Biomedical subjects

M W Steward

Publications and source records attributed to M W Steward.

At least 37 records · Page 2Linked to original sources

PubMed1997

Definition of the primary structure of hepatitis B virus (HBV) pre-S hepatocyte binding domain using random peptide libraries.

The pre-S-specific monoclonal antibody MA 18/7 has been shown to inhibit the binding of HBV to HepG2 cells and liver membranes. This antibody can thus be used to identify the critical residues of the pre-S region involved in the hepatocyte-binding domain. Using overlapping 7-mer peptides representing the pre-S region of HBV, the epitope recognized by MA 18/7 was shown to contain sequences from both the pre-S1 and pre-S2 regions, thus indicating that the hepatocyte-binding domain is conformationally dependent. To further characterize the primary structure of the hepatocyte-binding domain on the pre-S protein, a phage-displayed 15-mer peptide library and a 8-mer solid phase peptide library were used to analyze the fine specificity of the monoclonal antibody MA 18/7. Several mimotopes were identified with the phage-displayed peptide library, the majority of which possess a central motif with at least three identical residues present within the native pre-S1 sequence. No significant consensus sequences were found when these mimotopes were compared to the pre-S2 sequence. Mimotopes identified using the solid-phase peptide library also contained a similar motif. All phage mimotopes and a single mimotope from the solid-phase peptide library competed with recombinant HBsAg particles containing the pre-S1 region for binding to MA 18/7. Mouse antisera raised against four mimotopes from the phage display library reacted with HBsAg particles containing pre-S sequences. The data show that the structure of the pre-S molecule around the conserved DPAF motif in the pre-S region may have a functional role in binding HBV to cellular receptors, and that the central motif identified in mimotopes of this region may offer a novel strategy target for the improvement of existing hepatitis B vaccines which, at present, are mostly devoid of pre-S specificities.

Amino Acid Sequence↗

PubMed1997

CTL responses induced by a single immunization with peptide encapsulated in biodegradable microparticles.

A synthetic peptide representing a measles virus (MV) cytotoxic T cell epitope (CTL) when encapsulated in poly (D,L-lactide co-glycolide) (PLG) 50:50 microparticles induced a strong CTL response after a single intraperitoneal immunization of mice which was greater than that following administration of the peptide in Freund's complete adjuvant. A 100 micrograms dose of encapsulated peptide was shown to be more effective for CTL priming than 50 and 25 micrograms doses. A vaccine formulation prepared by simply mixing empty 50:50 PLG microparticles with the peptide resulted in the induction of CTL responses comparable to those induced by the encapsulated peptide. Moreover, a CTL response against MV-infected target cells was observed. These findings highlight the potential immunostimulatory effect of PLG microparticles for the induction of MV and peptide-specific CTL responses.

Amino Acid Sequence↗

PubMed1997

Peptide mimics of a conformationally constrained protective epitopes of respiratory syncytial virus fusion protein.

AIMS: To identify peptides that mimic (mimotopesi conformational and protective epitopes of RSV fusion protein and to assess their efficacy as immunogens and potential vaccines. MATERIAL AND METHODS: An 8-mer solid-phase (TG resin) library was screened with a neutralising and protective RSV fusion protein specific monoclonal antibodies (Mab-19). After selection of positive beads, reactive sequences were identified by microsequencing and 8-mer peptides were synthesised. Improvement of binding was analysed by amino acid replacement using the SPOTs method. RESULTS: Mabs were not able to bind to the free and soluble peptides, nor did these peptides induce anti-RSV specific antibodies. However, several peptides re-synthesised on a TG resin (to produce de-protected 8-mer peptides linked to the resin) or as SPOTs reacted specifically. Therefore it was critical to be able to reproduce this conformation in order to use these mimotopes as immunogens and potential vaccines. Using C-terminal constrained versions of the mimotopes, strong binding of one of the Mabs to the peptides was demonstrated by surface-plasmon resonance. Immunisation of Balb/c mice with these peptide-mimics produced anti-sera that: (1) reacted specifically with RSV; (2) inhibited the binding of the Mab to the virus; (3) neutralised RSV in vitro with high titres (range: 80-640); and (4) reduce significantly the viral load in the lungs of mice challenged with RSV (P < 0.01). CONCLUSIONS: This report demonstrates for the first time that: (1) a protective epitope of the conserved RSV fusion protein can be mimicked by synthetic peptides; and (2) immunisations with these mimotopes induced specific anti-RSV neutralising antibodies and reduced viral load in vivo. These results represent a novel concept for the development of a vaccine against RSV.

