Safety and immunogenicity of varied doses of R21/Matrix-M™ vaccine at three years follow-up: A phase 1b age de-escalation, dose-escalation trial in adults, children, and infants in Kilifi-Kenya

Background Falciparum malaria remains a global health problem. Two vaccines, based on the circumsporozoite antigen, are available. RTS, S/AS01 was recommended for use in 2021 following the advice of the World Health Organisation (WHO) Strategic Advisory Group of Experts (SAGE) on Immunization and WHO Malaria Policy Advisory Group (MPAG). It has since been pre-qualified in 2022 by the WHO. R21 is similar to RTS, S/AS01, and recently licensed in Nigeria, Ghana and Burkina Faso following Phase 3 trial results. Methods We conducted a Phase 1b age de-escalation, dose escalation bridging study after a change in the manufacturing process for R21. We recruited healthy adults and children and used a three dose primary vaccination series with a booster dose at 1–2 years. Variable doses of R21 and adjuvant (Matrix-M ™) were administered at 10µgR21/50 µg Matrix-M™, 5µgR21/25µg Matrix-M™ and 5µgR21/50µg Matrix-M™ to 20 adults, 20 children, and 51 infants. Results Self-limiting adverse events were reported relating to the injection site and mild systemic symptoms. Two serious adverse events were reported, neither linked to vaccination. High levels of IgG antibodies to the circumsporozoite antigen were induced, and geometric mean titres in infants, the target group, were 1.1 (0.9 to 1.3) EU/mL at day 0, 10175 (7724 to 13404) EU/mL at day 84 and (following a booster dose at day 421) 6792 (5310 to 8687) EU/mL at day 456. Conclusion R21/Matrix-M™ is safe, and immunogenic when given at varied doses with the peak immune response seen in infants 28 days after a three dose primary vaccination series given four weeks apart. Antibody responses were restored 28 days after a 4 th dose given one year post a three dose primary series in the young children and infants. Registration Clinicaltrials.gov (NCT03580824; 9 th of July 2018; Pan African Clinical Trials Registry (PACTR202105682956280; 17 th May 2021).

antibodies to the circumsporozoite antigen were induced, and geometric mean titres in infants, the target group, were 1.1 (0.9 to 1.3) EU/mL at day 0, 10175 (7724 to 13404) EU/mL at day 84 and (following a booster dose at day 421) 6792 (5310 to 8687) EU/mL at day 456.Conclusions: R21/Matrix-M™ is safe, and immunogenic when given at varied doses with the peak immune response seen in infants 28 days after a three dose primary vaccination series given four weeks apart.Antibody responses were restored 28 days after a 4 th dose given one year post a three dose primary series in the young children and infants.Registration: Clinicaltrials.gov(NCT03580824; 9 th of July 2018; Pan African Clinical Trials Registry (PACTR202105682956280; 17 th May 2021).

