Monoclonal and polyclonal antibodies both block and enhance transmission of human Plasmodium vivax malaria

Antibodies against gametes of the malarial parasite inhibit the development of the parasite in the mosquito and curtail the transmission of malaria. We now report that a monoclonal antibody against gametes of the human malaria pathogen Plasmodium vivax and antibodies induced during natural infections of P. vivax in humans which suppress infectivity of the parasites to the vector at high concentrations can, at lower concentrations, have the opposite effect and enhance the level of malaria infection in the mosquitoes. Infectivity enhancing effects of up to 12-fold were demonstrated when a transmission blocking monoclonal antibody and immune human sera were diluted, in some undiluted immune human sera, and in the sera of vivax malaria patients during convalescence after drug cure.     

Anti-gamete antibodies block transmission of human vivax malaria to mosquitoes

Antibodies were raised in rabbits by immunizing against fresh unfixed or cryopreserved female gametes of the human malaria pathogen Plasmodium vivax. The antibodies were shown to react with the surface of gametes by the indirect immunofluorescent test. When parasite isolates from P. vivax infected individuals were fed through a membrane to Anopheles tessellatus mosquitoes in the presence of immune rabbit sera, they completely blocked the infectivity of the parasite isolates to the vector. Immunoglobulins separated from these sera also blocked infectivity to the same extent as did the immune sera indicating that antibodies were responsible for the transmission blocking effect of the sera. This study indicated that P. vivax like other malaria parasites is highly susceptible to anti gamete transmission blocking immunity.     

The primary antibody response of malaria patients to Plasmodium falciparum sexual stage antigens which are potential transmission blocking vaccine candidates

Thirty serum samples collected from adult patients attending the Hospital for Tropical Diseases, London, with P. falciparum malaria, were studied. Sera were screened by indirect immunofluorescence for anti-gametocyte antibodies. Twelve of the serum samples taken from 14 patients with primary infections were found to have both IgM and IgG antibodies to gametocyte antigens and total Ig titres comparable with those of patients who had had previous malaria attacks. Sera of individuals from hyperendemic areas have been found to immunoprecipitate the 230 and 48/45 kD gametocyte surface antigens which are known targets of transmission blocking antibodies. To investigate the epitope specificity of the serum samples from our adult patients, competitive ELISAs with 3 monoclonal antibodies (MAbs) that block transmission and recognize different epitopes on the 48/45 Kd antigen, were carried out. Specific antibodies for these epitopes were found in 60% of the sera while nearly a third were able to inhibit the binding of at least two MAbs.     

The primary antibody response of malaria patients to Plasmodium falciparum sexual stage antigens which are potential transmission blocking vaccine candidates

Summary Thirty serum samples collected from adult patients attending the Hospital for Tropical Diseases, London, with P. falciparum malaria, were studied. Sera were screened by indirect immunofluorescence for anti-gametocyte antibodies. Twelve of the serum samples taken from 14 patients with primary infections were found to have both IgM and IgG antibodies to gametocyte antigens and total Ig titres comparable with those of patients who had had previous malaria attacks. Sera of individuals from hyperendemic areas have been found to immunoprecipitate the 230 and 48/45 kD gametocyte surface antigens which are known targets of transmission blocking antibodies. To investigate the epitope specificity of the serum samples from our adult patients, competitive ELISAs with 3 monoclonal antibodies (MAbs) that block transmission and recognize different epitopes on the 48/45 Kd antigen, were carried out. Specific antibodies for these epitopes were found in 60% of the sera while nearly a third were able to inhibit the binding of at least two MAbs.     

Plasmodium falciparum malaria transmission-blocking immunity under conditions of low endemicity as in Sri Lanka

Sera from acute primary Plasmodium falciparum patients in Sri Lanka were tested for the presence of antibodies against gamete antigens and for their functional effects of transmission blocking activity. Comparisons were made with corresponding data from a previous study from sera of patients from Papua New Guinea where malaria is more highly endemic. Although the prevalence of anti-gamate antibodies in the two groups were broadly similar, the prevalence of infectivity suppressive effects in the Sri Lankan sera (56%) was less than in Papua New Guinea sera (75%), suggesting that the generation of functionally effective transmission blocking antibodies requires prolonged exposure to multiple inoculations of malaria. In Papua New Guinea sera there was a good correlation between transmission blocking effects and antibody responses to Pfs 230, a known target of transmission blocking antibodies. Among the Sri Lankan sera no strong correlation was found between transmission blocking effects and the presence of antibodies to gamete surface antigens Pfs 230 nor Pfs 48/45 as detected by immunoprecipitation of radio-iodinated gamate proteins; a strong correlation was however, found between the intensity of response to gamete surface antigens by IFA and transmission blocking effects of these sera. It is possible therefore, that the antigens identified by IFA include non-protein moieties and that these may be the targets of transmission blocking antibodies in sera from acute primary infections of P. falciparum.     

