Development of a Humanized HLA-A2.1/DP4 Transgenic Mouse Model and the Use of This Model to Map HLA-DP4-Restricted Epitopes of HBV Envelope Protein

A new homozygous humanized transgenic mouse strain, HLA-A2.1+/+HLA-DP4+/+ hCD4+/+mCD4−/−IAβ−/−β2m−/− (HLA-A2/DP4), was obtained by crossing the previously characterized HLA-A2+/+β2m−/− (A2) mouse and our previously created HLA-DP4+/+ hCD4+/+mCD4−/−IAβ−/− (DP4) mouse. We confirmed that the transgenes (HLA-A2, HLA-DP4, hCD4) inherited from the parental A2 and DP4 mice are functional in the HLA-A2/DP4 mice. After immunizing HLA-A2/DP4 mice with a hepatitis B DNA vaccine, hepatitis B virus-specific antibodies, HLA-A2-restricted and HLA-DP4-restricted responses were observed to be similar to those in naturally infected humans. Therefore, the present study demonstrated that HLA-A2/DP4 transgenic mice can faithfully mimic human cellular responses. Furthermore, we reported four new HLA-DP4-restricted epitopes derived from HBsAg that were identified in both vaccinated HLA-A2/DP4 mice and HLA-DP4-positive human individuals. The HLA-A2/DP4 mouse model is a promising preclinical animal model carrying alleles present to more than a quarter of the human population. This model should facilitate the identification of novel HLA-A2- and HLA-DP4-restricted epitopes and vaccine development as well as the characterization of HLA-DP4-restricted responses against infection in humans

Analysis of mutant HLA-A2 molecules. Differential effects on peptide binding and CTL recognition.

Previous studies have identified several residues lining the groove of the HLA-A2.1 molecule that are critical for Ag presentation. However, it is not clear whether these residues are critical for binding of the peptide epitope per se or for determining the appropriate conformation of bound peptide. To distinguish between these possibilities, mutations at eight of these residues have been tested for their effects on the ability of the molecule to bind and present two known peptide epitopes--one derived from the influenza A matrix protein, the other from HIV pol. With only one exception, the mutations were found to affect the binding of the two peptides similarly. Most of the mutations resulted in intermediate deleterious effects on binding, with the B pocket mutant F9Y having the most dramatic negative effect on binding for both peptides. Two of the mutations significantly enhanced binding of both peptides and a peptide-specific effect on binding was seen with the substitution, Y99H, which enhanced binding of the matrix peptide yet diminished binding of the pol peptide. In contrast to the effects on binding, the effects of the mutations on presentation differed considerably for the two peptides. The most striking difference was seen with two alpha 2 alpha helix mutants that are fully recognized by pol peptide-specific CTL but not recognized by matrix peptide-specific CTL even though levels of binding were comparably diminished for the two peptides. These results suggest that some interactions, although not critical for binding per se, are critical for functional binding and the importance of these interactions differs among peptide epitopes

Different MHC class I alleles compete for presentation of overlapping viral epitopes.

We previously identified an HLA-B8+ donor, NW, whose lymphoblastoid cells failed to present a B8-restricted epitope from the influenza A nucleoprotein following viral infection, although added peptide could still be presented. The failure to present through HLA-B8 following viral infection appears to be specific for the NP epitope. Here, we report that donor NW makes an HLA-B2702-restricted influenza-specific CTL response to an epitope in the nucleoprotein that overlaps the B8-restricted epitope by 8 aa. Two mechanisms for the failure of this cell line to present the B8-restricted epitope following viral infection are investigated. One is that there is an antigen processing polymorphism specific to the NW cell line, so that there is either preferential generation or preferential transport of the B2702 epitope. The other is that B8 and B2702 compete for a common peptide fragment in the ER and this leads to suboptimal loading of HLA-B8

Genetic evidence for difference between intracellular and extracellular peptides in influenza A matrix peptide-specific CTL recognition.

