Critical Care Medicine:
Influenza pathogenesis: Lessons learned from animal studies with H5N1, H1N1 Spanish, and pandemic H1N1 2009 influenza
Meunier, Isabelle MSc; Pillet, Stéphane PhD; Simonsen, J. Neil BSc, MD, DTM&H, ABIM, FRCPC; von Messling, Veronika DrMedVet
From INRS-Institut Armand-Frappier (IM, SP, VvM), University of Quebec, Laval, Canada; Public Health Agency of Canada (JNS), National Microbiology Laboratory, Winnipeg, MB, Canada.
Dr. von Messling was supported by a New Investigator Award of the Canadian Institutes of Health Research. Ms. Pillet received a postdoctoral fellowship from the Fonds de la Recherche en Santé du Quebec. Ms. Meunier holds a scholarship from the Armand-Frappier Foundation.
The authors have not disclosed any potential conflicts of interest.
For information regarding this article, E-mail: firstname.lastname@example.org
Because cases of highly pathogenic influenza are rare, no systematic clinical studies have evaluated different therapeutic approaches. Instead, treatment recommendations are aimed at the alleviation of clinical signs and symptoms, especially the restoration of respiratory function, and at the inhibition of virus replication, assuming viral load is responsible for disease phenotype. Studies of highly pathogenic influenza in different animal models, especially nonhuman primates and ferrets, reproduce many of the key observations from clinical cases. Host-response kinetics reveal a delayed but broad activation of genes involved in the innate and acquired immune responses (innate responses produce inflammatory responses), which continue after the virus has been cleared and may contribute importantly to the clinical signs observed.
Experimental animal models point to an important role for immune dysregulation in the pathogenesis of highly pathogenic influenza. The use of these models to develop and validate therapeutic approaches is just beginning, but published studies reveal the importance of early treatment with antivirals and show the potential and limitations of approaches aimed at the host response.
The infection of 18 individuals in Hong Kong in 1997 with H5N1 highly pathogenic avian influenza strain, which resulted in six deaths, marked the first reported fatal outbreak of an avian influenza virus (1). Since then, H5N1 viruses have been associated with a broad range of clinical outcomes, from mild infections, primarily in children younger than 12 yrs, to severe respiratory illness and death, mostly in healthy adults (2, 3). Complications in severe cases included acute respiratory distress syndrome, leukopenia, lymphopenia, hemophagocytosis, and multiorgan dysfunction/failure. The unusual frequency of gastrointestinal symptoms, hematologic disorders, and hepatic and renal dysfunction in combination with viremia and detection of viral RNA in extrapulmonary tissues and fluids suggest a systemic dissemination in some patients (2–4). Severe and fatal cases also were associated with high chemokine and cytokine levels, indicating that immunopathology may contribute to the disease phenotype (4, 5).
H5N1 HIGHLY PATHOGENIC AVIAN INFLUENZA
In animal models, the different H5N1 strains are characterized by a wide range of infectivity, tropism, clinical signs, and mortality rate (6–11). Generally, the virulence of a given isolate correlates with its ability to replicate efficiently in the lower respiratory tract of the respective species, including mice, in which these viruses cause lethal disease without the previous adaptation generally required for human influenza A viruses (7, 8, 12). In mice, ferrets, and nonhuman primates, severe disease is associated with spread beyond the respiratory tract, especially to the gastrointestinal tract and the central nervous system (7, 9–12). The gastrointestinal tropism and the ability to infect mice and ferrets via the digestive system suggest a potential for fecal–oral transmission of these viruses (13), although human epidemiologic studies do not support an important role for fecal–oral transmission in influenza epidemics (Table 1).