Animals↗

PubMed1997

The potential of combinatorial peptide libraries for the identification of inhibitors of TNF-alpha mediated cytotoxicity in vitro.

The use of random peptide libraries has recently received considerable attention as a new tool for identifying novel therapeutic molecules. The libraries represent an enormous number of peptide sequence displayed either on the virion surface of filamentous phage clones or on a solid phase synthetic support. In this minireview we summarize our recent findings on the use of combinatorial peptide libraries to identify antagonists of the biological activities of TNF-alpha, a cytokine which plays a key role in immune and inflammatory responses.

Animals↗

PubMed1997

Use of a solid-phase random peptide library to identify inhibitors of TNF-alpha mediated cytotoxicity in vitro.

Tumour necrosis factor (TNF) is a pleiotropic cytokine which plays a central role in infection, inflammation and autoimmune diseases. Its functions are mediated through binding to high affinity cell surface receptors. An approach to modulate excessive levels of TNF-alpha in the serum is the use of soluble receptors. In this study the potential of a solid-phase combinatorial peptide library was investigated to identify peptide mimics of the binding site of the TNF-alpha receptor. One of the identified mimotopes was shown to inhibit TNF-alpha-mediated cytotoxicity in mouse L929 and in a human KYM-1D4 cell lines in a dose-dependent fashion. Characterization of the mimotope sequence by high-performance liquid chromatography and mass spectroscopy has shown that the inhibitory effect of the mimotope was dependent upon the presence of a protective MTR group on the side chains of the arginine residues in the mimotope sequence. Furthermore, antibodies to the mimotope were shown to recognize the recombinant human TNF 55-kDa receptor in an ELISA assay. These findings highlight the potential of combinatorial peptide libraries in identifying peptide mimics of the binding site of TNF-alpha receptor, which can be used as competitive inhibitors of ligand-receptor interactions.

Animals↗

PubMed1997

Fine specificity of the antibody response to a synthetic peptide from the fusion protein and protection against measles virus-induced encephalitis in a mouse model.

A synthetic peptide representing residues 397-420 from the measles virus (MV) fusion (F) protein was tested for its structure, immunogenicity and protective capacity against intracerebral challenge with a neuroadapted strain of MV. Analysis of the peptide by mass spectrometry showed that it was linear, despite the presence of two cysteine residues in the sequence. Circular dichroism spectroscopy highlighted a weak preference for the peptide to adopt an alpha-helical conformation. The peptide was shown to be immunogenic in BALB/c and C57BL/6 mice after intraperitoneal immunization in Freund's adjuvant, and anti-peptide antibodies from both strains of mice reacted with the MV as a solid phase antigen on an ELISA plate. When the fine specificity of the anti-peptide antibody response was examined using overlapping 8-mer peptides, serum antibodies from BALB/c mice recognized the region between residues 407-417 whereas antibodies from C57BL/6 mice recognized the region 408-420 of the 397-420 peptide sequence. Although anti-397-420 antibodies had no demonstrable neutralizing activity, protection against challenge with a neuroadapted strain of MV was demonstrated following active immunization with peptide in C57BL/6 mice or after passive transfer of anti-peptide antibodies in BALB/c mice. These findings highlight the importance of the 397-420 region in the induction of protective antibodies in the MV encephalitis mouse model, and suggest that this epitope might be a good candidate for inclusion in a future MV synthetic peptide vaccine.

Animals↗

PubMed1997

Identification of an immunodominant neutralizing and protective epitope from measles virus fusion protein by using human sera from acute infection.

Polyclonal sera obtained from African children with acute measles were used to screen a panel of 15-mer overlapping peptides representing the sequence of measles virus (MV) fusion (F) protein. An immunodominant antigenic region from the F protein (p32; amino acids 388 to 402) was found to represent an amino acid sequence within the highly conserved cysteine-rich domain of the F protein of paramyxoviruses. Epitope mapping of this peptide indicated that the complete 15-amino-acid sequence was necessary for high-affinity interaction with anti-MV antibodies. Immunization of two strains of mice with the p32 peptide indicated that it was immunogenic and could induce antipeptide antibodies which cross-reacted with and neutralized MV infectivity in vitro. Moreover, passive transfer of antipeptide antibodies conferred significant protection against fatal rodent-adapted MV-induced encephalitis in susceptible mice. These results indicate that this epitope represents a candidate for inclusion in a future peptide vaccine for measles.

