Bone Marrow Is a Major Parasite Reservoir in Plasmodium vivax Infection

P. vivax gene annotation through ortholog mapping.We mapped P. vivax transcripts by using the stage and cluster annotations of P. falciparum (29) after identification of their orthologs in PlasmoDB (http://www.plasmodb.org). Only syntenic ortholog mappings were considered. For asexual annotation, 3,990 out of 4,396 asexual P. falciparum genes had P. vivax orthologs. Most variant genes were excluded due to lack of syntenic orthologs. P. vivax genes were then labeled for cluster and stage according to their P. falciparum gene ortholog; 244 out of 249 asexual clusters had at least one corresponding P. vivax gene. Out of the 591gametocyte genes annotated as gametocyte specific in a previous report (29), 527 had corresponding P. vivax orthologs. All 29 gametocyte clusters had at least one P. vivax gene representative, and 27 clusters had at least five genes. These annotations were used for stage-specific P. vivax gene expression analysis.

Microarray data analysis.We obtained time course and cross-sectional microarray P. vivax expression profiles of infected blood samples from references 31 and 34, respectively. The expression values from reference 31 were already filtered, normalized, and transformed. Expression values from reference 34 were also normalized and only log-transformed and averaged across technical replicates. For comparison of gene expression levels of different stages (Fig. 1C, box plots), expression levels were averaged across all genes with the same stage annotation. P values were assigned based on two-sample paired t tests. Similarly, gene expression levels were averaged clusterwise for comparison of different clusters (Fig. 1C, heat maps).

Animals, infection protocol, and sample collection for bloodstream P. vivax analysis. (i) Animals.Fourteen spleen-intact male and female laboratory-bred Aotus lemurinus lemurinus monkeys karyotypes VIII and IX (65), with body weights between 758 and 829 g, were used as donors, experimental subjects, or controls. The animals were housed at Gorgas Memorial Institute of Health Studies (ICGES) in Panama and cared and maintained as described elsewhere (38). All animals received daily veterinary care, which included regular physical examinations and body weight determinations at least four times a year, deworming (mebendazol, Pantelmin; Janssen-Cilag, New Brunswick, NJ), as well as vitamin B₁₂ (Catosal; Bayer, Shawnee Mission, KS) and vitamins A, D, and E (Vigantol; Bayer) injections. Rooms had climate control with 12 air changes per hour, min-max temperatures set to 70 to 76°F and 70 to 80% humidity, with a fluorescent red/white light 12-h cycle starting at 03:00 p.m. A balanced mixture of fruits and laboratory primate chow was provided to meet their caloric requirements. Water was administered daily in plastic bottles fitted with a zip tube (Girton; Millville, PA). The animals were kept in pairs in stainless steel 4-unit quad cages (Lab Products Inc., Seaford, DE) with dimensions of 27 by 23.5 by 29.5 inches. Each cage was fitted with a 3/4-inch-diameter polyvinyl chloride (PVC) pipe perch placed across 2/3 of the length of the cage and a 6-inch-diameter, 14.5-inch-long PVC T pipe nest box attached to the roof and back of the cage with cable zip ties. Cages were routinely cleaned and sterilized at 180°F at weekly intervals in a cage washing machine (Steris, Erie, PA). During experimental infections, the animals did not receive analgesics, nor were they trained for special procedures. The experimental protocol was approved by the Gorgas Memorial Institute of Health Studies, Institutional Laboratory Animal Care and Use Committee (CIUCAL) in accordance with procedures described in the Guide for the Care and Use of Laboratory Animals (76) (protocol approval number 2011/02). All experiments described here were performed in accordance with the approved guidelines.

(ii) Infection protocol.A frozen parasite stabilate of P. vivax SAL-I or AMRU-I was thawed and inoculated into the saphenous vein of experimental spleen-intact Aotus monkeys as described previously (66). Giemsa-stained thick blood smears were then examined daily from a prick in the marginal ear vein, starting on day 5 postinoculation (p.i.), and parasitemia was evaluated and reported as the number of parasites per microliter using the method described in 1932 by Earle and Perez (67).

