Neutralizing activity of α-ANDV duck IgY/IgYΔFc <i>in vitro</i> and <i>in vivo</i>.

<p>A) Western blot analysis of IgY components recognized by α-duck IgY antibodies. A) represents IgY/IgYΔFc by Western blot with SDS-PAGE run under non-reducing conditions and probed with an α-duck IgY antibody recognizing the heavy chain of both IgY and IgYΔFc. HC is the heavy chain of IgY. B) Percent neutralization of α-ANDV FFP and α-ANDV duck IgYΔFc measured by ANDV PRNT. Dotted line represents 80% neutralization (PRNT). C) Neutralizing antibody bioavailability was determined by ANDV PRNT performed on hamster serum samples collected after passive transfer of α-ANDV duck IgYΔFc (12,000 NAU/kg and 64,000 NAU/kg) by the s.c. route on day 0, through 21 days. PRNT₈₀ titers (solid lines) and PRNT₅₀ titers (dashed lines) are plotted. D) Survival curve of hamsters challenged with 4,000 PFU of ANDV i.n. on day 0 and passively transferred with 5,000 NAU/kg of α-ANDV duck IgYΔFc on day 8 postinfection.</p

α-ANDV FFP effectively neutralizes ANDV <i>in vitro</i> and is detectable in hamsters after passive transfer.

<p>A) Neutralizing antibody titers were determined by ANDV PRNT₈₀ performed on α-ANDV FFP and normal human serum. * indicates results are statistically significant. B) Neutralizing antibody bioavailability was determined by ANDV PRNT₈₀ performed on hamster serum samples collected after passive transfer of α-ANDV FFP (64,000 NAU/kg) by either s.c. or i.m. route (3 hamsters per group) on day 0, through 21 days. PRNT₈₀ titers represent the lowest serum dilution neutralizing 80% of the plaques relative to the control (no serum). PRNT₅₀ values of a single group are denoted by a dashed line.</p

5,000 NAU/kg of α-ANDV FFP is sufficient to protect hamsters from lethal HPS disease.

<p>A) Survival curve of hamsters challenged with 4,000 PFU of ANDV i.n. on day 0 and passively transferred with dilutions of α-ANDV FFP on day 8. P-values were determined by comparing FFP dilution to no antibody control. B) α-N ELISA endpoint titers (log₁₀) were conducted with sera from surviving hamsters challenged with ANDV in A). GMT for each group are shown.</p

12,000 NAU/kg of anti-ANDV duck IgY/IgYΔFc protects hamsters from lethal HPS disease.

<p>A) Survival curve of hamsters that were challenged with 200 PFU i.m. of ANDV on day 0 and passively transferred with α-ANDV FFP (8 hamsters per group) or α-ANDV duck IgY/IgYΔFc (16 hamsters per group) on day 5 postinfection. * indicates statistical significance when compared to normal IgY/IgYΔFc treatment. B) α-N ELISA endpoint titers (log₁₀) were conducted with sera from surviving hamsters challenged with ANDV in A). GMT for each group are shown.</p

α-ANDV FFP passively transferred on days 5 and 8 protects hamsters from lethal disease and infection.

<p>A) Survival curve of hamsters challenged with 4,000 PFU i.n. of ANDV on day 0, then passively transferred with α-ANDV FFP (30,720 NAU/kg) on days 5, 8, 12 or 15 postinfection. Rabbit sera (administered at 1,920 NAU/kg) were collected 102 days post DNA vaccination <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035996#pone.0035996-Hooper2" target="_blank">[16]</a>. P-values were determined based on comparison to normal serum on matching day. B) α-N ELISA endpoint titers (log₁₀) were conducted with sera from surviving hamsters challenged with ANDV in A). GMT for each group are shown. * indicates results are statistically significant when compared to rabbit sera positive control.</p

12,000 NAU/kg of α-ANDV FFP and α-ANDV duck IgY/IgYΔFc protects hamsters from lethal HPS disease.

<p>A) and B) Survival curve of hamsters that were challenged with 4,000 PFU i.n. of ANDV on day 0 and passively transferred with α-ANDV FFP or α-ANDV duck IgY/IgYΔFc on day 5 postinfection (A) or day 8 postinfection (B). * indicates statistical significance when compared to normal IgY/IgYΔFc treatment. C) α-N ELISA endpoint titers (log₁₀) were conducted with sera from surviving hamsters challenged with ANDV in A) and B). GMT for each group are shown. * indicates results are statistically significant when compared to no antibody controls.</p

Antiviral Biologic Produced in DNA Vaccine/Goose Platform Protects Hamsters Against Hantavirus Pulmonary Syndrome When Administered Post-exposure