Amino Acid Sequence↗

PubMed1997

Oral delivery of poly(lactide-co-glycolide) encapsulated vaccines.

The induction of mucosal immune responses by oral delivery of vaccines is highly desirable. However vaccines to be used in this context will require protection from degradation in the gut and the use of specialised vehicles for their delivery and presentation. Using the biodegradable and biocompatible polymer, poly(lactide-co-glycolide), we have encapsulated bacterial and viral proteins, synthetic peptides and plasmid DNA in microparticles, and compared the immune responses resulting from their oral and parenteral administration to mice. The successful induction of specific systemic and mucosal humoral immune responses, as well as cell-mediated immune responses, demonstrates the potential of this polymer formulation as a vehicle for the oral delivery of vaccines.

Administration, Oral↗

PubMed1996

Mucosal immunization with a measles virus CTL epitope encapsulated in biodegradable PLG microparticles.

The immunogenicity of a cytotoxic T cell epitope (CTL) representing residues 52-60 from measles virus (MV) nucleoprotein, encapsulated in poly(lactide-co-glycolide) (PLG) microparticles was evaluated after mucosal immunization. After intranasal administration of the encapsulated CTL epitope linked at the carboxyl terminus of two copies of a T-helper epitope (TT-NP6), peptide-specific and MV-specific CTL responses were detected in splenocytes. However, these responses were lower than the responses observed when the TT-NP6 peptide was administered intranasally in saline or using CTB as an adjuvant. Intranasal coadministration of the encapsulated TT-NP6 peptide with CTB did not result in any significant potentiation of the CTL responses. The effectiveness of biodegradable PLG microparticles for mucosal delivery of CTL epitopes, combined with their excellent tissue compatibility and biodegradability suggests that they represent a valuable delivery system for synthetic immunogens. However, further work is needed to define the requirements for effective absorption by the nasal epithelium.

Animals↗

PubMed1996

Efficient binding to the MHC class I K(d) molecule of synthetic peptides in which the anchoring position 2 does not fit the consensus motif.

Peptides eluted from the MHC class I K(d) molecule are generally nonamers that display a strong preference for Tyr in position 2 and Ile or Leu in position 9. We investigated the binding ability of several synthetic peptides which did not fit this consensus motif. In our peptides, Tyr(2) was substituted by other amino acids, i.e. LeU, Ile or Met. These peptides were variants of the 252-260 K(d)-restricted peptide SYIPSAEKI derived from the Plasmodium berghei circumsporozoite protein. They bound to purified K(d) molecules in vitro with intermediate affinity. One of them was tested for in vivo stimulation of T cells and induced a cytotoxic response. These results demonstrate the importance of binding motif refinement to discover new binding characteristics and new ligands such as low-affinity peptides.

Amino Acid Sequence↗

PubMed1996

Priming of measles virus-specific CTL responses after immunization with a CTL epitope linked to a fusogenic peptide.

In this study, the potential of the amino-terminal sequence from the F1 polypeptide responsible for the fusion of measles virus (MV) with cell membranes as a carrier system for a CTL epitope from the MV nucleoprotein was examined. The addition of the fusion sequence (FP) at either the amino or the carboxyl terminus of the CTL epitope peptide rendered it immunogenic after intraperitoneal immunization in mice. The CTLs induced were able to lyse target cells pulsed with the peptide or persistently infected with MV. After intranasal administration of a FP-CTL chimera with or without cholera toxin B subunit (CTB) as an adjuvant, CTL responses to the peptide pulsed and to MV-infected target cells were detected. Responses in groups of mice where CTB was used as an adjuvant were stronger. However, intranasal administration of the CTL epitope did not induce a protective response against intracranial challenge with a neuroadapted strain of MV. These findings highlight the potential of fusion sequences as a carrier system for CTL epitopes and the potential of the intranasal route for administration of synthetic peptides representing MV sequences.

Amino Acid Sequence↗

PubMed1996

Biodegradable microparticles as a delivery system for measles virus cytotoxic T cell epitopes.