Introduction
Malaria remains a major public health burden with 247 million malaria cases estimated in 2021, and 95% of these occurring in the African region 1 .Malaria can manifest as mild or more severe disease, with severe cases leading to an estimated 619,000 deaths per annum globally 1 .Vulnerable groups outside of early childhood include women with approximately one-third of the 40 million pregnancies in Africa affected.Malaria in pregnancy and early childhood is associated with anemia [2][3][4] , severe disease, poor birth outcomes (low birth weight, prematurity, and stillbirth), and has an adverse socio-economic impact.Until recently, public health measures targeting malaria control and elimination were primarily limited to long-lasting insecticidetreated bed nets, intermittent preventive treatment, and chemoprevention.A vaccine targeting one of the key life stages of the P. falciparum parasite is needed that dramatically reduces the incidence of blood stage infection clinical malaria (e.g., 90% over 12 months of follow up post immunization) 5 .Pre-erythrocytic vaccines target antigens from the P. falciparum sporozoite.They induce antibodies against surface antigens that clear sporozoites from the skin or bloodstream or block the invasion of hepatocytes.Of the 24 vaccine candidates in active clinical development, 38% target the pre-erythrocytic stage (CSP) with R21 being the most advanced in clinical development 6 .
Recommendation by the World Health Organisation (WHO) for the RTS, S/AS01 vaccine to be used in October 2021 and pre-qualification were both major milestones in advancing malaria control.However, additional measures including highly effective vaccines will be required to meet malaria control targets to reduce global malaria incidence and mortality by at least 90% by 2030 7 .R21 is a circumsporozoite protein (CSP) vaccine 8 , similar to RTS,S/AS01 and adjuvanted with Matrix M™, a naturally occurring saponin from Quillaja Saponaria.R21 was designed by the Jenner Institute, University of Oxford using the C-Tag affinity purification process (R21c) 9 at The Clinical Biomanufacturing Facility (CBF), UK.Addition of the C-tag was necessitated to overcome technical difficulties in the purification process.Evaluation of R21c in phase 1 trials in the United Kingdom and Burkina Faso demonstrated the R21c vaccine to be safe and immunogenic with less reactogenicity at comparable doses in West African adults 10 .Due to a change in the manufacturing process on transfer to Serum Institute of India, bridging phase 1 results in adults and children were required.The changes included removal of the additional four amino acids E-P-E-A, comprising the C-tag sequence and expression of the R21 protein in Hansenula polymorphaat rather than Pichia pastoris.Safety and immunogenicity data following the primary series of this phase 1b trial informed the progression and dose choice to ongoing phase 2 11,12 and 3 trials.Results reported here are following completion of the phase 1b, where a booster (4 th dose) was given up to two years following a three dose primary series and follow-up was for 12 months post boosting.

Study design and participants
We conducted a phase 1b, open-label, age de-escalation, dose escalation, trial to evaluate the safety and immunogenicity of R21/Matrix-M™ in healthy Kenyan adults; aged 18-45 years (group 1) then children aged 1-5 years (group 2) and finally infants 5 months to ˂1 year (group 3).Participants were recruited from Junju village within the Kilifi Health and Demographic Surveillance System (KHDSS) 13 .
The trial was conducted in accordance with the International Conference on Harmonisation (ICH)-Good Clinical Practice (GCP) guidelines 14  Participants were given ample time to consider participation.Informed consent was documented by signature or thumbprint by all participating adults and parents or authorised guardians of children and infants before screening for eligibility.Enrolment was after a test of understanding where a score of >90% was required prior to fully informed consent.
Stakeholder engagement was conducted, and all approvals were in place prior to recruitment.Recruitment occurred following community meetings and in participants' homes.All participants were assessed with a screening questionnaire, clinical assessment, safety bloods, and a viral (Human Immunodeficiency Virus-HIV, Hepatitis B, and C) and haemoglobinopathy screen to determine enrolment eligibility.Urine samples were taken in adults to assess for diabetes in this cohort and pregnancy in women.Full trial screening and eligibility criteria are included in the trial protocol 15 .
R21 is a pre-erythrocytic protein-in-adjuvant malaria vaccine candidate.It is adjuvanted with Matrix-M™ based on the circumsporozoite protein (CSP) produced by using recombinant Hepatitis B surface antigen (HBsAg) particles expressing the central NANP repeat and the C-terminus.It spontaneously forms a particle, but without the need for excess Hepatitis B antigen required for particle formation with RTS, S/AS01.

