Plasmodium vivax malaria causes significant morbidity and mortality worldwide, and only one drug is in clinical use that can kill the hypnozoites that cause P. vivax relapses. HIV and P. vivax malaria geographically overlap in many areas of the world, including South America and Asia. Despite the increasing body of knowledge regarding HIV protease inhibitors (HIV PIs) on P. falciparum malaria, there are no data regarding the effects of these treatments on P. vivax's hypnozoite form and clinical relapses of malaria. We have previously shown that the HIV protease inhibitor lopinavir-ritonavir (LPV-RTV) and the antibiotic trimethoprim sulfamethoxazole (TMP-SMX) inhibit Plasmodium actively dividing liver stages in rodent malarias and in vitro in P. falciparum, but effect against Plasmodium dormant hypnozoite forms remains untested. Separately, although other antifolates have been tested against hypnozoites, the antibiotic trimethoprim sulfamethoxazole, commonly used in HIV infection and exposure management, has not been evaluated for hypnozoite-killing activity. Since Plasmodium cynomolgi is an established animal model for the study of liver stages of malaria as a surrogate for P. vivax infection, we investigated the antimalarial activity of these drugs on Plasmodium cynomolgi relapsing malaria in rhesus macaques. Herein, we demonstrate that neither TMP-SMX nor LPV-RTV kills hypnozoite parasite liver stage forms at the doses tested. Because HIV and malaria geographically overlap, and more patients are being managed for HIV infection and exposure, understanding HIV drug impact on malaria infection is important. Plasmodium vivax malaria causes significant morbidity and mortality worldwide Plasmodium vivax malaria geographically overlap in many areas of the world, including South America and Asia, and impact of drugs used in HIV management on P. vivax remains understudied. In addition, treatment of P. vivax is complicated by the ability of the parasite to assume a hypnozoite form in the liver stages, which leads to relapsing malaria P. falciparum in vitro, whereas NNRTIs do not P. vivax in ex vivo culture P. falciparum malaria is prevalent Plasmodium parasites in rodent malaria models P. falciparum in vitroThe World Health Organization (WHO) recommends HIV management with combination antiretroviral therapy (ARV), with first line therapy including a non-nucleoside reverse transcriptase inhibitor (NNRTI) and 2 nucleoside reverse transcriptase inhibitors (NRTIs) with few exceptions, and second line therapy including an HIV protease inhibitor (HIV PI) and 2 NRTIs P. vivax malaria, we set out to investigate the effects of the HIV PI lopinavir , the most potent antimalarial HIV PI with other Plasmodium species P. cynomolgi liver stage hypnozoite forms, an established animal model for P. vivaxGiven the overlap of HIV and malaria with HIV-infected and exposed patients in endemic areas receiving these drugs, and given the shortage of drugs available that offer radical cure for All studies described herein were performed on an animal study proposal approved by the NIAID Institutional Animal Care and Use Committee. Macaca mulatta (rhesus macaque) monkeys of Indian origin maintained in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care were used, and all experiments were performed in accordance with the guidelines of the NIAID Division of Intramural Research (DIR) Animal Care and Use Committee (Permit Number ASP LMIV 9E). For the pilot experiments  ), where quinine 20 mg/kg base dose IM BID for 7 days with a single dose of artesunate 8 mg/kg IV times one is used for high parasitemias. From this, we derived a dose of 32 mg/kg quinine dihydrochloride , or from our own prepared stocks, were used to infect a splenectomized monkey to test the quinidine gluconate regimen used as described below. This monkey also served as a donor (as described below) for a mosquito bite infection study in which the quinidine regimen used was also tested. In the former, the splenectomized monkey was infected and tested positive for parasites by D4–D9 after infection and was cured with quinidine gluconate 26 mg/kg IM BID x 7 days with smears remaining negative for 6 weeks. In the latter, two monkeys were infected by the bites of mosquitoes and one had a primary attack D9, relapse #1 D29, #2 D46; the second had Primary attack D11, relapse #1 D29, #2 D45  were allowed to feed on a splenectomized donor rhesus monkey that had been infected with P. cynomolgi B strain by intravenous (IV) injection of 1×106 frozen infected RBCs  thawed from frozen stock. When mature sporozoites reached the salivary glands at day 10 to 12, the infected mosquitoes were allowed to feed on 9 malaria-naive monkeys for 30 min, with 34 to 51 bites recorded used to infect each monkey . After feeding, mosquitoes not engorged with blood were removed, and mosquito bite counts represent the number of mosquitoes with blood , and 10,000 red cells per smear were examined before being declared negative.Based on the pharmacokinetic profiles of the drugs used in this investigation, and the assumption that 4–5 half-lives are required to both reach steady state and for drug elimination to occur An increase in the time to detection of parasites in the presumed first and second relapses as detected by smear were each used to deduce liver stage reductions, and appearance of parasites after treatment with quinine and quinidine were considered relapses, as previously described By smear, all control monkeys had time to first detection of blood stage parasites of 9–12 days (first parasitemias detected on D9–12 post infection) . Only 2/The TMP-SMX-treated monkeys did not have a first relapse at all, but did have second relapses Day 43, D41, D45.LPV-RTV-treated monkeys all had first and second relapses which occurred at parallel time points  compared with the control monkeys, suggesting that there was no impact on hypnozoites at this dose.P. cynomolgi rhesus macaque model. This is the first evaluation of LPV-RTV and TMP-SMX in vivo in non-human primates against relapses of P. cynomolgi as a model for P.vivax.We have demonstrated that TMP-SMX and LPV-RTV do not have significant effect on relapse in the Time to first detection of blood stage parasites, or prepatent period, was 9-12 days in 2 control monkeys, consistent with previous reports Plasmodium species and life cycle stages, including P. vivax ex vivo P. falciparumP. vivax, have not been fully described LPV-RTV did not increase prepatent period, nor did it prevent relapse. LPV-RTV-treated monkeys all had first and second relapses which occurred at parallel time points  compared with the control monkeys, suggesting that there was no impact of the HIV PI on hypnozoites. HIV PIs have been shown to kill a multitude of P. falciparum in vitro, and in P. knowlesi in vivoP. cynomolgi in vivo, primary parasitemia itself could be delayed and relapses still occur. These antifolates have also been shown to kill the exoerythrocytic stages of P. cynomolgi and P. knowlesi in vitroP. cynomolgi indicates TMP-SMX would offer no benefit in treating or preventing P. vivax malaria.The TMP-SMX-treated monkeys did not have a first relapse at all, but did have relapses Day 43, D41, and D45. As for TMP-SMX, delay to the first appearance of parasitemia but no difference between relapse in treated compared with control suggests there was killing of the actively dividing liver stage parasites, but not hypnozoites P. cynomolgi asexual stage An alternative explanation is that delay to the first appearance of parasitemia in this TMP-SMX treated group could be due to incomplete effectiveness of a shortened regimen of quinine fully killing asexual stage parasites, with TMP-SMX killing asexual stages. Other antifolates can kill In either case, the parasites seen on D43, 41, 45 are relapses because all animals were treated for the first parasitemia observed after the primary attack with quinidine, and this regimen was proven effective at eradicating asexual stages in our pilot experiments . TherefoBecause HIV and malaria overlap, further studies are required to validate whether PIs and TMP-SMX offer benefit in reducing clinical malaria through anti-liver stage, although possibly not anti-hypnozoite, effect. If drugs used in HIV management can reduce malaria burden, their administration can be tailored or timed to maximize those benefits.


