Immunological considerations for developing antibody therapeutics for Influenza A

Abstract

Influenza infection can give rise to serious illness leading to complications and hospitalization of patients. The efficacy of current standard of care is very limited and provides little relief for patients hospitalized with serious flu. Human monoclonal antibodies (mAb) against influenza are being developed as new treatment options for this patient population. When developing antibody therapeutics, it is important to consider all possible immunologic effects of the antibodies on viral infection and disease progression including those other than the postulated therapeutic mechanisms. An area of concern is the potential of antibody-dependent enhancement (ADE) of illness. ADE of viral infections has been extensively described for Dengue virus (DENV) but not for influenza. Recently, preliminary results from clinical viral challenge studies of anti-HA-stalk mAbs suggested the possibility of enhanced viral shedding, raising concerns for ADE when utilizing mAbs as therapeutic intervention for influenza although viral shedding was not enhanced in the clinical viral challenge of anti-M2 mAb TCN-032. We herein discuss the known mechanisms of ADE and their relevance to developing mAbs such as anti-HA and anti-M2 for influenza disease.

Keywords:

• antibody dependent enhancement
• antiviral
• clinical development
• Hemagglutinin HA
• Influenza
• matrix protein M2
• monoclonal antibodies
• therapeutics

Abbreviations

mAb	=	monoclonal antibodies
ADE	=	antibody-dependent enhancement
DENV	=	dengue virus
RSV	=	respiratory syncytial virus
M2	=	matrix protein 2
M2e	=	matrix protein 2 ectodomain
HA	=	hemagglutinin
ADCP	=	antibody-dependent cellular phagocytosis.

Influenza is a highly communicable acute respiratory disease and can be a serious health threat of epidemic and in instances pandemic proportions. Children, the elderly and immune compromised individuals are at the greatest risk of clinical deterioration particularly in the presence of additional risk factors such as immunosuppression, chronic respiratory conditions and co-morbidities. Annual vaccination may not adequately provide protection; epidemic strains may drift from vaccine strains and the natural immune response of the high risk patient population may not be as effective as in healthy adults. Recently reported incidences of narcolepsy upon administration of certain flu vaccines have added to concern over vaccinations.¹ For seasonal, uncomplicated flu, the current standard of care is the oral administration of oseltamivir. It inhibits viral neuraminidase activity and reduces virus shedding from infected cells but the therapeutic window for oseltamivir is very narrow with less than 48 hours upon onset of symptoms.^2-4 It has been reported that oseltamivir reduces the rate of hospital admissions among adults and adolescents.⁵ However, the efficacy of oseltamivir in disease alleviation has been subject to controversy as the meta-analyses findings vary depending on the randomized controlled trials, the patient populations and the disease severity included in the analyses.^6-8 For patients admitted to hospitals with severe influenza, the disease can progress with uncontrolled viral replication and systemic cytokine storm accompanied by lung tissue damage;⁹ the use of corticosteroids or single inflammatory mediator antagonists to undermine the cytokine storm has not demonstrated any clinical efficacy.¹⁰ Practically, therapeutic treatment options for hospitalized patients diagnosed with serious flu are lacking which puts the development of efficacious drugs specifically for this patient population at a level of utmost importance. In recent years, much effort has been invested in developing new therapeutic candidates to reduce viral titers with mechanisms of action alternative to oseltamivir and a growing list of antibodies are at different stages of clinical development.

Human monoclonal antibodies (mAbs) have been clinically proven as safe therapeutics for multiple disease categories including viral disease. For viral infections, the host humoral response can be an important protective mechanism working in concert with the innate and adaptive cellular immune response. Antibodies raised as part of the humoral response can directly neutralize virus and mediate killing of virus and infected cells via Fc effector functions.^11-13 Palivizumab is the only anti-viral mAb currently on the market for respiratory syncytial virus (RSV) prophylaxis. It has significantly reduced the rate of RSV hospitalization of premature infants and infants with chronic lung disease or congenital heart disease.¹⁴ The use of palivizumab is clinically safe without any causal link between treatment and serious or fatal adverse effects; in principle, antibody therapeutics can also be safe components of treatment regimen for other respiratory diseases including serious hospitalized influenza. To date, mAbs in development to combat severe influenza A have advanced through early stage clinical evaluation.

