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* Center for Global Health and Diseases and
Division of Pediatric Infectious Diseases, Case Western Reserve University, Cleveland, OH 44106;
Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia; and
Kenya Medical Research Institute, Kisian, Kenya
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Abstract |
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Introduction |
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The multiple malaria infections experienced by residents of areas of Africa where transmission is perennial or has predictable seasonal fluctuations generally result in decreased prevalence and density of asexual parasitemia and less morbidity in adults relative to infants (4, 5). These age-related changes are less pronounced in highland areas where transmission is episodic (6, 7). T cell and B cell immunity are important in vaccine-induced resistance against blood-stage P. falciparum (8, 9), and several lines of evidence indicate that Abs to merozoite surface proteins (MSP)4 elicited by natural infection mediate the nonsterilizing immunity that characteristically develops in many residents of malaria endemic areas. Adoptive transfer of the Ig fraction of pooled sera obtained from malaria-immune adult Africans to malaria-infected children leads to dramatic, albeit transient, decreases in the level of asexual parasitemia (10, 11, 12, 13). Sera from persons with recent malaria infection impair the growth of P. falciparum in vitro, although the magnitude of this inhibition is variable (14, 15, 16). MSP-1 is one of several targets of growth-inhibitory Abs elicited by natural infection (15, 17), and based on studies of vaccine studies of mouse models (18, 19) and Aotus monkeys (20, 21, 22) challenged with P. falciparum, is a leading candidate for inclusion in a human blood-stage vaccine. MSP-1 is expressed initially as a 
200-kDa protein during the late stages of erythrocytic schizogony. Following rupture of the schizont and release of merozoites into the plasma, a 42-kDa and ultimately 19-kDa fragment of MSP-1 (MSP-119) is produced and carried into the newly infected erythrocyte during the process of invasion (23). Considerable evidence indicates that the two cysteine-rich epidermal growth factor (EGF)-like domains included in MSP-119 have an essential role in blood-stage growth. These domains appear to be prime targets of protective immune responses (24, 25).
Participants in vaccine trials in malaria endemic areas cannot be deliberately challenged with P. falciparum on practical and ethical grounds. A surrogate of blood-stage immunity under conditions of natural transmission would thus facilitate the screening and evaluation of MSP-1 in anticipated vaccine trials. In addition, because clinical morbidity specifically attributable to malaria is infrequent even in areas where transmission is high, a quantitative functional assay of MSP-1 related immunity might reduce the number of participants needed to achieve statistical power to detect significant differences between vaccinated and control groups. Some (but not all) population-based surveys have shown that IgG Abs to MSP-119, particularly those of the IgG1 and IgG3 subclasses, correlate inversely with the incidence and density of asexual parasitemia and frequency of uncomplicated morbidity (26, 27, 28, 29, 30). This correlation is however imprecise, possibly because recombinant MSP-119 proteins used in serologic assays may not have conformations similar to the tertiary structure of the native molecule and because polyclonal responses elicited by natural infection may not include Abs with specificities that directly interfere with MSP-1-related function or that impair processing of the 42-kDa to the 19-kDa fragment that is essential for invasion (31, 32, 33). Egan et al. (15) first demonstrated that invasion-inhibitory Abs (IIA) specific for MSP-119 were present in IgG affinity purified from pooled sera of west African adults. The recent development of a transgenic P. falciparum line in which the region of the native msp1 gene sequence encoding MSP-119 is replaced with the orthologue of the distantly related mouse malaria P. chabaudi has enabled quantification of Abs that have P. falciparum MSP-119 (PfMSP-119)-specific IIA (34). Taking advantage of this newly developed in vitro assay, we aimed to determine in a field setting in the highlands of western Kenya whether the functionally relevant IIA that recognize this molecule are a better predictor of protection from blood-stage parasitemia than are MSP-119-specific IgG Abs measured by standard enzyme immunoassays (ELISA). The study was conducted in a highland, epidemic-prone area during a malaria outbreak as part of a larger study investigating correlates of protective immunity in individuals residing in an area of unstable, episodic malaria transmission. Adults and children were enrolled in the study, as studies in other epidemic-prone highland areas have demonstrated that adults and children in these areas are equally susceptible to infection and disease with P. falciparum (6).
