Assessment of antibody isotype profiles following vaccination and CHMI. Isotype profiles of serum antibody responses were assessed by ELISA.

<p>(A) Anti-MSP1₁₉ responses in the VAC037 Phase Ia clinical trial (open symbols) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy1" target="_blank">[19]</a> and the VAC039 Phase IIa clinical trial (closed symbols) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy3" target="_blank">[21]</a>. Responses are shown at baseline (d0); following ChAd63 MSP1 priming immunization (d28); following the MVA MSP1 boost (d84 in the Phase Ia trial or dC−1 in the Phase IIa trial); or following CHMI at day of diagnosis, dC+DoD, or at first follow-up post drug treatment, dC+35. (B) Anti-AMA1 responses in the VAC036 Phase Ia clinical trial <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy2" target="_blank">[20]</a> and VAC039 Phase IIa trial <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy3" target="_blank">[21]</a>, reported as in panel A. In all panels, individual and median responses are shown, n = 4<b>–</b>13 depending on sample availability for each tested time-point.</p

Associations between IgG antibody responses and CHMI outcome measures.

<p>Associations are reported between anti-MSP1₁₉ or anti-AMA1 total IgG ELISA titer readouts at various time-points and/or fold-change post-CHMI (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone-0107903-g001" target="_blank">Figure 1E</a>), as well as with time to malaria diagnosis by thick-film microscopy during CHMI, and parasitemia at time of diagnosis (measured by qPCR in terms of parasites/mL blood). In all cases, Spearman’s rank correlation coefficient and <i>P</i> value are shown. n/a = not applicable; n.d. = not done. *For these analyses, relevant vaccine groups were included: for the MSP1₁₉ analysis, data were combined from the MSP1-only vaccination group and from the MSP1+AMA1 and MSP1+ME-TRAP co-administration groups; and for the AMA1 analysis data were combined from the AMA1-only vaccination group and from the MSP1+AMA1 co-administration group.</p><p>Associations between IgG antibody responses and CHMI outcome measures.</p

Associations between IgG antibody avidity and total IgG ELISA titer.

<p>Associations are reported between anti-MSP1₁₉ ETSR allele or anti-AMA1 3D7 allele total IgG ELISA titer readouts (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone-0107903-g001" target="_blank">Figure 1B, D</a>) and the corresponding IgG avidity measurement by NaSCN-displacement ELISA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone-0107903-g003" target="_blank">Figure 3E, F</a>). Groups were assessed according to vaccination and/or parasite exposure status as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone-0107903-g003" target="_blank">Figure 3E, F</a>. In all cases, Spearman’s rank correlation coefficient and <i>P</i> value are shown. n.d. = not done (too few positive responders). *Data from vaccinees receiving only a single vaccine (i.e. MSP1-only or AMA1-only) were used for this analysis.</p><p>Associations between IgG antibody avidity and total IgG ELISA titer.</p

Assessment of antibody isotype profiles following vaccination, CHMI and natural exposure.

<p>Isotype profiles of serum antibody responses were assessed by ELISA. (A) Individual and median anti-MSP119 serum antibody isotype responses are shown for MSP1-only vaccinees at the peak after the MVA MSP1 boost (“Vaccine”, <i>n</i> = 12) and at dC+35 following CHMI (“Vaccine+CHMI”, <i>n</i> = 11) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy3" target="_blank">[21]</a>; at dC+35 for 18 infectivity control volunteers from three separate CHMI studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy3" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Ewer1" target="_blank">[22]</a>; and from 20 naturally-exposed immune adults from Kilifi, Kenya. (B) Individual and median anti-AMA1 serum antibody isotype responses are shown for AMA1-only vaccinees at the peak after the MVA AMA1 boost (<i>n</i> = 9) and at dC+35 following CHMI (<i>n</i> = 9) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy3" target="_blank">[21]</a>; and for infectivity control volunteers and naturally-exposed immune adults from Kilifi, Kenya as in panel A.</p

Correlations between anti-MSP119 and –AMA1 IgG1 and IgG3 isotype ELISA responses in different exposure populations.

<p>Serum IgG1 and IgG3 isotype responses were assessed by ELISA against (A) MSP119 (ETSR allele) and (B) AMA1 (3D7 allele). Correlations are shown: following MSP1 (n = 17) or AMA1 (<i>n</i> = 13) viral vectored vaccination only d84/dC−1 sera (open circle and square symbols respectively); following MSP1 (<i>n</i> = 11) or AMA1 (<i>n</i> = 9) vaccination followed by CHMI dC+35 sera (closed circle and square symbols respectively); following CHMI only (<i>n</i> = 18 for MSP119 only) in control volunteers’ dC+35 sera (closed diamond symbols); and for immune sera from Kenyan adults (<i>n</i> = 20; cross symbols).</p

Assessment of IgG antibody responses post-CHMI.

