Polyclonal breast milk–derived secretory IgA confers broad antiviral protection against multiple respiratory viruses

Abstract

Passive immunization is an important tool for protecting individuals with primary immune deficiencies from infectious diseases and offers a rapid response strategy for protecting vulnerable populations, particularly during the interval between pathogen emergence and vaccine deployment. Current approaches rely on intravenous delivery of IgG-enriched plasma or monoclonal antibodies, which limits accessibility, scalability, and effectiveness at mucosal surfaces, the primary entry point for respiratory viruses. Here, we tested LCTG-001, a polyclonal secretory IgA preparation derived from pooled human breast milk, to address these limitations. LCTG-001 demonstrated broad antiviral activity against clinically relevant respiratory viruses, including respiratory syncytial virus, adenovirus, and influenza A virus. LCTG-001 effectively recognized both group 1 and group 2 influenza A subtypes, including the highly pathogenic H5N1 clade 2.3.4.4b, and engaged nonneutralizing effector mechanisms such as neuraminidase inhibition and neutrophil activation. When administered intranasally, LCTG-001 conferred complete protection against influenza A challenge in both immunocompetent and antibody-deficient murine models, preventing morbidity and mortality through a transient early reduction in viral burden followed by sustained improvements in lung pathology and lung barrier integrity. These findings highlight the potential of breast milk–derived secretory IgA as a scalable, mucosa-targeted passive immunization strategy capable of addressing both endemic and emerging respiratory viral threats.

INTRODUCTION

Immunoglobulin replacement therapy, which can take the form of intravenous immunoglobulin (IVIG) or subcutaneous immunoglobulin, is essential for mitigating infectious diseases and is especially critical in treating primary immunodeficiencies such as common variable immunodeficiency (CVID). CVID is characterized by impaired antibody production, manifesting as reduced levels of immunoglobulin G (IgG), IgA, and/or IgM (1, 2). Regular IVIG infusions are administered to patients with CVID to replenish immunoglobulin levels, thereby reducing infection frequency and severity (3). Despite its effectiveness, IVIG therapy is costly, invasive, and requires frequent medically assisted administration (1, 4).

Beyond management of immunoglobulin immunodeficiencies, IVIG and convalescent plasma have also been used as passive immunization strategies to protect populations against viral infections, including in pandemic settings where vaccines are either unavailable or in limited supply. This was exemplified during the 1918 and 2009 influenza A virus (IAV) pandemics, as well as during the initial stages of COVID-19 (5–8). However, these strategies require intravenous administration of antibodies derived from the circulation, which are rich in IgG and monomeric IgA but differ markedly from the predominantly dimeric and secretory IgA (SIgA) present at mucosal surfaces (9). Thus, these approaches do not fully recapitulate the protective immune landscape of the respiratory mucosa found in healthy individuals (4, 10). In addition, antibodies administered intravenously must transudate from the circulation to the mucosal surfaces to exert their protective effects, which limits availability at respiratory surfaces (11). This limitation highlights the need for mucosally delivered passive immunization strategies which can deliver antibodies directly to the site of pathogen entry.

Sourcing SIgA from human biospecimens, such as those found in bronchoalveolar lavage (BAL) fluids, is not practical due to the invasiveness of the procedures and the limited yield of antibodies. In contrast, breast milk is a more attractive source that is more plentiful, accessible, and rich in mucosal SIgA (12). Previous work has demonstrated that breast milk–derived SIgA has antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is a viable prophylactic strategy in preclinical models (13). This study used breast milk from human milk donors post–SARS-CoV-2 infection, thereby enriching the antibody pool for anti–SARS-CoV-2 antibodies. Building on these observations, we hypothesized that purifying IgA from a large sample of donors without screening milk samples for neutralizing antibodies against any particular pathogen would yield a polyclonal SIgA product with broad antiviral activity against a diverse array of respiratory pathogens due to varied donor exposures to infections and/or vaccinations, akin to IVIG.

Here, we investigate a polyclonal, breast milk–derived SIgA product, LCTG-001. Our findings demonstrate that LCTG-001 exhibits broad antiviral activity in vitro against a panel of respiratory pathogens. We further demonstrate that LCTG-001 has neutralizing and nonneutralizing activity against both group 1 and group 2 IAVs, in addition to activity against emerging zoonotic strains. Last, we demonstrate that when delivered directly to the respiratory mucosa, LCTG-001 was protective in vivo against IAV challenge in both immunocompetent and immunocompromised hosts. Collectively, these results highlight mucosal delivery of breast milk–derived IgA as a passive immunization strategy capable of addressing limitations inherent in traditional intravenous antibody therapies, offering potential protection against both endemic and pandemic respiratory infections.

RESULTS

Assessment of broad antiviral activity against a panel of common respiratory viral pathogens

Unlike IgG in conventional IVIG or convalescent plasma, which is peripherally sourced from the circulation, SIgA is exclusively found at mucosal sites. Sourcing from the respiratory mucosa is not feasible due to procedural complexities and limited antibody yield. To circumvent these limitations, we explored breast milk as an alternative source of mucosal SIgA. Breast milk is a scalable and sustainable source of mucosal SIgA, and donations of extra breast milk are already collected by over 700 human milk banks worldwide. To produce purified SIgA, breast milk samples were collected from 120 lactating donors through collaborations with milk banks in Indianapolis and Pittsburgh. Initial sample shipments included 85 donor contributions received on 11 October 2022, followed by an additional 35 samples on 29 November 2022. The samples, collected throughout 2022, were pooled to yield a total volume of 15,585 ml. Approximately 1 liter of this pooled milk was processed using a previously validated and patented purification method (13) to yield a polyclonal SIgA product, designated LCTG-001. The purity of LCTG-001 was confirmed by Coomassie staining (fig. S1) and was consistent with previous reports (13). The donor cohort was not screened for recent infection or vaccination status, thereby aligning with conventional IVIG collection practices in which donors are not selected on the basis of prior antigenic exposures (3, 14).

We next evaluated whether the polyclonal SIgA pool, LCTG-001, could recognize and neutralize a panel of respiratory viruses, including respiratory syncytial virus (RSV), human adenovirus (HuAd), and IAV. Representative strains and serotypes were selected for each virus to assess LCTG-001’s binding and neutralizing potency. To assess LCTG-001’s antiviral activity, we performed enzyme-linked immunosorbent assays (ELISAs) to evaluate antibody binding, alongside microneutralization (MNT) assays to measure functional neutralization.

We first assessed binding of LCTG-001 to RSV fusion protein (RSV-F), the antigen used in licensed RSV vaccines (15). LCTG-001 demonstrated robust binding to RSV-F (AUC = 62.02 ± 1.273; Fig. 1A), and these antibodies were able to neutralize RSV at physiologically relevant concentrations [median inhibitory concentration (IC₅₀) = 320 μg/ml; Fig. 1B].

Fig. 1. LCTG-001 has broad antiviral activity against a panel of common respiratory viral pathogens.

(A, C, and E) LCTG-001 binding responses to RSV-F (A), HuAd5 lysate (C), and Cal/09 HA (E). (B, D, and F) LCTG-001 neutralizing antibody responses to RSV (B), HuAd5 (D), and Cal/09 (F), measured by percent (%) neutralization using MNT assays. Data presented in (A) to (F) represent mean ± SEM. Data presented in (A), (C), and (E) represent three technical replicates. Data presented in (B), (D), and (F) are from three independent experiments.

We next assessed the antiviral activity of LCTG-001 against adenovirus, focusing on HuAd type 5 (HuAd5), one of the most prevalent HuAd serotypes (16, 17). ELISAs were performed using the lysate from HuAd5-infected HeLa cells as a source of antigen (18). Again, LCTG-001 demonstrated specific binding to the lysate of infected cells (AUC = 62.28 ± 2.309; Fig. 1C) and neutralized HuAd5 with considerable potency (IC₅₀ = 180 μg/ml; Fig. 1D).

Last, we assessed the antiviral activity of LCTG-001 against IAV. IAV is a respiratory virus responsible for both seasonal epidemics and sporadic pandemics, with five pandemics in the last century (19). We first investigated the ability of LCTG-001 to recognize and neutralize A/California/07/2009 H1N1 (Cal/09). Cal/09 (H1N1) is an isolate from the 2009 H1N1 pandemic, which then circulated seasonally and was a recommended seasonal influenza vaccine strain for more than 5 years in North America. Hence, most of the population has been exposed through infection and/or vaccination. Similar to RSV and HuAd5, LCTG-001 showed specific binding to Cal/09 (H1N1) hemagglutinin (HA) (AUC = 54.76 ± 4.886; Fig. 1E) and neutralization activity (IC₅₀ = 670 μg/ml; Fig. 1F). Together, these findings demonstrate that LCTG-001, a breast milk–derived polyclonal SIgA product, exhibits broad-spectrum antiviral activity against multiple clinically relevant respiratory pathogens.