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Materials and Methods |
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Review and approval of the study was obtained from the Kenya Medical Research Institute National Ethical Review Committee and the Institutional Review Board for Human Studies at University Hospitals of Cleveland, Case Western Reserve University (Cleveland, OH). Written informed consent was obtained from all participants aged 16 years or older and from guardians of participants younger than this age.
Participants were recruited from residents of Kabobo, an isolated village located at an altitude of 2134 meters in the Uasin Gishu district in Kenya. Transmission of P. falciparum in this highland area is episodic and does not occur on a regular seasonal basis. Epidemics of malaria in various highland areas of western Kenya were first reported in the early to mid-20th century (7, 35, 36). Malaria infection surveys conducted in Kabobo during periods of no or little rainfall have shown that the prevalence of blood-stage P. falciparum in symptom-free 5- to 10-year-old school children is 8.9%. The prevalence increased to 45.5% during a period of heavy rainfall (37, 38).
The treatment-infection study commenced in May 1997, at the end of a 6-mo period of little rainfall, when increasing rain related to an El Niño event led to an epidemic in the area. Inclusion criteria included life-long residency in Kabobo, inhabiting a local domicile for the previous year, and lack of malaria morbidity, e.g., fever, chills, myalgia, or other self-identified illnesses within the previous 2 wk. Exclusion criteria were pregnancy and use of antimalarial drugs within the previous 2 wk. Recruitment was done after a series of community and individual meetings were held to explain the nature and purpose of the study. Persons agreeing to participate were given a single dose of sulfadoxine-pyrimethamine (300 mg), the government recommended drug for treatment of uncomplicated malaria in Kenya in 1997. Blood for laboratory studies (see below) was obtained by venipuncture before treatment with sulfadoxine-pyrimethamine. Subjects were followed weekly for the next 12 wk for symptoms of malaria morbidity and diagnosis of malaria infection by microscopic inspection of blood smears obtained by pricking the finger with a lancet (see below). Health care workers remained in the area during the entire study period. Clearance of parasitemia was documented 2 wk after sulfadoxine-pyrimethamine administration, and time to reinfection was calculated as time to infection after parasite clearance, leaving a total of 10 wk of follow-up time.
We intended to obtain blood immediately from persons who self-identified with fever, chills, or any symptoms consistent with malaria. A single dose of sulfadoxine-pyrimethamine was to be given when a positive blood smear was observed. Persons with positive blood smears within 2 wk of administration of the initial dose of sulfadoxine-pyrimethamine were given quinine plus doxycycline and excluded from further analysis. A total of 84 subjects (34 children ages 1–8 years old, 50 adults ages 16–80 years old) were recruited.
Laboratory studies
Malaria infection was diagnosed by microscopic inspection of thick and thin blood smears. Blood smears were prepared and stained with Giemsa, and slides examined by two experienced microscopists used by the Division of Vector Borne Diseases of the Kenya Ministry of Health. The microscopists were blinded to the study protocol and read each of the slides twice. A smear was deemed negative when no parasites were observed after counting microscopic fields that included at least 200 leukocytes. The density of parasitemia was expressed as the number of asexual P. falciparum per microliter of blood assuming a leukocyte count of 8000/µl. Malaria species identification was made by inspection of thin blood smears.
Abs to MSP-119 were measured by ELISA with plasma diluted 1/100 and 10 ng/ml protein applied to microtitration plates (15, 37). Recombinant MSP-119 proteins corresponding to the four major described variants of MSP-119 (the Wellcome/K1 (E-TSR) and MAD20 (Q-KNG) alleles as well as the E-KNG and Q-TSR variants) were expressed in Saccharomyces cerivisiae (39), obtained from the Malaria Research and Reference Reagent Resource Center (Manassas, VA). The Wellcome/K1 and MAD20 alleles were also expressed in Escherichia coli as GST fusion proteins (40). Ab levels were expressed in arbitrary units, which were calculated by dividing the OD generated by the test sample by the mean OD + 3 SD generated by samples from 40 North American or Australian individuals who had never been exposed to malaria. Values that were 
1.0 arbitrary units were considered positive. OD values representing 1 arbitrary unit for IgG, IgG1, IgG2, IgG3, and IgG4 Abs to the MAD20 variant of MSP-119 expressed in S. cerivisiae were 0.138, 0.157, 0.213, 0.070, and 0.105, respectively.
