Efficacy trial of malaria vaccine SPf66 in Gambian infants

Efficacy trial of malaria vaccine SPf66

Summary SPf66

malaria vaccine is a synthetic protein with aminoacid sequences derived from pre-erythrocytic and asexual blood-stage proteins of Plasmodium falciparum. SPf66 was found to have a 31% protective efficacy in an area of intensive malaria transmission in Tanzanian 1-5 old. We report a randomised, doublechildren, years blind, placebo-controlled trial of SPf66 against clinical P falciparum malaria in Gambian infants. 630 children, aged 6-11 months at time of the first dose, received three doses of SPf66 or injected polio vaccine (IPV). Morbidity was monitored during the following rainy season by means of active and passive case detection. Cross-sectional surveys were carried out at the beginning and at the end of the rainy season. An episode of clinical malaria was defined as fever (≽37·5°C) and a parasite density of 6000/µL or more. Analysis of efficacy was done on 547 children (316 SPf66/231 IPV). No differences in mortality or in health centre admissions were found between the two groups of children. 347 clinical episodes of malaria were detected during the three and a half months of surveillance. SPf66 vaccine was associated with a protective efficacy against the first or only clinical episode of 8% (95% Cl -18 to 29, p=0·50) and against the overall incidence of clinical episodes of malaria of 3% (95% Cl -24 to 24, p=0·81). No significant differences in parasite rates or in any other index of malaria were found between the two groups of children. The findings of this study differ from previous reports on SPf66 efficacy from South America and from Tanzania. In The Gambia, protection against clinical attacks of malaria during the rainy season after immunisation in children 6-11 months old at time of the first dose was not achieved. Lancet 1995; 346: 462-67

Medical Research Council Laboratories, Banjul, The Gambia (U D’Alessandro MD, A Leach MRCP, B O Olaleye MBBS, G W Fegan MSc, M Jawara BTEC, P Langerock LSCh, B M Greenwood FRCP); Departments of Medical Parasitology (C J Drakeley MIBiol, G A T Targett DSc) and Epidemiology and Population Sciences, London School of Hygiene and Tropical Medicine, London (S Bennett PhD); and Ministry of Health and Social Welfare, Banjul, The Gambia (M O George DDS)

Correspondence to: Dr Umberto D’Alessandro, Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK 462

in Gambian infants

Introduction The development of an effective vaccine against Plasmodium falciparum will be a major public health advance, as malaria remains an important cause of mortality and morbidity in young children, particularly in sub-Saharan Africa. SPf66 malaria vaccine is a polymeric synthetic peptide with aminoacid sequences derived from three P falciparum asexual erythrocytic stage proteins linked by the Asn-Ala-Asn-Pro motif derived from the circumsporozoite protein of P falciparum.1 This vaccine has been studied in Aotus monkeys2 and in young adult human volunteers3 in whom it was safe and immunogenic.3’4 Community-based studies in Colombia, and Ecuador confirmed Venezuela, safety, and clinical attacks immunogenicity, ’,-8 protection9 against of malaria. In Tanzania, in an area of intense perennial malaria transmission, it was shown that SPf66 had a protective efficacy of 31% in children aged 1-5 years. 10 in The Gambia, a trial in children 6-11 months old at the second dose, time of the first dose showed that, after the I the vaccine was safe and immunogenic." We report a phase III trial carried out to determine the protective efficacy of SPf66 against clinical episodes of malaria due to P falciparum in children 6-11 months old.

Subjects and methods Study area, population, and study design The study was carried out in the Upper River Division of The Gambia. Malaria is seasonal with moderate transmission (1-10 infective bites per year) occurring during the rainy season (July-December).’2 Anopheles gambiae is the dominant vector and compared to other parts of The Gambia, Upper River Division has the highest entomological inoculation rate (EIR) as well as the highest parasite (57-71%) and spleen rates (40-56%) in children 1-4 years old.13 Our objective was to determine whether three doses of SPf66 reduced the incidence of clinical episodes of malaria in children aged 6-11 months at the time of the first vaccination. All children were living in non-Primary Health Care (non-PHC) villages, which are villages of less than 400 people without a resident Village Health Worker and without easy access to antimalarial drugs. Although bednet usage is relatively high, none of the villages involved in the trial received insecticide for bednet treatment, as the Gambian National Impregnated Bednet Programme is currently limited to PHC villages." The target sample size of 600 children was chosen to give the study at least 90% power to detect a 40% reduction in the incidence of clinical malaria after the administration of the third dose, at a 5% significance level. Randomisation lists for children living on the north and south banks of the river Gambia were produced. Children were randomised to receive either SPf66 or injected polio vaccine (IPV). Entomology An

