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How Broadly Neutralizing Antibodies are Redefining Immunity to Influenza

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16 October 2024

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17 October 2024

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Abstract
Recent avian influenza outbreaks have heightened global concern over viral threats with the potential to significantly impact human health. Influenza is particularly alarming due to its history of causing pandemics and zoonotic reservoirs. In response, significant progress has been made toward the development of universal influenza vaccines, largely driven by the discovery of broadly neutralizing antibodies (bnAbs). bnAbs have the potential to neutralize a broad range of influenza viruses, extending beyond the traditional strain-specific response. This could lead to longer-lasting immunity, reducing the need for seasonal vaccinations, and improve preparedness for future pandemics. This review offers a comprehensive analysis of these antibodies, their application in clinical studies, and both their potential and possible shortcomings in managing future influenza outbreaks.
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Subject: Biology and Life Sciences  -   Virology

1. Introduction

Influenza is a globally endemic respiratory virus typically associated with upper respiratory tract infection, cough, and accompanying fever [1]. While generally not lethal, influenza poses a significant health burden on geriatric, paediatric, or otherwise immunocompromised individuals [2]. The World Health Organization (WHO) estimates around a billion seasonal infections, 3-5 million cases of severe disease outcomes, and up to 650,000 annual deaths can be attributed to influenza each year [3].
Human infections are primarily caused by influenza types A and B; however types C and D are also known. Influenza can be broadly classified by the composition of its major surface glycoproteins; the entry protein, hemagglutinin (HA), and exit protein neuraminidase (NA). The specific combination of HA and NA not only defines the virus's preferred host target and virulence, but also influences its zoonotic potential and pandemic threat [1].
Despite circulating for centuries [4], influenza remains a public health threat. The ability to continue evading existing immune responses is heavily linked to two phenomena; antigenic drift and antigenic shift. Antigenic drift describes the accumulation of glycoprotein mutations in response to selective pressure of acquired immune responses. Antigenic shift describes sudden introductions of new or recombined viral strains. The dramatic rearrangement of the antigenic landscape frequently has a devastating effect on immunologically naïve populations [5]. This has been demonstrated by the four historical flu outbreaks: the 1918 H1N1 Spanish Flu that killed an estimated 40 million people, the 1957 H2N2 Asian Flu, and the 1968 H3N2 Hong Kong Flu affecting 700,000 and 1 million people respectively, and the 2009 H1N1 Swine Flu affecting 16,000 people worldwide [6,7]. All except the 1918 Spanish Flu are attributed to antigenic shift, whereas the Spanish Flu is thought to be a zoonotic avian virus that underwent unusually rapid antigenic drift [6,7].
The fact that mildly antigenically drifted seasonal strains tend to be more immunologically tolerable suggests that a person’s infection history significantly influences disease severity. In the early 1980s, distinctions were made regarding “strain-specific” and “cross-reactive” antibodies, with the latter being mentioned as a possible explanation for the ability to tolerate mildly mutated strains [8]. This was corroborated in 1993, when Okuno and colleagues observed that mice immunized with A/Okuda/57 (H2N2) gained immunity to all H1 and H2 strains through the generation of a singular broadly neutralizing antibody (bnAb), termed C179 [9]. Here we review trends and treatments relating to antibodies capable of neutralizing multiple antigenically drifted, chronologically distinct viruses (intrastrain bnAbs), different viruses within the same Influenza Group (intrasubtypic bnAbs) and viruses within different influenza groups (intergroup bnAbs).

1.1. Hemagglutinin

HA is the primary immunologic target in influenza. Influenza A features 18 different HAs (H1-H18), while Influenza B has two HAs (Yamagata and Victoria). HA is synthesized as an immature HA0 chain, which is proteolytically cleaved by endoplasmic reticulum host proteases into disulphide-linked subunits – HA1 and HA2. HA1 primarily comprises the globular head domain, forming functionally critical structures including the receptor binding site (RBS). HA2, together with a portion of HA1 forms the stem domain. During viral infection, the HA1 RBS binds to sialic acid, inducing viral endocytosis. Upon endosomal acidification, the HA2 subunit undergoes a conformational change leading to the insertion of a hydrophobic fusion peptide into the host membrane. Alongside further conformational changes, this leads to endosomal collapse and the introduction of the viral genome to the host cell (Figure 1) [10].
Antibody potency against HA is influenced by both the mutation frequency of the epitope and its functional significance. As such antibodies targeting the head domain, particularly the RBS tend to be highly immunogenic; however epitopes in the head domain are less stable and tend to drift seasonally [11]. Conversely, antibodies targeting the stem must significantly impede conformational changes during acidification of the endosome or the fusion peptide [11]. Only few such epitopes have been characterised, yet the lower mutational rate of the HA stem means that functional antibodies are more likely to also be broadly neutralising (Figure 2) [12,13,14].

2. bnAbs against the HA Stem

The discovery of C179 in 1993 demonstrated that broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem exist [9]. However, the reduced accessibility of the stem, combined with the immunodominance of the accessible HA head, has been suggested as a barrier to the development of bnAbs targeting the stem [15]. It has been shown that enhancing immune focus away from the head by hyperglycosylating variable regions significantly increases the production of stem-targeting antibodies [16]. To date, only two main immunological stem epitopes have been identified: 1) the Central Stem (CS) Epitope, and 2) the Anchor Epitope/Fusion Peptide (Figure 2). Whether the limited identification of other stem epitopes is due to the functional importance of HA sites, steric constraints, or the dominance of other HA regions remains an open question.
Most stem antibodies characterized to date are IGVH1-69 somatically hypermutated antibodies, that predominantly bind via the heavy-chain complementarity determining region 3 (CDRH3) (Table 2).