P. falciparum gametocytes are sterilized within hours, whereas clearance from blood takes days. Gametocytaemia AUC and clearance times are determined predominantly by the more numerous female gametocytes, which are generally less drug sensitive than the minority male gametocytes, whereas transmission-blocking activity and thus infectivity is determined by the more sensitive male forms. In choosing doses of transmission-blocking drugs there is no substitute yet for mosquito-feeding studies.Indirect clinical measures assessing anti-malarial drug transmission-blocking activity in falciparum malaria include measurement of the duration of gametocytaemia, the rate of gametocyte clearance or the area under the gametocytaemia-time curve (AUC). These may provide useful comparative information, but they underestimate dose-response relationships for transmission-blocking activity. Following 8-aminoquinoline administration  Plasmodium falciparum differs from the other malaria parasites of humans in that the emergence of gametocytaemia is delayed with respect to asexual parasitaemia. The anti-malarial drugs which are used to treat falciparum malaria also kill young developing sexual stage parasites but they have little or no activity against the mature transmissible gametocytes. Treated infections are an important source of transmission especially in areas of low, unstable malaria transmission. Artemisinin combination treatment (ACT), the first-line treatment for falciparum malaria, reduces gametocyte carriage more than do other anti-malarial treatments [P. falciparum gametocytes [The objective of anti-malarial drug treatment is to cure the malaria infection as rapidly, reliably and safely as possible . In endeeatments –6, but eetocytes –9. Preveetocytes , 10. Prietocytes . Primaquetocytes . G6PD deetocytes .P. falciparum gametocytocide (given together with an ACT) should be reduced to 0.25 mg base/kg as this lower dose is safer and, on the basis of the available data from direct transmission-blocking assessments from mosquito-feeding studies, was considered to be as effective as higher doses in reducing transmissibility [P. falciparum gametocytaemia clearance [P. falciparum gametocytocides (notably primaquine).In efforts to contain and eliminate artemisinin-resistant falciparum malaria it is particularly important that all possible measures to reduce malaria transmission are taken. The World Health Organization (WHO) has recently recommended that the single primaquine dose for use as a sibility –14  but “for the currently recommended low dose regimen, there is little direct evidence to be confident that the effect of reduction in gametocyte prevalence is preserved” [P. falciparum gametocyte clearance following primaquine given in doses less than 0.4 mg/kg. The first study was a comparative assessment of asexual and sexual parasite clearance times following seven-day regimens of quinine given either alone or together with one of the following: tetracycline, primaquine 0.25 mg base/kg, primaquine 0.5 mg/kg, or with artesunate given alone or together with primaquine 0.5 mg/kg  the transmission-blocking effects are more evident in the mosquito  which would not be reflected in indirect assessments of transmission-blocking activity from serial gametocyte counts. Malaria gametocytes arise from blood stage schizonts, which have committed to sexual stage development, so they arise from asexual parasites –20. In fP. falciparum gametocytes. The biology of male and female gametocytes is very different. Since the early days of malariatherapy it was recognized that successful transmission of malaria to mosquitoes required fully mature gametocytes, and microgametocytes which readily exflagellated. Crucially it is these minority male (micro)gametocytes which are usually more drug sensitive [P. falciparum: “in our cases female gametocytes almost invariably make their appearance several days before males and throughout the period of their presence the females outnumber the males from between 5 and 20 to 1 or more, and then when the gametocytes begin to diminish it is the males which fall off in numbers, and the females appear to persist for a longer period in the peripheral blood” [The ratio of female to male gametocytes is unequal in falciparum malaria with a strong female bias in most studies , 23, 24.ensitive . In 1928ensitive . Shute al blood” . Thus ifP. falciparum asexual parasites which are sequestered, immunity, and the counting methods used [Serial asexual parasite counts in blood are used to assess therapeutic responses to antimalarial drugs. Asexual parasite clearance times are dependent on several factors in addition to the drug treatment including initial parasite counts, proportion of ods used . These fods used . Live gaods used . Microscods used , 36–38. ods used , 38, 39.Gametocyte clearance time estimates depend on the detection methods used. Times derived from the more sensitive QT-NASBA are obviously longer than those based on microscopy. Times derived from thick film counts per 1000 white cells will be longer than those per 200 white cells. Fitting a mathematical model of gametocyte dynamics to measurements of sub-microscopic gametocyte prevalence and density  to estimsolely by the less drug-sensitive females  [The relationship between e below) , 18.The area under the gametocytaemia time curve (AUC), whilst providing a model independent measure, assumes that density and time are equally important in terms of infectivity to mosquitoes, which clearly they are not. Without logarithmic transformation a gametocyte density of 1,000/uL for three days provides the same AUC as a density of 100/uL for 30 days. With logarithmic transformation this becomes 4.5 days. High gametocyte densities in patients presenting with acute symptomatic falciparum malaria are often not infectious at all, comprising the first wave of young Stage V gametocytes, whereas low densities later on are usually infectious , 34, 43.8-aminoquinolines sterilize malaria infections within hours, but gametocyte clearance does not start to increase for > one day  Figure , yet theUse of the AUC also assumes a linear relationship between gametocyte density and infectivity whereas in reality this relationship is sigmoid , 43, 44.The limited available evidence suggests that gametocyte clearance is a first order process. Ideally in drug comparisons or dose-response assessments this would be assessed by a first order rate constant or half-life, as for asexual parasite clearance , but gamThe Cochrane review  used theAUC (days 1 to 15) = ((8-1)*(G1 + G8)/2) + ((15-8)*(G15 + G8)/2)/14 for days 1 through 15.AUC (days 1 to 29) = ((8-1)*(G1 + G8)/2) + ((15-8)*(G15 + G8)/2) + ((29-15)*(G29 + G15)/2)/28 for days 1 through 29.AUC (days 1 to 43) = ((8-1)*(G1 + G8)/2) + ((15-8)*(G15 + G8)/2) + ((29-15)*(G29 + G15)/2) + ((43-29)*(G43 + G29)/2)/42 for days 1 through 43where Gx = mean gametocyte density on day X . The log(10) AUC values were estimated using geometric mean gametocyte density.Assessment of the reduction in infectivity of gametocytes to female anopheline mosquitoes by direct or membrane feeding measures the pharmacological effect that is intended –49. DireGametocyte sterilization is rapid but killing is slower.Clearance of dead gametocytes from the blood is slow.8-aminoquinolines affect predominantly the minority male gametocyte population .Comparison of changes in gametocytaemia and infectivity reveals important differences. Whereas 8-aminoquinolines sterilize falciparum malaria infections rapidly, usually within hours of exposure, gametocyte densities in blood do not decline for at least one day . This suClearly if drug effects are directed predominantly against a minority male gametocyte population  then dosAlthough sub-microscopic densities of gametocytes are an important source of malaria transmission , 44, 50,P. falciparum to anopheline mosquitoes [P. falciparum infectivity to mosquitoes by the third and fifth days of treatment, which was before any decline in gametocytaemia [P. falciparum within hours  was shown to clear gametocytaemia more rapidly than asexual stages in falciparum malaria . By 1929squitoes , 56. Thesquitoes , 57–60. cytaemia   but they are difficult and laborious, and there are few data as a consequence. Fortunately there is renewed interest in these assessments and more information to guide evaluations of transmission-blocking effects drugs and vaccines will soon become available. Meanwhile indirect measures based on quantitation of gametocytaemia provide a guide to the comparative activity of different drugs or different drug dosages - but they make a number of assumptions. Critically current gametocyte clearance measures probably underestimate significantly the inhibitory effects of 8-aminoquinolines on infectivity. This compromises dose-response assessments both for the time of onset and the magnitude of transmission-blocking activity. The unique studies of Rieckmann et al. in which volunteers were infected with P. falciparum and managed to a steady state of parasitaemia highlights the discrepancies between total gametocytaemia, mosquito oocyst development, sporozoite formation, and the infectivity of these mosquitoes to naïve volunteers [P. falciparum infections  are an advance [P. falciparum malaria infections from patients presenting with patent gametocytaemia. Unfortunately the 8-aminoquinolines require hepatic bioactivation which compromises ex vivo assessments. Characterizing sex-specific markers and assessing the pharmacokinetic-pharmacodynamic relationship for primaquine’s gametocytocidal activity on male and female gametocytes in vivo is a research priority. Ideally dose-response assessments should be performed by comparing the infectiousness of gametocytaemic blood to anopheline mosquitoes before and after different doses, preferably with assessment of both oocyst and sporozoite formation, plasma primaquine concentration measurement (and CYP2D6 genotyping) [More direct assessments of drug effects on male and female gametocytes individually such as the  advance , 63, and advance –49. Primotyping) . Given tDespite their difficulty, mosquito-feeding studies still remain the ‘gold standard’ for the assessment of transmission-blocking dose-response relationships. More information is needed on the relative susceptibility of male and female gametocytes and the kinetics of male and female gametocyte killing, together with direct comparisons with infectivity assessments, before indirect measures can be used confidently to make dosage recommendations.


