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A major obstacle in influenza eradication is the viruses' ability to change its main antigenic components, hemagglutinin (HA) and neuraminidase (NA), through antigenic shift/drift or interspecies reassortment (1). Influenza A strains have been isolated from many mammals and wild birds, all of which can reassort and sometimes crossed species boundaries. Because of the appearance of the highly pathogenic avian H5N1 virus (2003) and the pandemic H1N1 in 2009, new efforts, including treatment and surveillance, are being induced to better prepare for the threat of new emerging infectious influenza. This paper will focus on the influenza A life cycle, due to human disease disease, and focus on current and new theraupic/phophylaxis options.
Biology of Influenza
Influenza viruses belong to the Orthomyxovirus group and include three types A, B, and C. All types contain negative RNA segmented genomes of eight (A/B) or seven segments (C). Influenza A gene segments encode: nucleoprotein(NP), RNA-dependent RNA polymerase (PB1/PB2/PA), non-structural proteins NS1/2, HA, NA, and M1/M2. The viral envelop of influenza A/B is covered with HA/NA proteins (book).
Influenza infection starts with the binding of viral HA, a trimer protein composed of three HA1/ HA2 polypeptide chains, to sialic acid residues on the glycoproteins/lipids on the cell surface. Sialic acid moieties can have different steric conformations, namely Î±-2,3- or Î±-2,6-linkages. While in the human airway Î±-2,6-linkages predominate, Î±-2,3-linkages are more common in avian gut epithelium, explaining specific influenza species tropism. These receptors bind to the HA in a shallow indent at the top of the protein, composed of residues that are conserved throughout all influenza viruses. Here three side-chains of HA form hydrogen bonds to the sialic acid receptor, which induces endocytosis of virus(2).
The acidity of the endosomal compartment causes a conformational change in the HA protein, exposing a fusion peptide that joins the viral envelope with the endosomal membrane. The M2 ion channel is also important for this uncoating process as it pumps hydrogen ions into the virus particle disrupting structural protein-protein interactions, and allowing viral RNP complex (vRNA/NP/ RNA polymerase) to escape into the cytoplasm(1, book). The RNPs contain nuclear localization signals, which regulate their trafficking into the host cell nucleus. Here, the viral RNA genome can be transcribed into positive sense mRNAs that contains a 5' cap and 3' poly-A tail and thus are able to be exported out of the nucleus for expression. Capping occurs through the PB1 and PB2 proteins, which "take" the 5â€² capped primers from host pre-mRNA transcripts and initiate mRNA transcription (1). The polymerase also transcribes postive cRNAs from which the vRNA can be further copied. These then associated with NP proteins that have returned to the nucleus, forming new RNP complexes. Since the viral genome duplicates are not capped or polyadenylated, its nuclear export is mediated by the viral proteins M1 and NEP (nuclear export protein)/NS2. While NS2 forms a complex with NEP, it also binds to MI-RNP, bridges export proteins and RNP+M1 together for export (3).
The envelope proteins HA, NA, and M2 are translated by ribosome's, and later modified by the ER and Golgi. All three proteins have sorting signals that direct them to the cell membrane for virus assembly. Although vRNA packaging mechanisms are unknown, new research has shown that packaging signals on all vRNA segments could ensure full genome/all components are included into most virus particles .
The final process, namely budding, is mediated by an accumulation of M1 protein at the cell membrane. Once budding is complete, the HA spike continues to bind to its receptor, sialic acid, until the transmembrane sialidase, NA, cleaves the sialic acid residues, releasing the virus. In addition, NA also prevents viral aggregation by cutting the sialic acids present on the viral envelope and disintegrates mucins in respiratory tract secretions, allowing better viral penetration into the respiratory epithelium.
Current antiviral compounds in use or currently in trials
One of the most effective means of preventing influenza infection is through vaccination. Influenza vaccines are offered in both an inactivated trivalent form and a live attenuated influenza nasal spray. Currently, a third option for people over 65 years of age is offered called Fluzone High-Dose, which contains more antigen than the regular inactivated vaccine, producing a stronger immune response in older individuals (although it is not known whether this effect is more protective against influenza) (19). Although all these options might protect against influenza infection, their efficacy has several limitations including low efficiency in the immunocompromised and infants (1). Even more important is that antigenic drift/shift in both the HA and NA influenza proteins could generate virus strains resistant to neutralization.
A recent novel approach, however, has been taken to address the problem of varying influenza strains and vaccination. Wei et al. is one of the first to devise a strategy for a "universal" influenza vaccine by using a combination of gene-based vaccination and seasonal boaster shots. In this study, the group showed that combinations of H1N1 gene/booster vaccination increases neutralization of diverse H1N1 strains in mice and ferrets. Furthermore, sera from mice immunized also defused other influenza strains such as H2N2 and H5N1. One of the main differences in this vaccine strategy was that the antibodies produced were aimed at the conserved stem region of HA, thus providing a basis for a universal vaccine and subsequent clinical trials (2).
In contrast to vaccines, antiviral compounds can be used for treatment and prophylaxis of influenza. Two classes currently in the market include: M2 inhibitors, such as rimantadine and amantadine (adamantanes) and neuraminidase inhibitors (NAIs) oseltamivir and zanamivir (60). Adamantanes are only effective in influenza A cases, but their use is not recommended due to the high influenza resistance seen since the 2003-2004 season. This resistance has only grown with 100% resistance among influenza A (H3N2) and 2009 pandemic H1N1 viruses tested in 2009-2010(3). It has been found that a mutation at amino acid 31 in the M2 gene conferred influenza resistance to adamantanes (4).
