In vitro pan-influenza characteristics of the anti-hemagglutinin stem antibody CR9114

In this study, we used the human mAb CR9114, which binds all known influenza A subtypes and both influenza B lineages that cause influenza in humans and animals^6,7,8. CR9114 targets the conserved central stem region on the surface membrane fusion glycoprotein hemagglutinin (HA) of influenza A and B viruses (Fig. 1A,B, and Fig. S1). It uses residues on three heavy chain complementary determining regions (HCDR1-3) and one framework region (FR3), which form flexible loops that can flip into the hydrophobic groove on HA or have polar side chains that can form H-bonds with a variety of influenza HAs. The polar side chain of tyrosine residues ⁹⁸YYYY^100A is crucial for forming H-bonds with H5 (residues on CR9114: Y⁹⁸, Y^100A), H3 (Y⁹⁸, Y^100A), and B/Vic (Y⁹⁸; Fig. 1C, Table S1)²³. For influenza H1 and B/Yam, no crystal structures in complex with CR9114 are available, but molecular dynamics simulations show a key role for the same tyrosine residues of CR9114 for interacting with H1 (Y⁹⁸, Y⁹⁹, Y^100A) and B/Yam (Y⁹⁸; Fig. 1B,D, Table S1). The residues W21 and Q42 in the hydrophobic groove of HA, and D19 on the fusion peptide domain are conserved between influenza group A1 and A2 viruses and form H-bonds with CR9114 residues. CR9114 demonstrates a greater propensity to form hydrogen bonds with the hemagglutinin of influenza A compared to influenza B. This difference is evident in higher binding free energies calculated in silico for influenza A subtypes H1 and H3 in contrast to B/Yam and B/Vic. Analysis of the binding affinity of CR9114 to H1, H3, B/Yam, and B/Vic reveals that it binds strongest to H1, followed by H3, B/Vic, and B/Yam, consistent with the results of binding free energy simulations (Fig. 1E).

Pan-recognition characterization of the anti-hemagglutinin stem antibody CR9114. (A) Dendrogram of all known hemagglutinin (HA) subtypes and hemagglutinin esterase fusion proteins (HEF) belonging to four genera of influenza viruses: influenza A (group 1 and group 2; IAV), influenza B (Yamagata and Victoria lineages; IBV), influenza C (ICV) and influenza D (IDV). (B) 3D structures of CR9114 Fab in complex with H3 and B/Vic HA, and in silico models for CR9114 Fab in complex with ICV HEF and IDV HEF. (C) Surface representation of the CR9114-Fab paratope (yellow) and residues on CR9114-Fab that constitute binding hotspots (red) with H5, H3, and B/Vic HA. (D) In silico models of the CR9114-Fab paratope (yellow) that constitute binding hotspots (red) with H1 and B/Yam HA, as well as ICV and IDV HEF. (E) Binding Free Energy (kcal/mol), binding affinity (K_D, M), and live virus and pseudotyped virus microneutralization potency (IC₅₀, µg/mL) of CR9114 against H1, H3, B/Yamagata and B/Victoria HA, as well as ICV and IDV HEF (- : not neutralized). Affinity measurement experiments were performed with CR9114 Fab, IgG, IgM, IgA, and dIgA formats. Only the affinity for Fab is shown here, while the affinity for the other formats is presented in Fig. S2. IC₅₀s derived from the live virus neutralization assay represent four technical replicates.

Using molecular dynamics simulations, we found that hotspots for CR9114 binding are partially present in the stem regions of the functionally analogous hemagglutinin-esterase-fusion (HEF) glycoprotein from influenza C and D viruses (ICV and IDV)^29,30,31, potentiating wider breadth than previously thought (Fig. 1B,D,E, Table S2). While no detectable affinity for HEF by CR9114 Fab was found by bio-layer interferometry, a low apparent affinity (> 100 µM) was observed for the bivalent IgG and multivalent IgM formats of this antibody (Fig. 1E).

