The Anti-Circumsporozoite Antibody Response of Children to Seasonal Vaccination With the RTS,S/AS01E Malaria Vaccine

Abstract Background A trial in African children showed that combining seasonal vaccination with the RTS,S/AS01E vaccine with seasonal malaria chemoprevention reduced the incidence of uncomplicated and severe malaria compared with either intervention given alone. Here, we report on the anti-circumsporozoite antibody response to seasonal RTS,S/AS01E vaccination in children in this trial. Methods Sera from a randomly selected subset of children collected before and 1 month after 3 priming doses of RTS,S/AS01E and before and 1 month after 2 seasonal booster doses were tested for anti-circumsporozoite antibodies using enzyme-linked immunosorbent assay. The association between post-vaccination antibody titer and incidence of malaria was explored. Results A strong anti-circumsporozoite antibody response to 3 priming doses of RTS,S/AS01E was seen (geometric mean titer, 368.9 enzyme-linked immunosorbent assay units/mL), but titers fell prior to the first booster dose. A strong antibody response to an annual, pre-malaria transmission season booster dose was observed, but this was lower than after the primary vaccination series and lower after the second than after the first booster dose (ratio of geometric mean rise, 0.66; 95% confidence interval [CI], .57–.77). Children whose antibody response was in the upper tercile post-vaccination had a lower incidence of malaria during the following year than children in the lowest tercile (hazard ratio, 0.43; 95% CI, .28–.66). Conclusions Seasonal vaccination with RTS,S/AS01E induced a strong booster antibody response that was lower after the second than after the first booster dose. The diminished antibody response to the second booster dose was not associated with diminished efficacy. Clinical Trials Registration NCT03143218.


INTRODUCTION
Malaria transmission is highly seasonal in six of the ten African countries where malaria is not well controlled identified by WHO [1]. Widespread deployment of Seasonal Malaria Chemoprevention (SMC) has had a substantial impact on malaria in children in these areas [2] but in many parts of the Sahel and sub-Sahel, malaria remains the most frequent cause of death and hospital admission in young children [3]. Taking advantage of the high initial efficacy of the RTS,S/AS01 E malaria vaccine [4,5], we have suggested that RTS,S/AS01 E could be deployed in these areas as a seasonal vaccine [6]. This concept has been tested in a trial undertaken in 5,920 children in Burkina Faso and Mali during 2017-2020. Seasonal vaccination with RTS,S/AS01 E was non-inferior to SMC in preventing clinical episodes of malaria and the combination of RTS,S/AS01 E, with SMC was markedly superior to either intervention given alone in preventing uncomplicated cases of malaria, severe malaria requiring hospital admission, and death from malaria [7]. This paper reports, anti-circumsporozoite (anti-CSP) antibody titers measured in a sub-set of trial children sampled before and after three priming doses of RTS,S/AS01 E and before and after two subsequent booster doses given just prior to the malaria transmission season, together with the correlation between anti-CSP antibody titer following vaccination and the incidence of episodes of uncomplicated clinical malaria during the subsequent year.

Trial design
Blood samples for serologic testing were collected during the course of an individually randomised, controlled trial designed to determine whether seasonal vaccination with the RTS,S/AS01 E malaria vaccine was non-inferior to SMC in preventing clinical episodes of malaria and/or whether the combination was superior to either intervention given alone. The primary trial end-point was the incidence of uncomplicated, microscopically-confirmed Plasmodium falciparum malaria with a Downloaded from https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciab1017/6459764 by guest on 13 December 2021 density of 5,000 parasites per µl or more. There were a number of additional secondary endpoints [8].
The three main objectives of the serologic sub-study were determination of (a) P. falciparum anti-CSP antibody titers before and after three priming doses of RTS,S/AS01 E and before and after two subsequent annual booster doses, (b) whether the magnitude of the anti-CSP antibody response to priming or booster immunization influenced the risk of malaria during the subsequent malaria transmission season, and (c) whether the anti-CSP antibody titer response to annual booster doses of RTS,S/AS01 was influenced by administration of SMC in the previous malaria transmission season.

Trial sites and population
The trial was conducted in Bougouni and Ouélessébougou districts, Mali and in Houndé district, Burkina Faso. All households within the study areas with children aged 5-17 months on April 1 st 2017 were enumerated in February-March 2017. Eligible children whose parent or guardian provided consent for their child to join the trial were allocated randomly to an SMC alone, RTS,S/AS01 E alone, or SMC+RTS,S/AS01 E group by an independent statistician.

Interventions
Children in the RTS,S/AS01 E alone or RTS,S/AS01 E + SMC group received three doses of RTS,S/AS01 E vaccine (GSK, Rixensart, Belgium) at monthly intervals in April -June 2017 followed by fourth and Infants received half of these doses. All doses were administered by project staff under direct observation. All study children were given an insecticide treated bednet at enrolment in 2017.

