Developing Plasmodium vivax Resources for Liver Stage Study in the

Article Cite This: ACS Infect. Dis. XXXX, XXX, XXX−XXX

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Developing Plasmodium vivax Resources for Liver Stage Study in the Peruvian Amazon Region Pamela Orjuela-Sanchez,† Zaira Hellen Villa,‡ Marta Moreno,§ Carlos Tong-Rios,‡ Stephan Meister,† Gregory M. LaMonte,† Brice Campo,∥ Joseph M. Vinetz,‡,§,⊥ and Elizabeth A. Winzeler*,† †

Division of Host-Microbe Systems and Therapeutics, Health Sciences Center for Immunology, Infection and Inflammation, Department of Pediatrics, School of Medicine, University of California, San Diego, 9500 Gilman Drive #0760, La Jolla, California 92093-0760, United States ‡ Laboratorio ICEMR-Amazonia, Laboratorios de Investigacion y Desarrollo, Facultad de Ciencias y Filosofia, Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, San Martín de Porres, Lima, 15102, Peru § Division of Infectious Diseases, Department of Medicine, University of California, San Diego, 9500 Gilman Drive #0760, La Jolla, California 92093-0760, United States ∥ Medicines for Malaria Venture (MMV), International Center Cointrin, Block G, 20 Route de Pre-Bois, POB 1826, Geneva, CH-1215, Switzerland ⊥ Instituto de Medicina Tropical “Alexander von Humboldt”, Universidad Peruana Cayetano Heredia, Lima, 15102, Peru ABSTRACT: To develop new drugs and vaccines for malaria elimination, it will be necessary to discover biological interventions, including small molecules that act against Plasmodium vivax exoerythrocytic forms. However, a robust in vitro culture system for P. vivax is still lacking. Thus, to study exoerythrocytic forms, researchers must have simultaneous access to fresh, temperaturecontrolled patient blood samples, as well as an anopheline mosquito colony. In addition, researchers must rely on native mosquito species to avoid introducing a potentially dangerous invasive species into a malaria-endemic region. Here, we report an in vitro culture system carried out on site in a malaria-endemic region for liver stage parasites of P. vivax sporozoites obtained from An. darlingi, the main malaria vector in the Americas. P. vivax sporozoites were obtained by dissection of salivary glands from infected An. darlingi mosquitoes and purified by Accudenz density gradient centrifugation. HC04 liver cells were exposed to P. vivax sporozoites and cultured up to 9 days. To overcome low P. vivax patient parasitemias, potentially lower mosquito vectorial capacity, and humid, nonsterile environmental conditions, a new antibiotic cocktail was included in tissue culture to prevent contamination. Culturing conditions supported exoerythrocytic (EEF) P. vivax liver stage growth up to 9 days and allowed for maturation into intrahepatocyte merosomes. Some of the identified small forms were resistant to atovaquone (1 μM) but sensitive to the phosphatidylinositol 4-kinase inhibitor, KDU691 (1 μM). This study reports a field-accessible EEF production process for drug discovery in a malaria-endemic site in which viable P. vivax sporozoites are used for drug studies using hepatocyte infection. Our data demonstrate that the development of meaningful, field-based resources for P. vivax liver stage drug screening and liver stage human malaria experimentation in the Amazon region is feasible. KEYWORDS: Peruvian Amazon region, Plasmodium vivax, Anopheles darlingi, sporozoite, exoerythrocytic stage, in vitro culture, drug evaluation f the five species of malaria parasites affecting humans, Plasmodium vivax is the most widely distributed, with more than 2.5 billion people at risk of infection.1 P. vivax malaria is the predominant malaria parasite in South America, with the majority of the cases reported in the Amazon region. In 2015, the Peruvian Amazon contributed approximately 20% of the total malaria cases on the continent, and 80% of these episodes was caused by P. vivax (reviewed in ref 2). P. vivax malaria is characterized by periodic relapses of symptomatic blood stage parasite infections, which are triggered by the liver-resident, dormant stage of the parasite,

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the hypnozoite. This unique biological feature of P. vivax hinders global efforts to control and eliminate malaria because of delayed reintroductions of this parasite long after primary infection. Mechanisms of hypnozoite genesis and development are not understood, and the only radical cure treatment available to eradicate them is primaquine.3 Special Issue: Drug Discovery for Global Health Received: October 30, 2017

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DOI: 10.1021/acsinfecdis.7b00198 ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases

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Despite the global public health importance of P. vivax, investigations oriented toward the study of liver stages, including hypnozoites, are limited because of the challenging logistics in gaining access to the biological material in a controlled-laboratory setting. To date, P. vivax liver stage in vitro culture has been described using human immortalized liver cell lines such as HC04,4 hepatoma cell lines including HepG2,5 primary hepatocytes,6,7 and pluripotent stem cells.8 A human liver-chimeric mouse supporting P. vivax liver stage development was recently described.9 Although these advances are promising, the fact remains that the study of P. vivax exoerythrocytic forms relies on the cell culture systems being coincubated with P. vivax sporozoites. Obtaining sufficient quantities of infectious sporozoites is challenging (reviewed in refs 10−12). P. vivax sporozoites can still only be obtained by dissecting the salivary glands of mosquitoes that have fed on fresh P. vivax-infected blood. Because P. vivax blood stages cannot be continuously cultured, new sources of P. vivax must be continually obtained from patients or from nonhuman primates.13,14 There are ethical considerations associated with use of primates in research, and patient samples depend on uncontrollable clinical situations. In addition, patients may have different levels of antibodies against parasite transmission stages, and the parasites from different infections may be genetically heterogeneous. In addition to the challenges of establishing a continuous production of P. vivax sporozoites, an uninterrupted availability of mosquitoes is also required. While rearing of some mosquito species, such as Anopheles stephensi, have been standardized, this species cannot be brought to malaria endemic regions because of potential release. An. darlingi is the main malaria vector in the Americas, and continuous colonies of these mosquito species have been recently established in the Peruvian Amazon region.15,16 P. vivax sporozoites have been successfully obtained using this An. darlingi colony since 2012. In this work, we evaluated the ability of these P. vivax sporozoites to infect HC04 liver cells and to differentiate into liver stages. We also report the standardization of a culture medium that inhibits polymicrobial contamination and allows P. vivax liver stage culture progression without affecting the parasite development.

Initial experiments used sporozoites (22 500 sporozoites per well) added to HC04 cells (plated at a density of 45 000 cells per well) and incubated with DMEM media supplemented with 10% fetal bovine serum (FBS). These preliminary experiments showed that polymicrobial contamination of the cultures was a problem. In contrast to experiments with An. stephensi and P. berghei (where the culture medium is only supplemented with 100 units of penicillin and 100 μg/mL streptomycin), even after 1 day of P. vivax exoerythrocytic (EEF) culture, bacterial contamination was evident, as confirmed by Gram stain. A critical difference may be the infectivity mosquito rate: typically, more than 15 000 sporozoites per mosquito are obtained with P. berghei and An. stephensi. To create clean conditions for P. vivax EEF culture, we next tested two different strategies: (i) the use of a density gradient to purify P. vivax sporozoites from mosquito debris and microorganisms; (ii) standardization of a culture medium supplemented with antibiotics and antifungals to reduce contamination. (i) Accudenz density gradient: comparison of 40 independent dissections did not show significant difference (p = 0.89) in sporozoite yield from unpurified (1671 ± 1274 sporozoites/μL, standard deviation (SD)) versus Accudenz-purified (1617 ± 2066 sporozoites/μL, SD) samples. Regardless of the method, hepatocyte cultures using unpurified or purified sporozoites showed contamination 24 h after infection. Thus, density gradient centrifugation by itself did not prevent contamination. (ii) A new medium to prevent contamination of Plasmodium vivax EEF cultures: our second strategy tested different antibiotic combinations that would allow parasite development but prevent polymicrobial contamination. To establish the minimum concentrations of antibiotics and antifungals that would enable successful P. vivax EEF development, serial dilutions (100 to 0.01 μM, 2-fold dilutions) of neomycin and gentamicin (antibiotics already in use for P. falciparum blood stage culture21,22) were tested. Antibiotics were added to the culture medium at the moment of sporozoite dissection and during the 9 day P. vivax EEF culture; fresh culture medium with antibiotics was added every other day, and an aliquot of the culture medium was examined by microscopy every 24 h. We found that optimum concentrations of neomycin and gentamicin were 100 and 21 μM, respectively. Bacterial growth inhibition was sustained over the total incubation period (9 days) only when both antibiotics were used in combination. Once bacterial growth was controlled in the P. vivax EEF cultures, yeast contamination became a problem. A battery of antifungal compounds with diverse mechanisms of action was tested (100 to 0.01 μM, 2-fold dilutions) using the approach described earlier. To verify that yeast-killing antifungals were safe for P. vivax EEF culture, drug response assays were conducted in parallel with P. berghei EEF (Table 1) using a previously published luciferase assay.17 Two antifungals prevented yeast growth without affecting P. berghei EEF or HepG2 cell viability: 5-fluorocytosine and posaconazole. When used together, 5-fluorocytosine and posaconazole synergistically and completely inhibited yeast growth in P. vivax EEF cultures over 9 days. Neither 5fluorocytosine nor posaconazole had activity against P. falciparum (asexual blood stages) at similar concentrations used in the

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RESULTS AND DISCUSSION Elimination of Bacterial and Fungi Contamination from Plasmodium vivax Liver Stage in Vitro Cultures. To establish a liver stage culture system, donor patients were recruited and asked to provide blood samples for use in standard membrane feeding assays. Over the course of this study, 55 patients were enrolled with an average asexual parasitemia of 4989 ± 775 (standard error of mean, SEM) parasites per μL of blood and 791 ± 127 for sexual stages, detected by light microscopy. After blood feeding, 4900 ± 1106 (SEM) sporozoites per mosquito were obtained from the salivary-gland dissection of 500 ± 52 (SEM) infected mosquitoes per assay. These numbers are substantially lower than those observed for An. stephensi infected with P. berghei where sporozoites per mosquito numbers typically reach 17 020 ± 1594 (SEM).17 The number of sporozoites obtained per mosquito was also lower than with P. cynomolgi (54 309)18 and P. falciparum (45 233 ± 24 624, SEM)19 in An. stephensi or P. vivax in An. cracens (26 112).20 This low sporozoite yield is most likely the result of low, and largely unavoidable, parasitemias in the patients that were enrolled in our study or, possibly, a potentially lower vectorial capacity of An. darlingi. B