MSP-119-specific IIA was performed using D10 P. falciparum (which encodes the MAD20 allele) and an isogenic D10-P. chabaudi mouse EGF (PcMEGF) parasite line in which the P. chabaudi orthologue replaces the region of P. falciparum msp1 encoding the two EGF-like domains of MSP-119 (34). Ring-stage parasites were synchronized twice by sorbitol lysis and allowed to mature to trophozoite/schizont stages. Purified parasites were adjusted to 4% hematocrit with 1% infected RBC, and 50 µl of aliquots were placed in 96-well microtitration plates with an equal volume of 1/10 prediluted plasma in culture medium (final sera dilution 1/20). The same batch of prediluted plasma was added to the two parasite lines in the same assay. The cultures were incubated for 26 h to allow for schizont rupture and merozoite invasion. Thin smears were made, fixed with methanol, and stained with Giemsa. The number of ring-stage parasites per 500 (assay 1) or 1000 (assay 2) RBC were counted. The mean parasitemia for duplicate wells was calculated and results expressed as a percentage of the parasitemia in parallel cultures that contained nonimmune sera from people who had not been exposed to malaria. The percentage of invasion-inhibition specifically due to anti-MSP-119 Ab (MSP-119 IIA) was calculated with the following formula: percentage of invasion of D10 PcMEGF parasites relative to nonimmune controls – percentage of invasion of D10 isogenic parasites relative to nonimmune controls.
Statistics
Differences in the proportion of individuals with Abs to MSP-119 determined by serology (ELISA) were evaluated by the 
2 test. The significance of differences in Ab levels was evaluated by the nonparametric Mann-Whitney U test. Correlations between continuous variables, e.g., Ab levels to the two alleles of MSP-119, were assessed by Spearman’s rank correlation.
Kaplan-Meier survival analysis was used to compare the time to development of P. falciparum parasitemia in weeks according to whether an individual had high-level or low-level MSP-119-specific IgG Abs (as measured by ELISA) or high-level or low-level MSP-119-specific IIA activity. For assessment of time to infection, levels of IgG Abs to the MAD20 variant of MSP-119 expressed in S. cerivisiae were used. Risk of infection after administration of sulfadoxine-pyrimethamine at the beginning of the study was assessed by Cox proportional hazards analysis. Age and malaria infection status before sulfadoxine-pyrimethamine were adjusted for in the final Cox model. We hypothesized that a high level of MSP-119 IgG Abs or a high degree of PfMSP-119-specific IIA activity would correlate most strongly with protection from infection and therefore chose the 75th percentile of overall IgG Abs or IIA of the group to distinguish high vs low activity. An initial sample size of 100 individuals was calculated to give the study 85% power to detect a 60% decrease in the risk of infection in individuals with high-level Abs or IIA (>75th percentile), assuming an infection rate of 60% in the baseline (low activity) group. Our final sample size in the treatment-reinfection study, after exclusion of individuals who did not wish to have blood samples taken for malaria smear testing, individuals with persistent infection, and individuals with insufficient serum for testing, was 76 persons. We calculated that with this sample size the study had 71% power to detect a 60% decrease in risk of infection assuming an infection rate of 60% in the baseline group.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight of 84 people recruited to participate were excluded from analysis. They developed positive blood smears within 2 wk of taking sulfadoxine-pyrimethamine (indicative of liver-stage infection at the time of drug administration) because blood-stage P. falciparum was not completely eliminated (two individuals) or because insufficient plasma was obtained (six individuals). Thirteen of the remaining 76 persons (17.1%) included in the analysis of time to infection had blood-stage P. falciparum infection before administration of sulfadoxine-pyrimethamine.
