Safety and immunogenicity of BK-SE36/CpG malaria vaccine in healthy Burkinabe adults and children: a phase 1b randomised, controlled, double-blinded, age de-escalation trial

Background BK-SE36/CpG is a recombinant blood-stage malaria vaccine candidate based on the N-terminal Plasmodium falciparum serine repeat antigen5 (SE36), adsorbed to aluminium hydroxide gel and reconstituted, prior to administration, with synthetic oligodeoxynucleotides bearing CpG motifs. In healthy Japanese adult males, BK-SE36/CpG was well tolerated. This study assessed its safety and immunogenicity in healthy malaria-exposed African adults and children. Methods A double-blind, randomised, controlled, age de-escalating clinical trial was conducted in an urban area of Ouagadougou, Burkina Faso. Healthy participants (n=135) aged 21-45 years (Cohort 1), 5-10 years (Cohort 2) and 12-24 months (Cohort 3) were randomised to receive three vaccine doses (Day 0, 28 and 112) of BK-SE36/CpG or rabies vaccine by intramuscular injection. Results One hundred thirty-four of 135 (99.2%) subjects received all three scheduled vaccine doses. Vaccinations were well tolerated with no related Grade 3 (severe) adverse events (AEs). Pain/limitation of limb movement, headache in adults and fever in younger children (all mild to moderate in intensity) were the most frequently observed local and systemic AEs. Eighty-three of BK-SE36/CpG (91%) recipients and 37 of control subjects (84%) had Grade 1/2 events within 28 days post vaccination. Events considered by the investigator to be vaccine related were experienced by 38% and 14% of subjects in BK-SE36/CpG and control arms, respectively. Throughout the trial, six Grade 3 events (in 4 subjects), not related to vaccination, were recorded in the BK-SE36/CpG arm: 5 events (in 3 subjects) within 28 days of vaccination. All serious adverse events (SAEs) (n=5) were due to severe malaria (52-226 days post vaccination) and not related to vaccination. In all cohorts, BK-SE36/CpG arm had higher antibody titres after Dose 3 than after Dose 2. Younger cohorts had stronger immune responses (12–24-month-old > 5-10 years-old > 21-45 years-old). Sera predominantly reacted to peptides that lie in intrinsically unstructured regions of SE36. In the control arm, there were no marked fold changes in antibody titres and participants’ sera reacted poorly to all peptides spanning SE36. Conclusion BK-SE36/CpG was well-tolerated and immunogenic. These results pave the way for further proof-of-concept studies to demonstrate vaccine efficacy. Clinical trial registration https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=1921, PACTR201701001921166.


Background information on the BK-SE36 vaccine
There is a strong justification for blood-stage vaccines since protection by the antisporozoite RTS,S/AS01 vaccine candidate, currently the most advanced malaria vaccine, is not complete and long lasting.Asexual-stage parasites cause symptomatic malaria and blood-stage antigens are targets of acquired immunity.Controlling parasite density may reduce disease severity and P. falciparum gametocyte density, hence, potentially reducing disease transmission.
The P. falciparum serine repeat antigen-5 (SERA5) is an abundant blood-stage antigen secreted in large amounts into the lumen of the parasitophorous vacuole.It plays an essential role in the parasite life cycle and was among the first physiological substrate identified for a serine protease implicated for parasite egress.A recombinant form of SERA5 N-terminal domain (SE36) was selected for clinical development on the basis of the following: (i)epidemiological studies showing high antibody titres that inversely correlate with malaria symptoms and severe disease; (ii) in vitro studies demonstrating induction of antibodies that are inhibitors of parasite growth, exert antibody-dependent complement-mediated lysis of schizonts, or antibody-dependent monocyte-mediated parasite growth inhibition; and (iii) animal studies demonstrating protection against P. falciparum challenge in non-human primates.
SE36 was prepared under current Good Manufacturing Practice (cGMP) constraints and formulated with aluminium hydroxide gel (AHG) to yield BK-SE36.The safety and immunogenicity of BK-SE36 was demonstrated in a phase 1a trial in malaria naive Japanese adults; and in a phase 1b trial conducted in healthy subjects aged 6-32 years from a malaria endemic area in Northern Uganda.The trial promising results justified the conduct of a phase 1b trial of BK-SE36 in younger cohorts aged from 1 -5 years old in Burkina Faso.The trial aimed to test the immune response in younger cohorts that has so far not been included in BK-SE36 malaria vaccine clinical trials.The trial results provided additional data on safety and immunogenicity.