LCTG-001 engages nonneutralizing antibody effector functions against H1N1 IAV

While vaccination remains the primary strategy for control of seasonal influenza, antigenic drift, particularly in the HA head domain, limits vaccine effectiveness and necessitates regular vaccine strain updates. Antibody responses generated by infection or vaccination primarily target the immunodominant HA head, which can significantly vary between strains. Unlike the variable HA head, the stalk domain is relatively conserved, with stalk-specific antibodies capable of providing protection against diverse IAV strains (20). However, stalk-specific antibody responses are typically low in magnitude but tend to increase with age and influenza virus exposures (21, 22). Despite this, these antibodies can provide broad protection by targeting conserved stalk epitopes, particularly in the context of vaccine mismatch or exposure to novel pandemic strains where HA head-specific immunity is lacking. Hence, we next sought to determine whether stalk-specific anti-IAV antibodies were present in LCTG-001 through the use of a chimeric HA protein (cH6/1) composed of the conserved stalk domain of A/Puerto Rico/8/1934 (H1N1) and the head domain from the avian A/mallard/Sweden/81/02 (H6N2) strain (23). The use of an avian H6 head, which is rarely encountered in humans, allows for measurement of stalk-specific binding antibodies (24). Using this approach, LCTG-001 demonstrated stalk-specific binding antibody responses (AUC = 46.35 ± 9.036; Fig. 2A) that were not statistically different (P = 0.54) from antibody responses to full-length Cal/09 (H1N1) HA (AUC = 54.76 ± 4.886; Fig. 1E), suggesting that a large proportion of H1 HA-specific antibodies present in LCTG-001 recognizes the H1 stalk. These data indicate that LCTG-001 contains stalk-binding antibodies against IAV.

Fig. 2. LCTG-001 engages nonneutralizing antibody effector functions against H1N1 IAV.

(A) LCTG-001 binding responses to cH6/1 HA. (B) Anti–neuraminidase (NA) activity of LCTG-001 against Cal/09 (H1N1), measured by percent (%) inhibition with an enzyme-linked lectin assay (ELLA). (C) Primary human neutrophils were isolated and assessed for their ability to produce reactive oxygen species (ROS) in an in vitro luminol-based assay with LCTG-001 and Cal/09 (H1N1). ROS production was measured using relative light units (RLU). Data presented in (A) represent mean ± SEM of three technical replicates. Data presented in (B) represent mean ± SEM of three technical replicates. Data presented in (C) are a representative time course data for a single experiment with three technical replicates with mean ± SEM.

Antibodies that bind near the enzymatic site of neuraminidase (NA) exert antiviral activity by inhibiting NA function (25). In addition, previous reports indicate that stalk-specific antibodies can prevent or interfere with viral infection through a variety of mechanisms, including NA inhibition (26). Antibodies that bind to the HA stalk can interfere with NA activity through steric hindrance (27). Therefore, we next assessed whether the antibodies present in LCTG-001 could inhibit the enzymatic activity of NA. An enzyme-linked lectin assay (ELLA) was performed to measure inhibition of NA-mediated cleavage of terminal sialic acids from fetuin, a sialylated glycoprotein substrate (28). LCTG-001 potently inhibited the NA activity of Cal/09 (H1N1) (IC₅₀ = 8.7 μg; Fig. 2B).

Stalk-binding antibodies also induce Fc-dependent effector functions, including through Fc receptor engagement on the surface of immune cells. Previous work has demonstrated that IgA can induce neutrophil activation through an Fc-dependent pathway (29, 30). Published data from our group have shown that this activation leads to preferential induction of NETosis by IgA, a unique form of neutrophil cell death that has antimicrobial and antiviral functions (29). Hence, we next assessed whether LCTG-001 could engage Fc effector functions using a luminol-based assay as a surrogate for neutrophil activation (30). This well-established assay produces luminescence in the presence of reactive oxygen species, a by-product of neutrophil activation. Immune complexes formed using Cal/09 (H1N1) virus and LCTG-001 were used to stimulate neutrophils isolated from healthy donors. In comparison to virus- and neutrophil-only controls, LCTG-001 immune complexes were able to potently induce neutrophil activation (Fig. 2C). Collectively, these results show that LCTG-001 is capable of inducing a diverse array of antibody-mediated antiviral mechanisms against Cal/09 (H1N1) virus, including NA inhibition and neutrophil Fc-mediated effector functions.

LCTG-001 demonstrates broad activity against group 1 and group 2 IAVs

Seasonal IAV infections are predominantly caused by H1N1 viruses (which belong to group 1) and H3N2 viruses (which belong to group 2), both of which are included in current seasonal vaccination formulations (19). Given that LCTG-001 is composed of breast milk–derived SIgA from a pooled donor population with diverse histories of vaccination and exposures and that we have demonstrated the functional activity of LCTG-001 against group 1 IAV, we next assessed whether this activity extended to group 2 viruses by evaluating its efficacy against H3N2. By implementing similar experimental procedures as those described above, LCTG-001 demonstrated specific binding to the HA of A/Hong Kong/1/1968 (HK/68), an H3N2 virus (AUC = 40.49 ± 1.622; Fig. 3A). In addition, LCTG-001 demonstrated neutralizing activity against X-31 (IC₅₀ = 599 μg/ml; Fig. 3B), a recombinant virus with the HA and NA of HK/68 (H3N2) and the internal genes of PR8 (H1N1).

Fig. 3. LCTG-001 demonstrates broad activity against group 1 and group 2 IAVs.

(A) LCTG-001 binding responses to HK/68 HA. (B) LCTG-001 neutralizing antibody responses to X-31 (H3N2), measured by percent (%) neutralization using an MNT assay. (C) Anti-NA activity of LCTG-001 against X-31 (H3N2), measured by percent (%) inhibition with an ELLA. (D) Primary human neutrophils were isolated and assessed for their ability to produce ROS in an in vitro luminol-based assay with LCTG-001 and X-31 (H3N2). ROS production was measured using RLU. Data presented in (A) represent mean ± SEM of three technical replicates. Data presented in (B) represent mean ± SEM from three independent experiments. Data presented in (C) represent mean ± SEM of three technical replicates. Data presented in (D) are a representative time course data for a single experiment with three technical replicates with mean ± SEM.

To further characterize the nonneutralizing activity of LCTG-001 against H3N2, we next assessed whether these antibodies could inhibit the enzymatic activity of NA by ELLA. Similar to our findings with H1N1, LCTG-001 effectively inhibited NA enzymatic activity of X-31 (H3N2) (IC₅₀ = 0.58 μg; Fig. 3C). We next assessed whether LCTG-001 could induce Fc-dependent effector functions against X-31 (H3N2). Immune complexes were formed with X-31 (H3N2) virus and LCTG-001 and used to stimulate neutrophils isolated from healthy donors. In comparison to virus- and neutrophil-only controls, LCTG-001 immune complexes were able to potently induce neutrophil activation (Fig. 3D). Collectively, these results show that LCTG-001 is capable of binding and neutralizing both circulating subtypes of IAV in humans and is also capable of inducing nonneutralizing mechanisms against both group 1 and group 2 IAV.

LCTG-001 recognizes and induces nonneutralizing effector functions against the highly pathogenic influenza A (HPAI) H5N1 clade 2.3.4.4b virus

We next speculated that LCTG-001 might have antiviral activity against emergent strains of IAV that pose a major threat to public health, such as avian highly pathogenic H5N1 IAV. H5N1 belongs to the group 1 family of IAV, against which LCTG-001 was shown to have potent activity. We focused on the H5N1 clade 2.3.4.4b due to its unprecedented spread amongst poultry and mammals, including recent outbreaks in US dairy cattle, combined with several instances of zoonotic spillover into humans, raising concern over its pandemic potential (31, 32).

