SummaryTransgenic murine lines have been constructed that express a chimeric class I molecule composed of the cx1 and u2 domains of HLAA2.1 and the 0, transmembrane, and cytoplasmic domains of H-2Kb. Upon immunization with influenza virus, transgenic mice developed a strong A2.1Kb-restricted cytotoxic T lymphocyte (CTL) response specific for the same matrix protein epitope that serves as the dominant A2.1-restricted determinant in the equivalent human response. Fine specificity analysis of CTL clones using truncated peptides revealed strong similarity between the response repertoire of transgenic mice and that previously reported using influenza-specific A2.1-restricted CTL clones from humans. This suggests that even when considering T cell responses by different species, the a1 and ot2 domains of the restriction element play a dominant role in determining the CTL specific repertoire. Thus, substituting the a3 domain of A2.1 with a murine counterpart has permitted development of a transgenic strain that should serve as an excellent model system in studies of HLArestricted responses .

Antiviral cytotoxic T-cells are critical for control of lymphocytic choriomeningitis virus (LCMV) infection in mice. In H-2b mice, the antiviral response is directed against three Db-restricted epitopes in the viral nucleoprotein (NP396-404) and glycoprotein (GP276-286 and GP33-41). Our present data revealed a clear hierarchy among these three epitopes, in which NP396-404 is immunodominant, followed by GP33-41 and GP276-286, respectively. In order to identify additional CTL epitopes in the LCMV nucleoprotein and glycoprotein, we used the motifs for Db2- and Kb-binding peptides, combined with MHC class I-binding assays. Out of 23 Db motif-fitting peptides, we identified 4 Db binders, one of which (GP92-101) turned out to be a new CTL epitope. Among 28 Kb motif-fitting peptides, 12 bound Kb, and one of these (NP205-212) was a CTL epitope. Both newly identified CTL peptides were recognized by LCMV-immune splenocytes after secondary in vitro stimulation. Both peptides bound their MHC class I molecules with intermediate affinity (470 and 170 nM for GP92-101 and NP205-212, respectively). Responses against these peptides were weaker than the responses against the three major epitopes. None of the high affinity binders were new epitopes, suggesting that high affinity binders are either immunodominant epitopes or no epitopes at all. Thus, analysis of 51 Kb and Db motif-fitting peptides yielded 2 new, subdominant epitopes. Immunization of C57BL/6 mice with these peptides, or vaccinia virus recombinants expressing these epitopes as minigenes, protected against chronic LCMV infection, demonstrating that immunization with subdominant epitopes can confer protection against chronic viral infection.

We recently described human leukocyte antigen (HLA) A2, A3 and B7 supertypes, characterized by largely overlapping peptide-binding specificities and represented in a high percentage of different populations. Here, we identified 17 Plasmodium falciparum peptides capable of binding these supertypes and assessed antigenicity in both vaccinated and naturally exposed populations. Positive cytotoxic T lymphocyte recall and cytokine (interferon-gamma and tumor necrosis factor alpha) responses were detected for all peptides; all were recognized in the context of more than one HLA class I molecule; and at least 12 of the 17 were recognized in the context of all HLA alleles studied. These data validate the concept of HLA supertypes at the biological level, show that highly degenerate peptides are almost always recognized as epitopes, and demonstrate the feasibility of developing a universally effective vaccine by focusing on a limited number of peptide specificities.

We have previously experimentally analyzed the structural requirements for interaction between peptide antigens and mouse major histocompatibility complex (MHC) molecules of the d haplotype. We describe here two procedures devised to predict specifically the capacity of peptide molecules to interact with these MHC class II molecules (IAd and IEd). The accuracy of these procedures has been tested on a large panel of synthetic peptides of eukaryotic, prokaryotic, and viral origin, and also on a set of overlapping peptides encompassing the entire staphylococcal nuclease molecule. For both sets of peptides, IAd and 1Ed binding was successfully predicted in =75% of the cases. This suggests that definition of such sequence "motifs" could be of general use in predicting potentially immunogenic peptide regions within proteins.There is at present compelling evidence in support of the concept that T cells recognize a complex formed between major histocompatibility complex (MHC) molecules and "processed" antigen. The structural requirements for this interaction have been the object of intense investigation. In a previous study (1), the effect of a large number of single amino acid substitutions of an ovalbumin-derived peptide that binds strongly to ] on its capacity to bind purified IAd molecules was examined. Significant changes were only detected in a six-amino acid core region ] that had also been strongly implicated as crucial for binding by analysis of a set of N-and C-terminal truncated analogs. In a subsequent study (2), it was demonstrated that unrelated peptides that are good lAd binders contained regions ("motifs") that were structurally similar to each other and to . More recently, it was shown (A.S., L. Adorini, S.B., E.A., and H.M.G., unpublished data) that a different amino acid sequence pattern characterizes the interaction between peptide antigens and the other class II MHC molecule, lEd. The motif recognized by IEd molecules appears to be independent of the motif recognized by IAd and is defined by the presence of positively charged amino acids (refs. 3 and 4; A.S., L. Adorini, S.B., E.A., and H.M.G., unpublished data).In the present study, this information was used to derive a procedure that identifies motifs. Using a large panel of peptides, the presence of either an IAd or IEd motif as determined by this procedure was compared to the experimentally determined capacity of a given peptide to bind IAd and IEd. The peptide data base contained 62 peptides and, in addition, a set of peptides encompassing the entire staphylococcal nuclease molecule. In these sets ofpeptides, IAd and IEd binding was successfully predicted in ==75% of the cases, suggesting that similar procedures should be useful in selecting peptides capable of interacting with other MHC specificities, identify possible autoantigens in self molecules or specific blockers of autoimmune diseases, and because ofthe previously demonstrated strong correlation between MHC binding and immunogenicity (5) be useful in selecting potentially immun...

Chronic administration of protein therapeutics may elicit unacceptable immune responses to the specific protein. Our hypothesis is that the immunogenicity of protein drugs can be ascribed to a few immunodominant helper T lymphocyte (HTL) epitopes, and that reducing the MHC binding affinity of these HTL epitopes contained within these proteins can generate drugs with lower immunogenicity. To test this hypothesis, we studied the protein therapeutic erythropoietin (Epo). Two regions within Epo, designated Epo 91–120 and Epo 126–155, contained HTL epitopes that were recognized by individuals with numerous HLA-DR types, a property common to immunodominant HTL epitopes. We then engineered analog epitopes with reduced HLA binding affinity. These analog epitopes were associated with reduced in vitro immunogenicity. Two modified forms of Epo containing these substitutions were shown to be bioactive and nonimmunogenic in vitro. These findings support our hypothesis and demonstrate that immunogenicity of protein drugs can be reduced in a systematic and predictable manner.