Detection of Molecular Markers of Drug Resistance in 2009 Pandemic Influenza A (H1N1) Viruses by Pyrosequencing

The M2 blockers amantadine and rimantadine and the neuraminidase (NA) inhibitors (NAIs) oseltamivir and zanamivir are approved by the FDA for use for the control of influenza A virus infections. The 2009 pandemic influenza A (H1N1) viruses (H1N1pdm) are reassortants that acquired M and NA gene segments from a Eurasian adamantane-resistant swine influenza virus. NAI resistance in the H1N1pdm viruses has been rare, and its occurrence is mainly limited to oseltamivir-exposed patients. The pyrosequencing assay has been proven to be a useful tool in surveillance for drug resistance in seasonal influenza A viruses. We provide a protocol which allows the detection of adamantane resistance markers as well as the I43T change, which is unique to the H1N1pdm M2 protein. The protocol also allows the detection of changes at residues V116, I117, E119, Q136, K150, D151, D199, I223, H275, and N295 in the NA, known to alter NAI drug susceptibility. We report on the detection of the first cases of the oseltamivir resistance-conferring mutation H275Y and the I223V change in viruses from the United States using the approach described in this study. Moreover, the assay permits the quick identification of the major NA group (V106/N248, I106/D248, or I106/N248) to which a pandemic virus belongs. Pyrosequencing is well suited for the detection of drug resistance markers and signature mutations in the M and NA gene segments of the pandemic H1N1 influenza viruses.In the spring of 2009, an antigenically novel influenza A virus (H1N1) was detected in North America (7). The rapid widespread transmission of the virus resulted in the declaration of an influenza pandemic by the World Health Organization (WHO) (42). The 2009 pandemic influenza A (H1N1) virus (H1N1pdm) was determined to be a reassortant with a combination of gene segments that had not been previously described (12, 21). Phylogenetic analysis of the full genome sequences revealed that in the late 1990s, reassortment between seasonal influenza A virus (H3N2), classical swine influenza virus, and North American avian influenza viruses led to the appearance of triple-reassortant H3N2 and H1N2 swine influenza viruses that have since circulated in pigs in North America (40). The pandemic virus was a result of further reassortment between a triple-reassortant swine influenza virus and a Eurasian avian influenza virus-like swine influenza virus, resulting in the acquisition of two gene segments, coding for the M protein and neuraminidase (NA), from the Eurasian avian influenza virus-like swine influenza virus lineage. Recent genome sequence analysis performed with pandemic viruses collected in different regions found variants with characteristic amino acid changes, including 2 amino acid changes in the NA (21, 29). The reports identified three NA variants among the H1N1pdm viruses: one variant group has V106 and N248 (referred to as the A/California/04/2009 group); the second variant, named the A/Osaka/164/2009 group, is characterized by I106 and N248; and the third NA variant group contains I106 and D248, such as the A/New York/18/2009 strain.Currently circulating triple-reassortant swine influenza viruses in the United States do not contain any known markers of adamantane resistance (L26F, V27A, A30V, A30T, S31N, and G34E) (10, 25), whereas the Eurasian avian-like influenza viruses as well as the pandemic virus contain the adamantane resistance-conferring change S31N in the M2 protein. Currently, two classes of antiviral drugs are approved for use by the FDA for the control of influenza virus infections: adamantanes (M2 blockers) and neuraminidase inhibitors (NAIs). Resistance to adamantanes makes the NAIs oseltamivir and zanamivir the only pharmaceutical options available for use for the control of infections caused by the pandemic virus. Monitoring of resistance to NAIs is mainly based on the NA inhibition assay (23, 39, 41), which allows the detection of resistance conferred by known and novel mutations. However, the NA inhibition assay requires virus isolation and propagation, and the detection of resistance by the NA inhibition assay requires confirmation by sequencing of the NA gene segment to identify the markers of resistance and their presence in the original clinical material.Prior to the 2007-2008 influenza season, the frequency of resistance to NAIs had been very low (<0.5%) among field isolates (28, 35, 36). During the 2007-2008 influenza season, seasonal H1N1 viruses resistant to oseltamivir emerged and spread globally (3, 17, 31, 39), and by April of 2009, the majority of the H1N1 viruses were resistant to oseltamivir but sensitive to zanamivir. Of note, nearly all of the 2009 pandemic H1N1 viruses were sensitive to NAIs (8); only sporadic cases of oseltamivir-resistant viruses with the H275Y mutation in the NA gene segment were reported to the WHO, and they were mainly detected following antiviral drug treatment (5, 6, 42). The H275Y mutation is equivalent to the H274Y mutation in the N2 subtype amino acid numbering. Throughout the text, amino acids are described with the N1 numbering, and the corresponding N2 amino acid numbering is shown in parentheses, when it differs from the N1 numbering. Recent reports on the emergence of oseltamivir resistance highlight the need for close monitoring of the susceptibility of the pandemic H1N1 virus to the available drugs (5, 6, 42). Such information is needed to make informed decisions on measures aimed at managing pandemic virus infections.The molecular markers of NAI resistance are type and subtype specific and are also drug specific (1, 23). The H275Y (H274Y) change is the most commonly reported mutation conferring resistance to oseltamivir in the N1 subtype of NA. This change has been reported not only in seasonal H1N1 viruses but also in highly pathogenic H5N1 viruses (13, 22, 23, 31, 33). The H275Y (H274Y) mutation is also known to reduce susceptibility to the investigational NAI peramivir (23). The amino acid replacement N295S (N294S) in N1 has also been shown to reduce susceptibility to oseltamivir and zanamivir (33, 43). In addition, recent studies have demonstrated that mutations in other residues located in and around the NA active site can alter the susceptibilities of viruses to NAIs. For instance, changes at residues V116, I117, E119, Q136, D199 (D198), and I223 (I222) were associated with reduced susceptibility to NAIs in both seasonal and H5N1 viruses (26-28, 30, 32, 39). Moreover, crystal structure studies with the NAs of H1N1 and H5N1 viruses (9, 37) suggested that mutations at amino acids Q136, K150, and D151 (37) may affect susceptibility to oseltamivir and zanamivir, presumably by interfering with the binding of the drug to the NA. Changes at these residues were reported to reduce the susceptibilities to NAIs of viruses with the N1 enzyme (34; CDC, Atlanta, GA, unpublished data).It is important to develop the tools necessary for the rapid detection of NA markers known or suspected of affecting susceptibility to NAIs. Pyrosequencing has previously been shown to provide a rapid and high-throughput method for the detection of molecular markers of drug resistance in seasonal as well as highly pathogenic avian influenza viruses (4, 8, 15, 16, 19, 30, 31, 38).Here we report on the design and validation of pyrosequencing assays for the detection of signature markers in the M2 and NA gene segments of the pandemic H1N1 viruses.     