Study procedures
Potential participants were screened until the target enrolment was reached based on eligibility criteria including age.Participants were sequentially allocated to one of nine age de-escalating and dose-escalating groups.All participants received a three-dose primary series of vaccinations four weeks apart followed by a 4 th dose.The 4 th dose was administered two years after completion of the primary vaccinations in adults and children and one year after primary series in infants.All four vaccinations were at the same dose.Follow-up was 12 months after the 4 th dose in all groups.R21 was thawed to room temperature then administered intramuscularly after mixing with Matrix-M™.One of three doses was administered: 10µgR21/50µg Matrix-M™ (Groups 1A, 1B, 2B, 3B or 3D), 5µgR21/25µg Matrix-M™ (2A, 3A and 3C) or 5µgR21/50µg Matrix-M™ (3E).Vaccination was into the non-dominant deltoid for adults and children and the right arm for infants.Participants were observed for one hour post-vaccination and adverse events solicited in the week following vaccination using diary cards.Diary card completion was by field workers in participants' homes for the primary series and over the phone following 4 th dose due to COVID-19 pandemic restrictions.During the pandemic, following an amendment, participants were invited to attend the study clinic in the event of an adverse event (AE) of severity grade 3 or higher, or where clarification was required of AE grading.Unsolicited adverse events were collected in the 28 days after vaccination.Post-enrolment follow-up visits were conducted on days 28, 42, 56, 84, 238, 421, 456, 786, 814 and 1151.Additionally, safety bloods were taken on days two and seven post-primary series vaccinations.Safety oversight was by two local safety monitors and an independent Data Safety Monitoring Board (DSMB).Decisions to progress within and across groups were determined by the DSMB.Following an initial introductory meeting, the DSMB had six scheduled meetings.Scheduled reviews included cumulative adverse events for all vaccinees.Each group had a sentinel cohort with infants receiving variable doses and the adult doses fixed.DSMB meeting 1 occurred 72 hours after the first participant in group 1A was vaccinated.DSMB meeting 2 occurred no earlier than three weeks after the first dose was administered to the first participants in group 1B, DSMB meeting 3 occurred 10 days after the first dosing of group 2A, DSMB meeting 4 at three weeks after the dosing of the first participant in group 2B.DSMB meeting 5 was 10 days after the first vaccination in group 3A and DSMB meeting 6 was 10 days after the first dose in group 3B.Groups 3C, 3D, and 3E were vaccinated concurrently 10 days following the first vaccination in group 3B and review of cumulative safety data.One unscheduled DSMB meeting was held to review a rising alanine transaminase (ALT) in an adult.Stopping rules included one or more participants experiencing a SAE related to the investigational products or the occurrence of a suspected unexpected serious adverse reaction (SUSAR).
IgG enzyme-linked immunosorbent assay (ELISA) Anti-NANP antibodies were measured by ELISA just prior to and 28 days post each vaccination (Baseline, days 28, 56, 84, 421,456,786, 814).IgG responses were also ascertained seven days post first vaccination (Day 7).Additional immunology bleeds occurred 12 and 24 months post primary series (days 421 and 786) and one year post 4 th dose (days 786 and 1151).Additional immunogenicity timepoints were assessed at 42 days after first vaccination and six months post primary series (Day 238).ELISA methods used were as previously described 10,16 .Briefly, 96-well plates (NUNC) were coated with a synthetic peptide comprising 6 repeats of the amino acid sequence NANP (ProImmune) at a concentration of 0.2 µg/mL.After incubation and washing with PBS with 0.05% Tween, wells were blocked with casein (Sigma) for at room temperature for 1 hour.After washing, samples (diluted at between 1:100 and 1:5000) were added in duplicate and incubated at room temperature for 2 hours.After washing, alkaline phosphatase conjugated goat anti-human IgG (Sigma) secondary antibody was added at a dilution of 1:1000 and incubated at room temperature for 1 hour.After washing, P nitrophenyl phosphate (Sigma) and diethanolamine substrate (Pierce) buffer was added and the plate read at 405nm using Gen 5 software.A reference pool of positive serum formed a standard curve on each plate and was used to calculate ELISA Units (EU) for each.An internal control was included on each plate to standardise between assays.

Objectives and outcomes
The primary objective was to evaluate the safety and tolerability of R21 with the adjuvant Matrix™ in healthy adults then children and finally infants.The secondary objectives were to assess the cellular and humoral immunogenicity of R21 with the adjuvant Matrix-M™ in healthy adults, children, and infants.Outcomes included the occurrence of solicited local and systemic reactogenicity in the seven days following vaccination, unsolicited adverse events in the 28 days after vaccination, changes in baseline laboratory measures and the occurrence of SAEs throughout the study.Secondary outcomes included the comparison of antibody responses and longevity of responses across dose groups up to 25 months following booster vaccinations.

