Gal-binding wild-type strains. We present data which corroborate the concept that papA genes are highly homologous and encode proteins which exhibit greater than 70% homology among pili of different serotypes. The differences primarily occur in the cysteine-cysteine loop and variable regions and constitute the basis for serological diversity of these pili. Although there are differences in primary structures among these pili, antisera raised against pili of one serotype cross-reacted frequently with many other Gal-Gal pili of different serotypes. Furthermore, antisera raised against pili of the F13 serotype cross-reacted strongly or moderately with 52 (86%) of 60 wild-type Gal-Gal-binding E. coli strains. These data suggest that there are common immunogenic domains among these proteins. These additional data further support the hypothesis that broadly cross-protective PapA pilus vaccines for the immunoprophylaxis of pyelonephritis might be developed.     

Conserved and nonconserved epitopes among Haemophilus influenzae type b pili.

We investigated the binding of antibodies raised against four different Haemophilus influenzae type b (Hib) plus antigen preparations to the native pili and denatured pilins of 21 Hib isolates. Antibodies against live piliated Hib M43p+, adsorbed with a nonpiliated variant to remove nonpilus antibodies, bound to 18 of the 21 piliated Hib isolates in immunodot assays but failed to recognize the denatured pilins from any of the strains in Western immunoblot assays. Similarly, antibodies against purified native pili of strain E1ap+ bound to 11 of 21 piliated strains in immunodot assays but to only 2 of 21 piliated strains in Western blot assays. The native pili of all 21 strains were recognized by one or both of the antisera. These observations suggest that the immunodominant epitopes of native Hib pili are dependent on conformation and are moderately conserved. In contrast, antibodies against denatured M43p+ pilin or against a peptide derived from amino acids 5 through 17 of M43p+ pilin failed to bind to native pili from any of the 21 piliated isolates on immunodot assay. However, both sera recognized the denatured pilins from all the piliated strains on Western blot assay. These data indicate that the immunodominant epitopes of denatured pilins are highly conserved among different strains of Hib but are unavailable on intact pili for antibody binding.     

Mapping of the T-cell recognition sites of Pseudomonas aeruginosa PAK polar pili.

The polar pili of Pseudomonas aeruginosa consist of a subunit protein, pilin, which is a 144-residue polypeptide that contains a hydrophobic N-terminal region and eight hydrophilic regions distributed throughout the remainder of the molecule. T cells from mice immunized with pili or whole bacteria gave good pilus-specific T-cell proliferation responses. To delineate the T-cell antigenic regions of the pilin, T-cell blasts were generated from lymph nodes of pilus-primed BALB/c mice. These blasts were tested in vitro in T-cell proliferation assays for reactivity against the fragments of the pilin subunit prepared by enzymatic digestion. Citraconylation followed by trypsin digestion (cT) of the pilin subunit cleaved the protein into four fragments, cTI (residues 1 to 30), cTII (residues 31 to 53), cTIII (residues 54 to 120), and cTIV (residues 121 to 144). The ability to stimulate the T cells was found to reside in the cTI and cTIII regions, but not in the cTII or cTIV regions. A subfragment of cTIII, containing residues 82 to 104, was identified as the major T-cell recognition site within the cTIII region of the pilin molecule. A cross-reactivity was observed between pili from two strains of P. aeruginosa, namely, PAK and PAO, at the T-cell level. This cross-reactivity probably resulted from the sequence homology in the hydrophobic N-terminal region of these two molecules.     

Multiple sequence-reactive antibodies induced by a single peptide immunization with hypervariable region 1 of hepatitis C virus

Hypervariable region 1 (HVR1) of hepatitis C virus (HCV) is known to contain neutralizing epitopes. We previously found that murine antibodies against HVR1-#6 captured a different isolate, HCV-#7, and cross-reacted with the HVR1 peptide of HCV-#7. We investigated the inducibility and generality of cross-reaction of animal anti-HVR1 antibody responses in this study. Anti-HVR1-#7 antibodies, which were induced in mice and a chimpanzee by immunization, were found to be cross-reactive to HVR1-#6 peptide. Antibody responses against HVR1-#6-1 and HVR1-#7 peptides were detected in 11/165 (6.7%) and 26/165 (15.8%) HCV-infected individuals, respectively. Nine HVR1 sequences from six individuals, who were strongly positive for anti-HVR1-#7 antibodies, were only 50-64.5% identical to that of HVR1-#7. All nine of these HVR1 peptides were reactive to sera from the six patients and/or to antisera against HVR1-#6 and HVR1-#7 produced in mice and chimpanzees. Cross-inhibition tests of chimpanzee antisera indicated that a given species of anti-HVR1 antibodies was reactive to multiple HVR1 sequences. Fine epitope mapping of polyclonal and monoclonal anti-HVR1 antibodies showed that conserved subregions in HVR1 sequences determined the observed immunological cross-reactivity. Our data demonstrate that cross-reacting anti-HVR1 antibodies are inducible by a single peptide immunization.     