DAS181 Inhibits H5N1 Influenza Virus Infection of Human Lung Tissues

DAS181 is a novel candidate therapeutic agent against influenza virus which functions via the mechanism of removing the virus receptor, sialic acid (Sia), from the adjacent glycan structures. DAS181 and its analogues have previously been shown to be potently active against multiple strains of seasonal and avian influenza virus strains in several experimental models, including cell lines, mice, and ferrets. Here we demonstrate that DAS181 treatment leads to desialylation of both α2-6-linked and α2-3-linked Sia in ex vivo human lung tissue culture and primary pneumocytes. DAS181 treatment also effectively protects human lung tissue and pneumocytes against the highly pathogenic avian influenza virus H5N1 (A/Vietnam/3046/2004). Two doses of DAS181 treatment given 12 h apart were sufficient to block H5N1 infection in the ex vivo lung tissue culture. These findings support the potential value of DAS181 as a broad-spectrum therapeutic agent against influenza viruses, especially H5N1.Since 1997, the highly pathogenic avian influenza virus H5N1 subtype has been causing epidemics in wild and domesticated birds. From 2003 to March 2009, 411 cases of human infections with the avian H5N1 virus have been confirmed and over 60% of the cases were lethal (http://www.who.int/csr/disease/avian_influenza/country/cases_table_2009_03_11/en/index.html). These events have heightened public awareness of an impending influenza pandemic. However, investigation of H5N1 virus pathogenesis has been hampered not only by the strict biosafety requirement for handling the virus but also by the lack of a readily available experimental model that faithfully replicates the human respiratory system, as animal models differ in their similarity of binding and infection for human and avian influenza viruses (18, 19) Several experimental models have been used to study influenza virus infection in humans, including human cell lines; short-term cultures of in vitro differentiated epithelia, e.g., well-differentiated human airway epithelia (5, 16); and ex vivo cultures of human tissues (8, 11). Each of these has its advantages and disadvantages. We (8) and others (11) have demonstrated that short-term cultures of normal nasopharyngeal tissue, primary pneumocytes, and lung tissue sections can support H5N1 virus infection. These ex vivo human airway tissues or primary cells can potentially be more relevant models for investigating viral tropism and new antiviral agents since they are directly derived from normal human airway epithelia with little in vitro manipulation.The existing influenza virus therapeutic agents recognize various viral components as molecular targets. In recent years, modifying host cells as a way to interrupt the life cycle of a pathogen has emerged as a novel approach to tackle infectious diseases, and DAS181 is the first anti-influenza virus therapeutic candidate that targets the host cells rather than the virus. Evidence accumulated since the 1940s has indisputably demonstrated the key role of sialic acid (Sia) as the receptor for influenza virus infection (2, 10, 12, 14). DAS181 is designed to inhibit influenza virus infection by inactivating the viral receptor, Sia. It is a recombinant sialidase fusion protein composed of the active domain of Actinomyces viscosus sialidase and the heparin binding sequence derived from the human protein amphiregulin for anchoring to epithelial surfaces (4). The A. viscosus sialidase domain selectively cleaves Sia from host cells, rendering them inaccessible to influenza viral particles. By binding to the negatively charged glycosaminoglycans on the airway epithelial cell surface, the cationic C-terminal amphiregulin tag anchors DAS181 on the respiratory epithelium, thereby improving the potency of the molecule.We previously reported potent in vitro and in vivo efficacy of DAS181 against multiple influenza virus A and B strains (4), including the highly pathogenic H5N1 influenza virus infection of mice (A/Vietnam/1203/2004 or VN/1203) (1). Daily DAS181 treatment of mice at 1 mg/kg/day beginning 1 day preinfection with VN/1203 has been shown to protect 100% of the mice tested from this fatal disease, prevented viral dissemination to the brain, and effectively blocked infection in 70% of the treated mice. DAS181 at 1 mg/kg/day was also therapeutically effective, conferring enhanced survival of H5N1 virus-challenged mice when treatment began 72 h postinfection (1). To further evaluate DAS181 efficacy against the highly pathogenic H5N1 virus in model systems that closely mimic the human respiratory tract in vivo, we conducted studies with ex vivo cultures of human lung tissue and primary pneumocytes. The study results demonstrate that DAS181 effectively removes the influenza virus Sia receptors and inhibits H5N1 infection within human lung tissue.     

In Vitro Antiviral Activity of Favipiravir (T-705) against Drug-Resistant Influenza and 2009 A(H1N1) Viruses

Favipiravir (T-705) has previously been shown to have a potent antiviral effect against influenza virus and some other RNA viruses in both cell culture and in animal models. Currently, favipiravir is undergoing clinical evaluation for the treatment of influenza A and B virus infections. In this study, favipiravir was evaluated in vitro for its ability to inhibit the replication of a representative panel of seasonal influenza viruses, the 2009 A(H1N1) strains, and animal viruses with pandemic (pdm) potential (swine triple reassortants, H2N2, H4N2, avian H7N2, and avian H5N1), including viruses which are resistant to the currently licensed anti-influenza drugs. All viruses were tested in a plaque reduction assay with MDCK cells, and a subset was also tested in both yield reduction and focus inhibition (FI) assays. For the majority of viruses tested, favipiravir significantly inhibited plaque formation at 3.2 μM (0.5 μg/ml) (50% effective concentrations [EC₅₀s] of 0.19 to 22.48 μM and 0.03 to 3.53 μg/ml), and for all viruses, with the exception of a single dually resistant 2009 A(H1N1) virus, complete inhibition of plaque formation was seen at 3.2 μM (0.5 μg/ml). Due to the 2009 pandemic and increased drug resistance in circulating seasonal influenza viruses, there is an urgent need for new drugs which target influenza. This study demonstrates that favipiravir inhibits in vitro replication of a wide range of influenza viruses, including those resistant to currently available drugs.In the United States alone, seasonal influenza is responsible annually for infecting between 5 and 20% of the American population, resulting in more than 200,000 hospitalizations and 36,000 deaths (8). Globally, seasonal influenza causes between 250,000 and 500,000 deaths every year (60). Influenza is not only a disease of great medical importance but also of economic importance. Despite available vaccines, a recent study predicted that in the United States influenza results in direct medical costs of the order of $10.4 billion each year, with the total economic burden for the United States being projected at $87.1 billion each year (44). It is widely accepted that vaccination remains the most effective approach for the prevention of viral infections (48). Although there is a safe and effective annual trivalent influenza vaccine, a large proportion of the global population does not receive the yearly influenza vaccine. This can be due to a variety of reasons, including the lack of access to adequate health care, unavailability of vaccine supply, allergies, and adverse reactions. During the 2009 pandemic (pdm), in addition to the vaccination and epidemiological control measures being exerted by health care officials, antivirals targeting influenza offer an essential tool in treating infected patients, in addition to protecting those at high risk of infection, such as the young, elderly, and health care workers.Currently, there are two classes of anti-influenza drugs licensed in the United States for use in the treatment and management of influenza infections in humans: M2 ion channel blockers (also known as adamantanes) and neuraminidase (NA) inhibitors (NAIs) (30). Influenza antivirals are highly effective in the treatment of influenza infections if used promptly following the onset of symptoms or following exposure (45, 46). Both the M2 blockers amantadine and rimantadine are taken by the patient orally (45). However, of the two available NAIs, only oseltamivir is available as an oral formulation (zanamivir has to be inhaled [14, 53]), although other routes of administration have been investigated (31). The use of the M2 blockers amantadine and rimantadine is limited due to the rapid emergence of transmissible drug-resistant mutant viruses and the fact that they offer protection only against influenza A virus infections (32). The high prevalence of adamantane resistance in seasonal A(H3N2) viruses and oseltamivir resistance in seasonal A(H1N1) viruses is reflected in the CDC recommendations for the use of influenza antivirals (6).The majority of adamantane-resistant A(H3N2) and A(H1N1) viruses circulating globally in recent years share the same mutation, S31N, in the M2 protein (20), although other resistance-conferring mutations have been detected also (including A30T, L26F, and V27A) (20, 49). The globally spread oseltamivir-resistant seasonal A(H1N1) viruses share the same mutation, H275Y (H274Y in N2 subtype amino acid numbering), in the drug-targeted enzyme neuraminidase, although other mutations are known to cause reduced susceptibility in vitro (19, 47, 50).Seasonal A(H1N1) viruses resistant to both the adamantanes and the NAI oseltamivir have previously been reported, without an apparent link to treatment (12, 50). Currently, zanamivir is the only drug effective against both adamantane-resistant and/or oseltamivir-resistant influenza viruses, but due to the fact that it has to be inhaled, it is less suitable for use with several high-risk groups, including the severely ill (41), infants (33), and the elderly (22). Furthermore, zanamivir may decrease pulmonary function, so it is not recommended for the treatment of infections in individuals with chronic underlying lung and heart disease conditions (23).Since 1997, there have been several outbreaks of highly pathogenic avian influenza A(H5N1) infections in poultry, with a substantial number of infections occurring in humans (1). The overall case fatality of A(H5N1) infections in humans is over 60% and, unlike seasonal influenza, is most deadly in the young and healthy (ages 10 to 19 years) (59). Oseltamivir is the medication of choice for treating individuals infected with A(H5N1) (17). However, resistance in A(H5N1) viruses has been detected following the treatment of patients with oseltamivir (18, 38). In addition, naturally occurring reduced susceptibility to oseltamivir (35, 40) and possibly to zanamivir (29) has been documented for circulating A(H5N1) viruses, including novel mutations in the NA (29, 35). Adamantane resistance is widely spread among A(H5N1) viruses that carry mutations at amino acid residues 26, 27, and 31 in the M2 protein (13, 35) and among swine viruses circulating in Eurasia (27).In April 2009, a novel reassortant A(H1N1) virus was first identified as circulating in humans in both Mexico and the United States (7, 9). Since April, the virus has continued to transmit among humans, and on 11 June 2009 the World Health Organization classified the outbreak as the first influenza pandemic of the 21st century (58). The 2009 A(H1N1) pandemic viruses consist of a unique combination of gene segments, including those of the North American (triple reassortants) and Eurasian swine lineages (27, 54). The 2009 A(H1N1) pandemic viruses are resistant to the adamantanes and sensitive to the NAIs (3, 16). Yet, concerns exist about the possibility of acquisition of resistance to the NAI oseltamivir, since the majority of A(H1N1) viruses which have been circulating predominantly worldwide during the 2008-2009 influenza season are oseltamivir resistant due to the resistance-conferring H275Y mutation in the NA. Such an acquisition of resistance by the 2009 A(H1N1) pandemic viruses would be a major setback and would further limit the already sparse therapeutic options (15, 57). There have been laboratory-confirmed cases of oseltamivir-resistant 2009 A(H1N1) pandemic viruses (each carrying the H275Y resistance-conferring mutation in the NA) in the United States (5).Collectively, these recent findings emphasize not only the need for new effective antivirals to control and treat influenza infections but also the need to identify new molecular targets (47).One such compound which is currently being investigated and undergoing clinical trials for the treatment of influenza infections is favipiravir (T-705), a pyrazine derivative (2, 26, 31). Favipiravir targets the RNA-dependent RNA polymerase (RdRp), a component of influenza virus different from that of currently licensed influenza antivirals (24, 25). It was shown that favipiravir can inhibit the viral replication of influenza type A, B, and C viruses (24, 25, 55). Favipiravir reduces influenza virus replication by selectively inhibiting the viral RdRp, since it does not affect the synthesis of host cellular DNA and RNA (25). Favipiravir has also shown great potential to act as a broad-spectrum antiviral against many RNA viruses, as reviewed by Furuta and coworkers (26).The purpose of this study was to evaluate the ability, in vitro, of favipiravir to inhibit the viral replication of contemporary influenza viruses as well as viruses with pandemic potential, including viruses resistant to the currently available and licensed anti-influenza drugs. In this report we demonstrate that favipiravir is a potent inhibitor of seasonal influenza A and B virus replication, including that of drug-resistant and drug-sensitive viruses. In addition, favipiravir was shown to effectively inhibit influenza A viruses of other antigenic subtypes, including A(H2N2), viruses of avian origin [A(H4N2), A(H7N2), and A(H5N1)], and viruses of swine origin [A(H1N1) and A(H1N2)], as well as the 2009 A(H1N1) pandemic viruses.     