entomological survey in four representative non-PHC villages (two on the north bank and two on the south bank) was done 9 Aug-22 Nov, 1993. The man-biting rate was calculated from the geometric mean numbers of human-fed An gambiae collected from pyrethrum spray and exit trap catches, divided by the mean number of age-adjusted occupants in the room." Another

estimate was made in which fortnightly outdoor man-landing catches (1900 h-2200 h) were combined with indoor manlanding catches (2200 h-0700 h) in each village. Total counts of mosquitoes for each night were log transformed (n+1) and the geometric mean for all nights were determined. Abdomens of An gambiae were squashed onto filter paper for later blood meal identification,15 and head and thorax were stored for sporozoite analysis." EIR was calculated by multiplying the human biting rate by the sporozoite rate." EIR for children was calculated by dividing that of adults by 4-4.’

Vaccine Clearance was obtained from the Medical Research Council, UK, the Gambian Government, and the Gambian Ethical Committee. The trial started on Dec 6, 1993, after the monitors’ confirmation that the vaccine had been safe and immunogenic after two doses in preliminary studies." The Monitoring Committee recommended the use of SPf66 produced in Colombia at the dose of 1 mg on the grounds that it was as safe 1 as the other vaccines tested in the pilot trial" and more immunogenic. However, between the second and third dose of vaccine, children in the pilot trial who had received Colombian SPf66 showed a higher incidence of clinical attack of malaria than control children. For this reason, the Monitoring Committee and the Gambian Ethical Commitee recommended increased surveillance for these children and for those enrolled in the efficacy trial. SPf66 peptide (lot 0394A) was synthesised at the Instituto de Immunologia, Bogota, Colombia. The vaccine passed abnormal toxicity and bacterial endotoxin tests carried out by the National Institute for Biological Standards and Control (NIBSC), South Mimms, Herts, UK, and sterility testing by the PHLS Centre for Applied Microbiology and Research, Porton Down, UK. The polio vaccine was IMOVAX POLIO (Pasteur Merieux, Lyon,

Further doses were given after 4 and 26 weeks. Children were under surveillance for one hour after each dose, with access to

portable resuscitation equipment. Active and passive surveillance From Aug 1, 1994, children were visited

at

home twice

a

week

and parents questioned about any sickness, attendance at a Health Centre, or self-medication. The axillary temperature was taken; if it was 37-5°C or more, two thick-blood films were prepared. If blood films showed malaria parasites, antimalarial treatment was given within 24 h. Passive case detection was at 6 health centres (Diabugu, Yorobawol, Baja Kunda, Gambissara, Basse, Fatoto) and at the MRC clinic in Basse. Children in the trial who came to a health centre had their axillary temperature taken and blood films prepared if their temperature was 37-5°C or more. Symptoms, signs, probable diagnosis, and treatment were recorded. Children could be admitted to 3 of the Health Centres and to MRC Basse. All administrations were recorded and a weekly report was sent to the local monitor. Active and passive surveillance ended on Nov 20, 1994.

Cross-sectional surveys Two cross-sectional surveys were done, the first 3 weeks after the third dose had been given and the second at the end of November, 1994. Questions on bednet usage and insecticide treatment of the net were asked at the November survey. Spleen size was estimated by abdominal palpation, axillary temperature measured, and a blood sample collected for parasitological examination and for determination of the packed cell volume (PCV). Serum was separated and stored at -20°C and the red cell pellet was kept for PCR studies. Children with a positive blood film and/or a PCV of 25% or less were treated with chloroquine and/or iron and folic acid.