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because these cells have been shown to support both P. falciparum and P. vivax infection.4,23 A disadvantage of hepatoma cells, however, is that, once the cells reach confluency, cell overgrowth can interfere with parasite identification and promote hepatocyte apoptosis (reviewed in ref 24). Experiments were conducted in P. berghei-infected hepatocytes aiming to optimize cell biomass during the incubation time. First, we established the starting number of HC04 cells to be seeded 24 h before the sporozoite infection. Starting cell biomasses (384-well plates) of 1250 to 5000 cells per well were tested. After 24 h of incubation, a hepatocyte confluency of 85% (±10%, SEM) was achieved when 5000 cells/well were seeded. Next, we evaluated the effects of different concentrations of fetal bovine serum (FBS) (0, 1%, 5%, and 10%) on HC04 cell growth from day 1 to 10 postseeding. Concentrations of FBS below 10% significantly reduced cell growth; concentrations of FBS below 1% arrested cell growth and significantly interfered with cell morphology (Figure 2A). The use of DMEM media supplemented with 5% FBS significantly decreased cell growth without affecting parasite infection rate or development (Figure 2B,C). For this reason, 5% FBS was selected for further experiments.

Table 1. List of Compounds Screened to Control Plasmodium vivax EEF Culture Contaminationa antibiotic/ antifungal amphotericin B caspofungin 5-fluorocytosine fluconazole gentamicin sulfate itraconazole neomycin sulfate naftifine nystatin posaconazole terbinafine tolnaftate voriconazole a

antibacteria/ yeast (μM)

P. berghei IC50 (μM)

95% confidence interval

>0.5 >10 >10 >10 >20

0.07 6.09 47.65 8.89 >100

0.03−0.16 0.09−15.32 30.23−124.3 3.56−15.89

0.06 12.13 >50 NE >100

>10 >100

0.06 >500

0.01−10.56

5.829 >500

>100 >10 >0.5 >100 >100 >100

NE 10.45 3.56 NE 24.42 NE

NE 8.56−24.56 1.31−6.26 NE 12.39−48.16 NE

NE >50 >50 NE 37.64 NE

HepG2 IC50 (μM)

NE = not evaluated; bold compounds are currently in use.

liver assay. The half maximal inhibitory concentrations (IC50) of posaconazole and 5-fluorocytosine against asexual blood stage P. falciparum were 1.8 and >50 μM, respectively (Figure 1). Described IC50’s for posaconazole and 5-fluorocytosine in

Figure 1. Dose−response curve of posaconazole and 5-fluorocytosine in Plasmodium falciparum (asexual blood stage). Chloroquine and artemisinin were used as positive controls. Error bars represent the standard error of the mean of three independent experiments.

P. falciparum blood stage and P. berghei liver stage were not significantly different (Figure 1, Table 1). Supplementation of DMEM medium with 5-fluorocytosine (12 μM), posaconazole (0.5 μM), gentamicin (21 μM), and neomycin (110 μM) prevented contamination and allowed successful P. vivax EEF culture. Hepatocyte Biomass and Viability during Plasmodium vivax EEF Culture. Although P. vivax can be cultured in primary human hepatocytes,7 primary hepatocytes are problematic for a number of reasons. First, there can be lot-to-lot variability6,23 making them less suitable for drug development where assays need to be reproducible over time. Second, primary hepatocytes are expensive and may retain different levels of metabolic activity that may result in lower apparent compound activity, which could be particularly problematic with primary screens and compounds that have not been “optimized” to improve metabolic stability. Finally, primary hepatocytes do not self-regenerate, demanding that new lots be transferred to our laboratories in the Amazon periodically. For this reason, we decided to use hepatoma cell lines in our experiments. We decided to work primarily with HC04 cells

Figure 2. Effect of different concentrations of FBS in hepatocyte biomass, parasite replication, and growth. (A) Hoechst-stained HC04 nuclei were imaged and quantified using the Operetta high-content imaging system. Measurements were performed over a 10 day incubation period. (B) P. bergheiGFP infection rates at 48 h postinfection were similar in DMEM medium supplemented with 5% or 10% FBS. (C) P. bergheiGFP EEF growth estimated by GFP intensity 48 h postinfection as measured by flow cytometry. The standard error of the mean of three experimental replicates is shown. C



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Figure 3. HC04 cell viability over time. (A) Percentage of live HC04 cells over the incubation time. Data was extracted from fluorescence measurements using a microplate reader. The average of three different experimental replicates is shown. (B) LIVE/DEAD HC04 cell staining after a 9 day incubation period. Green-fluorescent calcein-AM indicates intracellular esterase activity (live cells), and red-fluorescent ethidium homodimer-1 detects loss of plasma membrane integrity (dead cells). Magnification, 20×. Scale bar = 100 μm.