Plasmodium falciparum human malaria parasite – MSP1 and AMA1. These antigens were delivered to healthy malaria-naïve adult volunteers in Phase Ia clinical trials using recombinant replication-deficient viral vectors – ChAd63 to prime the immune response and MVA to boost. In subsequent Phase IIa clinical trials, immunized volunteers underwent controlled human malaria infection (CHMI) with P. falciparum to assess vaccine efficacy, whereby all but one volunteer developed low-density blood-stage parasitemia. Here we assess serum antibody responses against both the MSP1 and AMA1 antigens following i) ChAd63-MVA immunization, ii) immunization and CHMI, and iii) primary malaria exposure in the context of CHMI in unimmunized control volunteers. Responses were also assessed in a cohort of naturally-immune Kenyan adults to provide comparison with those induced by a lifetime of natural malaria exposure. Serum antibody responses against MSP1 and AMA1 were characterized in terms of i) total IgG responses before and after CHMI, ii) responses to allelic variants of MSP1 and AMA1, iii) functional growth inhibitory activity (GIA), iv) IgG avidity, and v) isotype responses . These data provide the first in-depth assessment of the quality of adenovirus-MVA vaccine-induced antibody responses in humans, along with assessment of how these responses are modulated by subsequent low-density parasite exposure. Notable differences were observed in qualitative aspects of the human antibody responses against these malaria antigens depending on the means of their induction and/or exposure of the host to the malaria parasite. Given the continued clinical development of viral vectored vaccines for malaria and a range of other diseases targets, these data should help to guide further immuno-monitoring studies of vaccine-induced human antibody responses.The development of protective vaccines against many difficult infectious pathogens will necessitate the induction of effective antibody responses. Here we assess humoral immune responses against two antigens from the blood-stage merozoite of the  Plasmodium falciparum is the preeminent cause of human malaria disease and a leading example of a parasite with a complex multi-host multi-stage lifecycle where a number of steps in the infectious process have been shown to be antibody-susceptible. These include the invasion of liver cells by sporozoites delivered from the mosquito bite; the invasion of red blood cells (RBC) by merozoites; clearance of infected RBC; and sexual-stage development within the blood meal inside the mosquito midgut P. falciparum continues to exert a huge burden on global public health in terms of morbidity and mortality in vivo in humans One leading strategy for many years has sought to develop an effective antibody-inducing vaccine against the blood-stage merozoite, seeking to neutralize RBC invasion P. falciparum MSP1 and AMA1. In a series of Phase Ia clinical trials, we demonstrated that the use of a recombinant chimpanzee adenovirus serotype 63 (ChAd63) prime, followed by a boost with the recombinant orthopoxvirus, modified vaccinia virus Ankara (MVA), was safe in healthy UK adults and capable of inducing strong cellular and humoral immune responses against both antigens de novo antigen-specific responses induced in control volunteers as opposed to those induced by a lifetime of exposure in naturally-immune Kenyan adults. These data provide the first in-depth assessment of the quality of adenovirus-MVA vaccine-induced antibody responses in humans, along with assessment of how these responses can be modulated by subsequent parasite exposure.Recently we assessed this approach for targeting the blood-stage malaria antigens P. falciparum 3D7 clone sporozoites delivered by five infectious mosquito bites. In the case of the Phase IIa studies, vaccinated volunteers were immunized with vaccines encoding MSP1, AMA1 or the liver-stage malaria antigen ME-TRAP (a multi-epitope string fused to the thrombospondin-related adhesion protein) 9–5×1010 viral particles (vp)), followed eight weeks later by a boosting vaccination i.m. with the MVA vector (doses ranging from 1×108–5×108 plaque forming units (pfu)) recombinant for the same antigen, all as previously described Serum samples used throughout this study were obtained from Phase Ia safety and immunogenicity clinical trials for the MSP1 All necessary ethical approvals  and regulatory approvals  were granted as previously described, and the four trials were registered with ClinicalTrials.gov  Serum from adults living in Kenya was collected during adult cross-sectional surveys between 2006 and 2008 from the villages surrounding the Chonyi area in Kilifi, Kenya which experiences moderate malaria transmission with an entomological inoculation rate (EIR) of 10–100 infective bites/person/year 42/sequence blocks 16+17) fused in tandem and preceded by the naturally conserved regions of MSP1 sequence . The MSP142 region is composed of an N-terminal 33 kDa region  followed by a C-terminal 19 kDa region . In the case of ME-TRAP, the construct consists of the T9/96 allele of P. falciparum TRAP fused to a multi-epitope (ME) string The composition of the bi-allelic vaccine inserts for MSP1 19  was produced in and purified from Escherichia coli as a soluble glutathione S-transferase (GST) fusion protein The ELISA antigens used in this study have been previously described 19 and AMA1 405) = 1.0. The standard curve was then used to convert absorbance values of individual test sera  into AU . The limit of detection in both ELISA assays was 10 AU, and we assigned the AU value of 1.0 for any test samples with less than 10 AU. Any values more than 10 AU are considered as positive responses in the analyses reported here.Standardized ELISAs were conducted according to previously described protocols for both MSP1405 = 1.0, and plated at 50 µL/well in 16 wells of a 96 well plate. Following incubation and washing, an ascending concentration of the chaotropic agent NaSCN was added in duplicate wells (50 µL per well) down the plate (0 to 7 Molar (M) NaSCN). The NaSCN gradient was prepared beforehand by diluting an 8 M stock solution (made using water) into PBS. Plates were incubated for 15 min at room temperature (RT) before washing and development as for total IgG. The intercept of the OD405 curve for each sample with the line of 50% reduction of the OD405 in the NaSCN-free well for each sample (i.e. the concentration of NaSCN required to reduce the OD405 to 50% of that without NaSCN = IC50) was used as a measure of avidity.IgG antibody avidity was assessed by sodium thiocyanate (NaSCN)-displacement ELISA. The assays were performed exactly as for total IgG except sera were individually diluted in Casein block solution   to a level calculated to give an ODAntibody isotype ELISAs were performed exactly as for total IgG except sera were individually diluted 1∶100 in Casein block solution and 50 µL added to duplicate wells of six 96 well plates . After incubating for 2 h the plates were washed and the following secondary antibodies added (one per plate) at 1∶1000 dilution, 50 µL per well, in Casein block solution: biotin-conjugated mouse anti-human IgG1 Fc (clone HP6070) ; biotin-conjugated mouse anti-human IgG2 Fc (clone HP6002) ; biotin-conjugated mouse anti-human IgG3 (clone HP6050) ; biotin-conjugated mouse anti-human IgG4 (clone HP6025) ; alkaline phosphatase-conjugated goat polyclonal anti-human IgA α-chain ; and biotin-conjugated goat polyclonal anti-human IgM µ-chain . After 1 h, all the plates were washed before addition of 50 µL per well ExtrAvidin alkaline phosphatase  diluted 1∶5000 in Casein block solution . After 30 min, all plates were washed again before development as per the total IgG ELISA. Blank wells and internal development controls were included on each plate.19 or AMA1 antigen, a number of controls  were included on each plate. Instead of coating with recombinant malaria protein, these controls wells were coated instead with native human IgG1, IgG2, IgG3 and IgG4  at 2 µg/mL in PBS; or a hyperimmune Kenyan adult serum sample diluted 1∶10000 in PBS. During the ELISA, these wells were filled with Casein block solution during the steps where test sera were added to the rest of the plate. In the case of the MSP119 ELISAs, further controls included the addition of epitope-matched monoclonal antibodies (mAbs) in the place of test sera (again 2 wells per control) at 0.2 µg/mL in Casein block solution. These mAbs were against the e9 epitope of P. falciparum MSP119 and possessed the human IgG1 et al., in preparation) or IgA1 When coating the plates with recombinant MSP1P. falciparum parasites for 48 h and relative parasitemia levels were quantified by biochemical determination of parasite lactate dehydrogenase.GIA was assessed as previously described P≤0.05.Data were analysed using GraphPad Prism version 5.04 for Windows  and non-parametric analyses. To compare responses between selected time-points in a group a Wilcoxon matched-pairs signed rank test was used. Spearman’s rank correlation was used to analyse correlations. Statistical significance was considered at 19 and AMA1 antigens were shown to peak 4 weeks after the MVA boost  P. falciparum parasites, volunteers were typically challenged 16 days after the MVA boost , but showed a significant increase at the first follow-up visit after drug treatment (dC+35/d107), P = 0.02  , blood-stage parasite exposure led to a 1.9 fold increase  in the anti-MSP119 IgG antibody titer at dC+35  in volunteers receiving ChAd63-MVA immunization encoding MSP1 alone, AMA1 alone, MSP1+AMA1 co-administered or MSP1+ME-TRAP co-administered . dC−1 isstudies) . Very sistudies) , althougat dC+35 .19 antigen, with 5/6 in the first study , blood-stage parasite exposure led to a 1.8 fold increase  in the anti-AMA1 IgG antibody titer at dC+35  between the pre-challenge IgG antibody titer at dC−1 in volunteers receiving the MSP1 vaccine  and the post-challenge IgG titer measured at dC+35. A similar trend was observed in AMA1 vaccinees, but did not reach significance (P = 0.06). However, there was no clear correlation between the dC−1 titer and subsequent fold-change in IgG titer , there was a significant positive correlation between their dC+35 IgG titers against MSP119 and AMA1. Both the MSP1 and AMA1 viral vectored vaccines expressed bi-allelic constructs. In the case of MSP1 both major alleles of MSP119 were encoded 19 still showed a strong significant correlation with those measured against the heterologous QNKG allele, but the relationship was slightly weaker in comparison to the vaccinees who had been previously immunized against both. A similar result was observed in the immune sera from the Kenyan adults where a strong correlation in ELISA reactivity was observed against both alleles of MSP119 and AMA1 in agreement with other studies We also assessed cross-reactivity by ELISA of these sera against other alleles of MSP1de novo IgG antibody responses, we also sought to assess the impact on functional activity. In vitro GIA was measured against challenge homologous 3D7 clone parasites using 10 mg/mL purified IgG from the dC−1 and dC+35 time-points. The dC−1 data have been previously