On infection with clinical isolates, ferrets closely reproduce the disease severity and clinical signs observed in the similarly infected patient, including high fever, weight loss, anorexia, extreme lethargy, diarrhea, and, in some cases, neurologic signs. The morbidity of avian isolates, however, varies from highly pathogenic to asymptomatic (9, 10, 14). Anorexia, fever (>40°C), depression, coughing, signs of acute respiratory distress syndrome, and diarrhea also have been observed in macaques infected with highly pathogenic H5N1 viruses (6, 11, 128). As observed in other animal models, pathogenesis and lethality in mice are strongly strain-dependent and, to a lesser extent, dose-dependent (7, 10). Mice infected with a lethal dose begin to lose weight within 2 days, showing signs of illness (such as ruffled fur and listlessness) during the first week of infection, and they die after 7 to 9 days (10).
Regardless of the animal model, histopathological changes in the lung are characterized by extensive bronchiolitis and alveolitis, edema, and focal hemorrhage starting as early as 24 hrs after infection (6). Type II pneumocytes are the primary target of infection, and antigen-positive epithelial cells in the lung are generally found in close proximity to damaged, necrotic bronchi, either lining the bronchi or extracellularly within the bronchiolar lumen in association with necrotic debris (7, 9, 15). At later disease stages, focal immunostaining of inflammatory cells, mainly mononuclear cells, is found in subepithelial tissues in the pulmonary interstitium and in association with hemorrhage (7, 9).
Outside the respiratory tract, diffuse vacuolization of the hepatocellular cytoplasm, consistent with fat, portal tract biliary duct necrosis, mononuclear infiltrates, periportal hemorrhage, and hepatocellular necrosis were observed in the liver of ferrets infected with a highly pathogenic strain (9). In the neuropil of the olfactory bulb, cerebrum, and brainstem of these ferrets, scattered foci of marked neuronal degeneration and neuronophagia associated with inflammatory cell infiltrates were observed (9), whereas viral antigen was found glial cells and neurons of infected mice (7).
In all animal models, highly pathogenic H5N1 strains cause a massive and sustained infiltration of macrophages and neutrophils associated with a strong transcriptional induction of pro-inflammatory cytokines and chemokines in the lung, especially high levels of IL-6, tumor necrosis factor-α, interferon (IFN)-γ, and CXCL-10 (6, 10, 16, 17). In mice, sustained expression of monocyte chemoattractant protein-1 and macrophage inflammatory protein-1α also was detected (7) and although macrophage inflammatory protein-1α was not critical for virus replication and spread in this animal model (18), it has been associated with fatal outcomes in human infections (4). In addition to the increase of local cytokine and chemokine expression in the lung, high cytokine levels are also detected in the blood, indicative of a general and possibly excessive immune activation (6).
All influenza infections cause a transient lymphopenia, but the extent is more pronounced in animals infected with highly pathogenic strains (6, 10, 19). In macaques, circulating CD4+ and CD8+ T cells decrease within the first 2 days after infection (6), and cell suspension analysis reveals a reduction of cellularity and an alteration of the relative proportion of CD4+ and CD8+ cells in the thymus of mice infected with highly pathogenic H5N1 strains (6, 19).
H1N1 1918 SPANISH INFLUENZA
The 1918 to 1919 H1N1 pandemic killed as many as 50 million people worldwide and remains unprecedented in its severity. A first, mild wave in the spring of 1918 was replaced by a second wave in September to November 1918 that resulted in mortality rates >2.5%, compared to <0.1% typically recorded for seasonal influenza outbreaks. A third wave with equally high mortality rates swept around the world in 1919 (1). Histopathological analysis of lung tissues from individuals who died of primary influenza pneumonia in 1918 frequently showed severe pulmonary edema and/or hemorrhage with acute alveolitis and bronchopneumonia accompanied by a rapid destruction of the respiratory epithelium (20, 21). Genomic RNA of the 1918 virus was recovered from archived formalin-fixed lung tissues and from an Alaskan influenza victim who was buried in permafrost in November 1918 (22, 23), allowing the generation of a recombinant H1N1 1918 (H1N1 1918/rec) virus using plasmid-based reverse genetics (24). This recombinant virus is now used to investigate the determinants of the exceptional virulence of the Spanish influenza in different animal models.
