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, 8 (1), 4374

A Highly Potent and Broadly Neutralizing H1 Influenza-Specific Human Monoclonal Antibody

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A Highly Potent and Broadly Neutralizing H1 Influenza-Specific Human Monoclonal Antibody

Aitor Nogales et al. Sci Rep.

Abstract

Influenza's propensity for antigenic drift and shift, and to elicit predominantly strain specific antibodies (Abs) leaves humanity susceptible to waves of new strains with pandemic potential for which limited or no immunity may exist. Subsequently new clinical interventions are needed. To identify hemagglutinin (HA) epitopes that if targeted may confer universally protective humoral immunity, we examined plasmablasts from a subject that was immunized with the seasonal influenza inactivated vaccine, and isolated a human monoclonal Ab (mAb), KPF1. KPF1 has broad and potent neutralizing activity against H1 influenza viruses, and recognized 83% of all H1 isolates tested, including the pandemic 1918 H1. Prophylactically, KPF1 treatment resulted in 100% survival of mice from lethal challenge with multiple H1 influenza strains and when given as late as 72 h after challenge with A/California/04/2009 H1N1, resulted in 80% survival. KPF1 recognizes a novel epitope in the HA globular head, which includes a highly conserved amino acid, between the Ca and Cb antigenic sites. Although recent HA stalk-specific mAbs have broader reactivity, their potency is substantially limited, suggesting that cocktails of broadly reactive and highly potent HA globular head-specific mAbs, like KPF1, may have greater clinical feasibility for the treatment of influenza infections.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Isolation and molecular characterization of KPF1 hmAb. (a) Gating strategy to isolate peripheral blood plasmablasts (CD19 + IgD-CD38 + CD27++) 7 days after immunization. (b) Alignment of KPF1 VH and Vk with presumed germline amino acid sequences. (c) Phylogenic analysis of KPF1 lineage members based on amino acid sequence. Lineage members defined as same heavy chain V and J gene usage, HCDR3 length, and ≥85% HCDR3 similarity. Germline sequence is represented by green diamond, sequences obtained from single-cell plasmablast sequencing are represented by orange squares, the KPF1 mAb sequence is represented by the red square, sequences obtained by MiSeq-based deep sequencing of bulk total B cells are represented by blue squares, inferred intermediate sequences are represented by blue circles. Red line indicates the inferred pathway from germline to KPF1 hmAb. Size of symbols are proportional to the number of identical sequences obtained of an individual lineage member (N = 1–55), with the exception of the germline and KPF1 mAb sequences.
Figure 2
Figure 2
KPF1 hmAb is highly specific for H1 influenza. KPF1 hmAb and IgG isotype control hmAb were tested by ELISA for binding to (a) diverse recombinant influenza A H1 (A/Brisbane/59/07 and A/California/04/09), H3 (A/New York/55/04, A/Wisconsin/67/05 and A/Brisbane/10/07), H5 (A/Vietnam/1203/04), H7 (A/Netherlands/219/03), H9 (A/Hong Kong/1073/99) and influenza B (B/Brisbane/60/08) HAs and negative control protein (RSV-F) and (b) H1 (A/Brisbane/59/07 and A/California/04/09) HA proteins in increasing concentrations of urea. Symbols represent triplicate ± SEM. (c) Purified KPF1 was captured on a Protein G chip with the pH1N1 HA at decreasing concentrations passed over each channel. The data points are shown in black and the fit to a 1:1 binding model are shown in red. The results of one representative experiments of two are presented.
Figure 3
Figure 3
mPLEX-Flu binding profile. The patient’s plasma from before immunization (D0) and 7 days (D7) and 3 months (M3) post-immunization, and KPF1 hmAb were tested in decreasing concentration by multiplex assay for binding to the indicated recombinant influenza A H1, H2, H3, H5, H6, H7, H9 and influenza B HA proteins.
Figure 4
Figure 4
Potent in vitro neutralizing activity of KPF1 hmAb. Virus neutralization was determined using a fluorescent-based microneutralization assay,. MDCK cells were infected with mCherry-expressing pH1N1, PR8 H1N1, H3N2 and IBV, which were pre-incubated with two-fold serial dilutions of KPF1 hmAb. At 24 h p.i., virus neutralization was evaluated and quantified using a fluorescence microplate reader (a), and the percentage of infectivity calculated using sigmoidal dose response curves (b). Mock-infected cells and viruses in the absence of hmAb (No hmAb) were used as internal controls. Percent of neutralization was normalized to infection in the absence of hmAb. Data show means ± SD of the results determined for triplicates. (c) NT50 of KPF1 hmAb by fluorescent-based assay. *Highest amount of hmAb without detectable neutralizing effect.
Figure 5
Figure 5