2.1. The Central Stem Epitope

Most stem bnAbs, including C179, target the central stem (CS) epitope (Figure 2, Table 1) [17]. This epitope broadly consists of a conserved hydrophobic pocket, which affects conformational changes related to membrane fusion and HA0 processing [18,19,20,21]. CS antibodies also frequently mediate antibody dependent cellular cytotoxicity (ADCC) (Table 1).
The first human serum-derived bnAb to the CS was discovered in 2008 [12]. This antibody, called CR6261, elicited broad protection in pandemic H5N1 and H1N1 lethally challenged mice [12]. The potency of this site became apparent with F10, a human antibody targeting the CS, capable of neutralising H1N1, H2N2, H5N1, H6N1, H6N2, H8N4 and H9N2 [19]. This was rapidly followed by the discovery of FI6, an antibody that was able to bind to all 16 hemagglutinin subtypes in influenza A, but not influenza B [21]. FI6 has since been shown to elicit in vivo protection in mice, ferrets, and pigs against a panel of Influenza A viruses (Table 1)[21,22,23].

2.2. The Fusion Peptide and Anchor Epitope

A second antigenic site has been identified below the CS epitope and closer to the viral membrane (Figure 2, Table 1). It was initially thought to be unique to Group 2 influenza A viruses, [17,24,25] with CR8020 [24] and CR8043 [25] both eliciting robust protection against H3N2 and H7Nx viruses. However, recently Group 1 influenza has also been found to contain a relevant site near the viral membrane [26]. This site is reported to possess a strong polyclonal response upon H1N1 vaccination or infection, with classified antibodies – 047-09 4F04, 241 IgA 2F04 and 222-1C06 – recognizing a well conserved epitope amongst Group 1 viruses consisting of W343, H354, Q356, S361 and Y363 [26].

3. bnAbs against the HA Head Domain

The low mutational rate of the HA stem has historically made it more heavily investigated vaccine and bnMab therapy target, yet this usually came with the trade-off of lower potency Mabs [11] and frequently sterically occluded antigenic sites [51]. In contrast, the head domain is mutationally volatile, and immunodominant, yielding a more potent and diverse set of antibodies [15]; both with respect to their germline sequences and their binding mechanisms (Table 2). Many bnAbs against the head have been characterised, but their epitopes can be roughly grouped into four distinct sites; the Receptor Binding Site (RBS), the Lateral Patch, the Vestigial Esterase (VE) and the Occluded Site (Figure 2).

3.1. Receptor Binding Site

Broadly neutralising antibodies against the Receptor Binding Site (RBS) have been described in the literature for almost as long as stem antibodies (Table 2, Figure 2) [52]. These are usually characterized by hemagglutination inhibition (HAI), as antibodies that directly compete with sialic acid for the RBS [52,53,54,55].
Antibodies that neutralize the RBS frequently do so through molecular mimicry, sterically and electrostatically mimicking sialic acid [54,56]. Curiously, while the germline-encoded IGHVs differ widely between different RBS bnAbs, the dominant loop associated with RBS binding tends to be CDRH3. This CDRH3-centric mechanism makes RBS antibodies particularly susceptible to mutations in and around the RBS as small steric or electrostatic constraints in the CDR insertion path can completely abolish antibody binding [30,40,54,57,58,59].

3.2. Lateral Patch

The lateral patch is a region on the HA head that is offset from the RBS (Figure 2). However, reports on HAI activity [60] indicate active or passive inhibition of sialic acid binding. The original study that coined the term “lateral patch” characterized CL6649, an antibody with H1N1 intrastrain activity. However mechanistic insight into the mode of neutralization was lacking [60,61].
A recent study on H7N9 lateral patch antibodies may offer further insights on the mechanism involved with this site. Jia and colleagues found that sialic acid binding is passively inhibited by their antibody, H7.HK1. This antibody inhibits the HA 220-loop (G218-G228 in H7 numbering, or G228-238 in H3 numbering), which makes hydrophobic contacts with sialic acid [62]. It is worth noting, however, that these mechanisms may not translate between influenza subtypes and further research is needed to understand how CL6649 and H7.HK1 compare.

3.3. Vestigial Esterase

The Vestigial Esterase (VE) is a region located in the HA head between the RBS and the start of the HA stem (Figure 2)[63]. Its sequences are highly conserved within subtypes, but not across subtypes [62,63]. VE-specific antibodies tend to lack HAI activity [64,65], but display ADCC through Fc-FcγR responses [62,63,65,66,67] and may be involved in crosslinking and thereby conformationally restricting different HA trimers [64].
VE-specific bnAbs have been described against Group 1[63,68], Group 2 [64,69] and Influenza B [55,67], with ADCC consistently being described as a crucial mechanism for in vivo protection (Table 2).