Cytotoxic T-cell (CTL) responses are likely to be important for the clearance of a measles virus (MV) infection. To induce CTL responses. replicating vectors have generally been used but the use of such vectors in humans mav be problematic, and immunization with synthetic peptides may be more appropriate. We have investigated the potential of poly(lactide-co-glycolide)(PLG) microparticles as a delivery system for a CTL epitope representing residues 51-59 from MV nucleoprotein. After a single intraperitoneal injection in saline of the encapsulated epitope, CTL responses to the homologous peptide and MV were detected over a period of 4 months. Responses reached a maximum 30 days after priming and were maintained at high levels for 120 days. These responses were higher than those observed when the CTL epitope was administered in saline or as an emulsion in Incomplete Freund's Adjuvant. The pronounced immunostimulatory effect of microparticles, combined with their excellent tissue compatibility and biodegradability suggests that they represent a valuable delivery system for synthetic peptide immunogens.

Animals↗

PubMed1996

Immunological analysis of the protective responses to the chimeric synthetic peptide representing T- and B-cell epitopes from the fusion protein of measles virus.

The role of different adjuvant formulations and routes of immunization on the antibody responses and protection induced in mice was determined by a synthetic peptide representing T- and B- cell epitopes from the measles virus (MV) fusion (F) protein (TTB peptide) which has previously been shown to induce protective responses against MV encephalitis in mice. When the peptide TTB was administered in Freud's complete adjuvant (FCA), Freud's incomplete adjuvant (FIA), alum or with IL-2, anti-peptide antibody responses and anti-MV antibody responses were detected. Interestingly, immunization with TTB without adjuvant resulted in the induction of anti-peptide antibody responses which did not cross react with the MV. The use of FIA as an adjuvant led to a significantly higher IgG2a antibody response compared with FCA and alum, whereas alum led to a significantly lower IgG3 response. Immunization with TTB in FCA, FIA and alum led to the generation of high affinity antibodies (with alum generating the highest affinity), whereas immunization of peptide with IL-2 or in PBS resulted in the induction of antibodies of lower affinity. Only the FCA, FIA and alum formulations led to the induction of protective responses in mice against MV-induced encephalitis. When the subcutaneous route (s.c.) of immunization was compared with the intraperitoneal route (i.p.), s.c. immunization with the TTB peptide led to higher anti-peptide and anti-MV antibody responses and higher affinity antibodies compared to those induced following i.p. immunization. Mice receiving the TTB peptide via the s.c. route had a higher percentage survival after MV challenge than those immunized via the i.p. route. These results show that the nature of the adjuvant used as well as the route of immunization play an important role in the induction of protective anti-TTB peptide antibody responses against MV-induced encephalitis in mice.

Animals↗

PubMed1996

Linkage of a fusion peptide to a CTL epitope from the nucleoprotein of measles virus enables incorporation into ISCOMs and induction of CTL responses following intranasal immunization.

The introduction of soluble protein antigens into the endogenous processing pathway is a prerequisite for the efficient induction of MHC class-1 restricted cytotoxic T-lymphocytes (CTLs). Antigens incorporated into immunostimulating complexes (ISCOMs) containing lipids and Quil-A are able to induce CD8+ CTL responses in vivo. Furthermore, lipopeptides have also been used to raise peptide-specific CTLs and bypass the requirement for the use of an adjuvant. Although conventional ISCOM technology is in general restricted to the use of hydrophobic proteins or fatty acid-derivitized proteins or peptides, we have demonstrated that the linkage of a conserved paramyxovirus fusion peptide to a CTL epitope NP29 (residues 281-290 of measles virus nucleoprotein) resulted in the incorporation of this hydrophilic CTL epitope into ISCOMs and the in vivo priming of peptide specific CTLs following intranasal immunization. In addition, the fusion peptide-CTL epitope chimera was able to efficiently sensitise P815 target cells for lysis by nucleoprotein specific CTLs induced following immunization of mice with recombinant RAd-68 adenovirus, suggesting the efficient introduction of the peptide into the class-1 restricted antigen processing pathway. Furthermore, immunization of mice with this fusion peptide chimera in saline was able to prime an NP29-specific CTL response despite the absence of adjuvant.

3T3 Cells↗

PubMed1996

Structural requirements for synthetic immunogens to induce measles virus specific CTL responses.

