Biomedical subjects
Publications and source records attributed to M W Steward.
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The affinity of anti-gag antibody was studied for up to 9 years (1984-1993) in sera from 15 HIV-1+ patients with haemophilia. On the basis of their 1993 clinical status patients were divided into two groups: (i) patients who remained asymptomatic (n = 9); and (ii) those who progressed to AIDS between late 1987 and 1993. The affinity constants of antibody for p24 and p17 were determined by a double isotope fluid-phase radioimmunoassay; and the relationships between antibody affinity and titre, patient clinical course, CD4 cell counts and p24 antigenaemia were analysed. The affinity of p24- and p17-specific antibody was up to 100 times greater in asymptomatic patients than in patients who progressed to AIDS. Patients who developed AIDS either lost or failed to develop high-affinity antibodies early in the infection. Asymptomatic patients maintained high-affinity antibodies for several years; however, in some of these patients the affinity of anti-p24 and p17 antibodies subsequently fell later in the study period. The presence of low-affinity antibody and progressive reduction in the titre of specific antibody were earlier predictors of disease onset than CD4 cell counts. The failure to either develop or maintain high affinity gag-specific antibody suggests an early impairment of T helper function in individuals who progressed to AIDS. The presence of antibody of high affinity could be essential in controlling virus replication and the onset of AIDS.
A solid-phase 8-mer random combinatorial peptide library was used to generate a panel of mimotopes of an epitope recognized by a monoclonal antibody to the F protein of measles virus (MV). An inhibition immunoassay was used to show that these peptides were bound by the monoclonal antibody with different affinities. BALB/c mice were coimmunized with the individual mimotopes and a T-helper epitope peptide (from MV fusion protein), and the reactivity of the induced anti-mimotope antibodies with the corresponding peptides and with MV was determined. The affinities of the antibodies with the homologous peptides ranged from 8.9 x 10(5) to 4.4 x 10(7) liters/mol. However, only one of the anti-mimotope antibodies cross-reacted with MV in an enzyme-linked immunosorbent assay and inhibited MV plaque formation. Coimmunization of mice with this mimotope and the T-helper epitope peptide induced an antibody response which conferred protection against fatal encephalitis induced following challenge with MV and with the structurally related canine distemper virus. These results indicate that peptide libraries can be used to identify mimotopes of conformational epitopes and that appropriate immunization with these mimotopes can induce protective antibody responses.
Synthetic peptides representing T- and B-cell epitopes from the fusion (F) protein of measles virus (MV) were tested for their ability to induce a protective immune response against intracerebral challenge with neuroadapted strains of MV and canine distemper virus (CDV) in mice. Of the panel of peptides tested, only a chimeric peptide consisting of two copies of a promiscuous T-cell epitope (representing residues 288 to 302 of MV F protein) synthesized at the amino terminus of a B-cell epitope (representing residues 404 to 414 of MV F protein) was able to induce a protective response against challenge with MV and CDV in inbred mice. The protective response induced by this peptide (TTB) was associated with a significant reduction in mortality, histological absence of acute encephalitis, and greatly reduced titers of virus in the brains of TTB-immune mice following challenge compared with the results for nonimmunized controls. A chimeric peptide comprising one copy of the T-cell epitope and one copy of the B-cell epitope (TB) did not induce a protective response. A comparison of the antibody responses induced by the two chimeras suggested that differences in protective efficacy following immunization may be a result of the higher affinity of the antibody induced by the TTB peptide than that of the antibody induced by the TB peptide. In addition, differences in the immunoglobulin G subclass of the antipeptide antibody responses were observed, and these may play a role in the differences in protection observed. These results indicate that appropriately designed synthetic peptides have potential as vaccines for the induction of cross-reactive protection against morbilliviruses.