reported Having shown the impact of CHMI on vaccine-induced and 19 and -AMA1 antibodies using a NaSCN-displacement ELISA. Initially this was measured in serum from volunteers in the Phase Ia clinical trials of these vaccines 19 the median avidity of the antibodies tended to increase slightly over time following ChAd63 MSP1 priming (from d28 to d90) (19.We next assessed the avidity of the anti-MSP1 to d90) . Adminis to d90) . After t19    and anot, n = 9) . Followi, n = 9) .19 and AMA1 in 20 adults from the cohort in Kilifi, Kenya as well as in the infectivity control volunteers from the CHMI studies. The antigen-specific IgG in the Kenyan adults showed a much greater range of avidity in comparison to the vaccinees, ranging from 0–5 M for both MSP119  and vaccination plus 3D7 clone parasite exposure in the context of CHMI (dC+35), immune sera showed a significant positive correlation between IgG1 and IgG3 antibodies  . Interestingly, a very contrasting relationship was observed in the Kenyan adults, where a very dichotomous response was observed – immune sera showed strong IgG1 or IgG3 responses, but not both  . A similnot both .19, we observed a significant correlation between IgG1 and IgG3 responses following vaccination . Following CHMI, this relationship was not as strong and did not reach significance  Similar to MSP119, a different result was also observed in the Kenyan adults with all showing detectable anti-AMA1 IgG1 responses, but only about half of these also positive for anti-AMA1 IgG3. Overall these results suggest a propensity of the ChAd63-MVA viral vectored vaccines to induce a balanced IgG1 and IgG3 response, in comparison to parasite exposure where more skewed IgG isotype profiles appear evident.In the case of AMA1, as for MSP119 and AMA1. Initial data showed that a single CHMI  led to increased serum IgG antibody responses against both antigens in vaccinees post-infection and drug-treatment. On average, serum IgG responses to both antigens showed a similar 2-fold increase by 35 days post-CHMI. This kinetic and short-term re-boosting is consistent with modest incremental increases in antibody levels with age that are observed in repeatedly exposed individuals P. falciparum malaria antigens reported an increase in sero-reactivity to MSP1 and AMA1 in vaccinees post-challenge + T cell help to B cells responding to the infection. We have previously characterized in detail the T cell responses induced by these vaccines following vaccination and CHMI + T cells with a T follicular helper (Tfh) cell-like phenotype This study has explored the impact of CHMI on qualitative and quantitative vaccine-induced antibody responses against two blood-stage malaria antigens – MSP119 IgG. Similar results have been reported in travellers returning with P. falciparum infection 19 sero-positivity in AMA1 vaccinees was also more pronounced, suggesting that pre-existing immune responses against a blood-stage antigen (in this case either anti-AMA1 antibodies or AMA1-specific CD4+ T cell help) may aid the induction of de novo responses against other blood-stage antigens. A previous report from a different series of CHMI studies suggested PfEMP1 and the R2 region of glutamate-rich protein (GLURP) may be similarly immuno-dominant to MSP119, whilst responses against the R0 and R1 regions of GLURP, MSP3 C-terminus, F2 region of EBA175, and RIFINs are more akin to those observed against AMA1 As previously observed in our study of B cell responses de novo responses in malaria-naïve controls, and boosting of vaccine-induced responses in the MSP1 and AMA1 vaccinees, there was no significant impact on overall functional GIA activity of purified IgG assessed at the peak of the responses 35 days post-CHMI. Negative GIA in the serum of control volunteers after CHMI is consistent with a previous Phase IIa study report 19 responses measured using an alternative assay with a chimeric parasite de novo responses against other blood-stage parasite antigens. A previous AMA1 vaccine study in Mali suggested that malaria-specific non-AMA1 IgGs can interfere with GIA mediated by AMA1-specific IgGs de novo antibodies against other antigens during the CHMI that interfered with AMA1-associated GIA. Nevertheless, given the overall low levels of GIA induced by ChAd63-MVA MSP1 or AMA1 immunization  showed that neither adjuvant nor vaccine dose significantly influenced average avidity measured in the same manner as here 19 response (in comparison to that induced by vaccination), suggesting the IgG response underwent minimal affinity maturation in this context of primary low-density infection. In contrast, a wide range of avidities was observed for both antigens in the sera from Kenyan adults, similar to observations in rodent malaria models 19 measured in this Phase IIa vaccine trial did not, by themselves, impact on parasite multiplication rates following CHMI with homologous 3D7 clone parasites delivered by mosquito bite in vitroThe role of IgG avidity in protection against the blood-stage merozoite remains a poorly understood attribute of antibody function. The NaSCN-displacement ELISA employed here represents a relatively crude measure of the overall avidity (sum of affinities) of the polyclonal antigen-specific IgG response. Interestingly, increased avidity was clearly evident following the MVA booster vaccination for anti-MSP1in vivo in humans remains widely debated. Complicated interplay has also been described between different antibody isotype Fc regions, activatory and inhibitory Fc receptors on immune cells, the complement system, and arrayed antigen within soluble immune complexes The analysis of serum antibody isotypes showed that ChAd63-MVA MSP1 or AMA1 immunization in healthy UK adults induced predominantly IgG1, IgG3, IgA and IgM against both antigens. No role has been described for IgA in protection against blood-stage merozoites 42 vaccine given in the same manner 19, and substantial levels of IgM to both antigens in agreement with a primary immune response. The Kenyan adults showed mixed IgG1 and IgG3 responses against AMA1 that were more comparable to vaccination, in contrast to MSP119 where a dichotomous relationship was observed. Data are conflicting from other endemic populations, but in general IgG1 and/or IgG3 are most consistently observed for anti-AMA1 and anti-MSP119 serum antibody responses With regard to vaccine-induced AMA1- and MSP1-specific antibody isotype responses, a bivalent AMA1 protein vaccine administered in Alhydrogel elicited predominantly IgG1 Overall these data describe complex differences in qualitative aspects of human antibody responses against malaria antigens depending on the means of their induction and/or exposure of the host to the malaria parasite. Given the continued clinical development of viral vectored vaccines for a wide variety of diseases Figure S1Trial timelines. (A) In Phase Ia studies, the ChAd63 priming vaccination was administered on day 0 (d0) and the MVA booster vaccination on day 56 (d56). Blood samples were taken for exploratory immunology at the indicated time-points. (B) In Phase IIa studies, volunteers were immunized in the same manner. Subsequently, controlled human malaria infection (CHMI) followed the MVA booster vaccination on average 16 days later (range 13–25 days). Blood samples were taken at the indicated time-points, including the day before CHMI (dC−1); 35, 90 and 150 days post-CHMI ; and at the time-point of malaria diagnosis (dC+DoD), nominally d85. (C) Phase IIa infectivity control volunteers underwent CHMI without previous vaccination in parallel with vaccinated volunteers. Non-protected and control volunteers were diagnosed with microscope-patent blood-stage malaria infection on average 10–11 days after CHMI (dC+10/11) at which point they received anti-malarial drug treatment (TIF)Click here for additional data file.Figure S2Assessment of MSP119 IgG antibody responses post-CHMI. Mean anti-MSP119 serum IgG responses were assessed over time by ELISA and are shown for a second Phase IIa CHMI trial (TIF)Click here for additional data file.


Plasmodium vivax malaria, mechanisms that trigger transition from uncomplicated to fatal severe infections are obscure. In this multi-disciplinary study we have performed a comprehensive analysis of clinicopathological parameters and serum proteome profiles of vivax malaria patients with different severity levels of infection to investigate pathogenesis of severe malaria and identify surrogate markers of severity. Clinicopathological analysis and proteomics profiling has provided evidences for the modulation of diverse physiological pathways including oxidative stress, cytoskeletal regulation, lipid metabolism and complement cascades in severe malaria. Strikingly, unlike severe falciparum malaria the blood coagulation cascade was not found to be affected adversely in acute P. vivax infection. To the best of our knowledge, this is the first comprehensive proteomics study, which identified some possible cues for severe P. vivax infection. Our results suggest that Superoxide dismutase, Vitronectin, Titin, Apolipoprotein E, Serum amyloid A, and Haptoglobin are potential predictive markers for malaria severity.In  Plasmodium vivax has the most extensive global distribution. Although, generally P. vivax is known to cause benign infections, recent incidences of involvement of this species in complicated and severe malaria in different parts of the world suggest a drastic shift in the clinical paradigm for vivax malaria. In verity, except the occurrence of a very high level of parasitemia all the other complications of severe falciparum malaria including cerebral syndromes and fatal outcomes have been observed in acute P. vivax infectionsP. vivax has an ability to cause severe and fatal manifestations even at very low-grade parasitemias6Malaria caused an estimated 198 million cases worldwide in 2013, leading to 5,84,000 deathsBlood biomarkers and surrogate host markers for malaria can be used for early diagnosis, discrimination from other infections with overlapping clinical manifestations as well as aid in scrutinizing response to therapy and predicting outcomes8101215In this study serum samples from non-severe (uncomplicated) vivax malaria (NSVM) and severe vivax malaria (SVM) patients along with healthy community controls (HC) and two other febrile infectious diseases, dengue fever (DF) and leptospirosis (LEP) from three different endemic regions of India were investigated. 2D-differential in gel electrophoresis (2D-DIGE) and isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics in combination with electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) and Q-Exactive mass spectrometry platforms were used in the discovery phase of the study and selected targets were validated by enzyme-linked immunosorbent assay (ELISA) . Quite aAmong the 200 vivax malaria patients enrolled in the study, 34  were classified as severe cases of malaria and 166  were non-severe malaria patients. 