We also tested the neutralizing activity of CR9114 against influenza A and B viruses. CR9114 shows neutralization activity for group 1 and group 2 influenza A (H1 and H3, respectively), but not for influenza B (Fig. 1E), consistent with prior studies^6,7. Notably, neutralizing activity against influenza B becomes detectable if the apparent affinity provided by bivalent IgG is further increased by reconfiguring CR9114 as dimeric IgA (four antigen binding sites) or as pentameric IgM (ten antigen binding sites; Fig. S2), akin to the antibody isotype-associated avidity effects previously reported for neutralization of both influenza virus^32,33,34 and SARS-CoV-2³⁵.

Furthermore, we examined the ability of these CR9114 isotypes to interfere with the catalytic activity of neuraminidase (NA), the other surface glycoprotein of influenza virus A and B. As shown previously^{23,36,37,38,39}, CR9114 inhibits neuraminidase activity in an enzyme-linked lectin assay (ELLA, Fig. S2); the antibody sterically hinders the active site of neuraminidase and prevents cleavage of sialic acid by N1 and N2, and B/Yam NA. However, CR9114 IgG does not inhibit B/Vic in an ELLA. We show that avidity effects extend to neuraminidase inhibition, in which high avidity formats of CR9114 more potently inhibit NA than their IgG1 counterpart (Fig. S2). In conclusion, CR9114 is unique in its ability to bind influenza A, B, C and D viruses. As it has different mechanisms of action in vitro, it is the perfect candidate for investigating the contribution of various mechanisms of action to in vivo protection.

Dose-sparing effect of intranasal administration of CR9114 compared to intravenous administration, independent of in vitro microneutralization or neuraminidase inhibition

In mouse models, passive transfer of anti-influenza antibodies is typically performed through a peripheral, systemic injection route^6,7,11,21. We hypothesized that relative to direct application to the respiratory mucosa, peripheral administration would lower prophylactic efficacy due to a reduced antibody concentration at the site of viral infection. To test this principle, we delivered de-escalating doses of CR9114 IgG (15–0.2 mg/kg) via intravenous injection or intranasal instillation 24 hours before challenging the animals with A/H1N1 (A/WSN/1933, Fig. 2A,B) or B/Victoria (B/Malaysia/2506/2004, Fig. 2C,D). We compared survival proportions at day 21 and change in body weight to the control groups, where animals received phosphate-buffered saline (PBS) either via intranasal or intravenous administration.

Intranasal administration of CR9114 at the site of viral entry has dose-sparing effect compared with intravenous administration, for neutralized influenza A/H1N1 and A/H3N2 as well as non-neutralized B/Victoria. Kaplan–Meier survival curves showing in vivo protection by intravenous or intranasal CR9114 in a murine lethal challenge model against influenza A or B viruses. Efficacy of (A) intravenous or (B) intranasal CR9114 against lethal challenge with A/WSN/1933 (A/H1N1). Efficacy of (C) intravenous or (D) intranasal CR9114 against lethal challenge with B/Malaysia/2506/2004 (Victoria lineage). Efficacy of (E) intravenous or (F) intranasal CR9114 against lethal challenge with A/Hong Kong/1/1968 (H3N2). Survival curves (top panels) and change in body weight (bottom panels) of mice treated with indicated doses of CR9114 via intravenous or intranasal administration or PBS control are shown. Grey panels (left) show challenge strain, challenge dosage and indicate whether CR9114 can bind, neutralize (‘VNA’), and inhibit NA activity of viruses (‘NAI’) with yes (‘+’) or no (‘−’). Asterisks indicate significant differences in survival proportions at day 21 and bodyweight change relative to day 0 compared with PBS control (P < 0.05). “n.s.” indicates not significant compared with PBS control. These experiments were performed with 8–10 mice per group.