3.4. Interface and Occluded Epitope

Finally, the occluded epitope is a name given to an epitope either sitting between two HA monomers, or within the HA core. Despite being characterized as early as 1993 [70], the value of this low variability site for bnAbs had not been realized until three independent research teams demonstrated its ability to provide therapeutic protection against a range of different Influenza A viruses including H1N1, H3N2, H5N1, and/or H7N9 [71,72,73].
Despite its occluded nature, the epitope is available for binding even in intact, trimeric HAs[73], likely through protein dynamics. However, the mechanism by which protection is achieved has been reported to differ widely.
FluA-20 is a bnAb that has been shown to be protect mice against viral challenge with H1N1, H3N2, H5N1 or H7N9 subtypes. It has been found to bind the occluded epitope in uncleaved immature HA0. Trypsin-based treatment destabilizes the FluA-20 contacts, implying that FluA-20 may be disrupting HA trimerization. No HAI activity or significant ADCC has been observed [71].
Conversely, 8H10 bnAb has been shown to bind mature trimeric HAs and elicit protection against various H3N2 strains, and to bind to H4 viruses. Protection is thought to be primarily governed by ADCC [73]. These findings are corroborated in another study that shows a panel of antibodies targeting the occluded epitope to be eliciting protection primarily through ADCC [72].
It is worth noting that broadly neutralising sites have also been reported at an exposed site of the HA multimerization interface (Figure 2)[74]. This bnAb, termed F005-126, lacks HAI activity and protects HA from trypsin-based cleavage against a panel of H3N2 viruses, indicating inhibition of endosomal rearrangements [74].
Table 2. Overview of Extensively Studied Head-Targeting Broadly Neutralizing Antibodies (bnAbs). This table provides details on some of the most thoroughly researched head-targeting bnAbs, adding to previously published work [17]. It includes information on their in vitro binding affinity, in vitro neutralization capacity, and in vivo protective efficacy. The table also lists the immunoglobulin heavy-chain variable region (IGHV) gene used, the primary complementarity-determining region (CDR) recognition mode, and whether the antibody exhibits antibody-dependent cellular cytotoxicity (ADCC) as a significant protection mechanism. Additionally, it specifies whether the antibody was isolated from mice or humans, any known escape mutations, and the IgG subtype used in generating the findings. A – is shown if the information was not provided.
Table 2. Overview of Extensively Studied Head-Targeting Broadly Neutralizing Antibodies (bnAbs). This table provides details on some of the most thoroughly researched head-targeting bnAbs, adding to previously published work [17]. It includes information on their in vitro binding affinity, in vitro neutralization capacity, and in vivo protective efficacy. The table also lists the immunoglobulin heavy-chain variable region (IGHV) gene used, the primary complementarity-determining region (CDR) recognition mode, and whether the antibody exhibits antibody-dependent cellular cytotoxicity (ADCC) as a significant protection mechanism. Additionally, it specifies whether the antibody was isolated from mice or humans, any known escape mutations, and the IgG subtype used in generating the findings. A – is shown if the information was not provided.
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4. bnAbs in Clinical Trials

In the 1970s, Köhler and Milstein pioneered hybridoma technology, facilitating the production of monoclonal antibodies [101]. This breakthrough has significantly advanced the therapeutic use of monoclonal antibodies, particularly in autoimmune diseases, oncology, and infectious diseases. Broadly neutralising monoclonal antibodies (bnMAbs) hold promise as a therapeutic alternative to existing influenza treatments, particularly in combating Influenza A. Ongoing clinical trials aim to evaluate the efficacy and safety of stem-targeting bnMAbs in treating influenza infections (Table 3). Initial findings suggest that bnMAbs may provide significant clinical benefits in managing uncomplicated influenza A, as evidenced by several trials. For instance, in a clinical trial (NCT02071914), patients treated with CT-P27 at doses of 10 mg/kg or 20 mg/kg exhibited a statistically significant reduction in the area under the curve (AUC) of viral load compared to placebo. Another bnMAb, VIS410, administered at doses of 2000 mg or 4000 mg, demonstrated significant improvement in the signs and symptoms of influenza infection on days 3 and 4, along with a reduction in the time required to resolve peak viral load (NCT02989194).
However, when VIS410 was combined with oral oseltamivir at doses of 3600 mg or 8400 mg, it failed to show a statistically significant reduction in the time to oxygen cessation or in viral load in nasopharyngeal samples compared to oseltamivir with a placebo in hospitalized patients with influenza A infection (NCT03040141).
Similarly, the bnMAb MHAA4549A, at a dose of 3600 mg, resulted in a statistically significant reduction in viral AUC in nasopharyngeal samples in a challenge model involving H3N2 influenza (NCT01980966). Despite this reduction in viral load, MHAA4549A, whether at 3600 mg or 8400 mg, did not improve clinical outcomes over oseltamivir alone in patients hospitalized with severe influenza infections (NCT02293863).
Another bnMAb, MEDI8852, was assessed for its efficacy in treating acute, uncomplicated influenza A infections. However, it did not provide statistically significant improvements over oseltamivir alone and was even associated with potentially worsened disease progression compared to oseltamivir (NCT02603952). Consequently, a subsequent clinical trial to evaluate the dosing regimen of MEDI8852 was withdrawn by the sponsoring company (NCT03028909).
The bnMAb CR6261, while able to significantly reduce the percentage of participants exhibiting influenza symptoms following H1N1 challenge, failed to significantly impact viral shedding, AUC, or disease severity (NCT02371668). Additionally, a trial combining CR6261 with CR8020 was withdrawn due to unsatisfactory preliminary efficacy results (NCT01992276). Similarly, a trial involving CT-P27 was terminated due to the inactivation of the bnMAb (NCT03511066).
These findings emphasize the potential of bnMAbs as a therapeutic strategy against influenza, while also underscoring their limitations and the need for further research. Current clinical trials for influenza predominantly utilize IgG1 bnMAbs. IgG1 and IgG3 are the major subclasses generated during viral infections, with distinct functional differences and characteristics. IgG1 has a shorter 15 amino acid hinge region with only 2 disulfide bonds, providing a longer half-life and potentially greater therapeutic efficacy [102,103]. In contrast, IgG3, with its shorter half-life has been overlooked for its therapeutic applications. However, with its longer hinge region of 62 amino acids and 11 disulfide bonds [102,103] IgG3 may be able to overcome steric hindrance, a limitation observed in certain stem-targeting bnMAbs such as CR8020 [104]. Moreover, IgG3 has a higher affinity for the Fc receptors FcγRIIa, FcγRIIIa, and FcγRIIIb in its monomeric form compared to IgG1, while its binding efficiency in complex form exceeds IgG1 for all receptors [102], making it particularly effective at activating CDC and ADCC [103] mechanisms employed by multiple head and stem targeting bnAbs (Table 1 and Table 2) to indirectly neutralize influenza [105,106,107]. Switching the subclass of MAbs has been seen to have beneficial effects for SARS-CoV-2, with the switch from an IgG1 to an IgG3 enhancing both Fc mediated phagocytosis and the triggering of the classical complement pathway [108]. Additionally, Bolton and colleagues found that IgG3 antibodies exhibited superior binding and neutralisation capacity to antigenically drifted influenza and SARS-CoV-2 viruses relative to other IgG subclasses [109].
To enhance the therapeutic efficacy of IgG1 bnMAbs, modifications to the Fc region could be explored to improve ADCC initiation. Ocaratuzumab is an anti-CD20 mAb which has Fc modifications, P247I/A339Q, which have been shown to increase its binding to lower-affinity FcγRIIIa allowing it to have increased ADCC activity [110,111,112]. Fc modifications can also be used to increase the half life of antibodies to improve their therapeutic efficacy. The MAb therapy Sotrovimab, which was approved for use against SARS-CoV-2, has the Fc modifications M428L/N434S to extend the half life of the antibody [113].
Another promising approach involves the development of chimeric IgG1/IgG3 antibodies, which may enhance ADCC and CDC as well as improving binding to sterically hindered stem regions. Natsume and colleagues generated a chimeric form of rituximab, an anti-CD20 antibody, that consisted of the CH1 and hinge regions from IgG1, with the Fc region of IgG3 with the COOH terminal CH3 domain of IgG1. This chimeric antibody showed enhanced CDC and ADCC activities compared to the wild type [114]. Chimeric antibodies could combine the favourable pharmacokinetics of IgG1 with the functional advantages of IgG3, potentially overcoming the limitations associated with IgG3 monotherapy [114,115]. These strategies highlight avenues for optimizing bnMAb design to improve their utility in influenza treatment. Further research into IgG subclass-specific characteristics and their impact on bnMAb efficacy is warranted.