146 HC , 31 DF patients  and 13 LEP patients  were enrolled in this study . Kolmogop < 0.05), among which 75 spots exhibited increased abundance, while the remaining 56 spots were down-regulated  of 35 (27 up-regulated and 8 down-regulated) and 51 proteins (35 up-regulated and 16 down-regulated) were observed in NSVM and SVM, respectively  using Ingenuity Pathway Analysis (IPA) identified six and eight overlapping interaction networks in NSVM and SVM, respectively.   analysis also indicated involvement of the differentially abundant proteins in blood coagulation system. In addition, plasminogen activating cascade, apoptosis signaling, inflammation mediated by chemokine and cytokine signaling pathways were identified as the related physiological pathways with statistical significance ( < 0.05) . In DAVI < 0.05) . Taken tgulation ; Fig. 3.p < 0.05)  ( < 0.05) . HP and  < 0.05) . Intrigu < 0.05) . However < 0.05) . Importa < 0.05) .p < 0.005). Analysis of individual samples also presented similar results as obtained with the pooled samples (p < 0.005)  using SPR, which allows the measurement of functionally active target proteins, also indicated a progressive increase in serum abundance of SAA with increasing disease severity . SAA con samples . Nearly < 0.005) .In ELISA analysis, nearly 2-fold up-regulation in serum level of SOD was observed in SVM as compared to NSVM and HC. Further analysis of SOD activity in HC, NSVM and SVM sera also indicated an elevated level (more than 2-fold) of this antioxidant enzyme in severe infection . HoweverP. falciparum infection represents the major cause of malaria associated fatality, P. vivax can also cause severe infections with deadly complications18P. vivax can cause severe and fatal manifestations even at a very low parasite biomassIn plasmodial infections, mortality occurs mainly due to the severe disease syndromes leading to cerebral, renal, pulmonary or hepatic involvement and often multi-organ dysfunctions. Although, P. falciparum infection, while in case of vivax malaria the cause for coma is not precisely known. Since P. vivax does not sequester, neurological syndromes could be due to the release of toxic molecules in the circulationPathogenesis of severe vivax malaria is still largely a black box since the mechanisms that trigger the transition of uncomplicated malaria into severe-complicated manifestations are merely unidentifiedP. vivax infections. Aberrations in multiple essential physiological processes can provide a glimpse of the pathogenesis of severe manifestations of vivax malaria , cerebral manifestation and multiple organ involvement were observed in severe vivax malaria. Comparative proteomics analysis of HC, NSVM and SVM patients indicated alteration in serum levels of diverse classes of proteins associated with the coagulation pathway and hemostasis, complement cascade, muscle contraction and cytoskeletal regulation, signaling in immune system and acute phase response and lipid metabolism and transport in  malaria . Serum pP. falciparum infection, which could be due to the invasion of P. falciparum parasites to red cells of all ages even as early as orthochromatic erythroblasts, whereas P. vivax has specific predilection to infect only young RBC’s (reticulocytes) limiting the parasitemia level and RBC lysisClinicopathological analysis and proteomics profiling indicated alterations in different hematological parameters and serum abundance of some components of the coagulation system in malaria patients. Thrombocytopenia is observed almost invariably in malaria infection and can be used as a sensitive but non-specific marker of acute infectionSevere malaria is associated with systemic inflammation, increased capillary permeability, hypoxia, acidosis, endothelial activation and microvascular coagulopathy. Plasmodial infections influence blood coagulation by various pathobiological mechanisms which could be the critical components of malaria pathogenesiset al. demonstrated increased levels of multiple muscle-specific proteins including creatine kinase, carbonic anhydrase III and myosin XVA in children with cerebral falciparum malaria. Interestingly, we have identified elevated serum levels of NEB, VTN, and TTN in SVM patients, while in non-severe infection, serum levels of these candidates were found to be nearly similar compared to the healthy controls. This finding may raise the possibility of endoplasmic reticulum stress and cytoskeletal involvement in malaria. Earlier studies reported significant alterations of the red cell cytoskeleton in falciparum malaria patientsP. vivax infection. A previous study by Baker et al. presented evidences for the involvement of circulating neutrophil extracellular traps (NETs) in a protective mechanism against falciparum malariaElevated serum levels of various muscle proteins in circulation indicate the possibilities of muscle damage and microvasculature lesions in severe malaria. A recent study by Bachmann P. vivax malariaet al. demonstrated that measurement of Glutathione-S-transferase, lipid peroxidation and Catalase could be implemented as reliable biochemical markers for vivax malaria38Oxidative damage of platelets plays a crucial role in the pathogenesis of thrombocytopenia found in P. vivax infection, which is often observed in falciparum malaria. Serum abundance of proteins involved in cytoskeletal system and apoptotic pathways were highly modulated in severe P. vivax infection, which was not observed in the non-severe cases. Additionally, serum levels of a few proteins identified in this study including SAA, CRP, SOD, HP, Apo E, Apo A-I and TTN exhibited sound correlation with disease severity, and thereby could serve as potential indicators for severity of malarial infection. Hitherto, there is a serious lack of a “gold standard” definition for severe vivax malaria. Parameters for severe falciparum malaria defined by WHO cannot be merely extrapolated to define severe P. vivax infection, since there are considerable differences between the pathogenesis of these two plasmodial infections. We speculate that apart from clinicopathological parameters, serum/plasma levels of protein candidates identified in this study such as SOD, Apo E, VTN, TTN, SAA, and HP are potential predictive markers for malaria severity and could be studied further for improving case definitions for severe malaria.In summary, results obtained from this comprehensive proteomics analysis revealed activation of the oxidative stress and counteractive pathways, as well as elevated serum levels of cytoskeletal proteins as the possible cues contributing towards SVM. Noticeably, blood coagulation cascade was not affected adversely in This multi-centric study was approved by the Institutional Ethics Committees of Seth GS Medical College & KEM Hospital, Mumbai; Grant Govt Medical College and Sir JJ Group of Hospitals, Mumbai; PD Hinduja National Hospital & Medical Research Centre, Mumbai; Medical College Hospital Kolkata, Kolkata; and Malaria Research Center, S.P. Medical College, Bikaner. Experiments involving human subjects were performed in accordance with relevant guidelines and regulations. Prior to the sample collection process written informed consent was obtained from each participant after giving detailed explanations about the experimental procedure in the language best understood by them.P. vivax and P. falciparum) or co-infection(s) with any other infectious disease were excluded from this study. Female patients who were pregnant at the time of enrolment and participants below 18 and above 65 year age were not enrolled in this study. Subjects with a history of significant systemic diseases such as autoimmune disorders, chronic liver diseases and bleeding disorders or psychiatric illness as judged by history and physical examinations were also excluded. Demographic, epidemiological and clinical details, together with past history of diseases of all the malaria patients and controls enrolled for this study were documented.This study was conducted by recruiting malaria patients and controls from both the urban and rural populations from three different malaria endemic regions of India: Mumbai, Kolkata and Bikaner. Recruitment of the subjects was carried out during 2010 to 2013 at different study sites. Patients  with SVM and NSVM were diagnosed by clinical manifestations and microscopic examination of peripheral smear, and results were confirmed through RDT. Patients were classified as “severe malaria” based on the clinical description of severe falciparum malaria as per the WHO guidelines for malariaBlood samples (5.0 mL) were collected from the antecubital vein of the patients (during the febrile stage of infection) and HC subjects using serum separation tubes . Serum separation and storage was performed as described previouslyp-value < 0.05 was considered as statistically significant.After evaluation of clinical manifestations and confirmation of diagnosis, hematological and biochemical parameters including complete blood count, total bilirubin, creatinine, ALT, AST and ALP were measured in the study groups. Hematological investigations were carried out using a fully automated cell counter and biochemical measurements were performed using automated chemical analyzers. ESR was measured by using the Westergren methodDepletion of the high abundant serum proteins, protein extraction, and 2D-DIGE were performed as described previouslyp < 0.05) differentially abundant protein spots identified in 2D-DIGE experiment and subsequent enrichment of the digested peptides using Zip-Tip C18 pipette tips  were performed as described previouslyversion 3.5.3) was used for data acquisition using the following parameters: Mode- reflectron & positive, mass window range- 800–4000 Da, Laser shots- 2000 for MS, 4000 for MS/MS, precursor selection- 20 peaks and S/N > 6. Mascot version 2.1 (http://www.matrixscience.com) was used for protein identification using the following settings: database- Swissprot, species- Homo sapiens, missed cleavage- one, enzyme - trypsin, variable and fixed modification- oxidation of methionine and carbamido-methylation of cysteine, respectively and MS tolerance - 75 ppm and MS/MS tolerance - 0.4 Da.In-gel digestion of the statistically significant  were divided into three separate pools (n = 10) for iTRAQ-based quantitative proteomics analysis. Protein samples (in rehydration solution) were exchanged to TEAB buffer using Amicon Ultra 0.5 mL centrifugal 3 kDa filters . After buffer exchange, quantification of protein content in each sample was performed using QuickStart Bradford reagent . In-solution digestion (75 μg proteins from each sample) and subsequent iTRAQ labeling of the digested peptides were performed following the manufacturer’s instructions . HC samples were labeled with the 114 iTRAQ reagent, while NSVM and SVM samples were labeled with 115 and 116 iTRAQ labels, respectively. OFFGEL fractionation of the labeled peptides was performed using a 3100 OFFGEL fractionator  with high resolution  IPG strips. In-solution digestion, iTRAQ labeling, and OFFGEL fractionation protocols are described in details elsewhereversion 2.2 . Searches were performed against UniProt database specifying Homo sapiens taxonomy . Data were extracted between MH + 600 and 4000. IAA for cysteine and iTRAQ  were selected as fixed modifications and oxidized methionine was specified as the variable modification. 