William Pearson's LALIGN program, which implements a linear-space local similarity algorithm, was used to perform regional alignments. Sequence and structural comparisons were performed for the V3 loop of SU of HIV-1 strain MN, accession: AAT67509; P. vivax DBP, ACD76813; P. knowlesi DBP, XP_002261904; P. falciparum erythrocyte binding protein EBA-175 (F1), accession AAA29600. Plasmodium proteins are members of pfam05424 (a member of the superfamily cl05146).
Blood was collected in 10% citrate phosphate dextrose (CPD) and stored at 4°C unwashed for up to 4 weeks, or washed in RPMI with malaria supplements and stored in malaria culture medium at 50% hematocrit for up to 2 weeks. The DARC+ human erythrocytes used in the erythrocyte binding assay and the P. knowlesi erythrocyte invasion assay had the phenotype Fy(a-b+) as determined by standard blood banking methods using anti-Fya and anti-Fyb antisera (Gamma Biologicals, Houston, TX). Erythrocytes were washed three times in DMEM (Gibco BRL) and resuspended to a hematocrit of 10% in complete DMEM for the erythrocyte binding assay. Erythrocytes used in the P. knowlesi erythrocyte invasion assay were washed three times and resuspended to a hematocrit of 10% using malaria complete RPMI.
Cell Culture and Transfection of COS-7 Cells
COS-7 cells (ATCC CRL 1651; Rockville, MD) were cultured in DMEM with 10% heat inactivated FBS (Gibco BRL) in a humidified 5% CO2 incubator at 37°C. Cells were seeded in polystyrene dishes with 3.5-cm diameter wells and grown for 24 h to 30-50% confluence before transfection with 1 mg of pHVDR22 plasmid DNA and 10 ml of Lipofectamine (Gibco BRL).
P. knowlesi in vitro culture
Whole blood from rhesus macaques was collected in 10% CPD and allowed to separate overnight at 4°C. The erythrocyte phase was washed in RPMI with L-glutamine and supplemented with 25 mM HEPES, 300 mM hypoxanthine, 10 mM thymidine, 1.0 mM sodium pyruvate, and 11 mM glucose. This RPMI with malaria supplements was then used to prepare malaria culture medium by adding to a final concentration of 0.24% sodium bicarbonate and 0.2% Albumax-I (Life Tech, Gibco BRL). Cultures were maintained at a hematocrit of 10% in malaria culture medium under an atmosphere of 5% O2, 5% CO2, balanced N2 (Air Liquide, Houston, TX) at 38°C.
Percoll Purification of Schizont-infected Erythrocytes
Cultures of P. knowlesi at 5-10% infected erythrocytes were washed three times in RPMI with malaria supplements and 10% FBS and brought up to a hematocrit of 10%. A 50% Percoll solution was made by adding 0.45 volumes 1X PBS, 0.05 volumes 10X PBS and 0.5 volumes Percoll (Sigma). Two ml of the washed culture was overlaid on 2 ml of the 50% Percoll solution in a 4 ml polystyrene tube and centrifuged for 20 min at 2100 RPM in a Sorvall centrifuge. The ring of cells at the interface was removed, pooled and washed three time in 1X PBS. The pellet was brought up in malaria culture medium to 2 × 107 cells/ml.