Population biology and antimalarial resistance: The transmission of antimalarial drug resistance in Plasmodium falciparum

Malaria morbidity and mortality continue to increase across sub-Saharan Africa. This is largely as a result of the continued use of chloroquine and sulfadoxine-pyrimethamine, despite widespread resistance. Although eliminating the asexual stages of Plasmodium falciparum is the focus of treatment of individual symptomatic patients, at a population level, reducing the carriage of gametocytes - the sexual stage responsible for infection of the mosquito vector - is necessary to limit the transmission of malaria parasites and the spread of antimalarial resistance. The probability of a mosquito being infected depends on the prevalence, duration and density of viable gametocyte carriage in the human host, although additional humoral and leukocyte factors also affect transmissibility. There is a log-sigmoid relationship between gametocyte density in the patients' blood and infectivity to the mosquito. The infectivity and thus transmission potential associated with a particular antimalarial treatment can be characterised as a function of blood gametocyte density and time, summing these over the acute and all subsequent recrudescences of that infection. Gametocyte carriage and infectivity to mosquitoes is consistently higher in patients infected with drug resistant compared with drug sensitive malaria parasites. It is the ratio of transmission potential in drug resistant versus sensitive infections that drives the spread of resistance. Early access to highly effective antimalarial treatment reduces the risk of disease progression and limits gametocyte carriage. The remarkable spread of sulfadoxine-pyrimethamine (SP) resistance across vast regions results from the very high post-treatment prevalence and density of gametocyte carriage following SP treatment. In areas of low intensity malaria transmission, the gametocyte-reducing effect of widespread use of artemisinin-based combination therapy has resulted in a sustained decrease in malaria transmission and a decrease in the spread of resistance. Malaria treatment policy should be based primarily on therapeutic efficacy against asexual stages, but should also consider transmission reduction potential. Artemisinin-based combination therapies are the only antimalarials currently available which rapidly reduce both asexual and gametocyte stages of the P. falciparum lifecycle.     

Infectious reservoir of Plasmodium vivax and Plasmodium falciparum malaria in an endemic region of Sri Lanka

The infectious reservoir of Plasmodium vivax and P. falciparum in a malaria endemic region in Sri Lanka was defined in a population of 3,625 by directly feeding mosquitoes on a sample of infected individuals during a period of 17 months. The malaria case incidence in this population was concurrently monitored. P. vivax gametocyte densities were highest in the youngest age groups, and decreased steadily with increasing age. However, the infectivity per gametocyte appeared to be lower in the younger age groups than in the older ones. There was no significant correlation between the age of patients and their gametocyte densities for P. falciparum, to which this population was only recently exposed, nor was there a discernible trend in the infectivity per gametocyte in different age groups. The average infectivity of patients was lowest in the youngest (0-5 years) and the oldest (greater than 50) age groups. The contribution made by P. vivax patients in the different age groups to the reservoir of infection was estimated. Patients in the 6-25 year age groups made the largest contribution to the reservoir, followed by those in the 26-50 year age group. Patients in the youngest and the oldest age groups contributed least to the infectious reservoir. When population sizes in the different age groups were taken into consideration, the age groups between 6 and 50 years contributed almost equally to approximately 87% of the infectious reservoir. The reservoir of P. falciparum malaria was very small, being confined to 9% of the patients, and this appears to be a characteristic of epidemic malaria, as was the case with P. falciparum.     

Stage-specific effects of host plasma factors on the early sporogony of autologous Plasmodium falciparum isolates within Anopheles gambiae