The H1N1 1918/rec virus replicates efficiently in the upper and lower respiratory tract of all tested animal models (25–28); as in highly pathogenic H5N1 viruses, H1N1 1918/rec does not require adaptation to infect mice (24). In nonhuman primates, infectious virus is detected up to 8 days after infection in tissues of the upper and lower respiratory tract, whereas seasonal strains are restricted to the upper respiratory tract and are generally cleared by day 6 (26). Similar to seasonal viruses, H1N1 1918/rec is restricted to the respiratory tract in mice (24) and ferrets (25), and only low amounts of viral RNA were detected in the heart and spleen of some infected macaques (26) (Table 1).
In mice, ferrets, and nonhuman primates, infection with H1N1 1918/rec results in the onset of severe clinical signs within 1 to 2 days after infection and mortality rates ranging from 50% to 100% (24, 29, 30). Clinical signs in ferrets and nonhuman primates include lethargy, anorexia, rhinorrhea, sneezing, severe weigh loss, high fever, and, ultimately, respiratory distress syndrome leading to death in 50% to 75% of the animals within 2 wks (26, 31). In primates, an increased respiration rate and a decrease in blood oxygen saturation of as much as 36%, compared to preinfection levels, are also observed (26).
Infection with the H1N1 1918/rec virus results in widespread lung lesions, including necrotizing bronchitis, moderate-to-severe peribronchial and alveolar edema, alveolar hemorrhage, bronchiolitis, and moderate-to-severe alveolitis in all animal models (25–28, 32). The distribution of the alveolitis varies from peribronchial to diffuse, and it is composed of neutrophils and macrophages (32). Neutrophils are the predominant inflammatory cells, but alveolar macrophages are also frequently found (32). In macaques, widespread detection of antigen in plump alveolar cells and desquamation of these cells into the alveolar space is a prominent characteristic of the H1N1 1918/rec virus-infected lung tissue (26). However, in contrast to H5N1, no viral antigen is detected outside the respiratory tract for any of the animal models.
Although they are a natural host and may play an important role in influenza epidemiology, pigs infected with H1N1 1918/rec did not have severe respiratory signs or die (27), indicating that the relevance of this animal model for human influenza pathogenesis may be limited.
The infiltration of inflammatory cells in the lungs is accompanied by a significant increase of pro-inflammatory cytokines and chemokines, which correlates with the disease severity observed in the different animal models (15, 17). The global gene expression profiles in bronchi of infected macaques revealed that both a seasonal H1N1 strain and the highly pathogenic H1N1 1918/rec virus activate the transcription of several inflammatory chemokine and cytokine genes, including IL-6 (26); however, the IL-6 messenger RNA was expressed at a high level until day 8 only in the H1N1 1918/rec-infected macaques. Higher levels of IL-6 also were observed in the serum of the H1N1 1918/rec-infected animals, which reflects a systemic alteration of the immune response (26), suggesting a prominent activation of cells of the monocyte/macrophage lineage. Comparison of the H1N1 1918/rec virus with a highly pathogenic H5N1 strain in mice demonstrated higher levels of cytokines and chemokines in the lungs of the H5N1 group, whereas overall lung cellularity, virus growth, and patterns of immune cell subpopulation dynamics over time were very similar for both viruses (17), suggesting a common immunopathogenetic mechanism (Table 1).
PANDEMIC H1N1 2009 INFLUENZA
The ongoing H1N1 pandemic started in the Mexican town of La Gloria, Veracruz, in mid February 2009. This new strain is thought to be a reassortment of recent North American H3N2 and H1N2 swine viruses with Eurasian avian-like swine viruses (33).