Assembly and antigenicity of the Neisseria gonorrhoeae pilus mapped with antibodies.

The relationship between the sequence of Neisseria gonorrhoeae pilin and its quaternary assembly into pilus fibers was studied with a set of site-directed antibody probes and by mapping the specificities of antipilus antisera with peptides. Buried and exposed peptides in assembled pili were identified by competitive immunoassays and immunoelectron microscopy with polyclonal antibodies raised against 11 peptides spanning the pilin sequence. Pili did not compete significantly with pilin subunits for binding to antibodies against residues 13 to 31 (13-31) and 18-36. Pilus fibers competed well with pilin protein subunits for binding to antibodies raised against peptides 37-56, 58-78, 110-120, 115-127, 122-139, and 140-159 and competed weakly for antibodies against residues 79-93 and 94-108. Antibodies to sequence-conserved residues 37-56 and to semiconserved residues 94-108 preferentially bound pilus ends as shown by immunoelectron microscopy. The exposure of pilus regions to the immune system was tested by peptide mapping of antiserum specificities against sets of overlapping peptides representing all possible hexameric or octameric peptides from the N. gonorrhoeae MS11 pilin sequence. The immunogenicity of exposed peptides incorporating semiconserved residues 49-56 and 121-126 was revealed by strong, consistent antigenic reactivity to these regions measured in antipilus sera from rabbits, mice, and human and in sera from human volunteers with gonorrhea. The conservation and variation of antigenic responses among these three species clarify the relevance of immunological studies of other species to the human immune response against pathogens. Overall, our results explain the extreme conservation of the entire N-terminal one-third of the pilin protein by its dominant role in pilus assembly: hydrophobic residues 1-36 are implicated in buried lateral contacts, and polar residues 37-56 are implicated in longitudinal contacts within the pilus fiber.     

Carboxy-terminal peptides from the B subunit of Shiga toxin induce a local and parenteral protective effect

Two synthetic peptides corresponding to overlapping sequences from the C-terminus of the B chain of Shiga toxin were prepared and characterized. These peptides consisted of residues 54-67 and 57-67 in the protein sequence. This region coincides with the major peak of surface area residues, as predicted from a computer-derived plot. For the purpose of immunization, the peptides were either conjugated with a protein or a synthetic carrier, or were polymerized. Polyclonal antibodies against these peptides derivatives, induced in rabbits, recognized the homologous peptides and cross-reacted with the intact toxin. These antibodies were capable of neutralizing the various biological activities of the toxin, namely the cytotoxic, enterotoxic and neurotoxic activities. Active immunization of mice with the peptide derivatives protected them from the lethal effect of the toxin. Moreover, oral immunization of rats led to inhibition of fluid secretion in ligated ileal loops into which toxin was injected. This effect was paralleled by the induction of high levels of specific anti-peptide IgA antibodies in the serum after bile duct ligation.     

Antibodies against active-site peptides common to glucosyltransferases of mutans streptococci.

Polyclonal antibodies were raised against peptides derived from an active-site sequence common to the family of mutans streptococcal glucosyltransferases (GTFs). The sequence contains an aspartic acid residue that functions in formation of the enzyme transition state in catalysis. Two GTFs were targeted with similar but not identical sequences in this region: one that synthesizes an alpha-1,3-linked water-insoluble glucan and a homologous GTF that synthesizes an alpha-1,6-linked water-soluble glucan. For each enzyme, an 8-mer and 22-mer peptide were prepared. The two peptide lengths were chosen in order to increase the likelihood of the peptides folding in a conformation similar to that of the native enzyme. Each peptide immunogen produced high titers of antibody in rabbits, and all antisera cross-reacted with all peptides, albeit to various degrees. Native enzyme showed weak interaction with antisera, which, on the basis of enzyme denaturation experiments, likely reflects binding to a small but finite population of denatured enzyme in the sample. GTF was assayed for inhibition in the presence of protein A-purified immunoglobulin G from each antiserum. Given the mass of the antibody and catalytic importance of the peptide, any enzyme-antibody complex formation would result in enzyme inhibition. No significant inhibition was observed, which demonstrates that either polyclonal antibodies raised against each of the four peptides cannot access this active-site region, or antibodies do not recognize the native enzyme conformation. The advantages and challenges of generating antibodies against enzyme active-site peptides are discussed in the context of the crystal structure of Aspergillus oryzae alpha-amylase, which has a homologous peptide segment which serves the same catalytic function.     

