Malaria transmission-blocking vaccines Pfs230D1-EPA and Pfs25-EPA in Alhydrogel in healthy Malian adults; a phase 1, randomised, controlled trial

Abstract

Background: Malaria transmission-blocking vaccines target mosquito-stage parasites and will support elimination programmes. Gamete vaccine Pfs230D1-EPA/Alhydrogel induced superior activity to zygote vaccine Pfs25-EPA/Alhydrogel in malaria-naive US adults. Here, we compared these vaccines in malaria-experienced Malians.

Methods: We did a pilot safety study then double-blind, block-randomised, comparator-controlled main-phase trial in malaria-intense Bancoumana, Mali. 18-50-year-old healthy non-pregnant, non-breastfeeding consenting adult residents were randomly assigned (1:1:1:1) to receive four doses at months 0, 1, 4·5, and 16·5 of either 47 μg Pfs25, 40 μg Pfs230D1 or comparator (Twinrix or Menactra)-all co-administered with normal saline for blinding-or 47 μg Pfs25 plus 40 μg Pfs230D1 co-administered. We documented safety and tolerability (primary endpoint in the as-treated populations) and immunogenicity (secondary endpoint in the as-treated populations: ELISA, standard-membrane-feeding assay, and mosquito direct skin feed assay). This trial is registered at ClinicalTrials.gov, NCT02334462.

Findings: Between March 19, and June 2, 2015, we screened 471 individuals. Of 225 enrolled for the pilot and main cohorts, we randomly assigned 25 participants to pilot safety cohort groups of five (20%) to receive a two-dose series of Pfs25-EPA/Alhydrogel (16 μg), Pfs230D1-EPA/Alhydrogel (15 μg) or comparator, followed by Pfs25-EPA/Alhydrogel (16 μg) plus Pfs230D1-EPA/Alhydrogel (15 μg) or comparator plus saline. For the main cohort, we enrolled 200 participants between May 11 and June 2, 2015, to receive a four-dose series of 47 μg Pfs25-EPA/Alhydrogel plus saline (n=50 [25%]; Pfs25), 40 μg Pfs230D1-EPA/Alhydrogel plus saline (n=49 [25%]; Pfs230D1), 47 μg Pfs25-EPA/Alhydrogel plus 40 μg Pfs230D1-EPA/Alhydrogel (n=50 [25%]; Pfs25 plus Pfs230D1), or comparator (Twinrix or Menactra) plus saline (n=51 [25%]). Vaccinations were well tolerated in the pilot safety and main phases. Most vaccinees became seropositive after two Pfs230D1 or three Pfs25 doses; peak titres increased with each dose thereafter (Pfs230D1 geometric mean: 77·8 [95% CI 56·9-106·3], 146·4 [108·3-198·0], and 410·2 [301·6-558·0]; Pfs25 geometric mean 177·7 [130·3-242·4] and 315·7 [209·9-474·6]). Functional activity (mean peak transmission-reducing activity) appeared for Pfs230D1 (74·5% [66·6-82·5]) and Pfs25 plus Pfs230D1 (68·6% [57·3-79·8]), after the third dose and after the fourth dose (88·9% [81·7-96·2] for Pfs230D1 and 85·0% [78·4-91·5] Pfs25 plus Pfs230D1) but not for Pfs25 (58·2% [49·1-67·3] after the third dose and 58·2% [48·5-67·9] after the fourth dose). Pfs230D1 transmission-reducing activity (73·7% [64·1-83·3]) persisted 10 weeks after the fourth dose. Transmission-reducing activity of 80% was estimated at 1659 ELISA units for Pfs25, 218 for Pfs230D1, and 223 for Pfs230D1 plus Pfs25. After 3850 direct skin feed assays, 35 participants (12 Pfs25, eight Pfs230D1, five Pfs25 plus Pfs230D1, and ten comparator) had transmitted parasites at least once. The proportion of positive assays in vaccine groups (Pfs25 33 [3%] of 982 [-0·013 to 0·014], Pfs230D1 22 [2%] of 954 [-0·005 to 0·027], and combination 11 [1%] of 940 [-0·024 to 0·002]) did not differ from that of the comparator (22 [2%] of 974), nor did Pfs230D1 and combination groups differ (-0·024 to 0·001).

