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. 2013;9(12):e1003840.
doi: 10.1371/journal.ppat.1003840. Epub 2013 Dec 26.

Overcoming antigenic diversity by enhancing the immunogenicity of conserved epitopes on the malaria vaccine candidate apical membrane antigen-1

Sheetij Dutta  1 , Lisa S Dlugosz  1 , Damien R Drew  2 , Xiaopeng Ge, Diouf Ababacar  3 , Yazmin I Rovira  1 , J Kathleen Moch  1 , Meng Shi  1 , Carole A Long  3 , Michael Foley  4 , James G Beeson  2 , Robin F Anders  4 , Kazutoyo Miura  3 , J David Haynes  1 , Adrian H Batchelor  1
Affiliations

Overcoming antigenic diversity by enhancing the immunogenicity of conserved epitopes on the malaria vaccine candidate apical membrane antigen-1

Sheetij Dutta et al. PLoS Pathog. 2013.

Erratum in

  • PLoS Pathog. 2014 Jan;10(1). doi:10.1371/annotation/be0d67d1-314f-4654-8a2f-b668d1e32bbd. Ge, Xiopeng [corrected to Ge, Xiaopeng]

Abstract

Malaria vaccine candidate Apical Membrane Antigen-1 (AMA1) induces protection, but only against parasite strains that are closely related to the vaccine. Overcoming the AMA1 diversity problem will require an understanding of the structural basis of cross-strain invasion inhibition. A vaccine containing four diverse allelic proteins 3D7, FVO, HB3 and W2mef (AMA1 Quadvax or QV) elicited polyclonal rabbit antibodies that similarly inhibited the invasion of four vaccine and 22 non-vaccine strains of P. falciparum. Comparing polyclonal anti-QV with antibodies against a strain-specific, monovalent, 3D7 AMA1 vaccine revealed that QV induced higher levels of broadly inhibitory antibodies which were associated with increased conserved face and domain-3 responses and reduced domain-2 response. Inhibitory monoclonal antibodies (mAb) raised against the QV reacted with a novel cross-reactive epitope at the rim of the hydrophobic trough on domain-1; this epitope mapped to the conserved face of AMA1 and it encompassed the 1e-loop. MAbs binding to the 1e-loop region (1B10, 4E8 and 4E11) were ∼10-fold more potent than previously characterized AMA1-inhibitory mAbs and a mode of action of these 1e-loop mAbs was the inhibition of AMA1 binding to its ligand RON2. Unlike the epitope of a previously characterized 3D7-specific mAb, 1F9, the 1e-loop inhibitory epitope was partially conserved across strains. Another novel mAb, 1E10, which bound to domain-3, was broadly inhibitory and it blocked the proteolytic processing of AMA1. By itself mAb 1E10 was weakly inhibitory but it synergized with a previously characterized, strain-transcending mAb, 4G2, which binds close to the hydrophobic trough on the conserved face and inhibits RON2 binding to AMA1. Novel inhibition susceptible regions and epitopes, identified here, can form the basis for improving the antigenic breadth and inhibitory response of AMA1 vaccines. Vaccination with a few diverse antigenic proteins could provide universal coverage by redirecting the immune response towards conserved epitopes.

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Conflict of interest statement

SD has been named on an AMA1 related US patent. This does not alter our adherence to all PLOS policies on sharing data and materials.