To evaluate the antiviral activity of LCTG-001 against clade 2.3.4.4b (H5N1), we generated virus-like particles (VLPs) using plasmids encoding HA and NA from A/seal/QC/FAV869-3/2022 (H5N1) and structurally validated them by transmission electron microscopy (fig. S2A). Functional activity of HA and NA in the VLPs was validated by hemagglutination (HA) assay and ELLA, respectively (fig. S2, B and C). LCTG-001 demonstrated specific binding to VLPs (AUC = 39.82 ± 0.5062; Fig. 4A), thereby suggesting that this breast milk–derived polyclonal SIgA pool is capable of recognizing IAV strains to which there is unlikely to have been previous exposure among humans. To further elucidate the source of this cross-reactivity, we measured the binding of LCTG-001 to both recombinant H5 HA (AUC = 27.64 ± 1.51; Fig. 4B) and N1 NA (AUC = 45.57 ± 1.13; Fig. 4C) from clade 2.3.4.4b (H5N1). Notably, LCTG-001 showed binding activity to both proteins. Although LCTG-001 exhibited minimal neutralization of the recent clade 2.3.4.4b (H5N1) virus as measured by MNT (Fig. 4D) and exhibited no HA inhibition activity (Fig. 4E), it was capable of engaging nonneutralizing effector functions, including inhibition of NA enzymatic activity (IC₅₀ = 26.14 μg; Fig. 4F) by ELLA and neutrophil activation by luminol assay (Fig. 4G). When compared across all three IAV strains tested (H1N1, H3N2, and H5N1), there were no significant differences in the ability of LCTG-001 to induce neutrophil activation when complexed with virus (fig. S3). These data demonstrate that even in the absence of neutralizing antibodies, LCTG-001 contains antibodies that recognize the HA and NA of a clade 2.3.4.4b (H5N1) virus. The antibodies that bind to HA seem to be directed against the conserved group 1 HA stalk domain, and these antibodies are capable of inducing nonneutralizing functions.

Fig. 4. LCTG-001 recognizes and induces nonneutralizing effector functions against the highly pathogenic influenza A (HPAI) H5N1 clade 2.3.4.4b virus.

(A) LCTG-001 binding responses to 2.3.4.4b (H5N1) VLPs. (B) LCTG-001 binding responses to purified 2.4.3.4b (H5N1) HA. (C) LCTG-001 binding responses to purified 2.3.4.4b (H5N1) NA. (D) LCTG-001 neutralizing antibody responses to 2.3.4.4b (H5N1), measured by percent (%) neutralization using a plaque reduction (PRNT) assay. (E) LCTG-001 HAI activity against 2.3.4.4b (H5N1) VLP. (F) Anti-NA activity of LCTG-001 against 2.3.4.4b (H5N1) VLPs, measured by percent (%) inhibition with an ELLA. (G) Primary human neutrophils were isolated and assessed for their ability to produce ROS in an in vitro luminol-based assay with LCTG-001 and 2.3.4.4b (H5N1) VLPs. ROS production was measured using RLU. Data presented in (A) to (C) represent mean ± SEM of three technical replicates. Data presented in (D) represent mean ± SEM of three independent experiments. Data presented in (F) represent mean ± SEM of three technical replicates. Data presented in (G) are a representative time course data for a single experiment. Data presented represent three technical replicates with mean ± SEM.

Respiratory mucosal passive immunization with LCTG-001 protects against IAV infection in immunocompetent hosts

Given that LCTG-001 is a polyclonal pool of SIgA, an isotype primarily found in mucosal sites, we evaluated its in vivo efficacy against IAV following intranasal delivery. We first demonstrated that intranasal administration of LCTG-001 resulted in antibody deposition in the respiratory mucosa. Eight- to 12-week-old female BALB/c mice were administered 1 mg of LCTG-001 intranasally (fig. S4A). LCTG-001 was detectable in both the lung tissue and BAL fluid of the mice as early as 30 min postadministration (fig. S4, B and C). Antibody titers were stable for at least 120 min postadministration, with no significant differences in the amount of antibody detected at the two assessed time points. Next, as proof of concept, we used an immunocompetent BALB/c mouse model of IAV infection with A/Netherlands/602/2009 H1N1 (NL/09), a well-characterized mouse-adapted H1N1 IAV strain, nearly identical to Cal/09 (H1N1), which we evaluated in vitro (33). To this end, 8- to 12-week-old female BALB/c mice were administered 1 mg of LCTG-001 intranasally 2 hours before and 24 hours after intranasal infection with a sublethal dose [450 plaque-forming units (PFU)] of NL/09 (H1N1) (Fig. 5A). Animals were monitored for 14 days for clinical signs of disease, weight loss, and survival. A subset of animals was sacrificed 6 days postinfection (DPI), and lungs were collected for quantification of viral titers, cytokine analysis, and histopathology.

Fig. 5. Intranasally delivered LCTG-001 is protective against NL/09 challenge in immunocompetent mice.

(A) Experimental schema of intranasal (i.n.) LCTG-001 delivery and IAV challenge in female BALB/c mice. (B) Changes in body weight over 14 days post-IAV infection (DPI). (C) Percent (%) survival over 14 days post-IAV infection. (D) Clinical scores of LCTG-001– and PBS-treated BALB/c mice over 14 days post-IAV infection. (E) Bar graphs depicting levels of cytokines and chemokines present in lung tissue homogenates of BALB/c mice 6 days post-IAV infection. (F) Bar graphs depicting levels of chemokines involved in polymorphonuclear cell (PMN) and lymphocyte cell recruitment present in lung tissue homogenates of BALB/c mice 6 days post-IAV infection. (G) Representative H&E-stained histopathological images of lungs collected 6 days post-IAV infection of LCTG-001– and PBS-treated BALB/c mice. Red star indicates area with epithelial destruction. Red arrow indicates area of cellular infiltration. Scale bars, 200 μm. (H) Representative H&E-stained histopathological images of lungs collected 14 days post-IAV infection of LCTG-001– and PBS-treated BALB/c mice. Red arrow indicates area of cellular infiltration. Scale bars, 200 μm. Data presented in (B), (E), and (F) represent mean ± SEM. Statistical significance in (B) was determined using a two-way analysis of variance (ANOVA) with Šídák’s test for multiple comparisons. Statistical significance in (E) and (F) was determined with multiple t tests between LCTG-001– and PBS-treated mice with no correction for multiple comparisons. *P < 0.05 and ****P < 0.0001. Data in (B) to (H) are representative of one independent experiment, n = 5 to 10 mice per group.

Phosphate-buffered saline (PBS)–treated mice had a peak weight loss of 11.76 ± 3% by 8 DPI, with all animals subsequently recovering to baseline weight values (Fig. 5, B and C). In comparison to PBS-treated mice, LCTG-001 treatment fully protected animals from weight loss (peak weight loss 1.72 ± 0.85%, P < 0.0001; Fig. 5, B and C). In alignment with these observations, whereas PBS control mice showed clinical signs of disease, LCTG-001–treated mice were wholly protected and exhibited no clinical signs of disease (Fig. 5D).

Unexpectedly, there was no statistically significant difference in viral titers in the lungs of PBS- and LCTG-001–treated mice at 6 DPI (fig. S4A). Despite this, when examining cytokine responses in the lungs at the same time point, there were significantly increased levels of proinflammatory cytokines interferon-γ (IFN-γ) and interleukin-6 (IL-6) and the anti-inflammatory cytokine IL-10 in the lungs of PBS-treated mice compared to LCTG-001–treated mice (Fig. 5E). Cytokines and chemokines involved in immune cell chemotaxis, including granulocyte colony-stimulating factor (G-CSF), MIG, macrophage inflammatory protein 1α (MIP-1α), and MIP-1β, were also significantly higher in the lungs of PBS-treated mice compared to LCTG-001–treated mice (Fig. 5F). Histopathological analysis of lung tissues stained with hematoxylin and eosin (H&E) revealed that PBS-treated animals experienced significant tissue damage by 6 DPI, with visible areas of epithelial destruction and cellular infiltration (Fig. 5G). In contrast, the lungs of LCTG-001–treated animals showed preserved epithelial architecture and minimal cellular infiltration. Even by 14 DPI, lungs of PBS-treated animals showed signs of persistent epithelial destruction (red star) and cellular infiltration (red arrow), which were absent in LCTG-001–treated animals (Fig. 5H). Thus, the data presented above indicate that intranasal administration of LCTG-001 is protective against clinical signs of IAV infection in immunocompetent mice.