In contrast to adamantanes, NAI's remain the antiviral of choice for prevention/treatment of both influenza A/B; resistance has only been found in those isolates with a His274Tyr mutation in the NA enzyme. CDC reports show that circulating strains in this flu season (2010-2011) - 2009 pandemic H1N1, H3N2 and influenza B viruses- were mostly sensitive to oseltamivir and zanamivir, with detected resistance having limited public health impact (5,6). In the search to treat all types of influenza disease, advances in the administration of NAI are being sought to include patients with pneumonic influenza. This is especially important for zanamivir, as the typical inhalant form of the drug may be difficult for these patients. Currently, intravenous zanamivir has been evaluated in phase 2 trials, where healthy individuals exposed to viral challenge were protected against experimental infection (7). In addition, intravenous zanamivir was protective in a primate model of H5N1 infection , resulting in the Southeast Asia Influenza Clinical Research Network developing a protocol to study it in patients infected with H5N1(8).
Other NAIs in development include Peramivir and CS-8958. Peramivir has a longer half-life and binds to NA for longer than current NAIs, resulting in less frequent dosing. Further clinical trials will determine whether this translates into greater clinical effect (8). CD-8958, a long acting NAI, is currently in phase III clinical trials after phase II trials in Japan showed that a single inhaled dose was as effective as a standard 5-day treatment with oseltamivir in influenza infection. CD-8958 also showed efficacy against H5N1 avian influenza virus as well as influenza A and B (9).
Another novel strategy in phase II trial is T-705, a nucleoside like peptide, whose primary mechanism of action is the inhibition of viral RNA polymerase. Not only does T-705 display less toxic side effects than the similar acting ribavirin in human cells, but T-705 has shown inhibition of activity for many influenza B, A and C viruses in vitro, as well as in vivo/in vitro activity against pandemic swine originating H1N1 and H5N1(10-12).
While pathogen-targeted approaches has shown promise for treatment and prophylaxis of influenza, a host-targeting approach might be more appealing due to lower resistance potential and wider specificity. One such development is DAS181, a recombinant protein containing both a sialidase catalytic domain and an anchoring domain, which ensure its accumulation at its intended site of action (52,8).DAS181 would act by cleaving sialic acids residues and inhibiting viral entry into airway epithelium . By targeting a host component used by all strains, DAS181 has wide spectrum activity against both pandemic and seasonal influenza. This was proven by studies showing both in vitro and ex vivo inhibition of influenza virus replication in human airway epithelium cultures (HAE), human bronchial tissue sections, human lung biopsy tissue sections, and Madin-Darby kidney cells (56). In vivo data also demonstrated DAS181 efficiency by its inhibition of influenza replication or/and reduction of disease severity in mouse and ferret models for both seasonal and pandemic H1N1, H5N1, and H3N2, and influenza virus B. It is particularly important to mention that DAS181 resistant mutants were unstable and had low level resistance against DAS181 (58,59). This last fact is vital because it means that even if DAS181 induces selective pressure on influenza strains, these will most likely have reduced fitness.
Eventhough DAS181 is currently in phase II clinical trials (60), there are still potential concerns with its use. Hong Zhang, raised the issue that sialidase treatment could result in increased secondary bacterial infection, especially that by Streptococcus pneumoniae, since the NA protein ( a sialidase) has been linked to this effect. Studies by Hedlund et al. supported refuted this by showing that DAS181 did not increase S.pneumoniae colonization and actually protected mice against pneumonia compared with control animals (63).
All current approaches and potential interventions run the risk of resistance. In order to avoid this, many papers have raised the possibility of using combination chemotherapy. Support for reduced resistance for M2 inhibitors when combined with NAIs was seen in studies with combination therapy using zanamivir and rimantadine (clinical) or amantadine and oseltamivir (in vitro)(76,77). Additionally combination therapy of oseltamivir with amantadine or rimantadine was more efficient than monotherapy with oseltamivir in preventing death by H5N1 or H9N2 in mice (50). Furthermore, lethal challenge of H5N1 in mice treated with combination therapy with amantadine and oseltamivir produced a 90% survival rate compared to 60% and 30% with monotherapies (43). All these studies provide some evidence that combination therapy can not only be more efficacious, but can also reduce resistance among influenza strains.
Novel Approaches not yet in clinical trials
With the emerging threat of a new infectious influenza strain, inexpensive and efficient adjunctive therapies are constantly being discovered. One such intervention is a low pH nasal gel, created and evaluated by Rennie et al. for its safety and efficiency against influenza viruses. It is thought that exposure of the HA protein to low pH could mimic the environment of the endosome and cause untimely conformational changes that inhibit HA-receptor binding. Accordingly, this group found that both H3N2 and avian H3N2 were quickly inactivated by contact with solutions of pH 3.5 in vitro. Further in vivo studies with the ferret model showed that the intranasal spray, with an mucoadhesive gel, reduced the severity of acute influenza infection. The addition of the muchoadhesive gel increased the nasal retention of the solution, an important limitation to nasal delivery, and could be applied once a day. Moreover, because this product is non-specific and simple to make, there is less probability of resistance and could be readily available in an epidemic (13).
A second strategy for the treatment/prevention of influenza is RNA interference, where synthetic siRNA can result in specific degradation of target viral RNA (14). Experiments performed in the mouse model with anti-NP and anti-PA siRNA, showed that these inhibited the growth of H1N1, H5N1, H7N7, and Avian H9N2 and protected mice from lethal influenza challenge (15, 16). Clinical application of siRNA however, needs to address issues such as siRNA triggering of some immune reactions, safe transfecting agents, and stability of siRNA in body fluids (14)(64).