KPF1 hmAb restricts pH1N1 replication in vivo. Female C57BL/6 mice (N = 11) were treated i.p. with 0.1, 1 or 10 mg/kg of KPF1 hmAb, or with 10 mg/kg of an isotype control (IgG isotype control), or PBS 24 h before infection. Mice were then challenged with 10x MLD50 of pH1N1 and monitored daily for 2 weeks for body weight loss (a) and survival (b). Mice that lost 25% of their body weight were sacrificed. Data represent the means ± SD (N = 5). *Indicates p < 0.05 (when the differences between mice treated with 1 or 10 mg/kg of KPF1 hmAb and mice treated with PBS or IgG were significant using a one-tailed Student’s t test (body weight) or Mantel-Cox test (survival). To evaluate viral replication in the lungs (c), mice were sacrificed at 2 (N = 3) and 4 (N = 3) days p.i. and whole lungs were used to quantify viral titers by immunofocus assay (FFU/ml). *Indicates p < 0.05 using one-way ANOVA and Dunnett’s test for multiple comparison correction. Ns, no statistically significant differences. (d) Evaluation of KPF1 for its prophylactic activity against multiple H1 influenza strains. Female C57BL/6 mice (N = 6) received 10 mg/kg of KPF1 or IgG isotype control (IC) 24 h before viral infection. Mice were then challenged with 10x MLD50 of PR8 H1N1 (circles), TX H1N1 (squares), or NC H1N1 (triangles) and viral replication in lungs at 2 (N = 3) and 4 (N = 3) days p.i. (black and grey symbols, respectively) was evaluated as indicated above. For (C) and (D), symbols represent data from individual mice. Bars, geometric mean lung virus titers; dotted line, limit of detection (200 FFU/ml). & indicates virus was not detected or was detected only in 1 of 3 mice.
Figure 6
Figure 6
Therapeutic activity of KPF1 in infected mice. Female C57BL/6 mice (N = 5) were infected with 10x MLD50 of pH1N1 and then treated i.p. with 10 mg/kg of KPF1 hmAb at 6, 24 or 72 h p.i., or an isotype control hmAb (IgG isotype control; at 6 h p.i.) or PBS (at 6 h p.i.). Then, mice were monitored daily for 2 weeks for body weight loss (a) and survival (b). Mice that lost 25% of their body weight were sacrificed. Data represent the means ± SD (N = 5). *Indicates p < 0.05 (when the differences between mice treated with KPF1 hmAb and mice treated with PBS or IgG were significant using a one-tailed Student’s t test (body weight) or Mantel-Cox test (survival).
Figure 7
Figure 7
Generation and characterization of MARMs. (a) Amino acid mutations in the HA and NA of pH1N1 WT or mAb-resistant mutants (MARMs 1, 2 and 3) after 5 rounds of selection in the presence (MARMs) or absence (WT) of hmAb KPF1. The mutations effects on reactivity with the hmAb KPF1 were also evaluated in a microneutralization assay (NT50) and HAI. (b) Characterization of MARMs of pH1N1 by immunofluorescence. MDCK cells were mock infected (Mock) or infected (MOI 0.01) with pH1N1 WT or the MARMs (1, 2 and 3). At 36 h p.i., cells were fixed and protein expression was evaluated by IFA using the hmAb KPF1, or the mouse mAbs 29E3 (anti-HA) and HB-65 (anti-NP). DAPI was used for nuclear staining. Merge from representative images (10x magnification) are included. Scale bar, 50 nm. (c) Multicycle growth kinetics of pH1N1 WT and MARMs in MDCK cells. Virus titers in TCS of MDCK cells infected (MOI, 0.001) with pH1N1 WT or MARMs viruses were analyzed at the indicated h p.i by immunofocus assay (FFU/ml) using the anti-NP mouse mAb HB-65. Data represent the means ± SDs of the results determined for triplicate wells. *Indicates p < 0.05 (WT versus MARM 3) using a one-tailed Student’s t test. (d) Tridimensional protein structure for the globular head of HA of pH1N1. The image was created using the software program PyMol and the published structure for the HA of pH1N1 (3LZG,). Positions of amino acid substitutions in the MARMs (E129 and K180) are colored in yellow. The residues at each antigenic site are colored as red for the Sa site, orange for the Sb site, green for the Ca site, and magenta for the Cb site. The receptor binding site (RBS) location in the structure is indicated. (e) Generation of MARMs for TX H1N1. Amino acid changes in the HA and NA of TX H1N1 WT or MARMs (1, 2 and 3) after 5 rounds of selection in the presence (MARMs) or absence (WT) of hmAb KPF1. The effect of E129K mutation on reactivity with KPF1 was also evaluated in a HAI assay, using WT TX H1N1 as an internal control.
Figure 8
Figure 8
Relevance of amino acids 129 and 180 for the binding of KPF1 hmAb. (a) Binding of KPF1 hmAb to WT and mutant HA proteins. HEK293T cells were transiently transfected with the pCAGGS plasmids expressing WT or amino acid substitutions E129K, K180N, K180Q or E129K/K180N mutant HAs. Mock transfected cells were used as internal control. At 24 h post-transfection, cells were fixed and protein expression was evaluated by IFA using the hmAb KPF1, or a goat pH1N1 anti-HA polyclonal antibody as a control. DAPI was used for nuclear staining. Merge from representative images (10x magnification) are included. Scale bar, 50 nm. (b) Frequency of amino acid changes found in IAV H1N1 HA over time. Publicly available sequences of IAV H1N1 HA protein (Influenza Research Database) isolated between 2000–2009 (n = 8,586; black) or 2010–2018 (n = 8,417; grey) were analyzed and plotted according to the percentage of sequences containing the indicated amino acids at positions 129 (right) or 180 (left).

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