Rapid mutability

Like all RNA genomes, the influenza virus genome lacks a proofreading mechanism and thus mutates relatively frequently. Mutations may offer the virus various selective advantages, such as resistance to existing vaccines and antiviral drugs [36] - even small changes in the viral HA and NA antigen sequences, known as antigenic drift, may allow the virus to escape from the host's adaptive immunity [37]; increased infectivity and virulence; and greater horizontal spread (that is, one individual to another in the same species) and vertical spread (that is, crossing of the host species, such as from pig to man, generally due to a 'variant' virus). Larger diversity and more extensive changes can be rapidly generated by genetic reassortment, as described below.

Genomic reassortment

As the influenza genome is segmented (multiple pieces), new strains can quickly appear by reassortment in co-infection. Known as antigenic shift, this often leads to hybrid strains that are markedly different [25]. For example, co-infection by human and swine (pig) influenza viruses can generate reassortant viruses that will have genomic RNA segments from the two viral species [38]. Such a hybrid can cause a major epidemic because the human population will lack any natural immunity to it. It has been speculated that the 1918 influenza virus, which caused the largest influenza pandemic recorded in history, was caused by such a reassortant virus [39, 40]. The 2009 Mexican swine influenza is likely a product of multiple assortments between swine, human and European avian-like strains [41, 42]. Obviously, reassortment may result in a new combination of the HA and NA segments, thus changing the subtype name of virus as well.

Vulnerable population groups

Influenza can be particular deadly to specific groups in the population, such as the elderly and individuals with diabetes or immune deficiency (such as those with AIDS). In fact, people aged 65 years or older account for 90% of seasonal influenza-associated deaths, even though this group makes up only approximately 15% of the population [1, 2]. Thus, this group is in the greatest need of prophylaxis or more intensive treatments against influenza but, unfortunately, they are also generally less tolerant to aggressive treatments.

The brief background presented above should make it clear that reliable prevention and treatment of influenza is a critical need in public health. In this review, we start by summarizing the various current and now-defunct treatments for influenza (amantadine, oseltamivir (Tamiflu), zanamivir (Relenza)), as there are lessons to learn from their success and failure. We then discuss and critically review the prospective future anti-influenza treatments that are at different stages of development (newer NA inhibitors, sialidase, defensins, cathelicidin, statins, siRNA and host proteins).

M2 ion channel inhibitors: adamantanes

The influenza viral M2 protein acts as an ion channel that allows proton translocation through the virion envelope (Figure 1). This leads to acidification of the viral core, its resultant dissociation and the release of the viral NP-RNA complex in the infected cell cytoplasm, which is essential for viral RdRP function and viral gene expression [4]. The M2 inhibitors are adamantanes, characterized by three condensed cyclohexane rings fused in the chair conformation. Two M2 inhibitors, amantadine and rimantadine, were widely used against influenza but are now largely discontinued and replaced by NA inhibitors [43]. As one would predict from the role of M2 in the viral life cycle, these drugs were effective only when administered soon after diagnosis [43]. Nonetheless, their efficacy is limited to influenza A only, since influenza B viruses lack M2. Moreover, essentially all influenza strains have now developed high resistance against both amantadine and rimantadine. This has been attributed to their easy over-the-counter availability in highly populated nations such as China and Russia, and their large-scale use in the poultry for protection against 'chicken flu'.

Oseltamivir (Tamiflu)

The most popular influenza treatment regimen, developed nearly three decades after the M2 inhibitors, targets the viral NA. The NAs possess glycoside hydrolase activity that cleaves the glycosidic linkages of neuraminic acids. The influenza virus uses viral HA, a virion surface protein, to bind to sialic acid groups (Figure 2) on cell surface glycoproteins (Figure 1) [45]. For the progeny virions to be released from the cell, the NA activity must cleave the sialic acid groups from the host glycoproteins, and this is essential for viral spread and reinfection (Figure 1). Thus, blocking the function of NA with specific inhibitors is an effective way to treat influenza.

The influenza NA is a classic example of rational drug design based on the crystal structure of NA [46, 47]. Currently, two NA inhibitors are used in clinical practice: oseltamivir (Tamiflu; Roche/Genentech) and zanamivir (Relenza; GlaxoSmithKline) (Figure 2). The 2009 H1N1 pandemic witnessed record sales of both drugs, together exceeding US $4 billion, partly due to stockpiles for fear of a forthcoming epidemic. Both drugs bind the catalytic pocket of NA and function as competitive inhibitors of NA activity.

