Influenza Viruses: Targetting Conserved Viral Ha-Stem, Matrix and Nucleo-Proteins to Disarm a Resilient and Recurring Pandemic

Abstract

Much to the current worldwide pandemic caused by the SARs-Cov-2 virus, common flu caused by Influenza virus remain a long-standing mayhem to global health. Influenza viruses are important human pathogens responsible for substantial seasonal and pandemic morbidity and mortality. Despite the efficiency of widely available antiviral neuraminidase (NA) inhibitor drugs, and multiple formulations of the influenza vaccines, including inactivated influenza vaccines (IIV); a recombinant inactivated vaccine (RIV); and a live, attenuated influenza vaccine (LAIV), Influenza virus infection still remains an ongoing health and economic burden causing epidemics with pandemic potential keeping scientist on their toes in researching to combat the complexity often associated with the pathogenesis of these viral infection and perhaps its associated genetics. Most recent strides and advances within the global research landscape has seen efforts channeled towards the discovery and production of universal vaccines in a bid to address the unique challenge associated with the multiple viral strain explosion often encountered with influenza viruses. An important strategy for accomplishing this is to provoke an immune response to the virus’s “Achille’s heel”, i.e., conserved viral proteins, through targeting the hemagglutinin (HA) glycoprotein or protein domains shared by seasonal and pre-pandemic strains.

1. Introduction

Influenza viruses are RNA viruses that cause infectious respiratory diseases that are majorly characterized by fever, congestion, and myalgia, which ranges in severity from mild to life-threating, and they are estimated to cause about 250,000 to 500,000 deaths globally per year [1]. They are single-stranded, helically shaped, and belongs to the orthomyxovirus family consisting of 5 influenza virus genera, ranging averagely from 80 to 120 nm in size [2]. They often contain 8 gene segments that encodes 11 proteins (Figure 1). These segments encode viral proteins including hemagglutinin (HA), neuraminidase (NA), nonstructural 1 (NS1), NS2, matrix 1 (M1), M2, nucleoprotein (NP), nuclear export protein (NEP), polymerase acid (PA), polymerase basic 1 (PB1) and PB2 [3]. Influenza viruses are uniquely known to express spike glycoproteins such as hemagglutinin (HA) which facilitates viral recognition of host receptor binding site and neuraminidase (NA) which also aids viral release after replication within the host cells [2, 4]. HA binds Sialic acid bonded with galactose, in avian influenza (H5N1) affinity binding occurs with the α-2,3 sialic acid galactose receptor complex of birds in contrast with the α-2,6 binding in human Influenza virus A infections [1, 4, 5].

Figure 1.

Showing all the eight gene segments and encoded proteins of influenza A virus. Influenza virus’s genome is eight-segmented and encodes for two surface glycoproteins which includes neuraminidase (NA) and hemagglutinin (HA); matrix protein 2 (M2) ion channel that are securely buried into the viral lipid envelope; matrix protein 1 (M1) which lies beneath the membrane; protein-basic protein (PB1, PB2) protein-acidic protein (PA) which makes up the RNA polymerase complex that is associated with the encapsilated genome; nucleoprotein (NP) which coats the viral genome and nonstructural proteins (NS1 and NS2) which suppresses host cell’s mRNA production and serves as interferon antagonism.

Till date, three types of influenza virus have been known to cause infection in humans: A, B, and C. Type A influenza has subtypes determined by the surface antigens hemagglutinin (HA) and neuraminidase (NA). There are 18 different H subtypes and 11 different N subtypes. Eight H subtypes (H1, H2, H3, H5, H6, H7, H9, H10) and six N subtypes (N1, N2, N6, N7, N8, and N9) have been detected in humans. Type B influenza is classified into two lineages: B/Yamagata and B/Victoria [2]. Influenza B commonly affects children while Influenza C is rarely reported as a cause of human illness, which is probably because most cases are subclinical. Influenza C has still not been associated with any epidemic disease outbreak so far. WHO currently classifies influenza A(H1N1) and A(H3N2) as circulating seasonal influenza A virus subtypes, while also classifying avian influenza virus subtypes A(H5N1) and A(H9N2) and swine influenza virus subtypes A(H1N1) and (H3N2) as zoonotic or variant influenza [2, 6].