5. Broadly Protective Vaccines in Clinical Trials

bnMAbs represent a promising secondary defence mechanism against influenza, complementing the primary defence provided by vaccination. Current influenza vaccines require annual updates to address antigenic drift and shift. However, the induction of bnAbs via vaccination could offer cross-protection against multiple strains of influenza, irrespective of antigenic variations. Vaccines designed to elicit bnAbs may provide long-term immunity and reduce the need for frequent vaccine reformulations. Several clinical trials are currently investigating novel influenza vaccines aimed at inducing bnAb formation (Table 4). These trials employ various strategies to stimulate bnAb production specifically targeting the hemagglutinin protein of influenza A.
One promising approach involves presenting only the stem region of the influenza virus, aiming to elicit bnAbs against conserved stem epitopes across diverse influenza strains. UFluA, a stabilized stem nanoparticle vaccine currently in Phase 1 trials (NCT05155319), exemplifies this strategy. By stabilizing the HA stem into a nanoparticle format, UFluA aims to induce bnAbs effective against both group 1 and group 2 influenza A viruses, offering broad cross-protection.
Another vaccine candidate, H1ssF, employs the stem domain from Influenza A/New Caledonia/20/1999 (H1N1) genetically fused to the ferritin protein from Helicobacter pylori. This design is intended to enhance the presentation of the stem region to the immune system, thereby inducing bnAbs targeting the stem. Initial results from a Phase 1 trial demonstrated that H1ssF generated an increased IC80 concentration in a pseudoviral neutralization assay against the homologous H1N1 A/New Caledonia/20/99 virus (NCT03814720), indicating promising immunogenicity.
The G1 mHA vaccine utilizes a ‘mini-HA,’ a stabilized form of the HA stem trimer [121]. Previous studies have shown that this design can induce stem-targeting antibodies against various group 1 viruses in non-human primates [122]. Currently, G1 mHA is undergoing a Phase 1/2 trial (NCT05901636) to further evaluate its efficacy and safety in humans.
Chimeric vaccines represent another innovative approach to induce stem-targeting bnAbs. These vaccines employ a prime-boost regimen, using vaccines with different HA head domains but a consistent stem domain to focus the immune response on the stem. GSK3816302A, a chimeric vaccine currently in Phase 1 trials (NCT03275389), incorporates cH8/1 N1, cH5/1 N1, and cH11/1 N1 constructs to elicit bnAbs targeting the H1 stem. Initial results indicate an increase in anti-H1 stem antibodies post-vaccination, with a statistically significant humoral immune response. Notably, increased antibody titres against H2 and H18 subtypes were also observed, suggesting potential cross-reactivity [123].
A specific subset of bnAbs target the HA stem region in its post-fusion form (Table 1). To induce these bnAbs, vaccines must present the stem in a non-native post-fusion conformation. Previous studies have demonstrated that vaccines designed to present this form can induce protective bnAbs in mice, offering cross-protection against mismatched influenza A strains [124]. A clinical trial evaluating a post-fusion hemagglutinin antigen is currently in the recruitment phase (NCT06460064).
In parallel, a universal influenza vaccine, M-001, which incorporates conserved epitopes from the M1 matrix protein, NP, and HA of both influenza A and B, has advanced to Phase 3 trials [125]. Despite initial promise, results from this trial indicated no statistically significant difference between control and vaccine groups regarding the prevention of influenza illness or reduction in symptom severity (NCT03450915). These results show that while inducing the production of bnAb’s against hemagglutinin is an attractive route forward for the formation of a universal influenza vaccine, many challenges exist for these vaccines, and continued research into this area is required.

6. bnAbs in Current and Future Directions

bnAbs present a promising avenue for the development of universal influenza vaccines and therapeutic interventions for influenza infections. However, bnAbs do have potential downfalls within these areas.
Although several bnAbs have been identified, thus far no bnAb possesses complete universality (Table 1 and Table 2). This limitation necessitates precise identification of the influenza strain prior to bnAb administration, which is not a common practice in clinical settings, reducing their practicality for routine treatment.