Previously published work has shown that a cytotoxic T-cell epitope (CTL) representing residues 82-90 of the M2 protein of respiratory syncytial virus (RSV) is the target for a protective response against the virus. In this report, we demonstrate that a synthetic peptide representing residues 81-95, when covalently linked to a fusion peptide derived from the conserved N-terminal 19 residues of the F1 protein of measles virus efficiently primes RSV-specific CTLs in vivo following immunization without adjuvant.
In this study, a panel of 55 synthetic peptides representing 92.2% of the haemagglutinin (H) glycoprotein of measles virus (MV) were used to study the antigenic profile of the H molecule of anti-MV antibodies raised in mice and late convalescent human sera. In addition the immunogenicity of these peptides was tested in two mouse strains. Mouse anti-MV antibodies had different fine specificity of binding to the peptides depending on the mouse strain. Thus in BALB/c (H-2d) mice, anti-MV antibodies recognised six peptides representing residues 103-117; 123-137; 242-255; 293-307 and 463-477. In TO (H-2s) mice, anti-MV antibodies recognised peptides representing residues 49-72 and 463-477. When the immunogenicity of the peptides was tested, 29 were immunogenic in BALB/c mice and 34 were immunogenic in TO mice. Several of the anti-peptide antisera were found to cross-react with MV, depending on the solid phase assay system used but none were able to inhibit virus infectivity in vitro. The reactivity of a panel of late convalescent human sera with the peptides was heterogeneous and the extent of the binding to the peptides was related to the titre of anti-MV. However, human sera recognized certain peptides more frequently than others, in particular peptides at the carboxyl-terminus.
In this paper, we describe the results of experiments designed to test the hypothesis that immunogenic synthetic peptides representing non-immunodominant B- and T-cell epitopes of measles virus (MV) proteins can overcome the suppressive effect of maternal antibodies and induce anti-MV antibodies in young mice in the presence of maternal antibody to the virus. We have established a mouse model of immunosuppression by maternal antibody of both anti-MV and anti-peptide antibody responses in the young. Results obtained with this model immunization of young mice with a cocktail of synthetic peptides can overcome the suppression by maternal anti-MV antibodies and results in the induction of anti-peptide antibodies which recognize the virus. This work indicates that appropriately selected synthetic peptides have potential as vaccines which can be immunogenic and induce antibodies reactive with the virus-virus antibodies. in the presence of maternal anit-virus antibodies.
We have assessed the influence of different T-helper cell epitopes on the level and affinity of antibody to B-cell epitopes induced following co-immunization with free peptides mimicking epitopes from measles and respiratory syncytial virus envelope proteins. The responses obtained following co-immunization have been compared to those obtained following immunization with chimeric synthetic peptide immunogens in which the epitopes were covalently coupled. The results show that covalent linkage of the B- and T-cell epitopes is not necessary for the generation of T-cell dependent antibody responses to non-immunogenic B-cell epitopes. In addition the induction of memory B-cells required adjuvant but subsequent stimulation of these memory cells did not. The responses obtained were non-MHC restricted since co-immunization resulted in the production of antibody responses to B-cell epitopes in a panel of five inbred mouse strains but there were differences in the ability of different T-cell epitopes to provide help for antibody production to the same B-cell epitope. The affinity of antibodies to the B-cell epitopes induced following immunization with chimeric T:B peptides was higher than that obtained following co-immunization. These results indicate the value of co-immunization for the induction of antibody responses to B-cell epitopes across MHC differences and suggest that this strategy may be of value in the development of synthetic peptide vaccines. However, modifications of the approach need to be developed to ensure the production of antibody of the highest possible affinity.
The amino acid sequence 139-147 from hepatitis B surface antigen (HBsAg) has previously been shown to represent a B-cell epitope with potential as a component of a synthetic peptide vaccine against hepatitis B. In this paper, two regions of HBsAg which act as T-cell epitopes in inbred mice have been identified (residues 23-34 and residues 160-171). The ability of synthetic peptides representing these epitopes to provide help for the production of antibody against the 139-147 epitope has been assessed following their co-linear synthesis with the B-cell epitope and following co-immunization of the peptides in an uncoupled form. Both these strategies result in the induction of anti-peptide antibodies which specifically react with recombinant HBsAg. The results presented give further support to the concept that synthetic peptides representing appropriately chosen B- and T-cell epitopes from HBsAg could form the basis of a synthetic vaccine against hepatitis B.
