Intranasal antibody gene transfer in mice and ferrets elicits broad protection against pandemic influenza

Abstract

The emergence of a new influenza pandemic remains a threat that could result in a substantial loss of life and economic disruption worldwide. Advances in human antibody isolation have led to the discovery of monoclonal antibodies (mAbs) that have broad neutralizing activity against various influenza strains, although their direct use for prophylaxis is impractical. To overcome this limitation, our approach is to deliver antibody via adeno-associated virus (AAV) vectors to the site of initial infection, which, for respiratory viruses such as influenza, is the nasopharyngeal mucosa. AAV vectors based on serotype 9 were engineered to express a modified version of the previously isolated broadly neutralizing mAb to influenza A, FI6. We demonstrate that intranasal delivery of AAV9.FI6 into mice afforded complete protection and log reductions in viral load to 100 LD₅₀ (median lethal dose) of three clinical isolates of H5N1 and two clinical isolates of H1N1, all of which have been associated with historic human pandemics (including H1N1 1918). Similarly, complete protection was achieved in ferrets challenged with lethal doses of H5N1 and H1N1. This approach serves as a platform for the prevention of natural or deliberate respiratory diseases for which a protective antibody is available.

Fig. 1. AAV9 transduction of airway epithelia and protection against a mouse-adapted strain of H1N1

BALB/c mice were dosed with AAV9 vectors expressing either LacZ or ffLuc and subjected to histological and imaging analyses. (A to J) The vector was administered in either 20 μl (A to E) or 50 μl (F to J), and analyses of LacZ expression were performed 14 days later including en face of nasal septum (A and F) and histological sections of nasal septum (B and G) and lung (C and H). Mice were imaged for ffLuc expression at multiple days with a representative image at day 14 shown in (D) and (I) and a time course shown in (E) and (J). Background luminescence was about 4 × 10⁵ photons/s. (K, L, O, and P) BALB/c mice (n = 5 per group) were dosed intranasally with AAV9.FI6 and subsequently challenged with 10 LD₅₀ of A/PR8 delivered intranasally. Animals were followed for weights and survival as depicted in the Kaplan-Meier plots. (K) and (L) present mice treated with neuraminidase (NA) and dosed with 10¹¹ GC/50 μl of a control AAV9 vector expressing an irrelevant human IA (PG9), as well as those dosed with 10¹¹ GC of AAV9.FI6 in either 20 or 50 μl. (M and N). BALB/c mice (n = 5 per group) were treated with NA as above and then dosed with increasing amounts (3 × 10⁹, 10¹⁰, 3 × 10¹⁰, and 10¹¹ GC per mouse) of AAV9.FI6 vector to determine the minimal protective dose. Fourteen days later, vector-treated and naïve mice were challenged with 10 LD₅₀ of A/PR8 delivered intranasally in 50 μl. The weight of challenged mice was monitored daily. (O) and (P) present animals dosed with 10¹¹ GC of AAV9.FI6 in 50 μl and challenged 1, 3, 4, or 7 days after vector dosing; naïve mice were dosed with phosphate-buffered saline (PBS). A potentiator of AAV9-airway transduction, NA, did not enhance protection in this model (fig. S3) and was therefore excluded from subsequent experiments. *P ≤ 0.05, Student’s t test; ^####P ≤ 0.0001, Mantel-Cox test; ****P ≤ 0.0001, Dunnett’s test. n.s., not significant. Dotted lines denote 30% weight loss.

Fig. 2. AAV9.FI6 protects mice against pandemic strains of H5N1 and H1N1

BALB/c mice were given 10¹¹ GC of AAV9.FI6 vector in 50 μl intranasally (n = 15 per group). (A to O) Fourteen days later, vector-treated and naïve (n = 10) mice were challenged intranasally with 100 LD₅₀ of three different strains of H5N1 [A/Indonesia/5/2005 (A to C), A/Vietnam/1203/2004 (D to F), and A/Hong Kong/156/1997 (G to I)] and two different strains of H1N1 [A/Mexico/InDRE4114/2009 (J to L) and A/South Carolina/1/1918 (M to O)]. The weights of the animals were followed daily (first column), and mice were euthanized when they appeared in distress or their body weight declined >30% as depicted in the Kaplan-Meier plots (second column). Four mice from the naïve and vector-treated groups were necropsied at day 6 to quantify viral load in the lung (right column). The viral load was also quantified at the conclusion of the experiments in surviving animals (day 28). Diamonds and squares represent AAV9.FI6 vector–treated and naïve mice, respectively. *P ≤ 0.05, ****P ≤ 0.0001, Mann-Whitney test; ^#P ≤ 0.05, ***P ≤ 0.001, Mantel-Cox test.

Fig. 3. AAV9-mediated transduction of the ferret nasal airway

(A) Distribution of luminescence in the nasal cavity of a representative ferret 7 days after intranasal delivery of 10¹² GC of AAV9.ffLuc. (B) En face view of the nasal septum (S) of a ferret administered 10¹² GC of AAV9.nLacZ. (C) rhAFP expression in the NLF of ferrets collected 7 days after the intranasal administration of 10¹² GC of AAV9.rhAFP. For the influenza challenge experiments, ferrets were dosed with 10¹² GC of AAV9.FI6 vector given intranasally in 400 μl (200 μl per nare) to target the nasopharynx. (D and E) Seven days later, the ferrets were challenged with a lethal dose of either A/California/07/2009 (H1N1) (D) given under ABSL2 conditions or A/Vietnam/1203/2004 (H5N1) (E) given under ABSL3 conditions. The challenged animals were followed for weight loss and signs of respiratory distresses per guidelines. Circles, treated; triangles, naïve. (F) Fitch ferrets (n = 8 for the AAV9.FI6 group, n = 6 for naïve controls) were administered intranasally with 10¹² GC AAV.FI6 and challenged intranasally with 100 LD₅₀ (10⁶ PFU) of Mx10 7 days after treatment, and survival was monitored. Diamonds, treated; squares, naïve. (G) Viral load in nasal washes was monitored after challenge. Each data point represents viral load from an individual animal. Closed circles and triangles depict vector-treated and naïve ferrets, respectively. *P ≤ 0.05, ***P ≤ 0.001, Mann-Whitney test.

Fig. 4. AAV9-mediated transduction of the rhesus macaque nasal airway

An AAV9 vector (10¹³ GC) expressing rhesus-derived AFP was administered into the left nostril of two rhesus macaques. Sequential nasal lavage samples were harvested and analyzed for total protein and rhAFP.