To assess the kinetics of LCTG-001–mediated protection, 8- to-12-week-old female BALB/c mice were administered 1 mg of LCTG-001 intranasally 2 hours before and 24 hours following intranasal infection with a sublethal dose of NL/09 (H1N1) IAV (450 PFU). Animals were sacrificed at 2, 6, and 8 DPI, and BAL fluid and lungs were collected for quantification of BAL albumin, lung viral titers, and histopathology (fig. S5A). At 2 DPI, there were significantly lower viral titers present in the lungs of LCTG-001–treated mice compared to PBS-treated controls (fig. S5B). However, despite this early control, there were no differences in the viral titers of LCTG-001–treated and PBS-treated animals at 6 or 8 DPI (fig. S5B). Histopathological analysis and scoring of lung tissues stained with H&E demonstrated that LCTG-001–treated animals were better protected from tissue damage than PBS-treated animals (fig. S5, C to E), with PBS-treated animals experiencing significantly higher histopathological scores at 2, 6, and 8 DPI (fig. S5, F to H). Last, we quantified albumin levels in BAL fluid. As albumin is abundant in serum but is normally restricted from the airway/alveolar lumen by an intact epithelial-endothelial barrier, increased BAL albumin reflects increased lung barrier permeability and can therefore be used as a surrogate measure of lung barrier integrity (34). At 2 DPI, there was no difference in the amount of albumin present in the BAL fluid of LCTG-001–treated and PBS-treated mice. However, at 6 and 8 DPI, there was a significant increase in the amount of albumin in the BAL fluid of PBS-treated mice compared to LCTG-001–treated animals, suggesting that administration of LCTG-001 maintains the structural integrity of the airways (fig. S5I). Together, these data demonstrate that intranasal administration of LCTG-001 limits IAV-associated lung pathology and preserves airway barrier integrity, despite comparable viral burdens at later stages of infection.

Respiratory mucosal passive immunization with LCTG-001 protects against IAV infection in immunodeficient hosts

A common use of passive immunization strategies, including IVIG, is as replacement therapy for immunocompromised individuals, such as those with CVID. Patients with CVID have reduced or undetectable levels of IgA, including mucosal IgA (4, 10). Our previous experiments with immunocompetent BALB/c mice do not capture this immunocompromised status and therefore do not allow us to determine the ability of LCTG-001 to protect against IAV infection in these vulnerable populations. Hence, we next sought to assess the protective efficacy of LCTG-001 using the Jh^−/− knockout mouse model. This model lacks all antibody isotypes, including IgA, and represents one of the most relevant available systems to assess therapeutics in an immunocompromised system specifically lacking antibodies (35).

To this end, 8- to 12-week-old female Jh^−/− mice were administered 1 mg of LCTG-001 intranasally 2 hours before and 24 hours postinfection with 450 PFU of NL/09 (H1N1) (Fig. 6A). Animals were monitored for 14 days for clinical signs of disease, weight loss, and survival, with a subset of animals sacrificed 6 DPI, where lungs were collected for quantification of viral titers, cytokine analysis, and histopathology. Relative to immunocompetent animals (Fig. 5, B and C), PBS-treated Jh^−/− mice were markedly more susceptible to NL/09 (H1N1) infection. PBS-treated mice experienced peak weight loss (22.79 ± 1.41%) by 9 DPI, with three of five mice reaching humane endpoint (Fig. 6, B and C). In comparison to PBS-treated mice, treatment with LCTG-001 protected animals from weight loss (peak weight loss of 7.67% ± 2.53%, P < 0.0001) with no animals reaching humane endpoint and all recovering to baseline weight values by 14 DPI (Fig. 6, B and C). Again, in alignment with these observations, PBS-treated mice showed signs of clinical disease, with animals experiencing up to three clinical signs for 7 DPI. In stark contrast, LCTG-001–treated mice were wholly protected and exhibited no clinical signs of disease (Fig. 6D).

Fig. 6. Intranasally delivered LCTG-001 is protective against NL/09 challenge in immunocompromised mice.

(A) Experimental schema of intranasal LCTG-001 delivery and IAV challenge in female Jh^−/− mice. (B) Changes in body weight over 14 days post-IAV infection (DPI). (C) Percent (%) survival over 14 days post-IAV infection. (D) Clinical scores of LCTG-001– and PBS-treated Jh^−/− mice over 14 days post-IAV infection. (E) Bar graphs depicting levels of cytokines and chemokines present in lung tissue homogenates of Jh^−/− mice 6 days post-IAV infection. (F) Bar graph depicting levels of chemokines involved in polymorphonuclear cell (PMN) and lymphocyte cell recruitment present in lung tissue homogenates of Jh^−/− mice 6 days post-IAV infection. (G) Representative H&E-stained histopathological images of lungs collected 6 days post-IAV infection of LCTG-001– and PBS-treated Jh^−/− mice. Red star indicates area with epithelial sloughing. Red arrow indicates area of cellular infiltration. Scale bars, 200 μm. (H) Representative H&E-stained histopathological images of lungs collected 14 days post-IAV infection of LCTG-001– and PBS-treated Jh^−/− mice. Red star indicates area with epithelial sloughing. Red arrow indicates area of cellular infiltration. Scale bars, 200 μm. Data presented in (B), (E), and (F) represent mean ± SEM. Statistical significance in (B) was determined using a two-way ANOVA with Šídák’s test for multiple comparisons. Statistical significance in (C) was determined using the Mantel-Cox test. Statistical significance in (E) and (F) was determined with multiple t tests between LCTG-001– and PBS-treated mice with no correction for multiple comparison *P < 0.05, ***P < 0.0005, and ****P < 0.0001. Data in (B) to (H) are representative of one independent experiment, n = 5 to 10 mice per group.

Similarly to what was observed in immunocompetent mice, there were no statistically significant differences in viral titers in the lungs of PBS- and LCTG-001–treated mice on 6 DPI (fig. S6B). Cytokine analysis of the lungs at 6 DPI revealed a significant difference in immune responses between treatment groups. PBS-treated mice had significantly up-regulated levels of cytokines, including the proinflammatory IFN-γ and anti-inflammatory IL-10 (Fig. 6E). Cytokines and chemokines involved in immune cell chemotaxis, including G-CSF, MCP-1, IP-10, MIG, MIP-1α, MIP-1β, and RANTES, were also significantly higher in PBS-treated mice compared to LCTG-001–treated mice, suggesting the LCTG-001–treated mice had more regulated immune responses (Fig. 6F).

Histopathological analysis of lung tissues taken on 6 DPI and stained with H&E revealed that PBS-treated animals experienced significant tissue damage, with marked epithelial sloughing and cellular infiltration, as indicated by the red star and arrow, respectively (Fig. 6G). However, the lungs of LCTG-001–treated animals appeared healthy, with no indication of epithelial damage and minimal cellular infiltration. By 14 DPI, lungs of PBS-treated animals showed signs of persistent epithelial sloughing and cellular infiltration, as indicated by the red star and arrow, respectively, which were absent in LCTG-001–treated animals (Fig. 6H). Hence, the data presented above indicate that intranasal administration of LCTG-001 is protective against IAV infection in immunocompromised mice.

In summary, Jh^−/− mice are more susceptible to IAV infection than their immunocompetent BALB/c counterparts, with more morbidity and mortality observed in PBS-treated Jh^−/− mice than PBS-treated BALB/c mice, underscoring the critical impact of antibody deficiency on the response to a common respiratory infection. Nonetheless, intranasal administration of LCTG-001 in Jh^−/− mice was capable of ameliorating disease and preventing mortality associated with IAV infection, demonstrating its potential as a treatment option for immunocompromised patients lacking antibodies.

DISCUSSION

In this study, we evaluated the antiviral activity and protective efficacy of LCTG-001, a polyclonal SIgA product derived from pooled human breast milk, as a novel mucosal passive immunization strategy that can address the inherent limitations associated with conventional intravenous, IgG-based treatments. IVIG and monoclonal antibody passive immunization strategies, while standard of care in the field for conferring prophylactic and/or therapeutic protection against respiratory pathogens, often have limited transudation to the respiratory mucosa (36–39). These therapies are also logistically burdensome due to the need for parenteral administration in health care settings and are associated with a variety of side effects, including infusion-related reactions (40).

In contrast to the periphery, the predominant antibody isotype found in the respiratory mucosa is SIgA. SIgA is actively transported across epithelial barriers and is highly resistant to proteolytic degradation, making it well suited for conferring protection within the respiratory mucosa (41). Moreover, SIgA is naturally localized to mucosa through polymeric immunoglobulin receptor–mediated epithelial transport, depositing IgA into the lumen where respiratory viruses establish infection, a targeted delivery that serum-derived IgG achieves only sparingly (11, 39, 41, 42).