Trial rationale
The immune response to BK-SE36 may still be improved with the use of DNA sequences containing CpG motifs that can selectively promote cellular and/or humoral immune responses.The phase 1b trial in Uganda demonstrated low seroconversion in malaria exposed adults.The use of CpG K3 ODN may be one approach to broaden immune responses as a robust immune response may overcome immune tolerance or help immunocompromised individuals through the activation of multiple innate receptors that could target redundant pathways of innate responsiveness.A phase 1a clinical trial using BK-SE36/CpG was conducted in healthy adults in Japan where the vaccine was deemed safe and elicited antibody titres 3-to 4-fold higher as compared to BK-SE36 alone.
This study assessed BK-SE36/CpG safety and immunogenicity in a population exposed to malaria.

Objectives Primary
Assess the safety and reactogenicity of 3 doses of malaria vaccine candidate BK-SE36 (100 µg SE36 + aluminium hydroxide gel) mixed with TLR9 ligand (CpG K3 ODN), via the intramuscular route, in healthy African adults and children exposed to the parasite P. falciparum.The adverse event grading for clinical abnormalities will be done according to the Brighton collaboration guidelines (www.brightoncollaboration.org).The primary objective is restricted to serious or severe adverse events.

Secondary
• Assess the humoral immune response to the vaccine antigens administered intramuscularly by measuring the level of IgG in all subjects • Assess the safety and reactogenicity of the BK-SE36/CpG vaccine restricted to mild or moderate adverse events.

Exploratory
• Assess the quality of the humoral immune response on all participants by mapping the protective epitope(s) in SE36; Rationale for the number of doses: 2 vaccinations (28 days apart) + 1 booster dose.Immunogenicity results from a Phase 1a trial in Japan with BK-SE36/ CpG suggest that 2 administrations was enough for 100% seroconversion in malaria naïve population.Antibody titres were 3-to 4-fold higher than BK-SE36.However, a booster dose may be needed in malaria endemic areas and thus, inclusion of a booster dose at week 16 is intended to increase immunogenicity in the malaria exposed population.
Safety and immunogenicity of doses 21-days apart was confirmed in a phase 1a trial.
A dosing interval of 28-days would be similar to schedules of inactivated vaccines.

Rationale for the age cohort:
As no safety data exist in malaria endemic areas for the BK-SE36/CpG formulation, a cautious age de-escalation trial is planned keeping the age limit to 12 months.The Expanded Programme on Immunisation (EPI) vaccines are given up to 11 months, children below 12 months receiving EPI vaccines will be excluded to avoid any vaccine interference.

GO/No GO
The age de-escalation GO/No GO criteria will be based on safety criteria: The No GO stopping rule will be: any SAE (Serious Adverse Event) possibly, probably or definitely related to vaccination or 50% or more of subjects having a Grade 3 adverse reaction persisting at Grade 3 for > 48 hours within 7 days following each vaccine dose (day of vaccination and 7 subsequent days).The threshold of 50% corresponds to a consensus decision in accordance with the African Malaria Network Trust (AMANET) Malaria vaccine guidelines and will be applied for the start of the vaccination of cohort 2 and 3.
For the age de-escalation to cohort 2, the Local Safety Monitor (LSM) and the Principal Investigator (PI) will review the safety report on data collected within 7 days after the third vaccination of cohort 1 and will authorise the age de-escalation if there is no safety concern.If there are any safety concerns, the unblinded safety report will be submitted to the members of the Independent Safety Monitoring Committee (ISMC) for their individual recommendations to the sponsor representative.
For the age de-escalation to cohort 3, a safety report will be prepared after the second vaccination of cohort 2 on the cumulative safety data from cohort 1 and cohort 2 collected within 7 days after the second vaccination of cohort 2. The blinded safety report will be directly submitted to the ISMC for their recommendation to the sponsor representative.
If any vaccinee experiences an SAE related to vaccination, then all the immunisations of the subjects in this group will be stopped.The vaccination will only be resumed upon the decision of the sponsor according to the recommendation of the LSM, PI and ISMC.
All safety data will be shared with the sponsor and sponsor's representative.
GO/No GO criteria for immunogenicity: There is no immunogenicity stopping rule with respect to moving ahead from cohort 1 to cohort 2 and from cohort 2 to cohort 3.