Zanamivir (Relenza)

Zanamivir is more effective than oseltamivir and is supplied for oral inhalation only [36]. It is sold as double-foiled 'blisters' that release the drug in an inhaler when pierced by the user. Each blister contains a powder mixture of 5 mg of zanamivir and 20 mg of lactose (plus milk proteins). The recommended dose for treatment of influenza in adults and pediatric patients aged 7 years and older is 10 mg twice daily (that is, two inhalations, one 5-mg blister per inhalation) for 5 days. For prevention of influenza (prophylaxis), the recommended dose in adults and pediatric patients 5 years and older is 10 mg once daily for 10 days, inhaled as above. Adverse drug reactions are rarer than with oseltamivir (Table 1).

Resistance to oseltamivir and zanamivir: what they tell us

Influenza virus mutants, resistant to either drug, have been characterized from cell culture as well as from patients [43, 48, 49] and are particularly well-studied for oseltamivir. Interestingly, resistant mutations were found not only in the NA gene, but also in HA. It appeared that, at least in cell culture, two HA or an HA and NA mutation can act synergistically to increase resistance [53]. As expected, the NA mutations were in conserved catalytic (Arg152, Arg292) and structural (Glu119, Asp198, His274, Asn294) residues. A few common mutations were: R152K, R292K, E119V, D198N, H247Y (highly prevalent) and N294S [54]. Double mutations with synergistic oseltamivir resistance phenotype have been noted as well. This includes the E119V+I222V double mutant, isolated from an immunocompromised child infected with H3N2 virus, and H247Y+I222V, from patients infected with the H1N1 virus of the 2009 pandemic.

For future drug design and resistance expectation, it is important to learn that the effect of these mutations is dependent both on the NA subtype and the drug used [55, 56]. Generally, catalytic site mutants exhibit drug resistance, but also show decreased NA activity, such that viral infectivity, pathogenicity and transmissibility are affected. By contrast, mutations of the structural residues exhibit drug resistance without a significant effect on NA functionality. Thus, in natural selection against these drugs, the structural mutations may be favored because they would retain viral fitness. Lastly, the HA mutations tend to map to regions associated with receptor binding of the HA, apparently lowering the affinity of the HA for the cellular receptor, such that NA is no longer required for virus release.

Readers interested in the detailed dosage of the existing drugs (amantadine, rimantadine, oseltamivir and zanamivir) are encouraged to read the highly comprehensive treatise of the Advisory Committee on Immunization Practices from the National Center for Immunization and Respiratory Diseases, Center for Disease Control and Prevention, USA [57].

New and future neuraminidase inhibitors

EB peptide

In an interesting report [61], a 20-amino-acid peptide (RRKKAAVALLPAVLLALLAP), derived from the signal sequence of fibroblast growth factor 4, specifically bound to the influenza viral HA protein (Figure 1) and exhibited broad-spectrum antiviral activity against influenza viruses including H5N1. Named EB for Entry Blocker, the peptide was also protective when administered post-infection, suggesting that it prevented reinfection, which underscored its therapeutic potential.

Peptide NDFRSKT

In a complementary approach [62], a heptapeptide phage display library was biopanned against purified avian influenza virions of subtype H9N2. Multiple rounds of panning and antiviral screening led to the identification of the peptide NDFRSKT with strong antiviral properties. The peptide inhibited the hemagglutination activity of the viruses but not the NA and hemolytic activities. Further studies confirmed that the peptide directly interacted with the HA protein. The therapeutic status of the peptide remains unknown.

Statins

Statins inhibit cellular 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA reductase), an enzyme essential for cholesterol biosynthesis in the liver, and are extensively prescribed to treat hypercholesterolemia [69]. Statins are relatively safe with rare incidents of myositis, myopathy and neuropathy. Thanks to the generally accepted correlation of high serum cholesterol levels and predisposition to atherosclerosis, multiple statins are currently blockbuster pharmaceuticals that include: atorvastatin (Figure 2) (Lipitor by Pfizer), lovastatin (Mevacor by Merck & Co.), and simvastatin (Zocor by Merck & Co.), as well as generic varieties.