So far, most human infections with pandemic H1N1 2009 viruses result in a self-limiting, uncomplicated disease with a clinical course similar to that observed for seasonal influenza. Clinical signs atypical for seasonal influenza have been reported, however, including vomiting and diarrhea in a relatively large proportion of cases (33). Furthermore, some patients have required hospitalization because of severe pneumonia and respiratory failure, with a fatal outcome occurring in 0.5% of laboratory-confirmed cases. In contrast to seasonal influenza, a substantial proportion of the cases of severe illness and death have occurred among previously healthy adults aged 18 to 50 yrs, as well as among adults with underlying disease and pregnant women (34). Additionally, the current H1N1 pandemic virus is spreading rapidly as seasonal influenza in the Northern Hemisphere disappears, suggesting a greater transmission efficacy (Table 1).
Tropism and Pathogenesis
The pandemic H1N1 2009 viruses readily infect ferrets and nonhuman primates, in which an efficient spread to the lower respiratory tract was observed. The viruses also replicate in mice without previous adaptation (34–36), thereby reproducing the tropism seen for highly pathogenic influenza viruses. No virus was detected in mice outside the respiratory tract, whereas viral RNA and low amounts of infectious virus were found in rectal swabs and intestinal tissue samples of ferrets (36). It remains unclear if viral replication occurred in the tissues of the lower gastrointestinal tract or if the detected virus originated from the nasopharynx.
In mice and ferrets, virulence and mortality is dose-dependent and strain-dependent, with more virulent strains reaching mortality rates of up to 100% in mice and 50% in ferrets if inoculated at higher doses (34–36). Infection of macaques with H1N1 resulted in a more prominent increase in body temperature than infection with seasonal strains, but no mortality was observed (35, 36).
Histopathology and Immune Responses
Infection of mice with pandemic H1N1 2009 viruses results in severe bronchitis and alveolitis with widespread detection of viral antigen on days 3 and 6 after infection, whereas only few antigen-positive bronchial epithelial cells, but not alveolar cells, were detected on day 3 after infection with a seasonal strain (35). In ferrets infected with more virulent isolates, multifocal necrotizing rhinitis, tracheitis, bronchitis, and bronchiolitis have been observed, as well as more severe bronchopneumonia, with prominent viral antigen expression in peribronchial glands and occasionally in alveolar cells (34, 35). In mice, pandemic H1N1 2009 viruses induce higher levels of pro-inflammatory cytokines and chemokines than seasonal strains. At later stages, strong induction of IFN-γ, IL-4, IL-10, and IL-5 is detected (35), suggesting a dysregulated host response.
APPROVED ANTI-INFLUENZA DRUGS
M2 Ion Channel Inhibitors
Influenza viruses enter cells by endocytosis, and the low pH of the endosome is necessary to activate fusion between viral and endosomal membranes, and subsequent release of the virus into the cytoplasm. The viral M2 protein, which forms an ion channel in the viral envelope, plays an essential role in this process by enabling the influx of H+ ions from the endosome into the virus particle. The class of M2 ion channel inhibitors includes the amantadanes, amantadine, and rimantadine, which sterically block the ion channel, thereby interfering with virus entry into the cell (37) (Fig. 1). Amantadine and rimantadine have been licensed in the US as antiviral drugs since 1966 and 1993, respectively. Historically, they have been mainly used for prophylaxis during outbreaks or to reduce the duration of uncomplicated influenza infections. Both drugs confer 80% to 90% protection against illness and lowered transmission (38–41). Furthermore, amantadanes decrease the duration of the clinical course even if given 48 hrs after infection (41). Today, rimantadine is the drug of first choice because of the gastrointestinal and central nervous system side effects of amantadine (38).
Because of widespread resistance in circulating strains, amantadanes are rarely used today. Resistant H3N2 viruses emerged in China during the 2003 season and quickly spread worldwide, reaching resistance rates >90% in Asia and the US, and nearly 50% in Europe for H3N2 subtypes; rates for H1N1 are slightly lower (42). Notably, most Asian H5N1 isolates are also resistant to this class of drugs, and the pandemic H1N1 2009 virus carries the resistance-conferring mutation (42, 43), rendering these drugs mostly obsolete for the treatment of circulating influenza strains.