Our results demonstrate that LCTG-001 has broad-spectrum antiviral activity mediated by multiple mechanisms. This polyclonal SIgA neutralized diverse respiratory viruses, including RSV, adenovirus, and representatives of both circulating human group 1 and group 2 IAV subtypes, including those with pandemic potential, thereby highlighting its exceptionally broad reactivity. Similarly, polyclonal SIgA compositions administered via the intranasal and intratracheal route were able to neutralize SARS-CoV-2 in K18⁺ hACE2 transgenic mice (13). This breadth of activity likely arises from the rich and diverse pool of SIgA isolated from pooled breast milk, capturing a wide array of antibodies with varying specificities within a single product.

Notably, LCTG-001 not only inhibited IAV viral entry in vitro but also exhibited robust NA inhibition. While NA antibodies may not be potently neutralizing, they can limit viral spread and have been shown to correlate with protection in influenza infection (43–45). Both HA stalk binding and NA-specific antibodies can contribute to NAI activity, the former through steric hindrance of the NA binding site and the latter by directly binding near the enzymatic site of the protein (26, 27). The presence of NAI activity suggests that LCTG-001 can impair virion release, even in the context of strains for which conventional neutralizing antibodies are absent. Furthermore, LCTG-001 induced Fc-dependent activation of neutrophils, as evidenced by a luminol-based assay. In humans, IgA is known to engage FcαRI (CD89) on myeloid cells, triggering processes like phagocytosis, respiratory burst, and NETosis (29, 30). Our data confirm that a complex SIgA pool from breast milk can harness this pathway. Thus, beyond classical neutralization, LCTG-001 is capable of activating innate immune cells to facilitate pathogen clearance. The ability of SIgA to activate neutrophils has important implications for mucosal defense. IgA is often viewed as a non-inflammatory “shield” that blocks pathogen entry. Here, we show that SIgA can also engage innate immunity, further facilitating pathogen clearance. This FcαRI-dependent mechanism adds a crucial layer of protection at mucosal surfaces.

Our use of a polyclonal human SIgA pool from breast milk reveals the breadth of Fc-mediated activity of LCTG-001 in the context of IAV. Prior studies have mostly examined serum-derived or engineered monomeric IgA (29, 30, 46). SIgA can engage the FcαR on cells; however, this interaction requires accessory molecules such as MAC-1 and results in a lower affinity compared to cross-linked monomeric IgA (47–49). Hence, the ability of SIgA to engage Fc-mediated neutrophil effector functions in an IAV antigen–specific context has not been fully examined. We demonstrated that the polyclonal SIgA which comprises LCTG-001 retains the ability to engage Fc effector functions on neutrophils, triggering a potent effector response against three different strains of IAV. This novel finding further broadens the paradigm of how SIgA contributes to antiviral immunity, indicating that SIgA-based interventions could actively modulate the innate immune milieu in the airways during respiratory viral infection.

Another notable aspect of LCTG-001’s efficacy is its breadth of antiviral activities against IAVs. Our data suggest that this polyclonal mixture appears to target multiple viral proteins and life cycle steps: We detected antibodies likely directed against the conserved HA stalk, those capable of inhibiting NA (mediating NAI), and antibodies capable of stimulating Fc-dependent effector functions. These modes of action are complementary, and the interplay between Fab-mediated and Fc-mediated functions is evident in our data. In essence, LCTG-001 may not rely on a single mechanism of action but rather uses a variety of effector functions to mediate protection against viral pathogens. This was evident after H1N1 challenge, where there was incomplete neutralization at the highest concentration of antibody tested. Despite this, we observed high binding activity to Cal/09 (H1N1) HA. The robust protection may be partially explained by the high level of NA inhibition and Fc-mediated neutrophil activity against Cal/09 (H1N1), which suggests the presence of antibodies with potent anti-IAV activity that may not demonstrate traditional neutralization in vitro. These diverse activities may be especially valuable for highly mutable viruses like IAV, where complete protection often requires neutralizing the virus as well as containing infection through immune effector mechanisms (3, 50–52).

The cross-reactivity of LCTG-001 against an H5N1 avian influenza strain (clade 2.3.4.4b), despite no known exposure of donors to this strain, was particularly encouraging. These antibodies appear to be directed against the conserved group 1 HA stalk of H5 and to the N1 protein. This result suggests that the pool of human donor SIgA contains antibodies recognizing conserved epitopes shared with emerging avian viruses. VLPs were used to allow for work to be completed under standard biosafety level 2 conditions. Although VLPs generally preserve many of the structural and antigenic features of the wild-type virus, differences in particle assembly, glycosylation patterns, protein stoichiometry, and membrane composition can alter the presentation and accessibility of antigenic epitopes (53). Still, likely candidates for such cross-reactive targets are the HA stalk and NA (54, 55), as was confirmed by the observed binding activity of LCTG-001 to both recombinant HA and NA from 2.3.4.4b (H5N1). Through repeated infections or vaccinations with common IAV strains, antibodies likely accumulate in the breast milk that recognize conserved elements of potential pandemic viruses, in agreement with studies reporting cross-reactive serum IgG in humans (54, 56, 57). While these cross-reactive antibodies may not potently neutralize a novel strain, they can contribute to protection via secondary mechanisms such as by inhibiting the NA activity of the virus or by engaging Fc receptors for virus clearance. LCTG-001 could serve as a readily deployable passive immunization strategy in the face of an emerging influenza pandemic by providing immediate partial immunity while strain-specific vaccines or monoclonals are being developed. Even partial protection, by constricting viral replication or dampening undesired inflammation, could be life-saving during an outbreak of a novel virus, especially for immunocompromised individuals. The H5N1 cross-reactivity in LCTG-001 underscores the value of polyclonal SIgA in covering antigenic space that goes beyond the donors’ direct immunological history. In a sense, a diverse SIgA product “banks” the collective immunological experience of the population, including responses to past infections that might cross-protect against future threats.

In vivo studies further reinforce the broad protective capacity of LCTG-001, while suggesting protection may be mediated through both early reductions in viral burden and sustained improvements in lung pathology and barrier integrity. When administered prophylactically to both immunocompetent mice and immunocompromised mice, LCTG-001 significantly reduced disease severity following IAV challenge and was able to control early viral replication in immunocompetent mice, but with minimal effects on overall peak viral burden in the lung. IAV infection can trigger aberrant neutrophilia and CD8 T cell responses, which can contribute to immunopathology if not properly regulated (58). LCTG-001–treated animals showed attenuated lung pathology at multiple time points during infection and lower proinflammatory cytokine and chemokine levels compared to controls. This decoupling of disease outcome from viral load suggests that LCTG-001 primarily mitigates the tissue damage associated either from direct viral infection or immune-mediated mechanisms. Albumin concentrations in the BAL fluid were significantly lower in LCTG-001–treated immunocompetent mice, indicating that LCTG-001 limits damage to the lung epithelial-endothelial barrier. Studies have shown that IVIG is capable of modulating aberrant inflammatory immune responses by decreasing levels of proinflammatory cytokines such as IFN-γ and TNF-α, inhibiting apoptosis, and decreasing cytokines and chemokines involved in cell adhesion processes (59–61). Prior work has noted that passive antibody therapies can protect mice from lethal influenza even when classical neutralization titers are low, implying that antibodies can modulate the host immune response (51). In our study, LCTG-001–treated immunocompetent mice had blunted cytokine storm, tissue damage, and airway albumin concentration, all hallmarks of disease tolerance. We also found that B cell–deficient mice displayed more severe disease and distinct weight loss kinetics compared to immunocompetent mice. Despite this heightened susceptibility to disease, LCTG-001 conferred complete protection in these mice, further highlighting its potential for therapeutic use in vulnerable Ig-deficient populations.

