Background: T cells recognize a complex between a specific major histocompatibility complex (MHC) molecule and a particular pathogen-derived epitope. A given epitope will elicit a response only in individuals that express an MHC molecule capable of binding that particular epitope. MHC molecules are extremely polymorphic and over a thousand different human MHC (HLA) alleles are known. A disproportionate amount of MHC polymorphism occurs in positions constituting the peptide-binding region, and as a result, MHC molecules exhibit a widely varying binding specificity. In the design of peptide-based vaccines and diagnostics, the issue of population coverage in relation to MHC polymorphism is further complicated by the fact that different HLA types are expressed at dramatically different frequencies in different ethnicities. Thus, without careful consideration, a vaccine or diagnostic with ethnically biased population coverage could result.

Continuing antigenic drift allows influenza viruses to escape antibody-mediated recognition, and as a consequence, the vaccine currently in use needs to be altered annually. Highly conserved epitopes recognized by effector T cells may represent an alternative approach for the generation of a more universal influenza virus vaccine. Relatively few highly conserved epitopes are currently known in humans, and relatively few epitopes have been identified from proteins other than hemagglutinin and nucleoprotein. This prompted us to perform a study aimed at identifying a set of human T-cell epitopes that would provide broad coverage against different virus strains and subtypes. To provide coverage across different ethnicities, seven different HLA supertypes were considered. More than 4,000 peptides were selected from a panel of 23 influenza A virus strains based on predicted high-affinity binding to HLA class I or class II and high conservancy levels. Peripheral blood mononuclear cells from 44 healthy human blood donors were tested for reactivity against HLA-matched peptides by using gamma interferon enzyme-linked immunospot assays. Interestingly, we found that PB1 was the major target for both CD4؉ and CD8 ؉ T-cell responses. The 54 nonredundant epitopes (38 class I and 16 class II) identified herein provided high coverage among different ethnicities, were conserved in the majority of the strains analyzed, and were consistently recognized in multiple individuals. These results enable further functional studies of T-cell responses during influenza virus infection and provide a potential base for the development of a universal influenza vaccine.

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.

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