Experimental infection and protection against ... - TI Pharma
Experimental infection and protection against ... - TI Pharma Experimental infection and protection against ... - TI Pharma
Humoral immune responses to a single allele PfAMA1 vaccine in healthy malarianaïve adults. Avidity ELISA The avidities of the antibodies were determined by sodium isothiocyanate (NaSCN) elution ELISA as previously described [16]. Briefly, microtitre plates were coated with AMA1 variant proteins (Figure 1) as described above, and after blocking, incubated with a pre-determined titre (1 AU) of sera for 1 h. Plates were then washed and incubated with an increasing concentration of NaSCN (ranging from 0 to 3.0 M in 0.25 M steps) in duplicate wells for 15 min. Plates were washed and developed with goat anti-human IgG alkaline phosphatase conjugate and substrate as previously described [16]. The avidity index is expressed as the concentration of NaSCN required for 50% dissociation of bound antibodies (relative to duplicate wells without NaSCN). Growth Inhibition Assay (GIA) Antibodies used for growth inhibition assays (GIA) were purified from CPT plasma on protein A columns (Immunopure Plus Pierce, St Louis, MO, USA), exchanged into RPMI 1640 using Amicon Ultra-15 concentrators (30 kDa cut-off, Millipore, Ireland), filter-sterilised and stored at -20°C until use. IgG concentrations were determined using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). P. falciparum strains FCR3, 3D7 and HB3 were cultured in vitro using standard culture techniques in an atmosphere of 5% CO2, 5% O2 and 90% N2. FCR3 AMA1 (accession no. M34553) differs by one amino acid in the pro-sequence (D36G) from FVO AMA1 (accession no. AJ277646). The ectodomain (amino acids 25-545) of 3D7 (accession no. U65407) differs by 26 amino acids (2, 17, 5 and 2 for prodomain and domains I, II and III, respectively) from FVO and the ectodomain of HB3 (accession no. U33277) differs by 21 amino acids (2, 12, 4 and 3 for prodomain and domains I, II and III, respectively) from FVO. The GIA was performed as previously described [14]. Briefly, the effect of purified IgG antibodies on in vitro parasite growth was evaluated at two IgG concentrations (5 and 10 mg/mL, respectively) and each participants preimmune IgG was used as negative control. A IgG concentration of 10 mg /ml approximates the amount of IgG (9.5 to 11.5 mg /ml) found in undiluted human plasma [21]. Samples were run in triplicate using 96 well flat-bottomed plates with alanine-synchronized cultures of P. falciparum schizonts at an initial parasitemia of 0.2–0.4%, a haematocrit of 2.0% and a final volume of 100 µL. After 40 to 42 hours, cultures were resuspended, and 50 µL was transferred into 200 µL ice-cold PBS. The cultures were then centrifuged, the supernatant 63
64 Chapter 3 AMA1 IgG (AU/mL) 65536 32768 16384 8192 4096 2048 1024 512 FVO Alhydrogel 10 Alhydrogel 50 ISA 720 10 ISA 720 50 AS02 10 AS02 50 3D7 Alhydrogel 10 Alhydrogel 50 ISA 720 10 ISA 720 50 AS02 10 AS02 50 Alhydrogel 10 Alhydrogel 50 ISA 720 10 ISA 720 50 AS02 10 AS02 50 Treatment removed and the plates were frozen. Parasite growth was assessed by measuring parasite lactate dehydrogenase levels with the lactate diaphorase APAD substrate system, and plates were read at 655 nm after 30 min incubation in the dark. Parasite growth inhibition was expressed as: 100 x (1 – (OD655 Day84 – OD655 RBC)/(OD655 Day0 – OD655 RBC)), Where: OD655Day84 is the OD655 for IgG purified from day 84 plasma, OD655Day0 is the OD655 for IgG purified from day 0 plasma and OD655RBC is the average OD655 of RBC control wells. The data are presented as the arithmetic mean % inhibition from each sample triplicate. Statistics Antibody titres (IgG and IgG subclasses) were log-transformed to obtain normality and are presented as geometric means with 95% confidence intervals. GIA titres, IgG avidity and levels of depletion were approximately normally distributed and are therefore presented as arithmetic means with 95% confi- HB3 CAMP Alhydrogel 10 Alhydrogel 50 ISA 720 10 ISA 720 50 AS02 10 AS02 50 Figure 2: IgG levels to the vaccine allele and three variant AMA1 alleles in serum obtained on day 84. IgG antibody levels to the vaccine antigen (FVO) and 3 heterologous AMA1 alleles (3D7, HB3 and CAMP). Values for each subject within a treatment group are indicated by a specific symbol (same symbol within each treatment group is same subject throughout graphs 2, 4, 5, 6 and 7), boxes indicate median and quartile ranges. Treatment groups are indicated by Adjuvant name and AMA1 dose, respectively.
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64 Chapter 3<br />
AMA1 IgG (AU/mL)<br />
65536<br />
32768<br />
16384<br />
8192<br />
4096<br />
2048<br />
1024<br />
512<br />
FVO<br />
Alhydrogel 10<br />
Alhydrogel 50<br />
ISA 720 10<br />
ISA 720 50<br />
AS02 10<br />
AS02 50<br />
3D7<br />
Alhydrogel 10<br />
Alhydrogel 50<br />
ISA 720 10<br />
ISA 720 50<br />
AS02 10<br />
AS02 50<br />
Alhydrogel 10<br />
Alhydrogel 50<br />
ISA 720 10<br />
ISA 720 50<br />
AS02 10<br />
AS02 50<br />
Treatment<br />
removed <strong>and</strong> the plates were frozen. Parasite growth was assessed by<br />
measuring parasite lactate dehydrogenase levels with the lactate diaphorase<br />
APAD substrate system, <strong>and</strong> plates were read at 655 nm after 30 min incubation<br />
in the dark. Parasite growth inhibition was expressed as: 100 x (1 – (OD655<br />
Day84 – OD655 RBC)/(OD655 Day0 – OD655 RBC)), Where: OD655Day84 is the<br />
OD655 for IgG purified from day 84 plasma, OD655Day0 is the OD655 for IgG<br />
purified from day 0 plasma <strong>and</strong> OD655RBC is the average OD655 of RBC control<br />
wells. The data are presented as the arithmetic mean % inhibition from each<br />
sample triplicate.<br />
Statistics<br />
Antibody titres (IgG <strong>and</strong> IgG subclasses) were log-transformed to obtain<br />
normality <strong>and</strong> are presented as geometric means with 95% confidence intervals.<br />
GIA titres, IgG avidity <strong>and</strong> levels of depletion were approximately normally<br />
distributed <strong>and</strong> are therefore presented as arithmetic means with 95% confi-<br />
HB3<br />
CAMP<br />
Alhydrogel 10<br />
Alhydrogel 50<br />
ISA 720 10<br />
ISA 720 50<br />
AS02 10<br />
AS02 50<br />
Figure 2: IgG levels to the vaccine allele <strong>and</strong> three variant AMA1 alleles in serum<br />
obtained on day 84. IgG antibody levels to the vaccine antigen (FVO) <strong>and</strong> 3<br />
heterologous AMA1 alleles (3D7, HB3 <strong>and</strong> CAMP). Values for each subject within a<br />
treatment group are indicated by a specific symbol (same symbol within each<br />
treatment group is same subject throughout graphs 2, 4, 5, 6 <strong>and</strong> 7), boxes indicate<br />
median <strong>and</strong> quartile ranges. Treatment groups are indicated by Adjuvant name <strong>and</strong><br />
AMA1 dose, respectively.
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