Enormous efforts are currently aimed at preventing and treating influenza infections, including seasonal and pandemic influenza, however, outbreaks still remain a major public health challenge globally [1, 4]. This is majorly due to influenza viruses rapidly undergoing genetic mutations that restrict the long-lasting efficacy of vaccine-induced immune responses and therapeutic regimens [1]. These major viral genetic changes involve Antigenic Drift, which is caused by point mutations in genes encoding HA and N glycoproteins spikes thereby allowing for viral immune invasion against host responses and generated antibodies like vaccines. Similarly, antigenic shift which occurs in influenza virus A, caused by viral genome reassortment/swapping mechanisms among two different subtypes of influenza A which are replicating within the same host causing a jump to new species of host, and a highly diverse structure of virus able to cause the occasional pandemics seen in the world [2]. A combination of antiviral agents and vaccines remains the general prevention and treatment measures for influenza-related morbidity and mortality, however complications arising from viral genetic changes has bolstered scientific efforts on a journey to the discovery of universal vaccines.

2. Pathophysiology

Following respiratory transmission, human influenza virus attaches to and penetrates the respiratory epithelial cells in the trachea and bronchi. Other cell types often affected in the respiratory tract includes several immune cells, which can be infected by the virus and initiate viral protein production. However, the efficiency of replication varies among various affected cell types, and, in humans, the respiratory epithelium is the only site where the hemagglutinin (HA) molecule is effectively cleaved [5, 7]. The primary mechanism of influenza pathophysiology is a result of lung inflammation and compromise that is caused by direct viral infection of the respiratory epithelium, combined with the effects of lung inflammation also caused by immune responses recruited to handle the spread of the virus [7]. Influenza-mediated respiratory tract damage is caused by a combination of events, including: a) intrinsic viral pathogenicity due to its affinity for host airway and alveolar epithelial cells; and b) a robust host innate immune response, which, while aiding in viral clearance, can aggravate the severity of lung injury [7].

The host cell is then destroyed as a result of viral replication. Viremia, or the presence of a virus in the blood, has, on the other hand, is seldomly observed and never widely documented. Virus is released in respiratory secretions for 5 to 10 days, peaking 1 to 3 days after disease start [5, 7]. Inflammation caused by influenza pathogenic events can extend systemically and appear as multiorgan failure, the most common of which are lung compromise and severe respiratory distress [8]. Some links have also been found between influenza virus infection and cardiac complications, such as an increased risk of myocardial illness in the weeks following infection. Beyond the basic inflammatory profile, several of these processes remain uncertain [9, 10]. Researchers find it theoretically helpful to divide the progression of IAV infection into three stages, with the idea that many of these processes occur concurrently throughout the injury. The first is viral infection and replication in the airway and alveolar epithelium, during which methods that restrict viral entrance or replication might prevent or reduce the severity of the infection. The innate immune response to the virus is followed by the adaptive immunological response, which is crucial for viral clearance but may also cause severe damage to the alveolar epithelium and endothelium. The third step is the establishment of long-term immunity to the infecting virus strain, which is followed by the resolution of infiltrates and regeneration of damaged lung tissue, during which time the patient is more vulnerable to secondary bacterial infection [4, 5, 7].

2.1 Acute respiratory distress syndrome

The influenza viruses are significantly important human pathogens. In humans, infection of the lower respiratory tract of can result in flooding of the alveolar compartment, development of acute respiratory distress syndrome and death from respiratory failure. The extent to which the virus infiltrates the lower respiratory tract is an important factor in determining the degree of associated disease complications [8]. Infection of alveolar epithelial cells appears to cause the development of severe illness by damaging important mediators of gas exchange and permitting viral exposure to endothelial cells. Early interactions between the influenza virus, alveolar macrophages in the lung airways, and the epithelial lining are significant determinants of alveolar disease development [9]. Once this delicate barrier is penetrated, cytokine and viral antigen exposure to the endothelium layer can exacerbate inflammation, with endothelial cells being a primary source of pro-inflammatory cytokines that influence the amount and nature of future innate and adaptive immune responses [10].

In the final pathological stages, just like in the SARS-CoV-2 infection, where reports from Lancet on COVID-19 pathogenesis reveals that acute respiratory distress syndrome (ARDS) is the main cause of death in most patients [11, 12, 13, 14], influenza virus infection also initiates hypoxia and progression to ARDS [15]. ARDS is majorly experienced as shortness of breath and it’s also a common immunopathological event in SARS-CoV and MERS-CoV infections [11]. Clinically, severe Influenza A Virus infection can cause bilateral lung infiltrates and hypoxaemia, which are symptoms of acute respiratory distress syndrome (ARDS), and death from hypoxaemic respiratory failure is a major contributor to mortality [16, 17, 18, 19, 20, 21]. The cumulative incidence of ARDS related to seasonal IAV infection has been estimated to be 2.7 cases per 100,000 person-years, accounting for 4% of all respiratory failure hospitalizations throughout the influenza season [22].