similar. On day 5 postinfection, the ratio of small to medium forms was approximately 1:1. On day 7 postinfection, this ratio was 1:2, and by day 9, it was approximately 1:0.5. Comparisons of size ratios among isolates could not be performed since P. vivax-infected An. darlingi from various malaria donors had to be pooled together in order to obtain enough sporozoites to perform the infections. The HC04 infection rate, based on the percentage of cells containing HSP70-expressing parasites at day 3 postinfection, was, on average, 5 EEFs per 100 000 hepatocytes. We also assessed parasitemias on day 5, 7, and 9 postinfection. While there was a trend of identifying more parasites at day 5 and day 7, this difference was not statistically significant. To provide further evidence that identified parasite-like forms were indeed real parasites, cultures were treated for at least 3 days (5 days optimal) with 1 μM of atovaquone (to which P. vivax hypnozoites are known to be relatively resistant) or KDU691 (to which P. cynomolgi EEFs are known to be susceptible27). The cultures were fixed (day 3, 5, 7, and 9 postinfection) and stained with a Plasmodium heat shock protein 70 (PHSP70) antibody, and the number of identified objects compatible with P. vivax EEFs were registered. KDU691-treated wells showed 90% fewer EEFs (small, medium, and large) than dimethyl sulfoxide (DMSO)-treated controls (Figure 5). In contrast, in atovaquone-treated wells, a few small EEFs were detected on day 1 with only 1 small EEF per well-replicate in most of the experiments performed (5 out of 6 total). On day 5 and 7, this number remained similar to approximately 2 small EEFs per well-replicate (5 out of 6 total infections). The highest number of small EEFs was identified on day 9 (average 4 ± 1 SE, 5 out of 6 total infections). The differences between the number of small EEFs recorded on day 3, 5, 7, and 9 were only significant between day 3 and day 9 (p < 0.05). Atovaquone-treated wells showed no presence of medium and large forms. Treated and uninfected wells were always included, and the number of identified objects compatible with P. vivax EEFs in uninfected controls was negligible (one or undetectable).

HepG2 cells have been successfully cultured on twodimensional surfaces for as long as 25 days. However, after 7 days in culture, some metabolic activity has been shown to be reduced.25 HC04 cells incubated for 14 days reported no significant (1.2%) cell detachment4 and high viability values 96 h postseed.26 To assess HC04 cell viability during our P. vivax EEF culture-incubation time (at least 9 days), we next assessed the viability of uninfected hepatocyte cultures using live/dead staining, which was performed in 384-well plates with starting biomass of 5000 cells per well. HC04 cells were grown using DMEM medium supplemented with 5% FBS, 5-fluorocytosine (12 μM), posaconazole (0.5 μM), gentamicin (21 μM), and neomycin (110 μM). By day 1, cell viability was 92% (±3.04%, SEM), and it slowly decreased to 70% (±2%, SEM) by day 9 (Figure 3A,B). These results show that most HC04 cells were viable during the entire incubation period. Plasmodium vivax Liver Stage Identification and Validation. Because of the possibility that the developmental time course could be different for P. vivax isolates from the Amazon region, we determined known growth milestones of P. vivax EEFs within in vitro infections. The diameter of parasite-like forms was registered on days 3, 5, 7, and 9 postinfection. The average and standard deviation of all measured forms was calculated, and three size groups were classified: small, medium (mainly on day 5 postinfection), and large (seen on days 7 and 9 postinfection). We found that the parasites developed with close proximity to host nuclei, in many cases reshaping it. On day 3, only small mononucleated EEFs < 10 μm2 (average 3 ± 2 SD) were present but were barely detectable (Figure 4A). By day 5, most EEFs were polynucleated and 10 times bigger (average 51 μm2 ± 22 SD) than on day 3 dpi (days postinfection). The largest multitnuclear EEF forms were observed after 7 dpi with sizes >90 μm2 (average 186 ± 64 SD) (Figure 4A,B). No significant changes were observed in EEF sizes from day 7 and day 9, suggesting that, by day 7 dpi, schizonts may have reached full development. Mononucleated small forms were detected during the entire incubation period (9 days). The number of different EEFs detected at each time point remained relatively D



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Figure 4. Plasmodium vivax EEF culture in HC04 cells. (A) Images of P. vivax EEFs in HC04 cells at different incubation times. PvUIS4 or PHSP70 antibodies were used for P. vivax EEFs identification (green); Hoechst 33342 was used for cell and parasite nuclei staining (blue). Scale bars are 15 and 115 μm (day 9, large forms, top panel). Objective, 60× oil. (B) P. vivax EEF size distribution at different days in culture. Error bars represent the standard error of the mean of six different experimental infections.

