Non-neutralizing antibodies to influenza A matrix-protein-2-ectodomain are broadly effective therapeutics and resistant to viral escape mutations

Teha Kim; Lynn Bimler; Sydney L Ronzulli; Amber Y Song; Scott K Johnson; Cheryl A Jones; S Mark Tompkins; Silke Paust

doi:10.1126/sciadv.adx3505

. 2025 Sep 10;11(37):eadx3505. doi: 10.1126/sciadv.adx3505

Non-neutralizing antibodies to influenza A matrix-protein-2-ectodomain are broadly effective therapeutics and resistant to viral escape mutations

Teha Kim ^1,^2,^†, Lynn Bimler ^2,^3,^4,^†, Sydney L Ronzulli ⁵, Amber Y Song ^2,³, Scott K Johnson ⁵, Cheryl A Jones ⁵, S Mark Tompkins ^5,^6,^*, Silke Paust ^1,^2,^3,^4,^*

PMCID: PMC12422200 PMID: 40929256

Abstract

Influenza A viruses remain a global health threat, yet no universal antibody therapy exists. Clinical programs have centered on neutralizing mAbs, only to be thwarted by strain specificity and rapid viral escape. We instead engineered three non-neutralizing IgG2a mAbs that target distinct, overlapping epitopes within the conserved N terminus of the M2 ectodomain (M2e). Combined at low dose, this “triple M2e-mAb” confers robust prophylactic and therapeutic protection in mice challenged with diverse human and zoonotic IAV strains, including highly pathogenic variants. Therapeutic efficacy depends on Fc-mediated effector activity via FcγRI, FcγRIII, and FcγRIV, rather than in vitro neutralization. Serial passaging in triple M2e-mAb–treated immunocompetent and immunodeficient hosts failed to generate viral escape mutants. Our findings redefine the influenza-specific antibody therapeutic design and support Fc-optimized, non-neutralizing M2e-mAbs as a broadly effective, mutation-resistant, off-the-shelve therapy with direct relevance to human pandemic preparedness.

Non-neutralizing antibodies to influenza A are broadly protective and resist viral escape, offering strong therapeutic potential.

INTRODUCTION

Each year, influenza virus infection causes about one billion infections, including 3 to 5 million cases of severe illness and the death of approximately 500,000 people worldwide (1). In the US, influenza virus is responsible for up to 65 million illness cases yearly, incurring an annual US economic cost of roughly 11.2 billion dollars (2). In humans, infections with influenza A viruses (IAVs) far outnumber those with influenza B and generally lead to more severe disease (1). Although exposure to a specific influenza strain causes robust immunity and homologous protection, IAV has substantial pandemic potential from the emergence of novel strains that evade preexisting immunity owing to antigenic shift and drift in two of the virus’s immunodominant epitopes, hemagglutinin (HA) and neuraminidase (3–5). These antigenic changes thwart the development of monoclonal antibody (mAb)–based therapeutics and create the need for seasonal vaccination. In addition, global public health continues to be threatened by outbreaks of highly pathogenic avian influenza (HPAI) viruses (e.g., H5N1 and H7N9) that can spill over from infected animals into human populations, resulting in a 40 to 65% mortality rate (6). Since vaccine manufacturing and distribution take at least 6 months (7), vaccine availability can lag peak infection rates by several months, as it did during the H1N1 pandemic in 2009 (8–11). During such an event or when seasonal vaccine efficacy is low, access to reliable IAV therapeutics is essential to save lives. However, for each of the six current Food and Drug Administration (FDA)–approved treatments for IAV infection (12, 13), viral escape mutants have been observed in clinical trials and/or during seasonal or pandemic outbreaks (14–22). Because of widespread resistance, two of these treatments (amantadine and rimantadine), which once blocked the essential M2 proton channel (M2) of IAV, are now ineffective (23). Thus, there is a pressing need to develop effective, universal, escape mutant-resistant “off-the-shelf” IAV therapeutic agents to enhance pandemic preparedness, compensate for low seasonal vaccine effectiveness and vaccine uptake (25 to 50%), and offer off-the-shelve treatments to severely ill patients with influenza, including the immunocompromised, very young, or older populations with reduced immune function.

Off-the-shelve IAV treatments must be universally effective and, thus, should target highly conserved viral epitopes that are resistant to mutations leading to viral escape. IAV’s M2 protein has been previously considered an excellent target for such an IAV therapeutic agent (24). M2 is present in IAV’s lipid envelope and is required for both IAV’s intracellular life cycle and viral budding, for which it is trafficked to the cell surface (24–26). Further, its extracellular region (M2e) is highly conserved across different IAV serotypes (27–29). Thus, M2 antibodies should have universally protective and therapeutic potential. However, because of its M2’s small size, low immunogenicity, and rarity, preexisting immunity to M2e is limited in humans after natural infection. Only 18% of naturally IAV-infected patients generate M2e-specific antibody responses (30); thus, most of the human population lacks the benefits of M2e-mAb–mediated immunity. While intravenously administered mAbs are FDA-approved antiviral therapeutics, the treatment of respiratory syncytial virus and severe acute respiratory syndrome coronavirus 2 infections (31–35), there are now no FDA-approved antibody-based therapeutics for preventing or treating IAV infections. No IAV mAbs have successfully been developed clinically, and those evaluated were either strain specific, susceptible to viral escape, had limited efficacy, or lacked statistical analysis (24, 36–43) and NCT02623322, NCT01992276, NCT01719874, and NCT01390025. These include a variety of highly anticipated neutralizing mAbs targeting the HA stalk or M2e and cocktails thereof.

Neutralizing antibodies are traditionally preferred for the treatment of viral infections as they can block the virus from infecting cells; thus, they can prevent infection altogether when given prophylactically. However, an antibody neutralizing function may be dispensable for intravenously infused IAV treatments of patients with influenza, as the virus replicates in lung endothelial cells on the apical side of the lung, from which infused antibodies are generally excluded (44), and intravenously infused mAbs—including mAbs deemed neutralizing in vitro (38, 39, 45–47)—depend entirely on Fc effector functions (45, 48–52) within infected tissues. These considerations could explain the therapeutic failure of neutralizing IAV-specific mAbs observed in clinical trials (42, 43, 53) and NCT02623322, NCT01992276, NCT01719874, and NCT01390025. Despite this, non-neutralizing mAbs have not yet been developed as therapeutic agents.

Antibody binding to Fc receptors (FcRs) triggers a multitude of effector functions in FcR expressing immune cells, and antibody isotypes and subclasses differ in their ability to elicit Fc-mediated effector functions. These include antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), and complement-mediated cytotoxicity (54). In mice, all gamma-chain containing FcRs can trigger ADCP, which is mediated by neutrophils, macrophages, monocytes, and dendritic cells, while FcγRIIIa (CD16) ligation triggers ADCC in natural killer (NK) cells, neutrophils, macrophages, and monocytes. These immune cells are all resident in murine and human lung tissue, and they respond to IAV infection with vigorous proliferation, FcR-mediated antiviral functions, and the secretion of cytokines and chemokines that recruit additional immune cells and enhance the lung’s antiviral state (54). Thus, non-neutralizing mAbs with robust antiviral Fc-mediated effector functions may be ideal for attacking IAV-infected cells within infected tissues and may be optimal as IAV therapy infusion products.

Here, we introduce the concept that non-neutralizing mAbs are effective, universally protective, and escape mutant-resistant IAV therapeutics. We evaluated three of our previously developed non-neutralizing mAbs specific to M2e (55) and demonstrate that they target overlapping epitopes in the highly conserved N terminus of M2e, do so competitively, and, as such, are broadly effective and more protective against lethal IAV challenge as a triple cocktail. We identified immunoglobulin G2a (IgG2a) as the most effective isotype and engagement of FcγRI (CD64), FcγRIII (CD16), and FcγRIV (CD16-2) as being required for therapeutic M2e-mAb efficacy and as key components of the antiviral immune response in mice. Because our M2e-mAbs target the highly conserved region of the M2e N terminus, they are also—individually and in a low-dose triple M2e-mAb therapy—viral escape mutant resistant when tested in immunocompetent or immunodeficient mice, setting them apart from all now FDA-approved IAV therapeutics.