20 ppm precursor mass tolerance and 50 ppm fragment mass error tolerance were specified. Peptides identified with confidence interval (CI) values above 95% were used for protein identification and quantification.iTRAQ-based quantitative proteomics analysis was performed using two mass spectrometry platforms; Agilent 6550 Q-TOF and Thermo Scientific Q-Exactive. Agilent 6550 iFunnel Q-TOF LC MS/MS instrument  equipped with a Chip-Cube controlled by the Mass Hunter acquisition software was set to perform data acquisition in a positive ion mode as described previously6 and an allowed ion accumulation time of 60 ms. All the raw .msf files were processed using proteome discoverer 1.4 (Thermo Fisher Scientific); Mascot 2.2.4 and SEQUEST were used for database searching against the Uniprot Homo sapiens FASTA. The database searching parameters included precursor ion mass tolerance of 5 ppm and fragment mass tolerance of 0.02 Da.iTRAQ labeled samples  were also analyzed using Q-Exactive mass spectrometer . Details for the LC conditions and MS data acquisition and analysis parameters have been described previously elsewhereversion 1.5.2.6). Reverse and contaminant database hits were filtered out during data processing in Perseus. Categorical annotation was applied to group reporter ion intensities, values were log2 transformed, and were normalized by “subtract (mean)” followed by Z score normalization. Proteins groups were filtered for valid values, and p-values obtained from a paired t-test were used to evaluate significance of differences in the protein abundances between HC and malaria (NSVM/SVM) study cohorts. P-values (adjusted) <0.05 were considered to be statistically significant. Venny 2.0.2 was used to generate the Venn diagrams (http://bioinfogp.cnb.csic.es/tools/venny/index.html)Normalization and statistical analysis of the Q-TOF datasets was performed using the Perseus workstation (vs. NSVM/SVM) satisfying the threshold of statistical significance  in 2D-DIGE and/or iTRAQ (Q-TOF) analysis were subjected to bioinformatics analysis for determining their molecular functions, cellular component annotations, and possible connections with various biological processes, physiological pathways and networks. Differentially abundant proteins (with >1.2-fold change in NSVM/HC and/or SVM/HC) identified in iTRAQ-based quantitative proteomics profiling using the Q-Exactive mass spectrometer were also included in pathway analysis. Bioinformatics analysis and functional clustering of the differentially abundant proteins was performed using IPA version 9.0 , PANTHER system, version 10 (http:// www. pantherdb.org)version 6.7 (http://david.abcc.ncifcrf.gov/ home.jsp)Differentially abundant serum proteins (HC p < 0.05). Efficiency of the classifier proteins for prediction of NSVM, SVM and other two infectious diseases (DF and LEP) was analyzed by plotting ROC curves using GraphPad Prism software package (version 6.02).Eleven differentially abundant proteins were validated further in bigger cohorts of vivax malaria patients and controls  by ELISA. ELISA was performed using commercially available kits following the manufacturers’ directions: SAA, HP, Apo E, Apo A-I, HPX, RBP4, CP, and PLS , SOD , TTN , and VTN . Selection of the proteins for immunoassay-based validation was carried out on the basis of their fold-change, correlation with disease severity, association with malaria pathogenesis and commercial availability of the required ELISA kits. Quantitative direct/ competitive enzyme assay was carried out where standard and serum samples  at a specific dilution were subjected to a microplate pre-coated with a polyclonal antibody specific for the target proteins. Optical densities were measured by using a SpectraMax M2e scanner . Statistical significance of the average ratio of abundance was analyzed by Mann-Whitney U test  following the same methodology as described earlierSOD activity in the serum samples of HC, NSVM and SVM was measured using a commercially available kit  following the manufacturer’s instructions. In brief, 10 μL of standard solution and samples (in duplicates) were added in the designated wells on a 96-well plate along with 200 μL of the diluted radical detector. Reactions were initiated by addition of 20 μL of diluted xanthine oxidase to all the wells. The plate was carefully vortexed for a few seconds and incubated on a shaker for 30 minutes at room temperature. The absorbance was measured at 440–460 nm using a SpectraMax M2e scanner .Serum levels of TBARS in HC, NSVM and SVM were measured using a commercially available kit . Reagents and colorimetric standards were prepared according to manufacturer’s instructions. 100 μL of samples and standards (in duplicates) were added to labelled 5 mL vials containing 100 μL of SDS solution. 4 mL of color reagent was added and the vials were placed in boiling water for an hour. The vials were then immediately incubated on ice for 10 mins to stop the reaction. 150 μL from each vial was loaded onto clear plates at room temperature. The absorbance was measured at 530–540 nm using a SpectraMax M2e scanner .How to cite this article: Ray, S. et al. Clinicopathological Analysis and Multipronged Quantitative Proteomics Reveal Oxidative Stress and Cytoskeletal Proteins as Possible Markers for Severe Vivax Malaria. Sci. Rep.6, 24557; doi: 10.1038/srep24557 (2016).


In this study, antibody anamnestic or memory response elicited by a single episode of P. falciparum infection was investigated.There is no approved vaccine for malaria, and precisely how human antibody responses to +  cells, and antibodies specific to synthetic peptides representing antigenic regions of the P. falciparum proteins GLURP, MSP3 and HRPII were measured.This study involved 362 malaria patients aged between 6 months to 60 years, of whom 19% were early-diagnosed people living with HIV/AIDS (PLWHA). On the day malaria was diagnosed and 42 days later, blood specimens were collected. Parasite density, CD4+ cell counts of PLWHAs and HIV negative individuals were not statistically different, and median post-infection increases in anti-peptide IgG were similar in both groups of patients.On the day of malaria diagnosis, Immunoglobulin (IgG) antibodies against GLURP, MSP3 and HRP II peptides were present in the blood of 75%, 41% and 60% of patients, respectively. 42 days later, the majority of patients had boosted their serum IgG antibody more than 1.2 fold. The increase in level of IgG antibody against the peptides was not affected by parasite density at diagnosis. The median CD4P. falciparum elicits at least 20% increase in level of anti-parasite IgG. This boost in anti-P. falciparum IgG is not affected by parasite density on the day of malaria diagnosis, or by HIV status.In the majority (70%) of individuals, an infection of  Plasmodium falciparum malaria is a leading cause of morbidity and mortality among children in sub-Saharan Africa [Plasmodium falciparum [P. falciparum infections work to build antibody responses to candidate vaccine molecules. We know very little about the extent to which natural infections will boost antibodies generated by candidate malaria vaccines. Candidate blood-stage vaccines include GMZ2 which has been tested in children and adults [n Africa . Humorallciparum  and playlciparum , 4. Howelciparum . Earlierlciparum , 7 and tlciparum , 9. In glciparum , 11. Howlciparum , 13. Thelciparum . Howeverlciparum . Neverthd adults , 17 and d adults , 18. MSPd adults , 20 and d adults , 22. Indd adults , 24.A few clinical trials where whole sporozoite or trophozoite forms were used as vaccine showed that development of host protective immunity against malaria can be achieved although such vaccination would require large numbers of parasites , 26.P. falciparum uncomplicated malaria, we investigated the antibody anamnestic response against three synthetic peptides [P. falciparum infected patients on day 0 (baseline), and collected venous blood for determination of parasite density, CD4+ cell counts and sero-prevalence of antibodies aganist the synthetic peptides and to antigens in PE lysate. The patients were treated with artemether—lumefantrine (co-artem) according to standard treatment guidelines. At day 42, blood was collected from the convalescent patients to determine post-infection levels of specific Immunoglobulin (IgG) and sero-prevalence to the three peptides. The relationship between the levels of anti-peptide antibodies for day 0 and day 42 was assumed to represent a reasonable measure of secondary anti-P. falciparum antibody boost or increase after an acute episode of uncomplicated malaria.To gain further insight into the boosting of parasite specific antibody response following acute peptides  represenP. falciparum mono-infection at any parasitemia, and informed written consent from participants or their parents or guardians for minors, agreement to come to the study clinic for 42 day follow up, age: more than 6 months. A detailed clinical examination was performed and data entered into a pre-tested study questionnaire. The first dose of Artemether-Lumefantrine was administered under direct observation to ensure compliance. If a patient vomited within 30 min, the dose was repeated. The remaining doses were administered at home unsupervised.The study was conducted at Kasangati Health Centre (KHC) which is about 20 km, north east of Kampala the capital city of Uganda. Patients were recruited from the peri-urban villages located within 10 km of KHC. Kasangati lies within a moderate transmission area, with a peak transmission after the two main rainy seasons (February-March and September-October) every year. This study was conducted from November 2010 to January 2011 after the September—October rains. Consecutive sampling was used to enrol a prospective cohort of participants. Eligibility criteria included: uncomplicated malaria as defined previously . In brieA written informed consent was sought from all study participants before they were enrolled into the study. The study was approved by the Makerere University, School of Medicine, Research and Ethics Committee (SOMREC) and the Uganda National Council of Science and Technology (UNCST), 2007–045.P. falciparum mono-infection were subsequently enrolled and a venous blood sample taken for sera and estimation of CD4+ cells. Follow-up of patients was arranged for day 42 and participants were asked to return to the clinic whenever they felt unwell within the subsequent 6 weeks. Follow-up consisted of history taking, a physical examination and blood smear examination.On the day of recruitment, a finger prick blood specimen was taken and thick smears prepared and treated with Giemsa stain. Participants found to be smear positive for P. falciparum infection [During the day 42 visit, a blood specimen was collected to re-assess the antibody levels. Standardized treatment was provided according to the national treatment guidelines. Patients who failed to return on day 42 were visited at their homes by our social worker and brought to the health unit for examination and medical care. Day 42 was selected as suitable for assessing the secondary antibody response after nfection  and detenfection , 30.