Ingrid Felger

A Recombinant Blood-Stage Malaria Vaccine Reduces Plasmodium falciparum Density and Exerts Selective Pressure on Parasite Populations in a Phase 1-2b Trial in Papua New Guinea

The malaria vaccine Combination B comprises recombinant Plasmodium falciparum ring-infected erythrocyte surface antigen and 2 merozoite surface proteins (MSP1 and MSP2) formulated in oil-based adjuvant. A phase 1-2b double-blind, randomized, placebo-controlled trial in 120 children (5-9 years old) in Papua New Guinea demonstrated a 62% (95% confidence limits: 13%, 84%) reduction in parasite density in children not pretreated with sulfadoxine-pyrimethamine. Vaccinees had a lower prevalence of parasites carrying the MSP2-3D7 allelic form (corresponding to that in the vaccine) and a higher incidence of morbid episodes associated with FC27-type parasites. These results demonstrate functional activity of Combination B against P. falciparum in individuals with previous malaria exposure. The specific effects on parasites with particular msp2 genotypes suggest that the MSP2 component, at least in part, accounted for the activity. The vaccine-induced selection pressure exerted on the parasites and its consequences for morbidity strongly argue for developing vaccines comprising conserved antigens and/or multiple components covering all important allelic types.

Analysis of Multiple Plasmodium falciparum Infections in Tanzanian Children during the Phase III Trial of the Malaria Vaccine SPf66

In the first phase III efficacy trial of the malaria vaccine SPf66 in Africa, MOIs in SPf66- and placebo-vaccinated children were analyzed by polymerase chain reaction-restriction fragment length polymorphism of the Plasmodium falciparum merozoite surface antigen 2 (MSA2). MOIs were significantly reduced in asymptomatic vaccine recipients compared with those in asymptomatic placebo recipients; however, no differences were observed among symptomatic children in the vaccine and control groups. These results show that immunization with SPf66 modulates the course of naturally occurring infections, as reflected by reduced MOIs. In placebo recipients, however, there was a significant negative correlation between numbers of infecting genotypes, as identified by MSA2, and morbidity. Asymptomatic placebo recipients had an average of 5 concurrent infections, whereas children with clinical cases had an average of 3.4 infections. These data provide further evidence that premunition from concurrent infections is important in immunity against clinical malaria. No such effect of multiple infections was found in the vaccinated group.

11. Premunition in Plasmodium falciparum infection: insights from the epidemiology of multiple infections

Epidemiological studies of multiple clone infections by Plasmodium falciparum in highly endemic areas have demonstrated age dependence in both the multiplicity of infection and the relationships between this multiplicity and the risk of acute illness. We hypothesize that, in infants, host defence against blood-stage infections with P. falciparum relies mainly on fever and cytokine activities, and the infections are of short duration. In older children, a high multiplicity of infection is characteristic of low-level chronic parasitaemia. This appears to confer cross-protection against newly inoculated parasites, via partially genotype-specific responses which are short-term, lasting little longer than the infections themselves. This has important implications for our understanding of immunity against P. falciparum, its ecological niche, and the epidemiological impact of interventions against it.

4. Age dependence of the multiplicity of Plasmodium falciparum infections and of other malariological indices in an area of high endemicity

The relationship between age and various malariological indices in the Kilombero valley of Tanzania were examined by compiling data from 6 different community studies carried out between 1989 and 1996. The rate of acquisition of Plasmodium falciparum infection was highest in children 1-5 years of age, while recovery rates were lowest between the first birthday and early adolescence. As a result, peak prevalence was reached in 3-5 years old children. However, the prevalence of clinical malaria (estimated from the excess risk of axillary temperatures > or = 37.5 degrees C attributable to parasitaemia) was highest in children under one year of age. The peak in multiplicity of infection (identified by polymerase chain reaction-restriction fragment length polymorphism of the msp2 locus) occurred in 3-7 years old children. There was a significant correlation between parasite density and multiplicity of infection in infants and young children (1-2 years of age) but not in older individuals.