The viral neuraminidase (NA) protein is involved in the early and late steps of infection. During virus entry, NA is thought to cleave sialic acid residues from mucin, a class of glycoproteins abundantly present in the airways, which may impede access to the target cell membrane. NA also plays a role in virus egress by removing sialic acids from the viral particle and surrounding cell surfaces to avoid aggregation of newly formed virions (44, 45). The class of approved NA inhibitors currently includes oseltamivir and zanamivir, which are small molecules that bind with high affinity to the active site of the NA enzyme (Fig. 1.). This region is highly conserved among influenza A and B viruses and, consequently, NA inhibitors are active against viruses from both genera (46), but oseltamivir is less effective than zanamivir against influenza B (47). Both drugs have been approved by the US Food and Drug Administration for the treatment and prevention of influenza since 1999.
In ferrets, the oseltamivir regimen effective against seasonal influenza does not control highly pathogenic H5N1 infections (48). Subsequent studies using H5N1 viruses revealed that the effective dose depends on the virulence and the time after infection. A low dose of oseltamivir is sufficient to protect ferrets against a lethal challenge when treatment is started within 4 hrs after infection, whereas the dose has to be almost tripled when the treatment starts after 24 hrs (48). In a H5N1 macaque model, prophylactic treatment with 10 and 20 mg/kg zanamivir intravenously resulted in an important reduction of gross pathology, pneumonia, and viral load, whereas treatment with the higher dose 4 hrs after infection was associated a similar effect on the viral load without improving lung pathology and pneumonia (49), illustrating the importance of rapid diagnosis and treatment. One concern unique to highly virulent influenza viruses, especially of the H5N1 subtype, is their possible dissemination to the brain. Drug distribution studies in rats suggest that oseltamivir is limited in its ability to cross the brain–blood barrier, and thus it may not be the drug of choice if neuroinvasion is suspected (50).
During the past 2 yrs, oseltamivir-resistant H1N1 viruses have emerged in Europe and Asia, and they are spreading rapidly across the world (51–53). So far, there is little resistance in the H3N2 subtypes, and most H5N1 subtypes remain NA inhibitor-sensitive (4, 54, 55). However, the first oseltamivir-resistant pandemic H1N1 2009 isolates have been reported in Asia and North America (56, 57), and these viruses are likely to spread because of the ongoing use of oseltamivir. In contrast to oseltamivir, resistance to zanamivir is less frequent, even though a recently identified mutation is associated with decreased susceptibility to zanamivir without affecting oseltamivir susceptibility. Viruses carrying this mutation accounted for 2.3% of isolates between 2006 and early 2008, and they were found to retain wild-type fitness in ferrets (58), suggesting that increased zanamivir use will also lead to the rapid emergence of resistance.
Ribavirin and Viramidine
Ribavirin is a guanosine analogue licensed for the treatment of hepatitis C in combination with IFN-α. It inhibits viral replication either indirectly by decreasing intracellular guanosine triphosphate levels through the inhibition of inosine 5′-monophosphate dehydrogenase or directly by interfering with transcription and genome replication (59) (Fig. 1.). Because of its multiple mechanisms of action, ribavirin inhibits a broad range of DNA and RNA viruses and resistance rarely develops (60).