+ T-cell counts were measured by a FACS Counter .Thick smears were stained with 10% Giemsa stain for 10 min, sexual and asexual parasites were counted against every 200 white blood cells (WBCs). Results were multiplied by 40 to determine the parasite density (parasites/μL), assuming a normal WBC count of 8000 per μl , 32. A sP. falciparum serum antibodies to peptides MSP3, HRP II and GLURP were measured by enzyme linked immunorsobent assay (ELISA) essentially as described before [3, 3’, 5, 5’-Tetramethylbenzidine) substrate. Optical density (OD) was read at 450 nm with a reference at 620 nm using a plate reader. Sera of known unexposed individuals were used as controls. All the ELISAs for a given antigen were performed on the same day and on the same ELISA plate for day 0 and day 42 antibody tests of the same individual to eliminate the day to day variations.d before  using reAll samples with optical density readings above 1.4 were diluted further up to 1:1000 to ensure that ODs were less than 1.4. The OD obtained multiplied by the dilution factor. All specimens were analyzed twice or thrice in duplicates and the means of the ELISA OD used in the analysis. If the ELISA OD of an individual’s day 42 serum was higher than the same person’s corresponding value for the day 0 specimen, the increase was regarded as reflecting a positive antibody anamnestic response or boost. The ratio (D42 ELISA OD)/ (D0 ELISA OD) as well as % increase {(100 x (Day42 ELISA OD—Day0 ELISA OD)/Day0 ELISA OD} was then calculated.P. falciparum antigens was determined by ELISA using synthetic peptides which were coated onto ELISA microtiter plate wells. Amino acid sequences of the synthetic peptides [P. falciparum asexual blood stage proteins were; GLURP: (NH2) CGDKNEKGQHEIVEVEEILPEGC (CONH2), HRP II: (NH2)GCAHHAADAHHAADAHHAADAHHAADGC(CONH2), and MSP3: (NH2) AKEASSYDYILGWEFGGGVPEHKKEEN (CONH2).Sero-reactivity to peptides of peptides , 33, 34 Data were analysed using STATA version 11.0  and Graph pad Prism 5. Data were tested for normality using Shapiro-Wilk W test. Differences between medians of IgG levels on day 0 and day 42 were evaluated by Mann-Whitney test while those among three or more groups were assessed by the non-parametric Kruskal-Wallis test. Correlations were identified by Spearman rank test using actual or logarithmically transformed values P values <0.05 were regarded as statistically significant.P. falciparum infected individuals suffering from acute uncomplicated malaria were obtained to determine i) level of CD4+ lymphocytes, ii) IgG sero-reactivity to antigens of lysed parasitized erythrocytes and synthetic polypeptides representing merozoite proteins GLURP, HRP II and MSP3 all of which are important parasite antigens or vaccine candidate molecules [P. falciparum antibody was then determined by comparing levels of peptide-specific IgG for day 42 and day 0 as estimated by ELISA. The effect of CD4+ T cell levels and parasite density on magnitude of the sero-reactivity to synthetic peptides was analyzed.In this study, peripheral blood and serum of olecules , 22. AntIn this study, 362 participants diagnosed with uncomplicated malaria were enrolled. About 58% (211/362) of the enrolled study participants were female . Forty tWe evaluated the association of all baseline factors and post-infection increase in parasite-specific IgG antibody among the study participants . Sex of On day 0, the frequencies of IgG-sero-reactivity against GLURP, MSP3 and HRP II peptides were 75% (271/360), 60% (217/360) and 41% (148/358), respectively. On the other hand, the corresponding levels for IgM sero-prevalence to GLURP, HRP II and MSP3 were 80% (207/259), 42% (108/257) and 22% (57/256), respectively. For day 0 blood specimens, anti-peptide IgG levels were significantly associated with age for all the three peptides “Figs P. falciparum infection. However, for any of the peptides or when the ELISA absorbance units for the three peptides were added together (graph designated ‘Summative’), the post-diagnosis fold-increase in anti-parasite IgG did not significantly vary among the three age groups. “For many subjects, there was a notable increase in the anti-peptide IgG levels from day 0 to day 42 for all the studied peptides GLURP, MSP3 and HRP II. However, the median anti-peptide IgG for HRP II was significantly reduced from day 0 to day 42. “P. falciparum densities between the two groups . Similarly “+ cell count for the HIV + individuals (n = 24) was 1210/mm3 (IQR: 719–1547), while for the HIV negative individuals whose CD4+ cells were counted (n = 91) the corresponding value was 1096 /mm3 .The median asexual parasite density for the HIV negative study participants was 6400 PE/μL (IQR: 3000–24000) while the corresponding value for HIV positive subjects was 5600 PE/μL (IQR: 3000–19531), thus indicating insignificant difference in blood + cells counts (results not shown), Spearman’s test revealed no significant relationship between the CD4+ cell counts and the memory increase of antibody against peptides MSP-3, GLURP and HRPII. As shown in “When the post-infection fold increase (absorbance ratio: day 42/day 0) was plotted against the CD4+ cell counts “P. falciparum acute infection. The mathematical ratio of ELISA OD units for blood collected on the day of malaria diagnosis and 42 days later were considered to represent a rational estimate of the antibody immune memory (anamnestic) response. It was assumed that patients infected with P. falciparum normally seek treatment at a medical clinic within 2 weeks or so, such that 42 days after diagnosis, the level of blood antibodies on days 0 and 42 would not only provide valid measure of antibody anamnestic response but also enable assessment of post-treatment clinical outcome [P. falciparum malaria, 2) the significant increase in GLURP and MSP3 specific antibodies from day 0 and day 42 following P. falciparum infection and initiation of anti-malarial drug treatment, 3) finding of comparable antibody anamnestic increases among different age groups  4) unimpaired antibody-producing anamnestic/memory response among early-stage HIV infected people living with HIV/ AIDS (PLWHA). Whereas there were detectable levels of IgG antibodies recognizing all tested peptides representing P. falciparum merozoite proteins GLURP, HRP II and MSP3 on day 0 and day 42 among most study subjects, the IgG levels and sero-prevalence for GLURP peptide were higher than the corresponding serum reactivity to MSP3 or to HRPII. This result corroborates earlier studies which have also reported higher IgG levels and sero-prevalence against GLURP in endemic areas [In work reported here, antibody levels to peptides representing three merozoite antigens  were assessed among residents of a moderately endemic area in Uganda. Two of these synthetic peptides are conserved epitopes of merozoite surface proteins (GLURP and MSP3) recombinant portions of which comprise GMZ2, a leading blood-stage anti-malarial vaccine candidate that was recently tested at clinical trial phases 1 and 2 in Africa , 35. In  outcome , 30, 36.ic areas . Due to ic areas  and futuIn the present study, it was found that individuals who showed high anti-GLURP antibody levels did not necessarily have high serum IgG against MSP3 or HRP II, suggesting that predisposing host factors may be the cause of the observed variable sero-reactivity to different antigens. But these results also suggest possible differences in immunogenicity of the peptide epitopes as has been previously observed by others . It is lP. falciparum “strain” may not be adequate to produce a response on exposure to different epitopes of another “strain” [Five subjects did not show memory to any of the antigens after clearing an acute malaria episode. It is unlikely that all these individuals have never been exposed to malaria before this episode as they reside in an area of moderate malaria transmission. Several different explanations have been offered by others to explain for an apparent failure to produce antibodies to a defined antigen after a malarial infection. Antigenic polymorphism is common in malarial parasites that could explain low frequencies of responses to certain antigens –45. Simi“strain” . Others P. falciparum natural infections or by vaccine antigens are little understood. There are many reports suggesting that following treatment with antimalarial drugs and resolution of clinical malaria, the levels of antibodies to specific parasite antigens rapidly decline in absence of repeated infections [P. falciparum-specific Ab immune memory is short-lived, surviving for only few weeks to months [P. falciparum can also induce and maintain long-lasting parasite specific memory B cells in humans.For most participants of this study, antibody anamnestic increase in level of serum anti-parasite IgG from day 0 to day 42 after malaria diagnosis was observed for at least one or two of the three tested peptides. However human B cell memory, mechanisms and durability of antibody immune memory induced either by fections , 13. Indo months , 46, 47 o months . Nevertho months  have nowP. falciparum antibody immunity increases with age [In the present study, children aged less than five years had considerably lower levels of specific IgG antibodies against all three peptides studied compared to older children and adults . These rwith age , 21, 23.P. falciparum and malaria is important [+ cell counts for HIV sero-reactive and sero-negative individuals, suggesting that the enrolled PLWHAs were early-diagnosed HIV cases [+ cell counts. Of these HIV positive individuals, only seven percent (5/68) reported taking Cotrimoxazole prophylaxis therapy and none was on any anti retroviral therapy (ARV). Whereas cell mediated immunity is believed to have a modest effect on the development of naturally acquired resistance to malaria [+ T-cells, B cells and plasma cells after primary acute malaria episodes [While only few (n = 68) people living with human immunodeficiency virus infection/ acquired immuno-deficiency syndrome (HIV/AIDS) (PLWHAs) were enrolled in this study, up to 10% of residents in malaria endemic areas comprise PLWHAs and co infection of mportant , 51. TheIV cases , 53 stil malaria –56, someepisodes , 57, 58.+ cell levels drop to less than 200/uL [+ T cell counts  possess comparable antibody immune memory towards cognate peptide epitopes of GLURP and MSP3, both constituents of the GMZ2 vaccine candidate [Accordingly, it was considered useful to compare the antibody post-infection response of early diagnosed PLWHAs and sero-negative subjects in this study. The results shown in n 200/uL . Therefoandidate , 37. If andidate , the resandidate .P. falciparum antigens were measured in this study. However, these synthetic short peptides are unlikely to possess conformation of the native protein and may thus not completely bind antibodies elicited by the native protein. For the same reasons these peptides may also bind non specifically to antibodies and may not exactly reflect the antibody response.We note that antibody responses to relatively short peptides representing P. falciparum malaria episode among residents of a moderate-transmission malaria endemic region. The enrolled subjects included predominantly HIV sero-negative individuals and few early-diagnosed HIV sero-positive cases. The results show that 42 days after diagnosis of acute P. falciparum malaria and initiation of CoartemTM treatment, all the convalescent subjects possess increased serum antibody IgG to at least one of the three tested polypeptides representing important parasite surface proteins GLURP, HRP II and MSP3. A leading blood-stage vaccine candidate, GMZ2 contains GLURP and MSP3 polypeptides. With high blood CD4+ cells counts, all PLWHAs enrolled in the study achieved as high antibody anamnestic responses as HIV sero-negative individuals following acute malaria. Detection of increases in P. falciparum specific IgG antibody in the study subjects after an acute malaria episode suggests that post-infection boosting of specific IgG antibody widely occurs among residents of moderate malaria-transmission endemic areas. This post-infection increase in parasite specific antibody is not influenced by parasite density of the booster infection episode or blood CD4 +cell counts (at least > 350 CD4 /uL) of the convalescent patient.In summary, this study set out to determine the antibody immune memory response elicited by an acute


Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect and affects more than 400 million people worldwide. This deficiency is believed to protect against malaria because its global distribution is similar. However, this genetic disorder may be associated with potential hemolytic anemia after treatment with anti-malarials, primaquine or other 8-aminoquinolines. Although primaquine is used for malaria prevention, no study has previously investigated the prevalence of G6PD variants and G6PD deficiency in the Republic of Korea (ROK).Two commercialized test kits (Trinity G-6-PDH and CareStart G6PD test) were used for G6PD deficiency screening. The seven common G6PD variants were investigated by DiaPlexC kit in blood samples obtained living in vivax malaria endemic regions in the ROK.Of 1,044 blood samples tested using the CareStart G6PD test, none were positive for G6PD deficiency. However, a slightly elevated level of G6PD activity was observed in 14 of 1,031 samples tested with the Trinity G-6-PDH test. Forty-nine of the 298 samples with non-specific amplification by DiaPlexC kit were confirmed by sequencing to be negative for the G6PD variants.No G6PD deficiency was observed using phenotypic- or genetic-based tests in individuals residing in vivax malaria endemic regions in the ROK. Because massive chemoprophylaxis using primaquine has been performed in the ROK military to kill hypnozoites responsible for relapse and latent stage vivax malaria, further regular monitoring is essential for the safe administration of primaquine. Plasmodium vivax, is the most widespread human malaria and is endemic to the tropical and subtropical countries of the Americas and Asia, including the Republic of Korea (ROK) P. falciparum, P. vivax is characterized by hypnozoites in the liver, which initiate secondary blood-stage infection P. vivax liver hypnozoites. For this reason, primaquine has been administered to soldiers serving in endemic regions in the ROK.Malaria is a devastating protozoan disease that afflicts several hundred million people annually. Vivax malaria, which is caused by Glucose-6-phosphate dehydrogenase (G6PD) is an X-linked essential enzyme that protects cells from oxidative stress, particularly red blood cells. G6PD deficiency, the most common known enzymopathy, is a hereditary genetic defect. It is one of the most prevalent polymorphisms in humans, particularly in males. Since the global distribution of G6PD-deficient variants resembles the geographical distribution of malaria, it has been postulated that G6PD deficiency confers some protection against malaria. Furthermore, a number of G6PD deficient variants have been reported in sub-Saharan Africa and Southeast Asia P. vivax to prevent relapse. They are also administered to patients infected with P. falciparum to reduce gametocyte carriage to block transmission. However, primaquine can cause intravascular hemolysis in individuals with G6PD deficiency, which, depending on the G6PD variant, may increase the risk of primaquine-induced hemolysis from mild to severe G6PD deficiency is of interest because primaquine-induced hemolysis increases in the presence of G6PD variants. 8-aminoquinolines, including primaquine, are administered to patients infected with In the ROK, vivax malaria endemic regions are concentrated near the demilitarized zone (DMZ), and thus, military personnel and residents living in the area are at risk of contracting vivax malaria. Since 1997, chloroquine and primaquine chemoprophylaxis has been administered to soldiers without any G6PD deficiency testing. To date, no study has been undertaken to ascertain the prevalence of G6PD deficiency in the ROK. This study evaluated G6PD enzymatic activity and the prevalence of G6PD variants in individuals living in the vivax malaria endemic Yeon-cheon and Pa-ju regions of the ROK.The study was approved by the ethics committee of the Korea National Institute of Health. For each participant, a written consent form was used to obtain informed consent and permission for the collection of a 5 ml blood sample.n = 298) and soldiers (n = 746)), living in the endemic area  from February 2012 to February 2013. EDTA preserved venous blood samples were obtained from 1,044 individuals  and the Trinity G-6-PDH kit , were used to screen for G6PD deficiency. The CareStart G6PD deficiency screening test is a qualitative enzyme chromatographic test in rapid diagnostic test (RDT)-format. In accordance with the manufacturer's instructions, two drops of buffer were added to 2 µl of each of the 1,044 blood samples (298 residents and 746 soldiers) in a 96-well plate in duplicate. Test results were read visually after 10 mins. Samples with normal G6PD activity produced a distinct purple color background, whereas no color change was observed for G6PD deficient samples. Samples that produced a pale purple color were classified as normal. The 1,031 samples (285 residents and 746 soldiers) received in fresh condition within a maximum of 48 hours after sample collection were quantitatively tested for G6PD activity using the Trinity G-6-PDH test kit. All tests were conducted following the manufacturer's instructions G6PD variants were detected using DiaPlexC G6PD Genotyping Kit , which enables the seven different variants of the G6PD gene to be screened by one-step PCR. The seven variants produce PCR products of different sizes, as follows; Vanua Lava , Mahidol , Coimbra , Viangchan , Union , Canton , and Kalping . After genomic DNA extraction using the Genomic DNA Prep Kit (Blood)  from the 298 blood samples obtained from residents, PCR mixtures were prepared using the DiaPlexC kit, and 2 µl of total genomic DNA (25–50 ng or 100 ng) was added to 23 µl of a master mixture containing premix (12.5 µl), G6PD  primer mixture, and distilled water (8.5 µl). The PCR cycling conditions used were as follows: initial denaturation at 95°C for 15 min, 30 amplification cycles , and a final extension at 72°C for 40 sec. The PCR products were resolved by electrophoresis on 1% agarose gel, stained with safePinky , and visualized under ultraviolet illumination. Each PCR reaction was confirmed by an internal control.Forty-nine blood samples (19 samples from Yeon-cheon and 30 samples from Pa-ju) with multiple bands  were selPhenotypic  and genetic techniques are currently available to determine G6PD activity status. Phenotypic tests are the most widely used and are the best indicators of G6PD function levels. Some commercialized G6PD phenotype deficiency-testing products are also suitable for point-of-care use in clinics because they are rapid and straightforward. DNA-based genotyping could also be applied to large-scale screening programs to provide potentially quantitative measures and details of geographical deficiencies. In present study, phenotypic and DNA-based genotypic testing was conducted to assess the prevalence of G6PD deficiency in the ROK.n = 158) and Pa-ju (n = 886), in which vivax malaria is endemic. Simultaneously with blood collection, hemoglobin levels were measured to determine whether subjects were anemic. Higher mean hemoglobin levels were measured in the Yeon-cheon group (14.5 g/dL vs. 13.6 g/dL). Additionally, mean hemoglobin levels were higher in males than in females (14.0 g/dL vs. 13.4 g/dL), which is consistent with findings reported elsewhere vs. 12.0 years).A total of 1,044 blood samples were collected in two cities, Yeon-cheon (n = 285) and soldiers (n = 746)) and 1,044 (residents (n = 298) and soldiers (n = 746)) blood samples, respectively. The Trinity G-6-PDH kit is suitable for the quantitative measurement of G6PD enzyme activity based on enzyme kinetics. Although this kit is most commonly used, it is labor-intensive and time consuming. The CareStart G6PD deficiency screening kit provides a rapid diagnostic test result and is an attractive alternative for G6PD enzyme assays. The CareStart kit detects moderate and severe enzyme deficiencies (<2.7 U/g Hb), but levels of <2 U/g Hb are not detected To screen for G6PD enzyme activity, the Trinity G-6-PDH kit and the CareStart G6PD deficiency screening kit were used to test 1,031  using the 298 samples except samples from soldiers. The kit provides a simple and rapid alternative to PCR and enzyme digestion-based assays As shown in P. vivax development is still controversial. Since vivax malaria re-emerged in the ROK in 1993, 29,692 cases of endemic vivax malaria have been reported between 1994 and 2010. Furthermore, approximately 20%  of all vivax malaria cases  reported between 2005 and 2013 were located in either the Yeon-cheon or Pa-ju region. On average, 253 cases of vivax malaria were occurred each year in the regions. The vivax malaria in the ROK has long latent periods followed by relapses that are caused by hypnozoites. To eliminate hypnozoites, massive primaquine chemoprophylaxis has been performed on military personnel stationed near the DMZ, although it is known that primaquine induces hemolysis in individuals with certain G6PD variants. More specifically, no treatment guidelines have been issued that account for this effect. In fact, high dose primaquine causes hemolysis in healthy individuals with G6PD wild type, and risk of hemolysis is very different in individuals with G6PD variants. For example, high dose primaquine cause only slight reductions in hemoglobin in those with the A-variant, but hemolysis of 25% of red blood cells occurs in those with the Mediterranean B-variant Malaria is considered as the single great natural selection criterion in malaria endemic regions Neither G6PD deficiency nor any G6PD variant was found in individuals residing in vivax malaria endemic regions in the ROK. This low incidence of G6PD deficiency is interesting because G6PD deficiency is prevalent in neighboring Asian countries. In view of the fact that primaquine chemoprophylaxis is performed routinely in military personnel stationed near the DMZ, regular screening for G6PD deficiency appears essential to ensure the safe administration of primaquine.