Statistical analysis
Descriptive statistics were used to summarise all parameters.The analyses were based on an intention-to-treat population, which included all participants who received at least one vaccination dose.Safety data were summarised as the number of participants that experienced any event after vaccination in the study group and as a percentage of the total number of participants within the group.Unsolicited AEs were categorised according to the Medical Dictionary for Regulatory Activities preferred terms (PT).We estimated the geometric means of the IgG antibody titres (GMTs) against the NANP at multiple time points, together with their 95% CIs separately for each study group.Log-transformed anti-NANP IgG titres at day 84 were compared across age, dose and age-dose groups using one-way analysis of variance (ANOVA).Significant ANOVA results were followed by a Tukey post-hoc test.To assess the impact of boosting we estimated GMT fold change (GMFC) at days 421 and 456 relative to day 84 and performed a paired t-test within each group.Efforts were made during the study to minimise missing data occurrence.At the analysis stage, numbers (percentages) of incomplete data were reported.Missing data were not imputed in any way.Analyses were performed using Stata version 17.0.Stata Statistical Software: Release 17. College Station, TX: StataCorp LLC, RRID:SCR_012763 was used (http://www.stata.com).R software version 3.6.2(R Core Team, 2019, https://www.r-project.org) was also used.Figures were produced using GraphPad Prism version 9.4.0 (GraphPad Software, San Diego, California USA, www.graphpad.com).

Results
The study was conducted between 28 th April 2019 and 14 th June 2022.Primary series and booster vaccinations were complete by December 2019 and June 2021, respectively.The trial protocol was amended to allow for a delayed fourth dose in groups 1 and 2 due to the COVID-19 pandemic.
One hundred and eighty-eight participants were screened, 97 were excluded, and 91 were enrolled and received at least one vaccination (Figure 1).Twenty adults were enrolled in group 1, 20 children to group 2, and 51 infants to group 3. Eighty-eight participants completed primary series vaccinations, with 18 participants in group 1, 19 in group 2, and 51 in group 3.In group 1, two participants were withdrawn before their second vaccination.One participant moved out of the study area and an undisclosed history of substance abuse was uncovered in the second participant.One was excluded after the fourth vaccination as they moved out of the study area.In group 2, one child was withdrawn following second vaccination as the child was no longer resident in the study area.One child was excluded after booster vaccination due to moving out of the study area.In group 3, three infants were withdrawn before their fourth vaccination: two had moved out of the study area and one withdrew consent.After the fourth vaccination, two infants withdrew consent not due to adverse

Safety assessments
Solicited local adverse events.A total of 1077 adverse events were reported, with each participant reporting at least an AE.One hundred and forty-five AEs were reported for group 1, 199 for group 2 and 733 for group 3 (Table 2).
Injection site pain occurred in 12 (60%) of 20 participants in group 1 and two (10%) of 20 participants in group 2 after the first vaccination (Table 3).Solicited injection site pain occurred in 13 (72%) of 18 participants in group 1, five (25%) of 20 participants in group 2, and three (6%) of 51 participants in group 3 after the second vaccination.Pain occurred in two *Data are number (percentage) participants in each group at vaccination 1, 2, 3 and 4. A dash (-) means that a particular event was not assessed for that group.All solicited local and systemic adverse events were collected for 7 days after each vaccination.In group 1, 20 participants received the first vaccination dose, 18 participants received a second and a third dose, and 15 participants received a fourth dose.In group 2, 20 participants received the first and a second vaccination dose, and 19 participants received a third and a fourth dose.51 participants in group 3 received the first, a second and a third vaccination dose, and 48 participants received a fourth dose.
(11%) of 18 participants in group 1 and three (16%) of 19 participants in group 2 after the third vaccination.
No pain was reported in group 1 after the fourth vaccination dose.Participants reporting solicited pain at the injection site decreased as vaccinations progressed in groups 2 and 3. Other local AEs occurring in small proportions of adults (group 1) were induration, injection site itch and injection site warmth (Table 3; Table S1 3).Most systemic solicited AEs (73%, n=8) in group 1 were classified as related to the investigational product.Fever was the most frequent solicited systemic adverse event in groups 2 and 3, accounting for 20 (71%) of 28 solicited AEs in group 2 and 59 (76%) of 78 AEs in group 3, with the proportion of participants reporting fever increasing with the vaccination number.One participant in group 2 reported severe fever after the third vaccination and one participant in group 3 reported severe fever after the second, third and fourth vaccinations; all other adverse events were graded as either mild or moderate (Table 3; Table S1 15 ).Eight (73%) solicited systemic AEs in group 1 and all AEs in groups 2 and 3 were self-resolving.