2. Genotypes of merozoite surface protein 2 of Plasmodium falciparum in Tanzania

The merozoite surface protein 2 (MSP2) of Plasmodium falciparum is extremely polymorphic: 82 different msp2 alleles were found in 4 studies of molecular epidemiology conducted in Tanzania. This diversity renders msp2 suitable as a marker gene for the genotyping of P. falciparum infections. Amplification of msp2 by the polymerase chain reaction (PCR), and subsequent restriction digests of the PCR product (PCR-restriction fragment length polymorphism genotyping), has proved to be an informative tool for enumerating multiple concurrent infections in a blood sample, and distinguishing individual alleles. Depending on the specific questions asked in a genotyping study, analytical techniques of different degrees of complexity are employed. The restriction fragments resulting from a single HinfI digest generally allow the enumeration of multiple concurrent infections and the determination of their allelic families. When a restriction pattern is too complex to be resolved, owing to the high number of concurrent infections, or due to the appearance of previously undescribed alleles, one or more additional digests (DdeI, RsaI, ScrfI) may be necessary. To determine individual alleles unequivocally, in particular in longitudinal studies, when several consecutive samples need to be compared with each other, a more detailed analysis involving all 3 additional digests is applied. The methodological experience and results gained in 4 epidemiological field studies involving msp2 genotyping are summarized. We also provide the HinfI restriction patterns and some nucleotide sequences of the alleles found so far in our studies in Tanzania.

Molecular epidemiology of Plasmodium falciparum infections among asymptomatic inhabitants of a holoendemic malarious area in northern Ghana.

Age dependence of malaria infection was assessed in an age‐stratified cluster sample of 308 individuals from Kassena‐Nankana District of northern Ghana during June and July 2000. Overall prevalence of Plasmodium falciparum by microscopy was 70%, with the maximum among 5–9u2003year olds. Parasite density was highest (geometric mean 1922/μl blood) in 1–2u2003year olds. Eighty‐two per cent of samples were positive by polymerase chain reaction (PCR), and restriction fragment length polymorphism typing of the P. falciparum msp2 revealed a mean msp2 multiplicity of 3.4 (range: 1–8) genotypes per PCR positive sample. Multiplicity increased with age until 5–9u2003years and then started to reduce again into adulthood. About 49.3% of infections belonged to the msp2 FC27 allelic family and 50.7% to the 3D7 family. On the day of the survey, only 3.6% of the participants had fever (axillary temperature ≥u200337.5u2003°C) and 2.3% had fever associated with parasitaemia. The correlation between parasite density and msp2 multiplicity was 0.42; highest among infants, and decreased with age to a minimum among 5–9u2003year olds. Contrasting with results from Tanzania, this correlation increased with age in adolescents and adults. Parasite multiplicity is very high in this community, and the patterns of age dependence are similar to those in other holoendemic sites in Africa, validating the use of the age–multiplicity relationship as an indicator of malaria endemicity.

6. Multiple Plasmodium falciparum infections in Tanzanian infants

Abstract Paired blood samples from 99 Tanzanian infants were analysed to examine the infection dynamics of Plasmodium falciparum during the first year of life. Infecting parasites were genotyped by polymerase chain reaction amplification of the polymorphic gene for the merozoite surface protein 2 and subsequent analysis according to the resulting restriction fragment length polymorphism pattern. The same samples served as controls in a parallel case-control study for which an additional blood sample was taken from each child during a fever episode. The relationship of the number of concurrent infections (multiplicity) with age and morbidity was analysed and results were compared to those of a similar study on older children between 2 and 7 years of age, carried out in the same village at the same time. The mean of 2 infecting genotypes per positive blood sample from community surveys was low compared to that in older children, and there was no significant age-dependency of multiplicity within the first year of life. Multiplicity of infection in fever cases was also independent of age. In infants, multiplicity was positively associated with parasite density and risk of clinical malaria, in contrast to the situation in older children (>2 years). The findings help in the understanding of infection dynamics, premunition, and development of semi-immunity in malaria.