(iii) Sample collection.For expansion of P. vivax parasite populations, we inoculated one donor animal each with AMRU-I and SAL-I, respectively, and passaged the parasites in four experimental animals once parasitemia reached 5 × 10³/μl (Table S2). Parasitemia was monitored, and the animals were bled once for experimental samples (Fig. S2); when parasitemia reached a peak of ≥8.0 × 10³ parasites per µliter (between days 12 and 15 postinfection), 3.5 ml of blood was collected from the femoral vein in sodium citrate anticoagulant and spun down, and the plasma was separated and frozen for later use. The iRBC pellet was washed three times with phosphate-buffered saline (PBS; pH 7.2) and further divided: one aliquot containing 500 µl of packed RBCs was preserved in Glycerolyte and stored in liquid nitrogen as a stock repository; the remaining 1.5 ml of packed RBCs was split into 4 aliquots for subsequent use in qRT-PCR, Western blot analysis, IFA, and ex vivo culture. For qRT-PCR, packed RBCs were resuspended in 2.5 ml of McCoy’s medium supplemented with 20% human serum and passed once through a CF11 cellulose column previously stabilized with PBS (pH 7.2) to exclude white blood cells from the sample (68). The recovered RBC eluate was further centrifuged, washed three more times in PBS, and adjusted to a 20% hematocrit with McCoy’s medium to a suspension volume of ~1.3 ml. At this point, blood was mixed with 5 parts of Trizol for mRNA preservation. For Western blot analysis, packed RBCs were treated with saponin at 0.15% and centrifuged, and the pellet was washed, resuspended in SDS sample buffer, and frozen until further use. For IFA, the RBC sample was diluted 1:1,000 in PBS, spotted onto slides, fixed with methanol, and frozen until used for either IFA or Giemsa staining. For ex vivo culture, RBCs were overlaid onto a 45% Percoll cushion as described previously (69–71) and then centrifuged, and the band containing enriched mature P. vivax-infected RBCs (trophozoites, schizonts, and gametocytes) was collected and processed for Western blotting and IFA smears as described above. The pellet containing mostly ring forms was washed in PBS, resuspended in 5 ml of McCoys’s complete medium, and cultured in Corning Costar tissue culture 6-well plates (Sigma-Aldrich, St. Louis, MO) at 37°C in a culture hermetic bubble in an atmosphere of 90% N₂, 5% CO₂, and 5% O₂ for 48 to 96 h, replacing the gas mixture every 24 h without changing the medium. Giemsa-stained smears were taken at 24 and 48 h, and at 48 h additional aliquots were collected for qRT-PCR and IFA experiments as described above. Finally, the animals were rescued by treatment with mefloquine (MQ) at 20 mg/kg of body weight orally once to end the experiment.

To measure the dynamics of infection and gametocytogenesis, we treated an animal with CQ. This approach is based on the observation that gametocytogenesis is enhanced when subjects are treated with CQ or pyrimethamine, as it has been demonstrated in the Plasmodium chabaudi rodent malaria model (72–75). Specifically, we used the CQ-resistant P. vivax AMRU-I strain for this experiment, assuming that CQ treatment would sufficiently stress the parasite population to increase gametocyte production without clearing the asexual infection. We infected one Aotus monkey with P. vivax AMRU-I, and the animal was then treated at peak parasitemia and at recrudescence with CQ at 15 mg/kg orally for 3 days. During infection follow-up for the last 50 days p.i., blood samples were collected at 9 time points, approximately 3 to 5 days apart, to measure dynamics of infection by qRT-PCR and Giemsa smear.

Nonhuman primate terminal studies.All NHP animal acquisition, transportation, housing, care, procedures, and release from studies were performed according to the National Institutes of Health (NIH) Animal Research Advisory Committee (NIH ARAC) guidelines, protocols approved by the National Institute of Allergy and Infectious Diseases (NIAID) Animal Care and Use Committee (NIAID ACUC), and in compliance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (76). Briefly, Saimiri and Aotus monkeys were socially housed, consistent with the experimental design, in accord with the NIAID DIR Animal Care and Use Program Policy on Social Housing of Nonhuman Primates. All husbandry was consistent with the current state of the art for New World nonhuman primate care, including maximum quiet, fully enclosed nesting areas, a 12-h light-dark cycle, and safe toys. Daily cleaning and technician health checks were conducted as quietly and unobtrusively as possible. Individual animal records were taken to reflect all procedures conducted. A balanced mixture of fruits and laboratory primate chow were provided to meet their caloric requirements.