The aim of this study was to define the helper T cell epitopes on the haemagglutinin (H) of measles virus (MV) in BALB/c (H-2d) and TO (H-2s) mice. A panel of 55 synthetic peptides (15-mers, overlapping by five amino acids) representing 92.2% of the H protein were synthesized and tested for immunogenicity and ability to stimulate MV-primed lymphocytes in vitro. The results obtained show that mouse lymphocytes respond to defined regions of the H protein which differ according to mouse strain. Virus-primed lymphocytes from BALB/c mice responded in vitro to peptides 7, 38, 39 and 44 whereas lymphocytes from virus-primed TO mice responded only to peptide 39. When mice of both strains were immunized with the peptides, a number of peptides induced proliferative responses, showing that the T cell repertoire for epitopes on the H protein is broader than that following immunization with virus. In BALB/c mice, lymphocytes primed to peptides 37, 39, 40, 42 and 43 responded in vitro to MV and in TO mice, lymphocytes primed to peptides 14, 32, 39, 40 and 49 responded to the virus. Thus in both strains of mice peptide 39 behaved as a dominant T cell epitope following immunization with virus or peptides. When the results obtained experimentally were compared with sequences predicted to be T cell epitopes by a number of algorithms, the concordance was limited.
In order to investigate the usefulness of polysaccharides as carriers for the induction of antibody to synthetic peptides, peptides representing residues 139-147 of the surface antigen of hepatitis B and residues 129-140 of the pre-S2 region of the protein were coupled to mannan and dextran via an aminocaproic spacer molecule. Of the two conjugates studied, only mannan was useful as a carrier for the efficient production of anti-peptide antibodies.
The role of dissociation of primary antigen-antibody bonds in the solubilization of immune complexes (IC) has been investigated using photo-affinity crosslinked IC comprising NAP15-BSA and murine monoclonal anti-DNP antibodies. Non-covalently linked IC were solubilized rapidly when incubated with normal human serum (NHS), whereas covalently-linked IC were solubilized poorly or not at all. The rate and extent of complement activation produced by incubating covalently-linked and non-covalently linked IC with NHS was similar as assessed by the production of the C1s:C1-inhibitor, C3:properdin and C5b-9 complexes and the anaphylatoxins C4a and C3a. Thus, the inability of serum to solubilize photo-affinity crosslinked IC must be due to failure of dissociation of primary antigen-antibody bonds.
Vaccination with hepatitis B surface antigen (HBsAg) has shown that antibody directed against the common 'a' determinant of this antigen is protective against infection with hepatitis B virus (HBV). In this study the antigenic epitopes of the 'a' determinant have been analysed by competitive inhibition assays and by binding studies to synthetic peptides using a panel of monoclonal antibodies prepared against HBsAg, all of which are shown to recognise the common group determinant. One murine monoclonal antibody used in this study, RFHBs1, has been shown previously to block infectivity of HBV in susceptible chimpanzees ((1983) J. Med. Virol. 16, 89-95). This antibody bound to a cyclical synthetic peptide analogue of amino acids 124 to 137 of the major HBsAg polypeptide.
A region of the fusion protein of measles virus (residues 240 to 252) was predicted to contain a B and a T epitope. A synthetic peptide representing this sequence was shown to induce both T and B cell reactivity in several inbred strains of mice, but the responses were clearly major histocompatibility complex-restricted. Elongation of this peptide by six residues at the C terminus on the basis of predictions for B cell epitopes resulted not only in increased peptide immunogenicity in some strains of mice but also produced strain-related positive and negative effects on the recognition of the peptide. BALB/c and SWR mice were non-responders to the short version of the peptide but responded well to the elongated form. On the other hand, the injection of the elongated peptide into C57BL/6 mice resulted in a loss of both B and T cell responsiveness seen with the short version. These results indicate the importance of flanking sequences on the immunogenicity and antigenicity of synthetic B and T cell epitopes and highlight the necessity to determine the most appropriate size of peptide to be used as an immunogen.