As expected, we observed a strong reduction in prophylactic efficacy against both influenza A and B strains (increased mortality and weight loss) during dose down-titrations in the intravenous but not intranasal groups: mice receiving CR9114 intravenously were fully protected from mortality at 1.7 mg/kg against influenza A/H1N1 virus (Fig. 2A) and at 5 mg/kg against influenza B/Vic virus (Fig. 2C), whereas mice receiving CR9114 via intranasal administration were fully protected at the lowest dose tested of 0.2 mg/kg against both influenza strains (Fig. 2B,D). Following intravenous administration of CR9114, mice suffered weight loss at some dosages that protected from mortality (1.7 mg/kg and 5 mg/kg), whereas no weight loss was observed in mice receiving intranasally-administered CR9114.

For protection against influenza A/H1N1 and influenza B/Vic, we could not precisely quantify the dose reduction, as intranasal CR9114 was still 100% protective at the lowest dosage tested. We therefore also tested protection against A/H3N2, titrating down to lower dosages. Following intravenous administration, CR9114 only provided significant protection against mortality and weight loss at a dose of 10 mg/kg (Fig. 2E). Following intranasal administration, CR9114 provided significant protection against mortality and weight loss at doses as low as 0.2 mg/kg and 0.067 mg/kg (Fig. 2F). The median effective dosage (ED₅₀) was 4.423 mg/kg for intravenous CR9114 and 0.083 mg/kg for intranasal CR9114 (Supplementary Table S6). Thus, intravenously-administered CR9114 was 53-fold less potent than intranasally-administered CR9114 (fold change in median effective dosage: 53.1; 95% CI: 25.1 – 112.3).

We also seized the opportunity to confirm whether the breadth of protection exhibited by CR9114 via the intravenous route, as previously demonstrated in the work of Dreyfus et al.⁶, is conserved when CR9114 was administered intranasally. To this end, we administered de-escalating doses of CR9114 (15–0.2 mg/kg) intranasally in mice challenged with A/H5N1 (A/Hong Kong/156/1997) or B/Yamagata (B/Florida/4/2006). Intranasal administration of CR9114 potently protected mice from mortality and weight loss (Fig. 3): all tested doses down to 0.2 mg/kg provided significant protection against mortality and weight loss from lethal challenge with H5N1 and B/Yamagata.

Intranasal administration of CR9114 potently protects against neutralized avian influenza A/H5N1 and non-neutralized B/Yamagata. Kaplan–Meier survival curves showing the in vivo protection by intranasal CR9114 in a murine lethal challenge model against (A) A/Hong Kong/156/1997 (A/H5N1) and (B) B/Florida/4/2006 (Yamagata lineage). Survival curves (left panels) and change in body weight (right panels) of mice treated with indicated doses of CR9114 via intranasal administration or PBS control are shown. Grey panels (left) show challenge strain, challenge dosage and indicate whether CR9114 can bind, neutralize (‘VNA’), and inhibit NA activity of viruses (‘NAI’) with yes (‘+’) or no (‘−’). Asterisks indicate significant differences in survival proportions at day 21 and bodyweight change relative to day 0 compared with PBS control (P < 0.05). The highest dosage of B/Yamagata challenge differ slightly from that in the H5N1 model. This did not have a scientific reason but was due to a miscalculation in the dilution process (animals in the B/Yamagata challenge model received 13.2 mg/kg—blue line in panel B; animals in the H5N1 challenge model received 15 mg/kg—blue line in panel A).

Taken together, these data demonstrate that i) direct administration of CR9114 to the respiratory mucosa requires a lower effective dose to ensure a protective effect; ii) intranasally-administered CR9114 protects at low dosage independent of microneutralization read-out (positive for H1N1, H5N1, H3N2; negative for B/Yam and B/Vic) and neuraminidase inhibition (positive for H1N1, H5N1, H3N2, B/Vic; negative for B/Yam).