RESULTS

M2e-specific antibody clones 472, 522, and 602 bind to M2e’s highly conserved N-terminal region

We recently reported the generation of several non-neutralizing murine M2e-mAbs and established their efficacy as IAV prophylactic agents (55); each broadly reactive to M2e peptides from human, avian, and swine IAV strains (PR8, CA07, FM1, VN1203, Anhui1, swNE, swTX, and swMO) and capable of binding the corresponding intact virions and infected cells. When prophylactically administered, these M2e-mAb clones protected highly influenza susceptible Balb/c mice against laboratory, circulating, and HPAI infection–associated lethality (PR8, CA07, VN1203, and Anhui1; Table 1) (55). To map out each M2e-mAb clone’s antigen-binding site, we used an M2e–consensus sequence (CS) alanine scanning peptide library (Fig. 1A and table S1) and an 18-mer overlapping M2e-truncation peptide library (Fig. 1B) to test the binding of biotinylated IgG1 isotype M2e-mAb clones 472, 522, and 602. When alanine mutations were introduced into M2e’s highly conserved N terminus, M2e-mAb binding was abrogated or reduced (Fig. 1A). Truncation mutants lacking serine at the first position also abrogated the binding of all three M2e-mAbs (Fig. 1B). Further, we compared M2e-mAb binding to M2e peptides with either the original serine (polar uncharged side chains), lysine (positively charged side chains), aspartic acid (negatively charged side chains), or alanine (hydrophobic uncharged side chains). We established that M2e-mAb clones 602 and 522 bound to M2e peptides with a serine in the first position, and clone 472 bound to M2e peptides with a serine or an alanine in the first position; however, none of the clones bound to M2e peptides with a charged amino acid in the first position (Fig. 1C). Our data demonstrate that M2e-mAb clones 472, 522, and 602 bind M2e’s highly conserved N-terminal region, mechanistically explaining why M2e-mAb clones 472, 522, and 602 bind broadly to many IAV serotypes and validate their strong potential as a universal IAV treatment.

Table 1. M2e sequences of the IAVs used in this study and compared to the M2e CS.

Indicated M2e sequences of IAVs, isolated from humans, birds, and swine (10). The CS is derived from seasonal influenzas viruses circulating since 1957 (H1N1, H2N2, and H3N2; shown). Mutations in IAV serotypes compared to the CS are shown in bold font.

Virus	Subtype	Strain abbreviations	Amino acid sequence
Virus	Subtype	Strain abbreviations	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23
CS			S	L	L	T	E	V	E	T	P	I	R	N	E	W	G	C	R	C	N	D	S	S	D
A/PR/8/1934	H1N1	PR8	S	L	L	T	E	V	E	T	P	I	R	N	E	W	G	C	R	C	N	G	S	S	D
A/CA/07/2009	pH1N1	CA07	S	L	L	T	E	V	E	T	P	T	R	S	E	W	E	C	R	C	S	D	S	S	D
A/FM/1/1947 (WT and MA)	H1N1	FM1	S	L	L	T	E	V	E	T	P	T	K	N	E	W	E	C	R	C	S	D	S	S	D
A/Vietnam/1203 /2004	H5N1	VN1203	S	L	L	T	E	V	E	T	P	T	R	N	E	W	E	C	R	C	S	D	S	S	D
A/Anhui/1/2013	H7N9	Anhui1	S	L	L	T	E	V	E	T	P	T	R	T	G	W	E	C	N	C	S	G	S	S	E
A/sw/NE/A01444614/2013	H1N1	swNE	S	L	L	T	E	V	E	T	P	T	R	N	G	W	E	C	K	C	N	D	S	S	D
A/sw/TX/A01049914/2011	H3N2	swTX	S	L	L	T	E	V	E	T	P	T	R	S	E	W	E	C	R	C	S	D	S	S	D
A/sw/MO/A01444664/2013	H1N2	swMO	S	L	L	T	E	V	E	T	P	T	R	N	G	W	E	C	K	C	N	D	S	S	D

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M2e-specific antibodies bind to M2e competitively

The M2e-binding sites for M2e-mAb clones 472, 522, and 602 are similar but not identical (Fig. 1A). Thus, to determine whether M2e-mAb clones 422, 572, and 622 compete for binding, we used inactivated IAV virions to perform competition assays, choosing IAV serotypes that include commonly used laboratory strains, circulating strains, and HPAI strains with pandemic potential. These included H1N1 A/PR/8/34 (PR8), pH1N1 A/CA/07/2009 (CA07), A/Vietnam/1203/2004 (VN1203), A/Anhui/1/2013 (Anhui1) (Table 1 and Fig. 2), A/FM/1/1947 (FM1), A/sw/NE/A01444614/2013 (swNE), A/sw/TX/A01049914/2011 (swTX), and A/sw/MO/ A01444664/2013 (swMO) (Table 1 and fig. S1). We evaluated competitive antibody binding by first coating plates with the indicated inactivated IAV virions, followed by incubation with an unlabeled competitor M2e-mAb, and then a biotinylated monitored antibody for detection by enzyme-linked immunosorbent assay (ELISA). In this assay, a reduction of the detected absorbance indicates interference by the unlabeled competing antibody with the monitored antibody’s binding. In general, a given antibody clone strongly competes with itself (Fig. 2). However, despite the M2e-mAb clones’ similar binding capacity to the M2e N terminus across the different IAV serotypes, we observed some competition. While recognition of PR8 and CA07 by clones 472, 522, and 602 was similar, we observed greater competition when we evaluated antibody binding with VN1203 and Anhui1 (Fig. 2, A to E). Clone 522 outcompeted clone 602 with a higher affinity for a shared VN1203-M2e epitope (Fig. 2, C and E) and was, in general, the most competitive (Fig. 2E and fig. S1E). We obtained similar results when we tested M2e-mAb binding to various contemporary IAV strains (fig. S1). Our data demonstrate that M2e-mAb clones 472, 522, and 602 bind the N terminus of the highly conserved M2e protein broadly but distinctly.

Fig. 2. — Inactivated virions from (A) PR8, (B) CA07, (C) VN1203, and (D) Anhui1 were used as coating antigens to determine the competitive binding of biotinylated M2e-mAb clones 472 (IgG2a), 522 (IgG1), and 602 (IgG2a) (2 μg/ml) by competition ELISA. The competing antibody was added before the biotinylated antibody at fourfold dilutions starting at 100 μg/ml. Absorbance was measured with a biotin-binding secondary antibody. (E) The concentration at which the absorbance dropped 0.1 below the average absorbance of the “no competitor” control (specific to the antibody and virus) was used to summarize the data.

M2e-mAbs are more protective against lethal IAV challenge when administered as a triple cocktail

Because of their competitive binding, we hypothesized that prophylactic therapy composed of two or three M2e-mAb clones is superior and efficient at a lower dose to mAb therapy. We tested our hypothesis by prophylactically treating BALB/c mice with a 30-μg dose per mouse of either a triple M2e-mAb therapy [10 μg each of clones 472 (IgG2a), 522 (IgG1), and 602 (IgG2a)] or a double M2e-mAb therapy (composed of either 15 μg each of clones 472 and 522, clones 472 and 602, or clones 522 and 602), followed by lethal IAV challenge with either PR8, CA07, VN1203, or Anhui1. Our hypothesis proved correct, as the protection afforded by 30 $μ$ g of the triple M2e-mAb therapy was significantly better (88%) than the protection observed with any combination of two M2e-mAbs (33 to 44%; Fig. 3A) or individual M2e-mAb therapy components [published in (55)]. A 60- $μ$ g dose of the triple M2e-mAb therapy was also significantly protective of BALB/c mice challenged with either laboratory (Fig. 3B) or pandemic IAV strains (Fig. 3, C to E). Also, the treatment significantly ameliorated disease severity, as it considerably reduced weight loss in IAV-challenged BALB/c mice (Fig. 3, A to E). We concluded that the protection provided by the triple M2e-mAb therapy results from the sum of its individual cross-protective M2e-mAb clones rather than attributable to a single mAb or the protective effects of two together. Also, at low doses, an M2e-mAb cocktail composed of three cross-protective M2e-mAbs is more protective against lethal IAV challenge than its individual component antibodies. Consistent with our previous report, where individual M2e-mAb clones 472, 522, and 602 generally did not demonstrate neutralizing activity in vivo (55), triple M2e-mAb therapy only modestly decreased lung viral titers in IAV-infected mice, with significant decreases observed only in PR8 and VM1203-challenged animals (Fig. 3F).