Summary Quantitatively assessing the impact of naturally occurring transmission-blocking (TB) immunity on malaria parasite sporogonic development may provide a useful interpretation of the underlying mechanisms. Here, we compare the effects of plasma derived from 23 naturally infected gametocyte carriers (OWN) with plasma from donors without previous malaria exposure (AB) on the early sporogonic development of Plasmodium falciparum in Anopheles gambiae. Reduced parasite development efficiency was associated with mosquitoes taking a blood meal mixed with the gametocyte carriers' own plasma, whereas replacing autologous plasma with non-immune resulted in the highest level of parasite survival. Seven days after an infective blood meal, 39.1% of the gametocyte carriers' plasma tested showed TB activity as only a few macrogametocytes ingested along with immune plasma ended up as ookinetes but subsequent development was blocked in the presence of immune plasma. In other experiments (60.9%), the effective number of parasites declined dramatically from one developmental stage to the next, and resulted in an infection rate that was two-fold lower in OWN than in AB infection group. These findings are in agreement with those in other reports and go further by quantitatively examining at which transition stages TB immunity exerts its action. The transitions from macrogametocytes to gamete/zygote and from gamete/zygote to ookinete were identified as main targets. However, the net contribution of host plasma factors to these interstage parasite reductions was low (5-20%), suggesting that irrespective of the host plasma factors, mosquito factors might also lower the survival level of parasites during the early sporogonic development.     

Plasmodium falciparum transmission blocking immunity under conditions of low and high endemicity in Cameroon

Transmission blocking immunity (TBI) was studied in relation to age, gametocyte density and transmission intensity. subjects with high gametocytaemias were selected in a hypo-endemic urban district and a hyper-endemic rural area in South Cameroon. TBI was determined in blood from gametocyte carriers in a bioassay (Direct Membrane Feeding Assay), with either autologous plasma (OWN) or control serum (AB). Mosquito infection rates (IR) were compared. infection rates correlated positively with gametocyte and oocyst densities. Three TBI indicators were analysed: the proportion of transmission reducers (IRAB > IROWN, P < 0.01), the mean intensity of TBI (IRAB - IROWN), and the contribution of TBI to total inhibition [(IRAB-IROWN)/(100-IROWN)]. we could not discriminate between areas with regard to either the proportion of transmission reducers (urban 15% and rural 29%) or the mean levels of TBI (urban 10% and rural 9%), or contribution of TBI to total inhibition (urban 10% and rural 13%). there was no relationship between TBI indicators and age, but a trend of increasing values was observed with rising gametocytaemia, which was considered as a confusing factor. a multivariable analysis showed that the probability of being a reducer was 4.6 fold higher in the rural area than in the urban district.     

Age-dependent distribution of Plasmodium falciparum gametocytes quantified by Pfs25 real-time QT-NASBA in a cross-sectional study in Burkina Faso

Sexual stages of Plasmodium falciparum play a key role in the transmission of malaria. Studies on gametocytes are generally based on microscopic detection, but more sensitive detection methods for P. falciparum gametocytes frequently detect sub-patent gametocytes. We used Pfs25 mRNA quantitative-nucleic acid sequence-based amplification (QT-NASBA) to quantify gametocytes in 412 samples from a cross-sectional study in Burkina Faso, covering all age groups, to determine age-related patterns in gametocyte carriage and gametocyte density. The more sensitive QT-NASBA technique gave estimates of gametocyte prevalence 3.3-fold higher than microscopy (70.1% versus 21.4%, respectively). Prevalence of gametocytes significantly decreased with age. Our data suggest that asexual parasite densities are primarily responsible for the age-related decrease of gametocyte prevalence, possibly because of developing asexual stage immunity. Gametocyte densities decrease also with age, primarily because of decreasing asexual parasite densities; only a small but significant age effect on gametocyte density may be caused by developing sexual stage-specific immunity.     

Plasmodium falciparum transmission blocking immunity in three areas with perennial or seasonal endemicity and different levels of transmission

Plasmodium falciparum transmission blocking immunity (TBI) was investigated in 3 different endemic areas. Reared Anopheles gambiae s.s. were experimentally infected with the blood of gametocyte carriers, either in the presence of autologous plasma (OWN) or after replacement of the OWN plasma with a nonimmune serum of AB blood group (control). Transmission reduction was defined by a lower level of mosquito infection in the OWN batch compared with the control. After controlling for the effect of gametocytemia, the proportion of "transmission reducers" was lower in the town of Yaounde in Cameroon (UC), (14%, N = 75) than in the two rural areas of South Cameroon (RC) (29%, N = 31) and Sénégal (RS) (44%, N = 32). The contribution of TBI relative to the total inhibition of the parasite development (including human, parasite, and mosquito factors) was higher in RS (49.6%) than in RC (12.6%) and UC (9.5%).     