6.1. Escape Mutations

Influenza undergoes antigenic drift and shift over time, resulting in mutations within the HA protein. These mutations can restrict the effectiveness of certain bnAbs. For instance, an amino acid insertion at position 133 induces a localized structural change in the 130-loop of the HA head domain. This insertion is observed in 100% of all H6 and H10, ~ 95% of all H5 and ~ 70% of all H1 sequences. This alteration inhibits the activity of multiple RBS-targeting antibodies (Table 2), such as S139/1 and C05, as demonstrated by Ekiert and colleagues, and Lee and colleagues [127,128]. As a result, these antibodies may have reduced efficacy as therapeutic agents. Furthermore, the antibody FluA-20, which targets an epitope within the HA trimer interface, has already encountered escape mutations (Table 2). A mutation from an arginine to an isoleucine at position 229, one of the five major epitope residues, has already been seen in a H3 strain and rendered FluA-20 ineffective [71].
Although the stem region of HA is less prone to antigenic drift and shift, and therefore more conserved than the head region, escape mutations to stem targeting bnAb’s have also been identified (Table 1). Ekiert and colleagues identified two escape mutations for the fusion peptide targeting antibody CR8020 [24] which had entered clinical trials (Table 3), while Okuno and colleagues identified two escape mutations for the central stem targeting antibody C179 [9]. Although these mutations have not yet been seen in naturally occurring viruses, the potential for escape viruses from stem targeting antibodies is possible.
A further concern is the potential for vaccines and therapeutic bnAb’s that target regions susceptible to escape mutations to drive antigenic drift, leading to the emergence of strains resistant to such therapies. The generation of bnAb’s through universal vaccines, or the therapeutic use of bnAb’s could potentially induce mutations in currently conserved regions. Chai and colleagues determined that although Ser301, a key epitope residue at the centre of the 46B8-binding site, is highly conserved currently, a mutation to a phenylalanine allows viruses to evade neutralisation without compromising their fitness [95]. Park and colleagues were also able to determine that the presence of stem-targeting antibodies may result in mutant virus selection to escape the immune response. Within human challenge models, a mutant virus of A338V H1N1 2009 was preferentially selected for in participants with higher levels of pre-challenge anti-HA stem antibodies [35] .

6.2. Immunogenicity of bnAbs

bnMAbs and universal influenza vaccines in current clinical trials predominantly focus on antibodies targeting the HA stem. However, stem-targeting antibodies have demonstrated reduced immunogenicity compared to head-targeting antibodies [15]. The exact mechanisms behind this difference are not yet fully understood, though several explanations have been proposed.
Andrews and colleagues demonstrated that neutralizing stem antibodies exhibited weaker binding to whole influenza virions compared to head-targeting antibodies, despite showing equivalent binding to recombinant HA [129]. One proposed explanation for this is that stem-targeting antibodies face steric hindrance, as their binding site on the HA stem may be located near the viral membrane, potentially limiting accessibility (Figure 2). Additionally, the negative selection pressures exerted on B-cell clones producing stem-targeting antibodies may also play a role in their reduced immunogenicity. Stem-targeting antibodies are more prone to autoreactivity and polyreactivity, which could lead to immune tolerance or elimination of these B-cell clones. Andrews and colleagues identified that neutralizing stem-targeting antibodies exhibited higher levels of polyreactivity compared to non-neutralizing stem-targeting antibodies or head-targeting antibodies [129]. This observation was further supported by Bajic and colleagues who tested 12 bnAbs—six targeting the HA head and six targeting the stem. Of the stem-targeting antibodies, five displayed some degree of autoreactivity, whereas three head-targeting antibodies exhibited polyreactivity. Notably, CR6261, a stem-targeting bnMAb that had entered clinical trials for use as a therapeutic bnMAb (Table 3), showed both autoreactivity and binding to phospholipids, suggesting potential off-target interactions [130]. The presence of polyreactivity and autoreactivity in stem targeting bnAb’s raises concerns about their safety and efficacy as therapeutic agents. Polyreactivity may induce immune clearance mechanisms, such as B-cell anergy, and increase the risk of adverse effects due to off-target binding. These factors underscore the need for careful consideration of autoreactivity and polyreactivity in the development of bnMAbs, whether for therapeutic applications or as a foundation for universal influenza vaccines.

6.3. Antibody-Dependent Enhancement

The potential for antibody-dependent enhancement (ADE) of infection represents a critical concern in the development of bnAbs as therapeutic agents or as a foundation for universal influenza vaccines. ADE occurs when virus-specific antibodies facilitate, rather than inhibit, viral infection. This phenomenon arises when antibodies are present at subtherapeutic concentrations or function in a non-neutralizing manner, leading to increased disease severity. In such cases, antibodies may promote viral entry into host cells, thereby exacerbating infection, or contribute to the formation of excessive immune complexes, intensifying inflammatory responses [131,132,133,134] .
Non-neutralizing antibodies have been implicated in ADE during various viral infections, including the early stages of HIV-1 infection. Willey and colleagues demonstrated that during the acute phase of HIV-1 infection, non-neutralizing antibodies targeting the viral envelope can initiate complement-mediated ADE, resulting in heightened levels of infection [131]. Similarly, studies on influenza have shown that certain non-neutralizing antibodies can enhance disease outcomes. Winarski and colleagues reported that two non neutralising mAbs that bind to the head domain of HA caused enhanced disease in mice after viral challenge with H3N2. Interactions of these mAb’s with HA led to the destabilization of the HA stem domain, resulting in increased viral load and pathogenicity in mice [135]. Historical data further supports the role of non-neutralizing antibodies in severe influenza outcomes. During the 2009 H1N1 pandemic, middle-aged individuals with preexisting immunity to seasonal influenza exhibited higher rates of severe illness than expected. Monsalvo and colleagues identified the presence of cross-reactive, non-neutralizing antibodies in this population, which were considered to be linked to an increase in immune complex-mediated disease, with this population also having markers for complement activation via immune complexes. A similar mechanism was identified in fatal cases of the 1957 influenza pandemic [132]. These observations highlight the potential risks associated with non-neutralizing antibodies within pandemic influenza cases.
In light of these concerns, the potential for bnAbs to trigger ADE is being carefully studied due to the ongoing clinical trials which are investigating the use of bnAbs as a therapeutic intervention following influenza infection. Rao and colleagues examined the human monoclonal IgG1 bnMAb MHAB5553A, which targets a conserved epitope in the vestigial esterase domain of HA and exhibits strong antiviral activity against multiple influenza B strains in murine models [95]. Notably, MHAB5553A did not enhance pathogenesis in female DBA/2J mice within this study [134]. In conclusion, while bnAbs hold promise for influenza therapy and vaccine development, the risk of ADE must be thoroughly evaluated to ensure the safety and efficacy of these approaches. Ongoing research into the mechanisms of ADE in the context of bnAbs will be crucial for the advancement of universal influenza vaccines and antibody-based therapies.

