Influenza Virus and its Pandemic Potential

Influenza is an enveloped, negative-sense RNA virus of the Orthomyxoviridae family, classified into, A, B, and C strains (Horimoto and Kawaoka, 2005; Julkunen et al., 2000; Julkunen et al., 2001), circulating in aquatic birds and occasionally transmitting to other species e.g. ferret, swine, horses, chickens, guinea pigs, mice, seals and humans (de Boer et al., 1990). Influenza A virus is relatively simple containing a segmented genome with 11 genes coding for 11 proteins. The genome is surrounded by several non-structural proteins, like the nuclear export protein, non-structural protein 1, nucleocapsid protein and polymerase components. The viral genome and protein complex is surrounded by matrix protein 1 and a lipid envelope covered with two surface exposed proteins, hamaglutinin and neuraminidase. Hemaglutinin mediate host cell attachment and entry through interaction with sialic acid on glycoproteins/glycolipids on the host cell membrane, while neuraminidase mediates release of progeny virus by enzymatically cleaving sialic acid from the receptor molecules. Several subtypes of both hemaglutinin (H) and neuraminidase (N) (16 H and 9 N) are known to circulate in the aquatic bird reservoir (Fouchier et al., 2005; Webster et al., 1992). However, mainly three subtypes of hemaglutinin (H1, H2 and H3) and two subtypes of neuraminidase (N1 and N2) have been found to circulate widely in the human population.

The viral RNA polymerase of influenza A virus lacks a proofreading mechanism. Thus, it is able to produce accumulating point mutations, which eventually results in amino acid substitutions. Changes in hamaglutinin and neuraminidase are the most crucial and may affect both the infection rate and the immunogenicity of the viral strain, explaining how influenza virus can cause epidemics year after year producing mild to severe respiratory illness in 30-50 million people (Julkunen et al., 2000; Julkunen et al., 2001; Suzuki, 2005).

In the last century, four pandemics have also plagued the world including the current H1N1 "swine flu" (Belser et al., 2009; Peiris et al., 2009; Uyeki et al., 2002). The pandemic strains arise through reassortment, introducing drastic changes in the strains' hemagglutinin and/or neuraminidase molecule (Fouchier et al., 2003; Khiabanian et al., 2009). Antigens from these reassorted strains will be unfamiliar to the immunologically naïve population, thus having the potential of causing severe and devastating effects in the infected individuals. Additionally, if the strains are able to effectively spread from human to human, it has the potential of becoming a serious widespread infection (pandemic) (Horimoto and Kawaoka, 2005). This was the case for the most renowned and most devastating pandemic, the"Spanish flu" of 1918 (A/H1N1) (Reid et al., 2004). It is speculated to have been of avian origin according to available data, as are the other subsequent pandemic strains too (Horimoto and Kawaoka, 2005). The "Spanish flu" came in two waves. The first one was mild in the sense that the transmission rate was very low, giving the virus time to mutate and optimize itself into its deadly form before it returned in the second wave. A similar profile can be seen for the current H1N1 "swine flu" pandemic, which has been reported to be a triple reassortant carrying genes from porcine, avian and human influenza strains (Schnitzler and Schnitzler, 2009). This strain has been circulating amongst pigs in the United States since 1999, causing sporadic influenza infections in humans who were in contact with the pigs (Dawood et al., 2009; Shinde et al., 2009; Trifonov et al., 2009). However, the widespread and effective transmission to humans did not start until May 2009.

A looming influenza pandemic has always been feared prior to the H1N1 "swine flu". Previously, subtypes H5N1, H7N3, H7N7 and H9N2 of avian origin were feared as potential human pandemic strains since they were all endemic in birds. (Cauthen et al., 2000; Fouchier et al., 2004; Hirst et al., 2004; Lin et al., 2000) The H9N2 strain was considered to be low pathogenic in birds, but has sporadically been transmitted to humans, (Cameron et al., 2000; Hossain et al., 2008) resulting in disease development and influenza-like symptoms, though no deaths have been reported (Uyeki et al., 2002). The H7N7 and H5N1 strains differ from H9N2 in that they are highly pathogenic in birds (Horimoto and Kawaoka, 2005). In humans, H7 avian influenza A commonly manifests as conjunctivitis and/or respiratory symptoms (Belser et al., 2009). In contrast, H5N1 can produce respiratory symptoms, encephalopathy and/or diarrhea, and in severe cases, multi-organ failure (Beigel et al., 2005; Cheung et al., 2002). There have been multiple reported cases of bird to human transmission of H5N1 and H7N7 in recent years and human to human transmission has been suggested in some households (Beigel et al., 2005; Horimoto and Kawaoka, 2005). Fortunately, these viruses have not attained the efficiency of human to human transmission that the "swine flu" has attained. Although it is feared that eventual mutations could lead to improved transmission of these viruses between humans, thus leading to a pandemic strain.