Cellular and humoral inhibition of Plasmodium falciparum growth in vitro and recovery from acute malaria

Both mononuclear cell cytotoxicity and serum inhibition of Plasmodium falciparum growth in vitro were found to vary according to the stage of infection in Gambian children with clinical malaria. Cytotoxicity was displayed by mononuclear cells and serum from children with acute malaria but this form of parasite killing was more effective in children with low grade P. falciparum infections of at least 10 days duration. Parasite inhibitory antibody was not evident in sera from acutely infected children but was found in sera from children recovering from malaria and reached a peak in convalescent children when P. falciparum growth was inhibited by at least 50%. The humoral response in convalescent children was strain related, being more effective against the most recent infecting parasite strain than against other 'wild' P. falciparum isolates. In contrast, mononuclear cell cytotoxicity was not strain related; when effective, multiplication of all parasite isolates tested was retarded to the same degree. The discussion considers the role of mononuclear cell cytotoxicity in the development of protective immunity and suggests that it may be a 'front line' defense mechanism during each malaria attack.     

Epidemiology of Plasmodium falciparum in a rice field and a savanna area in Burkina Faso: seasonal fluctuations of gametocytaemia and malarial infectivity.

For a better understanding of the epidemiology of Plasmodium falciparum in an African savanna area, the authors have: (a) defined the real gametocyte reservoir in the native population; (b) followed the fluctuations of gametocytaemia throughout the transmission period; and (c) measured the infectiousness of malarious individuals to mosquitoes. The transversal surveys, in different villages of this endemic area, have shown that gametocyte carrier rates decreased with age and malaria experience; 10.9% of the whole population were potentially infectious to mosquitoes, and of these 73% were children and only 27% were adults. The longitudinal studies have shown that the P. falciparum gametocyte rate depends on the equilibrium between the gametocyte conversion rates and the density of the asexual forms. When there are large numbers of children who become carriers of the sexual stage of the parasite and at the same time a small number who lose their gametocyte infection, the gametocyte rate increases in the population; and vice versa. The circumstances under which gametocytes are produced are not well-known. Two factors seem to be important: the level of the parasite density and immune mechanisms. The infectiousness of malarious individuals was estimated by the 'mosquito infection probability'. The percentage of mosquitoes infected after feeding on gametocyte carriers (which may partly reflect the infectiousness of a human population to mosquitoes) was multiplied by the percentage of gametocyte carriers in the population. This indicated that, in this endemic area, 4% of biting mosquitoes would become infected; but this theoretical mosquito infection probability is over-estimated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anti-mosquito midgut antibodies block development of Plasmodium falciparum and Plasmodium vivax in multiple species of Anopheles mosquitoes and reduce vector fecundity and survivorship

The mosquito midgut plays a central role in the sporogonic development of malaria parasites. We have found that polyclonal sera, produced against mosquito midguts, blocked the passage of Plasmodium falciparum ookinetes across the midgut, leading to a significant reduction of infections in mosquitoes. Anti-midgut mAbs were produced that display broad-spectrum activity, blocking parasite development of both P. falciparum and Plasmodium vivax parasites in five different species of mosquitoes. In addition to their parasite transmission-blocking activity, these mAbs also reduced mosquito survivorship and fecundity. These results reveal that mosquito midgut-based antibodies have the potential to reduce malaria transmission in a synergistic manner by lowering both vector competence, through transmission-blocking effects on parasite development, and vector abundance, by decreasing mosquito survivorship and egg laying capacity. Because the intervention can block transmission of different malaria parasite species in various species of mosquitoes, vaccines against such midgut receptors may block malaria transmission worldwide.     

Artesunate reduces but does not prevent posttreatment transmission of Plasmodium falciparum to Anopheles gambiae

Combination therapy that includes artemisinin derivatives cures most falciparum malaria infections. Lowering transmission by reducing gametocyte infectivity would be an additional benefit. To examine the effect of such therapy on transmission, Gambian children with Plasmodium falciparum malaria were treated with standard regimens of chloroquine or pyrimethamine-sulfadoxine alone or in combination with 1 or 3 doses of artesunate. The infectivity to mosquitoes of gametocytes in peripheral blood was determined 4 or 7 days after treatment. Infection of mosquitoes was observed in all treatment groups and was positively associated with gametocyte density. The probability of transmission was lowest in those who received pyrimethamine-sulfadoxine and 3 doses of artesunate, and it was 8-fold higher in the group that received pyrimethamine-sulfadoxine alone. Artesunate reduced posttreatment infectivity dramatically but did not abolish it completely. The study raises questions about any policy to use pyrimethamine-sulfadoxine alone as the first-line treatment for malaria.     