Surveillance for malaria
Project staff based in study health facilities identified and treated all cases of malaria who presented at these facilities using a Rapid Diagnostic Test (RDT) and obtained a blood film for subsequent microscopy [8]. All hospital admissions of study children were documented by trial staff [8]. Blood films were read by two independent microscopists and, in instances of a discrepancy in positivity or density, by a third reader with discrepancies being resolved as described previously [9]. A crosssectional survey of malaria prevalence was undertaken in all study children one month after the last round of SMC administration each year.

Serology
In 2017, approximately 200 children (100 per group) and in 2018 and 2019 approximately 300 children (150 per group) from the RTS,S/AS01 E alone or RTS,S/AS01 E +SMC groups, together with 30-40 children from the SMC alone group, were selected at random by an independent statistician using systematic random sampling after sorting by age and sex to ensure that treatment groups were approximately balanced between these two variables. The same children were sampled pre-and post-vaccination within a study year but different children were selected each year. The timing of the collection of blood samples in relation to vaccination is shown in Figure 1.
IgG anti-CSP antibody titers were measured using a standardised ELISA at the CEVAC laboratory Ghent University, Belgium [10]. The lower limit of quantification for the assay was 1.9 ELISA units/ml (EU/ml) and samples with a titer below this lower limit (i.e. titers with a value of 0) were assigned a titer of 0.95 EU/ml, half the lower limit of detection.

Sample size and statistical analysis
Based on the findings in previous RTS,S/AS01 E trials, large differences in anti-CSP titer between preand post-priming or booster vaccinations were anticipated. In order to determine whether prior administration of SMC influenced the immune response to booster vaccination, approximately 160 children who had received RTS,S/AS01 E with or without SMC were selected in 2018 and 2019 to give a study with 80% power to detect a difference of 25% -30% in GMT between children who had received SMC or placebo in the previous year. received SMC and those who had not; (d) comparison of the incidence of morbidity outcomes in relation to antibody response using Cox regression models with a robust standard error to account for multiple episodes per child and defining a potential cut off titer associated with protection from the reverse cumulative plots [11]; (e) comparison of the prevalence of P falciparum infection at the end of the malaria transmission season in relation to antibody response, using Poisson regression with a robust standard error. For comparisons (d) and (e), incidence and prevalence were compared between groups defined by terciles of anti-CSP titer.

Ethics and trial oversight
The

Study children
Two hundred and thirty one pre-and post-vaccination paired blood samples were obtained from  Table 1.

Baseline anti-CSP antibody titers and the anti-CSP antibody response to RTS,S/AS01 E vaccination
No child had an anti-CSP antibody titer above the lower limit of quantification (1.9 EU/ml) prior to vaccination. Two children in the SMC alone group, who had not received RTS,S/AS01 E , had a marked increase in anti-CSP antibody titer post vaccination (post-vaccination GMTs 300.7 EU/ml and 728.2 EU/ml respectively), probably resulting from a labelling error, and these children were excluded from the analysis. With the exception of these two children, only three children in the SMC alone group had a titer above the lower limit of quantification at any survey.
Among children in the RTS,S/AS01 E alone or RTS,S/AS01 E + SMC group, antibody titers increased markedly one month after the third of three priming doses with a GMT of 368. 9 4, 195.0) and the ratio between post-booster titers -pre-booster titers was 3.87 (95% CI: 3.40, 4.41). The GMT following the first booster dose was significantly less than after the priming doses and the GMT following the second booster dose was significantly lower than that seen after the first booster dose.
The ratio of the geometric mean rise in titer following the second booster compared to the first booster was 0.66 (95% CI: 0.57, 0.77) (Table S1) (Table S3).
Nearly all children (>99%) showed a ten-fold increase in anti-CSP antibody titer one month after three priming doses but this proportion fell to 27% after the first booster dose and to 11% after the second booster vaccination. The comparable percentages for a two-fold increase in GMT were 89% and 85% respectively (Table 2).

Anti-CSP titre and protection against malaria
Tertiles of the post-vaccination antibody response were defined separately for each year of the study (Table 3). Over the three years of the study combined, the hazard ratios comparing the incidence of clinical episodes of malaria between children in the highest or middle tercile, compared to children in the lowest tercile were 0.43 (95% CI 0.28, 0.66) and 0.66 (95% CI 0.44, 0.99), respectively. Incidence was consistently lowest among children in the highest tertile in each year of the study; the hazard ratios for children in the highest tercile compared to those in the lowest tertile Only eight children in the serology sub-study had an episode of malaria severe enough to require hospital admission; 3 after the first booster and 5 after the second booster. Three of these children had a post-vaccination titer in the lowest tercile, two in the middle and three in the highest tercile.
The relationship between post-vaccination anti-CSP antibody titer one month after priming or after each booster vaccination and the prevalence of asymptomatic malaria parasitemia approximately five months later at the end of the malaria transmission season survey that year is shown in Table 4.
In the first year of the study, only three malaria infections were detected among children in the serology sub-study. However, this number increased to 27 and 22 in years 2 and 3 respectively. In year 2, the prevalence ratio for parasitemia for those whose post booster vaccination titer was in the highest tercile compared with those whose titer was in the lowest tercile was 0.81 (95% CI 0.33, 2.0), and in year 3 (after the second booster) it was 0.40 (95% CI 0.15, 1.05). Identification of potentially protective cut-off titers in relation to the level of efficacy seen in each year of the study, deduced from reverse cumulative plots, gave figures of 266.8 EU/ml, 207.2 EU ml and 157.5 EU/ml in years one, two and three of the study respectively (Table S4).