Interpretation: Pfs230D1 but not Pfs25 vaccine induces durable serum functional activity in Malian adults. Direct skin feed assays detect parasite transmission to mosquitoes but increased event rates are needed to assess vaccine effectiveness.

Funding: Intramural Research Program of the National Institute of Allergy and Infectious Diseases and US National Institutes of Health.

Figure 1.. Trial Profile.

Trial included Pilot Safety Cohort then Main Cohort. Subjects in Pilot Safety Cohort were enrolled (April 2015) in a double-blind, comparator-controlled pilot study to receive single vaccinations (16μg Pfs25, 15μg Pfs230D1, TWINRIX) on days 0 and 28, or to receive co-administered (two syringes, separate arms) vaccinations (16μg Pfs25 + 15μg Pfs230D1, TWINRIX + normal saline) on the same schedule; pilot safety cohort participants were followed for 6 months post-dose 2 for safety and immunogenicity. For Pilot Safety Cohort, “completed study per protocol” was defined as completed through study day 196 (~6 months post vaccination #2). For analysis purposes, subjects in the TWINRIX and TWINRIX + normal saline arms (n=10) were combined as a single comparator arm. Subjects were then enrolled into the main double-blind, comparator-controlled study (n=200) and divided into 4 arms: 47μg Pfs25 + normal saline; 40μg Pfs230D1 + normal saline; 47μg Pfs25 + 40μg Pfs230D1; and comparator (TWINRIX, Menactra + normal saline). Main cohort participants were initially scheduled to receive vaccinations on a 0, 1, 6, 18-month schedule, but ultimately received these on a 0, 1, 4·5, 16·5-month schedule in order to complete dosing before the peak malaria transmission season (dose 3 = 15 Sep to 16 Oct 2015; dose 4 = 15 Sep to 17 Oct 2016). For the Main Cohort, completed Year 1= completed through study day 510 (~11 months post dose 3; end of Year 1); completed Year 2 = completed study day 730 (~6 months post dose 4). Year 2 is indicated in grey. DSF Year 1 was defined as completing at least 1 DSF from study day 175 (7 days post dose 3) to study day 213 (45 days post dose 3); maximum of 12 DSFs were completed in Year 1 (2015). DSF Year 2 was defined as completing at least 1 DSF from study day 547 (7 days post dose 4) to study day 585 (45 days post dose 4); maximum of 12 DSFs were completed in Year 2 (2016). Pfs25 = Pfs25-EPA/Alhydrogel^®; Pfs230D1 = Pfs230D1-EPA/Alhydrogel^®; μg = micrograms; Vax = vaccine; DSF = direct skin feeds. ^AOne subject randomized to 40μg Pfs230D1 + normal saline was erroneously administered comparator for vaccination #1, then continued to receive comparator throughout study (subject and clinical team remained blinded); for analysis considered comparator subject (for as-treated analysis) and Pfs230D1 subject (for ITT). ^BOne Pfs230D1-randomized subject was administered 47μg Pfs25 + 40μg Pfs230D1 for vaccination #2; considered Pfs230D1 subject for both as-treated and ITT analysis. ^COne subject (TWINRIX + normal saline) became pregnant just prior to vaccination #2 and was intentionally unblinded early for counseling of risk given vaccine received. ^DTwo subjects (one Pfs25; one Pfs25+Pfs230D1) did not complete a single DSF in Year 2 but completed the study. Analysis populations: Pilot Safety Cohort primary (safety, as-treated): Pfs25, 16μg (N=5), Pfs230D1, 15μg (N=5), TWINRIX (N=5), Pfs25, 16μg and Pfs230D1, 15μg (N=5), TWINRIX and Normal Saline (N=5) Pilot Safety Cohort secondary (ELISA, SMFA; 2 weeks post dose 2, as-treated): Pfs25, 16μg (N=5), Pfs230D1, 15μg (N=4), TWINRIX (N=5), Pfs25, 16μg and Pfs230D1, 15μg (N=5), TWINRIX and Normal Saline (N=5) Year 1 Main Cohort primary (safety, as-treated): Pfs25, 47μg and Normal Saline (N=50), Pfs230D1, 40μg and Normal Saline (N=49), Pfs25, 47μg and Pfs230D1, 40μg (N=50), TWINRIX and Normal Saline (N=51) Main Cohort secondary (ELISA, SMFA; 2 weeks post dose 3, as-treated): Pfs25, 47μg and Normal Saline (N=44), Pfs230D1, 40μg and Normal Saline (N=39), Pfs25, 47μg and Pfs230D1, 40μg (N=46), TWINRIX and Normal Saline (N=44) Main Cohort secondary (DSF, as-treated): Pfs25, 47μg and Normal Saline (N=44), Pfs230D1, 40μg and Normal Saline (N=43), Pfs25, 47μg and Pfs230D1, 40μg (N=44), TWINRIX and Normal Saline (N=44) Year 2 Main Cohort primary (safety, as-treated): Pfs25, 47μg and Normal Saline (N=42), Pfs230D1, 40μg and Normal Saline (N=40), Pfs25, 47μg and Pfs230D1, 40μg (N=39), TWINRIX and Normal Saline (N=40) Main Cohort secondary (ELISA, SMFA; 2 weeks post dose 4, as-treated): Pfs25, 47μg and Normal Saline (N=42), Pfs230D1, 40μg and Normal Saline (N=39), Pfs25, 47μg and Pfs230D1, 40μg (N=37), TWINRIX and Normal Saline (N=40) Main Cohort secondary (DSF, as-treated): Pfs25, 47μg and Normal Saline (N=41), Pfs230D1, 40μg and Normal Saline (N=40), Pfs25, 47μg and Pfs230D1, 40μg (N=37), TWINRIX and Normal Saline (N=40)