Antibiotics like gentamicin (21 μM) and neomycin at high concentrations (150 μM) have been used for culturing asexual blood stages of P. falciparum22,28 and P. berghei ANKA.29 In our experience, only when both antibiotics (gentamicin plus neomycin) were added to the culture medium was bacterial contamination controlled. Amphotericin B (0.2 μM) is a welldescribed antifungal with a broad spectrum used to control yeast contamination in Plasmodium EEF in vitro cultures.6,30 However, amphotericin B has high anti-Plasmodium activity

One of the main challenges associated with developing a culture system in the Amazon region was polymicrobial contamination. Despite the significant number of microorganisms introduced to the culture by the infection of liver cells with P. vivax sporozoites, the available literature regarding contamination issues is limited. Density gradients, like Accudenz, can efficiently remove mosquito debris, but microorganisms from mosquito contents and carcasses will also be collected during the sporozoite purification step. E



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antipolymicrobial contamination during Plasmodium liver stage culture without affecting the parasite development. Another difficulty we faced in this work was in creating hepatoma cell monolayers suitable for imaging. When Huh7.5.1 liver cells are grown on two-dimensional surfaces, cells predominately grow as flat monolayers and sometimes form small multicellular masses. We found that proliferating HC04 cells rapidly formed three-dimensional cell culture layer formats hindering automated P. vivax EEFs identification. By reducing FBS by half (to 5%) in the culture medium, we were also able to reduce HC04 cell biomass without affecting P. vivax EFFs growth or development. Sattabongkot et al. (2006)4 described an average hepatocyte infection rate of 0.066% using HC04 cells and P. vivax sporozoites derived from Thailand patients. More recently, in 2015, Dumoulin et al.34 used flow cytometry to accurately assess an infection rate of 0.007% using HC04 cells and a stable transgenic line of P. falciparum constitutively expressing GFP. Our findings are in accordance with the previous report by Dumoulin et al.34 The low infection rates observed here could be explained by low permissiveness of the HC04 cell to P. vivax infection; HC04 cell death during the incubation period, including parasite-infected cells; high cell confluency; HC04 3dimensional cell organization hindering parasite identification, especially in the case of small uninucleated P. vivax EEFs. It remains unknown whether genetic variation of P. vivax from different patients might interfere with liver cell infectivity and effectiveness. Despite the low parasitemias found in our P. vivax EEF cultures, the results from different experimental infections were robust and reproducible. For validation purposes, P. vivax EEFs were identified using at least two sets of antibodies: HSP70 and UIS4.9 We had previously reported the use of a HSP70 polyclonal antibody for P. yoelii high content imaging screening.35 Here, we also validated the use of a very similar (>90% sequence identity) HSP70 antibody detecting P. vivax EEFs from early trophozoites to merosomes and hypnozoites. P. vivax up-regulated in infective sporozoites gen (PvUIS4)9 antibody was also very useful for the visualization of EEFs with sizes >10 μm2 where the parasitophorous vacuole membrane could be easily identified. Both antibodies recognized P. vivax EEFs originating from different P. vivax isolates, and we did not observe significant changes in the number of identified P. vivax EEFs within experimental replicates. In the human liver-chimeric mice model, P. vivax liver stages complete maturation by day 9 or 10 postinfection.9 In our P. vivax EEF in vitro HC04 culture, we mostly detected mature liver stages with multiple nuclei on day 9 postinfection. The size and shape of P. vivax schizonts identified in this work were similar to those described by Mikolajczak et al.9 However, the size of the mononucleated small forms found here was approximately half the size (5 μm2) of those described in the liver-chimeric mice model (10 μm2). The insensitivity of small P. vivax EEFs to atovaquone treatment30 and the killing by KDU691 27 under the evaluated protocol support the hypothesis that KDU691 is able to affect hypnozoite formation. We did not observe any changes in the size of the small EEFs during the 9 day incubation period. Overall, these results suggest that the in vitro culture of Amazonian P. vivax EEFs in HC04 cells resembles the P. vivax liver stage cycle in the liverchimeric mice model.9 In conclusion, here, we described a sustainable platform, suitable for deployment in malaria-endemic areas of the developing world for the experimental infection of hepatocyte

Figure 5. Activity of atovaquone (ATQ) and KDU691 against Plasmodium vivax EEFs. Number of identified objects compatible with exoerythrocytic forms of P. vivax counted in fixed (PHSP70 staining) wells 3, 5, 7, and 9 days after P. vivax infection. Wells were treated with 1 μM of ATQ or KDU691 4 h after sporozoite invasion. Compounds were replaced every 48 h until day 5 postinfection. Wells with DMSO 0.1% and uninfected wells were also included as controls. Six different experimental replicates were performed; error bars show SD. Small: mononucleated EEFs with sizes 10 μm2 and 90 μm2 (average 186 ± 64 SD).