Only splenectomized animals that participated in multiple malaria studies and were eligible for final bleed were utilized for postmortem sample collection by NIH pathologists.

Approximately 10³ to 10⁵ P. vivax pRBCs from cryopreserved or fresh NHP blood were utilized to infect animals intravenously (i.v.). Animals were monitored weekly or daily based on blood smears, hematocrit, and weight. Animals in this study experienced only subclinical signs from the infection (namely, blood RBC parasitemia and minor hematocrit depression). Any monkey that reached a parasitemia of 5% or more or a hematocrit of 25% or less was immediately treated for cure of parasites, including on weekends and holidays, with MQ (25 mg, single dose, orally) or Malarone (weight-adjusted pediatric dose of 25 mg/kg/day atovaquone plus 10 mg/kg/day Proguanil, orally for 3 days). Criteria for consideration of euthanasia (at the discretion of the attending veterinarian, after consultation with the Principal Investigator) included the initial and current hematocrit, initial and current degree of parasitemia, other indicators of response to the antimalarial drug treatment, and the general health condition of the animal. No animal suffered unnecessarily. Euthanasia was performed in accordance with the Guide for the Care and Use of Laboratory Animals (76).

P. vivax ex vivo culture from human patient samples.Cryopreserved Brazilian P. vivax isolates were obtained through the “Efficacy of Chloroquine (CQ) Alone Compared to Concomitant CQ and Primaquine for P. vivax Infection” clinical trial (NCT02691910) with written informed consent from all patients (Institutional Review Board of the Institute of Biomedical Sciences, University of São Paulo, Brazil [1169/CEPSH, 2014]). Cryopreserved isolates were thawed as previously described (77). Subsequently, P. vivax ring-stage parasites were enriched on a 1.080 g/ml KCl-Percoll gradient. Briefly, 1.080 g/ml KCl-Percoll was achieved by combining KCl high isotonic Percoll (10 mM HEPES, 115 mM KCl, 12 mM NaCl, final) at 35.97% with the KCl high buffer at 64.03%, as described by Roobsoong et al. (78). Density was confirmed with a DMA 35 portable density meter (Anton Paar; Graz, Austria). Density gradients were assembled by layering one part thawed cells in incomplete Iscove’s modified Dulbecco’s medium (IMDM) (3 to 50% hematocrit) on one part density gradient and then centrifuged for 15 min at 1,200 × g. Enriched parasites were collected from the interface, washed twice with incomplete IMDM, and finally resuspended at 1 to 2% hematocrit in completed IMDM (10% heat-inactivated pooled AB⁺ human sera from Interstate Blood Bank, Inc. [Memphis, TN] and 50 mg/ml gentamicin) and moved into culture. Cultures were maintained at 37°C in 5% CO₂, 1% O₂, and N₂ to balance. Parasitemia and asexual and gametocyte maturation were assessed by either light microscopy analysis or methanol-fixed and Hemacolor-stained cytospins (Shandon Southern Instruments, Sewickley, PA).

Sample processing and RNA extraction.For blood samples (Aotus, ex vivo samples), RNA was extracted from Trizol samples by using a Qiagen RNeasy Plus kit including a gDNA eliminator column (Qiagen, Valencia, CA), according to the manufacturer’s protocols.

Tissue samples from monkeys were either fresh frozen or stored in RNAlater (Qiagen) for subsequent RNA analyses. All tissues were completely homogenized in RLT lysis buffer (Qiagen) and using a Polytron homogenizer (Kinematica). Homogenized tissue lysate was centrifuged at 15,000 rpm for 15 min to remove undigested material (pellet), and the resulting clear supernatant was subsequently used for RNA extraction using an RNeasy plus minikit (Qiagen). The lysate was passed through gDNA eliminator columns (Qiagen) to remove genomic DNA, and RNA was extracted according to the manufacturer’s protocol. RNA was eluted in nuclease-free water. An additional gDNA removal step was performed using Turbo DNase (Ambion) treatment for subsequent qRT-PCR analyses. RNA concentrations were measured in a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Cambridge, MA),and samples were stored −80°C until cDNA synthesis.

cDNA synthesis.Reverse transcription was performed with an Invitrogen SuperScript III first-strand system for RT-PCR (Life Technologies, Inc., Carlsbad, CA). Briefly, 8 µl containing ~1 pg to 5 µg of total RNA was extracted by the Trizol method described above and used as the template in a 20-µl volume. After the reaction was stopped, RNase H was added for 20 min and then the samples were stored at −20°C until use. One extra reaction mixture without reverse transcriptase (RT-) was carried out as a negative control for genomic DNA contamination in all samples.