We have studied the immunogenicity of a synthetic peptide representing a cytotoxic T cell epitope (CTL) from the nucleoprotein of measles virus (MV). For the induction of peptide and MV-specific CTL responses after subcutaneous immunization, covalent linkage of the CTL epitope to a T-helper epitope was required. The presence of two copies of the T-helper epitope at the amino terminus of the CTL epitope (TT-CTL) resulted in the induction of strong CTL responses after administration in saline. In contrast, a chimeric peptide with one copy of the T-helper epitope at the amino terminus of the CTL epitope (T-CTL) was weakly immunogenic when given in saline. Analysis of the structure of the TT-CTL chimeric peptide by CD spectroscopy revealed an alpha-helical conformation, as compared to the random coil conformation favored by the T-CTL chimeric peptide. In addition, the CD spectra of the TT-CTL peptide in the presence of small unilamellar vesicules (SUV) revealed an increased helicity, as compared to the spectra of the T-CTL chimera in the presence of SUV. This suggests that the amphipathic character of the TT-CTL chimeric construct favors its interaction with the cell membrane of antigen presenting cells, therefore, facilitating its cytosolic delivery for class I presentation. These findings highlight the importance of antigen structure for the in vivo induction of CTL responses and may have implications for the design of synthetic peptide vaccines.

Amino Acid Sequence↗

PubMed1996

Induction of measles virus-specific cytotoxic T-cell responses after intranasal immunization with synthetic peptides.

We have investigated the structural requirements for the induction of cytotoxic T-cell responses (CTL) in vivo after intranasal immunization with an immunodominant CTL epitope from the nucleoprotein of measles virus (MV). For the induction of CTL responses, covalent linkage of the CTL epitope to a helper T-cell epitope was required and the orientation of the epitopes influenced the immunogenicity of the CTL epitope. The presence of two copies as compared with one copy of a T-helper epitope, rendered the CTL epitope more immunogenic and resulted in the in vivo induction of MV-specific CTLs without the need for an adjuvant. The role of CTL responses to this epitope in protection after intranasal administration was evaluated in a mouse model against challenge with a neuroadapted strain of MV. Although a decreased mortality in the peptide immunized compared with that in unimmunized mice was observed, the protection achieved was not significant. These findings highlight the importance of the rational design of synthetic immunogens for the induction of CTL responses and the potential of the intranasal route for immunization.

Adjuvants, Immunologic↗

PubMed1996

The adjuvant effect of a non-toxic mutant of heat-labile enterotoxin of Escherichia coli for the induction of measles virus-specific CTL responses after intranasal co-immunization with a synthetic peptide.

The intranasal route has been shown to be effective for immunization. However, immunization via this route may require the use of potent and safe adjuvant. The construction of non-toxic mutants of heat labile enterotoxin of Escherichia coli (LT), which is a potent mucosal adjuvant, is a major breakthrough for the development of mucosal vaccines. In this study we have assessed the ability of an LT mutant (LTK63) to act as an adjuvant following intranasal co-immunization with a peptide corresponding to a measles virus cytotoxic T lymphocyte (CTL) epitope. LTK63 was more effective at potentiating the in vivo induction of peptide-specific and measles virus-specific CTL responses than was administration of the peptide in saline. A concentration of 10 micrograms/dose of LTK63 was found to be the most effective in potentiating the in vivo priming of peptide-specific and measles virus-specific CTL responses. These findings highlight the potential of the non-toxic mutant of LT as a safe mucosal adjuvant for use in humans.

Adjuvants, Immunologic↗

PubMed1995

Induction of systemic immune responses to measles virus synthetic peptides administered intranasally.

A systemic antibody response was induced when a chimeric peptide containing two copies of a promiscuous T-cell epitope and one copy of a B-cell epitope (TTB) from the fusion protein of measles virus (MV) was administered to mice intranasally without adjuvant. A higher antibody titre was produced when the peptide was administered intranasally with cholera toxin B subunit (CTB) as an adjuvant and these antibodies crossreacted with the MV. Furthermore, splenocytes from intranasally immunized mice proliferated in vitro in the presence of the TTB peptide. The immune response following intranasal immunization with the peptide was influenced by the MHC haplotype of the strain of mice used. Thus CBA and BALB/c mice were high responders whereas C57BL/6 mice were low responders. Although peptide administered intranasally with CTB to CBA mice induced an immune response, no significant protection was observed against intra-cranial challenge with canine distemper virus which is antigenically related to MV.

Administration, Intranasal↗