Specific inclusion criteria for cohort 1 (adults 21-45 years old)
• Female or male subjects aged 21 to 45 years inclusive at the time of the first vaccination • Residing within the Ouagadougou health region and planning to stay for the study duration • General good health based on medical history and clinical examination • Written informed consent obtained before any trial procedure • Female and male volunteers practicing/willing to practice contraceptive methods recommended by the national health system for at least four (4) weeks before the first vaccination (for female participants only) and up to four (4) weeks after the third vaccination.

Specific inclusion criteria for cohort 2 (children 5-10 years old)
• Female or male subjects aged 5 to 10 years inclusive at the time of first vaccination.

Specific inclusion criteria for cohort 3 (children 12-24 months)
• Female or male subjects aged 12 to 24 months inclusive at the time of first vaccination.

Common inclusion criteria for cohorts 2 and 3
• Residing within the Ouagadougou health region and planning to stay for the study duration • Appear to be in generally good health based on malnutrition index and clinical and laboratory investigations • Signed or thumb-printed informed consent obtained from the parent(s)/guardian(s) of the child.Where parent(s)/guardian(s) are not literate, the consent form will be countersigned by an impartial witness • Subjects for whom the investigator believes that their parents/guardians can and will comply with the requirements of the protocol (e.g. return for followup visits) will be enrolled in the study.The trial period for each subject is 14 months (12 months + 8 weeks screening).

Non-inclusion criteria
Specific non-inclusion criteria for cohort 1 • History of anaphylaxis or known severe hypersensitivity to any of the vaccine components (adjuvant or antigen or excipient) • Administration of gamma globulin: 4 weeks prior to and after each vaccination; if administration is necessary during the study period, the volunteer will be withdrawn from the study • Planned administration/administration of a vaccine not foreseen by the study protocol within 30 days of the first dose of vaccine(s) • Use of any investigational or non-registered drug or vaccine within 30 days preceding the first dose of study vaccine, or planned use during the study period • Current participation in another clinical trial, or within 12 weeks of this study • Any other finding which in the opinion of the investigators would increase the risk of an adverse outcome from participation in the trial or result in incomplete or poor quality data.

Holding and stopping rules
The trial may be placed on safety hold for the following reasons: • On advice of the ISMC • On advice of the LSM.
• On advice of the PI.
• One or more participants experience a serious adverse event (SAE) that is determined to be possibly related to BK-SE36/CpG vaccine administration.• In case of an SAE possibly related to BK-SE36/CpG vaccination, the immunisation of the remaining subjects will be immediately (but not finally) discontinued until the decision of the sponsor or the sponsor's representative according to the LSM, PI and ISMC recommendations.The LSM and PI will arrange a meeting within 48h following the SAE to assess whether the event was unrelated or related to the vaccine.They will recommend stopping, or pausing or continuing the immunisation to the ISMC.The ISMC members can individually inform the sponsor's representative by email or at its discretion call for a TC to discuss any safety concerns within 48h of receipt of the report from LSM and PI.The final decision on stopping, or pausing or continuing the vaccination will belong to the sponsor taking into account recommendations of the LSM, PI and ISMC.
The trial will be stopped if the following occurs:

Number of scheduled Visits
11 clinic visits on site (+ 18 contact visits).

Follow-up duration
Total duration: 12 months following the first vaccination.

Sample size justification
The sample size is calculated to address the primary objective (safety).In each cohort (N=29), if the underlying risk of an SAE is 10%, then the probability of seeing at least one SAE is 95%.If the risk is 5% then the probability of seeing at least one SAE is 77%.With 87 vaccinees the probability of detecting at least one SAE is >99% if the risk is 10%, is 99% if the risk is 5% and 58% if the risk is 1%.

Endpoints Primary
• Occurrence of solicited adverse events considered related to vaccination (possibly, likely or definitely) and that are severe (Grade 3) within 7 days following vaccination (day of vaccination and 7 subsequent days) • Occurrence of unsolicited adverse events (AEs) considered related to vaccination (possibly, likely or definitely) and that are severe (Grade 3) within 28 days following each vaccination (day of vaccination and 28 subsequent days), according to the Medical Dictionary for Regulatory Activities (MedDRA classification) • Occurrence of serious adverse events (SAEs) at any point during the study period, according to the Medical Dictionary for Regulatory Activities (MedDRA classification) Secondary • Anti-SE36 protein IgG antibody titre at Day 0, Week 4, 8, 16, 20 and 52.
• Occurrence of solicited adverse events reactions considered related to vaccination (possibly, likely or definitely) and that are mild or moderate (Grade 1 or Grade 2) within 7 days following vaccination (day of vaccination and 7 subsequent days) • Occurrence of unsolicited adverse events (AEs) considered related to vaccination (possibly, likely or definitely) and that are mild or moderate (Grade 1 or Grade 2) within 28 days following each vaccination (day of vaccination and 28 subsequent days), according to the Medical Dictionary for Regulatory Activities (MedDRA classification)