France) (lot J0665). Vaccines were drawn up in 1 mL plastic syringes masked with tape as SPf66 and IPV differ in appearance, numbered according to a randomisation list and put in numbered envelopes. Vaccine was kept at 2-8°C during transport and storage. Five of every 100 syringes were withdrawn and checked by the local monitor to determine whether they contained the right vaccine. A mistake in the coding of the syringes occurred at the time of preparation of the third dose of vaccine: syringes for children on the south bank were wrongly labelled with the code for the north bank. Before investigators realised this, 300 children in the south bank had been vaccinated, of whom 154 had received the wrong vaccine. Half of these had received IPV instead of SPf66 and the other half SPf66 instead of IPV With the permission of the Monitoring and the Gambian Ethical Committees, it was decided to revaccinate all the children who received the wrong vaccine, not just those who had received IPV instead of SPf66, in order to maintain the blindness of the investigators. The children who received SPf66 instead of the third dose of IPV were excluded from the analysis of efficacy.

Informed consent and immunisation Parents of children in non-PHC villages who would be in the right age group in December, 1993, were visited at home by field staff. The study was explained and they were given a leaflet about the trial printed in English and their local language. One to two weeks later, parents were asked for consent to enrol their child in the study. One to two weeks before the first and third doses of vaccine were given, children were treated with a single dose of to clear sulfadoxine-pyrimethamine blood-stage P falciparum infections. Before immunisation, children were reviewed by a study physician and excluded if weight for age was 60% or less or there was evidence of chronic disease such as heart disease or sickle cell anaemia. Infants were not immunised during an acute illness but when they had recovered. Injections were subcutaneous with a 25G needle into the left thigh for the first and third doses, and into the right thigh for the second dose.

Laboratory methods For each blood slide, 100 high power fields (HPF) were examined. For films with one parasite or more per HPF, parasite densities were recorded per HPF. One parasite per HPF was assumed to indicate a density of 500 parasites/ILL.18 All slides were read twice, independently, and a third time if there was any discrepancy in positivity or if the difference of the log-densities was more than 1-5. Agreement was reached after the slides had been re-checked. Antibody levels against SPf66 were measured on 98 randomly selected serum samples from the July survey. Microtitre plates (Immunolon-2, Dynatech, USA) were coated with 0-(JLg/welI Spf66 overnight at room temperature. After incubation for one hour with blocking buffer (0-5% boiled casein), serial dilutions of sera were added and tested in triplicate. After a 2-h incubation, plates were washed and an anti-human Ig conjugated to HRP (Southern Biotechnology, Birmingham, Alabama, USA) added. After a further 1-h incubation, plates were washed, ABTS substrate (Kierkegaard and Perry, Gaithesburg, MD, USA) added and the plates read at 405 nm. Mean optical density plus 3 standard deviations (at 1:100, 1:200, 1:400, 1:800 dilutions) of serum samples from children in the safety study collected before any vaccination, defined the cutpoint for positivity. The lowest dilution in which the optical density was greater than the cutpoint was taken as the end titre of the sample.

Case definition, data methods

processing, and statistical

routinely checked for range and consistency. resolved by field supervisors. A clinical Discrepancies of was malaria defined as an illness associated with an episode of 37-5°C or more and with a P falciparum axillary temperature parasite density of 6000/jjLL or more.1Q,20 The estimated sensitivity and specificity for the above case definition were both Records

were

were

86%.

Primary analysis considered cases detected by either active or passive surveillance from Aug 1, 4-6 weeks after the third dose of 463

for the next 28 days and was removed from numerator and denominator for that period. Protective efficacy against episodes of documented fever associated with P falciparum parasitaemia of any density was also calculated as above, excluding mixed infections with P ovale or P malariae. Vaccine efficacy against infection was estimated by comparing P falciparum asexual parasite, high density parasite (5000 parasites/jjbL), and spleen rates found at the November clinical survey, and mean PCV and the mean change in PCV between the July and November surveys. Children who received antimalarial drugs within 28 days of the survey were excluded from analysis. Adjustments were made for confounding factors using multiple logistic or normal regression as appropriate.