While these results are highly encouraging, we acknowledge limitations and areas for further investigation. First, our murine models do not fully recapitulate human IgA biology. Mice lack the FcαRI (CD89) receptor for IgA on myeloid cells. This is important because many of IgA’s effector functions—including neutrophil activation, phagocytosis, and respiratory burst—are mediated by CD89 in humans (29, 30, 47, 62). The absence of this receptor in mice means that the contribution of SIgA’s Fc-dependent mechanisms in our in vivo studies was likely underrepresented. Our demonstration of neutrophil activation by LCTG-001 was conducted ex vivo with human cells. To better capture the full protective potential of human SIgA, future studies should use CD89-humanized mouse models. A prior study using transgenic mice expressing human CD89 showed enhanced IgA-mediated protection against IAV using serum-derived monomeric IgA, underlining that CD89 is critical in IgA-mediated immunity (63). Thus, our current efficacy data in wild-type and B cell–deficient mice may be a conservative underestimate of what LCTG-001 could achieve in a human setting with functional FcαRI on airway immune cells. Second, our in vivo challenge studies used a combined prophylactic and therapeutic treatment regimen. Intranasal delivery of SIgA has the potential to provide localized protection at the respiratory mucosa without requiring the clinical infrastructure associated with intravenous antibody administration typically required for passive immunization strategies (11, 64). Prophylactic deployment could also be relevant in outbreak scenarios, including ring prophylaxis strategies designed to protect high-risk contacts or vulnerable individuals following exposure to infectious cases (65). Therapeutic delivery of passive immunization is often constrained by a relatively narrow therapeutic window, especially for rapidly replicating respiratory viruses (66–68). Third, as a donor-derived product, LCTG-001 is inherently polyclonal and subject to batch-to-batch variability. The composition of antibodies depends on the pool of donor milk used; different donor cohorts or collection times may yield SIgA with different specificity profiles. Such variability is a challenge reminiscent of convalescent plasma therapies, developed over 70 years ago, which also suffer from inconsistent neutralizing titers between batches (14, 54). Moreover, the relative contributions of individual antibody specificities within LCTG-001 remain undefined. Previous work suggests that SIgA may exhibit increased cross-reactivity compared to matched IgG due to increased avidity (69). A direct comparison of IgA and IgG derived from the same donors would be valuable to assess their relative capacity to neutralize antigenically drifted influenza strains; however, IgG samples from the donors used to generate the LCTG-001 SIgA pool were not available in this study. Future comparisons may help clarify whether this increased avidity confers enhanced cross-reactivity against emerging variants.

In summary, we have demonstrated that breast milk–derived SIgA represents a viable and broadly acting passive immunization strategy. It is effective against a diverse panel of respiratory viruses, operates through multiple mechanisms of action, and may offer practical advantages over monoclonal therapies, including accessibility, scalability, and mucosal compatibility. These advantages position LCTG-001 as a strong candidate against both seasonal disease and for rapid pandemic response and introduce a novel paradigm for immunoglobulin replacement therapy for the respiratory mucosa.

MATERIALS AND METHODS

Collection and purification of LCTG-001

SIgA was purified and pooled from the breast milk of 120 lactating individuals, as described previously (13). Briefly, frozen human milk was thawed and clarified through centrifugation and dialyzed in PBS. The supernatant was loaded onto a CaptureSelect IgA Affinity Matrix resin, and IgA was isolated as previously described (13). Pooled elution fractions were dialyzed in PBS and filtered. The final concentration was determined using a spectrophotometer.

SDS–polyacrylamide gel electrophoresis

Two micrograms of LCTG-001 was mixed with 4× lithium dodecyl sulfate loading dye and β-mercaptoethanol at 95°C for 10 min. Sample was loaded onto a 4 to 12% bis-tris gel (Thermo Fisher Scientific) and ran at 100 V for 90 min with Bolt Mops SDS running buffer (Invitrogen). The gel was stained with Coomassie blue for 30 min at room temperature and destained using 40% methanol and 10% acetic acid in water.

Neutrophil isolation

Neutrophils were isolated from peripheral blood of consenting healthy male and female donors as described previously, using density gradient centrifugation (29, 30). Studies were approved by the Hamilton Integrated Research Ethics Board. Briefly, blood was collected into EDTA-coated tubes via venipuncture. Three milliliters of Histopaque 1119 and 3 ml of Histopaque 1077 were layered in a 15-ml Falcon tube, followed by 6 ml of blood. Samples were centrifuged at 2000 rpm for 30 min at room temperature with no deceleration in an Allegra X-12R centrifuge (Beckman Coulter). The neutrophil layer was collected between the Histopaque layers and red blood cells (RBCs). Neutrophils were diluted in 50 ml of cold polymorphonuclear cell (PMN) buffer [0.5% bovine serum albumin (BSA; Sigma-Aldrich) and 0.3 mM EDTA (Invitrogen) in Hank’s balanced salt solution (Gibco)]. Diluted cells were centrifuged at 1400 rpm for 5 min at room temperature. The supernatant was discarded, the pellet was resuspended, and RBCs were lysed using ammonium-chloride-potassium (ACK) lysis buffer [NH₄Cl (8.3 g/liter), KHCO₃ (1 g/liter), and 0.05 mM EDTA, in sterile distilled H₂O]. Cells were incubated with ACK lysis buffer for 5 min with agitation every 30 s. Following incubation, cells were diluted in 50 ml of PMN buffer and centrifuged at 1400 rpm for 5 min at room temperature, followed by an additional wash with PMN buffer. Cells were resuspended in a final volume of 1 ml in PMN buffer.

Cells and viruses

Human adenocarcinoma alveolar basal epithelial (A549) cells [American Type Culture Collection (ATCC)] and human embryonic kidney (HEK) 293T cells (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) (Gibco), 1× GlutaMAX supplement (Gibco), 1% of 1 M Hepes, and penicillin-streptomycin (100 U/ml; Gibco) at 37°C and 5% CO₂. Madin-Darby canine kidney (MDCK) cells (ATCC) were maintained in Earle’s minimal essential medium (MEM Earles) containing 10% FBS (Gibco), 1× GlutaMAX supplement (Gibco), 1% of 1 M Hepes, 1% of 1 M sodium pyruvate, and penicillin-streptomycin (100 U/ml; Gibco) at 37°C and 5% CO₂. Hep-2 cells (ATCC) were maintained in minimum essential medium (MEM) containing 10% FBS (Gibco), 1× GlutaMAX supplement (Gibco), 1% of 1 M Hepes, and penicillin-streptomycin (100 U/ml; Gibco) at 37°C and 5% CO₂. Expi293 cells (Gibco) were maintained at 37°C and 8% CO₂ with regular agitation (150 rpm) in Expi293 expression medium (Gibco).

A/Netherlands/602/2009 H1N1 (NL/09) and X-31 (gifts from P. Palese, Icahn School of Medicine at Mount Sinai, NY) were propagated in 10-day-old embryonated chicken eggs as per standard protocols (24). X-31 is a reassortant virus with the internal genes of A/Puerto Rico/8/1934 H1N1 (PR8) and the HA and NA of A/Hong Kong/1/1968 H3N2 (HK/68). A/California/04/2009 H1N1 (Cal/09) (gift from P. Palese) and A/seal/QC/FAV869-3/2022 H5N1 (clade 2.3.4.4b) (gift from Y. Berhane, CFIA, MB) were grown in MDCK cells. Cells were infected at 70% viability at a multiplicity of infection of 0.1 and incubated for 1 hour at 37°C. Following incubation, virus was removed and replaced with 1× infection media [1× MEM (Sigma-Aldrich), 0.6% BSA (Sigma-Aldrich), 1× MEM amino acids (Gibco), 1× MEM vitamin solution (Gibco), 1× GlutaMAX (Gibco), 1× penicillin-streptomycin (Gibco), 0.6% sodium bicarbonate, 1× Hepes, and tolylsulfonyl phenylalanyl chloromethyl ketone (TPCK)–treated trypsin (1 μg/ml; Sigma-Aldrich)]. The supernatant was collected 48 hours following infection by centrifugation at 500g for 5 min. RSV A2 expressing green fluorescent protein (GFP; ViraTree) was grown in Hep-2 cells. E1/E3-deleted HuAd serotype 5 expressing GFP was a gift from Z. Xing (McMaster University, ON).

H5N1 2.3.4.4b VLP production

Plasmids encoding the HA and NA from A/seal/QC/FAV869-3/2022 H5N1 were a gift from M. Côté (University of Ottawa, ON). HEK 293T cells were seeded in T150 flasks and transfected with HA and NA plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Briefly, 240 μl of lipofectamine was incubated with 8 ml of Opti-MEM (Gibco) for 7.5 min. During the incubation, 32 μg of each plasmid was diluted with 8 ml of Opti-MEM. Lipofectamine and plasmid mixtures were combined and incubated at room temperature for 25 min. After the incubation, 10 ml of supplemented DMEM (2% FBS, 1% penicillin-streptomycin, 1% GlutaMAX, and 1% Hepes) was added to the transfection mixture and incubated on cells overnight at 37°C and 5% CO₂. The next day, media was replaced with DMEM containing 0.3% BSA (Sigma-Aldrich), 1% penicillin-streptomycin, and 1% GlutaMAX. The cell supernatant was collected 4 days posttransfection and centrifuged at 3000g for 2 min. The pellet was removed, and the supernatant was stored at −80°C.