Exploratory
• Determining protective epitopes on SE36 one month after booster dose (Week 20) • Incidence of clinical malaria from Day 56 to Day 365

Primary evaluation criteria
The safety profile will be assessed by the following criteria: • Immediate reactogenicity (reactions within 60 minutes after each vaccination) • Local and systemic reactogenicity measured from Day 0 to Day 7 after each vaccination • Any unsolicited adverse event within one month after each vaccination • Any Serious Adverse Event (SAE) occurring throughout the study duration starting from the first immunisation.The relationship of the adverse event to the vaccine will be established by the investigator as definitely, probably, possibly, unlikely related, or not related.

Interim Analysis
In order to obtain a preliminary assessment of the safety and immunogenicity of the vaccine without waiting until the end of the trial, an analysis of data up to and including visit 27 (28 days after the booster dose) will be performed for each cohort, after cohort 3 has completed visit 27.A statistician independent of the trial will perform the analysis to maintain the blinding of trial personnel.The statistician will generate simple, summary descriptive statistics by trial arm as follows: -numbers of adverse events possibly, probably or definitely related to vaccination, -the proportions of participants experiencing such events -the proportions of participants with detectable anti BK-SE36 IgG titres -geometric mean titres among participants with detectable titres No formal statistical testing will be performed to compare the trial arms

Final analysis
The safety analysis population will include all participants who received at least one injection.Data from individuals who do not complete the full follow-up period will be included up until drop out/withdrawal.Immunogenicity analyses will include all subjects who received at least one injection and will be performed on all available data at each-time point.Statistical analyses will be exploratory, as the design is not powered to demonstrate statistically significant differences in outcomes.Categorical variables will be summarised by trial arm as percentages and 95% confidence intervals.Geometric means and 95% confidence intervals of the antibody titres will be determined by trial arm.Continuous variables other than titres and concentrations will be summarised by trial arm and means, SEM, Medians, Minimums, Maximums, and inter-quartile ranges reported.The proportion of subjects that received the vaccine without experiencing a Grade 3 adverse event or SAE will be reported with exact binomial 95% confidence intervals.Malaria incidence by trial arm will be calculated as the number of malaria episodes divided by the person time of follow-up.

BK-SE36 + CpG (100 μg SE36) Control vaccine (Rabies) Cohort 1 Cohort 2 Cohort 3 Cohort 1 Cohort 2 Cohort 3
• Positive pregnancy test • Currently breastfeeding • Suspected or current known alcohol or drug abuse Previous participation in any malaria vaccine trial • History of blood transfusion within the last 3 months • Symptoms, physical signs or laboratory values suggestive of systemic disorders, including renal, hepatic, cardiovascular, pulmonary, skin, immunodeficiency, psychiatric and other conditions, which could interfere with the interpretation of the trial results or compromise the health of the volunteers • Any clinically significant laboratory abnormalities on screened blood samples outside the normal range, as defined at the clinical trial site.

•
Occurrence of clinically significant hematological and/or biochemical abnormalities by laboratory testing, one week and four weeks after each vaccination, Week 26 and 52 in reference with the baseline before the first dose, by measuring: o RBC, hemoglobin, MCV, MCH, MCHC, platelets, ESR and WBC with differential counts o AST, ALT, total bilirubin, creatinine • Occurrence of clinically significant change in assessments of anti-dsDNA, anti-neutrophil cytoplasmic antibody (p-and c-ANCA), anti-nuclear antibody (ANA) at Day 0, Weeks 20 and 52.Mapping of protective epitope(s) by ELISA against overlapping peptides derived from the SE36 protein at Week 20 • Incidence of clinical malaria assessed by microscopic examination of thick and thin blood smears in the event of fever (temperature ≥ 38.0°C) or history of fever in the past 24 hours, starting from Day 56 to Day 365