Thirty-four of 76 (44.7%) participants developed blood-stage P. falciparum infection 1–10 wk after documented clearance of blood-stage infection with sulfadoxine-pyrimethamine. None of the participants, irrespective of whether they had blood-stage infection, had fever or other symptoms of malaria morbidity. The range of densities was from 40 to 600 asexual P. falciparum per microliter of blood. Infection with other Plasmodium species was not observed. Age, gender, and pretreatment infection status had no significant effect on the time to or risk of infection (Cox proportional hazards analysis; data not shown).
Abs to recombinant MSP-119 proteins measured by ELISA
MSP-119-specific IgG Ab reacting with a yeast-expressed recombinant MSP-119 representing the MAD20 allele was detected in plasma from 32 of 76 (42.1%) individuals immediately before drug administration. There was a bias toward IgG1 and IgG3 subclass Abs although all four subclasses were detected. IgG1, IgG2, IgG3, and IgG4 Abs were detected in 25 (32.9%), 11 (14.5%), 24 (31.6%), and 12 (15.8%) persons, respectively. There was no difference in the prevalence or level of IgG or IgG1 and IgG3 subclass Abs among participants 8 years of age and younger vs those 16 years or older. The prevalence of IgG2 and IgG4 Abs was higher in the younger age group, e.g., 26.7 and 30.0% of subjects 8-years-old or younger vs 6.5% for both IgG2 and IgG4 for subjects 16 years of age or older (p < 0.05 using 2 and Wilcoxon rank-sum analysis).
The 76 samples were tested against yeast-expressed recombinant MSP-119 representing the Wellcome/K1 and MAD20 alleles. IgG Abs to the two variants correlated strongly with each other (
= 0.94, p < 0.0001; Fig. 1A). There was also strong concordance for IgG Ab reactivity with the MAD20 and Wellcome/K1 alleles when recombinant MSP-119 was expressed in E. coli as GST fusion proteins ( = 0.94, p < 0.0001; data not shown). Ab reactivity with the MSP-119 expressed in yeast and E. coli correlated with each other, but not as well as that observed for proteins in the same expression system. Data for the MAD20 allele are presented in Fig. 1B ( = 0.52, p < 0.0001). Finally, levels of IgG Abs to the two other major MSP-119 variants, E-KNG and Q-TSR expressed in yeast, correlated strongly with each other and with levels to the Wellcome/K1 and MAD20 alleles ( = 0.85–0.89, p < 0.0001). Age did not correlate with the prevalence or level of IgG or IgG subclass Abs to any of the four recombinant MSP-119 proteins expressed in yeast. The example of IgG Abs to MAD20 allele of MSP-119 is shown in Fig. 1C. Ab levels above and below the 75th percentile are separated by the dotted line in Fig. 1C.
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IIA were evaluated using the wild-type P. falciparum D10 and transgenic PcMEGF lines. The two lines are isogenic except for their MSP-119 domains (34). Plasma from the Kenyan subjects was considerably more effective at inhibiting invasion of wild-type D10 parasites than was the mutant D10 PcMEGF line (Fig. 2A). The median percentage of parasitemia for the D10 and D10-PcMEGF lines for all 76 Kenyan samples combined was 57.5% and 89.0% of nonimmune human sera. Controls in each assay included rabbit antisera specific for either P. falciparum MSP-119 or the antigenically distinct P. chabaudi MSP-119 domain that is expressed in the D10 PcMEGF line in place of the wild-type domain. These two monospecific sera differentially inhibited the D10 and D10 PcMEGF line as expected (34). The finding that a large proportion of the IIA activity in many samples from this Kenyan population is specific for MSP-119 is consistent with earlier observations in other malaria-exposed populations in Papua New Guinea and Africa (our unpublished observations).