Transmission electron microscopy

A 3-μl drop of sample was applied to a formvar-coated copper grid stabilized with a 5-nm carbon film and left for 10 min to allow for the VLPs to settle on the grid. Excess sample was blotted off with filter paper. The grids were then briefly rinsed by immersion in deionized water and blotted dry with filter paper. Then, 3 μl of 1% (aq) uranyl acetate was applied to the grid surface and left for 1 min. Excess stain was removed by blotting with filter paper, and the grid was allowed to dry completely. Imaging of the grids was performed using a TALOS L120C transmission electron microscope (Thermo Fisher Scientific) operating at an accelerating voltage of 120 kV. Images were acquired with a 4K CETA complementary metal-oxide semiconductor camera.

Influenza A virus purification

Clarified supernatants from IAV-infected MDCK cells or allantoic fluid from embryonated chicken eggs were layered on top of 8 ml of 20% sucrose in NTE [0.5 M NaCl, 10 mM tris HCl, and 1 mM EDTA (pH 7.5)] buffer in Ultra-Clear Ultracentrifuge tubes (Beckman Coulter). Samples were spun for 2 hours at 4°C at 76650g inside a SW 32i rotor using an Optima L-90K Ultracentrifuge (Beckman Coulter). Clarified supernatants from VLP-transfected 293T cells were layered on top of 3 ml of 20% glycerol in PBS in Ultra-Clear Ultracentrifuge tubes (Beckman Coulter). Samples were spun for 4 hours at 4°C at 135,000g inside a SW 32i rotor using an Optima L-90K Ultracentrifuge (Beckman Coulter). Purified viruses and VLPs were quantified using a bicinchoninic acid assay Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Recombinant antigens

cH6/1 HA was a gift from P. Palese (Icahn School of Medicine at Mount Sinai, NY). cH6/1 is composed of the H6 HA head domain from A/mallard/Sweden/81/02 H6N1 and H1 HA stalk domain from PR8. HK/68 HA was made using the Expi293 expression system. Expi293 cells (Gibco) were transfected with plasmid encoding HK/68 HA (gift from A. McDermott, NIAID, USA) according to the manufacturer’s instructions. Proteins were purified by Ni resin affinity chromatography. RSV A2 fusion protein (RSV-F), Cal/09 HA, A/American wigeon/South Carolina/AH0195145/2021 (H5N1) HA, and A/Texas/37/2024 (H5N1) NA were purchased from SinoBiological. Lysate from HuAd5-infected HeLa cells was a gift from Z. Xing (McMaster University, ON).

Enzyme-linked immunosorbent assays

The 96-well NUNC-MaxiSorp plates (Thermo Fisher Scientific) were coated overnight at 4°C with purified soluble Cal/09 HA, HK/68 HA, cH6/1 HA, H5 HA, N1 NA, RSV-F recombinant protein, or purified 2.3.4.4b H5N1 VLPs, diluted to 2 μg/ml in bicarbonate-carbonate coating buffer (pH 9.4). For HuAd5 ELISAs, plates were coated with 50 μl of Ad-5–infected HeLa cell lysate (100 μg/ml) in PBS. Plates were blocked for 1 hour at 37°C with reagent diluent [0.5% BSA (Sigma-Aldrich) and 0.02% sodium azide, in 1× tris-Tween buffer]. LCTG-001 was serially diluted 1:2 across each row. For in vivo biodistribution studies, homogenized lung tissue or BAL fluid was serially diluted 1:2 across each row. Following a 1-hour incubation at 37°C, plates were washed three times with 1× tris-Tween wash buffer. After washing, goat anti-human-biotin IgA (Southern Biotech) was diluted 1:2000 in reagent diluent and added to all wells. Plates were again incubated for 1 hour at 37°C, followed by three washes with 1× tris-Tween buffer. A streptavidin-alkaline phosphatase secondary antibody (1:2000, Southern Biotech) was added to all wells for 1 hour at 37°C. Plates were subsequently washed three times before addition of pNPP one component microwell substrate solution (Southern Biotech) to each well. Plates were developed for 10 min, and the reaction was quenched with an equal volume 3 N sodium hydroxide. The optical density (OD) at 405 nm was read on a Biotek Synergy H1 (Agilent). OD values were normalized on the basis of control wells with no sample.

MNT assays

Human adenovirus MNT

The day before infection, 96-well plates of A549 cells were seeded at a density of 2.5 × 10⁵ cells per well in clear flat-bottom TC-treated 96-well plates (Corning). Cells were subsequently incubated with serial dilutions of LCTG-001 and an E1/E3-deleted HuAd serotype 5 expressing GFP (gift from Z. Xing, McMaster University, Hamilton, ON, Canada) (1.6 × 10⁶ PFU per well) for 24 hours at 37°C and 5% CO₂ in α-MEM supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% GlutaMAX (Gibco). Postincubation, fluorescence (GFP) was read using Cytation 5 (Agilent, Biotek). Neutralization titer was determined as the highest antibody dilution that achieved a 50% reduction in fluorescence compared to the virus control wells.

Respiratory syncytial virus MNT

The day before infection, Hep-2 cells were seeded at a density of 2.5 × 10⁴ cells per well in clear flat-bottom TC-treated 96-well plates (Corning). Cells were subsequently incubated with serial dilutions of LCTG-001 and RSV GFP (ViraTree) (4 × 10³ PFU per well) for 1 hour at 37°C, 5% CO₂. The antibody/virus mixture was removed and replenished with fresh DMEM supplemented with 2% FBS, 1% penicillin-streptomycin, 1% Hepes (pH 7.3), and 1% GlutaMAX for 24 hours at 37°C, 5% CO₂. Postincubation, fluorescence (GFP) was read using Cytation 5 (Agilent, Biotek). Neutralization titer was determined as the highest antibody dilution that achieved a 50% reduction in fluorescence compared to the virus control wells.

Influenza A virus MNT

The day before infection, 96-well plates of MDCK cells were seeded at a density of 5 × 10⁴ cells per well in clear flat-bottom TC-treated 96-well plates (Corning). Serial dilution of LCTG-001 and viruses (3600 PFU Cal/09 or 24500 PFU X-31) were preincubated for 30 min at 37°C and 5% CO₂ in DMEM supplemented with 0.3% BSA (Sigma-Aldrich), 0.1% penicillin-streptomycin (Gibco), and TPCK (1 μg/ml)–treated trypsin (Sigma-Aldrich). Following incubation, MDCK cells were infected with the LCTG-001–virus mixture for 1 hour at 37°C and 5% CO₂. After the 1-hour infection, the infectious media was replaced with fresh supplemented DMEM. Plates were incubated for 18 hours and then fixed with 80% acetone for 20 min at −20°C before blocking with 5% nonfat milk in PBS for 30 min at room temperature, followed by an incubation in 3% hydrogen peroxide for 20 min at room temperature. Plates were washed three times with PBS-Tween wash buffer before a 1-hour incubation at room temperature with a 1:2000 dilution of biotin-conjugated mouse anti-NP (Millipore) in 5% nonfat milk. Following incubation, plates were washed three times with PBS-Tween wash buffer and incubated for 1 hour at room temperature with a secondary horseradish peroxidase (HRP)–conjugated streptavidin (1:5000, Millipore). Plates were washed three times with PBS-Tween wash buffer before the addition of SigmaFast OPD (Sigma-Aldrich) to each well. Plates were developed for 10 min, and the reaction was quenched with an equal volume of 3 M hydrochloric acid. The OD at 490 nm was read on a Biotek Synergy H1 (Agilent). OD values were normalized on the basis of uninfected control wells, and percent neutralization was calculated on the basis of the ratio between the sample OD value to infected control values.