<p>Mean anti-MSP1₁₉ serum IgG responses were assessed over time by ELISA in a Phase IIa CHMI trial <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy3" target="_blank">[21]</a>. Dashed vertical lines represent: day 72 (d72) = day of CHMI; and d85 = nominal day of diagnosis. The first follow-up time-point after CHMI = day 107 (dC+35). (A) VAC039: MSP1 vaccinees (n = 8<b>–</b>9 depending on time-point assessed); AMA1-only vaccinees (n = 9); MSP1+AMA1 vaccinees (n = 8) plus one volunteer who was steriley protected in this group (dashed line); MSP1+ME-TRAP (n = 10); and infectivity controls (n = 6). The second Phase IIa CHMI trial with MSP1 vaccinees (VAC037) is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903.s002" target="_blank">Figure S2</a>. (B) Individual and median anti-MSP1₁₉ serum IgG responses are shown for MSP1 vaccinees at the peak after the MVA boost (open symbols, n = 17) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy1" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy3" target="_blank">[21]</a> and at dC+35 following CHMI (closed symbols, n = 11) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy3" target="_blank">[21]</a>; at dC+35 for 18 infectivity control volunteers from three separate CHMI studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy3" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Ewer1" target="_blank">[22]</a>; at dC+35 for AMA1 vaccinees (n = 9) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy3" target="_blank">[21]</a> and non-sterilely protected ME-TRAP vaccinees (n = 11) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Ewer1" target="_blank">[22]</a>; from 40 naturally-exposed immune adults from Kilifi, Kenya; and 59 malaria-naïve UK adults (prior to immunization or CHMI). (C) Anti-AMA1 serum IgG responses for each group as in panel A. (D) Individual and median anti-AMA1 serum IgG responses are shown for AMA1 vaccinees at the peak after the MVA boost (open symbols, n = 13) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy2" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy3" target="_blank">[21]</a> and at dC+35 following CHMI (closed symbols, n = 9) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy3" target="_blank">[21]</a>; at dC+35 for 18 infectivity control volunteers from three separate CHMI studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy3" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Ewer1" target="_blank">[22]</a>; at dC+35 for MSP1-only vaccinees (n = 8) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy3" target="_blank">[21]</a>; from 50 naturally-exposed immune adults from Kilifi, Kenya; and 19 malaria-naïve UK adults (prior to immunization or CHMI). (E) The fold-change in IgG titer from dC−1 (d71) to dC+35 (d107) is reported. Individual responses and geomean are shown, with symbol colouring according to group in panels A and C. The limit of detection in both ELISA assays was 10 AU (dashed horizontal line), and we assigned the AU value of 1.0 for any test samples with less than 10 AU. Any values more than 10 AU are considered as positive responses. *P<0.05 (Wilcoxon matched-pairs signed rank test).</p

Assessment of functional GIA post-CHMI.

<p>(A) <i>In</i><b><i> </i></b><i>vitro</i> GIA of purified IgG was assessed at 10<b> </b>mg/mL against 3D7 clone <i>P. falciparum</i> parasites. Individual data and medians are shown for each vaccinated or control group at the dC−1 and dC+35 time-points (<i>n</i> = 8<b>–</b>12). Pre-immunization (d0) sera were either tested individually or pooled (<i>n</i> = 7). Responses >20% are typically regarded as positive. (B) Relationship between GIA and anti-AMA1 serum IgG responses measured by ELISA. Results for volunteers immunized with ChAd63-MVA AMA1 are shown on dC−1 (before CHMI) and dC+35 (after CHMI). Non-linear regression curve is also shown (<i>n</i> = 19). The level of AMA1 antibody measured in this ELISA assay that gave 50% GIA (EC₅₀, dashed black line) was 7294 AU (95% C.I. = 3981<b>–</b>13362).</p

Associations between IgG antibody ELISA responses against two alleles of MSP1₁₉ and AMA1.

<p>Associations are reported between anti-MSP1₁₉ or anti-AMA1 total IgG ELISA titer readouts between two allelic variants of each antigen – ETSR (3D7) and QKNG (K1/Wellcome) for MSP1₁₉, and 3D7 and FVO for AMA1. Responses were assessed following vaccination only, vaccination + CHMI, CHMI only (control volunteers) and in naturally-exposed Kenyan adults (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone-0107903-g001" target="_blank">Figure 1B, D</a>). In all cases, Spearman’s rank correlation coefficient and <i>P</i> value are shown. n.d. = not done (too few positive responders). *Relevant data were pooled for analysis from vaccinees receiving either a single vaccine or co-administered vaccines (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone-0107903-t001" target="_blank">Table 1</a>).</p><p>Associations between IgG antibody ELISA responses against two alleles of MSP1₁₉ and AMA1.</p

Antibody immunogenicity of ChAd63 AMA1 and ChAd63-MVA AMA1 immunization regimes.

<p>(<b>A</b>) Groups 1A and 2A and (<b>B</b>) groups 1B and 2B total IgG ELISA responses against 3D7 AMA1 as measured in the serum over time. The geometric mean response is shown for each group and individual responses are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031208#pone.0031208.s008" target="_blank">Figure S7</a>. The horizontal dotted line indicates the limit of detection of the assay. (<b>C</b>) AMA1-specific ELISA titers against 3D7 (circles) and FVO (squares) in µg/mL for group 1B (<i>n</i> = 4) and group 2B (<i>n</i> = 4) at the peak time-point (day 84). Individual data points and the median are shown. (<b>D</b>) % GIA against 3D7 and FVO parasites at day 0 and day 84 in group 1B (open symbols) and 2B (closed symbols). (<b>E</b>) Relationship between 3D7 strain % GIA and serum 3D7 AMA1-specific IgG ELISA titer at day 0 and day 84 in groups 1B (open symbols) and 2B (closed symbols).</p

Flow chart of the study.

<p>All vaccinations were administered intramuscularly. ChAd63 AMA1 was administered on day 0 and MVA AMA1 on day 56. Six volunteers were excluded following screening for the following reasons: psychiatric morbidity; recurrent severe urticaria; elevated alanine aminotransferase (87 IU/L); unexplained microscopic haematuria and proteinuria; and withdrawal of consent (two individuals).</p