Recombinant Human Anti-HA Antibody (FI6) (CAT#: HPAB-M0100-YC)

Figure 1 Broadly binding and neutralizing anti-HA stalk mAbs require Fc-FcγR interactions for protection in vivo.

(A) Broadly neutralizing anti-stalk (red lines), strain-specific anti-head (blue lines), broadly neutralizing anti-head (green lines), and non-neutralizing anti-head (purple lines) mAb binding to Neth09-infected cells by flow cytometric analysis and (B) neutralization of Neth09 virus (mean ± SEM) in duplicate samples. (C and D) WT mice were given the indicated doses of IgG2a (red circles) or DA265-mutant (blue squares) FI6 mAb (C), 2G02 mAb (D), or PBS (black triangles) before Neth09 virus infection. Values represent the mean ± SEM percentage of weight change compared with day-0 values (left panels) and percentage of survival (right panels). n = 4–5 mice per group. Significant differences between the IgG2a and DA265 samples are shown. *P < 0.05 and **P < 0.01, by 2-tailed Student's t test.

DiLillo, D. J., Palese, P., Wilson, P. C., & Ravetch, J. V. (2016). Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection. The Journal of clinical investigation, 126(2), 605-610.

Figure 2 Broadly neutralizing anti-NA mAb requires Fc-FcγR interactions to mediate protection in vivo, but strain-specific anti-NA mAb does not.

(A) Broadly neutralizing anti-HA stalk mAb FI6 (red circles), strain-specific anti-HA head mAb 4C04 (blue squares), strain-specific anti-NA mAb 3C02 (green circles), and broadly neutralizing anti-NA mAb 3C05 (green squares) neutralization of Neth09 virus. Values represent the mean ±SEM of duplicate samples. (B and C) Mice were given the indicated doses of IgG2a (red circles) or DA265-mutant (blue squares) 3C02 (B) or 3C05 (C) mAb or PBS (black triangles) before Neth09 viral infection. Values represent the mean ± SEM percentage of weight change compared with day-0 values (left panels) and percentage of survival (right panels). n = 4–5 mice per group. Significant differences between the IgG2a sample and DA265 sample are shown. **P < 0.01 by Student's t test.

DiLillo, D. J., Palese, P., Wilson, P. C., & Ravetch, J. V. (2016). Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection. The Journal of clinical investigation, 126(2), 605-610.

Figure 3 Anti-HA mAb binding characteristics.

(A-F) Broadly-neutralizing anti-stalk (red lines), strain-specific anti-head (blue lines), broadly-neutralizing anti-head (green lines), and non-neutralizing anti-head (purple lines) mAb binding to purified Cal09 Virus (A), Neth09-infected A549 cells (B), recombinant Cal09 H1N1 protein (C), 2014-2015 trivalent vaccine (D), recombinant Cal09 HA1 head domain protein (E), or chimeric H5-head/H1-stalk HA protein (F) by ELISA assay (A, C, D, E, F) or flow cytometry analysis (B). Values represent mean (± SEM) relative OD values or mean fluorescence intensities (MFI) from triplicate (A, C, F) or duplicate (E) samples. Dashed line in (B) represents the level of detection in the flow cytometry assay as determined by mean MFI of samples stained with secondary antibody alone.

DiLillo, D. J., Palese, P., Wilson, P. C., & Ravetch, J. V. (2016). Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection. The Journal of clinical investigation, 126(2), 605-610.

Figure 4 IgG2a and DA265 mutant antibodies bind HA similarly.

Mouse IgG2a (circles) and DA265 mutant (squares) mAbs were diluted as indicated and tested for binding to recombinant Cal09 HA by ELISA. Values represent mean (± SEM) relative OD values from duplicate wells.

DiLillo, D. J., Palese, P., Wilson, P. C., & Ravetch, J. V. (2016). Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection. The Journal of clinical investigation, 126(2), 605-610.

Figure 5 Viral load in nasal washes and broncho-alveolar lavage (BAL).

Pigs were infected with A/sw/Eng/1353/09 and administered the indicated antibodies either by the I.V. route (I.V. solid symbols) or by aerosol (aer, hollow symbols). Nasal swabs (NS) were taken at 0, 1, 2, and 3 day post infection (dpi) and pigs sacrificed at 4 dpi. Viral titers in the nasal swabs (A) and BAL (B) were determined by plaque forming assay or real-time quantitative reverse transcriptase polymerase chain reaction.

Morgan, S. B., Holzer, B., Hemmink, J., Salguero, F. J., Schwartz, J. C., Agatic, G.,... & Townsend, A. (2018). Therapeutic administration of broadly neutralizing FI6 antibody reveals lack of interaction between human IgG1 and pig Fc receptors. Frontiers in immunology, 9, 865.

Figure 6 Mucosal and systemic IgG responses following administration of antibodies.

Human IgG1 (A) and pdmH1N1-specific IgG (B) titers in serum at 0, 2, and 4 day post infection (dpi) and in broncho-alveolar lavage at 4 dpi. Note for pdmH1N1-specific antibody in serum—all five pigs had titers of 1:640 at 4 dpi, while two animals at 1:1,024, and three at 1:640 at 2 dpi.

Morgan, S. B., Holzer, B., Hemmink, J., Salguero, F. J., Schwartz, J. C., Agatic, G.,... & Townsend, A. (2018). Therapeutic administration of broadly neutralizing FI6 antibody reveals lack of interaction between human IgG1 and pig Fc receptors. Frontiers in immunology, 9, 865.