Enzyme-linked lectin assays

The 96-well NUNC-MaxiSorp plates (Thermo Fisher Scientific) were coated overnight at 4°C with fetuin (25 μg/ml; Sigma-Aldrich) diluted in 1× KPL coating buffer (SeraCare). In a separate plate, LCTG-001 was serially diluted in sample diluent [MES (Life Technologies) (pH 6.5), 20 mM CaCl₂, 1% BSA (Sigma-Aldrich) and 0.5% Tween 20]. Virus (Cal/09, X-31, or H5N1 VLPs) was diluted to 90% of the max OD (predetermined) in sample diluent. Fetuin-coated plates were washed three times with PBS-Tween wash buffer, and LCTG-001 dilutions and virus were transferred to the fetuin-coated plate. Plates were incubated overnight at 37°C. Following incubation, plates were washed six times with PBS-Tween wash buffer. Peanut agglutinin (PNA)–HRP (1 mg/ml) was diluted 1:500 in PNA-HRP diluent [MES (pH 6.5), 20 mM CaCl₂, and 1% BSA] and added to all wells for 2 hours at room temperature. Plates were then washed three times with PBS-Tween wash buffer before the addition of SigmaFast OPD (Sigma-Aldrich) to each well. Plates were developed for 10 min, and the reaction was quenched with an equal volume of 3 M hydrochloric acid. The OD at 490 nm was read on a Biotek Synergy H1 (Agilent). OD values were normalized on the basis of control wells without virus, and percent inhibition was calculated on the basis of the ratio between the sample OD value to control values with virus only.

Luminol assays

Luminol assays were performed as previously described (30). Briefly, immune complexes were formed by incubating 2.5 μg of virus with 5 μg of LCTG-001 in 50 μl for 30 min at room temperature. Luminol (Sigma-Aldrich) was added to all wells. Following isolation, 5 × 10⁵ cells of human neutrophils were added to each well. Kinetic measurements of luminescence [in relative light units (RLU)] were taken at 1-min intervals for 1 hour using a Biotek Synergy H1 (Agilent).

Mice

Eight- to 12-week-old female BALB/c mice were purchased from Charles River Laboratories (Saint Constant, QC, Canada). B cell–deficient mice C.Cg-Igh-J^tm1Dhu of BALB/c background were purchased from Taconic Biosciences (Germantown, NY, USA). Animals were housed under specific pathogen–free conditions at McMaster University, Hamilton, ON. All animal protocols were reviewed and approved by the institutional Animal Research and Ethics Board (Animal Utilization Protocol 23-62).

Antibody administration and IAV infection

Animals received LCTG-001, PBS, and NL/09 intranasally as previously described (70). For antibody administration, animals were anesthetized with isoflurane and received a single bolus of 40 μl containing either 1 mg of LCTG-001 diluted in PBS or PBS as a negative control. Mice were infected with 450 PFU of NL/09, administered intranasally in a final volume of 40 μl diluted in PBS. Mice were monitored for weight loss and clinical signs daily, including huddling, lack of grooming, and lethargy. A reduction to 80% of initial weight was considered humane endpoint in accordance with institutional guidelines.

Lung isolation and preparation for histology

Mice were euthanized by exsanguination. Whole lungs were isolated as previously described (70), with some modifications. Briefly, lungs were removed from the mice and cannulated. Following cannulation, the right lobe was tied off using an additional suture and removed, followed by homogenization using the Bullet Blender Gold High-Throughput Bead Mill Homogenizer (Next Advance). Samples were stored at −80°C. The remaining lobe was inflated and fixed in 10% formalin for 24 hours. Following fixation, the lobe was dissected into three equal pieces and mounted onto slides. Samples were subsequently stained with H&E. Images were taken with the Zeiss Imager M2 microscope.

Histopathology scoring

A trained researcher was blinded, and all lung slices were scored according to the following criteria. Vascular pathology was graded 0 to 3: 0, none; 1, some blood leaking into interstitium; 2, mild and moderate hemorrhage; 3, diffuse hemorrhage. Alveolar pathology was graded 0 to 3: 0, none; 1, mild inflammatory exudates, patchy areas of edema, some loss of airway integrity; 2, moderate inflammatory exudates, moderate alveolar thickening (25 to 50% of the lung area); 3, severe inflammatory exudates, severe alveolar thickening and loss of structure (>50% of the lung area). Bronchiolar pathology was graded 0 to 3: 0, none; 1, mild inflammatory infiltrates; 2, moderate inflammatory infiltrates, detachment of epithelium in some bronchioles; 3, complete loss of bronchiole structure, hyaline membrane formation, airway cellular debris, severe inflammatory infiltrates. Representative images from each group were selected for presentation in the manuscript.

Cytokine chemistry

Evaluation of cytokine and chemokine induction in the lungs was performed using a mouse 32-plex cytokine/chemokine array (MD32) (Eve Technologies, Alberta, Canada).

Plaque assays

MDCK cells were seeded in six-well plates at a density of 7 × 10⁵ cells per well. The next day, media was replaced. Two days after cells were seeded, cells were infected with serial dilutions of lung homogenates diluted in 1× infection media [1× MEM (Sigma-Aldrich), 0.6% BSA (Sigma-Aldrich), 1× MEM amino acids (Gibco), 1× MEM vitamin solution (Gibco), 1× GlutaMAX (Gibco), 1× penicillin-streptomycin (Gibco), 0.6% sodium bicarbonate, 1× Hepes, and TPCK-treated trypsin (1 μg/ml; Sigma-Aldrich)]. After a 1-hour infection at 37°C, infectious media was replaced with 1× infection media, 0.01% DEAE-dextran (MP Biomedicals), and 0.5% Oxoid agar (Thermo Fisher Scientific). Infected cells were incubated for 2 days at 37°C and 5% CO₂. After incubation, cells were fixed with 3.7% paraformaldehyde in PBS for 30 min at room temperature. The agar layer was removed, and cells were stained with crystal violet for 30 min at room temperature to visualize plaques.

Plaque reduction assays

MDCK cells were seeded in 12-well plates at a density of 2.5 × 10⁵ cells per well. The next day, media was replaced. Two days after cells were seeded, and LCTG-001 was serially diluted and incubated with an equal volume of 30 PFU of virus for 30 min at 37°C. Following incubation, MDCK cells were infected with the LCTG-001–virus mixture for 1 hour at 37°C and 5% CO₂. Infectious media was replaced with 1× infection media, 0.01% DEAE-dextran (MP Biomedicals), and 0.5% Oxoid agar (Thermo Fisher Scientific). Infected cells were incubated for 2 days at 37°C and 5% CO₂. After incubation, cells were fixed with 3.7% paraformaldehyde in PBS for 30 min at room temperature. The agar layer was removed, and cells were stained with crystal violet to visualize plaques. Percent neutralization (%) was calculated as the reduction of plaques in the LCTG-001–virus conditions compared to control wells containing virus only.

HA and HA inhibition assays

Hemagglutinin and HA inhibition assays were performed as described previously (24). Briefly, chicken RBCs diluted 1:1 in Alsever’s solution (CFIA) were diluted 1:10 in PBS and centrifuged at 300g for 5 min. The supernatant was removed, and 125 μl of the RBC pellet was added to 25 ml of PBS to make 0.5% chicken RBCs. Samples were diluted 1:2 across clear V-bottom plates to a final volume of 50 μl. Then, 50 μl of 0.5% chicken RBCs was added to each well. Plates were read after a 1-hour incubation at 4°C.

For HA inhibition, LCTG-001 was serially diluted 1:2 in V- bottom plates to a final volume of 25 μl per well. Four HA units of VLPs diluted in PBS (25 μl) were added to each well, followed by 50 μl of 0.5% chicken RBCs. Plates were read after a 1-hour incubation at 4°C.

Albumin quantification

BAL fluid albumin was measured using ELISA (Abcam, ab108792) according to the manufacturer’s instructions.

Statistical analysis

Graphs and statistical analyses were generated using GraphPad Prism (GraphPad Software v10.4.2, La Jolla, CA, USA). Statistical significance between indexed RLUs and antibody titers in the lungs and airways was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. For weight loss and survival, statistical significance was determined using a two-way ANOVA with Šídák’s test for multiple comparisons and a Mantel-Cox test. For viral titers at 6 DPI for immunocompetent and immunocompromised mice, statistical significance was determined with a Student’s t test. For cytokine analysis, histological scores, and quantification of albumin and viral titers, statistical significance was determined with multiple Student’s t tests between LCTG-001– and PBS-treated mice with no correction for multiple comparisons, with *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001; ns indicates not significant.

Supplementary Materials

This PDF file includes:

Figs. S1 to S6