Malaria parasite-inhibitory antibody epitopes on Plasmodium falciparum merozoite surface protein-1(19) mapped by TROSY NMR.

Plasmodium falciparum merozoite surface protein 1 (MSP1)(19), the C-terminal fragment of merozoite surface protein 1, is a leading candidate antigen for development of a vaccine against the blood stages of the malaria parasite. Many human and animal studies have indicated the importance of MSP1(19)-specific immune responses. Anti-MSP1(19) antibodies can prevent invasion of red blood cells by P. falciparum parasites in vitro. However, the fine specificity of anti-MSP1(19) antibodies is also important, as only a fraction of monoclonal antibodies (mAbs) have parasite-inhibitory activity in vitro. Human sera from malaria-endemic locations show strong MSP1(19) reactivity, but individual serum samples vary greatly in inhibitory activity. NMR is an excellent method for studying protein-protein interactions, and has been used widely to study binding of peptides representing known epitopes (as well as non-protein antigens) to antibodies and antibody fragments. The recent development of transverse relaxation optimized spectroscopy (TROSY) and related methods has significantly extended the maximum size limit of molecules that can be studied by NMR. TROSY NMR experiments produce high quality spectra of Fab complexes that allow the mapping of epitopes by the chemical shift perturbation technique on a complete, folded protein antigen such as MSP1(19). We studied the complexes of P. falciparum MSP1(19) with Fab fragments from three monoclonal antibodies. Two of these antibodies have parasite-inhibitory activity in vitro, while the third is non-inhibitory. NMR epitope mapping showed a close relationship between binding sites for the two inhibitory antibodies, distinct from the location of the non-inhibitory antibody. Together with a previously published crystal structure of the P. falciparum MSP1(19) complex with the Fab fragment of another non-inhibitory antibody, these results revealed a surface on MSP1(19) where inhibitory antibodies bind. This information will be useful in evaluating the anti-MSP1(19) immune response in natural populations from endemic areas, as well as in vaccine trials. It will also be valuable for optimizing the MSP1(19) antigen by rational vaccine design. This work also shows that TROSY NMR techniques are very effective for mapping conformational epitopes at the level of individual residues on small- to medium-sized proteins, provided that the antigen can be expressed in a system amenable to stable isotope labelling, such as bacteria or yeast.

Precise epitope mapping of malaria parasite inhibitory antibodies by TROSY NMR cross-saturation.

We have applied NMR cross-saturation with TROSY detection to the problem of precisely mapping conformational epitopes on complete protein antigen molecules. We have investigated complexes of the Fab fragments of two antibodies that have parasite inhibitory activity, bound to the important malaria vaccine candidate antigen, Plasmodium falciparum MSP1(19). The results indicate remarkable overlap between these epitopes for inhibitory antibodies, and will provide a basis for theoretical modeling of the antibody-antigen interface.

Azidodeoxythymidine and didehydrodeoxythymidine as inhibitors and substrates of the human herpesvirus 8 thymidine kinase.

Human herpesvirus 8 (HHV-8), the aetiological agent of Kaposi's sarcoma (KS), encodes many core genes that have been maintained during evolution of the Herpesviridae. Among these is a thymidine kinase (TK) homologue (ORF21), which has 12% homology to the related TK encoded by herpes simplex virus. We show that the HHV-8 TK is a functional deoxythymidine (dT) kinase, with Michaelis constants (K(m)) for dT and ATP of 18.5 and 6.6 microM, respectively. Using homology modelling coupled with site-directed mutagenesis, we identify Gly265, Asp362 and Phe372 as key amino acid residues involved in the catalytic process. The HHV-8 TK is competitively inhibited by azidodeoxythymidine (zidovudine) and didehydrodeoxythymidine (stavudine) and can also accept these anti-retroviral compounds as substrates. These data have implications for our understanding of changes in AIDS-KS incidence following the clinical licensing of these compounds and in the development of new therapies for KS.