In mice, prophylactic and postexposure oral or intraperitoneal ribavirin treatment results in decreased viral titers in the lungs and faster virus clearance but does not reduce mortality, whereas administration by aerosol greatly reduces viral load and increases survival rates (61–64). On aerosol administration, ribavirin is found in high concentrations in the lungs and lower concentrations in the blood, other organs, and the brain (65); this may be an advantage for the treatment of avian influenza viruses, which spread outside of the respiratory tract. In patients, oral administration of 1000 mg daily for 5 days, beginning 24 or 48 hrs after the clinical onset of influenza, did not improve clinical signs or viral shedding (66). In contrast, aerosol treatment alleviated symptoms and reduced the virus shedding from the respiratory tract (67, 68). Treatment of severe influenza with intravenous ribavirin resulted in reduction of viral shedding for one patient and complete virus clearance in two others (69). However, intravenous ribavirin may lead to hemolytic anemia, and its use is prohibited in pregnant women, one of the risk groups for influenza, because of its teratogenic properties.
Viramidine is a prodrug that is converted to ribavirin by adenosine deaminase. It is not taken up by red blood cells, which reduces the risk of hemolytic anemia (70). In mice, oral viramidine is as efficient as ribavirin in reducing H5N1 influenza morbidity and mortality (71), and it is better tolerated than ribavirin in phase I clinical trials.
APPROVED DRUGS WITH POTENTIAL ANTI-INFLUENZA ACTIVITY
In addition to drugs that are approved for treatment of influenza, several other Food and Drug Administration-approved drugs have been used to treat cases of severe influenza based on the known mechanism of action. Subsequent evaluation in animal models has contributed to the efficacy assessment of these drugs under more controlled circumstances and provides guidance for use in patients.
Interferons and Interferon Inducers
On infection, cells produce type I IFN, leading to the induction of an antiviral state and promoting the activation of the immune system. Type I IFN are commonly used to treat chronic hepatitis C and show promise for treatment of hepatitis B and severe acute respiratory syndrome (72–74). Earlier clinical studies showed that intranasal IFN treatment reduced clinical signs of influenza, but it did not prevent infection and may be associated irritation of the nasal mucosa and occasional nosebleed (75–77). More recently, the anti-influenza efficacy has been assessed systematically in animal models. In mice and guinea pigs, pretreatment with a single dose of IFN-α considerably reduced lung titers and prevented mortality from lethal challenge with H1N1 1918/rec or a highly virulent H5N1 (28, 78). In ferrets, IFN-α pretreatment was at least as effective as oseltamivir at a dose of 2.5 mg/kg twice daily, resulting in reduced nasal wash titers and milder clinical signs after infection with seasonal influenza strains, but it had no beneficial effect against H5N1 (79). In addition to IFN, the therapeutic effect of IFN inducers such as poly(I:C) against influenza is being investigated. Prophylactic treatment with liposome-encapsulated poly(I:C) protected mice against mouse-adapted influenza strains (80), and clinical trials are underway to evaluate the toxicity of the compound.
The use of immunomodulators, particularly steroids and their derivatives, has been examined mostly in the context of H5N1 infections, which cause severe-to-fatal disease that is characterized by fulminant pneumonia and multi-organ failure associated with an excessive inflammatory response (2, 81). Treatment with corticosteroids was not associated with increased patient survival during H5N1 outbreaks in Hong Kong, Vietnam, and Thailand (2, 81, 82). The World Health Organization does not recommend the use of corticosteroids except in cases of septic shock with suspected adrenal insufficiency.
Only triamcinolone doses >4 mg/kg resulted in notable reduction of pulmonary lesions and suppression of immune activation in cotton rats infected with seasonal influenza viruses (83), demonstrating that a high-threshold steroid concentration is necessary for efficient treatment. However, no improvement associated with corticosteroid treatment was seen in mice infected with a highly virulent H5N1 strain (84).
As mentioned, influenza viruses enter target cells by endocytosis, and a low pH is required to trigger fusion of viral and endosomal membranes. Chloroquine is a weak base that is commonly used to treat malaria. In the cell, the drug accumulates in acidic organelles, such as endosomes and lysosomes, where it increases the pH (85, 86) (Fig. 1). Chloroquine has been shown to inhibit many viruses that require low pH for entry and is considered a possible treatment for severe acute respiratory syndrome and human immunodeficiency virus (87). Even though in vitro studies show that chloroquine interferes with H1N1 and H3N2 replication (88), it does not prevent weight loss associated with infection in mice or result in decreased viral replication in the nose of the ferrets (89).