Host Antiviral Response to Influenza Infections

Typically, influenza infects the epithelial cells of the upper respiratory tract, but it is also capable of infecting monocytes/ macrophages and other leukocytes, though immune cell infections are less productive for the virus, as only a few viruses are produced prior to the virus-induced apoptosis of these cells (Julkunen et al., 2000; Julkunen et al., 2001; Kaufmann et al., 2001). Humans are equipped with the necessary mechanisms for clearance of the virus during influenza infections. Primarily, the innate immune system participates in this process by providing a strong T_h1 response induced by type 1 interferons (Fernandez-Sesma et al., 2006). This response would require activation of at least nuclear factor kappa B, interferon regulatory factor and signal transducers and activators of transcription pathways (Julkunen et al., 2000; Julkunen et al., 2001). The following pathways would then allow the affected cells to produce the chemokines and cytokines involved in the desired antiviral immune response. Influenza A virus infected macrophages/ monocytes secrete macrophage inflammatory protein-1a/β and -3α, chemokine C-C motif ligand 5, monocyte chemotactic protein-1/3, interferon-inducible protein-10, interferon-a/b, interleukin-1 and -6, and tumor necrosis factor-a while epithelial cells infected with influenza secrete chemokine C-C motif ligand 5, monocyte chemotactic protein-1, interleukin-8 and interferon-α/β (Julkunen et al., 2000; Julkunen et al., 2001). In addition to this proinflammatory response, the human body also expresses numerous host defence peptides, both constitutively and in response to stimuli.

Host defence peptides have been demonstrated to be key players in bridging the complex interplay between innate (germline encoded) and adaptive (antigen specific) immunity. These signature molecules of host defence are present in virtually all species of life and carry a broad spectrum of activities, i.e. direct antibacterial, antifungal, antiviral and antiparasitic activities (Jenssen et al., 2006b) as well as modulation of host cell immune responses (Hancock and Sahl, 2006; Oppenheim and Yang, 2005; Hancock, 2001; Zasloff, 2002). They are generally short (12 to 50 residues), with a net positive charge (+ 2 to 9) due to an excess of basic arginine/lysine residues, and contain up to 50% hydrophobic amino acids. Structurally they are sorted into four classes based on their amphiphilic conformations (i.e., β- structures with two to four β-strands, amphipathic α-helices, loop and extended structures). Consequently, due to their broad spectrum of activities, several of the novel drug candidates that are currently passing through clinical trials are tailored around host defence peptides.

Influenza Virus Evasion of the Immune System

Despite the measures taken by the body to fight off an influenza infection, the virus has its own mechanism for subverting the host immune system. Three different studies of clinical patients infected with influenza A H5N1 observed that infected patients had higher serum levels of proinflammatory cytokines and chemokines in their plasma compared to healthy controls (Beigel et al., 2005; Horimoto and Kawaoka, 2005; Seo et al., 2002). This has also been observed in vitro when macrophages from healthy blood donors were infected with H5N1. In comparison to the cells infected with influenza H3N2 or H1N1, it was found that H5N1 infected cells had greater up-regulation of proinflammatory cytokines and chemokines in response to the infection (Cheung et al., 2002). Hypercytokinemia and the subsequent reactive hemophagocytic syndrome have also been implicated as the cause of death of many H5N1 infected patients as it results in the observed sepsis syndrome, acute respiratory distress syndrome and multi-organ failure (Beigel et al., 2005; Cheung et al., 2002). Therefore, the exaggerated immune response is suspected of causing the severity that is observed in these infections (Cheung et al., 2002). Despite the surge of proinflammatory cytokines, H5N1 is able to avoid clearance as its non-structural protein 1 is able to interfere with maturation of the immune response by inhibiting dendritic cell maturation and interferon-α/β production in myeloid dendritic cells (Fernandez-Sesma et al., 2006).

