Enfuvirtide (T20) is the first U.S. FDA-approved HIV fusion inhibitor-based anti-HIV drug. Its clinical application is limited because of its low potency and short half-life. We previously reported that peptide HP23-E6-IDL, containing both N- and C-terminal anchor-tails, exhibited stronger potency and a better resistance profile than T20. Here we designed an analogous peptide, YIK, by introducing a mutation, T639I, and then a lipopeptide, YIK-C16, by adding palmitic acid (C16) at the C-terminus of YIK. We found that YIK-C16 was 4.4- and 3.6-fold more potent than HP23-E6-IDL and YIK against HIV-1IIIB infection and 13.3- and 10.5-fold more effective than HP23-E6-IDL and YIK against HIV-1Bal infection, respectively. Consistently, the ex vivo anti-HIV-1IIIB activity, as determined by the highest dilution-fold of the serum causing 50% inhibition of HIV-1 infection, of YIK-C16 in the sera of pretreated mice was remarkably higher than that of YIK or HP23-E6-IDL. The serum half-life (t1/2 = 5.9 h) of YIK-C16 was also significantly longer than that of YIK (t1/2 = 1.3 h) and HP23-E6-IDL (t1/2 = 1.0 h). These results suggest that the lipopeptide YIK-C16 shows promise for further development as a new anti-HIV drug with improved anti-HIV-1 activity and a prolonged half-life.

Sickle RBCs (SS) represent a naturally existing host-cell resistance mechanism to hemoparasite infections. Here, we investigate the basis of this resistance using Babesia divergens grown in sickle and sickle trait cells. We found that oxygenation and its corresponding effect on RBC-sickling, frequency of fetal hemoglobin (HbF+) cells, the cellular redox environment and parasite proliferation dynamics, all play a role in supporting or inhibiting Babesia proliferation. To identify cellular determinants that supported infection, an image-flow-cytometric tool was developed which could identify sickled-cells and constituent hemoglobin. We showed that hypoxic conditions impaired parasite growth in both HbSS and HbAS cells. Further, cell-sickling was alleviated by oxygenation which in turn relieved the inhibition of parasite proliferation in SS cells under hypoxic conditions. Interestingly, our tool identified HbF+-sickle cells as host-cells of choice under both hypoxia and hyperoxia, which was confirmed using cord RBCs which contain high amounts of HbF. In contrast to AA and AS cells, uninfected HbSS cells showed a higher ROS-containing environment, which was further perturbed on infection. We investigated the parasite's response in hostile SS cells and found that it alters its sub-population structure, with 1N dominance under hypoxic conditions yielding to equivalent ratios of all parasite forms at hyperoxic conditions favorable for its growth. Multiple factors, including oxygenation and its impact on cell-shape, HbF positivity, redox status, and parasite pleiotropy allow Babesia propagation in sickle RBCs. Our studies provide a cellular and molecular basis of natural resistance to Babesia which will aid in defining novel therapies against human babesiosis.

Babesiosis is a zoonosis and an important blood-borne human parasitic infection that has gained attention because of its growing infection rate in humans by transfer from animal reservoirs. Babesia represents a potential threat to the blood supply because asymptomatic infections in man are common, and blood from such donors can cause severe disease in certain recipients. Extracellular vesicles (EVs) are vesicles released by cells that contain a complex mixture of proteins, lipids, glycans, and genetic information that have been shown to play important roles in disease pathogenesis and susceptibility, as well as cell–cell communication and immune responses. In this article, we report on the identification and characterization of EVs released from red blood cells (RBCs) infected by two major human Babesia species—Babesia divergens from in vitro culture and those from an in vivo B. microti mouse infection. Using nanoparticle tracking analysis, we show that there is a range of vesicle sizes from 30 to 1,000 nm, emanating from the Babesia-infected RBC. The study of these EVs in the context of hemoparasite infection is complicated by the fact that both the parasite and the host RBC make and release vesicles into the extracellular environment. However, the EV frequency is 2- to 10-fold higher in Babesia-infected RBCs than uninfected RBCs, depending on levels of parasitemia. Using parasite-specific markers, we were able to show that ~50%–60% of all EVs contained parasite-specific markers on their surface and thus may represent the specific proportion of EVs released by infected RBCs within the EV population. Western blot analysis on purified EVs from both in vivo and in vitro infections revealed several parasite proteins that were targets of the host immune response. In addition, microRNA analysis showed that infected RBC EVs have different microRNA signature from uninfected RBC EVs, indicating a potential role as disease biomarkers. Finally, EVs were internalized by other RBCs in culture, implicating a potential role for these vesicles in cellular communication. Overall, our study points to the multiple functional implications of EVs in Babesia–host interactions and support the potential that EVs have as agents in disease pathogenesis.

Human babesiosis is caused by apicomplexan parasites of the Babesia genus and is associated with transfusion-transmitted illness and relapsing disease in immunosuppressed populations. Through its continuous cycles of invasion, proliferation, and egress, B. divergens radically changes the metabolic environment of the host red blood cell, allowing us opportunities to study potential chemical vulnerabilities that can be targeted by drugs.