Primers for the qRT-PCR assay.To quantify stage-specific gene expression of selected P. vivax markers by qRT-PCR, we designed forward and reverse primers by using the following criteria: (i) maximum size of 20 bp; (ii) GC content of 55%; (iii) melting temperature (T_m) of 60°C; (iv) PCR product length of 70 to 200 bp. In addition, primers for the candidate gametocyte markers and the constitutive marker PVX_091645 were designed such that one of the two primers spanned an exon-exon junction, in order to minimize amplification from gDNA. Primers were synthesized by GenScript (Piscataway, NJ) and optimized on synthetic cDNA templates as described in Results and for Fig. 2A. For standard controls, we used Pvs25 (PVX_111175) and Pv18s rRNA.

PCR amplification was performed in 20-µl volumes with a Fast SYBR Green qPCR master mix system from Life Technologies, Inc., and an Applied Biosystems ViiA 7 real-time qPCR thermocycler system.

Generation of peptide antibodies against PvLAP5 and PvAMA1.Peptides used for rabbit immunizations are listed in Table S3. Antisera were generated by immunization of one rabbit per construct using standard protocols (GenScript, Piscataway, NJ). Preimmune serum samples were harvested for each study animal on the day prior to immunization (day −1) to immunization. On day zero, each animal was immunized by intraperitoneal injection (i.p.) with a primary dose of 50 µg of recombinant protein in Freund’s complete adjuvant. This was followed by three boosts of 25 µg protein in Freund’s incomplete adjuvant administered i.p. on days 14, 25, and 56. Antisera were collected on day 63 and tested in an enzyme-linked immunosorbent assay for reactivity against the recombinant protein relative to preimmune control serum samples.

Western blot analysis.For immunoblot analysis, 375 µl of peripheral blood at a peak parasitemia of ~8.0 × 10³ parasites/µl was collected from Aotus monkeys infected with P. vivax SAL-I; the blood samples were treated with 0.1% saponin in 1× PBS buffer to release parasites. Saponin-lysed parasites (3.0 × 10⁶) were resuspended in 10 volumes (15 µl) of SDS lysis buffer, and 5 µl (1.0 × 10⁶) of each sample was used for Western blotting. Blood from an uninfected Aotus monkey was used as a negative control. Mouse anti-pLDH antibodies at a 1:5,000 dilution, rabbit anti-PvLAP5 antibodies (GenScript) at a 1:1,000 dilution, and rabbit anti-PvAMA1 antibodies (GenScript) at a 1:1,000 dilution were used. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) and goat anti-rabbit IgG (H+L) secondary antibodies were used for detection.

Immunofluorescence assays.P. vivax AMRU-I and SAL-I samples from each inoculated Aotus animal were collected for thick and thin blood smear examination by Giemsa staining and bright-field microscopy. Aotus blood and ex vivo samples were spotted onto 10-well slides, fixed in methanol, and snap-frozen until further use. For IFA, slides were thawed and processed for staining. For antibody optimization, slides were incubated in a humid chamber for 1 h with serial dilutions of 1:500 to 1:5,000 antibody titers in bovine serum albumin-PBS (pH 7.2) buffer and then washed thoroughly with PBS 3 times. Samples were further labeled with secondary goat anti-rabbit Alexa Fluor 488-fluorescein isothiocyanate–conjugated antibodies (Invitrogen Molecular Probes, Eugene, OR), diluted 1:500, and incubated for 30 min. After a final wash with PBS, samples were mounted and counterstained with Vectashield 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA) and analyzed with a Nikon Eclipse TE300 inverted fluorescence microscope.