Intranasal prophylaxis with CR9114 is independent of the Fc domain

The effect of the administration route provided the opportunity to examine the hypothesis that neutralizing activity is operable when the antibody titer is high, whereas Fc-dependent effector functions are essential for protection at low antibody titers. Accordingly, we again performed dose down-titrations of passively-administered antibody through the intravenous and intranasal administration routes 24 hours before challenge. This time, we varied the CR9114 configuration, using wild-type IgG (CR9114-WT), LALA⁴⁰ or N297Q⁴¹ mutants (CR9114-LALA, CR9114-N297Q; harboring substitutions that disrupt the binding of the Fc region to Fc receptors on immune cells, thereby reducing or ablating Fc-mediated effector functions) or the bivalent antigen-binding site only (F(ab′)₂) (Figs. 4 and 5).

Intranasal administration route reveals low dose protection against influenza A independent of the antibody Fc, conferred by F(ab’)₂ alone. (A, B) Kaplan–Meier survival curves showing the in vivo efficacy of (A) intravenous and (B) intranasal administration of CR9114, CR9114-LALA, CR9114-N297Q, and CR9114-F(ab′)₂ in protecting mice against lethal challenge with influenza A/H1N1 (A/Puerto Rico/8/1934). Mice were treated with indicated doses of CR9114 or PBS via intravenous or intranasal administration 24 h before challenge as shown in the legend. Asterisks indicate significant differences in survival proportions at day 21 compared with PBS control (P < 0.05). “n.s.” indicates not significant compared with PBS control. Table S4 provides detailed information on the dose titration for each mouse group. Changes in body weight are presented in Fig. S3 and Table S5. These experiments were performed with 8–10 mice per group.

Intranasal administration of CR9114—unlike intravenous administration—results in protection against influenza B independent of the antibody Fc, conferred by F(ab’)₂ alone. (A, B) Kaplan–Meier survival curves showing the in vivo efficacy of (A) intravenous and (B) intranasal administration of CR9114, CR9114-LALA, CR9114-N297Q, and CR9114-F(ab′)₂ in protecting mice against lethal challenge with influenza B/Yam (B/Florida/4/2006). Mice were treated with indicated doses of CR9114 or PBS via intravenous or intranasal administration 24 h before challenge as shown in the legend. Asterisks indicate significant differences in survival proportions at day 21 compared with PBS control (P < 0.05). “n.s.” indicates not significant compared with PBS control. Table S4 provides detailed information on the dose titration for each mouse group. Changes in body weight are presented in Fig. S3 and Table S5. These experiments were performed with 8–10 mice per group.

For intravenous protection against A/H1N1, the downregulation of Fc effector functions (CR9114-LALA and CR9114-N297Q) and the removal of the Fc tail (F(ab′)₂) resulted in a marked increase in the median effective dosage (ED₅₀) compared to wild-type (Supplementary Fig. 4 and Table S6). The ED₅₀ for CR9114-LALA (1.623 mg/kg) was 7.3-fold higher than CR9114-WT (0.222 mg/kg). This difference was statistically significant (95% CI: 2.32–23.5). The ED₅₀ for CR9114-F(ab′)₂ (71.419 nmol/kg) was 7.9-fold higher than CR9114-N297Q (9.055 nmol/kg; Fig. 6A, Supplementary Fig. 5 and Table S6), also a statistically significant difference (95% CI 3.50–17.8).

Administration route reveals Fc-dependent versus independent mechanisms of protection. (A) Schematic showing the observed differences in the median effective dose (ED₅₀) of CR9114 IgG, IgG Fc-mutants, or F(ab′)₂ when administered via the intravenous (left panels) or intranasal route (right panels). (B) Schematic showing the necessary and sufficient mechanisms of action mediated by intravenously-(left) or intranasally-administered (right) pan-influenza antibody CR9114.