During the course of extensive mutagenesis of HLA-A2.1, we examined influenza A matrix peptide (FMP)-specific CTL recognition of HMy2.C1R (C1R) cells expressing mutant HLA-A2.1 molecules, sensitized with synthetic peptide, FMP 58-66, (exogenous peptide), or infected with influenza A virus (endogenous peptide). Most mutants showed equivalent presentation of exogenous and endogenous peptides to FMP-specific CTL. However, five of the mutants differed in this property. Two of the five mutants, F9L and T134K, present exogenous peptide to FMP-specific CTL, but fail to present endogenous peptide to CTL. Western blot analysis using anti-matrix protein Ab indicates that the matrix protein is expressed in these mutants after infection with virus. Interestingly, transfection of these two mutants with a minigene encoding FMP 58-66 results in efficient lysis by FMP-specific CTL. Peptide-binding assays demonstrate that the two mutations dramatically decrease the binding of FMP. However, these mutants bind FMP as well as wild type in the presence of exogenously added human beta 2-m, suggesting that the lower affinity for beta 2-m leads to the inability to present endogenous peptide. The remaining three mutants, Y27N, Q32K, and S132C, fail to present exogenous peptide, but present endogenous peptide to FMP-specific CTL. Pulse-chase analyses followed by endoglycosidase-H treatment show that the rate of maturation and processing of the five mutant HLA-A2 molecules in C1R cells is identical to that of wild type. Overall, this study suggests that the assembly and subsequent recognition of endogenous peptide differs from that of exogenous peptide

Engagement of a T cell receptor by major histocompatibility complex irrespective of peptide.

T cell receptors (TCR) identify target cells presenting a ligand consisting of a major histocompatibility complex molecule (MHC) and an antigenic peptide. A considerable amount of evidence indicates that the TCR contacts both the peptide and the MHC components of the ligand. In fully differentiated T cells the interaction between the peptide and the TCR makes the critical contribution to eliciting a cellular response. However, during the positive selection of thymocytes the contribution of peptide relative to MHC is less well established. Indeed it has been suggested that the critical interaction for positive selection is between the TCR and the MHC molecule and that peptides can be viewed as either allowing or obstructing this contact. This predicts that a given TCR is capable of engaging multiple MHC/peptide complexes. In this study a system is described which detects simply engagement of the TCR by MHC/peptide complexes rather than the functional outcome of such interactions. Using this approach the extent to which peptides can influence contacts between the TCR and the MHC molecule has been examined. The results show that the TCR does in fact engage a wide range of ligands in an MHC-restricted but largely peptide-independent manner, suggesting that only a few peptides are able to prevent the TCR from contacting the MHC molecule

Induction of antigen-specific CD8+ T cells, T helper cells, and protective levels of antibody in humans by particle-mediated administration of a hepatitis B virus DNA vaccine.

A DNA vaccine against the hepatitis B virus (HBV) was evaluated for safety and induction of immune responses in 12 healthy, hepatitis-naïve human volunteers using the needle-free PowderJect system to deliver gold particles coated with DNA directly into cells of the skin. Three groups of four volunteers received three administrations of DNA encoding the surface antigen of HBV at one of the three dose levels (1, 2, or 4 microg). The vaccine was safe and well tolerated, causing only transient and mild to moderate responses at the site of administration. HBV-specific antibody and both CD4+ and CD8+ T cell responses were measured before and after each immunization. All the volunteers developed protective antibody responses of at least 10 mIU/ml. In volunteers who were positive for the HLA class I A2 allele, the vaccine also induced antigen-specific CD8+ T cells that bound HLA-A2/HBsAg(335-343) tetramers, secreted IFN-gamma, and lysed target cells presenting a hepatitis B surface antigen (HBsAg) CTL epitope. Enumeration of HBsAg-specific T cells producing cytokine indicated preferential induction of a Type 1 T helper cell response. These results provide the first demonstration of a DNA vaccine inducing protective antibody titers and both humoral and cell-mediated immune responses in humans