2.2 Clinical manifestations and complications

In most cases, influenza produces a simple respiratory illness with a cough, fever, myalgias, chills or sweats, and malaise that lasts two to eight days. The onset is usually quick. Children might have unusual gastrointestinal symptoms such as vomiting and diarrhea. A small percentage of patients, particularly elderly individuals, young children, and those with medical comorbidities, will develop severe illness from viral or secondary bacterial pneumonia, resulting in respiratory and multiorgan failure. Extrapulmonary events are extremely uncommon [23, 24].

Common symptoms such as running nose, sore throat, muscle pains, fever, headaches and fatigue can trigger the release of pro-inflammatory cytokines and chemokines such as tumor necrosis factor or interferon from infected cells might be capable of producing a life-threatening cytokine storm [25]. Influenza does cause tissue damage compared to common cold that is caused by rhinovirus and as such symptoms might not entirely depend on inflammatory response. Also, the large amounts of cytokines have been observed to be dependent on the levels of viral replication produced by the strains [26]. Flu epidemics are difficult to control due to their rapid spread. However, influenza virus has a short generation time of two days (the time from being infected and then to infect the next person). Individuals can become infectious before being symptomatic thus quarantines following noticeable sign and symptom of the infection is not an effective public health intervention [27]. The virus shedding in an average person peak on day two while symptoms becoming apparent on day three [28].

3. Prevention and treatment

Early anti-influenza drugs were synthesized by large scale screening methods without the knowledge of their chemical structures and mechanisms of action [29], whereas, the recent antivirals have been discovered based on the structure of influenza virus protein as drug targets using X-ray crystallography method. This is structure based, involving the use of organic compounds that are able to bind to viral target protein receptors [30]. These structures have high binding affinity to the viral target following chemical synthesis and effective antiviral screening using standard in vitro assays such as cell based antiviral screening [31] and biochemical evaluation [32]. Some cell based antiviral screening includes plaque assays for studying replication in virus, yield-reduction assays for quantifying specific viral antigens and dye uptake for measuring cytopathic effect. The application of bioinformatics, robotics, miniaturization strategies have led to an advanced and high-throughput drug screening of large drug libraries with unique chemical structures [33] and computational screening [34]. In vivo drug screening using various animal models such as chicken, mouse, ferret have been used to evaluate new drugs [35] this is followed by clinical trials to study its bio-safety, kinetics and tolerance in human [36].

Advances has since then seen the treatment of influenza virus infection basically through vaccines, monoclonal antibodies and antivirals drugs. Antiviral influenza drugs are mostly NA inhibitors; however, they generally have short therapeutic window and current show emerging drug resistance [37]. Till date, four (4) antiviral drugs have been approved for the treatment of influenza: the NA inhibitors oseltamivir (Tamifu), peramivir (Rapivab), zanamivir (Relenza), and the cap-dependent endonuclease inhibitor baloxavir (Xofuza) [23, 37]. Oseltamivir is the preferred treatment for patients with severe influenza. Intravenous peramivir is an option for these patients if there are contraindications to or concerns about reduced bioavailability of oral oseltamivir [24]. Baloxavir is preferred for the treatment of uncomplicated influenza in patients of age 12 years and older. A study was conducted to compare baloxavir with oseltamivir and placebo in 1436 healthy people between 12 to 65 years of age who had influenza, baloxavir and oseltamivir reduced symptom duration by approximately one day compared with placebo. Adamantanes (amantadine and rimantadine [Flumadine]) are also approved for influenza treatment but are not currently recommended because these medications are not active against influenza B, and most influenza A strains have shown resistance to adamantane for the past 10 years [24].

Vaccines remain extremely essential to the prevention of the infection. Vaccination is the most preferred method for prevention, and routine chemoprophylaxis within the community is not recommended. The first influenza vaccine was developed in 1945, and since seen several others produced. Multiple formulations of the influenza vaccine are available, including inactivated influenza vaccines (IIV); a recombinant inactivated vaccine (RIV); and a live, attenuated influenza vaccine (LAIV). LAIV shows one of the best efficacies at around 70% and tends to be more effective in children. It delivers more NA and M2 antigens, triggers mucosal responses including IgA, and has the potential for inducing CD8 T cell responses [38].

As a primary prophylactic countermeasure, annual influenza vaccination is engaged globally with the aim of limiting influenza burden. However, the effectiveness of the current available influenza vaccines is limited because they only confer protective immunity when there is antigenic similarity between the selected vaccine strains and circulating influenza isolates. The consequences of antigenic drift or shift, results in an antigenic mismatch between the current vaccines and circulating influenza isolates. Accumulation of mutations, especially at key antigenic sites in the HA globular head, due to the absence of the proofreading activity of the viral RNA polymerase and then to the selective pressure exerted by the host immune system is often responsible for the escape of influenza virus from pre-existing immunity in the case of antigenic drift [39]. There is therefore a crucial need to develop a more effective broadly-reactive (universal) influenza vaccine with the capability to confer protection against both seasonal and newly emerging pre-pandemic strains.

