DNA Vaccine-Generated Duck Polyclonal Antibodies as a Postexposure Prophylactic to Prevent Hantavirus Pulmonary Syndrome (HPS)

Andes virus (ANDV) is the predominant cause of hantavirus pulmonary syndrome (HPS) in South America and the only hantavirus known to be transmitted person-to-person. There are no vaccines, prophylactics, or therapeutics to prevent or treat this highly pathogenic disease (case-fatality 35–40%). Infection of Syrian hamsters with ANDV results in a disease that closely mimics human HPS in incubation time, symptoms of respiratory distress, and disease pathology. Here, we evaluated the feasibility of two postexposure prophylaxis strategies in the ANDV/hamster lethal disease model. First, we evaluated a natural product, human polyclonal antibody, obtained as fresh frozen plasma (FFP) from a HPS survivor. Second, we used DNA vaccine technology to manufacture a polyclonal immunoglobulin-based product that could be purified from the eggs of vaccinated ducks (Anas platyrhynchos). The natural “despeciation" of the duck IgY (i.e., Fc removed) results in an immunoglobulin predicted to be minimally reactogenic in humans. Administration of ≥5,000 neutralizing antibody units (NAU)/kg of FFP-protected hamsters from lethal disease when given up to 8 days after intranasal ANDV challenge. IgY/IgYΔFc antibodies purified from the eggs of DNA-vaccinated ducks effectively neutralized ANDV in vitro as measured by plaque reduction neutralization tests (PRNT). Administration of 12,000 NAU/kg of duck egg-derived IgY/IgYΔFc protected hamsters when administered up to 8 days after intranasal challenge and 5 days after intramuscular challenge. These experiments demonstrate that convalescent FFP shows promise as a postexposure HPS prophylactic. Moreover, these data demonstrate the feasibility of using DNA vaccine technology coupled with the duck/egg system to manufacture a product that could supplement or replace FFP. The DNA vaccine-duck/egg system can be scaled as needed and obviates the necessity of using limited blood products obtained from a small number of HPS survivors. This is the first report demonstrating the in vivo efficacy of any antiviral product produced using DNA vaccine-duck/egg system

Humoral immunogenicity of a Coronavirus Disease 2019 (COVID-19) DNA vaccine in rhesus macaques (Macaca mulatta) delivered using needle-free jet injection

A SARS-CoV-2 DNA vaccine targeting the spike protein and delivered by jet injection, nCOV-S(JET), previously shown to protect wild-type and immunosuppressed Syrian hamsters (Mesocricetus auratus), was evaluated via two needle-free delivery methods in rhesus macaques (Macaca mulatta). The methods included intramuscular delivery of 2 mg per vaccination with the PharmaJet Stratis device and intradermal delivery of 0.4 mg per vaccination with the PharmaJet Tropis device. We hypothesized that the nCOV-S(JET) vaccine would mount detectable neutralizing antibody responses when delivered by needle-free jet injection by either the intradermal or intramuscular route. When delivered intramuscularly, the vaccines elicited neutralizing and variant (Beta, Gamma, and Delta) cross-neutralizing antibodies against SARS-CoV-2 in all six animals after three vaccinations. The neutralizing response to Omicron was lower with only 4 of 6 animals responding. When delivered at a lower dose by the intradermal route, strong neutralizing antibody responses were only detected in two of six animals. This study confirms that a vaccine previously shown to protect in a hamster model can elicit neutralizing and cross-neutralizing antibodies against SARS-CoV-2 in nonhuman primates. We posit that nCOV-S(JET) has the potential for use as booster vaccine in heterologous vaccination strategies against COVID-19

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

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 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

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

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

Side-by-Side Comparison of Gene-Based Smallpox Vaccine with MVA in Nonhuman Primates

<div><p>Orthopoxviruses remain a threat as biological weapons and zoonoses. The licensed live-virus vaccine is associated with serious health risks, making its general usage unacceptable. Attenuated vaccines are being developed as alternatives, the most advanced of which is modified-vaccinia virus Ankara (MVA). We previously developed a gene-based vaccine, termed 4pox, which targets four orthopoxvirus antigens, A33, B5, A27 and L1. This vaccine protects mice and non-human primates from lethal orthopoxvirus disease. Here, we investigated the capacity of the molecular adjuvants GM-CSF and <em>Escherichia coli</em> heat-labile enterotoxin (LT) to enhance the efficacy of the 4pox gene-based vaccine. Both adjuvants significantly increased protective antibody responses in mice. We directly compared the 4pox plus LT vaccine against MVA in a monkeypox virus (MPXV) nonhuman primate (NHP) challenge model. NHPs were vaccinated twice with MVA by intramuscular injection or the 4pox/LT vaccine delivered using a disposable gene gun device. As a positive control, one NHP was vaccinated with ACAM2000. NHPs vaccinated with each vaccine developed anti-orthopoxvirus antibody responses, including those against the 4pox antigens. After MPXV intravenous challenge, all control NHPs developed severe disease, while the ACAM2000 vaccinated animal was well protected. All NHPs vaccinated with MVA were protected from lethality, but three of five developed severe disease and all animals shed virus. All five NHPs vaccinated with 4pox/LT survived and only one developed severe disease. None of the 4pox/LT-vaccinated animals shed virus. Our findings show, for the first time, that a subunit orthopoxvirus vaccine delivered by the same schedule can provide a degree of protection at least as high as that of MVA.</p> </div