Human pathogen subversion of antigen presentation.

Many pathogens have co-evolved with their human hosts to develop strategies for immune evasion that involve disruption of the intracellular pathways by which antigens are bound by class I and class II molecules of the major histocompatibility complex (MHC) for presentation to T cells. Here the molecular events in these pathways are reviewed and pathogen interference is documented for viruses, extracellular and intracellular bacteria and intracellular parasites. In addition to a general review, data from our studies of adenovirus, Chlamydia trachomatis and Coxiella burnetii are summarized. Adenovirus E19 is the first viral gene product described that affects class I MHC molecule expression by two separate mechanisms, intracellular retention of the class I heavy chain by direct binding and by binding to the TAP transporter involved in class I peptide loading. Coxiella and Chlamydia both affect peptide presentation by class II MHC molecules as a result of their residence in endocytic compartments, although the properties of the parasitophorous vacuoles they form are quite different. These examples of active interference with antigen presentation by viral gene products and passive interference by rickettsiae and bacteria are typical of the strategies used by these different classes of pathogens, which need to evade different types of immune responses. Pathogen-host co-evolution is evident in these subversion tactics for which the pathogen crime seems tailored to fit the immune system punishment.

The influence of exogenous peptide on beta2-microglobulin exchange in the HLA complex: analysis in real-time.

We used an optical biosensor to determine the relative binding affinity of peptides to purified HLA class I molecules. In this assay we monitor beta2-microglobulin (beta2m) exchange within the HLA-A2 molecule, whereby native beta2m in the complex is replaced by beta2m immobilized at the surface of the biosensor. Quantitative kinetic measurements permit us to obtain association rate (kass), dissociation rate (kdiss) and affinity constants (KA) for the beta2m exchange reaction, alone, (control) and in the presence of exogenous peptide. We tested a panel of six peptides which had been designed and synthesized with an HLA-A2 binding motif, and had also been tested by the T2-cell binding assay, along with control peptides. The biosensor results demonstrate that exogenous peptide influences the dynamics of beta2m exchange in a sequence-specific manner. Five of six peptides increased the association rate, decreased the dissociation rate, and significantly increased the affinity (KA=1. 55-1.88x10(9) M-1) of HLA-A2 for immobilized beta2m compared with the control (KA =1.14+/-0.04x10(9)M-1), demonstrating stabilization of the complex. One peptide was unable to stabilize the complex, as also shown in the T2 binding assay. However, analysis of peptide sequences demonstrated that the HLA-A2 secondary motif as well as primary motif residues are required for HLA-A2 stabilization. Further experiments demonstrated that beta2m exchange alone cannot stabilize the HLA class I complex at the cell surface until a peptide of sufficient binding affinity is bound. Hence kinetics equal to or below the control values in our biosensor assay probably represent an unstable complex in vivo. Unlike other methods described for the analysis of peptide stabilization, this approach is significantly faster, provides full kinetic analysis, and is simpler, since it requires no labeling of peptides. Furthermore, this may have important implications in the assessment of peptide vaccines.

Identification of three HLA-A*0201-restricted cytotoxic T cell epitopes in the cytomegalovirus protein pp65 that are conserved between eight strains of the virus.

The Ag specificity of the CTL response against CMV is directed almost entirely to a single CMV tegument protein, the phosphoprotein pp65. We report the identification of three peptides derived from the protein pp65 that displayed a high or intermediate binding to HLA-A*0201 molecules, which were also able to induce an in vitro CTL response in peripheral blood lymphocytes from CMV seropositive individuals. The peptide-specific CTLs generated were capable of recognizing the naturally processed pp65 either presented by CMV-infected cells or by cells infected with an adenovirus construct expressing pp65 in an HLA-A*0201-restricted manner. Thus, we were able to demonstrate responses to subdominant CTL epitopes in CMV-pp65 that were not detected in polyclonal cultures obtained by conventional stimulations. We also found that the amino acid sequences of the three peptides identified as HLA-A*0201-restricted CTL epitopes were conserved among different wild-type strains of CMV obtained from renal transplant patients, an AIDS patient, and a congenitally infected infant, as well as three laboratory strains of the virus (AD169, Towne and Davis). These observations suggest that these pp65 CTL peptide epitopes could potentially be used as synthetic peptide vaccines or for other therapeutic strategies aimed at HLA-A*0201-positive individuals, who represent approximately 40% of the European Caucasoid population. However, strain variation must be taken in consideration when the search for CTL epitopes is extended to other HLA class I alleles, because these mutations may span potential CTL epitopes for other HLA molecules, as it is described in this study.