M2e-mAb therapeutic efficacy depends on FcγRI (CD64), FcγRIII (CD16), and FcγRIV (CD16-2)

M2e-mAbs clones 472, 522, and 602 did not fully neutralize IAV in vivo (Fig. 3F), suggesting that FcR-mediated immune functions are responsible for the previously observed therapeutic effects. Thus, we expressed the M2e-mAbs as either IgG1 or IgG2a isotypes, which robustly activate FcR-mediated immunity in mice (52). IgG2a binds to three activating receptors: FcγRI, FcγRIII, and FcγRIV (56–58), while IgG1 also binds to the low-affinity inhibitory receptor FcγRIIb and the low-affinity activating receptor FcγRIII. To experimentally determine the most effective isotype for M2e-mAb therapy, we compared the prophylactic efficacy of the triple M2e-mAb therapy when expressed as either IgG1 or IgG2a isotypes (fig. S2). We identified the IgG2a isotype as a more effective prophylactic M2e-mAb therapy at any dose compared to the matching IgG1-M2e-mAbs (Fig. 4, A to D). Similarly, while the IgG2a triple M2e-mAb therapy robustly ameliorated weight loss and prevented death in PR8-challenged BALB/c mice, the IgG1 triple M2e-mAb therapy was largely ineffective (Fig. 4E). These data demonstrate that IgG2a-FcR–mediated effector functions are essential for M2e-mAb therapeutic efficacy and that this efficacy may involve the activation of the complement cascade, FcγRI, FcγRIII, and/or FcγRIV.

Fig. 4. — (A to E) On the day before infection, 6- to 8-week-old female BALB/c mice were infused intraperitoneally with [(A) and (B)] 25 μg, [(C) and (D)] 100 μg of the specified M2e-mAbs, or (E) 60 μg (each for 20 μg) of the indicated triple cocktail. At 0 dpi, the mice were infected intranasally with a lethal dose (5× LD₅₀) of H1N1 A/PR/8/34. Survival and weight loss were monitored for 21 dpi. N = 7 to 8. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, log-rank (Mantel-Cox) test for survival and one- or two-way ANOVA (Dunnett’s multiple comparisons test for percent weight loss). Percent weight data for survival analysis is shown to the right of each graph. The heatmap below each weight loss curve indicates significantly different percent weight from the control group on each day. For heatmap, * indicates significance compared to PBS control. [(A) to (D)] Twenty-five and 100 μg data are displayed as two sets for clarity with the shared PBS group. dpi, days prior to infection.

To identify the relevant IgG2a-dependent effector functions responsible for M2e-mAb therapeutic efficacy, we engineered clone 602 to carry the following Fc mutations in its IgG2a isotype: “LALA-PG,” which does not bind to any FcγRs nor C1q (59); “LEEA2KA,” which binds to FcγRIIb, FcγRIII, and FcγRIV but not to FcγRI nor C1q (60); and “L235E,” which abrogates binding to FcγRI (60) and will distinguish between FcγRI and C1q binding (Fig. 5, A and B). We compared the efficacy of the wild-type (WT; IgG2a) and indicated Fc-mutant M2e-mAbs (all clone 602) when administered as a prophylactic treatment to BALB/c mice 24 hours before lethal IAV challenge. We discovered that half of the M2e-mAb protective effects required FcγRI but not the activation of the complement cascade (Fig. 5, C to E). These results suggest that FcγRIII and/or FcγRIV may also be required for therapeutic efficacy. To further evaluate this, we prophylactically treated BALB/c mice with the M2e-LE-mutant-mAb and with blocking mAbs specific to FcγRIII (clone: 275003), FcγRIV (clone: 9E9) (56), or both, which we infused into mice before and during IAV challenge. The addition of either blocking antibody or the combination of both (to FcγRIII and FcγRIV) abrogated the survival of IAV-challenged M2e-LE-mutant-mAb–treated BALB/c mice (Fig. 5, C to E). These results demonstrate that FcγRI, in combination with FcγRIII or FcγRIV, is responsible for therapeutic M2e-mAb efficacy. To determine their potential roles in M2e-mAb–mediated protection, we chose to use blocking antibody administrations [mAb clone 275003 and clone 9E9 to block FcγRIII and FcγRIV, respectively (56)] as no effector function blocking mutations have been identified for FcγRIII or FcγRIV, and a genetic deficiency in either FcR results in significant compensatory up-regulation of the other FcR in mice, making results difficult to interpret (58, 61). In contrast, FcγRIV expression does not differ between FcγRI-deficient and control animals (61). Together, our robust mechanistic studies establish that protection from IAV lethality requires M2e-mAb–triggered FcγRI and FcγRIII and/or FcγRIV-mediated effector functions.

Fig. 5. — (A) Recombinant mouse FcγRI, FcγRIIB, FcγRIII, FcγRIV, or the M2e-CS peptide was used as a coating antigen. WT M2e-mAb clone 602 and clone 602 with either the LE, LEEA2KA, or LALAPG Fc domain mutation (50 μg/ml) were used to determine binding to recombinant FcRs or the M2e-CS peptide by direct ELISA. PBS was used as a control. To test the binding of the WT and Fc-mutant M2e-mAb clone 602 preparations to C1q, the WT or mutant antibodies were used as coating antigens, followed by mouse C1q protein. PBS was used as a control. Anti-mouse C1q-biotin and avidin–horseradish peroxidase (HRP) were then used to quantify C1q protein bound by the capture antibodies by indirect ELISA. N = 3 independent experiments. (B) Summary of the differential binding affinities as determined in (A). (C) Experimental outline: Groups of 6- to 8-week-old female BALB/c mice were prophylactically intraperitoneally injected with either the WT (IgG2a) or the indicated Fc-mutant M2e-mAbs (all clone 602) on day −1 (100 μg per mouse). Control mice received PBS. Additional groups of mice were intraperitoneally injected with blocking mAbs specific to FcγRIII (100 μg per mouse) and/or FcγRIV (200 μg per mouse) on days −2, 1, and 4 and additionally prophylactically treated with the M2e-mAb clone 602 with the LE mutation (100 μg per mouse, intraperitoneally) at day −1. All mice were challenged with a lethal dose (3× LD₅₀) of PR8 by aerosol inhalation on day 0, and (D) survival and (E) weight loss were monitored for 21 days postinfection. Statistical significance for weight loss is shown in the heatmap. N = 10 to 29 mice per group; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; survival: log-rank Mantel-Cox test; weight loss: two-way ANOVA with Dunnett’s multiple comparisons test.

A single therapeutic treatment of mice with the triple M2e-mAb significantly ameliorates disease severity and enhances survival in mice challenged with PR8 or Anhui/1 IAV

Although postexposure prophylaxis can be offered to those exposed to IAV, antiviral therapy is most commonly used to treat infected patients. Therefore, we examined whether treating mice with our triple IgG2a M2e-mAb therapy enhances their survival when the therapy is administered after lethal H1N1 A/PR/8/34 IAV infection (Fig. 6). We tested the therapeutic efficacy for the triple M2e-mAb therapy using two IAV challenge doses, one 100% lethal (Fig. 6A) and the other 75% lethal (Fig. 6B), as the IAV infectious dose is poorly understood in humans. We determined that survival of highly susceptible BALB/c mice improved in all therapeutically treated experimental groups (days 0 to 4 postexposure), achieving statistical significance for several time points. In addition, treatment with the triple IgG2a M2e-mAb therapy significantly enhanced the survival of BALB/c mice when it was administered on the day of infection or 1 or 2 days later. At the lower IAV challenge dose, disease severity, as determined by weight loss, was also significantly ameliorated when the triple M2e-mAb therapy was administered as late as day 3 after the IAV challenge.

Fig. 6. — (A to B) Groups of 6- to 8-week-old female BALB/c mice were infected with (A) a lethal dose (3× LD₅₀) of PR8 by aerosol inhalation or (B) a sublethal dose of (1× LD₅₀) of PR8 by intranasal instillation. All mice received 450 μg in total of the M2e-mAb triple cocktail one time at the indicated time point (150 μg each of clones 472, 522, and 602). Survival and weight loss were monitored for 21 days postinfection. Survival was analyzed using the Mantel-Cox log-rank test. One-way ANOVA (overall weight loss) or two-way ANOVA (daily weight loss) with Dunnett’s multiple comparisons test was used to compare experimental groups to the isotype control. (C to E) Groups of 6- to 8-week-old female BALB/c mice were infected with a lethal dose (10× LD₅₀) of H7N9 Anhui1 by intranasal instillation. All mice received either 450 μg of the M2e-mAbs triple cocktail or isotype control one time at the indicated time point. (C) Survival and (D) weight loss were monitored for 15 days postinfection, and (E) lung viral titers were measured on day 5 (on day 6 for the day 5 groups) by plaque assay. Statistical significance for weight loss is shown in the heatmap. N = 8 to 10 mice per group for survival and weight loss. N = 5 mice per group for lung viral titers. A Mantel-Cox log rank test was used for the survival analysis. A paired t test (overall weight loss) or a two-way ANOVA with a Sidak’s multiple comparisons test (weight loss on individual days postinfection). Mann-Whitney test was used for lung viral titers on the specified day. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