Laboratory safety bloods. Transient changes in safety bloods
were seen during the study.All clinically significant results had settled at the point of the final study visit except in one participant who was withdrawn due to substance misuse (alcohol) associated with high ALTs.

Unsolicited adverse events within 28 days of vaccination.
There was a total of 396 unsolicited AEs (36 in group 1, 74 in group 2, and 286 in group 3) in the month following each vaccination.Abnormal safety laboratory results were found in 22.5% (89/396).Three hundred and sixty-eight (92.9%) of the unsolicited AEs were mild in nature.A total of 94.2% (373/396) were classified as unrelated to the investigational product and almost all (389/396) resolved.Twenty-seven cases of malaria, none of which were severe, were reported within 28 days after vaccination, while 104 malaria cases were reported throughout the study across groups.
Serious adverse events.There were two serious adverse events during the study requiring hospitalisation, neither related to vaccination.The first SAE was in a 13-month-old who was diagnosed with a lower respiratory tract infection.The second SAE was also a lower respiratory tract infection following seven days of symptoms (cough, intermittent fevers associated with one febrile convulsion, and shortness of breathe).Both resolved uneventfully.There was one pregnancy reported (conception approximately 3.5 months post third vaccination) that resulted in a healthy female infant as assessed at birth, four and seven months of age.

NANP-specific IgG responses
Peak NANP-specific IgG responses were seen at day 84 (28 days following the primary series of vaccinations) across dose and age groups (Figure 2).and age-dose (F=13.9,df=5, 81; p<0.001).However, when the analysis by dose was restricted to the infant groups, these differences by dose were not statistically significant by the Tukey post-hoc test (Table S2 15 ).All groups elicited high GMTs 28 days post primary series which declined significantly prior to boosting one and two years later (days 421 and 786) except in group 2A (Table S3 15 ).At day 421 GMFC from day 84 were 0.25 (95% CI 0.15-0.44;p<0.001) in group 1 and 0.12 (95% CI 0.08-0.18;p<0.01) in group 3 (Table S3 15 ).At day 456 although the GMFC was 0.7 (95% CI 0.5 to 0.9) in group 3, for individual dose groups there were no significant decreases in GMTs when compared to day 84 (Table 4).
After day 28, Anti-NANP IgG reached their lowest levels at days 421 and 786 across groups and doses (Figure 2).The antibody kinetics in Figure 2 show substantial immune responses to the first vaccination at day 28 for infants and children which peak at day 84 then wane at days 421 and 786.Following a fourth dose of R21/Matrix-M™, however, anti-NANP antibody levels were restored at day 456 and 814 (28 days post 4 th dose) in groups 2 and 3 [Figure 2].The GMTs at day 456 and 814 rose post 4 th dose to levels similar to those seen at day 84 for all age-dose groups except groups 1A/B and 2A where a decline in GMTs was observed p=0.002 (Table 4).Immune responses were not assessed in 48 (53%) of 91 participants six months post primary series (day 238).A total of 45/48 of these were due to pandemic restrictions in group 3 infants and three withdrawals.