In relatively recent approaches, statins have been tested in influenza, based on the premise that they might reduce the mortality and morbidity caused by the cytokine storm [30, 65, 66, 70, 71]. However, these studies have generated contradictory claims. In one retrospective study [72], a database of 3,043 adults in the US hospitalized with laboratory-confirmed influenza during the 2007 to 2008 influenza season was analyzed. Of these patients, 1,013 received statins and 151 died within 30 days of their influenza test. The analysis revealed a positive correlation of statin use with reduced mortality. By contrast, when 1,520 patients in the UK [73] with confirmed 2009 pandemic influenza A (H1N1) infection were surveyed for pre-admission statin use and in-hospital severity, no significant correlation could be found. Another recent study in Spain [74] examined the use of corticosteroids, macrolides and statins among 197 patients with 2009 pandemic influenza H1N1, who also had complications from pneumonia, suggesting a role of the inflammatory response. Unfortunately, none of these immunomodulatory therapies was found to be associated with a lower risk for developing severe disease. The apparent variability among some of these studies may be due to a number of factors [75], such as subtle differences in the viral genome sequence between the two pandemics, the dose and frequency of statin use, and environmental factors. Clearly, a more detailed, focused and controlled clinical trial is needed to evaluate the benefit of statin use, perhaps in conjunction with an antiviral agent such as oseltamivir or zanamivir.

Sphingosine mimics

Sphingolipids are a family of lipid mediators, of which sphingosine and its phosphate (sphingosine 1-phosphate or S1P) have been recognized as modulators of diverse cellular activities. The sphingosine analog family is a group of recent immunosuppressants with high therapeutic potential for influenza. In the body, these compounds mimic natural sphingosine and are first phosphorylated by sphingosine kinase [76, 77]. The phosphoform then acts as an agonist of multiple types of sphingosine receptors, which leads to lymphopenia through the sequestration of lymphocytes in the lymph nodes, resulting in immunosuppression [76, 77]. Fingolimod (FTY720) (Figure 2), an early member of this family (trade name Gilenya, from Novartis), is derived from a fungal metabolite and currently approved for treatment of autoimmune conditions, multiple sclerosis, cardiac failure and arrhythmia. Later, removal of a hydroxyl group in FTY720 generated AAL-4, which was much more rapidly phosphorylated in humans, improving its efficiency [78]. A series of studies have now shown that AAL-4 provides significant protection against the cytokine storm in pathogenic influenza by limiting pulmonary injury [79, 80]. In a representative study [80], mice were intranasally infected with the pathogenic pandemic A/Wisconsin/WSLH34939/09 influenza virus and an hour later treated with AAL-R (0.2 mg/kg in 100 µL water intratracheally) or 100 µL water alone. These animals with compared with those receiving 5 mg/kg (also in water) of oseltamivir (Tamiflu) by gavage. The results showed that AAL-R administration alone significantly lengthened the survival time of the animals (82%) compared with those that received water. As expected, oseltamivir treatment alone significantly increased the number of survivors (50%) compared with just water (21%); however, protection was significantly less than that from AAL-R treatment (50% versus 82%). Interestingly, a combination of AAL-R and oseltamivir resulted in 96% survival, which is greater than either drug alone. Hence, a dual drug cocktail of a direct viral function inhibitor and a host immune response inhibitor may be a promising approach in the treatment of influenza.

Nuclear factor-kappaB inhibitors

NF-κB is a transcription factor of many genes of the cellular innate immune pathway and its activation underlies a variety of antiviral as well as inflammatory responses that range from septic shock to cancer. In fact, NF-κB has been considered a major target of immunomodulatory and anti-inflammatory therapy [81, 82]. In its interesting dual role, NF-κB is not only a critical contributor of cytokines and interferon synthesis in influenza infection, but also essential for the growth of the virus itself [83–87]. Although the exact mechanism for the latter remains unclear, the balance must be tightly regulated, as the influenza viral NS1 protein actually inhibits NF-κB [88]. Clearly, even after the inhibition, enough active NF-κB persists to promote viral growth and the inflammatory response. Thus, inhibitors of NF-κB may have a two-pronged beneficial effect in influenza: they will inhibit the virus directly and will also moderate the systemic inflammation. This has been shown in cell culture and mice [84–86], but specific and controlled studies in patients with influenza still need to be done. Nonetheless, the prospects of an anti-inflammatory therapy of influenza are real, because some anti-NF-κB drugs, such as acetyl salicylate (aspirin) are routinely sold in stores without prescription and widely used by the general public for many years without major side effects.

Defensins

The defensins typically contain six Cys residues, forming three intramolecular disulfide bonds that regulate their structure and function (Figure 3) [89]. They are subdivided into α-, β- and θ-defensins, depending on their molecular weight and location of the Cys-Cys bonds. A number of defensins inhibited influenza virus growth, but the mechanism remains a matter of debate.