Results

*Determined at the November morbidity survey. Table 1: Characteristics of the study cohorts was restricted to children who received all three doses of SPf66 or IPV. Children in the control group who received one dose of SPf66 in error and children who received chemoprophylaxis for suspected sickle-cell disease were excluded. Vaccine efficacy (VE) was derived from VE=100X(1IRR)%, where IRR is malaria incidence in the SPf66 group/incidence in the IPV group. IRR was estimated by Poisson regression, relating the occurrence of each child’s first or only malarial episode during the surveillance period to the number of child-days at risk. The regression allowed the incidence rate in each group to vary by a calendar month, whilst preserving a constant rate ratio. After an episode of malaria, a child was removed from both numerator and denominator. Children who were lost at follow-up, who withdrew, or who died were included up to the date of the event. Children who were not found by the field assistant and who had not attended one of the Health Centres at the time of the visit, but who were seen at a later visit, were excluded from the denominator for the period for which they were not under observation. Before breaking the code, potential confounders were considered to be: age at first vaccination, ethnic group, river bank (north or south), distance from the Health Centre, bednet usage reported at the November survey, and haemoglobin genotype. Estimates and confidence intervals (CI) for the vaccine efficacy were calculated with Poisson regression to adjust for the above factors. Kaplan Meier survival curves were used to show timing of malaria episodes. Protective efficacy was also calculated separately on clinical episodes detected by active or active/passive surveillance (children referred by a field assistant to the Health Centre), and for those detected by passive surveillance alone (mothers who spontaneously brought children to Health Centres). The total number of episodes of clinical malaria during the surveillance period was used to estimate the overall incidence rate ratio. After each recorded episode, a child was considered not to be at risk

vaccine, and

ACD: active case detection. PCD: passive case detection. APCD: *Episodes detected by PCD are ignored. tEp1sodes detected by ACD/APCD are ignored.

Table 2: Vaccine

464

efficacy against first

or

Screening and immunisation The census identified 711 children in 210 villages, aged 6-11 months between Dec 6 and 20, 1993 (447 on the south bank in 133 villages and 264 on the north bank in 77 villages). On the day of first vaccination, 18 children could not be identified, the parents of 5 refused to participate, and 19 were excluded (one had an abscess, five acute pneumonia, eight were severely malnourished, three had Down’s syndrome, one tuberculosis, and one was already participating in another research project). 669 children received the first dose, and 663 (99%) and 630 (94%) received second and third doses. 39 children received the first but not the third dose: three were withdrawn due to illness (1 SPf66/2 IPV), five died (2/3), and 31 (16/15) moved away. After discovering the syringe-coding error the local monitor produced a list of 154 children for re-vaccination, 152 (76 SPf66/76 IPV) of these were re-vaccinated. Study cohorts 76 children in the IPV group who had received one dose of SPf66 in error were excluded from analysis, as were two children (1 SPf66/1 IPV) who had moved out of the study area and could not be re-vaccinated, three (2/1) who died before the surveillance started, and two children (1/1) who had sickle-cell disease and chemoprophylaxis throughout the rainy season. The main analysis of efficacy was on 547 children (316 SPf66/231 IPV), 320 on the south bank and 227 on the north. There were no important differences between children in the two groups (table 1). Immunogenicity SPf66-antibody levels were measured in 98 serum samples obtained 3 weeks after the third dose of vaccine and selected randomly before breaking the code. Ten of these samples were from children in the IPV group who

active/passive case detection.

only clinical episode of P falciparum as established by passive and active

case

detection

SPF66 - - - IPV

*A child with Hb genotype SS is excluded. Table 4: Results of the November clinical survey

(%)*

Days of observation

Figure: Kaplan Meier curves established from active and passive case detection after the third dose of vaccine had received one dose of SPf66 by mistake and these were therefore excluded. 98% (55/56) of the children who had received a full course of SPf66 had detectable antibody (geometric mean of titres : 521-6), whereas none of the 32 children in the IPV group had measurable antibody.

