Experimental infection and protection against ... - TI Pharma
Experimental infection and protection against ... - TI Pharma Experimental infection and protection against ... - TI Pharma
A saturation of antibody avidity and concentration induced by malaria vaccine candidate Apical Membrane Antigen 1 Figure 3: AMA1 antibody concentration and association constant (ka) of pooled rabbit sera (A) or human sera (B). Dashed lines show inverse curve fit of data in panel A. A: Inverse correlation between concentration and ka of AMA1 antibodies (clear circles) and concentration and ka of antibodies measured against heterologous AMA1 sequences from strain 3D7 (grey) or HB3 (black) B: Inverse correlation between concentration and ka of AMA1 antibodies in naturally exposed semi-immunes (stars) and concentration and ka of AMA1 antibodies in human volunteers post-vaccination with FVO PfAMA1 [25-545] vaccine (construct Pf11.0) adjuvanted by Alhydrogel® (white), Montanide ISA 720 (grey) or AS02A (black). Parasite growth inhibition was calculated based on optical density (OD) measurements as follows: 100 - ((ODexperimental - ODbackground)/ (ODcontrol - ODbackground) x 100). IgG purified from pre- immunization plasma was used as a control and culture medium was used to measure the background OD. Statistical Analysis Statistical fitting of interaction kinetics was performed using BIAevaluation software 3.2, whereby the closeness of fit is represented by the statistical value chi2. Chi2 reduces to the average squared residual per data point. All other statistical analyses were performed in SPSS 16.0 (SPSS Inc.). For comparison between means of independent data sets the Kruskall-Wallis was used for three or more groups, the Mann-Whitney U for two independent groups and Wilcoxon singed ranks for paired samples. Non-parametric Spearman analyses were used to describe correlations. Curve fits were assessed by ANOVA. All statistical analysis were two-tailed and p values less than 0.05 were considered significant. Results Sera obtained from rabbit immunization studies and purified immunoglobulins (Ig) obtained from volunteers in a phase I clinical trial were tested for their 87
88 Chapter 4 Figure 4: Correlation between residue number of the vaccine construct and AMA1 antibody concentration (A) or association constant ka (B) post-vaccination in rabbit sera. binding kinetics to the full length homologous ectodomain of recombinant PfAMA1 by Surface Plasmon Resonance (construct Pf4mH, Figure 1). Association constants (ka) and dissociation constants (kd) of immunoglobulin-antigen (IgAg) interactions were determined separately by fitting kinetic curves to a simple 1:1 Langmuir binding model. Fits to the model showed an average chi2 of 2.98 for rabbit samples (n=38 measurements) and 0.85 for human samples (n=72 measurements). Two rabbit samples could not be fitted with the model and were excluded from analyses. Mean ka and kd values of all other measurements are shown in Table 1. Kinetic constants of rabbit and human samples covered a similar range. Values for kd were considerably smaller than for ka (~10 8 fold difference), reflecting a stable antigen-antibody complex. Antibody concentration (of binding antibodies) and ka values of pooled rabbit sera were inversely correlation (Figure 3A), approximated by inverse curve fit (p=0.002, R 2 = 0.871). The implication of these results are best illustrated by analysis of a simple non-covalent interaction system, whereby the increase in IgAg over time (δ[IgAg]/ δt) is described by concentration of immunoglobulin [Ig], antigen [Ag] and ka and kd as follows: δ[IgAg]/ δt = ka*[Ig]*[Ag] – kd*[IgAg]. In a state of equilibrium, when the increase in immunoglobulin-antigen complex over time is zero, concentration and binding constants are balanced (ka*[Ig]*[Ag] = kd*[IgAg]). Algebraic rearrangement will result in ka/kd*[Ig] = [IgAg]/[Ag], where [IgAg]/[Ag] represents the fraction of antigen bound by antibody. Assuming kd is negligible, the observed inverse relationship in rabbit sera (Figure 3A) thus suggests that the fraction of bound antigen is constant and identical for all investigated sera.
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88 Chapter 4<br />
Figure 4: Correlation between residue number of the vaccine construct <strong>and</strong> AMA1<br />
antibody concentration (A) or association constant ka (B) post-vaccination in rabbit sera.<br />
binding kinetics to the full length homologous ectodomain of recombinant<br />
PfAMA1 by Surface Plasmon Resonance (construct Pf4mH, Figure 1). Association<br />
constants (ka) <strong>and</strong> dissociation constants (kd) of immunoglobulin-antigen (IgAg)<br />
interactions were determined separately by fitting kinetic curves to a simple 1:1<br />
Langmuir binding model. Fits to the model showed an average chi2 of 2.98 for<br />
rabbit samples (n=38 measurements) <strong>and</strong> 0.85 for human samples (n=72<br />
measurements). Two rabbit samples could not be fitted with the model <strong>and</strong><br />
were excluded from analyses. Mean ka <strong>and</strong> kd values of all other measurements<br />
are shown in Table 1. Kinetic constants of rabbit <strong>and</strong> human samples covered a<br />
similar range. Values for kd were considerably smaller than for ka (~10 8 fold<br />
difference), reflecting a stable antigen-antibody complex.<br />
Antibody concentration (of binding antibodies) <strong>and</strong> ka values of pooled rabbit<br />
sera were inversely correlation (Figure 3A), approximated by inverse curve fit<br />
(p=0.002, R 2 = 0.871). The implication of these results are best illustrated by<br />
analysis of a simple non-covalent interaction system, whereby the increase in<br />
IgAg over time (δ[IgAg]/ δt) is described by concentration of immunoglobulin<br />
[Ig], antigen [Ag] <strong>and</strong> ka <strong>and</strong> kd as follows: δ[IgAg]/ δt = ka*[Ig]*[Ag] –<br />
kd*[IgAg].<br />
In a state of equilibrium, when the increase in immunoglobulin-antigen complex<br />
over time is zero, concentration <strong>and</strong> binding constants are balanced<br />
(ka*[Ig]*[Ag] = kd*[IgAg]). Algebraic rearrangement will result in ka/kd*[Ig] =<br />
[IgAg]/[Ag], where [IgAg]/[Ag] represents the fraction of antigen bound by<br />
antibody. Assuming kd is negligible, the observed inverse relationship in rabbit<br />
sera (Figure 3A) thus suggests that the fraction of bound antigen is constant <strong>and</strong><br />
identical for all investigated sera.
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