PvRII Erythrocyte Binding Assay
COS-7 cells were transfected by Lipofectamine with 1-2 mg of pHVDR22 DNA, a plasmid kindly provided by L. Miller which expresses region II of the DBP of P. vivax on the cell surface as a chimera with the HSV gD protein  Duffy Fy (a-b+) erythrocytes were washed three times in RPMI 1640, resuspended to a hematocrit of 1% in 1 ml of complete DMEM with the chemokines RANTES, MIP-1α, SDF-1 or AOP-RANTES at concentrations of 0, 0.1, 1, 10, and 100 nM for 1 h at 37°C (Peprotech, Gryphon Pharmaceuticals, San Francisco, CA). This suspension was swirled over aspirated COS-7 cells 40-60 h after transfection and allowed to settle over 2 h at 37°C. The COS-7 cells were then washed three times with 2 ml of PBS to remove nonadherent erythrocytes. The number of adherent erythrocyte rosettes was scored in 20 randomly chosen fields at a magnification of 40 using an inverted microscope. Percent inhibition was determined by dividing the number of rosettes in the presence of chemokines by the number at a concentration of 0 nM. The 50% inhibitory concentration (IC50) was determined by the mean of three separate experiments to use in a semi-log cubic spline curve fit with the DeltaSoft 3 software (Biometallics, Inc., Princeton, NJ).
P. knowlesi Erythrocyte Invasion Assay
Human Duffy Fy(a-b+) erythrocytes were washed in complete malaria medium and 2 × 107 washed cells were added to increasing concentrations of chemokines in malaria culture medium at final volume of 900 ml for 1h at room temperature. To each tube of chemokine-treated erythrocytes, 100 ml or 2 × 106 schizont-infected erythrocytes was added and placed in a well of a polystyrene 24-well plate (Becton-Dickinson). The cultures were maintained under a blood-gas atmosphere at 38°C for 8 h to allow the infected erythrocytes to rupture and release free merozoites capable of infecting new erythrocytes and developing to ring-stage trophozoites. The culture was centrifuged at 2100 RPM for 3 min and a thin smear was made from the pellet. The thin smear was fixed with methanol and stained with Leukostat Solution B (100 mg Eosin Y+300 ml 37% formaldehyde + 400 mg sodium phosphate dibasic + 500 mg potassium phosphate monobasic, q.s. to 100 ml with dH2O), rinsed, and stained with Leukostat Solution C (47 mg Methylene Blue + 44 mpp Azure A + 400 mg sodium phosphate dibasic + 500 mg potassium phosphate monobasic, q.s to 100 ml with dH2O). The percentage of erythrocytes infected with ring-stage trophozoites per 2000 erythrocytes was determined at 1000X. Inhibition of invasion expressed as % inhibition was determined by dividing the percentage of ring-stage parasites by the percentage of ring-stage parasites at 0 nM chemokine, multiplying by 100 and subtracting this value from 100 .
The software StatView (Brainpower, Inc., Calabasas, CA), was used to determine the statistical difference between the inhibitory concentrations of RANTES, AOP-RANTES, and MIP-1α, using a two-way ANOVA test.
The plasmids pHVDR22, pHKADR22, pHKBDR22 and pHKGDR22 encode for the region II (amino acids 198-522) of the P. vivax DBP and region II of the P. knowlesi α, β and γ genes, respectively, in the context of the HSV gD protein. These plasmids have been previously described and were kindly provided by the laboratory of Louis H. Miller. These plasmids were created from the plasmid pRE4, which contains an SV40 origin of replication, a Rous sarcoma virus LTR as a promoter, the coding region of the HSV glycoprotein D (HSV gD) inserted in the HindIII cloning site, and the SV40 early polyadenylation signal. The HSV gD features a 25 amino acid signal peptide at the amino terminus, a 24 amino acid hydrophobic transmembrane region, a 30 amino acid cytoplasmic tail at the carboxy terminus, and two epitopes at amino acids 11-19 and 272-279 that can be targeted specifically with the monoclonal antibodies ID3 and DL6, respectively. The region II sequences were inserted between the unique Apa I and Pvu II restriction sites.
Cloning and Site Directed Mutagenesis
Mutants of the region II expressing plasmids were generated by three strategies: inverse PCR, PCR and restriction digestion, or PCR-based site directed mutagenesis. Each mutant was sequenced by Research Genetics, Inc. (Huntsville, Ala.) to confirm proper construction at the site of mutation.