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= 0.86, p < 0.0001; Fig. 2B). MSP-119-specific IIA did not correlate with IgG measured by ELISA (the example of yeast-expressed MAD20 allele is shown in Fig. 2C) or with age (Fig. 2D). Interestingly, the two individuals shown in Fig. 2D with the highest MSP-119-specific IIA levels (72 and 75% invasion-inhibition, above the dotted line) did not have IgG Abs to MSP-119 detected by ELISA. MSP-119-specific IIA but not MSP-119 IgG Ab detected by serology correlates with delayed time to infection
Time to infection after administration of sulfadoxine-pyrimethamine was similar whether or not individuals had high levels of MAD20 MSP-119-specific IgG (above the 75th percentile) detected by ELISA (Fig. 3A). This lack of association was observed when age, gender, or pretreatment infection status were included as covariates in the analysis (adjusted hazard ratio approached 1 in all instances) and when ELISA was performed with E. coli-derived fusion proteins instead of the yeast-expressed Ags (data not shown). There was also no correlation between time to or risk of infection and total IgG, IgG1, and IgG3 Abs to MSP-119 (e.g., the hazard ratio for IgG was 1.33; 95% confidence interval 0.62, 2.87). Median levels of IgG and IgG subclass Abs were similar among individuals who developed parasitemia vs those who did not (IgG, 0.90 vs 1.04 arbitrary units, IgG1 0.48 vs 0.32, and IgG3 0.37 vs 0.44; all p values not significant).
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No clear dose-response association for IIA was seen. The protective effect appeared limited to individuals in the highest quartile of MSP-119-specific IIA, with hazard ratios (95% confidence intervals) of 1, 1.03 (0.43, 2.48), 0.93 (0.38, 2.25), and 0.33 (0.11, 1.06) when comparing the first through fourth quartiles.
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Discussion |
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The lack of association of total IgG or IgG subclass Abs to MSP-119 measured by ELISA with either invasion-inhibitory activity or protection against infection has several important implications. Firstly, the results demonstrate that MSP-119 recombinant proteins expressed in various systems do not react identically with human Abs elicited by natural infection. Secondly, as shown by others (15, 25, 41), there is partial but not complete cross-reactivity of IgG and IgG subclass Abs with the four major variants of MSP-119 defined by amino acid substitutions at positions 1644, 1691, 1700, and 1701, i.e., the E-TSR MAD20, Q-KNG Wellcome/K1, E-KNG, and Q-TSR alleles. These findings may be due to the possibility that the conformers/oligomers of the yeast- and E. coli-derived recombinant proteins differ from each other as well as native MSP-1. However, in prior studies in populations in malaria endemic areas of Africa, Abs to the E. coli-derived MSP-119 recombinant protein were associated with protection from clinical malaria (26). In addition, Abs to the yeast-derived MSP-119 recombinant protein were associated with protection from parasitemia and febrile illness (30). The E. coli- and yeast-derived MSP-119 recombinant proteins used in the present study would therefore be expected to have conformations similar to these previously tested Ags. Thirdly, mAbs that prevent merozoite invasion of erythrocytes by inhibiting proteolytic processing of the 42-kDa to 19-kDa fragment of MSP-1 as well as those that block this inhibitory activity have been described (42, 43). Similar to the findings presented in this study, examination of sera from Nigerian children showed no correlation between anti-MSP-119 Abs measured by serology and Abs that inhibited the processing of native MSP-142 to MSP-119 (44). Thus, although serologic assays presumably detect MSP-119 Abs with multiple specificities (given the caveats previously mentioned), the MSP-119-specific IIA assay used in this study measures inhibitory activity of Abs targeting the region of MSP-1 containing the two essential EGF-like domains that function in the presence of the aforementioned blocking Abs. Measurement of MSP-119-specific IIA, a functional assay, would thus be expected to correlate more strongly with protection from infection than with anti-MSP-119 Abs measured by ELISA even when the latter use optimally conformed MSP-119 recombinant constructs. The present study demonstrates that in a naturally exposed population, the invasion of inhibition assay was in fact a superior predictor of protection from infection. Although we did not determine the genotypes of MSP-119 alleles in infected study subjects, prior studies have demonstrated extensive cross-reactivity of IIA to the variant MSP-119 and MSP-142 epitopes (15, 45), suggesting that the levels of IIA to other alleles possibly circulating in this area would be similar to the MAD-20 allele tested in this study.
