Orientation of epitopes influences the immunogenicity of synthetic peptide dimers.

The immunogenicity of synthetic peptide dimers based on epitope sequences derived from the mycobacterial 65-kDa antigen and the foot and mouth disease virus (FMDV) VP1 protein was examined in inbred mice. The analysis was directed towards the potential helper role of a T cell stimulatory mycobacterial epitope (65-85) with respect to poorly immunogenic sites either from the same molecule (422-436) or from VP1 (141-160). The 65-85 repeat homodimer induced an antibody response in CBA/ca but not in C57BL/6 mice, both nonresponders to the 65-85 monomer, and amplified the antibody response in BALB/c, monomer-responder mice. Analysis of the immunogenicity of hybrid dimers in BALB/c mice showed that the orientation of peptides within the dimer is critical for the extent of the produced antibody response. Only the 422-436/65-85 but not the 65-85/422-436 induced antibodies binding to the 422-436 sequence which was nonimmunogenic when injected either as a monomer or dimer. Despite the striking difference in immunogenicity, both tested hybrid dimers reacted equally in the solid-phase immunoassay with antisera raised to 65-85-dimer or 422-436/65-85 peptides or with a monoclonal antibody to the 422-436 epitope. The described differences in antibody responsiveness also cannot be attributed merely to the extent of T cell stimulation since the proliferative responses were uniformly expressed for all relevant combinations of peptides. Antisera to 65-85 dimer and 422-436/65-85 hybrid also reacted with the native 65-kDa protein. Furthermore, the production of FMDV-neutralizing antibodies in response to the 141-160 (VP1-derived)/65-85 hybrid peptide in 141-160 nonresponder B10.D2 mice also confirmed the helper activity of the 65-85 epitope. Thus, combining heterologous peptides with the N-terminal of the mycobacterial 65-85 sequence may be generally applicable for the potentiation of peptide vaccines.

A high proportion of anti-peptide antibodies recognize foot-and-mouth disease virus particles.

Synthetic peptides representing the amino acid sequence 141-160 of the structural protein VP1 of foot-and-mouth disease virus (FMDV) elicit virus-neutralizing antibody. Absorption of anti-peptide sera with purified virus particles removed all detectable virus-binding and neutralizing activity, and reduced the ELISA titres against the homologous peptide by 31-41%. The proportion of anti-peptide antibodies that also recognized virus was unaffected by whether the peptide had been inoculated free, carrier-linked or as part of a fusion protein. The majority of these antibodies reacted with sites composed of residues 142-150. Peptides extended at the amino terminus, into regions shown to be poorly antigenic on the intact virus, induced greater neutralizing responses by increasing the proportion of virus-binding antibodies recognizing region 141-150 from 35% to 70%. However, the total proportion of activity against the longer homologous peptide removed by virus absorption remained within the range 31-41%.

Non-responsiveness to a foot-and-mouth disease virus peptide overcome by addition of foreign helper T-cell determinants.

Study of the immune response to synthetic antigens has shown that uncoupled peptides can realize their potential as vaccines only if they contain domains that react with helper T-cell receptors and Ia antigens in addition to antibody binding sites. Here we consider whether genetically restricted non-responsiveness to an uncoupled peptide could be overcome by synthesizing a peptide with an additional helper T-cell epitope from a different protein. We demonstrate that H-2d mice, which are non-responders to the 141-160 VP1 peptide of foot-and-mouth disease virus (FMDV), can be converted into responders by immunization with peptides containing the FMDV sequence with defined 'foreign' helper T-cell determinants from ovalbumin or sperm whale myoglobin. Furthermore, the virus-neutralizing activity of the antibody raised against peptide was dependent on the determinant used. Thus, FMDV peptides with the added sequences 323-339 from ovalbumin and 132-148 from sperm-whale myoglobin elicited a high degree of neutralizing activity in B10.D2 mice. The sera from mice which received the peptide with the added sequence 105-121 from sperm whale myoglobin did not neutralize the virus, although they had high levels of anti-141-160 FMDV peptide activity. Our data indicate that the T-cell help given by the 'foreign' epitopes is B-cell clone specific. These results are likely to have important implications for the design of peptide vaccines.

Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein.

Synthetic vaccines for viral diseases can use defined regions of viral proteins as immunogens: the peptide sequence of amino acids 141-160 of the VP1 protein of foot and mouth disease virus (FMDV) elicits virus-neutralizing antibodies to protect guinea pigs, cattle and pigs either when coupled to a carrier protein or when administered in liposomes or in incomplete Freund's adjuvant. The immune response to these peptides is much lower than that to complete virus particles and the same sequence fused to the N terminus of beta-galactosidase did not produce a more potent immunogen than synthetic peptide alone. We report here an expression system for immunogenic epitopes linked to a carrier protein, hepatitis B core antigen, to form part of a virus-like complex which can present these epitopes to the immune system at high density. The immunogenicity of these structures approaches that of FMDV particles.

A synthetic peptide which elicits neutralizing antibody against human rhinovirus type 2.

Synthetic peptides corresponding to six predicted immunogenic sites on human rhinovirus type 2 (HRV2) have been tested for their reactivity with an anti-virion antibody and for their ability to elicit neutralizing antibody. Four of the peptides reacted with HRV2 antiserum in an indirect ELISA. Rabbit antisera produced to three of these four peptides, one each from VP1, VP2 and VP3, reacted with the virus in an indirect ELISA and with the corresponding proteins by Western blotting. Furthermore, antiserum to one of the peptides, designed to cover the neutralization epitope NIm-II on VP2, not only reacted well in a sandwich ELISA and in an immunoprecipitation test but also neutralized virus infectivity.

Demonstration of neutralizing and non-neutralizing epitopes on the trypsin-sensitive site of foot-and-mouth disease virus.

The isolation of monoclonal antibodies directed against the trypsin-sensitive site on the 140S particle of foot-and-mouth disease virus (FMDV) has enabled the demonstration of at least three distinct epitopes within this site. Reaction with two of these resulted in neutralization of virus infectivity. None of the epitopes appeared to be present on the 12S particles, and one of the neutralizing epitopes was sensitive to even milder configurational changes of the particle.

Studies on the stability of foot-and-mouth disease virus using absorbance - temperature profiles.

The main immunogenic component of FMD virus harvests is the intact 140S virus particle. For the production of stable vaccines it is important to ensure that virus strains having a stable capsid should be used. The method of measuring the heat stability of FMD virus by following the increase in optical density of purified virus during capsid breakdown as the temperature is increased was described in 1964 (P. Bachrach, 1964, J. Mol. Biol 8. 348). We have investigated this technique in the hope that it might provide a means of screening virus isolates rapidly to eliminate unstable strains. Using this method we have been unable to follow the course of virus breakdown from the absorbance profile alone. A much more precise indication has been obtained by including ribonuclease in the virus sample. This enhances the optical density rise which occurs when the capsid disintegrates and the RNA becomes accessible. A sigmoid curve is obtained from which 50% breakdown temperature can be calculated. The presence of RNase does not appear to alter the temperature at which breakdown occurs since parallel samples heated in the absence of RNase showed the same behaviour when virus breakdown was measured by sucrose gradient analysis. With a given virus preparation the method gives highly reproducible results. Typical standard deviations for the 50% breakdown point were about 0.5 degrees C. However, the results are influenced by the method of purification of the virus. Virus purified by CsCl gradient centrifugation was less stable than virus purified on sucrose gradients. Thus the environmental conditions through which the virus passes during processing may affect its stability.(ABSTRACT TRUNCATED AT 250 WORDS)