Histological assays.Formalin-fixed tissues from monkeys were embedded in paraffin blocks, and sections of 5-µm thickness were obtained for each tissue. For immunohistochemistry analysis, sections were processed through deparaffinization steps in xylene followed by dehydration steps through a graded ethanol series (100% to 50%). Antigen retrieval was performed by incubating slides at 95°C in a steamer for 30 min and using a universal antigen retrieval reagent (R&D Systems). Following antigen retrieval, blocking was performed using Tris-buffered saline blocking buffer (Thermo Scientific) for 20 min, followed by 10 min each with avidin and biotin blocking buffers (Invitrogen) to block endogenous biotin and avidin, respectively. Double labeling of parasite-specific antigens (pLDH, PvLAP5, PvAMA1, and Pvs16) and blood vessels (CD31) was performed by the following protocol of antibody dilutions and incubations. Mouse monoclonal anti-pLDH antibodies were used at 1:1,000 dilution in combination with rabbit anti-CD31 antibodies (Abcam, Inc.) at 1:20 dilution. Rabbit anti-PvLAP5 antibodies (1:500), rabbit anti-PvAMA1 antibodies (1:500), or rat anti-Pvs16 antibodies (1:500) were each used in combination (1:20) with mouse anti-CD31 antibodies (Abcam, Inc.). Primary antibodies were diluted in blocking buffer and incubated overnight at 4°C. Secondary antibodies were selected to label parasites in red and blood vessels in brown. HRP-conjugated goat anti-rabbit or anti-rat–HRP antibodies and a biotin conjugate of the F(ab')2 fragment of goat anti-mouse IgG (H+L) antibodies were used (Invitrogen) (diluted to 1:500 in universal blocking buffer), followed by streptavidin conjugated to alkaline phosphatase (AP; Thermo Scientific) (diluted 1:3,000 in universal blocking buffer). For the development of signal, 3,3′-diaminobenzidine (DAB) chromogen reagent (Thermo Scientific) was added for 20 min for development of brown color. The slides were washed in water, and Fast Red 4 TR/naphthol AS-MX substrate reagent (Sigma-Aldrich) was applied for 5 min. Slides were subsequently rinsed in water and counterstained in Mayer’s hematoxylin and mounted in aqueous mounting medium. Images of the slides were captured using a whole-slide imaging system, and parasites were quantified by counting 500 consecutive high-power fields at 400× magnification. Intravascular and extravascular parasite localization was quantified with respect to CD31-positive blood vessels.

Calculation of tissue and blood parasite burdens. (i) Bone marrow.For calculation of parasite burdens, first the volume of the bone marrow from each animal was calculated using total body weight and the assumption it represented 4% of body weight and had a density of 1.03 g/cm³ (79). Second the volume of the slide measurement (500 high-power fields [hpf]) was calculated as follows: length (160 µm) × width (160 µm) × section depth (5 µm), i.e., 3.2 × 10⁻⁶ cm³. The volume of bone marrow was then divided by the volume of the slide to give the number of bone marrow units per animal. The resulting number was multiplied by the number of parasites per 500 hpf to give the total number of parasites in bone marrow for each animal.

(ii) Liver.For liver burdens, the volume of the liver was calculated using total body weight and an assumption it represented 27 ml per kg (79). The volume of the liver was then divided by the volume of the slide (see above, 3.2 × 10⁻⁶ cm³) to give the number of liver units per animal. The resulting number was multiplied by the number of parasites per 500 hpf to give the total number of parasites in liver for each animal.

(iii) Lung.For lung burdens, the volume of the lung was calculated using total body weight and an assumption it represented 7.7 ml per kg (79). The volume of the lung was then divided by the volume of the slide (see above, 3.2 × 10⁻⁶ cm³) to give the number of lung units per animal. The resulting number was multiplied by the number of parasites per 500 hpf to give the total number of parasites in lungs for each animal.

(iv) Peripheral blood.For peripheral blood parasite burdens, the total volume of blood was calculated using total body weight and an assumption it represented 50 ml per kg (79); based on that volume, the total RBC count was calculated using an estimate of 5 × 10⁶ red cells/µl. This number was multiplied by the measured parasitemia to give the total number of parasites in peripheral blood for each animal.