From the perspective of immunogenicity, our results raise the possibility that the protective epitopes of the native protein may be poorly represented in at least some recombinant constructs of MSP-1. It will thus be of interest to determine whether MSP-1 constructs that have been recently been found to protect Aotus monkeys against blood-stage P. falciparum also induce high levels of anti-MSP-119 Abs detectable by both ELISA and IIA (21, 46). The levels of anti-MSP-119 Abs in the present study were similar to those seen in earlier studies (26, 28) and suggest that the high-level anti-MSP-119 Abs detected by ELISA in vaccinated Aotus monkeys (46) differ in their specificities from those induced by natural infection of humans.
Studies of malaria-exposed populations in which time to infection after administration of antimalarial drugs or seasonal increases in transmission has been evaluated have generally been concerned with examination of immunity to Ags expressed by pre-erythrocytic P. falciparum (47, 48). Interpretation of this parasitologic endpoint is predicated on the assumption that immunity to pre-erythroyctic and liver-stage P. falciparum are the most important variables that determine time to development of blood-stage infection. A high degree of immunity to merozoites released from hepatic schizonts may however also result in delay of time to blood-stage infection. Indeed, our earlier studies in residents of the highlands of Kenya reported that T cell IL-10 responses to antigenic peptides of liver stage Ag-1 were weakly associated with time to infection, but found no correlation with T cell IFN-
or Ab responses to this Ag (37). Studies of mice challenged with P. yoelii sporozoites indicate that immunization with the C-terminal fragment of P. yoelii MSP-1 leads to protection against blood stage infection by immunity directed against liver-stage parasites (49). Using transgenic P. berghei that express the P. falciparum MSP-119 orthologue, we have observed that protection against blood stage challenge and peak parasitemia in mice rendered immune by repeated infection and drug cure correlates with MSP-119-specific invasion-inhibitory activity but not with the presence or level of Abs to MSP-119 measured by serology (50). It was not possible in the present study to determine whether MSP-119-specific IIA correlates with peak parasitemia or symptoms of malaria because treatment with antimalarial drugs was mandated when asexual parasitemia first became detectable. The relatively low frequency of infection during the first 4 wk of the study likely reflects the prolonged effect of prior sulfadoxine-pyrimethamine treatment. Although the exact rates of sulfadoxine-pyrimethamine resistance in this highland area at the time the study was done (1997) are unknown, the eradication of infection at 2 wk in 82 of 84 individuals treated with sulfadoxine-pyrimethamine suggests that high-frequency sulfadoxine-pyrimethamine resistance did not exist at the time. Other potentially protective factors, such as bednet use and the presence of sickle cell trait or glucose-6-phosphate dehydrogenase deficiency, are unlikely to have affected risk of infection in this population: only 2 of the 76 study participants (2.7%) used bednets, and studies from a nearby highland area of Kenya demonstrated that frequencies of sickle cell trait and glucose-6-phosphate dehydrogenase deficiency are low in these populations (3% and 1%, respectively) (51).
Our findings provide evidence that MSP-119-specific invasion-inhibitory activity as measured by an in vitro assay that uses transgenic P. falciparum is an immune correlate of protection against blood-stage P. falciparum. The study was conducted in an area where transmission of malaria is episodic and characterized by some as "mesoendemic" (7). Further investigations will be required to establish whether a similar relationship exists in endemic areas where transmission is stable and higher than in the Kenya highlands, such as in infants in the lowlands of western Kenya who experience higher levels of asexual parasitemia (29). A dose-response relationship, which was not observed in the present study, might be seen in a population with higher IIA levels. Clinical surveillance conducted in a setting where treatment is indicated only for symptomatic malaria will also be necessary to determine whether MSP-119-specific invasion-inhibitory activity is associated with a decrease in the peak level and incidence-density of parasitemia as well as malaria-attributable fever. Finally, it will be important to examine a larger number of individuals to have sufficient statistical power to determine whether lower levels of MSP-119-specific invasion-inhibitory activity correlate with protection against malaria.