The following constructs were made from the pHVDR22 plasmid:
pv22d32 This construct contains a deletion in amino acids 216-247 of the RII of P. vivax, which corresponds to the V3-like peptide region with similarity to the V3 loop and comprises cysteines C1 to C4 of region II. For lack of proper restriction enzyme sites, an inverse PCR strategy was use to amplify the entire pHVDR22 plasmid flanking the site to be deleted. The primers 5'TGT ATG AAG GAA CTT ACG AAT TTG G3' and 5'TTT CAT TAC AGT ATT TTG AAG3' were first phosphorylated with T4 kinase then used with the long range, high fidelity DeepVent polymerase (New England Biolabs, Inc., Beverly, MA) to amplify the product under the following thermocycling conditions: 5 minutes at 94°C initial denaturing, then 35 cycles at 94°C for 60 seconds, 55°C for 60 seconds, 72°C for 3 minutes. The product was digested with DPN I to eliminate methylated input plasmid DNA, then blunt-end ligated with high concentration ligase (Gibco BRL).
pv22MNV3 This construct replaces the 32 amino acid V3-like peptide of the P. vivax RII with the V3 loop of HIV-1 strain MN. To amplify the V3 loop of HIV-1MN by PCR, PM-1 cells were infected with HIV-1MN (donated by Dr. James Robinson, Tulane University Medical Center) and genomic DNA was isolated from infected cultures. This DNA includes proviral DNA and was used as template for a PCR with the primers P2 5'GAC GCT GCG CCC ATA GTG CTT CCT G3' and P5 5'ACA CAT GGAATT CGGCCAGTA GT3' which are homologous to conserved regions of the env gene of HIV and amplify the region between nucleotides 6884 and 7783, which includes the V3 loop.
This PCR product then served as template in a nested PCR of the HIV-1MN V3 loop using the primers HVMN-F 5'AATTGTACAAGACCCAACTAC3' and HVMN-r 5'ATGTGCTTGTCTTATAGTTCC3'. This nested PCR was carried out using the DeepVent enzyme to generate blunt ends. The product of the second, nested PCR was gel purified. The gel-purified amplicon was then re-amplified in a 300 ml PCR using HVMN-r and HVMN-F primers, which were first phosphorylated with T4 poly N kinase. This reamplification product was column purified and blunt-end ligated to the inverse PCR product described in the preparation of pvD32. The sequenced construct matched the MN V3 sequence as previously published.
pv22suf32 This construct was designed to determine if the 32-aa V3-like peptide of P. vivax RII is sufficient for DARC binding by deleting all flanking RII amino acids. The primers used to create this construct were 5'CAA AAT CAG CTG ATG AAA AAC TGT AAT TAT3' and 5'CAA ATT GGG CCC TTC CTT CAT ACA TAA TTG3' and contain the restriction sites for Apa I and Pvu II. The pHVDR22 plasmid was digested with Apa I and Pvu II, and the digested vector was separated from the insert by gel electrophoresis and extracted using the QIAEX II gel extraction kit (Qiagen Inc., Valencia, CA). The PCR product was also digested with Apa I and Pvu II and ligated to the digested vector.
pv22d5C1 This construct deletes amino acids 198-216, or the 5' flanking region to C1. This was created using the primers 5'TGT ATG AAG GAA CTT ACG AAT TTG G3' and 5' GGG GCC TTG GGC CCT GTC ACA AC3', the product of which was digested with Apa I and Pvu II and cloned into the digested vector as described for psuf32
pv22d3C4 This construct deletes amino acids 247 to 522 or the 3' flanking region to C4. This was created using the primers 5'CCG GTC CTG GAC CAG CTG ACG3' and 5'TTT CAT TAC AGT ATT TTG AAG3' the product of which was digested with Apa I and Pvu II and cloned into the digested vector as described in psuf32
pv22d5C4 This construct deletes amino acids 198 to 247 or the 5' flanking region to C4 This was created using the primers 5'CAA TTA CAG CTG AAG GAA CTT ACG AAT TTG3' and 5' GGG GCC TTG GGC CCT GTC ACA AC3' the product of which was digested with Apa I and Pvu II and cloned into the digested vector as described in pv22suf32
pv22KARA The Stratagene QuickChange kit (Promega) was used to mutate the heparin binding consensus site in PvRII at amino acids 217-226 from YKRKRRERDW to YARKAREADW using the primers 5' GTA ATT ATG CGA GAA AAG CTC GGG AAG CAG ATT GG3' and 5' CCA ATC TGC TTC CCG AGC TTT TCT CGC ATA ATT AC3'. These primers also introduce an Ava I site as a silent mutation for screening.
