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. 2016 May 31;15:301.
doi: 10.1186/s12936-016-1348-9.

The Biological Function of Antibodies Induced by the RTS,S/AS01 Malaria Vaccine Candidate Is Determined by Their Fine Specificity

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Free PMC article

The Biological Function of Antibodies Induced by the RTS,S/AS01 Malaria Vaccine Candidate Is Determined by Their Fine Specificity

Sidhartha Chaudhury et al. Malar J. .
Free PMC article

Abstract

Background: Recent vaccine studies have shown that the magnitude of an antibody response is often insufficient to explain efficacy, suggesting that characteristics regarding the quality of the antibody response, such as its fine specificity and functional activity, may play a major role in protection. Previous studies of the lead malaria vaccine candidate, RTS,S, have shown that circumsporozoite protein (CSP)-specific antibodies and CD4(+) T cell responses are associated with protection, however the role of fine specificity and biological function of CSP-specific antibodies remains to be elucidated. Here, the relationship between fine specificity, opsonization-dependent phagocytic activity and protection in RTS,S-induced antibodies is explored.

Methods: A new method for measuring the phagocytic activity mediated by CSP-specific antibodies in THP-1 cells is presented and applied to samples from a recently completed phase 2 RTS,S/AS01 clinical trial. The fine specificity of the antibody response was assessed using ELISA against three antigen constructs of CSP: the central repeat region, the C-terminal domain and the full-length protein. A multi-parameter analysis of phagocytic activity and fine-specificity data was carried out to identify potential correlates of protection in RTS,S.

Results: Results from the newly developed assay revealed that serum samples from RTS,S recipients displayed a wide range of robust and repeatable phagocytic activity. Phagocytic activity was correlated with full-length CSP and C-terminal specific antibody titres, but not to repeat region antibody titres, suggesting that phagocytic activity is primarily driven by C-terminal antibodies. Although no significant difference in overall phagocytic activity was observed with respect to protection, phagocytic activity expressed as 'opsonization index', a relative measure that normalizes phagocytic activity with CS antibody titres, was found to be significantly lower in protected subjects than non-protected subjects.

Conclusions: Opsonization index was identified as a surrogate marker of protection induced by the RTS,S/AS01 vaccine and determined how antibody fine specificity is linked to opsonization activity. These findings suggest that the role of opsonization in protection in the RTS,S vaccine may be more complex than previously thought, and demonstrate how integrating multiple immune measures can provide insight into underlying mechanisms of immunity and protection.

Keywords: Antibody; Epitope; Malaria; Phagocytosis; Protection.

Figures

Fig. 1
Fig. 1
Antibody responses stratified by protection status. Endpoint ELISA antibody titres are shown for recombinant full-length CS (a), NANP repeat peptide (b), and the C-terminal PF16 peptide (c) for non-protected (NP) and protected (P) individuals. p value determined using Student’s t test
Fig. 2
Fig. 2
CSP antibody epitope fine specificity. a Endpoint ELISA antibody titres to NANP and PF16 peptides compared against ELISA antibody titres to recombinant full-length CS antigen (CS). b Additive model of CS antibody titres as a linear combination of NANP and PF16 ELISA antibody titres compared to ELISA antibody titres to CS. The following model was fitted using linear regression: CS titre = β1 × NANP titre + β2 × PF16 titre, and obtained values of β1 = 20.5 and β2 = 389. c Relative antibody responses shown as NANP:CS antibody titre ratio and PF16:CS antibody titre ratio. R2 values from a Pearson correlation are shown if the statistically significance is at a cut-off of p < 0.01
Fig. 3
Fig. 3
Flow cytometry to measure antibody-mediated phagocytosis in THP-1 cells. Example histograms of THP-1 cells incubated with CSP-coated NeutrAvidin beads. Markers capture either cells that have taken up at least one bead and thus are fluorescent positive (M1) or cells that have taken up two or more beads (M2). The control is a serum pool from malaria naïve US donors, CSP-specific is a serum pool that is used as positive control by the Malaria Serology laboratory at WRAIR
Fig. 4
Fig. 4
Representative opsonization activity and fitting. Representative data for opsonization activity at serial dilutions (1:100, 1:500, 1:2500, 1:12,500, 1:62,500) for two measures of opsonization: the frequency of cells undergoing phagocytosis (Mfreq, top) and the mean fluorescence intensity (MFI, bottom) of cells undergoing phagocytosis. Opsonization activity (circles) is shown after subtracting pre-immune serum activity. Examples of low (left), medium (middle), and high (right) opsonization titres are shown. The four-parameter curve fitted to the data is shown in red, the horizontal line indicates the opsonization activity at 60 % of maximum activity, and the vertical line indicates the log dilution corresponding to 60 % maximum activity, as determined by the fitted curve
Fig. 5
Fig. 5
Scatterplot of opsonization titres and ELISA antibody titres. Scatterplots comparing Mfreq opsonization titre (top) and MFI opsonization (bottom) with endpoint ELISA titres for CS (a), NANP (b), and PF16 (c) antigens. The Pearson correlation coefficient (R2) and statistical significance (p) is shown
Fig. 6
Fig. 6
Opsonization titres separated by protection. Opsonization titres calculated from Mfreq (a) and MFI (b) opsonization activity measures is shown for non-protected (NP) and protected (P) individuals
Fig. 7
Fig. 7
Opsonization index and epitope fine specificity separated by protection. Mfreq opsonization index (a), MFI opsonization index (b), and log ratio of PF16 antibody titres to NANP antibody titres (c) for non-protected (NP) and protected (P) individuals. p values determined using Student’s t test
Fig. 8
Fig. 8
Opsonization index and epitope fine specificity combined with NANP antibody response. a Scatterplot of antibody fine specificity, based on PF16:NANP antibody titre ratios compared with antibody titre to NANP for non-protected (NP) and protected (P) individuals. b Scatterplot of MFI opsonization index and NANP antibody titre for non-protected and protected individuals. A 2-way ANOVA shows that both pairs of immune measures significantly distinguish between protected and non-protected subjects (p = 0.002 and p = 0.0002, for PF16:NANP and NANP titre, and MFI opsonization index and NANP titre, respectively)

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