ANTIVIRAL DRUGS IN DEVELOPMENT
All available influenza-specific drugs are limited by the rapid emergence of resistance once they are broadly used. Therefore, a global effort is ongoing to develop new antiviral drugs that either target different steps in the viral life cycle or modulate the immune system. Several of the more promising candidates are already undergoing preclinical evaluation or are in clinical trials.
To prevent virus attachment to the target cell, molecules can target the receptor-binding or fusion domains in the viral attachment protein HA or sialic acids, the cellular receptor. Different molecules, mainly multivalent substrate analogues and blocking peptides, have been designed that interfere with these initial virus–host cell interactions (90–92) (Fig. 1).
The recombinant fusion protein DAS181 (Fludase; NexBio, San Diego, CA), which is composed of the catalytic domain of Actinomyces viscosus and an epithelium-anchoring domain, prevents infection by cleaving-off sialic acids present at the cell surface of the airway epithelium. In mice, DAS181 pretreatment resulted in improved lung function, less pathology, significantly reduced lung titers, and inhibition of systemic dissemination after H5N1 infection (93, 94). Postexposure treatment was beneficial if initiated 48 hrs after infection in the case of H1N1 infection (94), whereas the control of H5N1 infection required treatment within 24 hrs (93). In ferrets, treatment with 1000 U/day (>1 mg/kg/day) for 7 days starting 2 days before infection greatly reduced virus shedding and prevented disease after challenge with a human seasonal virus without signs of toxicity (94). Recently completed phase Ia studies with this compound demonstrate that repeat doses of DAS181 are well-tolerated and have no toxic effects (95).
Because activation of the HA protein requires extracellular proteases, the antiviral activity of different protease inhibitors has been evaluated. Given intraperitoneally or intranasally, the trypsin-like protease inhibitor aprotinin reduced lung titers and resulted in a 35% mortality reduction on challenge with a mouse-adapted virus (96). Aprotinin inhalation also reduced clinical signs of influenza and parainfluenza infections in clinical trials (97). The development of inhibitors specifically targeting the HA receptor-binding domain is less advanced. Given prophylactically, a synthetic polymeric attachment inhibitor prevented H5N1 morbidity and mortality in mice. However, when treatment was initiated 6 hrs after infection, only a modest decrease in lung titers was observed (92).
Several new NA inhibitors are in advanced stages of development, such as peramivir (RWJ-270201, BCX-1182) and other cyclopentane and cyclopentane amide derivatives, pyrrolidines (A-192558, A-315675) and other pyrrolidine derivatives, and 2,3-disubstituted tetrahydrofuran-5-carboxylic acid derivatives.
Peramivir has been most extensively studied. A prophylactic dose of 10 to 20 mg/kg injected intramuscularly prevented or greatly reduced mortality in mice after H1N1 or H3N2 challenge, respectively (98). A single intramuscular dose followed-up by 7 days of oral treatment was as effective as oseltamivir in protecting against infection with a highly virulent H5N1 strain. When treatment was started 24 or 48 hrs after a lethal challenge, 78% and 56% of the mice survived, respectively (99). In ferrets, peramivir treatment initiated 1 hr after infection and daily for 4 days thereafter resulted in improved survival after infection (100). However, in humans, oral peramivir did not provide good protection, which was attributed to the low oral bioavailability (<5%) of the drug (101). The efficacy of intravenous or intramuscular routes of inoculation is being evaluated in phase III clinical trials.
RNA Polymerase Inhibitors
In addition to viral transcription and replication, the influenza polymerase complex also possesses an endonuclease activity that allows the virus to synthesize the viral messenger RNA using the capped primers of the host (102). Several compounds targeting the replication or endonuclease activities are in development (Fig. 1.).