Mean PCV (SPf66 299, IPV IPV -4 2, p=O’61j

28.8, p-0.50): Mean PCV difference (SPf66-3.9,

Table 5: Odds ratios and for confounding factors

means

for

SPf66/IPV after adjustment

protective efficacy of 8% (95% CI -18 to 29) (p=0-50). Kaplan-Meier plots (figure) of the estimated proportion free of malaria by days of observation show very little difference between the two groups. Vaccine efficacy was 13% (95% CI -43 to 47) (3, p=0-58) against episodes detected by passive case detection, and 5% (95% CI -21 to 27) (p=0’70) against episodes detected by active or active/passive case detection. Efficacy was similar for children aged 6-8 months and children aged 9-11 months at the time of first dose (data not shown). All clinical episodes detected by active and passive surveillance are shown in table 3: the adjusted vaccine efficacy against all episodes was 3% (95% CI -24 to 24) (p=0’81). There was no evidence of protection against episodes of fever associated with any level of parasitaemia (VE=-2%, 95% CI -26 to 18) (p=0’85). Those children who had received one dose of SPf66 in error were not significantly less or more at risk against first or only clinical episode of a

Entomology The entomological inoculation ratio for adults, estimated from pyrethrum spray and exit-trap collections was 0-034 infective bites per night; the rate estimated from manlanding catches was 0-456 infective bites per night. Therefore, during the 112-day surveillance period, children received O’8-l 1-6 infective bites.

Mortality and hospital admissions Five children (2 SPf66/3 IPV) who had received the first dose died before the third dose was due, 4 (2 SPf66/2 IPV) died after the third dose-3 (1 SPf66/2 IPV) before surveillance started on Aug 1. Hospital records and interviews with parents suggest that only one death (SPf66), which occurred in October, was due to cerebral malaria. the surveillance period, 93 study children were at least once for a total of 105 admissions to (61/44) MRC Basse, or to Yorobawol or Fatoto Health Centres (51 SPf66/41 IPV). Clinical malaria was diagnosed in 56 and was the most frequent cause of admission (30 SPf66/26 IPV). Two children (SPf66) were admitted for severe malaria, one died and the other received blood transfusion and recovered. 62-8% of admissions occurred during September and October.

During

admitted

Protective efficacy Analysis of vaccine efficacy against first or only clinical episode of malaria is shown in table 2. SPf66 vaccine had

’tplsoaes za aays apart or iess nave not been consiciereci as new clinical episoaes. VE against all episodes: unadjusted 2%; adjusted 3% (95 CI -24; 24) p=0.81.

Table 3: Clinical episodes of malaria detected by active and passive surveillance

malaria than other children on the south bank who received only IPV (VE=8%, 95% CI -40 to 40)

(p=0’70). Cross-sectional surveys 618 children were seen during the July, 1994, survey, shortly after completing their vaccination, and blood samples for determination of parasitaemia (614) and PCV (612) were collected. One child (SPf66) had a malariapositive slide and 2 had slightly enlarged spleens (1 SPf66/1 IPV). Mean PCV was similar in the SPf66 and in IPV groups (32’5% SPf66/32-9% IPV). In November, 1994, 600 children were seen and blood samples for parasitaemia (597) and PCV (592) were collected. 108 children who had had chloroquine or fansidar (68 SPf66/40 IPV) 28 days or less before the survey and those who received only one dose of SPf66 were excluded. No significant differences in the prevalence of parasitaemia, high-density parasitaemia, or splenomegaly, or in the mean-parasite density, mean PCV, and percentage of children with a PCV of 25% or less were found between the two groups (table 4). These results did not change after adjusting for confounding factors (table 5). Adjusted mean PCV was 28’9% in the SPf66 group and 28-8% in the polio group (p=0’50); the 465

adjusted mean difference in PCV between the July and the November surveys was -3-9% in children who received the malaria vaccine and —4-2% in those who received the polio vaccine (p=0-61). No differences in the anthropometric measures (mean z score weight for age, weight for height, and height for age) were found between the two groups of children at the November survey (data not

shown).