(IC50 = 0.1 μM) against blood and liver stages of P. falciparum31 and P. berghei (Table 1), respectively. We found that amphotericin B only inhibited yeast contamination at concentrations higher than 0.5 μM. For this reason, amphotericin B was not used in the culture medium. In a previous study,32 yeast contamination frequently occurred in EEF cultures of P. cynomolgi in primary hepatocytes of Macaca mulatta. Contamination was minimized by incorporating 5fluorocytosine (4 μM), but even at this concentration, cultures could not progress for more than 5 dpi. We found that, using a broad antifungal panel, the only other compound that showed activity against yeast besides 5-fluorocytosine and amphotericin B was posaconazole (0.5 μM). Posaconazole IC50 against P. falciparum asexual blood stages has been reported to be 2.6 μM.33 Because concentrations of posaconazole lower than 1 μM did not prevent yeast contamination, a mixture of both 5fluorocytosine (12 μM) and posaconazole (0.5 μM) was used. We were able to standardize a potent drug cocktail to prevent F



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cell lines with P. vivax sporozoites leading to the development of exoerythrocytic forms. This report also is the first to validate the infectivity and in vitro culture capabilities of P. vivax sporozoites derived from an An. darlingi mosquito source. Having a colony of An. darlingi (generation F52) in proximity to P. vivax infected human subjects will enable the use of this platform for drug discovery, vaccine development, and fundamental studies of P. vivax EEFs.

fresh media and slides were incubated for 3, 5, 7, or 9 days. Media culture was replaced every other day. Drug in vitro assays were performed in coated slide chambers seeded with HC04 cells (45 000 cells per well) and infected with Accudenz column purified P. vivax sporozoites (1:3 sporozoite to HC04 cell infection ratio).35 Four hours after the sporozoite infection, wells were treated with 1 μM of atovaquone (Santa Cruz biotechnology #sc-217675) or KDU691 (Genomics Institute of the Novartis Research Foundation27). Fresh compound was added every other day from day zero until day five, and the assay was ended on day 3, 5, 7, or 9 postinfection. Growth controls included solvent alone in the absence of drug (DMSO, 0.1%, v/v) and uninfected hepatocytes. Plasmodium vivax EEF Liver Stage Detection. Parasite detection was performed by indirect immunofluorescence. P. vivax infected slide chambers were fixed with PBS− paraformaldehyde (4% v/v, 20 min) (Affymetrix #19943), permeabilized with PBS-Triton X-100 (0.1% v/v, 5 min) (SIGMA #93443), and stained overnight (4 °C) using one of two sets of antibodies: (i) PvUIS4 (P. vivax up-regulated in infective sporozoites gene 4) mouse monoclonal antibody (1 mg/mL, dilution 1:150) kindly provided by Sebastian A. Mikolajczak (Seattle Biomedical Research Institute); (ii) PHSP70 (Plasmodium heat shock protein 70) mouse polyclonal antibody (1 mg/mL, dilution 1:250). PHSP70 antibody was developed by GenScript using a codon-optimized sequence of amino acids 335 to 627 (Sequence ID: XP_001614972.1). PHSP70 fragment was expressed in E. coli, double purified, and used for mice immunization according to GenScript protocols. To reduce nonspecific staining background, the Alexa Fluor 488 AffiniPure Goat Anti-Mouse IgG, Fcγ Fragment (Jackson ImmunoResearch Inc. #115-545-071) (1 mg/mL, dilution 1:500) secondary antibody was used. Nuclei and HC04 cell membrane were detected using Hoechst 33342 (Thermo Fisher Scientific Inc. #62249) (500 μM) and CellMask deep red (Thermo Fisher Scientific Inc. #C10046) (1×), respectively. After the staining was completed, Lab-Tek chambers were removed; slides were mounted with Vectashield (Vector Laboratories #H-1000), and #1.5 glass coverslips were mounted using nail polish. The number of parasites (small: uninucleated EEFs with sizes 10 μm2 and 90 μm2) per well was manually counted using a PerkinElmer UltraView Vox Spinning Disk confocal microscope (60× oil objective). P. vivax EEFs cell area (μm2) was calculated using the software Cellsens Standard version 1.2 from Olympus. Viability Assays. Viability assays on uninfected hepatocytes were performed using the LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells (Molecular Probes #L3224). After kit titration, concentrations of 4 μM for green-fluorescent calceinAM (CA_AM excitation/emission, 494 nm/517 nm) and 4.5 μM for red-fluorescent ethidium homodimer-1 (EthD-1 excitation/emission, 528 nm/617 nm) were used. Viability assays were performed in 384-well (growth area 10 mm2) plates (Greiner #82051-282) seeded with HC04 cells (5000 cells per well). DMEM media supplemented with FBS (5% v/v), antifungals, and antibiotics (same concentrations used for P. vivax assays) was replaced every other day. As dead controls, cells treated with 70% methanol for 30 min were included. Viability assays were performed on day 2, 4, 6, 8, and 10 after seeding. Fluorescence measurements were read using a