To date, two nucleoside analogues have been tested for anti-influenza activity: 2′-deoxy-2′-fluoroguanosine and T-705; 2′-deoxy-2′-fluoroguanosine has only been evaluated in vitro (103, 104), but T-705 (Toyama Chemical, Tokyo, Japan), which acts as a purine nucleoside analogue, is better-characterized (105). Unlike ribavirin, T-705 does not affect the host DNA or RNA synthesis and has a better therapeutic index in preclinical tests. Treatment of mice once, twice, or four times daily for 5 days starting 1 hr after H5N1 infection with 30 to 300 mg/kg daily prevented lung pathology and mortality. In a direct comparison, T-705 was less efficient than zanamivir and oseltamivir but better than ribavirin (106). Phase I/II trials with T-705 are ongoing in the US and Japan, and clinical data are accumulating (107).
Small Interfering RNA
Small interfering RNA are RNA duplexes of 21 t o 25 nucleotides that recognize specific RNA and trigger their degradation through a process called RNA interference (108). Directed against conserved regions of different viral proteins, small interfering RNA are very efficient in vitro (109) (Fig. 1.). In mice, pretreatment with influenza-specific small interfering RNA reduced the viral load in the lungs and protected against lethal challenge with H5N1, H1N1, and H7N7 viruses (110, 111). In a rhesus macaque severe acute respiratory syndrome model, small interfering RNA were efficient when used 5 hrs or 24 hrs after infection, highlighting their therapeutic potential against respiratory infections (112).
PASSIVE IMMUNOTHERAPY AND VACCINE DEVELOPMENT
To date, passive immunotherapy has been used for patients at high risk for several viruses, including rabies, hepatitis A, and respiratory syncytial virus (113). During the 1918 pandemic, severely ill patients treated with convalescent sera had a case fatality rate of 16%, whereas 37% of untreated patients died (114). More recently, treatment of two H5N1-infected patients with plasma from a convalescent patient resulted in fast recovery (115, 116).
Monoclonal antibodies against the HA of H1 and H2 viruses administered 2 days after infection increased survival in mice (117), and whole antibodies or Fab fragments protected SCID mice from lethal H1N1 infection (118, 119). Furthermore, humanized antibodies and human monoclonal antibodies developed from a patient with Vietnamese H5N1 protected mice against lethal challenge, even when administered up to 3 days after infection (120, 121). These animal studies demonstrate the potential of passive antibody transfer as influenza treatment, and much work is focusing on this therapeutic approach, especially with the emergence of biologically robust NA-resistant strains.
Because of the acute nature of influenza infections, prophylactic vaccines will remain the most efficient and cost-effective control measure. With the exception one cold-adapted live-attenuated vaccine, all North American influenza vaccines are inactivated and contain either detergent-split virions or further purified viral glycoproteins. All these vaccines are grown in embryonated chicken eggs and include one previously chosen H1N1 and H3N2 strain together with an influenza B virus, which are changed annually in response to the ongoing antigenic drift (122). In Europe, a subunit vaccine adjuvanted with MF59, an oil-in-water emulsion, has been in use since 1997 (123), and a cell culture-produced inactivated vaccine was approved in 2007 (124). It is thus likely that these or similar formulations will be approved in North America in the foreseeable future.
The worldwide effort to prepare for a possible influenza pandemic has resulted in the development of a broad range of candidate vaccines. Even though a universal vaccine that protects against all influenza subtypes remains elusive, the inclusion of more conserved internal viral proteins has resulted in protection against different strains from the same subtype and even a certain level of heterosubtypic immunity in different animal models (125, 126). To increase the level and duration of the immune response, various adjuvants have been developed, including specific stimulators of immune-signaling pathways and immunogenic proteins, in addition to the latest generation of oil-in-water emulsions (127). Several of these compounds have shown efficacy in different animal models and are being evaluated in clinical trials.
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