<div><p>Andes virus (ANDV) and ANDV-like viruses are responsible for most hantavirus pulmonary syndrome (HPS) cases in South America. Recent studies in Chile indicate that passive transfer of convalescent human plasma shows promise as a possible treatment for HPS. Unfortunately, availability of convalescent plasma from survivors of this lethal disease is very limited. We are interested in exploring the concept of using DNA vaccine technology to produce antiviral biologics, including polyclonal neutralizing antibodies for use in humans. Geese produce IgY and an alternatively spliced form, IgYΔFc, that can be purified at high concentrations from egg yolks. IgY lacks the properties of mammalian Fc that make antibodies produced in horses, sheep, and rabbits reactogenic in humans. Geese were vaccinated with an ANDV DNA vaccine encoding the virus envelope glycoproteins. All geese developed high-titer neutralizing antibodies after the second vaccination, and maintained high-levels of neutralizing antibodies as measured by a pseudovirion neutralization assay (PsVNA) for over 1 year. A booster vaccination resulted in extraordinarily high levels of neutralizing antibodies (i.e., PsVNA₈₀ titers >100,000). Analysis of IgY and IgYΔFc by epitope mapping show these antibodies to be highly reactive to specific amino acid sequences of ANDV envelope glycoproteins. We examined the protective efficacy of the goose-derived antibody in the hamster model of lethal HPS. α-ANDV immune sera, or IgY/IgYΔFc purified from eggs, were passively transferred to hamsters subcutaneously starting 5 days after an IM challenge with ANDV (25 LD₅₀). Both immune sera, and egg-derived purified IgY/IgYΔFc, protected 8 of 8 and 7 of 8 hamsters, respectively. In contrast, all hamsters receiving IgY/IgYΔFc purified from normal geese (n=8), or no-treatment (n=8), developed lethal HPS. These findings demonstrate that the DNA vaccine/goose platform can be used to produce a candidate antiviral biological product capable of preventing a lethal disease when administered post-exposure.</p></div

ANDV DNA vaccine is immunogenic in geese.

<p><b>A</b>. Eight geese were vaccinated i.m. with 1mg ANDV DNA vaccine pWRG/AND-M(opt) or pWRG/AND-M(1.1) at 2 week intervals (blue arrows) using the PharmaJet V1. One year later the same, geese were booster vaccinated again with 1mg ANDV DNA pWRG/AND-M(opt) or pWRG/AND-M(opt2). Sera was collected from the vaccinated geese during the times indicated by red arrows. Egg collection is indicated by yellow ovals. <b>B.</b> Goose serum titers following initial vaccination series and <b>C.</b> long-range boost by PsVNA. Black lines indicate geese boosted with pWRG/AND-M(opt) and red lines indicate hamsters boosted with pWRG/AND-M(opt2). <b>D.</b> Mean of geese vaccinated initially with pWRG/AND-M and boosted with pWRG/AND-M(opt) (Group A) and pWRG/AND-M(opt2) (Group B).</p

Identification of ANDV glycoprotein G_n and G_c epitopes.

<p>Microarray slides were incubated with IgY, IgYΔFc, or IgY/IgYΔFc isolated for egg yolks of vaccinated either after the initial or booster vaccination series, or serum from vaccinated rabbits (first column- amino acid peptide starts with, second column- IgY from egg yolks after initial vaccination series, third column- IgYΔFc from egg yolks after initial vaccination series, fourth column- IgY/IgYΔFc from egg yolks after initial vaccination series, fifth column- IgY/IgYΔFc from egg yolks after booster vaccination, sixth column- sera from vaccinated rabbit). Reactivity was measured based on a spectrum ranging from no activity in white, mild reactivity in gray, to strong reactivity in red. Epitopes regions marked with a (*) indicate epitopes of high interest after long-range booster vaccination. (::) indicate previously published amino acid regions with which human sera from HPS patients were strongly reactive [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003803#pntd.0003803.ref034" target="_blank">34</a>]. (♦) indicate unique epitopes that rabbit IgG recognized strongly compared to IgY/IgYΔFc from eggs of vaccinated geese.</p

α-ANDV IgY/IgYΔFc administered after the onset of viremia does not protect hamsters from lethal HPS.

<p><b>A.</b> Survival curve of hamsters that were challenged with 200 PFU i.m. of ANDV on day 0 and passively transferred with 40,000 NAU/kg α-ANDV IgY/IgYΔFc, 40,000 NAU/kg α-ANDV human FFP, normal IgY, or normal FFP on days 8 and 10 post-infection (grey arrows). n = 8 for all groups. <b>B.</b> Serum and lung isolated from ANDV-infected hamsters on day 10 were evaluated for viral genome by RT-PCR. Symbols represent lung tissue titers from different animals. The mean for each group is also shown (line).</p