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METHODS Anopheles darlingi Colony and Plasmodium vivax Donors. Laboratory-reared An. darlingi mosquitoes were obtained from the University of California, San DiegoUniversidad Peruana Cayetano Heredia Insectary, Satellite Laboratory (Iquitos, Peru).15 P. vivax-infected blood from volunteers (≥18 years old) enrolled in the city of Iquitos from 2015 to 2016 was used as the source of infectious gametocytes. After the P. vivax malaria infection was microscopically diagnosed by Giemsa staining, 10 mL of blood was collected and used within 1 h for standard membrane blood feeding (SMFA).36 An. darlingi salivary glands were subsequently dissected on day 14−15 of postfeeding, and the P. vivax sporozoite counting was performed using a Neubauer chamber. Plasmodium vivax Sporozoite Purification Method. The method described in Kennedy et al.37 was used. Briefly, a 17% w/v solution of Accudenz (Accurate Chemical #AN7050) dissolved in distilled deionized water (ddH2O; Mediatech #25055-CM) was filtered, sterilized, and stored at 4 °C. A 3 mL, 17% Accudenz solution was loaded into a 15 mL Falcon tube followed by the gentle addition of 1 mL of the sporozoite mixture of, at a minimum, 200 dissected mosquitoes. The 15 mL Falcon tube was centrifuged at 2500g for 20 min (no brake) at room temperature. The interface was loaded onto a 1.5 mL eppendorf tube and centrifuged at 12 000 rpm for 3 min. The supernatant was aspirated, and the pellet containing the sporozoites was resuspended in DMEM high glucose (Life Technologies #11965118). Sporozoites were counted using a hemocytometer before and after the Accudenz column purification protocol. Plasmodium vivax Liver Stage Culture and Drug in Vitro Assay. A modified protocol described by Cui et al.38 was used. Twenty-four hours prior to mosquito dissection, 8-well Nunc Lab-Tek (growth area 70 mm2) chamber slides (Thermo Scientific Nunc #125658) were coated with poly-L-lysine 0.01% (v/v) (SIGMA #P4707) and seeded with HC04 cells (45 000 cells per well). DMEM high glucose supplemented with 10% fetal bovine serum (FBS) (heat inactivated, Mediatech Inc. #35-011-CV), 100 IU/mL penicillin, and 100 μg/mL streptomycin (penicillin−streptomycin 10 000 IU/mL; Life Technologies #5140122) was used for HC04 cell Lab-Tek seeding and cell culture maintenance. Seeded Lab-Tek chambers were incubated at 37 °C in a 5% CO2 atmosphere. For drug assays, Accudenz purified sporozoites (see above) were resuspended in DMEM high glucose media supplemented with FBS (5% v/v) and a cocktail of antibiotics and antifungals: 12 μM 5-fluorocytosine (Cayman #11635), 0.5 μM posaconazole (Cayman #14737), 50 μg/mL gentamicin sulfate (Gemini Bio-Products #400-100P), and 100 μg/mL neomycin trisulfate salt hydrate (SIGMA #N1876). HC04 preseeded Lab-Tek chambers were infected with the Accudenz column purified sporozoites (1:3 sporozoite to HC04 cell infection ratio)35 and incubated for 4 h at 37 °C in a 5% CO2 atmosphere. After this initial incubation period, the infection media was replaced with G



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removed and a CellTiter-Glo reagent (Promega #G7570) was added. Bioluminescence was recorded immediately using an EnVision Multilabel reader (PerkinElmer). Two independent experiments in technical duplicates were performed. Asexual Blood Stage Plasmodium falciparum Culture Medium Sensitivity Assay. P. falciparum susceptibility to 5fluorocytosine and posaconazole was measured using the malaria SYBR Green I-based fluorescence assay.41 Each compound was tested in duplicate on a 10-point concentration curve prepared by a 3-fold dilution ranging from 50 μM to 48.8 nM. Chloroquine diphosphate salt (SIGMA #C6628) and artesunate (SIGMA #A3731) were tested as positive controls. Methanol (100%; posaconazole solvent) was also tested from 3.2% (the highest concentration presented in the above compounds) to 0.0015% in 2-fold dilution. Three independent experiments in technical triplicates were performed. Statistical Analysis. Descriptive statistics for determining mean, percentages, standard error/deviation, and plotting graphs were calculated with GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA, USA). A Wilcoxon matched-pairs test was used to compare the number of sporozoites obtained before and after the Accudenz purification step. Inhibitory concentrations of 50% (IC50) for liver and blood assays were obtained using the average normalized bioluminescence intensity of 4 wells per concentration and plate. A nonlinear variable slope four-parameter regression curve-fitting model was used. Ethics Statements. Study protocols were approved by the Human Research Protection Program from the University of California, San Diego (approval number 120652) and Universidad Peruana Cayetano Heredia (R-157-13-14). Written informed consent was obtained from all study participants.