Seasonal patterns of Plasmodium falciparum gametocyte prevalence and density in a rural population of Burkina Faso

Gametocytes are the malaria parasite stages that secure the transmission from the human host to the mosquito. The identification of natural parameters that influence gametocyte carriage can contribute to a better understanding of the dynamics of the sexual stage parasites for transmission reducing strategies. A total of 3400 blood slide readings were done during four cross-sectional surveys (2002-2003) including all age groups to determine the effect of season on Plasmodium falciparum gametocytes in a seasonal malaria transmission area of Burkina Faso. Entomological data were collected to determine the malaria transmission intensity in relation to seasons. Transmission intensity was estimated by monthly EIRs, averaging 28 and 32 infective bites/person/month in the wet seasons of 2002 and 2003, respectively. The EIR in the dry seasons was below one infective bite/person/month. The gametocyte prevalence was significantly higher at the start and peak of the wet season compared to the dry season when corrected for asexual parasite density and age. Gametocyte density significantly increased during the wet season after correction for asexual parasite density and age. In this study, season appears to be an independent parameter that determines gametocyte prevalence and density and should be considered to be included in epidemiological studies on malaria transmission.     

A high-throughput screen targeting malaria transmission stages opens new avenues for drug development

A major goal of the worldwide malaria eradication program is the reduction and eventual elimination of malaria transmission. All currently available antimalarial compounds were discovered on the basis of their activity against the asexually reproducing red blood cell stages of the parasite, which are responsible for the morbidity and mortality of human malaria. Resistance against these compounds is widespread, and there is an urgent need for novel approaches to reduce the emergence of resistance to new antimalarials as they are introduced. We have established and validated the first high-throughput assay targeting the red blood cell parasite stage required for transmission, the sexually reproducing gametocyte. This assay will permit identification of compounds specifically targeting the transmission stages in addition to the asexual stage parasites. Such stage-specific compounds may be used in a combination therapy, reducing the emergence of resistance by blocking transmission of resistant parasites that may be selected in a patient.     

Increased gametocytemia after treatment: an early parasitological indicator of emerging sulfadoxine-pyrimethamine resistance in falciparum malaria

BACKGROUND: Although malaria treatment aims primarily to eliminate the asexual blood stages that cause illness, reducing the carriage of gametocytes is critical for limiting malaria transmission and the spread of resistance. METHODS: Clinical and parasitological responses to the fixed-dose combination of sulfadoxine and pyrimethamine in patients with uncomplicated falciparum malaria were assessed biannually since implementation of this treatment policy in 1998 in Mpumalanga Province, South Africa. RESULTS: Despite sustained cure rates of > 90% (P = .14), the duration of gametocyte carriage increased from 3 to 22 weeks (per 1000 person-weeks) between 1998 and 2002 (P < .001). The dhfr and dhps mutations associated with sulfadoxine-pyrimethamine resistance were the most important drivers of the increased gametocytemia, although these mutations were not associated with increased pretreatment asexual parasite density or slower asexual parasite clearance times. The geometric mean gametocyte duration and area under the gametocyte density time curve (per 1000 person-weeks) were 7.0 weeks and 60.8 gametocytes/microL per week, respectively, among patients with wild-type parasites, compared with 45.4 weeks (P = .016) and 1212 gametocytes/microL per week (P = .014), respectively, among those with parasites containing 1-5 dhfr/dhps mutations. CONCLUSIONS: An increased duration and density of gametocyte carriage after sulfadoxine-pyrimethamine treatment was an early indicator of drug resistance. This increased gametocytemia among patients who have primary infections with drug-resistant Plasmodium falciparum fuels the spread of resistance even before treatment failure rates increase significantly.     

Infectiousness of the Human Population to Anopheles arabiensis by Direct Skin Feeding in an Area Hypoendemic for Malaria in Senegal

Direct skin feeding experiments are sensitive assays to determine human infectiousness to mosquitoes but are rarely used in malaria epidemiological surveys. We determined the infectiousness of inhabitants of a malaria hypoendemic area in Senegal. Gametocyte prevalence by microscopy was 13.5% (26 of 192). Of all individuals who were gametocyte positive, 44.4% (11 of 25) infected ≥ 1 Anopheles arabiensis mosquito and 10.8% (54 of 500) of mosquitoes became infected. Of all individuals who were gametocyte negative by microscopy, 4.3% (7 of 162) infected ≥ 1 mosquito and 0.4% (12 of 3240) of mosquitoes became infected. The 18.2% (12 of 66) of all mosquito infections was a result of submicroscopic gametocyte carriage and two individuals without asexual parasites or gametocytes by microscopy were infectious to mosquitoes. When infectivity and local demography was taken into account, children 5–14 years of age contributed 50.8% of the human infectious reservoir for malaria. Adults and submicroscopic gametocyte carriers may contribute considerably to onward malaria transmission in our setting.