The therapeutic antibody candidates in development for severe influenza A either target a highly conserved epitope on the N-terminal ectodomain of M2 (M2e)¹² or the stalk region of hemagglutinin (HA) common to all influenza A viruses. In contrast to the anti-HA-stalk mAbs, TCN-032 (anti-M2e) does not neutralize influenza virus; instead TCN-032 mediates the killing of infected cells.¹² TCN-032 has successfully completed a seasonal influenza viral challenge phase 2 study in healthy human subjects (Theraclone-Sciences, NCT01719874), and has demonstrated reduction in clinical symptoms and viral shedding when dosed 24 hours post challenge.¹⁵ No adverse effects or evidence of enhanced infection are detected. At least one viral challenge study testing an anti-HA stalk mAb has been completed (Crucell; CR8020, NCT01938352), another is ongoing (Visterra, VIS410, NCT02468115). Anti-HA-stalk MHAA4549A is being tested in combination with oseltamivir versus oseltamivir alone in hospitalized patients with severe influenza A (Genentech, NCT02293863). Clinical testing results on any anti-HA-stalk mAbs have not been published as of today. However, in contrast to the clinical viral challenge data for TCN-032, preliminary results from viral challenge phase 2 studies of anti-HA-stalk mAbs presented at the PAHO/WHO meeting held in Washington DC in November 2014 suggest the possibility of enhanced viral shedding in some treated human subjects. This unexpected clinical observation has led to speculations that mAbs, depending on dosing regimen and possibly the influenza challenge strain, may mediate antibody-dependent enhancement (ADE) of influenza infectivity potentially leading to disease exacerbation and representing a significant safety risk when treating patients hospitalized with serious flu.

The potential association of ADE with the innate and adaptive immune response has been described in the context of some viral diseases. For example, ADE reported in secondary infections of Dengue virus (DENV) is mediated by non-protective antibodies that were generated over the course of a prior primary heterosubtypic DENV infection; antibodies elicited against chronic infection of rapidly evolving HIV viruses may mediate ADE because they cannot neutralize the new viral quasispecies; and poorly affinity matured antibodies elicited by formalin-fixed inactivated virus vaccines such as RSV and measles have induced ADE in infants during primary infection. The ADE underlying mechanisms triggered by DENV and formalin-fixed inactivated RSV vaccine have been extensively studied.¹⁶ DENV antibodies enhance viral entry into macrophages and other myeloid cells via binding to Fcγ receptors leading to viral amplification and subsequent suppression of the innate antiviral signaling by infected cells. Low avidity non-protective antibodies elicited through formalin-fixed RSV vaccination of infants form immune complexes upon RSV infection leading to complement activation, deposition in lung and subsequent pulmonary tissue injury. As for influenza, the potential of ADE playing a role in exacerbation of disease has not been explored until recently.

Fcγ receptors mediated viral infection of myeloid cells described for DENV is one of the best-studied mechanisms of ADE.^17,18 This ADE mechanism however has not been described for influenza infection even though alveolar macrophages are relatively abundant in the lung. Influenza A has a broad cell tropism and severe seasonal or pandemic influenza A infections that lead to pneumonia often involve the lower respiratory tract and alveoli where multiple cell types support influenza viral replication. If left uncontrolled, infection of the lower airway and alveolar cells can cause loss of functions and contributes directly to development of pneumonia.¹⁹ Unlike other infected cell types and with the exception of high pathogenicity avian flu and 1918 pandemic H1N1, infection of alveolar macrophages by influenza is abortive, i.e. no viral progeny is produced;²⁰ it is unlikely that resident alveolar macrophages support Fcγ receptor mediated ADE. At an early stage of disease, infection of the alveolar macrophages can play a protective role by inducing interferon to limit viral replication and to attenuate the subsequent cytokine production by infected epithelial cells and leukocytes that are recruited to the lung in the later stage of infection.^21,22 At late stages of disease, excessive influenza infection of resident alveolar macrophages leads to their depletion and reduces their normal function of viral clearance by phagocytosis and antibody-dependent cellular phagocytosis (ADCP) which is critical for control of viral titers and disease resolution.^23,24 It is thus important for a mAb therapeutic to reduce virus titer in a relatively early stage of infection to avoid depletion of alveolar macrophages in order to effect disease resolution.