Long-Acting Neuraminidase Inhibitor Laninamivir Octanoate (CS-8958) versus Oseltamivir as Treatment for Children with Influenza Virus Infection

We conducted a double-blind, randomized controlled trial to compare a long-acting neuraminidase inhibitor, laninamivir octanoate, with oseltamivir. Eligible patients were children 9 years of age and under who had febrile influenza symptoms of no more than 36-h duration. Patients were randomized to 1 of 3 treatment groups: a group given 40 mg laninamivir (40-mg group), a group given 20 mg laninamivir (20-mg group), and an oseltamivir group. Laninamivir octanoate was administered as a single inhalation. Oseltamivir (2 mg/kg of body weight) was administered orally twice daily for 5 days. The primary end point was the time to alleviation of influenza illness. The primary analysis included 184 patients (61, 61, and 62 in the 40-mg group, 20-mg group, and oseltamivir group, respectively). Laninamivir octanoate markedly reduced the median time to illness alleviation in comparison with oseltamivir in patients infected with oseltamivir-resistant influenza A (H1N1) virus, and the reductions were 60.9 h for the 40-mg group and 66.2 h for the 20-mg group. On the other hand, there were no significant differences in the times to alleviation of illness between the laninamivir groups and oseltamivir group for patients with influenza A (H3N2) or B virus infection. Laninamivir octanoate was well tolerated. The most common adverse events were gastrointestinal events. Laninamivir octanoate was an effective and well-tolerated treatment for children with oseltamivir-resistant influenza A (H1N1) virus infection. Further study will be needed to confirm clinical efficacy against influenza A (H3N2) or B virus infection. Its ease of administration is noteworthy, because a single inhalation is required during the course of illness.Swine origin influenza A (H1N1) virus (2009 pandemic H1N1 virus) was first detected in Mexico in the spring of 2009, and the World Health Organization declared a pandemic caused by 2009 pandemic H1N1 virus in June 2009 (21). Many otherwise healthy children and adults, as well as members of high-risk populations, who became infected with 2009 pandemic H1N1 virus developed severe illness and died. Neuraminidase inhibitors have recently been reported to be effective in preventing severe illness in patients with 2009 pandemic H1N1 virus infection (2, 8), and the importance of early treatment with neuraminidase inhibitors has been emphasized. However, appearance of oseltamivir-resistant 2009 pandemic H1N1 virus strains has been reported worldwide, up to 225 strains as of February 2010 (22), and the spread of oseltamivir-resistant 2009 pandemic H1N1 virus has become a concern. In fact, since almost 100% of seasonal influenza A (H1N1) viruses have become resistant to oseltamivir (20), there is an urgent need to develop anti-influenza agents that are effective not only against 2009 pandemic H1N1 virus but also against oseltamivir-resistant 2009 pandemic H1N1 virus.Previous studies have reported the potential advantages of laninamivir octanoate (CS-8958; Daiichi Sankyo Co., Ltd., Tokyo, Japan), an octanoyl ester prodrug of laninamivir. Laninamivir has shown in vitro neuraminidase-inhibitory activity against various influenza A and B viruses, including subtypes N1 to N9 and oseltamivir-resistant viruses (23), and it has also been found to be effective against a swine origin H1N1 strain (7). Moreover, laninamivir octanoate has long-lasting antiviral activity. Preclinical studies of CS-8958 in mice showed that after intranasal administration it was rapidly converted to its active metabolite, laninamivir, that the laninamivir generated was retained in the lungs, where it had a long half-life of 41.4 h (10), and that a single intranasal dose of laninamivir octanoate exhibited efficacy similar to that of repeated doses of zanamivir or oseltamivir (12, 23). A study in healthy volunteers showed that laninamivir was slowly eliminated from the body over a period of up to 6 days after a single inhalation (6).Influenza virus infection is one of the major causes of pediatric hospitalizations in the winter season (15, 17), and schoolchildren and children who attend day care centers are the principal transmitters of influenza in the community (13). The purpose of this trial was to compare the efficacy and safety of laninamivir octanoate to those of oseltamivir in children.     

Immune history shapes specificity of pandemic H1N1 influenza antibody responses

Human antibody responses against the 2009 pandemic H1N1 (pH1N1) virus are predominantly directed against conserved epitopes in the stalk and receptor-binding domain of the hemagglutinin (HA) protein. This is in stark contrast to pH1N1 antibody responses generated in ferrets, which are focused on the variable Sa antigenic site of HA. Here, we show that most humans born between 1983 and 1996 elicited pH1N1 antibody responses that are directed against an epitope near the HA receptor–binding domain. Importantly, most individuals born before 1983 or after 1996 did not elicit pH1N1 antibodies to this HA epitope. The HAs of most seasonal H1N1 (sH1N1) viruses that circulated between 1983 and 1996 possess a critical K133 amino acid in this HA epitope, whereas this amino acid is either mutated or deleted in most sH1N1 viruses circulating before 1983 or after 1996. We sequentially infected ferrets with a 1991 sH1N1 virus and then a pH1N1 virus. Sera isolated from these animals were directed against the HA epitope involving amino acid K133. These data suggest that the specificity of pH1N1 antibody responses can be shifted to epitopes near the HA receptor–binding domain after sequential infections with sH1N1 and pH1N1 viruses that share homology in this region.Most influenza pandemics occur when a new subtype of virus enters the human population. Once introduced into the human population, influenza viruses typically accumulate mutations in the hemagglutinin (HA) and neuraminidase (NA) glycoproteins, a process called antigenic drift. An H1N1 influenza virus strain caused a pandemic in 2009 (Smith et al., 2009) even though H1N1 viruses have circulated in humans from 1918 to 1957 and then again from 1977 to 2009. The 2009 pandemic H1N1 (pH1N1) strain is antigenically distinct from recently circulating seasonal H1N1 (sH1N1) strains and is more closely related to older sH1N1 strains (Garten et al., 2009; Manicassamy et al., 2010; Skountzou et al., 2010).Sera isolated from influenza-infected ferrets are currently used for surveillance of antigenically drifted influenza strains (Stöhr et al., 2012). Anti-pH1N1 antibody responses elicited in ferrets are focused on the highly variable Sa antigenic site of HA (Chen et al., 2010). Conversely, the majority of monoclonal antibodies derived from humans infected or vaccinated with pH1N1 are directed against conserved regions of the HA stalk and receptor binding domain (Li et al., 2012; O’Donnell et al., 2012; Wrammert et al., 2011). Most of these monoclonal antibodies possess many somatic mutations and bind to sH1N1 viruses efficiently, which is consistent with the idea that these antibody responses were likely originally primed by sH1N1 infection and were later recalled during pH1N1 infection/vaccination (Settembre et al., 2011; Wrammert et al., 2011; Li et al., 2012; O’Donnell et al., 2012; Qiu et al., 2012). Understanding the precise events that promote the development of these cross-reactive antibody repertoires will aid in developing a universal influenza vaccine that targets conserved areas of HA.Here, we compared the specificity of pH1N1 antibody responses elicited in different aged humans. We find that most individuals born between 1983 and 1996 elicit pH1N1 antibody responses that are dominated against an epitope near the HA receptor–binding domain. Most sH1N1 viruses that circulated between 1983 and 1996 share homology with the pH1N1 virus in this region of HA. Antibody responses dominated against this HA epitope were induced after sequential infection of ferrets with a 1991 sH1N1 virus and a pH1N1 virus. Most humans born before 1983 or after 1996 did not mount anti-pH1N1 antibody responses against this HA region. Importantly, most sH1N1 viruses that circulated before 1983 or after 1996 have an amino acid mutation or deletion in this HA epitope.     