Antibodies against synthetic carboxy-terminal peptides distinguish H-ras and K-ras oncogene products p21

Synthetic peptides corresponding to the carboxy-terminal region of H-ras, K-ras, and N-ras oncogene product p21 proteins are used to obtain antibodies specific to each ras oncogene product. The synthetic peptides of 32 amino acids are immunogenic in rabbits without being coupled to carriers. Specific antibodies are purified by absorption of the antisera with the other peptides coupled to CH-Sepharose 4B, and antibodies reacting with all three peptides are obtained by affinity chromatography. These findings imply that antibodies specific to each peptide recognize the variable carboxy-terminal region while antibodies reacting with all three peptides recognize the constant region of the carboxy-terminal amino acid sequence of p21 proteins. The affinity-purified antibodies against H-ras and K-ras peptides are shown to react specifically with c-H-ras and v-K-ras p21 proteins expressed in E. coli and eukaryotic cells, respectively. These antibodies may be useful tools to study the functional roles of p21 carboxy-terminal domain and to detect differential expression of the family of ras oncogenes in cancerous tissues. The affinity-purified anti-N-ras peptide antibody, however, fails to react with N-ras p21 in spite of its positive reactivity with the N-ras peptide.     

Reactivity of synthetic peptides representing selected sections of hepatitis C virus core and envelope proteins with a panel of hepatitis C virus-seropositive human plasma

A series of 54 synthetic peptides, 15–20 residues long, that represented selected parts of the structural proteins of hepatitis C virus (HCV) were tested for immunoreactivity with a panel of 45 plasma samples from potential blood donors who were known to be seropositive for anti-HCV. Most of the ten peptides that represented the core protein showed reactivity with most of the panel samples. All except one of the 20 peptides that represented non-hypervariable regions of envelope proteins E1 and E2 showed little or no reactivity. In contrast, 18 of the the 24 peptides that represented variants of the hypervariable region 1 of the E2 protein reacted with at least one panel sample. Notably, 40% of the panel samples cross-reacted with two or more different peptides sequences some of which differed by more than 50%. Two panel samples each cross-reacted with seven different peptide sequences. The results suggest a broad anti-hypervariable region antibody specificity in many anti-HCV-seropositive samples and possible limits on the mutability of hypervariable region sequences. The work contributes to understanding the immunogenicity and persistence of HCV. J Med Virol 51:67–79, 1997. © 1997 Wiley-Liss, Inc.     

Comparison and analysis of the nucleotide sequences of pilin genes from Haemophilus influenzae type b strains Eagan and M43.

Previous studies have demonstrated antigenic differences among the pili expressed by various strains of Haemophilus influenzae type b (Hib). In order to understand the molecular basis for these differences, the structural gene for pilin was cloned from Hib strain Eagan (p+) and the nucleotide sequence was compared to those of strains M43 (p+) and 770235 b0f+, which had been previously determined. The pilin gene of Hib strain Eagan (p+) had a 648-bp open reading frame that encoded a 20-amino-acid leader sequence followed by the 196 amino acids found in mature pilin. The translated sequence was three amino acids larger than pilins of strains M43 (p+) and 770235 b0f+ and was 78% identical and 95% homologous when conservative amino acid substitutions were considered. Differences between the amino acid sequences were not localized to any one region but rather were distributed throughout the proteins. Comparison of protein hydrophilicity profiles showed several hydrophilic regions with sequences that were conserved between strain Eagan (p+) and pilins of other Hib strains, and these regions represent potentially conserved antigenic domains. Southern blot analyses using an intragenic probe from the pilin gene of strain Eagan (p+) showed that the pilin gene was conserved among all type b and nontypeable strains of H. influenzae examined, and only a single copy was present in these strains. Homologous genes were not present in the phylogenetically related species Pasteurella multocida, Pasteurella haemolytica, and Actinobacillus pleuropneumoniae. These data indicate that the pilin gene was highly conserved among different strains of H. influenzae and that small differences in the pilin amino acid sequences account for the observed antigenic differences of assembled pili from these strains.