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

Abstract

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.

Fig 1. ANDV DNA vaccine is immunogenic in geese.

A. 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. Goose serum titers following initial vaccination series and C. 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). D. 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).

Fig 2. IgY/IgYΔFc from eggs collected from vaccinated geese has high-titer neutralizing activity.

A. Total IgY was purified from eggs collected after the initial vaccination series and long-range boost and evaluated for α-ANDV neutralizing activity by PsVNA. The red box indicates samples that were pooled for future animal experiments. B. Purified IgY was visualized by Coomassie stain.

Fig 3. Identification of ANDV glycoprotein G_n and G_c epitopes.

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 [34]. (♦) indicate unique epitopes that rabbit IgG recognized strongly compared to IgY/IgYΔFc from eggs of vaccinated geese.

Fig 4. Bioavailability of α-ANDV IgY/IgYΔFc in Syrian hamsters.

Groups of three hamsters were injected with 64,000 NAU/kg (open circles) or 12,000 NAU/kg (crosses) α-ANDV IgY/IgYΔFc by the s.c. route. Sera collected on days 1, 3, and 6 were evaluated for ANDV neutralizing activity by PsVNA. Each point represents the mean PsVNA80 titer ±SE. The limit of detection for this assay is represented by a dashed line.

Fig 5. α-ANDV IgY/IgYΔFc administered prior to the onset of viremia protects hamsters from lethal HPS.

A. Survival curve of hamsters that were challenged with 200 PFU i.m. of ANDV on day 0 and passively transferred with 20,000 NAU/kg α-ANDV goose sera, 20,000 NAU/kg α-ANDV goose IgY/IgYΔFc, 20,000 NAU/kg α-ANDV rabbit sera, normal goose sera, or normal goose IgY on days 5 and 8 post-infection (grey arrows). n = 8 for all groups. 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. The limit of detection, a titer of 100, is shown as a dotted line.

Fig 6. α-ANDV IgY/IgYΔFc administered after the onset of viremia does not protect hamsters from lethal HPS.

A. 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. 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).