Impact of Extended Duration of Artesunate Treatment on Parasitological Outcome in a Cytocidal Murine Malaria Model.

Artemisinin-based combination therapies are a key pillar in global malaria control and are recommended as a first-line Plasmodium falciparum treatment. They rely upon a rapid 4-log-unit reduction in parasitemia by artemisinin compounds with a short half-life and the killing of remaining parasites by a partner compound with a longer half-life. Current treatment guidelines stipulate giving three 24-h-interval doses or six 12-h-interval doses over a 3-day period. Due to the short half-life of artesunate and artemether, almost all of the resulting cytocidal activity is confined within a single 48-h asexual P. falciparum cycle. Here, we utilized a luciferase reporter, Plasmodium berghei ANKA, in a cytocidal model in which treatment was initiated at high parasitemia, allowing us to monitor a greater than 3-log-unit reduction in parasite density, as well as 30-day survival. In this study, we demonstrated that increasing the artesunate duration from spanning one asexual cycle to spanning three asexual cycles while keeping the total dose constant results in enhanced cytocidal activity. Single daily artesunate doses at 50 mg/kg of body weight over 7 days were the minimum necessary for curative monotherapy. In combination with a single sub-human-equivalent dose of the partner drug amodiaquine or piperaquine, the three-asexual-cycle artesunate duration was able to cure 75% and 100% of mice, respectively, whereas 0% and 33% cures were achieved with the single-asexual-cycle artesunate duration. In summary, cytocidal activity of the artemisinin compounds, such as artesunate, can be improved solely by altering the dosing duration.

Human immunodeficiency virus type 1 Vpr polymorphisms associated with progressor and nonprogressor individuals alter Vpr-associated functions.

Following infection with Human immunodeficiency virus 1 (HIV-1) there is a remarkable variation in virus replication and disease progression. Both host and viral factors have been implicated in the observed differences in disease status. Here, we focus on understanding the contribution of HIV-1 viral protein R (Vpr) by evaluating the disease-associated Vpr polymorphism and its biological functions from HIV-1 positive rapid progressor (RP) and long-term nonprogressor (LTNP) subjects. Results presented here show distinct variation in phenotypes of Vpr alleles from LTNP and RP subjects. Most notably, the polymorphism of Vpr at R36W and L68M associated with RP shows higher levels of oligomerization, and increased virus replication, whereas R77Q exhibits poor replication kinetics. Interestingly, we did not observe correlation with cell cycle arrest function. Together these results indicate that polymorphisms in Vpr in part may contribute to altered virus replication kinetics leading to the observed differences in disease progression in LTNP and RP groups.

Quantification of multiple infections of Plasmodium falciparum in vitro.

BACKGROUND: Human malaria infections caused by the parasite Plasmodium falciparum often contain more than one genetically distinct parasite. Despite this fact, nearly all studies of multiple strain P. falciparum infections have been limited to determining relative densities of each parasite within an infection. In light of this, new methods are needed that can quantify the absolute number of parasites within a single infection. METHODS: A quantitative PCR (qPCR) method was developed to track the dynamic interaction of P. falciparum infections containing genetically distinct parasite clones in cultured red blood cells. Allele-specific primers were used to generate a standard curve and to quantify the absolute concentration of parasite DNA within multi-clonal infections. Effects on dynamic growth relationships between parasites under drug pressure were examined by treating mixed cultures of drug sensitive and drug resistant parasites with the anti-malarial drug chloroquine at different dosing schedules. RESULTS: An absolute quantification method was developed to monitor the dynamics of P. falciparum cultures in vitro. This method allowed for the observation of competitive suppression, the reduction of parasites numbers due to the presence of another parasite, and competitive release, the improved performance of a parasite after the removal of a competitor. These studies demonstrated that the presence of two parasites led to the reduction in density of at least one parasite. The introduction of drug to a mixed culture containing both a drug resistant and drug sensitive parasites resulted in an increased proportion of the drug resistant parasite. Moreover, following drug treatment, the resistant parasite experienced competitive release by exhibiting a fitness benefit greater than simply surviving drug treatment, due to the removal of competitive suppression by the sensitive parasite. CONCLUSIONS: The newly developed assay allowed for the examination of the dynamics of two distinct clones in vitro; both competitive suppression and release were observed. A deeper understanding of the dynamic growth responses of multiple strain P. falciparum infections, with and without drug pressure, can improve the understanding of the role of parasite interactions in the spread of drug resistant parasites, perhaps suggesting different treatment strategies.

Human immunodeficiency virus type 1 Vpr: oligomerization is an essential feature for its incorporation into virus particles.

HIV-1 Vpr, a nonstructural viral protein associated with virus particles, has a positive role in the efficient transport of PIC into the nucleus of non-dividing target cells and enhances virus replication in primary T cells. Vpr is a 96 amino acid protein and the structure by NMR shows three helical domains. Vpr has been shown to exist as dimers and higher order oligomers. Considering the multifunctional nature of Vpr, the contribution of distinct helical domains to the dimer/oligomer structure of Vpr and the relevance of this feature to its functions are not clear. To address this, we have utilized molecular modeling approaches to identify putative models of oligomerization. The predicted interface residues were subjected to site-directed mutagenesis and evaluated their role in intermolecular interaction and virion incorporation. The interaction between Vpr molecules was monitored by Bimolecular Fluorescence complementation (BiFC) method. The results show that Vpr forms oligomers in live cells and residues in helical domains play critical roles in oligomerization. Interestingly, Vpr molecules defective in oligomerization also fail to incorporate into the virus particles. Based on the data, we suggest that oligomerization of Vpr is essential for virion incorporation property and may also have a role in the events associated with virus infection.