Similarly, in some cases of H1N1 "swine flu" infected individuals, the symptoms, unlike seasonal influenza, are similar to those found in H5N1 infected patients (Peiris et al., 2009). The specific inflammatory responses to the virus have yet to be determined but hypercytokinemia and reactive hemophagocytic syndrome may also be suspected as the cause of death in these individuals. This outlines the crucial role the immune system plays in the pathogenesis of influenza and thus, must be addressed to be able to alleviate the symptoms or eliminate infections.

Influenza Vaccination

The technique of vaccination dates back more than two hundred years to when Edward Jenner successfully vaccinated his patients against smallpox virus through exposure to cowpox virus. Vaccination has since proven a very effective public health initiative, preventing the severity and magnitude of several infectious diseases. In principle vaccination is done through administration of an attenuated micro-organism or important components of a micro-organism (e.g. surface proteins) able to trigger an immune reaction in the host. The efficacy of a vaccine relies completely on the T_h cell regulated development of high affinity B cell memory and the consolidation of the response through antigen re-challenge. Resultant B cell memory is the key feature yielding immunity after vaccination. Influenza vaccines have been available since the 1940s and are without doubt the most important strategy to prevent influenza virus epidemics. Since the circulating influenza strains are constantly changing U.S. Food and Drug Administration annually recommend two of the influenza A strains (currently a wild type of H3N2 and H1N1) and one B strain most responsible for human infections to be included in the following seasons vaccine. There are several licensed manufacturers world wide, and all of them produce their vaccines in eggs, either formulated as a trivalent inactivated influenza vaccine for intramuscular injection or as a live attenuated influenza vaccine administered as an intranasal spray. Comparative studies of the two vaccine types have indicated that the live attenuated influenza vaccines offers a significantly better protections against both well matched influenza strains and against strains that have undergone antigenic drift (Belshe et al., 2007). This broad spectrum protection provided by the live attenuated influenza vaccine has later been confirmed to also apply to vaccine batches from other seasons (Piedra et al., 2007). The achieved vaccine protection is on average between 70-90% and is predominantly affected by the age and immune competence of the immunized individual. Lower protection can be expected in years with a suboptimal match between the vaccine strain and the circulating viral strains. However this does not always hold true, since the vaccine in general will reduce disease symptoms even though it initially fails to protect against the primary infection (Herrera et al., 2007; Nichol et al., 2007). This illustrates that one important focal point both for improved epidemic vaccine manufacturing and for increased pandemic preparedness would be to better understand the mechanism behind vaccine cross-protection (Boon and Webby, 2009). For a comprehensive overview of the current trends on seasonal influenza vaccination the readers are refereed to Fiore et al. (2009); Chen and Subbarao, 2009.

There are also strategies in place for prepandemic vaccine production, using an eight plasmid system (i.e. plasmid-based reverse genetics or reassrotment) (Hoffmann et al., 2002a; Hoffmann et al., 2002b) with six plasmids carrying attenuating internal protein genes from a stable master donor virus, and two plasmids with hemagglutinin and neuraminidase from the potential pandemic strain (for review see Chen and Subbarao, 2009; O'Neill and Donis, 2009). Though the initial prepandemic vaccine trials demonstrated rather disappointing immunogenicity, formulation and use of new adjuvant strategies has lately produced vaccine alternatives giving a more satisfactory protection (Leroux-Roels et al., 2008; Leroux-Roels et al., 2007; Treanor et al., 2006). Despite this success and great potential of the prepandemic vaccines program there is an ethical aspect that should also be mentioned. Vaccines are expensive and many nations will not have the economic means to stockpile these prepandemic vaccines types, given that there is no guarantee that the pandemic strain will match the strain the vaccine is tailored around. To illustrate this; in 1996 the first devastating reports came on the highly pathogenic avian flu (H5N1), and variants of this have since circulated in domestic and wild birds resulting in an imminent threat of a pandemic onset (de Jong et al., 1997; Yen and Webster, 2009). Despite several reported cases of avian to human transmission, the influenza strain has so far not been able to adopted the ability to efficiently spread amongst humans, and has not per definition caused a pandemic. However, in this same time period, investors and granting agencies have enabled initiation of more than 60 prepandemic clinical vaccine trials against the strain. Then, almost out of the blue came reports on an influenza strain (H1N1) from porcine origin that started to cause severe influenza-like respiratory illnesses in Mexico in Fraser et al., (2009) and by early May 2009 the World Health Organization classified this as an influenza pandemic (Neumann et al., 2009).