Laninamivir Prodrug CS-8958, a Long-Acting Neuraminidase Inhibitor,Shows Superior Anti-Influenza Virus Activity after a Single Administration

Two neuraminidase (NA) inhibitors, zanamivir (Relenza) and oseltamivir phosphate (Tamiflu), have been licensed for use for the treatment and prophylaxis of influenza. We have reported on laninamivir (code name, R-125489), a novel neuraminidase inhibitor, and have discovered that the laninamivir prodrug CS-8958 worked as a long-acting neuraminidase inhibitor in a mouse influenza virus infection model when it is intranasally administered. In this study, CS-8958 was administered just once 7 days before infection and showed significant efficacy in vivo. The efficacy of a single administration of CS-8958 after viral infection was then compared with that of repeated administrations of oseltamivir phosphate or zanamivir in mice and ferrets. CS-8958 showed efficacy superior or similar to the efficacies of the two licensed NA inhibitors. CS-8958 also significantly reduced the titers of an oseltamivir-resistant H1N1 virus with a neuraminidase H274Y substitution in a mouse infection model. These results suggest that since CS-8958 is characteristically long lasting in the lungs, it may be ideal for the prophylaxis and treatment of influenza.Influenza is a serious respiratory illness which can be debilitating and which causes complications that lead to hospitalization and death, especially in elderly individuals. This respiratory disease is caused by influenza A and B viruses, which are pathogens that are highly contagious for humans. Influenza A viruses are classified into subtypes on the basis of the antigenicities of hemagglutinin (HA) and neuraminidase (NA) molecules. To date, 16 HA subtypes (H1 to H16) and 9 NA subtypes (N1 to N9) have been reported. Seasonal influenza or influenza epidemics are caused by influenza A virus H1N1 and H3N2 and influenza B virus (22), and every year the global burden of influenza epidemics is believed to be 3.5 million cases of severe illness and 300,000 to 500,000 deaths (6), before the new pandemic in 2009.In the last 100 years, humans have experienced three influenza pandemics: the first in 1918 (H1N1), the second in 1957 (H2N2), and the third in 1968 (H3N2) (22). In 2009, a new swine-origin influenza virus (H1N1) infected humans (20) and caused a pandemic. WHO has reported more than 400,000 confirmed cases worldwide as of 18 October 2009 (http://www.who.int/csr/don/2009_10_23/en/index.html). Another possible concern is a pandemic caused by highly pathogenic avian influenza (HPAI) H5N1 viruses. Since 2003, the number of humans infected with the HPAI H5N1 virus has increased, and the fatality rate is high. More than 444 cases infected with the H5N1 virus and as many as 262 deaths were reported as of 27 November 2009 (http://www.who.int/csr/disease/avian_influenza/country/en/). Thus, there is considerable concern that such highly pathogenic viruses will cause sustained human-to-human transmission and the next global pandemic.Two countermeasures, vaccinations and treatment with antivirals, are available to control human influenza. Although vaccinations play a critical role in influenza prophylaxis, they are an insufficient tool both for prophylaxis and against a pandemic virus. Therefore, antivirals are an important tool that may be used to mitigate influenza pandemics. Currently, two types of anti-influenza virus drugs are available: M2 ion channel blockers (adamantane) (5) and NA inhibitors. However, adamantane-resistant viruses readily emerge and are already prevalent worldwide among the seasonal influenza viruses (both the H1N1 and the H3N2 subtypes) (1, 3). The pandemic 2009 H1N1 viruses are also adamantane resistant (9). Moreover, the emergence of adamantane-resistant HPAI H5N1 viruses has prevented the use of adamantane for the treatment of infections caused by these viruses (4). The adamantane drugs have not been recommended for use for the treatment or chemoprophylaxis of influenza in the United States since the 2005 influenza season (1, 2). The second and most recently developed class of drugs with activities against influenza A and B viruses are the NA inhibitors, which bind to the NA surface glycoprotein of newly formed virus particles and prevent their efficient release from the host cell (8). Two NA inhibitors, zanamivir (inhaled drug, 10 mg/dose; Relenza) and oseltamivir (oral drug, 75 mg/dose; Tamiflu), are currently licensed for use. Both drugs require twice-daily administration for treatment. Oseltamivir is predominant and is used worldwide for the treatment of influenza, and the generation and circulation of oseltamivir-resistant seasonal influenza viruses have become major concerns (10, 11, 15, 17, 18). In particular, the worldwide prevalence of neuraminidase H274Y oseltamivir-resistant mutants of seasonal H1N1 virus have been reported, and 95% of H1N1 isolates tested from the fourth quarter of 2008 to January 2009 (WHO, http://www.who.int/csr/disease/influenza/H1N1webupdate20090318%20ed_ns.pdf) and almost all the H1N1 isolates tested since October 2008 in the United States (CDC, http://www.cdc.gov/flu/weekly/) were reported to be oseltamivir resistant. As well, a number of oseltamivir-resistant pandemic 2009 H1N1 viruses (7) and HPAI H5N1 viruses (18) have already appeared, although their appearance is still sporadic. These epidemics of oseltamivir-resistant influenza viruses therefore necessitate the development of alternative antiviral agents.We found a new strong neuraminidase inhibitor, laninamivir (code name, R-125489), and reported that CS-8958 (laninamivir octanoate or the laninamivir prodrug) worked as a long-acting neuraminidase inhibitor (12, 16, 23). Laninamivir potently inhibited the neuraminidase activities of various influenza A and B viruses, including subtypes N1 to N9 and oseltamivir-resistant viruses (23), as well as pandemic 2009 H1N1 virus (14). Due to the long retention of R-125489 in mouse lungs after the intranasal administration of CS-8958 (16), the intranasal administration of a single dose of CS-8958 showed efficacy superior to the efficacies of zanamivir and oseltamivir in mouse models of infection with influenza A virus and seasonal and current pandemic strains (14, 23).In this report, the in vivo efficacy of a single administration of CS-8958 was compared with the efficacies of repeated administrations of zanamivir (intranasal) and oseltamivir (oral) in mouse or ferret models of influenza A and B virus infection and the administration of oseltamivir in a mouse model of H274Y virus infection. We demonstrate the great potential of the single administration of CS-8958 as an alternative treatment against influenza viruses, including oseltamivir-resistant mutants.     