Figure 2.. Antibody titres for single and combination immunogen arms by ELISA.

Anti-Main cohort participants received vaccinations on a 0, 1, 4·5, 16·5-month schedule from May to Oct 2015 (for dose 1, 2, 3) and Sep to Oct 2016 (for dose 4), and samples were collected at post-vaccination timepoints (schedule in Table S4B, Appendix, page 45) to assess anti-Pfs25 (Figure 2A) and anti-Pfs230D1 (Figure 2B) antibody titres by ELISA. Values are presented as ELISA EU. Geometric means are presented with error bars indicating 95% confidence interval. Differences in antibody titres induced by vaccines versus comparator were analyzed by Mann-Whitney test. For anti-Pfs25 titres, significant differences were observed 2 weeks after vaccinations 2, 3, and 4 (p<0·0001 for each Pfs25-containing group versus comparator). For anti-Pfs230D1 titres, significant differences were observed 2 weeks post-vaccination 1 (p=0·0001 for Pfs230D1 alone; p=0·0024 for Pfs25+Pfs230D1), and 2 weeks after each subsequent vaccination (p<0·0001 for each Pfs230D1-containing group). d730 = study day 730, ~6 months post dose 4 (end of study); comparator antibody titres to Pfs25 and Pfs230D1 were not completed for d730. NS = normal saline. Associated tables with anti-Pfs25 (Figure 2A) and anti-Pfs230D1 (Figure 2B) ELISA data at peak timepoints (2 weeks post vaccination) are provided below each associated figure. Seropositive is defined as EU > mean + 3SD of plate level of detection (Pfs25=55 EU; Pfs230D1=43 EU). Post vaccination receipt sample missingness (due to missed visit, off study post vaccination) ranged from 0-3 participants per each time point and was evenly disbursed between arms (Pfs25: pre dose 1 = 0, post dose 1 = 2 off study; post dose 2 = 3 missed visits; post dose 3 = 1 off study; post dose 4 = 0; Pfs230D1: pre dose 1 = 0, post dose 1 = 1 off study; post dose 2 = 3 missed visits; post dose 3 = 0; post dose 4 = 1 missed visit; Pfs25+Pfs230D1: pre dose 1 = 0, post dose 1 = 1 off study, 1 missed visit; post dose 2 = 2 off study; post dose 3 = 1 off study, 2 missed visits; post dose 4 = 1 off study, 1 missed visit) . Pfs25 = 47 μg of Pfs25-EPA/Alhydrogel^® + normal saline; Pfs230D1 = 40 μg of Pfs230D1-EPA/Alhydrogel^® + normal saline; Pfs25+Pfs230D1 = 47 μg of Pfs25-EPA/Alhydrogel^® + 40 μg of Pfs230D1-EPA/Alhydrogel^®; comparator = Twinrix (dose 1-3) or Menactra (dose 4) + normal saline.