Although there is no doubt that vaccination has proven extremely efficient in the control of influenza, the protection is never a hundred percent, hence supporting the need for good influenza drugs both for prophylactic and therapeutic use. Drug development could also be encouraged as an international preparation strategy for the next influenza pandemic, as these pandemics are caused by novel strains of influenza where human immunity is lacking and the protection from a prepandemic vaccines may be somewhat limited.

Influenza Drugs for Prophylactic and Therapeutic Use

Adamantane derivatives have long been in use as treatment alternatives against influenza virus infection. Amantadine was the first of these compounds that demonstrated the ability to inhibit replication of influenza (Figure 1) (Davies et al., 1964), possibly through blocking of the interior channel within the tetrameric helical bundle of the viral matrix 2 protein (Sansom and Kerr, 1993), thus inhibiting the influx of H+ ions into the virion, a process crucial for triggering the uncoating stage (Horimoto and Kawaoka, 2005). Another derivative in this drug family of matrix 2 protein ion-channel blockers is rimantadine (Figure 1). Both compounds have long been available for both prophylactic and therapeutic treatment of influenza A virus infections. However, their current usefulness is limited as many influenza strains easily develop resistance or have already developed resistance against this group of drugs (Englund et al., 1998; Hayden, 2006). Amongst the reported strains that have attained this resistance is the 2004 H5N1 strain (Beigel et al., 2005). Thus, the use of adamantane derivatives may not be sufficient in handling future influenza threats.

Figure 1: Structural small molecules approved or in clinical trails; (A) Amantadine, (B) Rimantadine, (C) Oseltamivir, (D) Zanamvir, (E) Peramivir, (F) Ribavirin, (G) Taribavirin and (H) T-705.

Neuraminidase inhibitors are a more successful class of drugs that have been approved for influenza prophylaxis and treatment. The viral neuraminidase mediates spread of influenza progeny after successful repl icat ion, by cleaving of Nacetylneuraminic acid from the cell surface glycoprotein. Thus, by inhibiting this cleavage, release of newly formed viral particles is prevented (Moscona, 2005a). Due to the important and highly conserved role of neuraminidase in the infection cycle of influenza, this enzyme is a prime target for anti-influenza therapeutics. Amongst the influenza drugs that inhibit this pathway are the commonly recommended oseltamivir (Kim et al., 1997) (Tamiflu; Roche) and zanamivir (von Itzstein et al., 1993) (Relenza; GlaxoSmithKline) (Figure 1). Both drugs prevent viral spread by mimicking the substrate of neuraminidase, and by binding to its active site, it inhibits the enzymatic role of the protein (Moscona, 2005b). However, oseltamivir requires a rearrangement in the neuraminidase active site to be effective. Thus, a mutation that prevents the structural rearrangement from occurring would render the drug ineffective. Not surprisingly, several influenza strains have already acquired such resistance against oseltamivir including several isolated cases of H5N1 (Beigel et al., 2005; Horimoto and Kawaoka, 2005; Moscona, 2005b). In contrast, zanamivir acts on neuraminidase without the conformational change oseltamivir requires to be effective (Moscona, 2005a; Moscona, 2005b). Zanamivir-resistant H3N2 influenza strains have been found to have poor viability as mutations that permit zanamivir resistance commonly reduces the neuraminidase activity (Zurcher et al., 2006). Thus, although resistance is emerging against oseltamivir, zanamivir is still a viable alternative for influenza treatment.

Novel Influenza Intervention Strategies and Drugs in Clinical Trial

There are several different drugs in clinical development for influenza treatment (i.e. peramivir, ribavirin, taribavirin, T-705, Fludase®), and common for all of them is that they are classified as small molecule drugs (Figure 1). Small molecules have been the drug class of choice from a pharmaceutical point of view for decades for several reasons, one being the low cost of synthesizing these drug molecules. In addition to the rather inexpensive final product, chemical modification and library generation is also fairly easy and inexpensive, enabling biologists to screen thousands of small molecule derivatives in their chase for a lead candidate. If that is not enough, computational solutions and in silico prediction models (e.g. Monte Carlo, molecular dynamics simulation, ligand docking, etc.) with small molecules are also relatively easy and accurate, as the molecular flexibility and variation is highly restrained by the size of the molecule (Christmann-Franck et al., 2004; Mizutani and Itai, 2004; Steindl and Langer, 2004). However, a big drawback with these types of small molecule drugs is that they are primarily designed to target and interfere with the viral entry, replication and release cycle. Hence, resistance development occurs rather easily over time, as has been illustrated throughout the history and evolution of antibiotic against influenza (Englund et al., 1998; Hayden, 2006; Beigel et al., 2005) and against other pathogens.