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Acknowledgments |
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Footnotes |
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2 C.C.J. and R.A.O. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. James W. Kazura, Center for Global Health and Diseases, Case Western Reserve University, Wolstein Research Building, 2103 Cornell Road, Cleveland, OH 44106. E-mail address: james.kazura{at}case.edu
4 Abbreviations used in this paper: MSP, merozoite surface protein; MSP-119, 19-kDa fragment of MSP-1; EGF, epidermal growth factor; IIA, invasion-inhibitory Ab.
Received for publication January 7, 2004. Accepted for publication April 28, 2004.
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References |
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globulin. Trans. R. Soc. Trop. Med. Hyg. 57:170.
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 K. E. M. Persson, C. T. Lee, K. Marsh, and J. G. Beeson Development and Optimization of High-Throughput Methods To Measure Plasmodium falciparum-Specific Growth Inhibitory Antibodies. J. Clin. Microbiol., May 1, 2006; 44(5): 1665 - 1673. [Abstract] [Full Text] [PDF]
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B. Mahajan, D. Jani, R. Chattopadhyay, R. Nagarkatti, H. Zheng, V. Majam, W. Weiss, S. Kumar, and D. Rathore Identification, Cloning, Expression, and Characterization of the Gene for Plasmodium knowlesi Surface Protein Containing an Altered Thrombospondin Repeat Domain Infect. Immun., September 1, 2005; 73(9): 5402 - 5409. [Abstract] [Full Text] [PDF]
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D. R. Drew, P. R. Sanders, and B. S. Crabb Plasmodium falciparum Merozoite Surface Protein 8 Is a Ring-Stage Membrane Protein That Localizes to the Parasitophorous Vacuole of Infected Erythrocytes Infect. Immun., July 1, 2005; 73(7): 3912 - 3922. [Abstract] [Full Text] [PDF]
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N. MINAKAWA, S. MUNGA, F. ATIELI, E. MUSHINZIMANA, G. ZHOU, A. K. GITHEKO, and G. YAN SPATIAL DISTRIBUTION OF ANOPHELINE LARVAL HABITATS IN WESTERN KENYAN HIGHLANDS: EFFECTS OF LAND COVER TYPES AND TOPOGRAPHY Am J Trop Med Hyg, July 1, 2005; 73(1): 157 - 165. [Abstract] [Full Text] [PDF]
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C. C. JOHN, A. M. MOORMANN, D. C. PREGIBON, P. O. SUMBA, M. M. MCHUGH, D. L. NARUM, D. E. LANAR, M. D. SCHLUCHTER, and J. W. KAZURA CORRELATION OF HIGH LEVELS OF ANTIBODIES TO MULTIPLE PRE-ERYTHROCYTIC PLASMODIUM FALCIPARUM ANTIGENS AND PROTECTION FROM INFECTION Am J Trop Med Hyg, July 1, 2005; 73(1): 222 - 228. [Abstract] [Full Text] [PDF]
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I. Malhotra, P. Mungai, E. Muchiri, J. Ouma, S. Sharma, J. W. Kazura, and C. L. King Distinct Th1- and Th2-Type Prenatal Cytokine Responses to Plasmodium falciparum Erythrocyte Invasion Ligands Infect. Immun., June 1, 2005; 73(6): 3462 - 3470. [Abstract] [Full Text] [PDF]
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P. H. Corran, R. A. O'Donnell, J. Todd, C. Uthaipibull, A. A. Holder, B. S. Crabb, and E. M. Riley The Fine Specificity, but Not the Invasion Inhibitory Activity, of 19-Kilodalton Merozoite Surface Protein 1-Specific Antibodies Is Associated with Resistance to Malarial Parasitemia in a Cross-Sectional Survey in The Gambia Infect. Immun., October 1, 2004; 72(10): 6185 - 6189. [Abstract] [Full Text] [PDF]
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