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Figure 1. Steps in Hemagglutinin Endosomal Collapse. The process begins when hemagglutinin (HA) binds to sialic acid on the host cell surface, facilitating viral endocytosis. In the endosome, HA initially exists in its a) prefusion state and is bound to sialic acid (not shown). Upon endosomal acidification, HA releases the bound sialic acid and undergoes a conformational change to its b) extended intermediate state, allowing the fusion peptide (shown in red) to insert into the endosomal membrane. The c) extended intermediate state collapses, and HA undergoes a "jackknife" motion (c1 → c2), drawing the viral and endosomal membranes together. As membrane fusion occurs, HA adopts its d) post-fusion state, forming a fusion pore that facilitates the release of viral genetic material into the cytoplasm.
Figure 1. Steps in Hemagglutinin Endosomal Collapse. The process begins when hemagglutinin (HA) binds to sialic acid on the host cell surface, facilitating viral endocytosis. In the endosome, HA initially exists in its a) prefusion state and is bound to sialic acid (not shown). Upon endosomal acidification, HA releases the bound sialic acid and undergoes a conformational change to its b) extended intermediate state, allowing the fusion peptide (shown in red) to insert into the endosomal membrane. The c) extended intermediate state collapses, and HA undergoes a "jackknife" motion (c1 → c2), drawing the viral and endosomal membranes together. As membrane fusion occurs, HA adopts its d) post-fusion state, forming a fusion pore that facilitates the release of viral genetic material into the cytoplasm.
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Figure 2. Approximate Locations of Stem Epitopes in Representative Influenza A Group 1 (A/South Carolina/1/1918(H1N1), PDB: 1RUZ), Influenza A Group 2 (A/Hong Kong/1/1968(H3N2), PDB: 4WE4) and Influenza B (B/Hong Kong/8/73, PDB: 3BT6). The central stem (CS) epitope (pink) and fusion peptide (cyan H3N2) or fusion peptide and anchor epitope (cyan H1N1) are in the stem. Conversely the RBS (blue), VE (green), lateral patch (yellow) are situated in the head domain. The occluded epitope and the interface epitope (orange) are marked in orange on a single rotated representative H3N2 monomer.
Figure 2. Approximate Locations of Stem Epitopes in Representative Influenza A Group 1 (A/South Carolina/1/1918(H1N1), PDB: 1RUZ), Influenza A Group 2 (A/Hong Kong/1/1968(H3N2), PDB: 4WE4) and Influenza B (B/Hong Kong/8/73, PDB: 3BT6). The central stem (CS) epitope (pink) and fusion peptide (cyan H3N2) or fusion peptide and anchor epitope (cyan H1N1) are in the stem. Conversely the RBS (blue), VE (green), lateral patch (yellow) are situated in the head domain. The occluded epitope and the interface epitope (orange) are marked in orange on a single rotated representative H3N2 monomer.
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Table 1. Overview of Extensively Studied Stem-Targeting Broadly Neutralizing Antibodies (bnAbs). This table provides details on some of the most thoroughly researched stem-targeting bnAbs, adding to previously published work [17]. It includes information on their in vitro binding affinity, in vitro neutralization capacity, and in vivo protective efficacy. The table also lists the immunoglobulin heavy-chain variable region (IGHV) gene used, the primary complementarity-determining region (CDR) recognition mode, and whether the antibody exhibits antibody-dependent cellular cytotoxicity (ADCC) as a significant protection mechanism. Additionally, it specifies whether the antibody was isolated from mice or humans, any known escape mutations, and the IgG subtype used in generating the findings. A – is shown if the information was not provided.
Table 1. Overview of Extensively Studied Stem-Targeting Broadly Neutralizing Antibodies (bnAbs). This table provides details on some of the most thoroughly researched stem-targeting bnAbs, adding to previously published work [17]. It includes information on their in vitro binding affinity, in vitro neutralization capacity, and in vivo protective efficacy. The table also lists the immunoglobulin heavy-chain variable region (IGHV) gene used, the primary complementarity-determining region (CDR) recognition mode, and whether the antibody exhibits antibody-dependent cellular cytotoxicity (ADCC) as a significant protection mechanism. Additionally, it specifies whether the antibody was isolated from mice or humans, any known escape mutations, and the IgG subtype used in generating the findings. A – is shown if the information was not provided.
Name In vitro Binding In vitro Neutralisation In vivo Protection Germline Encoded IGHV CDR Recognition Mode ADCC activity Source Escape Mutants IgG-type in Studies Ref
Central Stem C179 H1, H2, H5, H6, H9 H1, H2, H5, H6, H9 H1, H5 - - Yes Mouse T332K,