Peramivir (RWJ-270201) is the next generation cyclopentane derivatives of a neuraminidase inhibitor (Figure 1). It is currently undergoing Phase III clinical testing as a result of the collaborative effort of the U.S. Department of Health & Human Services and BioCryst Pharmaceut icals (http://www.biocryst.com/peramivir.htm). Studies comparing peramivir to traditional neuraminidase inhibitors has demonstrated a similar or greater inhibitory effects against influenza in both in vitro and in vivo models (Bantia et al., 2006; Bantia et al., 2001; Chand et al., 2005; Govorkova et al., 2001). Even more intriguing, comparative studies of peramivir with oseltamivir and zanamivir have also demonstrated that strains resistant to the latter two compounds, still were susceptible to peramivir (Mishin et al., 2005). However, in vivo protection with peramivir is highly dependent on the route of administration due to the low oral bioavailability (Barroso et al., 2005) and thus, new formulations are currently being made and tested by BioCryst Pharmaceuticals.

Ribavirin has long been recognized as a broad spectrum antiviral drug, with the potential for treating respiratory syncytial virus, hepatitis, influenza and herpes (Figure 1) (Eggleston, 1987). It targets inosine 5'-monophosphate dehydrogenase, which plays a role in GTP biosynthesis and viral RNA synthesis. Thus, as it targets a key enzyme for virus replication, there have been no reported cases of ribavirin-resistant influenza. Although ribavirin seems to be a highly effective drug against influenza in vitro and in vivo, it does not perform well in clinical trials and has been implicated as having potential teratogenicity and the ability to cause hemolytic anemia (Cohen et al., 1976; Rodriguez et al., 1994; Smith et al., 1980). Therefore, in the guidelines for H5N1 management of infected individuals, the World Health Organization strongly advises against the use of ribavirin.

Taribavirin (Viramidine) is a carboxamidine analog of ribavirin, which has been proven to have significant antiviral effects on influenza A and B infections in vitro and in vivo (Figure 1) (Sidwell et al., 2005). The drug has just finished Phase IIb test ing (Valeant Pharmaceuticals Int . ; http://www.valeant.com/) as an oral drug candidate for treatment of hepatitis C virus infections, demonstrating comparable activity as ribavirin with significantly lower toxic effects. Based on the non-toxic nature and oral bioavailability of taribavirin, it has been accredited with market potential for treatment of influenza virus infections (Sidwell et al., 2005).

T-705 (favipiravir) is a RNA polymerase inhibitor developed by Toyama Chemical Co. , Ltd., (http://www. toyamachemical.co.jp/en/index.html) which is currently being tested in Phase II clinical trials (Figure 1). A ser ies of pyrazinecarboxamide derivatives including T-705, have demonstrated very broad spectrum antiviral activity against a range of various RNA viruses, including influenza virus, arenaviruses, West Nile virus and yellow fever virus (Furuta et al., 2009). In particular, T-705 has demonstrated a potent and selective inhibitory activity against a panel of different influenza strains both in vitro and in vivo (Furuta et al., 2002). Though the efficacy was less than that exerted by oseltamivir and zanamivir, it demonstrated a remarkably better protection than ribavirin (Sidwell et al., 2007). In time of addition studies, it has been established that T-705 targets the early- to middle-stages of the viral replication cycle and that the compound neither has an effect on viral adsorption nor on viral release (Furuta et al., 2005). However, even in single dose treatment experiments where a 100% lethal dose of influenza A virus was administered to the mice, protection could be observed when T-705 was administered up to 60 hours after viral infection (Sidwell et al., 2007).

