Search

Primary proliferative responses can be initiated by adding dendritic cells pulsed with antigen to autologous T cells in 20-microliter hanging drop cultures. To identify primary T-cell epitopes of HIV gag, a series of 23 overlapping peptides, 15 amino acids long, spanning the p24 region were used. Significant proliferative responses were induced in cells from healthy HIV-negative donors by 11 of these peptides. One of two peptides that bound human leukocyte antigen (HLA)-A *0201 in a peptide-binding assay using the antigen-processing defective cell line T2 also induced a primary proliferative response. Primary T-cell proliferation was seen in response to some peptides of gag that have not previously been identified as T-cell epitopes in cells from infected individuals. These epitopes might be useful not only for vaccines in antigenically naive individuals but also might increase the breadth of immune responses in seropositive patients.

Understanding the factors which regulate the repertoire of a T cell response is important when selecting T helper cell epitopes for inclusion in synthetic viral vaccines. In this study we have examined the T cell response to human rhinovirus (HRV) type 1 A in a mouse model system, using a comprehensive set of synthetic peptides which span all four of the proteins which make up the HRV capsid. This constitutes the first study to use a set of peptides covering the entire sequence of all structural proteins of any virus. This study identifies the major proliferative (CD4) T cell epitopes within the minor receptor group HRV 1 A, and analyzes these epitopes with relation to their location within the three-dimensional structure of the virus. The proliferative response to HRV is highly selective, with strong responses to only a very small number of epitopes, many of which are grouped together within restricted areas of the primary structure of the HRV proteins. The repertoire of the response is almost entirely specific to the major histocompatibility complex haplotype of the host. The major T cell epitopes are spatially distinct from the sites of the major antibody recognition sites, and are buried within the viral capsid. In striking contrast to the antibody responses, the T cell responses are highly cross-reactive against a wide variety of viral serotypes.

Human rhinovirus serotypes (HRV) fall into two distinct groups, major and minor, by virtue of their cell receptor-binding ability. In this study minor receptor-binding group viruses are demonstrated to bind directly to cells of the murine immune system, including lymphoid dendritic cells which act as antigen-presenting cells, although they do not produce a productive infection in murine cells. This binding is specific and can be blocked by other serotypes of minor-group HRV. Pre-treatment of HRV 1A, a minor-group virus, with HRV 1A-specific antibodies inhibited the cellular proliferation of murine virus primed T helper cells, whereas antibody treatment of HRV 15, a non-binding major serotype, gave no inhibition. The cell binding ability of minor-group HRV played a role in the overall immunogenicity of this virus group, which was shown to be enhanced compared to the immunogenicity of major-group viruses in mice.

Lymphocytes from mice immunized with human rhinovirus (HRV) serotypes 1A or 15 proliferated in vitro in response to HRV and the activated cells were shown to be helper T (Th) cells. Lymphocytes from mice primed with HRV-1A responded to seven of eight heterologous virus serotypes, the responses to other minor cell receptor group viruses being greater than to those belonging to the major cell receptor group. A similar bias was seen with cells from mice primed with HRV-15 in that they responded preferentially to other major receptor group viruses. This pattern of cross-serotype recognition was shown to be similar in three inbred mouse strains and was not dependent upon the major histocompatibility complex haplotype. These results have revealed that there are determinants within the viral proteins of a number of serotypes of HRV that are recognized by Th cells primed against a single HRV serotype. Thus, at the level of Th cell recognition of HRV, a cross-serotype reactivity is seen which is not reflected in the B cell antibody response to virus, which is generally highly serotype-specific.

The presentation of heterologous amino acid sequences on the surface of hepatitis B core antigen (HBcAg) particles has been studied using a defined linear neutralization site from human rhinovirus (HRV). Previous work has shown that fusion particles, in which the HRV peptide sequence is linked to the amino terminus of the HBcAg protein, induce excellent immune responses in experimental animals. Using predictive models of HBcAg particulate structure and the approximate location of the major immunogenic regions we have designed and constructed bacterial expression vectors which direct synthesis of chimeric particles in which heterologous sequences are presented within an immunodominant area on the particle. Immunological responses to the heterologous peptide sequence are improved by at least tenfold when compared with amino terminal fusions of the same peptide sequence to HBcAg. Moreover, the restriction placed on the heterologous peptide by its linkage at both ends within the HBcAg protein results in a more constrained structure. In the case of the rhinovirus peptide sequence this results in an antigenic conformation more closely resembling that on the native virus particle. Such a system lends itself well as a general approach to the induction of high titre antibodies against defined epitopes.