To examine our M2e-mAb therapy’s efficacy against an IAV serotype of significant public health concern, we evaluated the therapeutic efficacy of M2e-mAb in mice challenged with the avian IAV H7N9 (Anhui), which causes severe disease and high mortality in poultry and humans (6). A single treatment of H7N9-challenged BALB/c mice with the triple M2e-mAb therapy significantly lowered lung viral titers, even when the treatment was administered as late as day 4 postinfection (Fig. 6C). The reduction in lung viral titers correlated with significantly improved survival: 100% of H7N9 challenged and therapeutically treated BALB/c mice survived when the M2e-mAb treatment was administered on the day of infection (day 0) or day 1 or day 3 postinfection (Fig. 6D). In addition, 80% of mice treated on day 2, 70% of mice treated on day 4, and 60% of mice treated on day 5 postinfection survived (Fig. 6D). It is noteworthy that we did not observe statistical significance at the later time points (days 3 to 5 postinfection), possibly due to the improved survival of the isotype control groups, which benefitted from hydration when IgG2a isotype control mAb was infused in 200 μl of saline on day 3, 4, or 5 post-H7N9 challenge. However, survival remained high in all M2e-mAb–treated experimental groups (days 0 to 5 postinfection) compared to those treated with IgG2a isotype control mAb infusions. Disease severity, as determined by overall weight loss, was also significantly ameliorated when the M2e-mAb triple cocktail therapy was administered as late as day 4 after the H7N9 IAV challenge (Fig. 6E, overall weights). These data establish our triple M2e-mAb therapy as robustly effective against one of the most lethal IAV serotypes and superior to FDA-approved treatments.

Treatment with M2e-mAb therapies does not drive the development of IAV viral escape mutants

Viral escape mutants have been reported in response to all FDA-approved IAV therapies, including those targeting M2 function (14–22, 62). These escape mutants have arisen as early as 48 hours after treatment in humans (14, 17). To determine whether our M2e-mAb therapy drives the development of viral escape mutants in IAV-challenged mice, we first passaged PR8 IAV in the presence of the M2e-mAb triple cocktail or phosphate-buffered saline (PBS; control) seven times in immunocompetent (BALB/c) mice over the course of 24 days (Fig. 7A). This number of passages and timeframe allows for a robust evaluation of whether viral escape mutants will form to antibody therapy, based on prior published studies (14, 15, 40, 51, 62, 63). We isolated viral RNA from the mice’s lungs to generate IAV M gene segment–specific cDNA, which we subjected to Sanger sequencing and compared to the M2 sequence of the original (day 0) PR8 virus isolate (Fig. 7A). Encouragingly, no mutations arose in the M gene in any of our treatment groups despite constant selective and immune pressure from M2e-mAb treatments (Fig. 7B). In addition, we evaluated whether single M2e-mAb treatments or alternating M2e-mAb treatments resulted in M region mutations. We detected no M region mutations in PR8 viruses isolated after 24 days of single or alternating M2e-mAb treatments (fig. S3).

Fig. 7. — PR8 “stock virus” was passaged through WT mice for 24 days, and the final viral isolates were analyzed by Sanger sequencing. (A) Outline of the time points for mouse-to-mouse passaging of lung PR8 isolates and the indicated M2e-mAb triple cocktail treatments [clones 472 (IgG2a), 522 (IgG1), and 602 (IgG2a)] in WT mice. At each passage, virus was isolated from lung homogenates of M2e-mAb triple cocktail or PBS control–treated mice and used to infect a group of naïve prophylactically M2e-mAb triple cocktail therapy or PBS (control) treated groups of mice. Intraperitoneal injections of the M2e-mAb triple cocktail or PBS (control) are indicated by a blue arrow. (B) Sequencing chromatograms from the viral isolates isolated from therapeutically or PBS control–treated groups of WT mice. (C) BALB/c mice were treated with 60 μg of the M2e-mAb triple cocktail [clones 472 (IgG2a), 522 (IgG1), and 602 (IgG2a)] 1 day before infection with a lethal dose of (5× LD₅₀) of PR8 that had been isolated from either M2e-mAb triple cocktail–treated or PBS control–treated WT mice. Then, their survival and percent weight loss were determined. N = 10 mice per group. Log-rank analysis (Mantel-Cox) test for survival, ***P < 0.001, **P < 0.01, and ns, not significant.

To experimentally address this possibility that mutations outside of the M region can enable viral escape by delaying M2e expression (39, 51), we challenged BALB/c mice with a lethal dose of the PR8 virus isolated from triple M2e-mAb therapy–treated BALB/c mice or with PR8 virus isolated from PBS-treated (control) animals and treated half of the mice in each group prophylactically with the triple M2e-mAb therapy. If the virus has not mutated in a way that allows it to evade the triple M2e-mAb treatment, then therapeutic effectiveness should not decrease over time. The triple M2e-mAb therapy maintained robust effectiveness against the PR8 virus isolated from both PBS control and M2e-mAb–triple therapy–treated mice (Fig. 7C), demonstrating that M2e-mAb therapy does not result in viral immune escape. Together, our sequencing and in vivo data demonstrate the virus’s failure to escape from our highly effective M2e-mAb–based antiviral therapy. Hence, M2e-mAb therapy is superior to now FDA-approved treatments for IAV infection (14–22, 62).

Some of the FDA-approved treatments for IAV infection have been observed to increase the transmissibility of resistant viruses (19). Thus, we examined whether our M2e-mAb therapy modulates IAV virulence. We found that passaging PR8 IAV in WT BALB/c mice in the presence of either individual M2e-mAbs or the triple M2e-mAb cocktail lowered its virulence. Twice as many plaque-forming units (PFU) were needed to reach a median lethal dose (LD₅₀) for IAV virus derived from the lungs of mice treated with the triple M2e-mAb cocktail (6 PFU) compared to IAV derived from isotype-matched control mAbs or PBS-treated mice (3 PFU) (fig. S3E). Single M2e-mAb treatments revealed that clone 472 most robustly reduced viral fitness (12.7 PFU = LD₅₀), followed by clones 602 and 522 (9.4 and 4.3 PFU = LD₅₀, respectively). Therefore, we hypothesize that, during a seasonal or pandemic outbreak, our M2e-mAb therapy may reduce virulence, resulting in lower transmissibility and reduced viral persistence.

Severely immunocompromised individuals comprise about 3% of the US population and are at high risk of substantial influenza-related morbidity and mortality (64). Recombinase activating gene 2 knockout (Rag2-KO) mice, which lack T and B cells but have innate immune cells and NK cells capable of FcR-mediated immunity, develop chronic or fatal IAV infections, recapitulating what is observed in immunocompromised individuals (65, 66). Notably, Rag2-KO mice rapidly develop viral escape mutants in response to therapies, including M2e-mAb treatments (14, 40). To determine whether our M2e-mAb triple cocktail ameliorates disease in immunocompromised hosts, we prophylactically and therapeutically treated PR8 IAV–challenged Rag2-KO mice with either individual M2e-mAbs, alternating M2e-mAb treatments, or the triple M2e-mAb cocktail (fig. S4). Individual treatments with M2e-mAb clones 472 and 602, but not 522, alternating single M2e-mAb treatments, and M2e-mAb triple cocktail therapy significantly improved the survival and ameliorated infection-induced weight loss of PR8 IAV–challenged Rag2-KO mice (fig. S4). Also, comparisons of PR8 IAV’s M gene sequences revealed that no mutant escape viruses developed in PR8 IAV–infected Rag2-KO mice, regardless of the therapy regimen (fig. S3, C and D, and table S2). Our data demonstrate that M2e-mAb therapy ameliorates disease in IAV-infected immunocompromised mice and does not elicit viral escape mutants.

Together, our results have established the triple M2e-mAb cocktail therapy as robustly effective and viral escape mutant-resistant therapy against IAV, including against one of the most lethal IAV subtypes. Our findings are in stark contrast to previously published M2e-mAbs and FDA-approved M2 inhibitors, which rapidly elicit escape mutants in WT and immunocompromised mice (38–40, 48).

DISCUSSION

In this study, we used non-neutralizing mAbs to develop a highly and broadly effective viral escape mutant-resistant therapy to generate a safe, effective, and universally protective off-the-shelf IAV treatment option. We selected the ectodomain of the IAV-encoded M2 protein as a suitable target for our therapy, as M2 assembles into a highly conserved proton channel expressed on influenza virions and infected cells and is required for viral entry and the viral life cycle, and its N terminus is highly conserved across different IAV subtypes (24–26). Our M2e-mAbs, which bind to the highly conserved N terminus of IAV-encoded M2e, are highly effective against a variety of laboratory and circulating IAV subtypes, including an HPAI; thus, they have universal potential and could enhance preparedness for seasonal and pandemic IAV outbreaks.