Humans have six α-defensins, of which α-defensin-1 (also known as human neutrophil peptide-1, HNP-1) and α-defensin-2 (HNP-2) were shown to increase neutrophil uptake of influenza virus [95, 96]. The β-defensins had a significantly lower activity in this particular mechanism. In another study, HNP-1 (Figure 3) exhibited anti-influenza activity in epithelial cell culture as well, inhibiting viral RNA and protein synthesis [97]. Pretreatment of the cells with HNP-1 also inhibited viral replication, showing that the inhibition was due to modulation of cellular pathways. Protein kinase C was shown to be inhibited by HNP-1 treatment, suggesting the involvement of the protein kinase C pathway in nonimmune cells.

The θ-defensins, also called retrocyclins, are uniquely circular 18-residue peptides formed by post-translational joining of the N- and C-termini of two nonapeptides, and they also occur exclusively in primates [89, 96]. The human θ-defensin genes are intriguing exceptions as they are pseudogenes harboring mutations that prevent the production of θ-defensin proteins. Studies of nonhuman primate θ-defensins have revealed that they are lectins with glycoprotein-binding properties that can inhibit fusion of HIV with the host cell, thus suggesting a novel antiviral regimen [98–101]. Human β-defensin 3, another lectin, also inhibited HA-mediated influenza viral fusion in a similar way [101]. Recently, retrocyclin-1 (Figure 3) and its various synthetic analogs, some with structural variations (for example, hapivirins, diprovirins) were tested against influenza virus in human cell culture and shown to block infection at low micromolar concentrations [99, 101, 102]. The success of synthetic peptides has opened the possibility that further engineering of the defensin sequences may lead to more optimized anti-influenza efficacy and pharmacological properties [100, 103]. In an interesting complementary approach, aminoglycosides (amikacin 40 µg/mL; gentamicin 5 µg/mL; tobramycin 10 µg/mL), which are known to promote suppression of termination by codon-misreading [104], were used to produce retrocyclin from the endogenous human genes, and this also resulted in resistance to HIV-1 [103]. Although aminoglycosides are sometimes prescribed to fight serious bacterial infections, nephrotoxicity and ototoxicity are relatively frequent [105]. It remains to be seen whether an optimized dose will generate enough retrocyclin to block influenza viral fusion without causing unacceptable toxic effects in the patient with influenza.

A few defensin mimetics are currently being developed by PolyMedix (Radnor, PA, USA) and are at various stages of preclinical and clinical trials, but apparently none is being tested against influenza.

Cathelicidins: LL-37

In humans, the cationic antimicrobial protein hCAP18 is cleaved between Ala103 and Leu104 to generate LL-37, a 37-residue peptide with two tandem Leu residues at the amino terminus (Figure 3). Recent studies are revealing these peptides are not just antibacterial molecules but have a variety of innate immune functions [92, 93]. LL-37, which is expressed in a number of cell types including epithelial, was recently shown to protect mice against influenza [94]. Using a lethal dose of the two different influenza A strains (A/PR/8/34 H1N1 and A/Udorn/307/72 H3N2), significantly higher survival and decreased weight loss was observed in LL-37-treated animals, which compared favorably with the Relenza-treated positive controls. Although a part of the better prognosis could be due to suppression of the inflammatory response by LL-37, the accompanying lower pulmonary viral titer, the short window of the acute infection, and the reproducibility of the viral inhibition in cell culture all point to a direct antiviral role of LL-37, the mechanism of which remains to be determined [94]. The LL-37 in these mouse experiments was administered with a respiratory nebulizer at a concentration of 500 µg per milliliter of saline and compared with the same concentration of Relenza [94]. At least in cell culture, the virus-inhibitory concentration of LL-37 approximated its natural concentration in the human lung [94]. LL-37, promising as it is, awaits further development for influenza treatment. It also remains to be seen whether other such antimicrobial peptides have antiviral, and specifically anti-influenza, properties. If so, this family may constitute an exciting and novel regimen for influenza treatment in the future.

Collectins

The collectins belong to the superfamily of collagen-containing C-type lectins (hence their name), and act as pattern recognition receptors for pathogenic molecules [106, 107]. Better known members include the mannan-binding lectin and the surfactant proteins A and D. There is a large body of literature documenting an innate antiviral role of these lectins, in which they neutralize viral infectivity by binding to viral fusion glycoproteins, such as influenza viral HA and NA proteins; however, their contributions in opsonizing viral antigens and triggering neutrophil oxidative respiratory burst and an inflammatory response suggest that their pharmacological potential must await a more detailed analysis of these diverse roles [96, 108–112].