Anti-HFRS Human IgG Produced in Transchromosomic Bovines Has Potent Hantavirus Neutralizing Activity and Is Protective in Animal Models

Abstract

We explored an emerging technology to produce anti-Hantaan virus (HTNV) and anti-Puumala virus (PUUV) neutralizing antibodies for use as pre- or post-exposure prophylactics. The technology involves hyperimmunization of transchomosomic bovines (TcB) engineered to express human polyclonal IgG antibodies with HTNV and PUUV DNA vaccines encoding G_nG_c glycoproteins. For the anti-HTNV product, TcB was hyperimmunized with HTNV DNA plus adjuvant or HTNV DNA formulated using lipid nanoparticles (LNP). The LNP-formulated vaccine yielded fivefold higher neutralizing antibody titers using 10-fold less DNA. Human IgG purified from the LNP-formulated animal (SAB-159), had anti-HTNV neutralizing antibody titers >100,000. SAB-159 was capable of neutralizing pseudovirions with monoclonal antibody escape mutations in G_n and G_c demonstrating neutralization escape resistance. SAB-159 protected hamsters from HTNV infection when administered pre- or post-exposure, and limited HTNV infection in a marmoset model. An LNP-formulated PUUV DNA vaccine generated purified anti-PUUV IgG, SAB-159P, with a neutralizing antibody titer >600,000. As little as 0.33 mg/kg of SAB-159P protected hamsters against PUUV infection for pre-exposure and 10 mg/kg SAB-159P protected PUUV-infected hamsters post-exposure. These data demonstrate that DNA vaccines combined with the TcB-based manufacturing platform can be used to rapidly produce potent, human, polyclonal, escape-resistant anti-HTNV, and anti-PUUV neutralizing antibodies that are protective in animal models.

Keywords: HFRS; hamster; hantaan; hantavirus; marmoset; passive transfer; puumala; transchromosomic bovine.

FIGURE 1

Neutralizing antibody responses in TcB vaccinated with either HTNV or PUUV DNA vaccines. (A) TcB #2034 (black symbols) was vaccinated with unformulated HTNV DNA vaccine and a veterinary adjuvant at the times indicated by the black arrows, TcB #2026 (blue symbols) was vaccinated with a LMP-formulated HTNV DNA vaccine at the times indicated by the blue arrows, and TcB #2303 (red symbols) was vaccinated with a LNP-formulated PUUV DNA vaccine at the times indicated by the red arrows. Sera was collected from the TcB at the indicated timepoints and represents the geometric mean titer ± standard deviation of 2–5 independent assays (Days 14, 21, 56, and 84, all others represent data from a single assay). (B) SAB-159 and SAB-159P were evaluated for neutralizing (solid bars) and cross neutralizing (open bars) activity by PsVNA. (C) SAB-159 purified material stored at 4°C for the indicated number of days was evaluated by PRNT. Limit of quantitation is 20 (gray shaded area).

FIGURE 2

SAB-159 neutralizes MAb escape mutants. Pseudovirions with wildtype G_nG_c, single MAb escape mutations, or a double escape mutation were used in PsVNA to determine the effect mutations have on the capacity of antibody to neutralize virion entry into cells. (A) Anti-HTNV MAb 3D5, MAb HCO2, and a 1:1 MAb cocktail of those antibodies were evaluated in the PsVNA. The IC50 were plotted. (B) The anti-HTNV purified human IgG (SAB-159) prediluted at 1:30 and control polyclonal antibodies were evaluated by PsVNA. The PsVNA₅₀ titers were plotted. The positive control was anti-HTNV rabbit (rab) sera; the negative control was normal rabbit sera; and the negative control was purified IgG antibody from a naïve TcB (Negative IgG). *Indicates that the mutation(s) in the GnGc resulted in a >10-fold reduction in PsVNA₅₀ titer (escape). Limit of quantitation is 20 (gray shaded area).

FIGURE 3

SAB-159 protects hamsters when administered prior to HTNV exposure. (A) Hamsters were administered 10 mg/kg SAB-159 subcutaneously and serum collected and analyzed by HTNV PsVNA. The results of two experiments are shown for hamsters that were either serially bled following SAB-159 administration (n = 6, closed symbols) or hamsters bled on the indicated day following SAB-159 administration (n = 8, open symbols). GMT ± SEM are plotted. (B) The data from (A) was used to calculate the half-life of 12.1 days based on PsVNA₅₀ data (closed symbols used for calculation). (C) Characterization of the protective efficacy of SAB-159 against HTNV challenge was determined by N-ELISA when SAB-159 was administered in decreasing concentrations on Day -1 prior to HTNV challenge, (D) at 10 mg/kg at increasing timepoints prior to HTNV challenge, and (E) at 10 mg/kg with increasing concentrations of HTNV challenge doses. PsVNA₅₀ titers (open circles) are shown for hamsters in (C–E). The shaded gray area represents the limit of detection for the PsVNA assay. N-ELISA log₁₀ titers are displayed for individual hamsters, ≥2 is positive.

FIGURE 4

SAB-159 limits HTNV infection in marmosets. (A) Experimental design. Two groups of 3 marmosets each were administered either 71,871 NAU/kg (12.23 mg/kg) SAB-159 or control human IgG on Day -1 prior to HTNV challenge. Sera collection dates are shown in gray arrows. Serum from indicated times was analyzed by (B) N-ELISA and (C) PsVNA. The shaded gray area represents the limit of detection for the PsVNA assay.

FIGURE 5

Human IgG products protect hamsters from infection when administered post-exposure. (A) Hamsters were administered decreasing concentrations of SAB-159P on Day -1 prior to a 1,000 PFU PUUV challenge. Sera collected on Day 0 was analyzed by PUUV PsVNA (open circles) and Day 35 by N-ELISA (black bars). Characterization of the protective efficacy of (B) 10 mg/kg SAB-159 and (C) 10 mg/kg SAB-159P administered on indicated days post infection. For (B,C), sera was collected 1 day following passive transfer for analysis in PsVNA (open circles) and Day 35 for analysis in N-ELISA (black bars). (A–C) The shaded gray area represents the limit of detection for the PsVNA assay. N-ELISA log₁₀ titers are displayed for individual hamsters, ≥2 is positive.