Using Fc-mutant M2e-mAbs and blocking FcR-specific antibodies, we identified Fc $γ$ RI, Fc $γ$ RIII, and Fc $γ$ RIV as essential for IgG2a M2e-mAb–mediated protection, suggesting that a variety of FcR-mediated effector functions, and as such, FcR-expressing immune cell types contribute to M2e-mAb–mediated protection. M2e-mAb–based therapies in humans would use a humanized M2e-mAb cocktail where human isotypes interact with human FcRs, thus allowing the engineering of the mAb-based therapy to optimize antibody half-life (67) and FcR-mediated effector functions (68). However, we did not attempt to identify which FcR functions are mediated by these receptors. Fc $γ$ RI, Fc $γ$ RIII, and Fc $γ$ RIV are involved in phagocytosis, degranulation, and ADCC. NK cells, monocytes, macrophages, neutrophils, dendritic cells, basophils, mast cells, and eosinophils express Fc $γ$ RIV. In contrast, Fc $γ$ RIII is expressed by NK cells, monocytes, and macrophages, and Fc $γ$ RI is expressed by monocytes, macrophages, and dendritic cells (69, 70). While others have been able to selectively deplete some tissue-resident subsets of immune cells, isolate and adoptively transfer them (48), markers exclusively expressed by a single immune cell type are rare. Thus, these approaches either fail to distinguish related and phenotypically similar immune cell types or result in too few cells available for adoptive transfer.

Previously developed mouse and human M2e-mAbs with protective potential are strain specific, have limited efficacy, or drive viral immune escape, and none have been successful in clinical trials (41, 51). To enable protection at lower doses, increase universality, and resistance to viral immune escape, we tested double and triple M2e-mAb cocktails composed of individual M2e-mAbs that are protective from lethal IAV challenge in mice. We demonstrated the broad therapeutic applicability, minimum effective dosage, and therapeutic administration time points for our M2e-mAb triple cocktail in mice challenged with human and zoonotic biosafety level (BSL)-2 and BSL-3 IAV strains.

While low-dose combinations of M2e-mAb pairs failed to protect IAV-challenged mice from lethality completely, a triple cocktail composed of M2e-mAbs with competitive binding sites in the M2-proteins N-terminal region was universally protective and highly effective at low doses. These results suggest that the protection provided by the triple M2e-mAb cocktail is not due to a dominant effect by one or two of the component antibodies but due to all three M2e-mAb cocktail component antibodies acting in synergy. The concept of combining multiple therapies or epitopes had been suggested (10, 71) but has not been evaluated extensively. Thus, combining mAbs and their combined FcR effector functions is a more effective IAV treatment than using individual mAbs.

Using a triple mAb cocktail allowed us to lower the dose. When administered prophylactically, the 60-μg dose (approximately 3.7 mg/kg) is 100% protective against lethal PR8 IAV challenge. Estimates based on our data for a starting prophylactic dose for a human clinical trial would be 0.3 mg/kg (72), a much lower dose than that of many clinical trials (24, 36–43) and NCT02623322, NCT01992276, NCT01719874, and NCT01390025. However, the efficacy and associated therapeutic dosage of our M2e-mAb triple cocktail still need to be determined in humans.

We observed partial neutralization of H1N1 and H7N9 in prophylactically triple M2e cocktail–treated IAV-infected mice, presumably due to clone 472, which has some neutralizing activity (55). However, all M2e-mAb–treated animals became infected with influenza as evidenced by their weight loss. Thus, our M2e-mAbs do not prevent infection despite their partial neutralizing activity. The therapeutic effect of the M2e-mAb cocktail was strongly dependent on FcR functions and most robust when M2e-mAbs were expressed as the IgG2a isotype, with therapeutic efficacy depending on Fc $γ$ RI-, Fc $γ$ RIII-, and Fc $γ$ RIV-mediated effector functions. Our data are supported by prior studies demonstrating most M2e-mAbs to be non-neutralizing (48, 50, 73).

Using Sanger sequencing of the M region and in vivo challenge studies in immunocompetent and immunodeficient mice, we demonstrated that our individual non-neutralizing M2e-mAbs and the resulting triple cocktail are viral escape mutant-resistant treatments. Our results demonstrated unchanged susceptibility of IAV to the M2e-mAb triple cocktail therapy after prolonged viral passage in the presence of therapy. Thus, despite IAV’s well-established ability to develop escape mutations to M2e-mAbs in vitro (39) and mice (38–40, 48), we have demonstrated that it is possible to target IAVs with M2e-mAbs without driving viral immune escape.

Another crucial attribute of our triple M2e-mAb cocktail therapy is its ability to effectively reduce lung viral titers, ameliorate disease severity, and reduce lethality when administered up to 4 days postinfection to BALB/c mice challenged with the virulent H7N9 HPAI virus. Until our study, therapeutic efficacy had not been evaluated for published M2e-specific mAbs administered later than 2 days postinfection (30, 52). Emerging, reemerging, and smoldering outbreaks of zoonotic IAVs, including H7N9, pose a substantial public health threat to the human population due to their high lethality and their ability to cause upper and lower respiratory tract disease, severe pneumonia with respiratory failure, encephalitis, and multiorgan failure (6). Thus, our results are impactful as they establish that robust and effective off-the-shelf mAb-based therapeutics, including those made of non-neutralizing mAbs, can be developed to protect us from future potential IAV pandemics. Our results are especially important considering that several highly anticipated HA stalk antibodies have failed to demonstrate efficacy in phase 2 clinical trials (74) and NCT05567783, albeit it is noteworthy that human IgG1, the isotype that would be used clinically, has weaker Fc effector function than murine IgG2a because it binds human FcγRs and C1q with lower affinity. In contrast, murine IgG2a’s hinge and glycosylation enable stronger FcγRI/III/IV engagement, receptor clustering, and downstream ADCC and phagocytosis, making it more potent in preclinical models (75). Thus, we have developed a universally effective and viral escape mutant-resistant M2e-mAb triple cocktail that significantly reduces lung viral titers and ameliorates disease severity even when administered as late as 4 days postinfection.

In summary, our study establishes a triple cocktail of cross-protective non-neutralizing M2e-mAbs to be (i) efficacious at preventing IAV lethality at low doses; (ii) consistently, universally protective, and therapeutic between IAV subtypes; and (iii) resistant to viral immune escape. These highly desirable attributes make the M2e-mAb cocktail a strong candidate for a universal off-the-shelf IAV therapeutic, ensuring rapid availability, consistent protection between strains, and prevention of viral resistance development. Broadly effective, escape mutant-resistant off-the-shelf therapeutics are likely our best option for reducing lethality during future IAV pandemics and may provide us with the necessary window to develop and disseminate a pandemic subtype–specific vaccine. Thus, we present a universally protective and viral escape mutant-resistant therapeutic agent utilizing a trio of newly generated non-neutralizing IAV-M2e-specific antibodies. Our data will critically shape future M2e-mAb–based IAV-therapeutic development strategies and provide precedence for embracing non-neutralizing M2e-mAbs for clinical development.

MATERIALS AND METHODS

Mice

Female 6- to 8-week-old BALB/c and Rag2-KO mice were obtained from Charles River Laboratories Inc., San Diego and Envigo RMS Inc., Indianapolis, Indiana, USA, respectively. Mice were housed in the animal facilities of the Texas Children’s Hospital, the Scripps Research Institute, the Jackson Laboratory, and the University of Georgia, Athens. All protocols involving the use of experimental animals in this study were approved by Baylor College of Medicine’s, The Scripps Research Institute’s (18-0014), the Jackson Laboratory’s (AP-200993-1126), and the University of Georgia, Athens’ (A2020 03-033) Institutional Animal Care and Use Committees and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Viruses

Influenza strains used in this study are H1N1 A/PR/8/34 (PR8), pH1N1 A/CA/07/2009 (CA07), A/Vietnam/1203/2004 (VN1203), A/Anhui/1/2013 (Anhui1), A/FM/1/1947 (FM1), A/sw/NE/A01444614/2013 (swNE), A/sw/TX/A01049914/2011 (swTX), and A/sw/MO/ A01444664/2013 (swMO). Before use in this study, all viruses were obtained, passaged, isolated, and quantified as previously described (55). All experiments using H7N9 or H5N1 IAV were reviewed and approved by the institutional biosafety program at the University of Georgia, Athens and were conducted in BSL3 enhanced containment. Work with HPAI virus H5N1 followed guidelines for using Select Agents approved by the US Centers for Disease Control and Prevention.