Fludase® (DAS181) is a recombinant sialidase fusion protein that is being developed by NexBio (http://www.nexbio.com). It works through cleavage of sialic acid residues on the host cells (Belser et al., 2007; Malakhov et al., 2006). Sialic acid is the entry receptor for influenza and is consequently crucial for the infection to occur. The typical receptor for avian and equine influenza viruses is a(2,3)-linked sialic acid while human influenza viruses traditionally use an a(2,6)-linked sialic acid (Ito, 2000). Both receptor types are found on human respiratory epithelial cells, though the majority are a(2,6)-linked sialic acids (Hassid et al., 1999; Matrosovich et al., 2004). Fludase® is capable of cleaving both types of sialic acids, as well as ?(2,8)- linked sialic acid, an entry receptor used by some laboratorygenerated influenza strains (Malakhov et al., 2006). Due to the variety of sialic acids that can be cleaved by Fludase®, this drug candidate has the potential of being effective against all circulating strains of influenza. Also, since it targets host cell components instead of the virus directly, drug resistance is hardly likely to occur (Malakhov et al., 2006). The drug is still in clinical Phase I trials, but has demonstrated very promising results both from a prophylactic and a therapeutic treatment point of view.

The drugs that have been discussed above are all effective or interesting drug candidates for influenza intervention and therapy. However, they all target different parts of the influenza infection cycle, rendering them susceptible for resistance development. This together with the constant threat of influenza epidemics and pandemics of various origins underscores the need for drugs that would be persistently effective against influenza in the future. A novel approach for treating influenza may be the use of innate defence regulators that can swing the host immune response towards a greater proinflammatory or anti-inflammatory route since the immune system plays a large role in an influenza infection. The benefit of using immunomodulators for antiviral intervention, overcomes the problem of resistance as these types of drugs target the host cells instead of the rapidly mutating viruses. Thus, it might be advantageous to explore the possibility of using host defence peptides or derivatives thereof in the prophylaxis and treatment of influenza.

Though the majority of the initial peptide drug candidates either failed approval by U.S. Food and Drug Administration or got terminated due to lack of efficacy, there is a growing body of evidence supporting their potential as therapeutic drugs targeting a variety of microbial- and immune-related disorders. However, there are still several strongest arguments against drug development around a peptide scaffold, one being the high cost of goods. By moving from traditional solid-phase synthesis to solution-phase- and hybrid methodologies, the cost of goods can easily be reduced, thereby alleviating the problem to some extent. Roche and Trimeris joint success in producing Enfuvirtide (Fuzeon, T-20), a 36 amino acid peptide that interacts with the HIV glycoprotein gp41 to block viral entry into CD4+ T-cells, is a great example that even large peptides can succeed in the clinic. Enfuvirtide is currently being produced by solid- and solution-phase hybrid synthesis at a level of 3.7 metric tons annually (in 2005) and is used as the drug of last resort for treatment of drug resistant HIV (Andersson et al., 2000; Schneider et al., 2005). From a drug development point of view there was (a decade ago) some truth in the high expense and complexity of generating peptide libraries, compared to small molecule libraries, but development of new synthesis strategies have revolutionized the way scientists look at peptide libraries, making them affordable even for small-size academic labs (Hilpert et al., 2005; Winkler et al., 2009).

Computer hardware has gone through an even more rapid evolution over the past decades, increasing the computing power of a standard machine significant ly, thus making chemoinformatics and computer-aided drug design more mainstream. Computational simulations and prediction models make use of chemical descriptors describing the biochemical and physical characteristics of a molecule. These models were originally restrained to small molecules, but through the development of new descriptors and the assistance of stellar processing capacity, robust and precise prediction models can now also be constructed for peptide molecules (Cherkasov et al., 2009; Fjell et al., 2009; Jenssen et al., 2008; Jenssen et al., 2006a; Jenssen et al., 2007). Peptides, as drugs, have also been faced with scepticism regarding their poor pharmacokinetic properties, short half-life and lack of oral bioavailability (Chatterjee et al., 2008). However formulation technologies and/or chemical modification (e.g. N-methylation) may significantly improve the peptides' pharmacokinetic profile. Multiple N-methylations has been demonstrated to significantly improve the oral bioavailability, metabolic stability and intestinal permeability of peptides (e.g. Veber-Hirschmann- and α-IIb-β3 intergrin-analogues) (Biron et al., 2008; Chatterjee et al., 2008). Pegylation of interferon-a for chronic hepatitis C virus treatment has prolonged the retention time of the drug in the body and so demonstrates that it is possible to improve circulation half-life of peptide drugs (Barnard, 2001). Advances in formulation and delivery systems, e.g. implantable scaffolds, hydrogels and micro- or nano- particle systems will also help expedite the progression of peptide drugs into clinical use (Kobsa and Saltzman, 2008).