In Vitro System for Modeling Influenza A Virus Resistance under Drug Pressure

One of the biggest challenges in the effort to treat and contain influenza A virus infections is the emergence of resistance during treatment. It is well documented that resistance to amantadine arises rapidly during the course of treatment due to mutations in the gene coding for the M2 protein. To address this problem, it is critical to develop experimental systems that can accurately model the selection of resistance under drug pressure as seen in humans. We used the hollow-fiber infection model (HFIM) system to examine the effect of amantadine on the replication of influenza virus, A/Albany/1/98 (H3N2), grown in MDCK cells. At 24 and 48 h postinfection, virus replication was inhibited in a dose-dependent fashion. At 72 and 96 h postinfection, virus replication was no longer inhibited, suggesting the emergence of amantadine-resistant virus. Sequencing of the M2 gene revealed that mutations appeared at between 48 and 72 h of drug treatment and that the mutations were identical to those identified in the clinic for amantadine-resistant viruses (e.g., V27A, A30T, and S31N). Interestingly, we found that the type of mutation was strongly affected by the dose of the drug. The data suggest that the HFIM is a good model for influenza virus infection and resistance generation in humans. The HFIM has the advantage of being a highly controlled system where multiplicity parameters can be directly and accurately controlled and measured.Each year thousands of people die from human H1N1 and H3N2 influenza A virus epidemics (38). In 2009, a swine-origin influenza A (H1N1) virus caused a pandemic (8). Fortunately, this virus causes a mild disease that either resolves on its own or, if caught in time, is amenable to treatment with the currently available neuraminidase inhibitors, oseltamivir carboxylate and zanamivir (8). In the past, human H1N1, H2N2, and H3N2 influenza A viruses have caused pandemics leading to many more deaths (25). Neuraminidase inhibitors, such as oseltamivir carboxylate and zanamivir, and M2 ion channel blockers, such as the adamantane derivatives, amantadine, and rimantadine, have been effective for the prevention and treatment of human influenza A virus infections (19, 22, 30-32, 39). However, with more frequent use of these inhibitors, influenza viruses resistant to the adamantanes or oseltamivir carboxylate have emerged in the human population (4, 5, 9, 16, 20, 26, 32). Amantadine resistance is so widespread that adamantane is no longer recommended for the treatment of human influenza A virus infections (20), and resistance to oseltamivir carboxylate in the currently circulating H1N1 human influenza viruses is essentially 100% (32).We wished to employ our hollow-fiber infection model (HFIM) to determine whether when influenza virus was exposed to amantadine in this in vitro circumstance (i) mutations could be generated in the M2 gene and (ii) these mutations would mimic those seen clinically. In this way, we would provide some validation that the system can be employed to identify clinically relevant mutations early for the development of new drugs and to explore the spacing of doses and administration schedule to determine if emergence of resistance can be suppressed.Sequencing the M2 genes of progeny viruses obtained from individual viral plaques of viruses grown in the HFIM system in the presence of amantadine showed that most of the viruses contained mutations identical to those found in clinical isolates obtained from patients treated with amantadine (5).(Portions of this paper were presented previously [29a].)     

Prophylactic Activity of Intramuscular Peramivir in Mice Infected with a Recombinant Influenza A/WSN/33 (H1N1) Virus Containing the H274Y Neuraminidase Mutation

Peramivir is a neuraminidase (NA) inhibitor (NAI) under development that must be administered by the systemic route. The prophylactic activity of intramuscular (IM) peramivir was evaluated with mice infected with wild-type (WT) and oseltamivir-resistant (H274Y NA mutant) recombinant influenza A/WSN/33 (H1N1) viruses. Treatment regimens consisted of IM injections starting 1 h before viral challenge that were single (45 mg/kg or 90 mg/kg) or multiple (45 mg/kg daily for 5 days). All peramivir regimens prevented mortality and weight loss while significantly reducing lung viral titers (LVT) in mice infected with the WT virus. For animals infected with the H274Y mutant, the multiple-dose regimen completely prevented mortality and was associated with significant reduction in weight loss and LVT compared to untreated animals. In contrast, both single-treatment regimens reduced mortality and weight loss but did not significantly reduce LVT. Although further experiments using different influenza A/H1N1 virus strains and other animal models are needed, our results suggest that 5-day IM peramivir therapy may be considered a prophylactic alternative to control influenza infections caused by oseltamivir-resistant viruses with the H274Y mutation.Neuraminidase (NA) inhibitors (NAIs) constitute one of the most valuable options for the control of influenza epidemics and pandemics. Two NAIs, inhaled zanamivir and oral oseltamivir, have been approved for the treatment and prevention of influenza infections in many countries (16). In addition, other NAIs are at different stages of development. Peramivir, which is a cyclopentane analogue compound, has shown potent in vitro activity against influenza A and B viruses (4). By the use of NAI assays, we previously demonstrated that peramivir 50% inhibitory concentration (IC₅₀) values for Canadian clinical influenza A/H3N2, A/H1N1, and B viruses were lower than those of zanamivir and oseltamivir (10). In other studies, mean IC₅₀ values of clinical influenza A/H1N1 viruses from untreated individuals against peramivir were also lower than those against oseltamivir and zanamivir (14, 15). Furthermore, on-site dissociation studies demonstrated that peramivir remained tightly bound to the NA enzyme with a half-time for the substrate conversion of >24 h compared to 1.25 h for both zanamivir and oseltamivir (5).In controlled trials of prophylaxis and treatment, oral peramivir was associated with reduced viral titers but no significant decrease in time to relief of symptoms, a feature that could be attributed to a low oral bioavailability in humans (6). The bioavailability of peramivir may be improved by using intravenous (IV) or intramuscular (IM) injections. Indeed, comparison of single IM versus oral peramivir with the same dose (10 mg/kg), administered 4 h prior to a lethal influenza A/WSN/33 (H1N1) virus challenge, demonstrated that the IM route was associated with a higher survival rate in mice than that of the oral route (100% versus 50%) (5). Also, a single IV injection of 3 mg/kg of peramivir provided a significant therapeutic effect that was superior to that of oral oseltamivir in a lethal mouse model of influenza A and B virus infections (18). The emergence and rapid dissemination of the seasonal A/Brisbane/59/2007 (H1N1) virus containing the NA mutation H274Y in N2 numbering (H275Y in N1 numbering), which is associated with a high level of resistance to oseltamivir and moderate cross-resistance to peramivir in vitro (9), are a major clinical concern. The aim of the present study was to evaluate the prophylactic efficacy of IM injections of peramivir in mice infected with a recombinant influenza A/WSN/33 (H1N1) virus containing or not containing the H274Y NA mutation, which has been associated with 427- and 48-fold increases in oseltamivir and peramivir IC₅₀ values, respectively, in NAI assays (1).     

Triple Combination of Oseltamivir,Amantadine, and Ribavirin Displays Synergistic Activity against Multiple Influenza Virus Strains In Vitro