Malaria infection and morbidity in infants in relation to genetic polymorphisms in Tanzania

To investigate the effects of host polymorphisms on malaria morbidity and infection, the frequency distributions of TNF α promotor gene and sickle cell trait were studied in infants in an area highly endemic for Plasmodium falciparum transmission in Tanzania. Differences in parasite prevalence, density, febrile episodes with and without parasitaemia, PCV levels and the frequencies of different MSP‐2 parasite infections were assessed by genotype. The frequency of the TNF α promotor allele 2 was 0.09, and the trait was in Hardy–Weinberg equilibrium. There were no differences in malariametric indices between infants with the normal TNF α promoter gene and those who were heterozygous for this trait. Infants heterozygous for the TNF α promotor gene appeared to have fewer febrile episodes when they were free of parasites. The two infants homozygous for the TNF α promoter allele 2 had both a much higher incidence of fever, independently of parasitaemia, than the average for the other genotypes. The frequency of the sickle cell allele S was 0.06 and the trait was also in Hardy–Weinberg equilibrium. Infants heterozygous for the sickle cell trait had significantly lower parasite densities, but similar prevalence, and MSP‐2 infections compared to infants with normal haemoglobin. PCV levels, and the incidence of febrile episodes both with and without parasitaemia were also similar. In contrast to the sickle cell trait, the TNF α promotor polymorphism appeared not to have any protective effect on malaria in this study, and its importance in other unspecified fever‐causing diseases in this population needs further investigation.

9. Effect of insecticide-treated bed nets on haemoglobin values, prevalence and multiplicity of infection with Plasmodium falciparum in a randomized controlled trial in Tanzania

A randomized controlled trial of insecticide-treated bed nets (ITNs) was conducted in an area of high malaria transmission in Tanzania in order to assess the effects of ITNs on infection and anaemia. One hundred and twenty-two children, aged 5 to 24 months, were randomly allocated to 2 groups, one of which received ITNs. Outcome measures were assessed in 6 consecutive months with monthly cross-sectional surveys. These measures were haemoglobin values, Plasmodium falciparum prevalence and density, and multiplicity of infection determined by polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) of the msp2 locus. There was a significant increase in mean heamoglobin values and a significant decrease of 16.4% in microscopically determined P. falciparum prevalence in children in the ITN group six months after the start of the trial. Both effects were more pronounced in younger children. However, no significant difference was observed in parasite density or multiplicity of infection among infected children. Comparison with PCR results indicated that microscopically subpatent parasitaemia was more frequently found in children in the ITN group. This, together with the observed similar multiplicity in the 2 groups, suggests that infections are maintained despite ITN use, owing to the chronicity of infections. This study shows that ITNs reduce the risk of anaemia in highly exposed young children. The virtually unchanged multiplicity of infection indicates that the potentially protective concomitant immunity is not compromised.

Prospective risk of morbidity in relation to multiplicity of infection with Plasmodium falciparum in São Tomé.

The prospective risk of acute morbidity was analysed in relation to multiplicity of Plasmodium falciparum infection in 491 individuals in a peri-urban community in São Tomé. In an initial cross-sectional survey, 40.5% of individuals were recorded by microscopy as infected with P. falciparum, and by PCR 60.5%, with the maximum prevalence in children aged 5-10 years. PCR-RFLP typing of the msp-2 gene of P. falciparum found a mean of 2.4 parasite genotypes per infected person, with little age dependence in this multiplicity and a total of 43 different msp-2 alleles identified. None of these were unique for São Tomé. Study participants were encouraged to report to a project worker whenever they suffered a febrile illness. During the 3 months following the parasitological survey the recorded incidence rates decreased with increasing baseline msp-2 multiplicity, both for P. falciparum-positive episodes and for fever without parasitaemia. While this is consistent with suggestions that multiple P. falciparum infections may protect against super-infecting parasites, confounding by patterns of health service usage is an alternative explanation. The incidence of clinical malaria episodes was only a little higher in children than in adults. This weak age-dependence in clinical immunity might be a consequence of a cohort effect resulting from resurgence of the disease after the breakdown of malaria control programs in the 1980s.