V395E *		 IgG2a [9,27,28]
27F3 H1, H2, H5, H6, H9, H11, H12, H13, H16, H3, H7, H10, FluB H1, H5, H6, H3, H7, H10 - IGHV1–69 CDRH2 - Humans - IgG1 [29,30]
FI6 H1-H16 H1, H5, H3, H7 H1, H5, H3 IGHV3–30 CDRH3
CDRL1		 Yes Humans R62K, D239G, R240Q T333K, A388T ° - [21,22,23,31,32]
CR6261 H1, H2, H5, H6, H8, H9 H1, H2, H5, H6, H8, H9 H1, H5 IGHV1–69 CDRH2 Weak Humans A388V IgG1 [18,30,33,34,35]
CR6323 H1, H2, H5, H6, H8, H9 H1, H2, H5, H6, H8, H9 - IGHV1–69 HCDR2 - Humans H357L/T* IgG1 [34]
09-2A06 H1 H1 - IGHV1–69 - - Humans - - [36]
09-3A01 H1 H1 - IGHV4–39 - - Humans - -
05-2G02 H1, H3, H5 H1, H3, H5 - IGHV1–18 - - Humans - -
A06 H1, H5 H1, H5 H1 IGHV1–69 - - Humans - IgG1 [37]
39.18 H1, H2 H1, H2 - IGHV1–69 - - Humans - - [38,39]
39.29 H1, H2, H3 H1, H2, H3 H1, H3 IGHV3-30 CDRH3 - Humans G387K, D391Y/G -
81.39 H1, H2, H3 H1, H2, H3 - IGHV3-15 - - Humans - -
36.89 H3 H3 - IGHV1–18 - - Humans - -
FE43 H1, H5, H6, H9 H1, H5, H6, H9 H1, H5, H6 IGHV1–69 - - Humans None found IgG1 [40]
FB110 H1, H2, H5 H1, H2, H5 - IGHV3-23 - - Humans None found IgG3
3Е1 H1, H5, H9, H3, H7 H1, H5, H9, H3, H7 H1, H5 IGHV4-4 Mostly Heavy Chain - Humans - IgG1 [41]
CT149 H1, H5, H9, H3, H7 H5, H9, H3, H7 H1, H5, H3, H7 IGHV1–18 CDRH3CDRH2 Yes Humans - IgG1 [42]
31.a.83 H1, H2, H5, H9, H3, H7 H1, H2, H5, H9, H3, H7 - IGHV3–23 Mostly CDRH3
CDRH2		 - Humans - - [43]
56.a.09 H1, H5, H3, H7 H1, H5, H3, H7 - IGHV6–1 Mostly CDRH3
CDRH2		 - Humans - -
CR9114 H1, H2, H5, H6, H8, H9, H12, H13, H16, H3, H4, H7, H10, H15, FluB H1, H2, H5, H6, H8, H9, H12, H3, H4, H7, H10 H1, H2, H3, H5, H9, FluB IGHV1–69 CDRH2 Weak Humans R62K, D239G, R240Q, L335V, D363G, A388T ° IgG1 [30,31,33,44,45]
F10 H1, H2, H5, H6, H8, H9, H11, H13, H16 H1, H2, H5, H6, H8, H9, H11 H1, H5 IGHV1–69 CDRH2 Yes Humans N460, S123, E190D+G225D, N203VHA + E329KNA* IgG1 [19,30,32,46]
MEDI8852 H1-H18 H1, H2, H5, H6, H9, H3, H7 H1, H5, H3 IGHV6-1 CDRH2
CDRH3
CDRL1		 Yes Humans - IgG1 [47,48]
CR9117 Mouse homologue of CR9114, presumed to have similar neutralization capacity - Yes Mouse - IgG2a [33]
Anchor Domain Polyclonal response (FISW84 / 222-1C06 were named) H1, H2, H5 H1, H2, H5 H1 IGHV3-23
IGHV3-30
IGHV3-30-3
IGHV3-48		 CDRk3
CDRH2
CDRH3		 No Humans - IgG1 [26]
Fusion Peptide CR8020 H3, H4, H7, H10, H14, H15 H3, H7, H10 H3, H7 IGHV1–18 CDRH1
CDRH3		 Weak Humans D372N, G376E * IgG1 [20,25,49,50]
CR8043 H3, H4, H7, H10, H14, H15 H3, H7, H10 H3, H7 IGHV1–3 CDRH1
CDRH3		 - Humans R378M, Q380R/T * IgG1 [25,50]
9H10 H3, H9 H3, H10 H3 - - - Mice R378M
T385R
Q387R/T
G386E *		 - [50]
°=numbering from methionine, *=H3 numbering.
Table 3. Table of stem-targeting bnMAb’s currently undergoing clinical trials for use in influenza infection. Information about the type of antibody, target, dose regime and results were taken from the corresponding study record listed on the NIH clinical studies site.
Table 3. Table of stem-targeting bnMAb’s currently undergoing clinical trials for use in influenza infection. Information about the type of antibody, target, dose regime and results were taken from the corresponding study record listed on the NIH clinical studies site.
Name Type and Target Dosage/ Infection Model Result Trial Registry ID/ Reference
CT-P27 CT-120 & CT-149 mAb’s targeting the stem region of group 1 and group 2 influenza hemagglutinin 10 mg/kg CT-P27, 20 mg/kg CT-P27, or placebo in an influenza challenge model Reduction of AUC of Viral Load, as measured by Quantitative PCR of Nasopharyngeal Swab for patients who received CT-P27 NCT02071914, [105]
90 mg/kg CT-P27, 45 mg/kg CT-P27, or placebo NCT03511066 was terminated due to CT-P27 inactivation NCT03511066.
MEDI8852 Human IgG1 kappa monoclonal antibody (MAb) targeting H1N1 and H3N2 viruses, as well as subtypes such as H2, H5, H6, H7, and H9 via the stem region 750 mg or 3000mg MEDI8852 given with oseltamivir or 3000 mg MEDI8852 on its own to patients with acute, uncomplicated influenza caused by Type A strains. MEDI8852 provided no statistically significant improvement over oseltamivir alone, potentially worsened disease in combination compared to oseltamivir alone NCT02603952,[116]
Low dose and high dose of MEDI8852 and oseltamivir in comparison to oseltamivir and placebo Withdrawn due to company decision NCT03028909
VIS410 Human immunoglobulin IgG1 monoclonal antibody engineered to bind to the stem region of group 1 and 2 influenza A hemagglutinins Influenza challenge with H1N1 followed by a single administration of VIS410 or placebo No results posted NCT02468115, [117]
2000mg or 4000mg VIS410 was given to patients with uncomplicated influenza A infection and compared to a placebo Statistically significant improvement in signs and symptoms of influenza infection on day 3 and 4 with VIS410 compared to placebo. Statistically significant reduction in time to resolution of peak viral load when patients were given VIS410. NCT02989194, [118]