The multiple antigenic peptide (MAP) system for presenting epitopes to the immune system has been studied with an immunogenic foot-and-mouth disease virus (FMDV) peptide comprising amino acids 141-160 of protein VP1. Neutralizing antibody responses known to protect guinea-pigs against challenge infection were obtained with a single inoculation of 0.8-4 micrograms of peptide, presented as an octamer or a tetramer, whereas 20 micrograms of a dimer were required to evoke a similar level of antibody. A monomeric preparation did not elicit measurable levels of neutralizing antibody at doses up to 20 micrograms. The octameric MAP was also immunogenic using an aluminum hydroxide adjuvant. Antibodies elicited by the octameric, tetrameric and dimeric constructs differed qualitatively in their reaction with sequences within the 141-160 peptide. Those against the octamer reacted poorly with peptides within the 141-160 sequence, whereas those elicited by the tetramer and dimer reacted preferentially with the peptides covering the N-terminal region. The levels of neutralizing antibody obtained with the octamer and tetramer compare favourably with those obtained when the FMDV peptide is attached to carrier proteins but are lower than those obtained when it is presented as part of a peptide-hepatitis B virus core particle. Nevertheless, the ability to elicit protective levels of neutralizing antibody without the use of a carrier protein would be a distinct advantage in the development of synthetic peptide vaccines.

Synthetic peptides representing amino acids 156 to 170 of virus capsid protein VP2 from human rhinovirus (HRV) type 2 have previously been used to elicit a neutralizing antibody response; polyclonal antisera against this peptide have been compared with a virus-neutralizing monoclonal antibody (8F5) which recognizes the same linear sequence. The neutralizing activity of both antibodies against virus is abolished by an amino acid substitution in VP2 at position 163 but only the peptide antiserum neutralizing activity is affected by a change in VP2 at position 158. The minimal binding site of 8F5 has been mapped, using overlapping peptides, to a sequence TRLNPD covering VP2 residues 160 to 165. Using affinity purification it has been shown that 8 to 10% of total neutralizing activity of polyclonal rabbit or guinea-pig antivirus antiserum is due to recognition of this linear VP2 156 to 170 determinant. Furthermore, a similar population of neutralizing antibodies appears to be associated with an immune response to recent HRV infection in humans. These results confirm the existence of linear determinants on the surface of HRV which may be mimicked by suitable synthetic peptides.

We recently reported the enhanced immunogenicity of a peptide epitope when it was presented as a fusion protein with hepatitis B core antigen. In those experiments the fusion protein was expressed in vaccinia virus. We have now refined the system so that large amounts of highly immunogenic particles can be produced using a simple bacterial expression system. We describe the expression of three different viral epitopes as chimeric particles that induce good antibody responses to each epitope after one dose of low amounts of antigen. Finally we demonstrate that the immunogenicity is a reflection of both T helper cell sites within the core protein and also the particulate nature of the immunogens.

The immunogenicity of a 19 amino acid peptide from foot-and-mouth disease virus has previously been shown to approach that of the inactivated virus from which it was derived after multimeric particulate presentation as an N-terminal fusion with hepatitis B core antigen. In this report we demonstrate that rhinovirus peptide-hepatitis B core antigen fusion proteins are 10-fold more immunogenic than peptide coupled to keyhole limpet hemocyanin and 100-fold more immunogenic than uncoupled peptide with an added helper T-cell epitope. The fusion proteins can be readily administered without adjuvant or with adjuvants acceptable for human and veterinary application and can elicit a response after nasal or oral dosing. The fusion proteins can also act as T-cell-independent antigens. These properties provide further support for their suitability as presentation systems for "foreign" epitopes in the development of vaccines.

Uncoupled peptides from all seven serotypes of foot-and-mouth disease virus (FMDV) protein VP1 have been used to elicit neutralizing antibody responses in guinea-pigs. The responses were largely serotype specific, although some significant cross-neutralization was observed. Dimeric tandem peptides have also been used to simultaneously elicit neutralizing antibodies to two different FMDV serotypes. The possible existence of structural features common to the B-cell neutralization sites or the guinea-pig helper T-cell sites within all seven peptides are analysed and discussed.

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.

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.