pv22KAKA The Stratagene QuickChange kit was used to mutate a second potential heparin binding consensus site at amino acids 364-373, between C5 and C6, from SVKKRLKGNF to SVKARLAGNF using the primers 5' GAT GTA CTC AGT TAA AGC AAG ACT TAA GGG G3'. These primers also introduce an Afl II site as a silent mutation for screening
pv22KA The Stratagene QuickChange kit was used to introduce a single alanine substitution in the heparin binding consensus site at amino acids 217-226 from YKRKRRERDW to YKRARRERDW. 5'CTC TTT CCC GAC GAG CTC TCT TAT AAT TAC AG3' and 5'CTG TAA TTA TAA GAG AGC TCG TCG GGA AAG AG3'. These primers also introduce a Sac I site as a silent mutation for screening.
The following mutants were made from the pHKADR22, pHKBDR22, or pHKGDR22 plasmids using the Stratagene QuickChange kit:
pkalpha22KARA This mutant was designed to change the heparin binding consensus site in pHKADR22 at amino acids 217-226 from DKRKRGERD to DARKAGEAD using the primers 5'GTC CCA ATC TGC TTC CCC GCG AGC TCT CGC ACT ACC ACA CTT G and 5'CAA GCG TAA TGA TGC GAG AGC TCG CGG GGA AGC AGA TTG GGA C3'. These primers also introduce a Sac I site as a silent mutation for screening.
pkbeta22KARA This mutant was designed to change the heparin binding consensus site in pHKBDR22 at amino acids 217-226 from NKRKRGTRD to NARKAGTAD using the primers 5' CAG TCC CAA TCT GCT GTC CCG CGA GCT TCT GCA TTA TTA CAC C3' and 5'GGT GTA ATA ATG CGA GAG CTC GCG GGA CAG CAG ATT GGG ACT G3'. These primers also introduce a Sac I site as a silent mutation for screening.
pkgamma22KARA This mutant was designed to change the heparin binding consensus site in pHKGDR22 at amino acids 217-226 from DKRKRGERD to DARKAGEAD using the primers 5'GTC CCA ATC TGC TTC CCC GCG AGC TCT CGC ACT ACC ACA CTT G and 5'CAA GCG TAA TGA TGC GAG AGC TCG CGG GGA AGC AGA TTG GGA C3'. These primers also introduce a Sac I site as a silent mutation for screening.
Transfected cells used in the erythrocyte binding assay were rinsed in PBS and incubated for 1 h at 37°C with monoclonal antibodies that bind to amino acids 11-19 and 272-279 of the mature HSV gD protein found in pHVDR22. These primary antibodies, ID3 or DL6 (provided by Drs. Gary Cohen and Roselyn Eisenberg), were used at a 1:2000 dilution in PBS containing 10% FBS. The cells were rinsed with PBS and incubated at 37°C with fluorescein conjugated anti-mouse antibodies at 1:100 in PBS containing 10% FBS. Untransfected COS-7 cells were also stained as a negative staining control. The cells were then fixed with 4% paraformaldehyde for 15 min, and observed for surface expression of the products of the transfected plasmids using an inverted fluorescence microscope.