A HECT ubiquitin-protein ligase as a novel candidate gene for altered quinine and quinidine responses in Plasmodium falciparum.

The emerging resistance to quinine jeopardizes the efficacy of a drug that has been used in the treatment of malaria for several centuries. To identify factors contributing to differential quinine responses in the human malaria parasite Plasmodium falciparum, we have conducted comparative quantitative trait locus analyses on the susceptibility to quinine and also its stereoisomer quinidine, and on the initial and steady-state intracellular drug accumulation levels in the F1 progeny of a genetic cross. These data, together with genetic screens of field isolates and laboratory strains associated differential quinine and quinidine responses with mutated pfcrt, a segment on chromosome 13, and a novel candidate gene, termed MAL7P1.19 (encoding a HECT ubiquitin ligase). Despite a strong likelihood of association, episomal transfections demonstrated a role for the HECT ubiquitin-protein ligase in quinine and quinidine sensitivity in only a subset of genetic backgrounds, and here the changes in IC50 values were moderate (approximately 2-fold). These data show that quinine responsiveness is a complex genetic trait with multiple alleles playing a role and that more experiments are needed to unravel the role of the contributing factors.

In silico analysis of HLA-1 and HLA-2 recognition of a designed recombinant human papillomavirus vaccine based on L1 protein HPV subtype 45.

BACKGROUND: Human leukocyte antigen (HLA) can bind and present the processed antigenic peptide derived from the vaccine to the T cell receptor, and this capability is crucial in determining the effectivity of the vaccine to terminate virus-infected cells, activate macrophages, and induce B cells to produce antibodies. A recombinant vaccine candidate based on protein L1 HPV45 was designed and analysed whether it is recognisable by T cells through the binding of their epitopes to HLAs. METHODS: The study consisted of two parts: part one was the analysis of the L1 recombinant protein binding to HLA-1 and 2 epitopes, whereas part two was the distribution analysis of HPV-linked HLA allele. HLA allele sets found at high frequency in the general population and in specific Indonesian population were listed for the binding analysis of the recombinant L1 HPV45 protein. In part one, immunoepitope servers from IEDB were used to predict the binding of the designed proteins to HLA alleles. The prediction method for MHC-I binding prediction was the NetMHCpan EL 4.1 whilst for MHC-II binding prediction was the Consensus approach. Antigenicity analysis for each peptide was conducted using VaxiJen 2.0 with the threshold 1.0 to select the highly antigenic peptides, and positions of these epitopes in the secondary and tertiary structure of the recombinant protein were also predicted. The percent population coverage of the alleles capable of binding to these epitopes worldwide was also estimated. In part two, the worldwide distribution and frequency of HPV-related HLA-1 and 2 were studied. RESULT: Two highly antigenic peptides (EEYDLQFIF and KLKFWTVDLK) were recognised by high-frequency HLA-1 alleles in both, the general and Western Javanese. In addition to these two epitopes, a few more peptides are also recognised by the high-frequency Western Javanese HLA-1 alleles, which are not in Weiskopf's list of high-frequency HLA-1 alleles in the general population. Analysis of the highly antigenic epitopes binding to HLA-DRB1 alleles in general (YIKGTSANM) and Western Javanese (LRRRPTIGP) populations showed that these peptide cores associate to HLA-DRB1*04, albeit the different sub-types, due to the presence of different allele in each population group. Analysis of the epitopes and the positive binding alleles showed on average 25.65% population coverage. CONCLUSION: The recombinant vaccine candidate based on protein L1 HPV45 is presumed to contain highly antigenic peptides that can bind to high-frequency HLA-1 and 2 alleles present in general and Western Javanese populations. It was expected that the protein is capable of eliciting T cell-mediated responses in both populations; however, in vitro study is needed to prove the protectiveness of the designed recombinant protein.

Genetic linkage analyses redefine the roles of PfCRT and PfMDR1 in drug accumulation and susceptibility in Plasmodium falciparum.

Resistance to quinoline antimalarial drugs has emerged in different parts of the world and involves sets of discrete mutational changes in pfcrt and pfmdr1 in the human malaria parasite Plasmodium falciparum. To better understand how the different polymorphic haplotypes of pfmdr1 and pfcrt contribute to drug resistance, we have conducted a linkage analysis in the F1 progeny of a genetic cross where we assess both the susceptibility and the amount of accumulation of chloroquine, amodiaquine, quinine and quinidine. Our data show that the different pfcrt and pfmdr1 haplotypes confer drug-specific responses which, depending on the drug, may affect drug accumulation or susceptibility or both. These findings suggest that PfCRT and PfMDR1 are carriers of antimalarial drugs, but that the interaction with a drug interferes with the carriers' natural transport function such that they are now themselves targets of these drugs. How well a mutant PfCRT and PfMDR1 type copes with its competing transport functions is determined by its specific sets of amino acid substitutions.