3600 mg or 8400 mg VIS410 combined with oral oseltamivir or placebo with oseltamivir in patients hospitalised with influenza A infection No statistically significant reduction in time to cessation of oxygen, or reduction of viral load in nasopharyngeal samples NCT03040141
MHAA4549A Human monoclonal antibody, IgG1, targeting the influenza A virus hemagglutinin stem across multiple subtypes Influenza challenge with H3N2 influenza virus followed by a dose of 400 mg, 1200 mg or 3600 mg Statistically significant reduction in AUC of virus in nasopharyngeal samples was seen at 3600mg compared to placebo. Influenza symptom scores, mucus weight, and inflammatory biomarkers were also reduced. NCT01980966
3600mg or 8400mg given either on its own or with oseltamivir to patients hospitalised with severe influenza infection MHAA4549A did not improve clinical outcomes over OTV alone. MHAA4549A+OTV did not further reduce viral load versus placebo+OTV.MHAA4549A did not alleviate symptoms quicker than a placebo. NCT02293863, [119]
3600mg or 8400mg given to patients with uncomplicated seasonal influenza A infection 3600mg dose was able to statistically reduce the number of days to alleviate symptoms compared to the control NCT02623322
CR8020 A mAb targeting the stem region of group 2 influenza A hemagglutinin 15 mg/kg CR8020 given before challenge with a H3N2 influenza virus. No results NCT01938352
CR6261 mAb that targets the stem region of group 1 and group 2 influenza hemagglutinin 50 mg/kg administered one day after challenge with H1N1 Statistically reduced percentage of participants who experienced influenza symptoms. No statistically significant reduction in AUC or viral shedding. NCT02371668, [120]
CR8020/ CR6261 Withdrawn due to preliminary efficacy results from an influenza challenge trial NCT01992276
Table 4. Table of broadly protective vaccines and vaccine antigens targeting the hemagglutinin currently undergoing clinical trials for use in influenza prevention.
Table 4. Table of broadly protective vaccines and vaccine antigens targeting the hemagglutinin currently undergoing clinical trials for use in influenza prevention.
Phase Name of vaccine Target/ Type of vaccine Dosage/ Infection model Results Trial Registry ID/ Reference
Recruiting fH1/DSP-0546LP Post-fusion hemagglutinin antigen Combination of 2 dose levels of fH1 (2 and 8 μg), 3 dose levels of DSP-0546LP (2.5, 5, and 10 μg), and placebo. Each dose level of fH1 will be combined with the low, medium, and high dose level of DSP-0546LP to assess safety, tolerability, and immunogenicity Active NCT06460064, [124]
Phase 1 EBS-UFV-001 Induction of antibodies against conserved stem antigens across group 1 and 2 via a hemagglutinin stabilized stem nanoparticle vaccine Testing the safety, tolerability and immunogenicity of 20 µg or 60 µg of UFluA as single dose or as two dose No results posted NCT05155319, [126]
H1ssF HA stem domain from Influenza A/New Caledonia/20/1999 (H1N1) genetically fused to the ferritin protein from H. pylori. 20 mcg was given to group 1, group 2 received 60 mcg on a prime boost schedule. All regimes generated an increased IC80 concentration when tested in a pseudoviral neutralization assay against the homologous H1N1 A/New Caledonia/20/99 virus NCT03814720
GSK3816302A Chimeric vaccines of D-SUIV cH8/1 N1, D-SUIV cH5/1 N1, and D-SUIV cH11/1 N1 to induce cross reactive stem targeting antibodies against H1 stem Chimeric H5, H8 and H11 with and without adjuvants AS03 or AS01 were tested for their reactogenicity, safety and immunogenicity. H8 and H5 were given with a placebo second dose, or all three were given. An increase in anti H1 stem antibodies, as measured by ELISA and MN assay, was seen across all dose schedules with adjuvant AS03 providing a statistically significant increase in humoral immune response for anti-H1 stem antibody by ELISA at Day 29 and Day 85. Increases in antibody titres against H2 and H18 were also identified. NCT03275389, [123]
Phase 1/2 G1 mHA Mini-hemagglutinin stem-derived protein vaccine antigen Single dose of influenza G1 mHA with or without Al(OH)3 adjuvant at two dose levels to evaluate safety, reactogenicity and immunogenicity Active NCT05901636, [121,122]
Phase 3 (M-001) A recombinant 45 kDa protein produced in Escherichia coli. consisting of three repetitions of nine linear, conserved influenza A and B epitopes to form a single recombinant protein. Epitopes were derived from: M1 matrix protein, NP and HA Vaccination with 1mg dose of M-001 twice: Once at Day 0, and once at Day 21 then followed for 2 years No statistical difference in prevention of influenza infection. Did not statistically reduce the number of patients with influenza like symptoms, or a reduction of severity of either qRT-PCR or culture-confirmed influenza illness NCT03450915, [125]
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