Figure 7 Entry neutralization activity of serum and broncho-alveolar lavage (BAL) following administration of antibodies.

(A) Individual 50% inhibition titers in the serum at 0, 2, and 4 day post infection (dpi) and BAL at 4 dpi. (B) Neutralizing antibody response measured in the serum of the pigs following FI6 I.V. administration at 2 and 4 dpi. (C) Antibody response in the BAL of animals given FI6 I.V. or by aerosol at 4 dpi. (D) Negative sera from pre-challenge samples and negative BAL from control animals are shown alongside positive control. The dashed line represents the 50% inhibition titer and FI the fluorescence intensity of green-fluorescent protein. The neutralizing titer of serum and BAL from animals infected with the same A/sw/Eng/1353/09 virus and sacrificed at 14 dpi is shown in red.

Morgan, S. B., Holzer, B., Hemmink, J., Salguero, F. J., Schwartz, J. C., Agatic, G.,... & Townsend, A. (2018). Therapeutic administration of broadly neutralizing FI6 antibody reveals lack of interaction between human IgG1 and pig Fc receptors. Frontiers in immunology, 9, 865.

Figure 8 Antibody-dependent cytotoxicity (ADCC) activity of pig and human PBMC.

Madin–Darby canine kidney cells expressing H1 HA were incubated with FI6, MPE8, immune, or normal pig sera in the presence of either pig or human PBMC. ADCC was measured in triplicate by lactate dehydrogenase release. FI6 and MPE8 mAbs were used at 10 µg/ml (left panel) or at a concentration range from 0.1 to 1,000 ng/ml (right panel). Representative of three experiments.

Morgan, S. B., Holzer, B., Hemmink, J., Salguero, F. J., Schwartz, J. C., Agatic, G.,... & Townsend, A. (2018). Therapeutic administration of broadly neutralizing FI6 antibody reveals lack of interaction between human IgG1 and pig Fc receptors. Frontiers in immunology, 9, 865.

Figure 1 Recombinant Human Anti-HA Antibody (HPAB-M0100-YC) in SDS-PAGE

Lane 1: Non-reduced mAb
Lane 2: Reduced mAb

Figure 2 Human Anti-IAV HA Recombinant Antibody (HPAB-M0100-YC) in ELISA

ELISA analysis of HPAB-M0100-YC was performed by coating with Influenza A H2N2 (A/Japan/305/1957) Hemagglutinin / HA Protein (His Tag). Then blocked with BSA and incubated with HPAB-M0100-YC. The HRP-conjugated goat anti-Human IgG as a secondary antibody (1: 2000). Detection was performed using TMB substrate and stopped with sulfuric acid. The absorbances were read on a spectrophotometer at 450 nm.

Figure 3 Human Anti-IAV HA Recombinant Antibody (HPAB-M0100-YC) in ELISA

ELISA analysis of HPAB-M0100-YC was performed by coating with Influenza A [A/Hong Kong/483/97 (H5N1)] Hemagglutinin (HA) Protein (His Tag). Then blocked with BSA and incubated with HPAB-M0100-YC. The HRP-conjugated goat anti-Human IgG as a secondary antibody (1: 2000). Detection was performed using TMB substrate and stopped with sulfuric acid. The absorbances were read on a spectrophotometer at 450 nm.

Figure 4 Human Anti-IAV HA Recombinant Antibody (HPAB-M0100-YC) in WB

Western blot analysis of HPAB-M0100-YC was performed with Influenza A H2N2 (A/Japan/305/1957) Hemagglutinin / HA Protein (His Tag) onto a 12% Tris-HCl polyacrylamide gel. Proteins were transferred to a CN membrane and blocked with 5% skim milk for at least one hour. Membranes were probed with HPAB-M0100-YC and HRP goat anti-Human IgG as a secondary antibody (1: 2000). Chemiluminescent detection was performed.

Lane 1: Reduced antigen (0.8 μg)

Figure 5 Human Anti-IAV HA Recombinant Antibody (HPAB-M0100-YC) in WB

Western blot analysis of HPAB-M0100-YC was performed with Influenza A [A/Hong Kong/483/97 (H5N1)] Hemagglutinin (HA) Protein (His Tag) onto a 12% Tris-HCl polyacrylamide gel. Proteins were transferred to a CN membrane and blocked with 5% skim milk for at least one hour. Membranes were probed with HPAB-M0100-YC and HRP goat anti-Human IgG as a secondary antibody (1: 2000). Chemiluminescent detection was performed.

Lane 1: Reduced antigen (0.8 μg)

Figure 6 Human Anti-IAV HA Recombinant Antibody (HPAB-M0100-YC) in DB

Dot Blot analysis of HPAB-M0100-YC was performed by coating with Influenza A H2N2 (A/Japan/305/1957) Hemagglutinin / HA Protein (His Tag).

HPAB-M0100-YC incubation concentration: 2 μg/mL.
HRP-conjugated goat anti-Human IgG as a secondary antibody (1: 2000)

Figure 7 Human Anti-IAV HA Recombinant Antibody (HPAB-M0100-YC) in DB

Dot Blot analysis of HPAB-M0100-YC was performed by coating with Influenza A [A/Hong Kong/483/97 (H5N1)] Hemagglutinin (HA) Protein (His tag).

HPAB-M0100-YC incubation concentration: 2 μg/mL.
HRP-conjugated goat anti-Human IgG as a secondary antibody (1: 2000)