Novel Camelid Antibody Fragments Targeting Recombinant Nucleoprotein of <i>Araucaria hantavirus</i>: A Prototype for an Early Diagnosis of Hantavirus Pulmonary Syndrome

<div><p>In addition to conventional antibodies, camelids produce immunoglobulins G composed exclusively of heavy chains in which the antigen binding site is formed only by single domains called VHH. Their particular characteristics make VHHs interesting tools for drug-delivery, passive immunotherapy and high-throughput diagnosis. Hantaviruses are rodent-borne viruses of the Bunyaviridae family. Two clinical forms of the infection are known. Hemorrhagic Fever with Renal Syndrome (HFRS) is present in the Old World, while Hantavirus Pulmonary Syndrome (HPS) is found on the American continent. There is no specific treatment for HPS and its diagnosis is carried out by molecular or serological techniques, using mainly monoclonal antibodies or hantavirus nucleoprotein (N) to detect IgM and IgG in patient serum. This study proposes the use of camelid VHHs to develop alternative methods for diagnosing and confirming HPS. Phage display technology was employed to obtain VHHs. After immunizing one <i>Lama glama</i> against the recombinant N protein (prNΔ₈₅) of a Brazilian hantavirus strain, VHH regions were isolated to construct an immune library. VHHs were displayed fused to the M13KO7 phage coat protein III and the selection steps were performed on immobilized prNΔ₈₅. After selection, eighty clones recognized specifically the N protein. These were sequenced, grouped based mainly on the CDRs, and five clones were analyzed by western blot (WB), surface plasmon resonance (SPR) device, and ELISA. Besides the ability to recognize prNΔ₈₅ by WB, all selected clones showed affinity constants in the nanomolar range. Additionaly, the clone KC329705 is able to detect prNΔ₈₅ in solution, as well as the native viral antigen. Findings support the hypothesis that selected VHHs could be a powerful tool in the development of rapid and accurate HPS diagnostic assays, which are essential to provide supportive care to patients and reduce the high mortality rate associated with hantavirus infections.</p></div

Kinetic analysis and ranking of selected llama anti-prNΔ₈₅ VHH clones; ranking by highest to lowest affinity.

<p>k_on - association rate constant; k_off - dissociation rate constant; K_D - equilibrium dissociation constant; R_max - response at saturation; Chi² - mean squared of the signal noise.</p><p>Kinetic analysis and ranking of selected llama anti-prNΔ₈₅ VHH clones; ranking by highest to lowest affinity.</p

Llama immunization schedule.

<p>The <i>Lama glama</i> was immunized with the recombinant nucleoprotein ARAUV, strain HPR 02-72. The blood was collected from the jugular vein seven days after the third immunization.</p><p>Llama immunization schedule.</p

Detection of hantavirus naturally infected rodents by the assay using anti-prNΔ₈₅VHH (KC329705) as captor.

<p>To detect viral antigens in rodent serum samples, about 500 ng/well of KC329705 anti-prNΔ₈₅ VHH were adsorbed in microwell plates. Unspecific sites were blocked and infected rodent sera, diluted 1∶100 in blocking buffer, were added to the wells. After washing, the Mab 432/6BF was used as tracers. The wells were washed and the goat anti-mouse IgG peroxidase conjugated incubated. During the detection of native viral antigen, the hantavirus recombinant nucleoprotein (200 ng/well) was used as a positive control. While the negative control was performed adding no anti-prNΔ₈₅VHHs to the plates.</p

Monitoring the llama immune response by ELISA.

<p>The animal showed a rapid and strong response against the prNΔ85 protein after the second immunization (i.e., by Day 21; see immunization schedule <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108067#pone-0108067-t001" target="_blank">Table 1</a>). A sharp decrease in the humoral response was observed at day 28, when the third immunization was carried out. The final bleed performed on day 35, showed an antiserum titre of 1.0×10⁻⁶. The arrows indicate the immunization days.</p

Surface plasmon resonance analysis to show specific binding of VHH and monoclonal antibody.

<p>Sensorgrams obtained after injection of purified VHHs (KC329704–329708) at concentrations of 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.063, 0.0313, 0.0156, 0.00781, 0.0039 µM.</p

ELISA using VHH for hantavirus recombinant viral antigen detection in solution.

<p>For detection of soluble prNΔ₈₅, 500 ng/well of KC329704, KC329705, and KC329706 anti-prNΔ₈₅VHHs were adsorbed to a solid surface. After blocking unspecific bind sites, 0, 100, and 200 ng/well of prNΔ₈₅ were added to the reaction. Subsequently, Mab 432/6BF was added and the reaction revealed ater incubation of goat anti-mouse IgG peroxidase conjugated and TMB substrate. To perform the negative control no anti-prNΔ₈₅VHHs were added to the plates. The clone KC329705 was able to detect up to 100 ng of recombinant antigen in solution.</p

<i>In vitro</i> inhibition of phospholipase activity.

<p>The quantification of phospholipase activity inhibition by selected VHHs was assayed using synthetic fluorescent phospholipid. To verify the ability of VHHs to inhibit the phospholipase activity of BthTX-II, the toxin was pre-incubated with selected VHHs for 30 minutes at 37°C in different proportions (1:5; 1:10 and 1:40 w/w). Inhibition of phospholipase activity by (A) KF498607; (B) KF498608; (C) KF329715; (D) KC329718. BthTX-II activity on the phospholipid, in the absence of VHH, was used as a positive control, and considered as having 100% activity. The negative controls were carried out using medium reaction without BthTX-II. All measurements were performed in duplicate. Error bars represent standard deviation.</p

Western blot of purified VHHs against BthTX-I.

<p>15% SDS-PAGE was carried out to resolve BthTX-I and BthTX-II under reducing conditions. After electrophoresis, BthTX-I and BthTX-II were electrotransferred to a nitrocellulose membrane and probed with selected VHHs (KF498607, KF498608, KC329715, and KC329718). Samples were incubated with mouse anti-His antibody followed by HRP-conjugated anti-mouse IgG produced in goat. Reactive signals were detected by DAB staining in the presence of hydrogen peroxide. <i>Lama glama</i> pre-immune serum was used as negative control (-), and <i>Lama glama</i> immune serum, as positive control (+).</p

VHHs affinity to BthTX-I and BthTX-II.

<p>Representative sensorgrams of the interaction were measured in a Biacore T200 system (GE Healtcare). Purified VHHs (KF498607, KF498608 and KC329718) at concentrations of 6.25 to 0.049 μg/mL were flowed over a BthTX-I-coated CM5 chip and 25 to 0.006 μg/mL were used for immobilized BthTX-II. (A) KF498607 x BthTX-I; (B) KF498607 x BthTX-II; (C) KF498608 x BthTX-I; (D) KF498608 x BthTX-II; (E) KC329718 x BthTX-I; (F) KC329718 x BthTX-II. Assays were injected in a flow rate of 30 μl/min at 37°C. RU indicates resonance units.</p