On the contrary, when CR9114 was instilled intranasally, low-dosage protection was also observed for all configurations. Neither downregulation of Fc-mediated effector functions nor removal of the Fc tail resulted in statistically significant increases in the ED₅₀ (S5 and Table S6), although there was a trend towards slightly higher ED₅₀ for the Fc-mutants and F(ab′)₂ compared to wildtype IgG. The difference in ED₅₀ between intranasally-administered CR9114-WT and CR9114-LALA was not statistically significant (1.82-fold; 95% CI 0.56–5.90), nor the difference between CR9114-N297Q and CR9114-F(ab′)₂ (1.75-fold; 95% CI: 0.66–4.61).

When mice were challenged with B/Yamagata, intravenous administration of CR9114-WT, -LALA, and -N297Q protected from mortality and weight loss only at the highest dosage(s) (Fig. 5). The downregulation of Fc effector functions heavily reduced the protective efficacy of intravenously-administered CR9114. The ED₅₀ for CR9114-LALA (5.530 mg/kg) was 29.8-fold higher than CR9114-WT (0.186 mg/kg). The difference was statistically significant (95% CI: 9.2–97.0). Moreover, none of the tested F(ab′)₂ doses (up to 45 mg/kg; molar equivalent of 68 mg/kg CR9114 IgG) yielded significant protection compared to PBS controls, hence no dosage provided 50% protection and no fold difference between ED₅₀s could be calculated.

In contrast, when CR9114 Fc mutants or F(ab′)₂ were administered intranasally, mice were protected against mortality and weight loss. Intranasal administration was also marked with a less prominent increase in median effective dosage. The ED₅₀ for CR9114-LALA (0.326 mg/kg) was 3.1-fold higher than CR9114-WT (0.104 mg/kg; Fig. 6A). This difference was statistically significant (95% CI: 1.16–8.50). CR9114-N297Q and CR9114-F(ab′)₂ protected more than 50% of mice from lethal challenge at the lowest dosages tested, so it was not possible to fit a dose–response curve, calculate ED₅₀ and determine the fold difference. These data indicate that intranasally-administered antibody is more potent if it contains the Fc domain (Supplementary Figs. 4 and 5), although the relative contribution of Fc is much lower than for intravenously-administered antibody.

We further extended our studies and antibody dosing ranges to evaluate protection against influenza A/H3N2, an influenza group A2 virus that is bound and neutralized by CR9114, albeit less potently compared to A/H1N1 (Fig. 1E; Supplemental Fig. 6). Intravenously-administered full-length CR9114 protected against A/H3N2 at 10 mg/kg, but CR9114-F(ab′)₂ did not protect even at the highest dosage tested (75 mg/kg; given its lower molecular weight, this is equimolar to a dosage of 113 mg/kg of CR9114 IgG).

In contrast, when administered intranasally, both full-length CR9114-WT and the CR9114-F(ab′)₂ protected against mortality (at 0.2 mg/kg and 1 mg/kg, respectively) and weight loss (at 0.067 mg/kg and 0.11 mg/kg, respectively). When comparing ED₅₀s from the dose–response curves, CR9114-WT provided 4.45-fold more potent protection compared to CR9114-F(ab′)₂ (95% CI: 2.03–9.72). This data further supports the observation that the Fc-tail is necessary for intravenous protection, but not for intranasal protection, although it does contribute to intranasal potency.

In summary, following challenge with influenza A and B, we recapitulated the impact of administration route as was observed for full-length, wild-type CR9114 (Fig. 2): efficacy of CR9114 was less affected by de-escalating the dose following intranasal administration compared to intravenous administration for all configurations of the antibody. Moreover, we demonstrate that in contrast to intravenous administration, where prophylaxis requires the antibody Fc-tail to protect against both influenza A and B, intranasal protection is Fc-independent. Without its Fc domain, the potency of CR9114 is generally lower than the other full-length IgG configurations, but F(ab′)₂ is sufficient to provide local prophylactic protection if administered directly to the site of infection.