Cell lines

FreeStyle 293-F cells were purchased from Thermo Fisher Scientific. FreeStyle 293 Expression Medium (Thermo Fisher Scientific) was used for culturing and transfecting the cells without any additional reagents in orbital shakers at 135 rpm, 37°C, and 8% CO₂. MDCK.2 cells were purchased from American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 25 mM Hepes, 4 mM l-glutamine, and penicillin-streptomycin (100 U/ml). Consensus and Vietnam matrix protein 2 (M2)–inducible human embryonic kidney (HEK) cells were generated using the Flp-In T-REx 293 Cell Line system (Invitrogen) and grown following the methods in the previous papers (55, 76). The HEK cells were cultured in DMEM containing 10% FBS, 25 mM Hepes, 4 mM l-glutamine, hygromycin B (100 μg/ml), blasticidin (15 μg/ml), and penicillin-streptomycin (100 U/ml). Both MDCK.2 cells and the HEK cells were maintained at 37°C and 5% CO₂.

Intranasal IAV challenge

PR8 IAV was administered intranasally in 20 μl of PBS to 6- to 8-week-old mice anesthetized with isoflurane. CA07 and VN1203 IAVs were administered intranasally in 30 μl of PBS to 6- to 8-week-old mice anesthetized with ketamine/xylazine. H7N9 IAV was administered intranasally to 6- to 8-week-old mice anesthetized with 2,2,2-tribromoethanol in tert-amyl alcohol (Avertin; Aldrich Chemical Co). For each challenge with CA07, VN1203, and H7N9, the viral inoculum was back-tittered on MDCK-ATL cells to confirm the administered dose. All animals were monitored daily for body weight and humane endpoints for euthanizing. Survival and weight loss were monitored for up to 21 days postinfection or until all animals recovered to at least 90% of the starting body weight. All intranasal IAV challenges were conducted inside a BSL-2 safety cabinet.

Aerosolized IAV challenge

PR8 IAV in 1× PBS was placed into a MiniHEART-HiFlo Continuous Nebulizer (SunMed). An Eisco Superior Stand and Rod Set with a three-prong clamp with a Boss Head (Thermo Fisher Scientific) was used to stabilize the nebulizer. The nebulizer inlet was connected to a Flow Gauge and EasyAir2 compressor (Precision Medical) using oxygen tubing (SunMed). Corrugated plastic tubing (Harvard Bioscience) was attached to the top of the nebulizer, and the other end was attached to the mouse container. The in-unit flow gauge of the compressor was set to 10 liters/min and the flow gauge, attached to the condenser, to 8 liters/min. Mice were exposed to PR8-containing aerosol vapor for 25 min for infection with the aerosolized PR8 IAV. Overflow IAV aerosol was disposed of into a waste container containing 10% bleach through the oxygen tubing. Intranasal and aerosolized IAV challenge results in similar viral titers and disease courses (fig. S5). All aerosolized IAV challenges were conducted inside a BSL-2 safety cabinet. To achieve an LD₅₀ upon inhalation of virus, we titrated the length of time the mice inhale aerosolized virus in the chamber and used plaque assays to confirm that the resulting viral titers in the lungs of mice infected with intranasal droplets or by aerosolized virus inhalation match on day 3 postinfection.

Viral titer quantification

Three days postinfection, a subset of IAV-infected mice was humanely euthanized, and lung tissues were collected for virus quantification. Lung tissue samples were homogenized and titrated by plaque assay. Briefly, lungs were placed in PBS on ice (0.75 ml per lung), homogenized, and centrifuged at 850g for 10 min at 4°C, after which the supernatant was placed in an Ultracel-100 tube (Amicon Ultra-15 centrifugal filter unit; Ultracel-100 regenerated cellulose membrane) and centrifuged at 4000g for 30 min at 4°C. Supernatants were serially diluted 10-fold in DMEM and added to MDCK.2 cells (ATCC) in 6- or 12-well tissue culture plates. After a 1- to 2-hour incubation period, 2 ml of a 0.27% agar in DMEM containing N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-trypsin (0.5 μg/ml) or 1.2% Avicel microcrystalline cellulose overlay [MEM supplemented with Hepes, l-glutamine, NaHCO₃, penicillin/streptomycin/amphotericin B, and TPCK-trypsin (2 μg/ml)] was overlaid. Plates were incubated for 48 to 72 hours at 37°C with 5% CO₂, washed, fixed with 4% paraformaldehyde or methanol:acetone (80:20), and stained with 0.4% crystal violet solution to visualize plaques. For H5 or H7 viruses, supernatants were diluted in DMEM + 2% FBS, and Avicel overlay included 2% FBS instead of TPCK-trypsin.

Neutralization assay

MDCK.2 cells (ATCC) were seeded at a density of 1 × 10⁶ cells per well in six-well plates (VWR International) and incubated overnight at 37°C 5% CO₂. A total of 20 to 50 PFU/ml PR8 IAV was incubated with the indicated single M2e-mAb or the triple M2e-mAb cocktail (25 μg/ml; 8.3 μg/ml of each cocktail component antibody) at 4°C for 30 min in PBS. MDCK.2 cells were washed twice with PBS before antibody-virus mixtures were added, and the plates were gently shaken every 10 to 15 min at 37°C for 1 hour. Then, cells were overlayed with 0.27% agar in DMEM containing TPCK-trypsin (0.5 μg/ml) and incubated at 37°C for 3 days before plates were fixed with 4% paraformaldehyde for 1 hour and stained with 0.4% crystal violet solution for 30 min. Clear plaque numbers were counted, and virus titers (log₁₀ PFU/ml) were calculated.

M2e-mAb production from hybridomas

Antibody production for 472, 522, and 602 clones was performed by expanding the hybridomas as previously described (55). IgG2a isotype control–matched antibody was purchased from BioXCell (#BE0085).

Biotinylation of M2e-mAbs

M2e-mAbs were biotinylated using EZ-Link Hydrazide Biocytin (Thermo Fisher Scientific) according to the manufacturer’s instructions for labeling glycoproteins with hydrazide biocytin. Biotinylated M2e-mAb was separated from a nonreacted material by dialysis [10 kDa molecular weight cut-off (MWCO); Thermo Fisher Scientific] in 1× PBS for 12 hours. Samples were removed from dialysis cassettes, aliquoted, and stored at 4°C until use.

Cloning, expression, and purification of isotype-switched M2e-mAbs

Variable region sequences of the M2e-mAbs were verified by Sanger sequencing. For the M2e-mAbs heavy chain and light chain variable regions, gBlock double-stranded DNA was synthesized and purchased from Integrated DNA Technologies (IDT, USA). AgeI-HF/Eco47III [New England Biolabs (NEB)] or AgeI-HF/BstAPI (NEB) enzyme sites were added at the end of the gBlock DNA for their cloning into mouse IgG1 or IgG2a heavy chain plasmids or the mouse kappa light chain plasmid (InVivoGen). The gBlock DNA and plasmids were cut with the specified enzymes, subjected to agarose gel electrophoresis, extracted using the Monarch DNA Gel Extraction Kit (NEB), ligated using T4 DNA Ligase (Promega), and transformed into NEB 10-beta Competent Escherichia coli cells (NEB), and colonies were screened with Zeocin for the heavy and light chain plasmids using blasticidin (InVivoGen). The plasmids were purified using the QIAprep Spin Miniprep Kit (QIAGEN), and their sequences were confirmed by Sanger sequencing (Genewiz). Log phase (0.3 to 3 × 10⁶ cells/ml) FreeStyle 293-F cells were cotransfected with the specified M2e-mAbs heavy and light chain variable region plasmids (InvivoGen) using polyethylenimine (Polysciences). After 5 days, cells were centrifuged, and antibodies were purified from the supernatants using protein G or A bead affinity chromatography by The Scripps Research Institute’s Antibody Core Facility.