An advantage for peptide drugs are their tolerability and relatively low toxic potential as a result of their susceptibility for proteolytic degradation. One may argue that this, in general, is a negative feature of peptide drugs. However, results indicate that the responses they trigger are so rapid that sufficient protection can be initiated prior to protease cleavage. A good example is the innate defence regulator-1 (IDR-1), an anti-infective peptide that selectively modulates the innate immune response (Figure 2) (Scott et al., 2007). The peptide, by itself, has no direct antibacterial activity in vitro. However, it demonstrates significant protection in an invasive murine S. aureus model even when administered 24 hours prior to bacterial challenge, indicating that it triggers a long lasting immunity (Scott et al., 2007). A 5- mer derivative of IDR-1 (IMX942) has been developed by Inimex Pharmaceuticals (http://www.inimexpharma.com/) and is showing promising results in the current Phase I safety testing in healthy volunteers. This success also demonstrates that although peptide drugs (Figure 2) originally were viewed as much larger chemical structures than the small molecule drugs (Figure 1), the current size difference is less obvious.

Figure 2: Selected peptides in clinical trails; (A) IM862, (B) SCV-07 and (C) IDR-1.

In a typical influenza infection, the non-structural protein 1 is able to interfere with the host immune system by preventing efficient type I interferon production (Fernandez-Sesma et al., 2006). Thus, the immune responses are suppressed and the proinflammatory response must be boosted in order to facilitate clearance of the virus. The idea of boosting the immune system has been visited previously with attempts at administering interferon intranasally for prophylaxis of influenza A infections, but this was not very effective (Isomura et al., 1982; Phillpotts et al., 1984). The recent success of pegylated interferon-a with ribavirin treatment for hepatitis C virus infections (Palumbo, 2009) has revitalized the possibility of using immunomodulation as a form of influenza therapy and it has even been suggested that the combined ribavirin and pegylated interferon-α therapy has a possible applicability in influenza infections. There are also many other hepatitis C virus drugs in clinical trials that exert immunomodulatory effects that could possibly be used against influenza such as a di-peptide, IM862 (Implicit Bioscience; www.implicitbioscience.com), isolated from the calf thymic peptide complex Thymalin (Anisimov et al., 2000) and a synthetic derivative of IM862, SCV-07 (SciClone;). Both drugs are currently in Phase II clinical trials and have demonstrated the ability to stimulate the production of immune cells and trigger a T_h1 response assisting in resolving the hepatitis C virus infections (Figure 2) (Orellana, 2002; Tulpule et al., 2000). SCV- 07 has also demonstrated significant reduction of recurrent lesions when administered orally in a guinea pig model of recurrent genital herpes simplex virus 2 (Rose et al., 2008), indicating the broad spectrum activity and immune modulation potential of these peptide drugs.

Due to the effectiveness of HIV and Mycobacterium tuberculosis combination therapies, there have been studies examining different combinations of antiviral drugs that could potentially be used in influenza infection. For example, in a study combining rimantadine and different neuraminidase inhibitors, synergistic and additive effects were observed at specific concentrations and combinations of the drugs (Govorkova et al., 2004). Immunomodulators could also potentially be used in combination with the conventional influenza antivirals as a supplement that would not only facilitate more efficient elimination of the virus, but also decrease the severity of the symptoms that manifest in infected patients. Further study is obviously warranted to determine whether such combined therapy truly is applicable for influenza infections. This approach may also be valuable in a pandemic situation where several of the conventional influenza drugs can experience reduced efficacy.

Influenza research has been a hot topic for the past decade, fuelled by the imminent public fear of a pandemic. We are starting to see the fruits of this focused research; novel drug candidates are being pursued and more detailed understandings of the immunological responses to influenza are being drawn up. However, fear as a motivator is dangerous in itself, as the general public rapidly will adapt and over time show decreasing interest to the problem at hand. Let us hope some of the enthusiasm in this field can progress and outlive the focused attention from the public media on this topic, thus leading to a diverse mix of influenza treatment strategies. Development of small molecules and peptide drugs targeting viral infections are now, more than ever, an innovative and very interesting strategy that if pursued with research and adaptation of new technology platforms, may one day bear significant fruits.