3.1 The journey to a universal flu vaccine

In influenza virus vaccine design, the major targets of the antibody response against the virus are the surface glycoprotein antigens hemagglutinin (HA) and neuraminidase (NA). As earlier stated, Hemagglutinin (HA) and neuraminidase (NA), are the main surface glycoproteins on influenza viral particles. NA is however less abundantly expressed on the virion in comparison HA expression, with HA to NA ratio often ranging from 4:1 to 5:1 [38]. The influenza HA is responsible for binding to sialic acid, the receptor on target host cells, and there are approximately 500 molecules of HA per virion [40]. The mature form of the HA glycoprotein exists as a homotrimer containing three HA monomers that are composed of a globular head and a stem/stalk region. The receptor binding site (RBS) is present in the globular head, which is however a hypervariable region of the protein, while the stem region is majorly involved in the pH-induced fusion event triggered by endosome acidification following viral adsorption. The stem/stalk region of the HA is more conserved among and across HA subtypes belonging to the same group [38]. Antibody response elicited against this stem/stalk region forms one of the major approaches towards developing a more responsive vaccine to both current and future strains of influenza viruses (Figure 2).

Figure 2.

Showing the phylogenetic trees of (a) hemagglutinin (HA) and (b) neuraminidase (NA). The primary surface glycoproteins of influenza viruses, HA and NA, are divided into several categories and subtypes. During the last century, only viruses producing H1, H2, or H3 HAs and N1 or N2 NAs (such as H1N1, H2N2, or H3N2; circled in purple) have spread widely in the human population. The scale bars represent a 6% change in amino acid levels (source: [41]).

3.1.1 Stem-based universal vaccine approaches

Influenza virus infection can elicit neutralizing antibodies against both the globular head and the stem structures of the HA viral protein. Currently, ongoing strategies for more efficacious vaccine development is aimed at eliciting antibodies that target the conserved stem region of HA since previously existing influenza vaccines only show minimal induction of stem-directed humoral immunity [3]. Several studies describe ongoing approaches to elicit stem-directed antibodies including sequential immunization with heterologous influenza strains, immunization with modified proteins by removing or glycan-masking the globular head, referred to as headless HA, through minimizing epitopes of the stem region, hyperglycosylated HA head domain, Chimeric HA, and Mosaic HA [3, 38]. Self-reactivity of this antibodies may occur due to their polyreactive profile and the proximity of the HA stem region to the cell membrane which is a crucial limitation described by scientists to this approach.

Nachbagauer et al. [40] recently presented a unique concept in the stem-based approach using the context of a LAIV with a H8 head domain and an H1 stem domain (cH8/1) and a split-inactivated vaccine with an H5 head domain and an H1 stem domain (cH5/1) [40]. Using preclinical ferret investigations, the scientists assessed protection against pandemic H1N1 virus challenge using several sequential prime-boost combinations and vaccination regimens. These studies show that a sequential live-attenuated followed by split-inactivated viral vaccination strategy provides superior protection against pandemic H1N1 infection. Scientists have characterized this notion as a sequential immunization and chimeric HA proteins approach to stem-based universal vaccine design.

Furthermore, in a stem-based immunogens approach to the universal influenza vaccine design, based on the H1 subtype, Impagliazzo et al. created stable mini-HA stem antigens, where the best candidate demonstrated structural and binding characteristics with widely neutralizing antibodies equivalent to full-length HA, indicating correct folding. This immunogen totally protected mice in lethal heterologous and heterosubtypic challenge scenarios and lowered fever in cynomolgus monkeys following a sublethal challenge [42]. However, determining the effectiveness of antibodies targeting conserved epitopes in the HA stem region to offer protection remains a critical challenge [43].

3.1.2 Consensus based approach: computationally optimized broadly reactive antigens (COBRAs)

Furthermore, in order to overcome the extreme variability of influenza HA, in particular at the head region, Giles and Ross, [44] described the generation of computationally optimized broadly reactive antigens (COBRAs) for the influenza HA. The COBRA-based approach is a classic reverse vaccinology approach based on multiple layering of consensus HA protein sequences, followed by the generation of a final consensus sequence capable of recapitulating, in a unique protein, amino acid changes undergone by influenza virus from the past years to the present [45]. In this approach, a phylogenetic tree is inferred using hemagglutinin (HA) amino acid sequences. Primary and secondary consensus sequences are constructed, and the secondary consensus sequences are subsequently aligned to provide the resultant consensus, known as COBRA. In multiple clinical investigations, a firm called Sanofi-Pasteur used this method with a mechanism called Elicite HAI+ antibodies [46, 47, 48, 49].