Discussion
R21/Matrix-M™ is safe when given as a three dose primary series with a 4 th dose one to two years later in adults, young children, or infants at a dose of :10µgR21/50µg Matrix-M™, 5µgR21/25µg Matrix-M™ or 5µgR21/50µg Matrix-M™.There were no serious adverse events related to the investigational product and most solicited adverse events were mild to moderate and self-limiting.Systemic adverse events following vaccination were uncommon in Kenyan adults and similar to profiles seen in Burkinabe adults vaccinated with three doses of R21c 10 .The frequency of adverse events was inversely related to the number of doses administered (Table 3).Pain was the most frequent AE seen in adults, particularly after the 1 st and 2 nd vaccinations.In children, pain and temperature were the most frequent AEs solicited.In vaccinated infants, local adverse events in the seven days following vaccination were infrequent with mild pain being most common in the 1-5 years age group.Mild to moderate temperatures were the most common systemic adverse events irrespective of dose in those aged under five years.AEs were most frequently seen in the infant groups but were mostly mild to moderate and similar in frequency across age-dose groups.
R21 elicited high anti-NANP IgG GMTs following a three dose primary series for all age groups and R21/Matrix-M™ doses received.GMTs were inversely related to age irrespective of dose administered.In the target population, children's immune responses were optimal in those vaccinated at an earlier age (5 to ˂12 months) with three dose primary series 28 days apart and boosting at 12 months post 3 rd dose.The optimal dose that induced the maximum response as measured by anti-NANP IgG response was seen in the infants vaccinated with 5µgR21/50µg Matrix-M™.Anti-NANP IgG GMTs in group 3 when 5µgR21/50µg Matrix-M™ was administered were 13,279 (7366-23,942) at day 84 and 6661 (4558-9735) at day 456, the highest in any dose or age group.Infants vaccinated with the higher adjuvant dose had higher GMTs post primary series and booster irrespective of R21 protein concentration.Infants vaccinated with half the protein/ adjuvant dose (5µgR21/25µg Matrix-M™ (3A/C) had almost 50% the response seen (GMTs) compared to infants vaccinated in Group 3E (5µgR21/50µg Matrix-M™) at day 84.GMFC at day 456 however in groups 3E and 3A/C were comparable; 0.7 (95% CI 0.4 to 1.3) and 0.7 (95% CI 0.4 to 1.2) respectively (Table 4).Given that 10µgR21/50µg Matrix-M™ elicited lower GMTs at day 84 than 5µgR21/50µg Matrix-M™ in the infant groups with no appreciable difference in adverse event profile given the group We observed a decay in anti-NANP IgG titres pre-boosting (days 421 and 786) most notable in the adults and children who were sampled two years post their 3 rd vaccination.There was, however, evidence of a boosting response when pre and post boosting GMTs were compared with GMTs rising seven-fold in groups 1 and 2.Although the boosting response in adults was modest, boosting was more immunogenic in children and in infants.A three dose primary series regime with one or more boosters may be an effective approach for sustained protection in early childhood.Further studies assessing antibody function and vaccine efficacy will help determine whether the apparent lower boosting in adults indicates reduced responsiveness due to multiple previous exposure to malaria.There were no solicited adverse events following the 4 th vaccination in adults which may be in keeping with a sub-optimal immune boosting response observed at day 84.Baseline antibodies were detectable in group 1 adults.Anti-NANP IgG GMTs at day seven were lower in the infants than the children and adults, this could be due to an anamnestic response in the 1 to 5 year olds and adults compared to the infants.
Retention of study participants was good despite the COVID-19 pandemic with only one participant lost to follow-up although eight were withdrawn as they no longer fulfilled the eligibility criteria.Limitations included the small size of the study and the open label design.Due to the COVID-19 pandemic restrictions the methods used for collection of data were also modified at the point of boosting to minimise risks of SARS-CoV-2 transmission to participants and staff and to comply with public health directives.Moderate and severe AEs are unlikely to have been missed due to daily telephone contact in the week post immunization and clinic attendance for grade 3 or 4 AEs.All SAEs were captured in hospital as recruitment was in the KHDSS where clinical episodes are linked to community members.

Conclusions
These data demonstrate that R21/Matrix-M™ is safe and should be evaluated in larger randomised controlled clinical trials to ascertain further safety, reactogenicity, immunogenicity and to assess its efficacy in a broader African paediatric and adult population.i) R21/Matrix-M™ is safe -Self-limiting adverse events were reported relating to the injection site and mild systemic symptoms; ii) R21/Matrix-M™ is immunogenic -High levels of IgG antibodies (anti-NANP peptides) were induced when the vaccine was given at varied doses, with the peak immune response seen in infants 28 days after a three-dose primary vaccination series given four weeks apart; iii) One of the most relevant pieces of information is that the antibody responses were restored 28 days after a 4th dose given one year post a three-dose primary series in young children and infants.
Minor comments for authors' consideration: Introduction: Please add the malaria data provided by the World Malaria Report 2023. 1.