The recurring emergence of influenza virus strains that are resistant to available antiviral medications has become a global health concern, especially in light of the potential for a new influenza virus pandemic. Currently, virtually all circulating strains of influenza A virus in the United States are resistant to either of the two major classes of anti-influenza drugs (adamantanes and neuraminidase inhibitors). Thus, new therapeutic approaches that can be rapidly deployed and that will address the issue of recurring resistance should be developed. We have tested double and triple combinations of the approved anti-influenza drugs oseltamivir and amantadine together with ribavirin against three influenza virus strains using cytopathic effect inhibition assays in MDCK cells. We selected A/New Caledonia/20/99 (H1N1) and A/Sydney/05/97 (H3N2) as representatives of the wild-type versions of the predominant circulating seasonal influenza virus strains and A/Duck/MN/1525/81 (H5N1) as a representative of avian influenza virus strains. Dose-response curves were generated for all drug combinations, and the degree of drug interaction was quantified using a model that calculates the synergy (or antagonism) between the drugs in double and triple combinations. This report demonstrates that a triple combination of antivirals was highly synergistic against influenza A virus. Importantly, the synergy of the triple combination was 2- to 13-fold greater than the synergy of any double combination depending on the influenza virus subtype. These data support the investigation of a novel combination of oseltamivir, amantadine, and ribavirin as an effective treatment for both seasonal and pandemic influenza virus, allowing the efficient use of the existing drug supplies.Influenza epidemics are responsible for significant morbidity, mortality, and economic burden annually in the United States, including an estimated 41,000 deaths, more than 290,000 hospitalizations, and 44 million days of lost productivity (34). Currently, two classes of drugs are approved for the treatment of influenza, the adamantanes and the neuraminidase inhibitors (NAIs). When used to treat susceptible seasonal influenza, these antiviral drugs provide a modest benefit by reducing symptoms by approximately 1.5 days in otherwise healthy patients if treatment is initiated within 48 h of symptom onset (22, 27, 37). However, the therapeutic benefit of these antiviral drugs in cases of severe infection by highly pathogenic avian influenza virus is less clear. In cases of sporadic H5N1 influenza virus infection, the data suggest that, while treatment with antivirals may provide some benefit, the mortality rate remains close to 60% (1, 28). Thus, as single agents, influenza drugs do not exhibit sufficient potency to treat severe influenza virus infections.The effectiveness of the adamantanes and NAIs has been eroded by emerging viral resistance, both treatment induced and naturally occurring. Resistance to the adamantanes, which block the M2 channel and prevent viral uncoating, emerges rapidly in treated patients (21), and resistant strains are transmissible (2). In recent years, the level of resistance to adamantanes has risen to such a high level globally that this drug class no longer is recommended as monotherapy (3, 12). Most recently, resistance to amantadine developed in the majority of A/H3N2 viruses in the United States, such that in the 2008 to 2009 influenza season, virtually 100% of the characterized A/H3N2 viruses were resistant to amantadine (6). Sporadic resistance to oseltamivir, the most widely used NAI, was reported as early as 1999 (8), and the development of drug resistance has been documented with the use of oseltamivir against both seasonal influenza virus (26, 29, 35) and avian influenza virus (11). Whether treatment induced or naturally occurring, widespread resistance to oseltamivir in A/H1N1 seasonal influenza virus emerged in Europe in early 2008 and now is dominant over large portions of Europe, Asia, North America, and the Southern Hemisphere (43, 51). In the 2009 influenza season, 99.4% of all A/H1N1 viruses isolated from patients in the United States were resistant to oseltamivir (6). As a result, virtually all circulating strains of influenza A virus in the United States currently are resistant to either of the two classes of anti-influenza drugs. In light of the widespread resistance patterns among H1N1 and H3N2 subtypes, and with no rapid diagnostic tools to characterize resistance being available, the continued use of these drugs as monotherapies may result in dual resistance, raising the specter of treatment-induced multidrug-resistant influenza virus. In fact, this phenomenon has been documented in severely immunocompromised patients, where the sequential use of NAIs and M2 inhibitors resulted in the generation of viruses resistant to both drugs (26, 49).The emergence of drug-resistant mutants is a significant problem not only for influenza virus but also for other rapidly mutating viruses (7, 30, 42, 48). For these viruses, the use of antiviral drugs in combination has proven to be an effective strategy for suppressing the development of drug resistance, resulting in the durability of treatment regimens. For example, it has been known since the mid- to late 1990s that the simultaneous use of three antiviral agents in combination for human immunodeficiency virus (HIV) will block viral replication and decrease the probability of the emergence of resistance, effectively establishing a chemicogenetic barrier to drug-resistant mutations (16, 17). With regard to the use of combination therapy for influenza virus, clinical studies have tested the safety and drug interactions of double combinations of available anti-influenza drugs (36), and a number of studies have looked at the effect of double drug combinations for the treatment of influenza virus in vitro (14, 19, 20, 23, 32, 44) and in animals (13, 25, 31, 44, 47). To date, there are no published studies of the effects of triple antiviral drug combinations for influenza virus.To address dual problems of potency and resistance in treating severe influenza, including avian influenza, we chose to optimize the use of existing antivirals and to determine the effectiveness of triple-drug combinations for treating influenza. We hypothesized that a triple combination of drugs with different mechanisms of action, and which act at three different stages of the viral life cycle, would result in synergistic antiviral activity. In this study, we evaluated the interactions between oseltamivir, amantadine, and ribavirin. To test our hypothesis that these drugs interact synergistically, we explored the in vitro antiviral activity and synergism of the single, double, and triple treatments against a panel of influenza A viruses. Our results show that these drugs act synergistically, with triple combinations showing greater synergy than any of the double combinations evaluated. Furthermore, the synergy of the triple combination was maintained across multiple strains representing different influenza A virus subtypes, including the three major subtypes that currently cause significant morbidity and mortality in humans (H1N1, H3N2, and H5N1). To our knowledge, this is the first time the antiviral activity and synergism of a triple combination of oseltamivir, amantadine, and ribavirin for influenza virus has been demonstrated.     

Influenza Vaccines: From Surveillance Through Production to Protection

Influenza is an important contributor to population and individual morbidity and mortality. The current influenza pandemic with novel H1N1 has highlighted the need for health care professionals to better understand the processes involved in creating influenza vaccines, both for pandemic as well as for seasonal influenza. This review presents an overview of influenza-related topics to help meet this need and includes a discussion of the burden of disease, virology, epidemiology, viral surveillance, and vaccine strain selection. We then present an overview of influenza vaccine—related topics, including vaccine production, vaccine efficacy and effectiveness, influenza vaccine misperceptions, and populations that are recommended to receive vaccination. English-language articles in PubMed published between January 1, 1970, and October 7, 2009, were searched using key words human influenza, influenza vaccines, influenza A, and influenza B.ACIP = Advisory Committee on Immunization Practices; CDC = Centers for Disease Control and Prevention; CI = confidence interval; COPD = chronic obstructive pulmonary disease; FDA = Food and Drug Administration; GBS = Guillain-Barré Syndrome; GISN = Global Influenza Surveillance Network; HA = hemagglutinin; HAI = hemagglutination inhibition; HIV = human immunodeficiency virus; LAIV = live-attenuated influenza vaccine; M = matrix; NA = neuraminidase; NI = NA inhibitor; NS = nonstructural protein; PB = polymerase basic; TIV = trivalent inactivated vaccine; WHO = World Health OrganizationThe current influenza A pandemic with novel H1N1 and the race to develop effective vaccines against it have increased the profile of influenza and influenza vaccination among the lay public and medical professional community, making them more likely to inquire about influenza strain surveillance and vaccine development. Furthermore, deaths attributed to novel H1N1 influenza infection have highlighted the substantial contribution of influenza infection to overall morbidity and mortality and the importance of vaccination against seasonal influenza.Although seasonal influenza is the most common vaccine-preventable cause of death in the United States, influenza vaccination rates remain unacceptably low for all categories of people at highest risk.^1-9 Both patient-related factors (eg, lack of awareness of need and concern over adverse effects) as well as physician-related factors (eg, failure of physician to recommend for it or recommendation by physician against it) contribute to poor vaccine uptake.^10,11 Because misinformation and lack of physician recommendation are among the most common reasons why susceptible people do not receive vaccination, this review aims to provide a better understanding of seasonal influenza vaccines to a wide medical audience. A PubMed search for relevant English-language articles published between January 1, 1970, and October 7, 2009, was performed to find pertinent literature using the key words human influenza, influenza vaccines, influenza A, and influenza B.     

Effects of the Combination of Favipiravir (T-705) and Oseltamivir on Influenza A Virus Infections in Mice