Chimeric peptides comprising B- and T-helper cell epitopes from the proteins of infectious agents represent immunogens with potential for use as new vaccines. However, it has become clear that the orientation of the epitopes, the presence of spacer residues and the number of copies of the epitopes influence the specificity, levels and affinity of the antibody produced following immunization with such constructs. Furthermore, the response to peptides is under genetic control leading to major histocompatibility complex (MHC)-linked non-responsiveness. In this study, we have investigated the potential of co-immunization of immunogenic peptides (to provide T-cell help) with non-immunogenic peptides (representing B-cell epitopes) to overcome the non-response to the latter. For this purpose, we have employed peptides representing T- and B-cell epitopes derived from the sequences of the fusion and haemagglutinin glycoproteins of measles virus. The results obtained show that simple co-immunization of a B-cell epitope with a T-cell epitope results in the production of antibody to the B-cell epitope without the requirement for covalent linkage of the two peptides. This approach could thus be used to overcome the problem of poor immunogenicity of peptides and will be of potential value in the design of immunization strategies using synthetic immunogens.
The influence of the epitope density of the antigen on antibody affinity values determined by fluid- and solid-phase immunoassays was assessed. The affinity of the interaction of a panel of monoclonal anti-DNP antibodies of different affinities (as determined by equilibrium dialysis) for DNP-protein conjugates of various hapten substitution ratios was used as the test system. The results obtained showed that the epitope density of the antigen markedly influences the observed affinity values obtained by both experimental approaches. However, the monoclonal antibodies were ranked in affinity terms by both assays in a similar order to that given by equilibrium dialysis. It is concluded that provided due care is exercised in choosing an appropriate epitope density for the test antigen, these methods can be used to provide rapid estimations of average antibody affinities.
The immunogenicity of chimeric peptides produced by collinear synthesis to contain both T and B cell epitopes from the fusion protein and the haemagglutinin of measles virus was studied in mice. The T cell epitope used was from the fusion protein (residues 288 to 302), which has been shown to be promiscuous in its binding to mouse major histocompatibility complex molecules. This epitope was coupled by (i) a glycine-glycine spacer to a B cell epitope from the fusion protein (residues 404 to 414) and (ii) either its amino or carboxy terminus to a neutralizing antibody epitope from the haemagglutinin (residues 188 to 199). The results obtained show that such chimeric peptides can indeed function as complete immunogens in a range of mouse strains of different H-2 haplotype, and can induce the production of antibodies which bind to the fusion protein and to measles virus. Furthermore, it was shown that the orientation of the T cell epitope with respect to the B cell epitope had a significant effect upon the immunogenicity and antigenic specificity of the chimera. This work gives further support to the concept of rationally designed synthetic peptide vaccines.
Previous work has shown that the synthetic peptide C-C-(200-213)-P-P-S-(141-158)-P-C-G, in which residues 200-213 and 141-158 correspond to immunogenic regions of the VP1 protein of foot-and-mouth disease virus (FMDV), is capable of inducing high levels of neutralizing antibody but only inconsistent protection of cattle against virulent FMDV challenge. The possibility exists that differences in affinity may well underlie the observed variations in biological effectiveness of the anti-peptide antibodies in immunized animals. This has been investigated by assessing the affinity for peptide and whole virus of the anti-peptide antibodies in sera from protected and non-protected cattle using both fluid-phase and solid-phase assays. The results obtained show that the affinities of serum antibodies for peptide and virus in protected cattle were significantly higher than those in non-protected animals. Thus in order to assess vaccine efficacy, particularly in the case where synthetic immunogens are employed, consideration should be given to the determination of antibody affinity in addition to other parameters of the antibody response.