More specifically, vaccination of mice with H1N1-based COBRA candidates resulted in broad HAI activity against a panel of 17 H1N1 virus strains. Furthermore, when inoculated mice were challenged, there was little or no detectable viral replication, as found in animals immunized with a matching approved vaccine [46]. Similarly, previous studies describing the design and generation of H5N1-based COBRA found that mice, ferrets, and nonhuman primates (Cynomolgus macaques) vaccinated with COBRA clade 2 HA H5N1 virus-like particles (VLPs) had higher HAI antibody titers recognizing different isolates representing divergent subclades [44, 49].

Aside from the COBRA-based strategy, there are several potential vaccines targeting the HA head. Song et al. [50] demonstrated the production of a fusion protein comprised of the globular HA head domains (HA1–2, spanning amino acids 62–284) from H7N9 and the Salmonella typhimurium flagellin (fliC) produced in Escherichia coli (E. coli). The authors chose fliC as a potent Toll-like receptor-5 (TLR5) ligand in order to induce an innate immune response with subsequent induction of cytokine production and dendritic cell activation, ultimately leading to higher titers of antigen-specific IgG recognizing different isolates representing divergent subclades [44, 49].

3.1.3 Vaccines targeting internal viral proteins

Internal influenza virus proteins are often highly conserved, making them viable targets for a universal vaccination. Although these proteins are rarely detected on virions or cell surfaces, making them inaccessible to antibodies, they are abundant in infected cells, where they are also processed and presented to T cells through major histocompatibility complex molecules. T cells have therefore been proven to play a significant role in influenza virus immunity. In this approach, NP and M1 have been widely studied as possible targets for universal T cell–based vaccine. Virus-based and DNA vaccination approaches have been shown in animal models to induce protective immune responses, and they are now being studied in a variety of clinical trials [41]. Over two consecutive influenza seasons, Evans et al. [51] conducted a phase 2b, randomized, placebo-controlled, double-blind trial of a recombinant viral-vectored vaccine (modified vaccinia Ankara expressing virus nucleoprotein and matrix protein 1; MVA-NP + M1), which has been shown to induce both CD4 and CD8 T cells, at eight outpatient clinical trial sites in Australia. They wanted to see if generating extra responses to conserved CD4 and CD8 T-cell antigens improves routine influenza vaccination. Based on their findings, they concluded that MVA-NP + M1 was well tolerated, with no vaccine-related major side effects. When administered within 28 days of normal QIV immunization, a vaccine intended to stimulate modest T-cell responses to cross-reactive internal proteins of influenza A did not result in an increase in incidence.

Finally, another notable vaccine technique is the epitope-based Multimeric-001 (M-001) candidate vaccine, which is now being tested in clinical trials. This vaccine, initially published by Ben-Yedidia et al. and later produced by BiondVax Pharmaceuticals Ltd., is made up of B- and T-cell epitopes taken from influenza A and B strains, containing nine conserved epitopes from the HA (including the globular head), NP, and M1 proteins [38, 52, 53]. To compensate for M-001 peptides’ poor immunogenicity and expensive cost, the epitopes are concatenated in triplicate into a single recombinant protein generated in E. coli. M-001 has been evaluated in both preclinical and clinical research, and it has been shown to protect mice against infection with various influenza strains while also being safe and generating both B- and T-cell specific immune responses [38, 53]. However, M-001 alone does not elicit HAI antibodies, which can only be generated when M-001 is followed by a boosting with seasonal or pandemic strain specific vaccinations [54].

4. Conclusion

Influenza virus infection is a continuing health and economic burden that causes epidemics with pandemic potential, impacting 5–30% of the world population each year and resulting in millions of hospitalizations and thousands of fatalities. Because of its great vulnerability to antigenic variation, influenza A is the type most responsible for pandemics. Influenza is extremely infectious, with symptoms including fever, cough, chills or sweats, myalgias, and malaise. The hypervariability of the amino acid sequences encoding HA and NA is primarily responsible for epidemic and pandemic influenza epidemics, which are the result of antigenic drift or shift. As a result, research on an effective broadly-reactive influenza vaccine capable of providing protection against both seasonal and pandemic influenza is now underway.

Conflict of interest

The authors declare no conflict of interests.