Discussion SPf66 did not protect young Gambian children against first attacks of clinical malaria, overall incidence of malaria attacks or infection, nor prevent the fall in PCV associated with malaria, seen in young Gambian children during the rainy season.21 Almost 350 clinical attacks were detected in three and a half months of passive and active surveillance, twice the number detected in the Tanzanian SPf66 trial’O in one year of surveillance in about the same number of children (aged 1-5 yr), in an area with a higher malaria-transmission rate, perhaps reflecting differences in the age distribution of the two cohorts or intensity of surveillance. About 50% of children under surveillance experienced at least one clinical attack of malaria and this gave the study the power to detect an even smaller level of protection than the 40% assumed in calculating sample size. Previous trials of SPf66 have shown some protection against clinical malaria.7-10 Why did this trial produce a different result? There was a mistake in coding syringes for the third dose. It was decided to revaccinate all of the children who received the wrong vaccine to maintain blindness of the study investigators and to exclude from analysis children in the control group who received one dose of SPf66. Raised antibody titres to SPf66 were found only in children in the trial who were in the SPf66 vaccine group, confirming that children retained in the trial had been correctly immunised. The intensity of infection in areas of South America where successful trials of SPf66 have been conducted is substantially less than in The Gambia, which may have contributed to the difference in results. A current trial in an area of Thailand with a level of infection between that in South America and tropical Africa will help to clarify this point. Higher level of exposure to malaria, however, cannot be the explanation for the different results obtained in the Gambian and Tanzanian studies because Gambian children were exposed to less than 12 infectious bites, substantially fewer than in the Tanzanian study. In Tanzania, SPf66 had no effect on prevalence of malaria infection, PCV, or number of clinical attacks of malaria-a statistically signficant result was obtained only for the incidence of first or only attacks of clinical malaria. Thus, the difference between the two trials may be more apparent than real. The lower 95% CI for vaccine efficacy against first clinical attacks of malaria was 0% in the Tanzanian trial, whilst the upper 95% CI for efficacy against first attacks of malaria in the Gambian study was 29%. Thus, both trials are compatible with a true level of protection against first attacks of malaria of 0-29%. The ages of the children who were vaccinated (1-5 years old in Tanzania and 6-11 months old in The Gambia) were different. It is possible that age influences immune response to SPf66, but this seems unlikely for a peptide vaccine given with alum. Nearly all children in the Gambian trials showed a good antibody response to 466

SPf66 although the mean antibody trial fell to near prevaccination levels after 15 months." Any difference in outcome is likely to be due to the relationship between age and the level of exposure to malaria. Children in the Gambian trial are likely to have had less prior exposure to malaria before immunisation than those in the Tanzanian trial and this could have determined their immune response to vaccination. Genetic factors are known to influence cellular and humoral immune response to malaria23 and to influence immune response to SPf66. Thus, it is possible that genetic differences in the two study populations influenced the outcome of the trials. It is also possible that efficacy of SPf66 may be influenced by the distribution of variants among the population of parasites in the area where the trial is undertaken, because one component of SPf66 corresponds to a variable region of the malaria antigen MSPl.=6 A further difference between the Gambian trial and other trials is the length of follow-up. In The Gambia, the malaria season is short and lasts only for a few months. Children were followed for about three and half months, as there is virtually no transmission after the rainy season. In most other studies, the minimum length of follow-up was one year. Furthermore, in the Tanzanian trial, the Kaplan Meier curves showing the proportion of children who had not yet had a clinical episode after the third dose, started to diverge only 20 weeks after the third dose.10 In Venezuela, a decrease in incidence was observed only 3 months after the third dose? and in Colombia,9 after five months after the third dose. If protective efficacy of the vaccine becomes apparent only a few months after the third dose, the Gambian trial would not have been able to detect any protection. Children in the trial will be followed during the 1995 malaria transmission season. We thank Dr M E Patarroyo and his colleagues at the Instituto de Inmunologia, Bogota, Colombia for providing SPf66 vaccine; the National Institute for Biological Standards and Control, UK, and the PHLS Centre for Applied Microbiology and Research, Porton Down, UK, for undertaking sterility and pyrogenicity assays; children and their parents, the staff of the 6 health centres and the Divisional Health Team of Upper River Division; field staff Kebba Keita, Alhagi Darbo, Pa Amadou Suso, and Waza NjaJ; Dr Margaret Pinder and Mr Kebba Jammeh for help with microscopy; Dr Madeleine Thomson for her help in analysis of entomological data; the MRC Trial Monitoring Committee (Prof M Molyneux, Prof M Hommel, Dr A Holder, Dr H Inskip); Dr J F Trape who assisted the Monitoring Committee; the Gambia Government/MRC Ethical Committee; the local Safety Monitor (Dr P T Corrah); and Mrs L Wijgergangs and Mrs M Wintermans for coding the syringes. The study was supported by the UK Medical Research Council.