During the 2009 pandemic of H1N1 influenza A, a significant number of otherwise healthy young to middle-age influenza infected adults without any known risk factors or pre-existing co-morbidities developed severe infection including, in some cases, respiratory failure. A study of a representative 2009 patient population revealed that the presence of high serum antibody titers against pandemic H1N1 virus in severely ill patients was concomitant with immune complex formation of low avidity, non-protective antibodies and in fatal cases, the deposition of complement C4d in the lung.²⁵ The immune repertoire of these patients had been shaped by seasonal circulating influenza strains; they had preexisting serum antibodies that cross-reacted with the 2009 pandemic influenza strain but did not protect against it. In contrast, very young children had none or only very little prior exposure to any influenza strains; and the elderly may had developed protective immune memory to the H1N1 influenza strains circulating prior to 1957. These observations are reminiscent of reports of pulmonary immune complex deposition found in the fatal cases of infected infants following vaccination with formalin-fixed inactivated RSV.²⁶ Moreover, during the 2009 H1N1 pandemic, a significantly higher proportion of severely ill patients had functionally impaired alleles of the complement inhibitory receptor CD55 when compared to patients with mild infection, further corroborating the contributory pathogenic role of excessive complement activation by low potency and low avidity non-protective antibodies in the lung.²⁷ While the risk contributed by pathogenic immune complex formation remains unclear, successful clinical development of mAb therapeutics for influenza will likely involve testing high potency protective antibodies and defining the optimal dose range before treating patients hospitalized with severe influenza. Likewise, development of universal vaccines will have to aim at eliciting high potency protective antibodies to mitigate the risk of enhanced disease progression.

Protective antibodies against HA-stalk present a unique mechanism of ADE of influenza infection that has not been described for other viral infections or other classes of antibodies. All current anti-HA-stalk clinical candidates target a common epitope region closely located to residues on the HA2 subunit that functionally mediates fusion of the viral and host cell membranes prior to transfer of viral genome into the host cell cytoplasm. In vitro analysis of these mAbs shows that their mechanism of action to neutralize influenza infection is based on blocking viral entry into cells. Consequently and due to the highly conserved nature of this epitope region, vaccines have been designed to specifically elicit antibodies against this epitope region; when tested in mice and ferrets, such vaccines have demonstrated prophylactic efficacy against challenge by heterologous viruses.^28-30 However, in a recent study, piglets immunized by UV irradiation inactivated H1N2 influenza developed enhanced respiratory disease upon challenge with a pandemic H1N1 virus. Specifically, the enhanced severity was caused by the predominant non-protective anti-HA-stalk antibodies that caused enhanced viral fusion and increased viral entry into host cells, whereas protective neutralizing antibodies against the challenge virus were absent.³¹ The finding of this particular study has raised concern and has questioned efforts of this approach of universal influenza vaccine development. Interestingly, despite the fact that both piglets and ferrets share various influenza disease characteristics with humans, a similar enhancement of disease has not been described for ferrets treated with HA-stalk based vaccines. It remains unclear to what extent the study in piglets will translate into humans. Until fully understood and considering the preliminary results of enhanced viral shedding in the recent viral challenge phase 2 study, the potential of ADE induced specifically by this class of anti-HA stalk therapeutics will certainly be closely watched; future development will require the mitigation of risk of disease enhancement and may have to take into consideration potential antibody-associated effects to ensure a safe treatment regimen. An optimal dose of high potency protective mAb may be able to tip the delicate balance between effective neutralization and enhanced fusion toward therapeutic efficacy.

Current clinical candidates such as TCN-032 (anti-M2e) and multiple versions of anti-HA (VIS410, CR8020, MHAA4549A and others) provide the opportunity to advance the development of therapeutic options for severe hospitalized influenza A. TCN-032 in particular, by targeting M2 that is abundantly expressed on influenza infected cells, likely reduces viral shedding by using alveolar macrophages for ADCP which has been shown to be a critical protective mechanism against influenza infection in mice treated with a M2-based vaccine.²⁴ In conjunction with other Fc-mediated effector mechanisms such as antibody-dependent cytocytoxicity and complement-dependent cytotoxicity,^12,32 TCN-032 may provide alternative universal therapeutic mechanisms of action against all influenza A strains without involving the HA-stalk.

With continuous generation of clinical safety and efficacy data on anti-influenza mAb therapeutics, we envision that mAb therapeutics will eventually provide an important improvement in treating severe hospitalized influenza A disease by reducing viral titers at the optimal dose without forming deleterious immune complex or causing enhanced infection. Therapeutic cocktails of more than one mAb with different antigen target specificities and mechanisms of action will also have the potential for synergy in clinical efficacy and risk mitigation.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.