Favipiravir (T-705 [6-fluoro-3-hydroxy-2-pyrazinecarboxamide]) and oseltamivir were combined to treat influenza virus A/NWS/33 (H1N1), A/Victoria/3/75 (H3N2), and A/Duck/MN/1525/81 (H5N1) infections. T-705 alone inhibited viruses in cell culture at 1.4 to 4.3 μM. Oseltamivir inhibited these three viruses in cells at 3.7, 0.02, and 0.16 μM and in neuraminidase assays at 0.94, 0.46, and 2.31 nM, respectively. Oral treatments were given twice daily to mice for 5 to 7 days starting, generally, 24 h after infection. Survival resulting from 5 days of oseltamivir treatment (0.1 and 0.3 mg/kg/day) was significantly better in combination with 20 mg/kg of body weight/day of T-705 against the H1N1 infection. Treatment of the H3N2 infection required 50 mg/kg/day of oseltamivir for 7 days to achieve 60% protection; 25 mg/kg/day was ineffective. T-705 was ≥70% protective at 50 to 100 mg/kg/day but inactive at 25 mg/kg/day. The combination of inhibitors (25 mg/kg/day each) increased survival to 90%. The H5N1 infection was not benefited by treatment with oseltamivir (≤100 mg/kg/day for 7 days). T-705 was 30 to 70% protective at 25 to 100 mg/kg/day. Survival improved slightly with combination treatments. Increased activity was seen against H5N1 infection by starting treatments 2 h before infection. Oseltamivir was ineffective at ≤40 mg/kg/day. T-705 was 100% protective at 40 and 80 mg/kg/day and inactive at 20 mg/kg/day. Combining ineffective doses (20 mg/kg/day of T-705 and 10 to 40 mg/kg/day of oseltamivir) afforded 60 to 80% protection and improved body weights during infection. Thus, synergistic responses were achieved with low doses of T-705 combined with oseltamivir. These compounds may be viable candidates for combination treatment of human influenza infections.The emergence of swine influenza H1N1 virus infections in 2009 (2) highlights the need for effective antiviral therapy in a largely immune-naïve population. Treatment options for influenza are becoming more limited because viruses, including the 2009 swine H1N1 virus, are resistant to the antiviral drugs amantadine and rimantadine (3, 4, 11, 13, 20). Oseltamivir-resistant viruses are also becoming more common in the environment, particularly within the last 2 years (1, 5, 19). Thus, more potent and effective treatments are needed to combat these growing threats.More potent antiviral therapy can be achieved by using drugs in combination, as demonstrated in mouse models (10, 14-17, 24, 26, 27). Such treatment can slow down the emergence of drug-resistant viruses (12). The reported animal studies have primarily focused on the known-active antiviral agents amantadine, rimantadine, oseltamivir, peramivir, zanamivir, and ribavirin. The kinds of studies that can be performed have been limited based upon the number of active antiviral compounds that are available.In 2002, Furuta et al. reported a novel pyrazine molecule, T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide, now named favipiravir), as an inhibitor of influenza virus infections in cell culture and in mice (8). T-705 inhibits both influenza A and B viruses (8, 23, 29). The compound converts to nucleoside mono- (T-705 RMP [ribosylated, monophosphorylated]), di-, and triphosphate (T-705 RTP [ribosylated, triphosphorylated]) forms in cells (9). The mode of action of T-705 RTP is similar to that of ribavirin triphosphate as an inhibitor of influenza virus RNA polymerase (6, 9). Unlike ribavirin monophosphate, T-705 RMP is only weakly inhibitory to cellular inosine monophosphate (IMP) dehydrogenase (9, 28), and thus, it is less cytotoxic. These properties make T-705 a viable candidate for the treatment of influenza virus infections in humans. The compound is currently undergoing phase II clinical trials.The use of T-705 in combination with other antiviral substances has not been reported. The purpose of the present work was to evaluate whether the combination of T-705 with the widely used antiviral drug oseltamivir is more beneficial than either substance used alone against influenza virus infections in mice. We chose three mouse-adapted influenza viruses for these comparisons, A/NWS/33 (H1N1), A/Victoria/3/75 (H3N2), and A/Duck/MN/1525/81 (H5N1). The A/NWS and A/Victoria viruses are of seasonal origin and are confined to the respiratory tract following infection. The A/Duck virus is a low-pathogenicity avian virus from the United States that also does not spread beyond the respiratory tract of mice. The experimental influenza A/Duck mouse infection does not fully reflect the type of pathogenesis of the highly pathogenic avian influenza H5N1 viruses from the Old World. This is because the A/Duck virus lacks the multibasic amino acid R-X-R/K-R motif in the hemagglutinin protein, whereas the highly pathogenic avian H5N1 viruses contain it (7). This motif allows for the highly pathogenic viruses to be proteolytically activated by ubiquitous subtilisin-like cellular proteases, allowing the virus to spread in vivo beyond the respiratory tract and to cause multiorgan failure. Nevertheless, the A/Duck virus induces rapid, severe lung infections that are difficult to treat with conventional antiviral therapy. Using these three models, H1N1, H3N2, and H5N1, in mice, we were able to demonstrate the benefits of using oseltamivir and T-705 in combination to treat influenza virus infections.     

Assessment of Pandemic and Seasonal Influenza A (H1N1) Virus Susceptibility to Neuraminidase Inhibitors in Three Enzyme Activity Inhibition Assays

The neuraminidase inhibitors (NAIs) zanamivir and oseltamivir are currently the only antiviral drugs effective for the treatment and prophylaxis of 2009 pandemic influenza A (H1N1) virus infections. The proven potential of these viruses to acquire NAI resistance during treatment emphasizes the need to assess their NAI susceptibility. The 50% inhibitory concentrations (IC₅₀s) are known to vary depending on the neuraminidase inhibition (NI) test used; however, few side-by-side comparisons of different NI assays have been done. In the present study, a panel of 11 isolates representing 2009 seasonal and pandemic influenza H1N1 viruses, including oseltamivir-resistant H275Y variants, were tested in three functional NI assays: chemiluminescent (CL), fluorescent (FL), and colorimetric (CM). The sensitivities of the viruses to zanamivir, oseltamivir, and three investigational NAIs (peramivir, R-125489, and A-315675) were assessed. All isolates with the exception of H275Y variants were sensitive to all five NAIs by all three NI assays. The H275Y variants showed substantially elevated IC₅₀s against oseltamivir and peramivir. The three NI assays generally yielded consistent results; thus, the choice of NI assay does not appear to affect conclusions based on drug susceptibility surveillance. Each assay, however, offers certain advantages compared to the others: the CL assay required less virus volume and the FL assay provided the greatest difference in the IC₅₀s between the wild type and the variants, whereas the IC₅₀s obtained from the CM assay may be the most predictive of the drug concentrations needed to inhibit enzyme activity in humans. It would be desirable to develop an NI assay which combines the advantages of all three currently available assays but which lacks their shortcomings.For the treatment and chemoprophylaxis of infections caused by influenza A viruses, the U.S. Food and Drug Administration (FDA) has approved four drugs: amantadine and rimantadine as well as zanamivir and oseltamivir. These drugs belong to two classes, adamantanes (i.e., M2 ion-channel blockers) and neuraminidase (NA) inhibitors (NAIs), respectively. In recent years, the effectiveness of M2 blockers has been greatly compromised, which limits their usefulness in clinical practice. This is largely due to the rapid emergence and widespread circulation of adamantane-resistant influenza viruses (1, 5, 6, 7, 14, 17). More recently, the emergence and worldwide spread of seasonal H1N1 viruses resistant to oseltamivir, currently the most widely used drug against influenza infections, became a considerable public health concern (15, 21, 25, 32). Monitoring the NAI resistance of influenza viruses is an ongoing public health issue since the emergence in 2009 of pandemic viruses that are resistant to M2 blockers.Cell culture-based assays are typically not used for assessment of virus sensitivity to NAIs because of the unpredictable effect of hemagglutinin (HA) receptor binding (2, 34). Instead, drug susceptibility can be monitored by functional (biochemical) NA inhibition (NI) assays, and subsequent genotypic methods are generally required to identify the molecular marker(s) of resistance in the NA. The principle underlying the functional methods relies on the enzymatic nature of the NA, a viral surface glycoprotein and antigen. NA acts by cleaving the terminal neuraminic acid (also called sialic acid) from receptors recognized by influenza viral HA, thus facilitating the release of progeny virions from infected cells and preventing self-aggregation (29). Structurally, NAIs mimic the natural substrate, neuraminic acid, and produce tight interactions, with conserved residues of the NA active site competing with neuraminic acid for binding (11, 23). Preincubation of virus with NAIs leads to the inhibition of enzyme activity, which is detected after the addition of enzyme substrate. Most NI assays commonly used for virus surveillance utilize as substrates small synthetic conjugates that produce either a luminescent or a fluorescent signal upon cleavage by the NA enzyme. The chemiluminescent (CL) assay uses the 1,2-dioxetane derivative of neuraminic acid substrate in the influenza neuraminidase inhibitor resistance detection (NA-Star) kit (8), while the fluorescent (FL) assay employs 2′-O-(4-methylumbelliferyl)-N-acetylneuraminic acid substrate (MUNANA) (30). The results of the NI assays are expressed as the 50% inhibitory concentration (IC₅₀), which represents the NAI concentration that inhibits 50% of the enzyme activity of the virus. As the NA activity of clinical specimens is usually insufficient for determining the IC₅₀ due to a low viral content, NI assays, using either the substrate provided with the NA-Star kit or the MUNANA substrate, require virus propagation in cell cultures or embryonated chicken eggs. It is noteworthy that IC₅₀s are specific to the virus type/subtype and to the individual NAI tested (8, 19, 20, 24, 32, 37). The IC₅₀s obtained can be used for assessment of virus susceptibility to NAIs, including detection of resistant viruses, as well as for comparing the potencies of antiviral drugs belonging to the NAI class. Although both the CL and FL assays allow reliable detection of NAI resistance, the more recently developed CL assay was reported to be about 70 times more sensitive in detecting NA activity and has a greater linear range than the FL assay (8). The CL assay was also selected for use in the global drug susceptibility surveillance program by the Neuraminidase Inhibitor Susceptibility Network (NISN) (37, 39) and by other surveillance laboratories (28, 32). It should also be noted that IC₅₀s may vary even for the same virus when the NI assay is done using the NA-Star substrate (CL assay) and the MUNANA substrate (FL assay), according to reports on seasonal viruses (37). Whether one of the two assays, the CL or FL assay, more reliably predicts the level of resistance and the drug concentration required for the NA activity inhibition in vivo are key points of interest and remain to be elucidated.A third assay, the colorimetric (CM) assay, which utilizes fetuin as the substrate of the NA, is typically used to determine the titer of anti-NA antibodies because small substrates do not effectively compete with antibodies (3, 31). This assay is not widely used for antiviral susceptibility testing. Unlike the NA-Star and MUNANA synthetic substrates, fetuin is a large, natural, and soluble bovine glycoprotein that contains abundant neuraminic acids at the ends of its oligosaccharide moiety (which include the presence of two residues of α2,3-linked sialic acid and one residue of α2,6-linked sialic acid) (4, 33) and has been used as a substrate in NA-catalyzed reactions (3). Given that NAIs compete with the enzyme substrate for binding to the active site, the structure of the substrate can potentially influence the outcome of the competition and, as a result, the IC₅₀. In this respect, fetuin may represent a better natural substrate for the enzyme-neuraminic acid attached via an α2,3 or α2,6 linkage to oligosaccharide chains on the cell surface. Furthermore, since the cleavage of each neuraminic acid is chemically converted, the CM assay can be a quantifiable method from which the resulting IC₅₀s would correlate more closely to the NA activity of the virus tested. Despite these apparent advantages to the use of fetuin, the CM method relies on chemical reactions that are time-consuming, cumbersome, and impractical for high-throughput use. In addition, the assay requires concentrated virus stocks for testing. Thus, fetuin is still considered an undefined substrate that does not confer sufficient sensitivity or specificity for use in routine NAI susceptibility assays (34). The potential usefulness of a large substrate such as fetuin for assessment of the NAI susceptibilities of novel H1N1 viruses or novel inhibitors remains largely unexplored.Resistance to NAIs is not defined as clearly as that to adamantanes. In NI assays, a drug-resistant virus should have IC₅₀s consistently greater than the threshold value that is determined for each viral type/subtype and drug tested (27, 32, 37). Since the 2007-2008 influenza season, about a decade after the introduction of NAIs into clinical use, an NA framework mutation, H275Y (H274Y in N2 numbering), was consistently and most commonly detected in oseltamivir-resistant H1N1 viruses isolated worldwide (15, 21, 25, 32). Although the H275Y substitution represents the most-defined oseltamivir resistance marker of influenza viruses carrying the NA of the N1 subtype (35), novel NAI resistance-associated mutations—determined by elevated IC₅₀s in NI assays—continue to be revealed (21, 22, 32). Importantly, oseltamivir-resistant viruses from the ongoing H1N1 pandemic have been detected and reported around the world (9, 10, 26, 38). Seasonal and 2009 pandemic H1N1 viruses have the same phylogenetically distant NA gene ancestors (16), which necessitates the comprehensive assessment of the drug susceptibilities of the new pandemic viruses. Therefore, it is necessary to evaluate existing NI assays in order to better understand which assay may be the most sensitive for the detection of NAI resistance and/or the most predictive of virus susceptibility to NAIs in vivo.In the present study, we assessed the susceptibilities of a panel of seasonal and pandemic H1N1 influenza viruses, including virus variants bearing the established oseltamivir resistance mutation, H275Y in the NA, against five NAIs: two FDA-approved NAIs, zanamivir and oseltamivir, and three investigational NAIs, peramivir, R-125489 (the bioactive metabolite of the prodrug CS-8958 [laninamivir]), and A-315675 (a bioactive form of the prodrug A-322278). In order to better characterize and assess the consistency of IC₅₀s and levels of susceptibility, these viruses were tested in the widely used CL and FL assays, as well as with the CM method.     