References 1

chimeric molecule used 585-91.

a

2

3

4

5

6

7

et al. Characterization of SPf(66)n: malaria vaccine. Vaccine 1994; 12:

Lopez MC, Silva Y, Thomas MC, as a

Patarroyo ME, Romero P, Torres ML, et al. Induction of protective immunity against experimental infection with malaria using synthetic peptides. Nature 1987; 328: 629-32. Patarroyo ME, Amador R, Clavijo P, et al. A synthetic vaccine protects humans against challenge with asexual blood stages of Plasmodium falciparum malaria. Nature 1988; 332: 158-61. Amador R, Moreno A, Valero A, et al. The first field trials of the chemically synthesized malaria vaccine SPf66: safety, immunogenicity and protectivity. Vaccine 1992; 10: 179-84. Amador R, Moreno A, Murillo L, Sierra O, et al. Safety and immunogenicity of the synthetic malaria vaccine SPf66 in a large field trial. J Infect Dis 1992; 166: 139-44. Patarroyo G, Franco L, Amador R, et al. Study of the safety and immunogenicity of the synthetic malaria SPf66 vaccine in children aged 1-14 years. Vaccine 1992; 10: 175-78. Noya O, Berti YG, de Noya BA, et al. A population-based clinical trial with SPf66 synthetic Plasmodium faciparum malaria vaccine in Venezuela. J Infect Dis 1994; 170: 396-402.

Sempertegui F, Estrella B, Moscoso J, et al. Safety, immunogenicity and protective effect of the SPf66 malaria synthetic vaccine against Plasmodium falciparum infection in a randomized double-blind placebocontrolled field trial in an endemic area of Ecuador. Vaccine 1994; 12: 337-42. 9 Valero MV, Amador LR, Galindo C, et al. Vaccination with SPf66, a chemically synthesised vaccine, against Plasmodium falciparum malaria in Colombia. Lancet 1993; 341: 705-10. 10 Alonso PL, Smith T, Armstrong Schellenberg JRM, et al. Randomised trial of efficacy of SPf66 vaccine against Plasmodium falciparum malaria in children in southern Tanzania. Lancet 1994; 344: 1175-81. 11 Leach A, Dzakeley CJ, D’Alessandro U, et al. A pilot safety and immunogenicity study of the malaria vaccine SPf66 in Gambian infants. Parasite Immunol 1995; 17: 441-44. 12 Greenwood BM, Pickering H. A review of the epidemiology and control of malaria in The Gambia, West Africa. Trans Roy Soc Trop Med Hyg 1993; 87 (suppl 2): 3-11. 13 Thomson MC, D’Alessandro U, Bennet S, et al. Malaria prevalence is inversely related to vector density in The Gambia, West Africa. Trans Roy Soc Trop Med Hyg 1994; 88: 638-43. 14 D’Alessandro U, Olaleye BO, McGuire W, et al. Reduction in mortality and in morbidity from malaria in Gambian children following the introduction of a National Insecticide Impregnated Bednet Programme. Lancet 1995; 345: 479-83. 15 Burkot TR, Goodman WG, DeFoliart GR. Identification of mosquito bloodmeals by enzyme linked immunosorbent assay. Am J Trop Med Hyg 1981; 30: 1336-41. 16 Wirtz RA, Burkot TR, Graves PM, Andre RG. Filed evaluation of enzyme-linked immunosorbent assays for Plasmodium falciparum and Plasmodium vivax sporozoites in mosquitoes, (Diptera: Culicidae) from Papua New Guinea. J Med Entomology 1987; 24: 433-37.

8

17 Port GR, Boreham PFL, Bryan JH. The relationship of host size to feeding by mosquitoes of the Anopheles gambiae Gilles complex (Diptera: Culicidae). Bull Entomol Res 1980; 70: 133-44. 18 Greenwood BM, Armstrong JRM. Comparison of two simple methods for determining malaria parasite density. Trans Roy Soc Trop Med Hyg 1991; 85: 186-88.

Armstrong-Schellenberg JRM, Smith T, Alonso PL, et al. What is clinical malaria? Finding a case definition for field research in highly endemic areas. Parasitol Today 1994; 10: 439-42. 20 Smith T, Armstrong-Schellenberg JA, Hayes R. Attributable fraction

19

estimates and

case

definition for malaria in endemic

areas.