Epitope mapping using M2e peptide libraries

M2e peptide libraries were synthesized on the basis of the M2e-CS peptide, N-SLLTEVETPIRNEWGCRCNDSSD, and purchased from GenScript. The specified M2e peptide library was coated in the 96-well plates and incubated in 15 mM Na₂CO₃ and 35 mM NaHCO₃ bicarbonate buffer (pH 9.6) at 4°C overnight. The plates were blocked with PBS containing 1% bovine serum albumin (BSA) for 1 hour at room temperature before the specified biotinylated IgG1 M2e-mAbs or biotinylated IgG1 control mAbs (clone: MOPC-21, BioXCell) diluted in PBS 0.1% Tween 20 (PBS-T) were added and incubated for 1 hour at room temperature. The plates were washed three times with PBS-T, and streptavidin–horseradish peroxidase (HRP; Pierce Chemical), diluted in PBS-T, was added to the plates. After 1 hour, the plates were washed four times with PBS-T, and the peroxidase substrate tetramethylbenzidine (TMB) solution (Thermo Fisher Scientific) was added. After 5 min, 2 N H₂SO₄ was added in the plates, and absorbance was detected at 450 nm using the SpectraMAX iD3 instrument (Molecular Devices).

M2e-mAb competition ELISA

Nunc Maxisorp Flat-Bottom plates (Thermo Fisher Scientific) were coated overnight at 4°C with purified inactivated IAV (55) at 0.5 μg/ml in bicarbonate buffer (pH 9.6). After washing the plates three times with PBS 0.05% Tween 20, the plates were blocked with 1% BSA in PBS for 2 hours and washed three times with PBS 0.05% Tween 20 before biotinylated M2e-mAbs were added at 2 μg/ml (the concentration resulting in ~50% saturation in the assay) to the plates and incubated for 1 hour at 37°C. Then, the indicated M2e-mAbs were added as the competing antibody in fourfold dilutions and incubated for 1 hour at 37°C. After washing the plates with PBS 0.05% Tween 20 three times, a 1:10,000 streptavidin-HRP dilution (Vector Laboratories) was added to the plates and incubated for 1 hour at 37°C. The plates were then washed with PBS 0.05% Tween 20 three times, TMB substrate was added for 10 to 15 min before the reaction was stopped with the addition of H₂SO₄, and absorbance was measured at optical density at 480 nm.

M2e-mAb analysis by SDS-PAGE and Coomassie staining

SDS–polyacrylamide gel electrophoresis (SDS-PAGE) of the indicated M2e-mAbs prepared in both reducing and nonreducing conditions was performed using NuPAGE Bis-Tris Welcome Pack, 4 to 12%, 10-well (Thermo Fisher Scientific). Protein size was compared to PageRuler Plus Prestained Protein Ladder (10 to 250 kDa). To visualize proteins, the SDS-PAGE gel was stained with Coomassie Stain Solution (Rockland) for 30 min and then destained with Coomassie Brilliant Blue R-250 Destaining Solution (Bio-Rad), and bands were visualized using a ChemiDoc XRS+ System (Bio-Rad).

Detection of M2e-mAb binding to M2-expressing HEK cells by flow cytometry

M2 expressing HEK cells were treated with tetracycline (2 μg/ml) to induce M2 expression. After 48 hours, the cells were detached with trypsin-EDTA (0.05%), washed with PBS containing 2% FBS, and stained with the specified antibodies for 30 min at room temperature. After washing with PBS containing 2% FBS, the cells were stained with Alexa Fluor 488–conjugated goat anti-mouse IgG secondary antibody (Invitrogen) for 30 min, washed, and fixed in 2% paraformaldehyde. Flow cytometry was performed to measure M2e-mAbs binding to M2-expressing HEK cells using Cytek Aurora (Cytek Biosciences).

The 602-IgG2a Fc variant development

The pFUSE-CHIg-mG2a plasmid (InvivoGen) containing the 602 heavy chain variable region was used for the generation of Fc variant antibodies. gBlock double-strand DNAs for the L235E (LE), L235E/E318A/K320A/K322A (LEEA2KA), and L234A/L235A/P329G (LALAPG) mutated CH2 domains were synthesized and purchased from IDT with BamHI/BsrGI (NEB) restriction enzyme sites at the ends. The region encoding for the 602-IgG2a WT CH2 domain was replaced with the LE, LEEA2KA, or LALAPG gBlock DNA, all plasmids were confirmed by Sanger sequencing, and each of the 602 Fc variants was produced and purified following the “Cloning, expression, and purification of isotype-switched M2e-mAbs” section.

Quantification of M2e-mAb binding to FcγRs, C1q, and M2e peptides by ELISA

The 96-well plates were coated with the indicated antigens, such as M2e peptides (5 μg/ml; M2e vaccine or CS), or recombinant mouse Fc gamma RI, RIIB, RIII, or RIV (5 μg/ml; R&D Systems) in 15 mM Na₂CO₃ and 35 mM NaHCO₃ bicarbonate buffer (pH 9.6) at 4°C overnight. The plates were then blocked with PBS + 1% BSA for 1 hour at room temperature before the specified M2e antibodies, mouse IgG1 isotype control (clone: MOPC-21, BioXCell), or mouse IgG2a isotype control (clone: C1.18.4, BioXCell) was added (all 50 μg/ml) in PBS-T (0.1% Tween 20) and incubated for 1.5 hours. The plates were washed three times with PBS-T, and goat anti-mouse IgG-HRP (Jackson ImmunoResearch, USA) was added and incubated for 1 hour. Peroxidase activity was measured with TMB solution (Thermo Fisher Scientific), which was added for 5 min before 2 N H₂SO₄ was added to stop the reaction. Absorbance was detected at 450 nm using a SpectraMAX iD3 instrument (Molecular Devices).

To perform the indirect ELISA to determine the binding of the M2e-mAbs to mouse C1q protein, 96-well plates were coated with the specified M2e antibodies in the bicarbonate buffer (pH 9.6) at 4°C overnight. The plates were blocked with PBS + 1% BSA. After 1 hour, the plates were washed three times with PBS-T and added with mouse (0.5 μg/ml) C1q protein (Complement Technology) in PBS-T. After a 1-hour incubation at room temperature, the plates were washed three times with PBS-T, added with biotinylated anti-mouse C1q antibody (Thermo Fisher Scientific), and incubated for 1 hour. Then, the plates were washed three times with PBS-T, added with HRP-conjugated streptavidin (Pierce Chemical), and incubated for 1 hour. The plates were washed four times with PBS-T after the incubation with HRP-conjugated antibody or streptavidin, and peroxidase activity was measured with TMB solution (Thermo Fisher Scientific), which was added for 5 min before 2 N H₂SO₄ was added to stop the reaction. Absorbance was detected at 450 nm using a SpectraMAX iD3 instrument (Molecular Devices).

Prophylactic M2e-mAb treatment

Twenty-four hours before infection of mice with either a 5× or 10× LD₅₀ of the specified IAV subtype, BALB/c mice were prophylactically treated by intraperitoneal injection with the specified dose of single M2e-mAb or the triple M2e-mAb cocktail as previously described (55).

Triple M2e-mAb cocktail treatment of influenza-infected BALB/c mice

BALB/c mice were challenged with 3× LD₅₀ PR8 IAV by aerosol inhalation or, alternatively, 1× LD₅₀ PR8 IAV by intranasal instillation. A total of 450 μg the triple M2e-mAb cocktail was administered once intraperitoneally, at the indicated time points after viral challenge. Survival and weight loss were monitored daily over 21 days postinfection. BALB/c mice were challenged with 10× LD₅₀ Anhui1 by intranasal instillation, and 450 μg the triple M2e-mAb cocktail or isotype control was administered in mice intraperitoneally at the indicated time points after viral challenge. Survival and weight loss were monitored daily over 15 days postinfection. A subset of animals was euthanized on day 5 or 6 postinfection (day 6 for groups receiving therapy on day 5), lungs were harvested, and homogenized and titers were measured by plaque assay.

