Broad protection against influenza infection by vectored immunoprophylaxis in mice

Abstract

Neutralizing antibodies that target epitopes conserved among many strains of influenza virus have been recently isolated from humans. Here we demonstrate that adeno-associated viruses (AAV) encoding two such broadly neutralizing antibodies are protective against diverse influenza strains. Serum from mice that received a single intramuscular AAV injection efficiently neutralized all H1, H2 and H5 influenza strains tested. After infection with diverse strains of H1N1 influenza, treated mice showed minimal weight loss and lung inflammation. Protection lasted for at least 11 months after AAV injection. Notably, even immunodeficient and older mice were protected by this method, suggesting that expression of a monoclonal antibody alone is sufficient to protect mice from illness. If translated to humans, this prophylactic approach may be uniquely capable of protecting immunocompromised or elderly patient populations not reliably protected by existing vaccines.

Figure 1

VIP results in expression of broadly neutralizing influenza antibodies in mouse serum. (a) Crystal structure of the HA trimer from the PR/34 strain of influenza (PDB code 1RU7). Residues are colored according to their variability in the five influenza strains used in this study, with the most variable residues shown in red and invariant residues shown in green. Variability is calculated as the site entropy. The binding of the V_H/V_L domains of the F10 (red) and CR6261 (blue) antibodies is modeled in the context of the HA trimer (PDB code 1RU7) based on alignment with PDB structures of antibodies bound to HA monomer (PDB codes 3FKU and 3GBN, respectively). These antibodies bind to the conserved stalk rather than the variable head of the HA. (b) Matrix of pairwise identities of the HA protein sequences used in this study calculated as the fraction of amino acids that were identical between two sequences and colored according to the degree of identity (green is most similar and red is least similar). PR/34: A/PR/8/1934 (H1N1); CA/09: A/California/7/2009 (H1N1); SI/06: A/Solomon Islands/3/2006 (H1N1); JP/57: A/Japan/305/1957 (H2N2); VN/04: A/Vietnam/1203/2004 (H5N1). (c) Human IgG in serum of BALB/c mice (as quantified by ELISA) at indicated times after intramuscular injection of 1 × 10¹¹ GC of optimized AAV encoding b12, F10 or CR6261. The plot shows mean and standard error of six recipient mice per antibody. (d) Neutralizing activity of sera taken from mice 5 weeks after they were injected with AAV encoding b12, F10 or CR6261 (just before infection with influenza virus) as measured against five strains of influenza (PR/34, CA/09, SI/06, JP/57 and VN/04) using a GFP reporter assay (Supplementary Fig. 2). Values are calculated as the fold dilution of serum that resulted in 50% neutralization of influenza infection. Solid line represents the lowest dilution tested (20-fold) and values below this line are extrapolated from the curve fit or are plotted at a value of 15 to represent no detectable neutralization activity. Each point represents an individual mouse serum sample tested against one strain of influenza. Each plot contains data generated from one individual experiment, and each experiment was performed once with the indicated number of mice.

Figure 2

VIP protects mice against influenza infection. (a) Total human IgG in serum was measured by ELISA in samples taken 5 weeks after intramuscular injection of AAV encoding b12, F10 or CR6261 (2 d before influenza infection) into BALB/c mice. Each point represents an individual mouse. (b,c) BALB/c mice that received VIP 5 weeks prior were infected intranasally with 1 × 10⁴ PFU of CA/09 influenza. Weight loss was monitored for the indicated time period in recipients of AAV encoding control (b12) or F10. The plot shows mean and standard error of eight recipient mice per antibody (b). Correlation between weight loss of individual mice 4 d post-infection with CA/09 and serum concentration of CR6261 (c). (d) BALB/c mice that received VIP 5 weeks prior were infected intranasally with 5 × 10⁴ PFU of SI/06 influenza. Weight loss was monitored for the indicated time period in recipients of AAV encoding control (b12) or F10. The plot shows mean and standard error of eight recipient mice per antibody. (e) BALB/c mice that received VIP 5 weeks prior were infected intranasally with 1,000 PFU of PR/34 influenza. Survival (left) and weight loss (right) were monitored for the indicated time period in recipients of AAV encoding control (b12) or F10. The plots shows mean and standard error of eight recipient mice per antibody. (f,g) Serum was taken from BALB/c mice 8 weeks after they were injected with AAV encoding b12, F10 or CR6261, and 3 weeks after they were infected with CA/09 (f) or SI/06 (g) influenza. In vitro neutralization of five strains of influenza (PR/34, CA/09, SI/06, JP/57 and VN/04) by serum was detected by GFP-reporter assays. Values are calculated as the fold dilution that resulted in 50% neutralization of influenza infection. Solid line represents the lowest dilution tested (20-fold dilution), and values below this line are extrapolated from the curve fit or are plotted at a value of 15 to represent no detectable neutralization activity. (h,i) Serum was taken from BALB/c mice 5 weeks after they were injected with AAV encoding b12 or F10 and either just before (naive) or 3 weeks after (post-) infection with CA/09 or SI/06. HAI performed with CA/09 virus (h) or SI/06 virus (i). HAI titers are expressed as the highest dilution of serum that allowed complete hemagglutination. Solid line represents the lowest dilution tested (16-fold or 40-fold dilution for CA/09 or SI/06, respectively), and values below this line are plotted along the axis to represent no detectable HAI activity. Each plot contains data generated from one individual experiment, and each experiment was performed once with the indicated number of mice.

Figure 3

VIP protects both young and old immunodeficient mice from influenza infection. (a) Young (3 months) or old (12 months) NSG mice were injected intramuscularly with 1 × 10¹¹ GC of AAV encoding luciferase. At indicated time points, luciferase was measured in vivo by bioluminescence imaging. The plot shows mean and s.d. of eight young and eight old recipient mice. (b) Young or old NSG mice were injected intramuscularly with 1 × 10¹¹ GC of AAV encoding F10. At indicated time points, serum human IgG was measured by ELISA. The plot shows mean and s.d. of eight young and eight old recipient mice. (c,d) Young (c) and old (d) NSG mice were injected intramuscularly with 1 × 10¹¹ GC of AAV encoding F10 or luciferase (Luc). Four weeks later mice were infected intranasally with 1,000 PFU of PR/34 influenza. Survival (left) and weight loss (right) were measured (n = 4–8). Error bars, mean and s.e.m. (e) Hematoxylin and eosin staining of representative lung sections from young NSG mice that received VIP encoding F10 or Luc, taken 5 d after infection with 1,000 PFU of PR/34 influenza. Scale bar, 100 μM. (f–h) BALB/c mice were injected with AAV encoding b12 or F10. Eleven months later, total human IgG in serum was quantified by ELISA (1 d before influenza infection) (f). Each point represents an individual mouse. (g,h) Eleven months after injection of AAV, mice were infected with 1,000 PFU of PR/34 influenza (n = 14–20). Weight loss was monitored (g). Error bars, mean and s.e.m. Immediately following infection and every 2 d thereafter, three mice were euthanized in each group and influenza RNA in the lungs was measured by qPCR (h). Samples were normalized to the endogenously expressed mouse ribosomal protein L32 gene and plotted relative to the signal of the lowest sample. Each plot contains data generated from one individual experiment, and each experiment was performed once with the indicated number of mice.