Stat Med

1994; 13: 2345-58. 21 Greenwood BM, Bradley AK, Greenwood AM, et al. Mortality and morbidity from malaria among children in a rural area of The Gambia, West Africa. Trans Roy Soc Trop Med Hyg 1987; 81: 478-86. 22 Greenwood B’M. The microepidemiology of malaria and its importance to malaria control. Trans Roy Soc Trop Med Hyg 1989; 83 (suppl): 595-98.

Riley EM, Olerup O, Troye-Blomberg M. The immune recognition of malaria antigens. Parastiol Today 1991; 7: 5-11. 24 Patarroyo ME, Vinasco J, Amador R, et al. Genetic control of the immune response to a synthetic vaccine against Plasmodium falciparum.

23

Parasite Immunol 1991; 13: 509-16.

25 Murillo LS, Tenjo F, Clavijo O, et al. A specific T-cell receptor genotype preference in the immune response to the synthetic P falciparum malaria vaccine. Parasite Immunology 1992; 14: 87-94. 26

Cooper JA, Merozoite surface antigen-1 1993; 9: 50-54.

of Plasmodium. Parasitol

Today

Vascular function in the forearm of hypercholesterolaemic patients off and on lipid-lowering medication Summary To study whether vascular dysfunction in hypercholesterolaemia is reversible, we investigated patients without overt arterial disease who were taking maintenance treatment for hypercholesterolaemia. Medication was stopped for 2 weeks, reinstituted for 12 weeks, and again stopped for 6 weeks. During both maintenance treatment and the 12 weeks of step-up medication the lipid profile was improved but did not return to normal. Dose-response curves for serotonin-induced vasodilatation, an index of nitric oxide-dependent vasodilatation, showed a comparable and significant rightward shift after a medication-free period of 2 and 6 weeks compared with control subjects, indicating endothelial dysfunction, which was already maximum after 2 weeks. After 12 weeks of lipid-lowering medication, the difference in endothelial function between controls and patients had disappeared. Co-infusion of L-arginine, the substrate for nitric oxide synthase, returned the impaired serotonin response during hypercholesterolaemia to normal, but had no effect on this response in controls or in patients while on lipid-lowering medication. Neither endothelium-independent vasorelaxation, assessed by sodium nitroprusside infusion, nor vasoconstriction induced

Departments of Nephrology and Hypertension (E S G Stroes MD, H A Koomans MD, T J Rabelink MD) and Lipid Clinic of the Department of Medicine (T W A de Bruin MD), University Hospital Utrecht, The Netherlands Correspondence to: Dr T J Rabelink, Department of Nephrology and Hypertension, Room F03.226, Heidelberglaan 100, 3584 CX The Netherlands

the nitric oxide blocker L-NMMA, were different between controls and patients, whether the latter were on or off lipid-lowering medication. Our results show an L-arginine-sensitive, impaired nitricoxide-mediated vascular relaxation of forearm resistance vessels in hypercholesterolaemia which is reproducible, and reversible after short-term lipid-lowering therapy. Demonstration of such changes in this readily accessible vascular bed will allow larger trials assessing vascular function during lipid-lowering therapy to be done.

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Lancet 1995; 346: 467-71

Introduction The vascular endothelium

helps prevent atherosclerosis by maintaining vasodilatation; and inhibiting platelet aggregation, leucocyte adhesion, and proliferation of smooth-muscle cells through the release of nitric oxide (NO) and other factors.’ Impaired endotheliumdependent vasodilatation has been demonstrated in coronary arteries of patients with atherosclerosis,2,3 and in peripheral arteries of patients with diabetes4 or hypercholesterolaemia.5,6 Six to 12 months of lipidlowering treatment prevent or attenuate acetylcholineinduced vasoconstriction in atherosclerotic coronary arteries.’-" Such a long period of lipid-lowering therapy was chosen on the assumption that it takes time for hypercholesterolaemia-associated vascular abnormalities to disappear. In one study, the endothelium-mediated responses of coronary arteries were still abnormal after 12 days of lipid-lowering medication, although the lipid

profile had greatly improved." The first objective of our study was to find out whether, in hypercholesterolaemic patients without overt arterial 467