Figures

Figure 1
Figure 1. AMA1 sequence diversity and allelic coverage of four monovalent vaccines as compared to the QV.
(A) A dendrogram constructed with full-length AMA1 sequences from the 26 target strains tested in GIA and 175 field strain sequences obtained from Genbank (Table S1). P. berghei (rodent) and P. reichenowi (chimpanzee) sequences were also included. (+) indicates that this allelic AMA1 protein was included in QV. The colored boxes were the 8 target strains used in the WRAIR GIA. (B) ELISA titers (×1000) for the five vaccine groups tested against 7 allelic proteins. Symbols are mean of three rabbits and lines are median titer across strains. (C) Box-and-whiskers plot using individual rabbit ELISA data grouped on the basis of whether the coat antigen-antisera combinations were homologous or heterologous and whether monovalent or QV rabbits were tested. The number under each box represents the total number of protein-antisera combinations included. (*) indicates, p<0.01, (**) p<0.001, (***) p<0.0001 and (****) p<0.0001 for ANOVA followed by Tukey's multiple comparisons test. (D) One-cycle GIA of the five vaccine groups against four non-vaccine and four vaccine strains using 1∶5 whole serum dilution. Symbols in Fig. 1B and 1D and 1F are matched, except strain SA250 that was only tested in the GIA. Each symbol is mean of three rabbits tested in two experiments and lines are median inhibition across strains. (E) GIA data from individual rabbits from three experiments grouped similar to the ELISA data, except the groups were made based on homologous and heterologous parasite-antisera combinations. (F) GIA activity of pooled QV sera was compared to a pool of the highest titer rabbit sera in the four monovalent vaccine groups 3D7+FVO+HB3+W2mef (4-Way pool). Lines are median inhibition across 8 target parasite strains; representative of 2 experiments is shown.
Figure 2
Figure 2. Quantification of broadly inhibitory antibodies.
(A) IC50 values against three target strains for the monovalent anti-3D7 and anti-QV IgG, affinity purified from pools of 3 rabbit sera, using a 3D7 AMA1 column (GIA in Fig. S2). (B) GIA reversal comparing the ability of AMA1 allelic proteins to reverse the inhibition of 3D7 parasite invasion by pools of anti-QV or anti-3D7 sera. P. berghei AMA1 was used as the negative control (Pb). The data is representative of 2 experiments. (C) GIA of pooled anti-3D7 and anti-QV IgG (3 rabbits each) that was bound and eluted from a M24 AMA1 affinity column and tested at 0.15 mg/ml against 3D7 or three non-vaccine parasite strains (7G8, M24 and 102-1). Mean+s.e.m from 3 experiments is shown.
Figure 3
Figure 3. Inhibitory coverage against laboratory and field isolates.
(A) One-cycle GIA at 1.25 mg/ml total IgG pool from 3 rabbits tested by the NIH pLDH assay. GIA of anti-QV was compared to trivalent and bivalent vaccine groups and antibodies against P. berghei AMA1 (PbAMA1) were tested as the negative control. Strains CP803, CP806, CP830, CP845, and CP887 were recent culture adapted Cambodian isolates and HB3, GB4, MT/S1, C2A, W2mef were laboratory strains. Lines are median inhibition across strains. (B) One-cycle GIA at 1 mg/ml total IgG pool from 3 rabbits conducted by the WRAIR flow-cytometric method against 8 parasite strains. (*) indicates, p<0.05; (***) p<0.0001 (corrected for multiple comparisons). (C) Two-cycle GIA at 1 mg/ml pooled IgG conducted by the Burnet Institute flow-cytometric method. Strains CSL-2, HCS-E5, 2006, 2004, XIE were recently culture adapted field isolates from Africa, Asia and isolates E8B07, CAMP, D10, K1, T996 were laboratory strains .
Figure 4
Figure 4. Mapping inhibitory monoclonal antibodies.
(A) View of the hydrophobic trough and the surrounding loops showing approximate spatial location of mAb epitopes. (B) Polymorphic and conserved face of AMA1. Domain-1 residues (light blue); domain-2 (yellow); domain-3 (magenta); C-terminal processing site at residue Thr517 (black); mAb 4G2 binding residues (orange); mAb 1F9 epitope centered on the C1L-loop (dark blue); and mAb 1B10, 4E8 and 4E11 epitopes on the 1e-loop (purple). (C) Coomassie blue stained and western blot panels showing chimeric proteins (Fig. S5) used to map representative mAbs. (D) Dot blot reactivity pattern of inhibitory mAbs against diverse P. falciparum AMA1 allelic proteins and P. berghei AMA1 control. (E) A cross-strain GIA against 7 parasite strains at 1 mg/ml mAb concentration.
Figure 5
Figure 5. Biological activity of monoclonal antibodies.
(A) Binding of 3D7 AMA1 (OD450) to immobilized RON2 peptide inhibited by serial dilutions of the mAbs. Negative control mAb 5G8 binds to the N-terminal prosequence. (B) Western blot of a 3D7 parasite processing inhibition assay using 200 µg/ml mAbs. Top panel shows the merozoite bound full-length (83 kDa) 3D7 parasite AMA1 and the product of N-terminal processing (66 kDa). Bottom panel shows the co-migrating products of normal shedding (48+44 kDa) and the product of anomalous AMA1 processing (52 kDa). These fragments were captured from the culture supernatant using a sub-inhibitory concentration of polyclonal anti-3D7 AMA1 sera (1∶2500) in the processing assay . (C) GIA against 3D7 target strain, using 1×IC30 dose of individual mAbs (black), 2×IC30 dose of individual mAbs (gray), 1×IC30+1×IC30 mixture of two 1e-loop mAbs (green) or 1e-loop+domain2-loop mAb (blue) or 1e-loop+domain-3 mAb (orange) or domain2 loop+domain-3 mAb (red). Mean+s.e.m. of 3 experiments; (*) p<0.05 comparing the mean of each group to the mean of 2×IC30 dose of individual mAbs (gray bars). (D) GIA against the 3D7 parasite strain using increasing concentrations of mAb 1E10, with (red line) or without (blue line) the addition of 1×IC30 concentration of mAb 4G2 (1.8 mg/ml, expected 30% GIA in green). Predicted inhibition for additive interaction (black line) was calculated according to “Bliss independence” as has been applied to determine synergy by Williams et al. ; data are mean+s.e.m. of triplicate wells. (E) Inhibition of 7 parasite strains using 2 mg/ml of the RON2 inhibitory mAb or a mixture of 1 mg/ml each of the RON2 inhibitory mAbs and processing inhibitory mAb 1E10; a representative of two experiments is shown.
Figure 6
Figure 6. Mapping inhibitory activity of polyclonal antibodies.
(A) Distribution of high frequency polymorphisms on the three domains (D1, 2, 3), the polymorphic face and the conserved face of AMA1. (B) Region-specific inhibitory contributions determined by adding chimeras to reverse anti-3D7 or anti-QV serum pool mediated GIA activity (approximately 60% starting GIA activity) against 3D7 parasites. Reversal using chimeric proteins CryD1, CryD2, CryD3, Cry D1+CryD2, CryD2+CryD3, CryD1+CryD3, POLY and CONS (4 µM, ∼200 µg/ml final concentration) was determined with respect to P. berghei AMA1 as the control. Mean+s.e.m. of 3 experiments and (*) indicates statistical significant p value of t-tests comparing anti-3D7 and anti-QV reversals. (C) Region-specific ELISA response with pooled sera (% of total) calculated as the ratios of end-point titers against a 3D7 chimera relative to the end-point titer against 3D7 AMA1 protein (mean+s.e.m. of triplicates in a representative of three experiments). (D) Competition ELISA shows the binding of HRP labeled mAbs to heterologous 102-1 AMA1 protein. The mAb binding was competed out using serial dilutions of polyclonal anti-3D7 or anti-QV or pre-immune rabbit serum pools (x axis). Shown is mean OD405 of 2 wells from a representative of two experiments.
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