Multidetector microplate reader Synergy HTX (BioTek Instruments). Results were expressed as a percentage of relative fluorescence at 530 nm of samples treated with CA_AM + EthD-1 as compared with the fluorescence at 530 nm of control groups treated with CA_AM and EthD-1, separately. The percentage of live cells at each time point was estimated using the formula: % live cells = [F(530)sam − F(530)min/ F(530)max − F(530)min] × 100%. Formula parameters are defined as follows: F(530)sam = fluorescence at 530 nm in the experiment/day HC04 cell sample, labeled with calcein acetoxymethyl (calcein AM) and EthD-1; F(530)min = fluorescence at 530 nm in control HC04 cells where all (or nearly all) cells were alive, labeled with EthD-1 only; F(530)max = fluorescence at 530 nm in HC04 cells, where all (or nearly all) cells were alive, labeled with calcein AM only. For F(530)min and F(530)max controls, we used HC04 cells seeded 24 h before the assay at a cell concentration projected (considering 30 h as cell doubling time) for the evaluated time point. Three independent experiments in technical triplicates were performed. On the day of the viability evaluation, some wells were also stained with 500 μM Hoechst 33342 (Thermo Fisher Scientific #H3570) for nuclei counting. Plates were scanned on the Operetta CLS (PerkinElmer) using the 10× objective. For nuclei counting, the Harmony High Content Imaging and Analysis Software (PerkinElmer) was used. Viability assays on infected hepatocytes were performed on 384-well (growth area 10 mm2) plates (Greiner #82051-282) infected with P. bergheiGFP39 sporozoites. Parasite growth was evaluated in DMEM media supplemented with antifungals/ antibiotics (same concentrations used for P. vivax assays) and different concentrations of FBS (0, 5, and 10% v/v). Infection rates and parasite GFP intensity values were measured 48 h postinfection using a FACSCanto flow cytometer (Becton Dickinson); data was analyzed using the Diva software 6.0 (Becton Dickinson). Culture Medium Plasmodium berghei EEF Toxicity Assay. HepG2-A16-CD81EGFP cells were seeded into 1536well plates 24 h prior to P. berghei sporozoite infection.17 Twelve hours later, 12-point serial 3-fold dilutions of 5fluorocytosine, posaconazole, gentamicin sulfate, and neomycin trisulfate hydrate dissolved in DMSO or water were transferred into the cell-seeded plate. Atovaquone (10 μM) and DMSO 0.1% were used as positive and negative controls of growth, respectively. Twenty-four hours after the hepatocyte cells were seeded, P. berghei sporozoites expressing luciferase40 were dissected and used to infect (1 × 103 sporozoites per well) the plate with compounds. After a 48 h incubation period, the parasite growth was quantified by bioluminescence measurement using a BrightGlo reagent (Promega #E2610) and the EnVision Multilabel plate reader (PerkinElmer) as previously described.17 Two independent experiments in technical duplicates were performed. Culture Media Cytotoxicity Assay. A liver cell cytotoxicity assay of the standardized media cocktail (5-fluorocytosine, posaconazole, gentamicin sulfate, and neomycin trisulfate hydrate) was performed using HepG2-A16-CD81EGFP cells.17 First, 1536-well plates were seeded with hepatoma cells 24 h before the assay. Each cocktail compound was dissolved in DMSO or water, diluted (12-point serial 3-fold dilutions), and transferred into a cell-seeded plate. Puromycin (10 μM) (Santa Cruz biotechnology #sc-205821) and DMSO (0.1%) were used as positive and negative controls of toxicity, respectively. Fortyeight hours after incubation (37 °C, 5% CO2), the media was

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Pamela Orjuela-Sanchez: 0000-0002-1093-9105 Joseph M. Vinetz: 0000-0001-8344-2004 Elizabeth A. Winzeler: 0000-0002-4049-2113 Author Contributions

P.O.-S. designed experiments, performed assays, analyzed data, and wrote the manuscript. Z.H.V. performed assays and analyzed data. M.M. coordinated An. darlingi colony work. C.T.-R. carried out mosquito and field work. S.M. performed P. berghei luciferase assay. G.M.L.M. conducted P. falciparum blood stage assay. B.C. provided advice. J.M.V. coordinated Iquitos field study site, gave advice, and wrote the manuscript. E.A.W. conceived the study, designed experiments, and wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was funded by Medicines for Malaria Venture (MMV120094/95/96) to E.A.W. and the US National Institutes of Health Cooperative Agreement U19AI089681 and D43TW007120 to J.M.V. We are indebted to all malaria patients for donating their blood samples to this study. We acknowledge Lutecio Torres, Gerson Guedez, Juan Michi, and Christian Rodriguez for their assistance in the insectary management and experiments. We thank Dionicia Gamboa H



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from the Instituto de Medicina Tropical Alexander von Humbolt, Universidad Peruana Cayetano de Heredia, for supervising the work carried out at the field study site. We would like to thank Paula Maguina, Jan Economy, and Rosa Alban for administrative assistance. This publication has been possible thanks to the authorization and permits N. 0424-2012AG-DGFFS-DGEFFS from Direction de Gestión Forestal y de Fauna Silvestre y la Dirección General Forestal y de Fauna Silvestre del Ministerio de Agricultura de la Republica del Peru.

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ABBREVIATIONS ATQ, atovaquone; calcein AM, calcein acetoxymethyl; dpi, days postinfection; DMSO, dimethyl sulfoxide; EEF(s), exoerythrocytic form(s); FBS, fetal bovine serum; IC50, half maximal inhibitory concentration; PHSP70, Plasmodium heat shock protein 70; PvUIS4, P. vivax up-regulated in infective sporozoites gen; EthD-1, red-fluorescent ethidium homodimer-1; SD, standard deviation; SEM, standard error of mean; spz(s), sporozoite(s)

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Developing Plasmodium vivax Resources for Liver Stage Study in the

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