Figure 3.. Pfs230D1 shows superior transmission reducing activity (TRA), transmission-blocking activity (TBA) and durability.

TBV functional activity was assessed by standard membrane feeding assay. Transmission reducing activity (TRA; Figures 3A, 3C, 3E) was defined as ((mean oocyst count in control sera – mean oocyst count in test sera)/mean oocyst count in control sera) x 100. Transmission blocking activity (TBA; Figures 3B, 3D, 3F) was defined as ((mean infection prevalence in assay control – mean prevalence in the test sample)/mean prevalence in the assay control) x 100. For 2 weeks post-each vaccination (post dose 3, Figures 3A, 3B; post dose 4: Figures 3C, 3D), differences between groups were analysed by Kruskal-Wallis test with Dunn’s correction for multiple comparisons; at 10 weeks post-dose 4 (Figures 3E, 3F), differences between Pfs230D1 group and comparator was analyzed by Mann-Whitney test. Significant p-values are presented. Transmission reducing plotted as a function of ELISA titre for Pfs25 and Pfs230D1 single antigen arms at 2 weeks post-dose 3 (Figures 3G, 3I), and 2 weeks post-dose 4 (Figure 3H, 3J). Results for the Pfs25+Pfs230D1 combination arm are presented in Figure S12, Appendix, page 67. Results for transmission reducing activity at 10 weeks post dose 4 for Pfs230D1 single antigen arm can be found in Figure S13, Appendix, page 68. Empty circles represent participants with negative DSF results; closed circles indicate participants with positive DSF results; green diamonds indicate DSFs that were positive for a non-falciparum Plasmodium species detected by PCR analysis of a single midgut selected from the feed. Dotted horizontal lines represent no difference from assay control (non-immune sera).

Figure 4.. DSF assay results for the 35 participants who transmitted parasites in at least one DSF assay.

Of 175 individuals who underwent at least one DSF assay, 35 transmitted parasites on at least one occasion. One of these 35 DSF+ participants yielded positive transmission events in both years, 17 in Year 1 only, and 17 in Year 2 only. Each subject is depicted by timelines over two seasons that indicate DSF timepoints and their outcomes, stratified by trial arm. Feeds used 60 mosquitoes in Year 1 and 30 in Year 2. Dissections were performed 7 days after feed to assess transmission, with each surviving mosquito surveyed for the presence of oocysts with feeding and survival rates per arm available in Table S11, Appendix, page 70. The dots and colored circles in the figure represent DSF assays, with small black dots denoting negative DSF where no dissected mosquitoes had oocysts. Red circles are positive DSF transmission events, with the circle size proportional to the percentage of dissected mosquitoes that contained oocysts.