Methods:
The authors should explain why only 0.2 µg/mL was used to coat plates and provide the purity levels of the NANP peptides used.

Results:
The authors should explain if these anti-CSP antibodies are inhibitory in in vitro assays and if they recognize the C-terminal portion of PfCSP.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

Are sufficient details of methods and analysis provided to allow replication by others? Partly
If applicable, is the statistical analysis and its interpretation appropriate?I cannot comment.A qualified statistician is required.

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Malaria vaccine (P.vivax) I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.clinical trials to ascertain further safety, reactogenicity, immunogenicity and to assess its efficacy in a broader African paediatric and adult population".

Introduction
Here the focus is on children and adults, and in the abstract, we have adults, children and infants and other part of the paper young children etc. Can we ensure consistence of the above on the manuscript?

Methods
The authors should include a brief description of the selection of study participants.Was this done in the village or community units etc? Why a sample size of 20, 20 and 51 participants?Why healthy adults of 18-45 years ( not a much narrow age like <18 or close to 18 years eg 18-24 years) with a sample size of 20? Table 1:-group females 75% and males 5% this should be corrected The age distribution of 18-45 (median of 27 years) of the study participants should be considered as a limitation or may bring some biases

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Epidemiology of malaria I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 2 .
Figure 2. Geometric mean for NANP IgG antibody titres by age at dose administered.
Irene S SoaresDepartment of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil The most advanced P. falciparum circumsporozoite protein (PfCSP)-based malaria vaccine, RTS,S/AS01 (RTS,S), confers partial protection but with antibody titers that wane relatively rapidly, highlighting the need to elicit more potent and durable antibody responses.This malaria vaccine was recommended by the WHO for use in African countries in October 2021.R21/Matrix-M™ is similar to RTS,S/AS01 and was recently licensed in Nigeria, Ghana, and Burkina Faso following Phase 3 trial results.The manuscript by Sang et al. reports on a phase 1b age de-escalation, dose-escalation trial in adults, children, and infants in Kilifi, Kenya.The authors recruited healthy adults and children and used a three-dose primary vaccination series with a booster dose at 1-2 years.Variable doses of R21 and adjuvant (Matrix-M™) were administered at 10µg R21/50 µg Matrix-M™, 5µg R21/25µg Matrix-M™, and 5µg R21/50µg Matrix-M™ to 20 adults, 20 children, and 51 infants.The study is very carefully planned and executed.The immunological methods of analysis are up to date and provide important information.
. Ethical approval was obtained from the Kenya Medical Research Institute (KEMRI) Scientific and Ethics Review Unit (CGMR-C/116/3711) on the 26 th of September 2018 and the Oxford Tropical Research Ethics Committee (OxTREC-6-18) on 27 th September 2018, and regulatory approval from the Pharmacy and Poisons Board of Kenya (PPB/ECCT/18/12/01/2019) on 18 th March 2019.

Table 1 . Demographic and baseline characteristics.
event, two infants moved out of the study area, and one infant was lost to follow-up.All enrolled participants were included in the safety and immunogenicity analyses.Three in four of those in group 1 were males.Groups 2 and 3 were generally balanced with respect to gender.The median age at enrolment was

Table 2 . Summary of all adverse events for consented subjects.
[1]Subjects who experience one or more AEs or SAEs are counted only once[2]Subjects are counted only once within a particular severity grade *Percentages are based on number of AEs reported in each study group **Percentages are based on N for each treatment arm

Table 4 . Immunological outcomes by age, dose, and age-dose category.
Tukey post-hoc test is presented in TableS215 .†GMFC is anti-NANP geometric mean titre fold ratio at day 456 relative to day 84.† †P-values comparing anti-NANP IgG titres at days 84 and 456 for each age, dose and age-dose category.