Viral M2e-mAb therapy escape mutant resistance assay and M region sequencing

Twenty-four hours before IAV infection (day −1), groups of BALB/c mice were injected intraperitoneally with 60 μg of the indicated M2e-mAbs or the triple M2e-mAb cocktail (20 μg/each, 60 μg in total). On day 0, mice were anesthetized with isoflurane and intranasally infected with a 5× LD₅₀ dose of PR8 IAV. On day 3 or 4 postinfection, mice were euthanized with an isoflurane overdose, and lungs were harvested and homogenized to isolate the virus. IAV was isolated and purified from lung homogenates as previously described (55). The PR8-containing solution (remaining in the top of the filter tube) was placed in a new 15-ml tube and centrifuged at 850g for 5 min at 4°C. Then, 20 μl of the purified virus preparation was used to intranasally infect naïve BALB/c mice that had been treated with the indicated M2e-mAbs or the triple M2e-mAb cocktail 24 hours before infection. Alternatively, Rag2-KO mice were injected intraperitoneally with 60 μg of the indicated M2e-mAbs or the triple M2e-mAb cocktail (20 μg/each, 60 μg in total) 24 hours before and 3 days after intranasal infection with a 5× LD₅₀ dose of PR8. Mice were euthanized with an isoflurane overdose on day 7, lungs were harvested, and virus was isolated as described above. PR8 IAV was passaged six times through groups of seven BALB/c mice or twice through groups of three Rag2-KO mice before its isolation for Sanger sequencing. Total RNA was isolated using the QIAamp Viral RNA Mini Kit (QIAGEN), cDNA was synthesized using the M2-2 primer (5′-GCGAAAGCAGGTAGATATTG-3′), which binds to a 3′ noncoding region of influenza’s viral RNA segment 7 (vRNA7), and the Omniscript RT Kit (QIAGEN). The cDNA was amplified by polymerase chain reaction (PCR) using KAPA HiFi HotStart ReadyMix (Roche), using M2-2 and SEQ7 (5′-ATATCGTCTCGTATTAGTAGAAACAAGGTAG-3′) primers. The SEQ7 primer binds to a 5′ noncoding region of influenza vRNA7. The expected size of the PCR product is 1042 bp, and this was confirmed by gel electrophoresis. Genewiz Inc. performed sequencing analysis with M2SeqN1 (5′-ATGTTATCTCCCTCTTGAGC-3′) and SEQ7 primers. M2SeqN1 anneals to 331 to 351 of M1 cDNA, and SEQ7 anneals to a 5′ noncoding region of the cDNA. To identify potential viral escape mutants, the sequence of the “passaged” viruses was compared to the input PR8 virus (day 0), which matched the original M2e sequences (40).

Identification of M2e-mAb–mediated Fc-effector functions required for the protection of PR8-challenged mice

To block the activities of FcγRIII and FcγRIV in vivo, BALB/c mice were intraperitoneally infused on days −2, 1, and 4 post–PR8 IAV infection with commercially available anti-FcγRIII antibody (100 μg per mouse; clone: 275003, R&D Systems) and/or anti-FcγRIV antibody (200 μg per mouse; clone: 9E9 BioLegend), based on a published protocol (77). Indicated groups of mice were also therapeutically treated with a single intraperitoneal injection of either the 602-Fc-WT, LE-, or LEEA2K, or LALAPG M2e-mAbs (100 μg per mouse), or PBS as a control, on day −1 post-PR8 infection, which was performed using a dose of 3× LD₅₀ PR8 by aerosol inhalation on day 0.

Statistics

GraphPad Prism 9 was used for all statistical analyses. A Mantel-Cox log rank test was used for the survival analysis. To compare percent weight loss, a one-way analysis of variance (ANOVA) (overall weight loss) or a two-way ANOVA (weight loss on individual days postinfection) with Dunnett’s multiple comparisons test was used to compare multiple experimental groups with one isotype control group. A paired t test (overall weight loss) or a two-way ANOVA with a Sidak’s multiple comparisons test (weight loss on individual days postinfection) was used to compare a single experimental group with its corresponding isotype control group. A one-way ANOVA with Turkey’s multiple comparisons (comparing multiple experimental groups to a shared isotype control group) or Mann-Whitney test (comparing a single experimental group to its isotype control group) was used for lung viral titers on the specified day. All statistics are indicated in the figure legends. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05.

Study approval

All institutions, each institutions’ Animal Care and Use Committees approved all protocols for animal experiments, and all institutions follow the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research.

Acknowledgments

We thank D. Kubitz and acknowledge the Center for Antibody Development and Production at The Scripps Research Institute for producing hybridomas and monoclonal antibodies used in this study. We thank S. Epstein (Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, MD) for providing A/PR/8/1934 (H1N1) and A/FM/1/1947- MA (H1N1). We also thank E. G. Brown (University of Ottawa, Ottawa, Canada) for approving the sharing of A/FM/1/1947- MA (H1N1). We thank T. Ross (University of Georgia, Athens, GA) for providing A/CA/07/2009 (H1N1) and R. Webby (St. Jude Children’s Research Hospital, Memphis, TN) for providing A/Anhui/1/2013 (H7N9) and A/Vietnam/1203/2004 (H5N1). We also thank J. Yewdell (National Institutes of Health, Bethesda, MD) for sharing the NP-specific hybridoma H16-L10. A/Anhui/1/2013 (H7N9) was provided via the WHO Global Influenza Surveillance and Response System (GISRS).

Funding: This work was supported by the Albert and Margaret Alkek Foundation, Houston TX, 2015 (to S.P.), National Institutes of Health grant R01AI130065 (to S.P. and S.M.T.), and National Institute of General Medical Sciences of the National Institutes of Health grant AI053831 (to S.L.R.).

Author contributions: S.P.: Conceptualization, methodology, investigation, funding acquisition, project administration, supervision, writing—original draft, and writing—review and editing. S.M.T.: Conceptualization, methodology, investigation, funding acquisition, project administration, supervision, and writing—review and editing. L.B.: Methodology, investigation, visualization, validation, formal analysis, writing—original draft, and writing—review and editing. T.K.: Methodology, investigation, visualization, validation, formal analysis, and writing—review and editing. S.L.R.: Methodology, investigation, formal analysis, and funding acquisition. A.Y.S.: Investigation. S.K.J.: Investigation. C.A.J.: Investigation.

Competing interests: The authors declare that they have no competing interests.

Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.

Supplementary Materials

This PDF file includes:

Figs. S1 to S5

Tables S1 and S2

sciadv.adx3505_sm.pdf^{(3.8MB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figs. S1 to S5

Tables S1 and S2

sciadv.adx3505_sm.pdf^{(3.8MB, pdf)}

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Non-neutralizing antibodies to influenza A matrix-protein-2-ectodomain are broadly effective therapeutics and resistant to viral escape mutations

Teha Kim

Lynn Bimler

Sydney L Ronzulli

Amber Y Song

Scott K Johnson

Cheryl A Jones

S Mark Tompkins

Silke Paust

Roles

Abstract

INTRODUCTION

RESULTS

M2e-specific antibody clones 472, 522, and 602 bind to M2e’s highly conserved N-terminal region

Table 1. M2e sequences of the IAVs used in this study and compared to the M2e CS.

Fig. 1. Clones 472, 522, and 602 bind M2e in M2e’s highly conserved N-terminal region.

M2e-specific antibodies bind to M2e competitively

Fig. 2. M2e-specific antibodies bind to M2e competitively.

M2e-mAbs are more protective against lethal IAV challenge when administered as a triple cocktail

Fig. 3. M2e-mAbs are more protective against lethal influenza virus challenge as a cocktail.

M2e-mAb therapeutic efficacy depends on FcγRI (CD64), FcγRIII (CD16), and FcγRIV (CD16-2)

Fig. 4. M2e-mAbs expressed as IgG2a isotypes are more protective than IgG1 isotypes.

Fig. 5. IgG2a M2e-mAbs mediate critical effector functions through FcγRI, III, and IV when administered systemically to influenza A virus–challenged mice.

A single therapeutic treatment of mice with the triple M2e-mAb significantly ameliorates disease severity and enhances survival in mice challenged with PR8 or Anhui/1 IAV

Fig. 6. A single therapeutic treatment with the triple M2e-mAb significantly ameliorates disease severity and enhances survival in mice challenged with H1N1 PR8 or with the pathogenic avian influenza strain A/Anhui/1/2013 (H7N9).

Treatment with M2e-mAb therapies does not drive the development of IAV viral escape mutants

Fig. 7. M2e-mAb triple cocktail therapy does not drive the development of viral escape mutants.

DISCUSSION

MATERIALS AND METHODS

Mice

Viruses

Cell lines

Intranasal IAV challenge

Aerosolized IAV challenge

Viral titer quantification

Neutralization assay

M2e-mAb production from hybridomas

Biotinylation of M2e-mAbs

Cloning, expression, and purification of isotype-switched M2e-mAbs

Epitope mapping using M2e peptide libraries

M2e-mAb competition ELISA

M2e-mAb analysis by SDS-PAGE and Coomassie staining

Detection of M2e-mAb binding to M2-expressing HEK cells by flow cytometry

The 602-IgG2a Fc variant development

Quantification of M2e-mAb binding to FcγRs, C1q, and M2e peptides by ELISA

Prophylactic M2e-mAb treatment

Triple M2e-mAb cocktail treatment of influenza-infected BALB/c mice

Viral M2e-mAb therapy escape mutant resistance assay and M region sequencing

Identification of M2e-mAb–mediated Fc-effector functions required for the protection of PR8-challenged mice

Statistics

Study approval

Acknowledgments

Supplementary Materials

This PDF file includes:

REFERENCES AND NOTES

Associated Data

Supplementary Materials

ACTIONS

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RESOURCES

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