Polyclonal antisera with pre-determined specificities for a range of rat IgE epitopes were produced by immunizing rabbits with KLH-conjugates of five different synthetic peptides representing sequences 378-396, 414-428, 491-503, 522-535 and 560-571 in the CH3 and CH4 domains of rat IgE. Each rabbit elicited peptide-specific antibodies which were capable of binding affinity-purified rat IgE (IR162) (titres 1/1000-1/10,000) and IgE in rat immunocytoma serum (IR162) either immobilized on microtitre-plates or in free-solution as assessed by ELISA. Heating a solution of rat IgE at 56 degrees C for 1 hr, a treatment known to abolish the cytophilic activity of rat IgE and also induce irreversible conformational changes in the CH3 and CH4 domains, resulted in enhanced binding of the immunoglobulin to antibodies directed against IgE sequences represented by two of the synthetic peptides 414-428 and 491-503, but not to the three other peptides. The five anti-peptide sera together with two previously studied antisera specific for rat IgE sequences 459-472 and 542-557 were tested in functional assays designed to investigate the mode of interaction between rat IgE and its receptor on rat mast cells. Each anti-peptide serum was capable of inhibiting the binding of IgE to mast cells and furthermore, able to initiate the secretion of histamine from cells sensitized with rat IgE in an "anti-IgE"-induced manner. In view of the evidence implicating the CH3 and/or CH4 domains as the location of the mast cell receptor-site on rat IgE, we propose a model to describe the mode of interaction between IgE and its mast cell receptor.

A mouse monoclonal antibody (mAb) has been produced by conventional cell fusion methods against a synthetic peptide, p123, representative of a portion of the CH4 domain of rat immunoglobulin E (IgE). This monoclonal antibody was reactive with both peptide and purified rat IgE (p.rat IgE) by indirect enzyme immunosorbent assay (ELISA), and its binding to p.rat IgE was found to be inhibitable by pre-incubation with rat ascitic fluid containing the immunocytoma 162 (IR162) IgE. Heating of the immunocytoma IgE in solution at 56 degrees for 1 hr resulted in its enhanced binding of the mAb. The effect of this treatment was investigated further using p.rat IgE heated at 56 degrees for various time intervals between 0 and 60 min. The mAb showed enhanced binding to IgE heated for as little as 10 min, a similar level of binding being shown by samples heated for 30 and 60 min. The degree of aggregation of the IgE molecules brought about by the heat treatments was measured by differential UV absorption. This revealed a decrease in the proportion of monomeric IgE with an accompanying increase in the percentage of dimer and larger aggregates with increased time of heating at 56 degrees. These absorption data, together with the ELISA inhibition data, suggest that, rather than inducing changes mediated by aggregation of the IgE molecules in solution, heating at 56 degrees causes subtle alterations in the conformation of individual IgE molecules at specific sites within their CH4 domains, one of which is detected by this mAb.

Two peptides, P123 and P124, representing amino acid sequences His 542-Lys 557 and Tyr 459-Arg 472, respectively, of the CH4 domain of rat IgE and predicted to be located on accessible regions of the protein were synthesized by a solid-phase procedure. Rabbits were immunized with the peptides conjugated to KLH and their antisera were tested for reactivity with free peptide and rat IgE by inhibition-ELISA. Each animal produced antibodies which reacted specifically with its immunizing peptide (titre greater than 1/62,500), but not with other synthetic peptides of similar chain-length and composition. Antisera directed against peptides P123 and P124 specifically bound purified rat IgE (IR 162) and IgE in whole myeloma serum (greater than 1/6400), but showed no reaction with normal rat serum proteins and only very low binding to purified human IgE. In addition the binding of anti-peptide sera to rat IgE could be completely inhibited with either homologous peptide or purified rat IgE, but not by other peptides or purified human IgE. Heating rat IgE for 1 hr at 56 degrees C enhanced its binding to anti-peptide antibodies by between 4- and 60-fold, but markedly reduced its reactivity with a rabbit anti-rat IgE (Fc) serum. These results suggest that antibodies directed against the synthetic peptides employed recognize and specifically bind to sites within the CH4 domain of rat IgE represented by their respective immunizing peptides. Furthermore, these antibodies are capable of detecting subtle alterations in structural conformation resulting from heating at 56 degrees C. Epitopes represented by peptides P123 and P124 may contribute to the heat-sensitive cytophilic region of rat IgE.

As an alternative strategy to the use of proteolytic and chemical cleavage in the production of fragments of immunoglobulins retaining Fc effector functions, peptides representative of amino acid sequences constituting the putative active sites have been synthesized and assessed for biological activity in various in vitro systems. This approach has been adopted in attempts to define more precisely the autoantigenic epitope on human IgG against which anti-gamma-globulin antibodies (the so-called general 'rheumatoid factors'), found in the sera and joint fluids of patients with rheumatoid arthritis, are directed. Synthetic peptides representative of epsilon-chain sequences are being used in the production of antibodies (polyclonal and monoclonal) directed against specific epitopes within the Fc regions of human and rat IgE. The ability of these antisera to influence the in vitro functional properties of IgE anaphylactic antibodies is now under investigation, with particular attention being focused on cytophilicity and mast cell triggering. Preliminary findings suggest that certain of the antisera might be capable of inhibiting mast cell sensitization by IgE antibodies, and therefore might form the basis of a new type of anti-allergy compound.