DISCUSSION
The anti-CSP antibody response of children in this trial to vaccination with three priming doses of RTS,S/AS01 E followed by a booster dose was similar to that seen in several previous studies in African children [12,13]. However, it was lower than that recorded in the phase 3 RTS,S/AS01 E trial in whom the GMT one month after three priming doses in those vaccinated between the ages of 5-17 months, was 570.3 (95% CI 543.7, 589.3] EU/ml for all centres combined. A range of responses was seen between the 11 centres in the phase 3 trial with a high post-vaccination GMT of 686 (95% CI 604, 778) EU/ml being found at the Nanoro center in Burkina Faso [14,15] but similar GMTs to those found in the present study were found in Lambarene, Gabon and Lilongwe, Malawi. Half of the children in the current trial also received SMC. This is unlikely to have directly influenced the response to vaccination as this was given a month prior to SMC administration. Furthermore, there was no difference between the anti-CSP antibody response to booster immunisation between children who had received SMC in the previous transmission season and those who had not.
However, it is possible that reduction in the burden of malaria through administration of SMC enhanced the ability of RTS,S/AS01 E to induce more protective non-anti-CSP immune responses in the RTS,S/AS01 E combined group.
Relatively little is known about the impact of a booster dose of the RTS,S vaccine on either efficacy or on the immune response. In the first clinical trial of RTS,S undertaken in Africa, a booster dose of RTS,S/AS02A given to adults one year after three priming doses gave an approximately four-fold higher titer than that seen after priming and a partial return of protection against malaria infection [16]. However, in the phase 3 RTS,S/AS01 E trial, in which a booster dose was given 18 months after priming, the post booster anti-CSP antibody GMT in children who entered the trial when aged 5-17 months was 318.2 (95% CI 295.1, 343.0) EU/ml [14], substantially less than that seen after priming.
In the current trial, the response to a second booster dose was less than that seen after the first, raising a concern that further booster doses might result in a progressive decline in the immune response. A challenge study conducted in healthy adult volunteers showed a higher level of protection against challenge when a fractional dose of vaccine was given for the third priming and booster doses than in volunteers given three or four full doses [17] suggesting that the immunogenicity and efficacy of repeated seasonal vaccine booster doses of RTS,S/AS01 E might be improved if a fractionated dose was used. However, in a subsequent volunteer challenge study, no significant differences in efficacy were seen between groups in which various combinations of full and fractionated doses were explored [18].
The fact that the lower anti-CSP antibody response to a second booster dose compared to the first was not associated with any detectable loss of efficacy [7] is reassuring and suggests that the second booster dose may have increased the quality of the antibodies produced in some way, for example by altering their avidity and/or isotype, inducing protective antibodies to the C terminal of the CSP molecule, or antibodies binding to a Fcγ receptor promoting phagocytosis or activation of NK cells [19][20][21][22][23][24][25][26][27] or even to an increase in antibodies to blood stage antigens [28]. The RTS,S/AS01E vaccine induces a strong cellular immune response [29][30][31][32][33][34][35][36][37], including CD4 immune responses associated with protection, and we hypothesise that repeated boosting might enhance T cell mediated immune responses more markedly than that of the anti-CSP antibody responses. A systems biology approach is helping to characterise the relative importance of individual immune responses in the protection provided by RTS,S/AS01 E in human challenge studies [38,39]. A weakness of this study is that it did not include cell mediated immune assays but further serologic assays are now planned. Furthermore, children were also older after the second than after their first booster dose and this could have influenced their overall immune response and influenced comparisons between years.
As noted in most, but not all, previous human challenge or field trials with RTS,S/AS01 E a correlation was found between anti-CSP antibody titer and incidence of clinical episodes of malaria but this association could be influenced by confounding so this comparison is not sufficient to establish anti-CSP as a correlate of protection [40] . Moreover, the declining anti-CSP antibody response with successive boosters was not matched by a corresponding decline in efficacy, suggesting the potential protective role of other vaccine induce immunological responses, both humoral and cellular acting together.
In view of the encouraging efficacy results obtained with seasonal RTS,S/AS01 E vaccination given with SMC, the trial described in this paper is being extended until children reach the age of five years and the impact of third and fourth booster doses on the immune response and on efficacy against malaria is being followed.      Cox regression models for the pooled analysis over all three years of the study were adjusted for study year and the age of the child. The overall analysis aggregates person time and events for the tertiles defined separately in each year, and children above and below the specific threshold in each year of the study. * PYAR = person years at risk. Poisson regression models for the pooled analysis over all three surveys were adjusted for study year and the age of the child. The overall analysis aggregates the number of children testing positive for P. falciparum and the total number of children for the tertiles defined separately in each year, and for children above and below the specific threshold in each year of the study.