Fig 1.
The schedule for immunization (inverted triangle), sample collection and final bleeding (rhombus) is shown. The evaluation of NoV Ab response in serum during the time course of immunization is depicted for: a) Norwalk and b) MD2004. Antibody titers were measured by ELISA (lines) and the number of ASC for each llama were measured by ELISPOT (bars) using recombinant NoV VLPs Norwalk and MD2004.
Table 1.
Selection of VHH for characterization.
Table 2.
Specificity of the VHHs to the P or S domain of VP1
Table 3.
Reactivity of the VHHs against VLPs representing strains from different genotypes.
Fig 2.
Surrogate virus neutralization tests were performed using different sources of carbohydrates: (a) synthetic carbohydrates H1 for Norwalk GI.1 VLPs or H3 for GII.4 MD145 VLPs; (b), PGM type III for Norwalk GI.1 or MD2004 GII.4 VLPs and (c) saliva for Norwalk GI.1 or MD145 GII.4 VLPs. Sigmoidal curves were fit to the mean percent control binding calculated by comparing the amount of VLP bound to each source of carbohydrate in the presence of VHH pretreatment to the amount of VLP bound in the absence of pretreatment.
Fig 3.
Hemagglutination inhibition (HAI) assay.
The HAI titer of each VHH was defined as the lowest antibody concentration that completely prevented NoV VLP-mediated hemagglutination of human RBCs. Norwalk or P7-587 GI.1 VLPs hemagglutinated 0 Rh- RBC (a); MD2004 or MD145 GII.4 VLPs hemagglutinated B Rh+ (b).
Table 4.
Summary of VHH activity in different surrogate neutralizing assays.
Fig 4.
Hyperimmune llama serum blocking assay.
The percentage of VHH binding to hyperimmune serum pretreated VLPs was calculated compared to the positive control of each VHH binding to the VLPs without pretreatment. Bars represent the average of two independent assays.
Fig 5.
Competition assay between VHHs.
The images show immunofluorescence staining of Vero cells expressing NoV VP1 from Norwalk strain (a) or MD2004 strain (b). Ten μg of an unlabeled VHH together with 1 μl of Alexa Fluor 568 labeled VHH were incubated on the fixed cells. A decrease in the fluorescent signal indicates a competitive binding with the labeled VHH.
Table 5.
Kinetic constants for different VHH determined by Biacore binding assays.
Fig 6.
a) Association (light gray block) and dissociation (dark grey block) profiles for VHH M1 or M5 binding to MD2004 VLP coupled on the sensor surface. VHH M1 concentrations spanned from 45 to 1226 nM and VHH M5 concentrations spanned from 6 to 167 nM. The surface density of VLPs was 3000 RU for both VHHs. Red lines correspond to the fit of a 1:1 (one step) binding model. b) Sequential binding of VHH M1 and VHH M5 to coupled MD2004 VLPs. VHH M1 injected at 450 nM completely inhibited the binding of 5.57 nM. Inhibition was overcome with 150 nM VHH M5. VHH M5 was injected at 450 nM but did not modulate VHH M1 binding as shown in the case of the 5.57 or 150 nM injection of VHH M1. Contact times for both injections were 180 s. The black profiles correspond to the kinetics of binding of VHH (corresponding case) binding to MD2004 while the red profiles highlight the binding profile in the presence of VHH.
Fig 7.
a) ELISA with overlapping peptides corresponding to the P domain of VP1 NoV. b) Putative linear epitope of VHH M6 shown in the structure of a GII.4 P dimer. Subdomains of norovirus VP1 are represented in different colors, P1 in light grey and P2 in dark grey. The putative M6 epitope is shown in green.
Fig 8.
Epitope mapping of clone M7 specific for the GII.4 MD2004 strain.
a) Expression of MD2004 VP1 variants in Vero cells. The images show immunofluorescence staining of Vero cells transfected with pCI constructs carrying VP1 from wild type MD 2004 and five P domain mutants: E376Q, G340A, N368T, V3891I, and V340/E376. VHH M7 binding was detected with rabbit anti-VHH polyclonal serum and anti-rabbit IgG labeled with Alexa Fluor 488 dye. Labeled MAb TV20 directed to the S domain was used as positive control. b) P domain structure showing the amino acids involved in VHH M7 epitope (light blue). Subdomains of norovirus VP1 are represented in different colors, P1 in light grey and P2 in dark grey.