Hypoglycemia and Outcome in Critically Ill Patients

OBJECTIVE: To determine whether mild or moderate hypoglycemia that occurs in critically ill patients is independently associated with an increased risk of death.PATIENTS AND METHODS: Of patients admitted to 2 hospital intensive care units (ICUs) in Melbourne and Sydney, Australia, from January 1, 2000, to October 14, 2004, we analyzed all those who had at least 1 episode of hypoglycemia (glucose concentration, <81 mg/dL). The independent association between hypoglycemia and outcome was statistically assessed.RESULTS: Of 4946 patients admitted to the ICUs, a cohort of 1109 had at least 1 episode of hypoglycemia (blood glucose level, <81 mg/dL). Of these 1109 patients (22.4% of all admissions to the intensive care unit), hospital mortality was 36.6% compared with 19.7% in the 3837 nonhypoglycemic control patients (P<.001). Even patients with a minimum blood glucose concentration between 72 and 81 mg/dL had a greater unadjusted mortality rate than did control patients (25.9% vs 19.7%; unadjusted odds ratio, 1.42; 95% confidence interval, 1.12-1.80; P=.004.) Mortality increased significantly with increasing severity of hypoglycemia (P<.001). After adjustment for insulin therapy, hypoglycemia was independently associated with increased risk of death, cardiovascular death, and death due to infectious disease.CONCLUSION: In critically ill patients, an association exists between even mild or moderate hypoglycemia and mortality. Even after adjustment for insulin therapy or timing of hypoglycemic episode, the more severe the hypoglycemia, the greater the risk of death.APACHE = Acute Physiology and Chronic Health Evaluation; AUC = area under the curve; CI = confidence interval; ICU = intensive care unit; IIT = intensive insulin therapy; NICE-SUGAR = Normoglycemia in Intensive Care Evaluation-Survival Using Glucose Algorithm Regulation; OR = odds ratioUntil recently, intensive insulin therapy (IIT) had been recommended to improve patient outcome^1-3 despite its association with an increased risk of hypoglycemia.^4-11 However, hypoglycemia, like hyperglycemia,^12-16 has emerged as a possible predictor of mortality and morbidity in critically ill patients.^5,17-21The NICE-SUGAR (Normoglycemia in Intensive Care Evaluation-Survival Using Glucose Algorithm Regulation) trial found that IIT increased 90-day mortality compared with conventional treatment in critically ill patients.^22,23 In that trial, the incidence of severe hypoglycemia (blood glucose level, ≤40 mg/dL (to convert to mmol/L, multiply by 0.0555) was significantly higher with IIT. Furthermore, the relative risk of severe hypoglycemia was 13.7, more than twice that seen in prior randomized controlled trials.^5,9-11 Thus, the incidence of hypoglycemia might be a key element of blood glucose control in critically ill patients, although no causal link between hypoglycemia and mortality has been demonstrated. However, no consensus exists on the definition of hypoglycemia in patients with critical illness.²⁴ Studies thus far have mainly focused on severe hypoglycemia.For editorial comment, see page 215We sought to determine the epidemiology and independent association of hypoglycemia in the intensive care unit (ICU). We hypothesized that mild or moderate hypoglycemia would be common and would be independently associated with an increased risk of death.     

Characterization of Human Influenza Virus Variants Selected In Vitro in the Presence of the Neuraminidase Inhibitor GS 4071

An oral prodrug of GS 4071, a potent and selective inhibitor of influenza neuraminidases, is currently under clinical development for the treatment and prophylaxis of influenza virus infections in humans. To investigate the potential development of resistance during the clinical use of this compound, variants of the human influenza A/Victoria/3/75 (H3N2) virus with reduced susceptibility to the neuraminidase inhibitor GS 4071 were selected in vitro by passaging the virus in MDCK cells in the presence of inhibitor. After eight passages, variants containing two amino acid substitutions in the hemagglutinin (A28T in HA1 and R124M in HA2) but no changes in the neuraminidase were isolated. These variants exhibited a 10-fold reduction in susceptibility to GS 4071 and zanamivir (GG167) in an in vitro plaque reduction assay. After 12 passages, a second variant containing these hemagglutinin mutations and a Lys substitution for the conserved Arg292 of the neuraminidase was isolated. The mutant neuraminidase enzyme exhibited high-level (30,000-fold) resistance to GS 4071, but only moderate (30-fold) resistance to zanamivir and 4-amino-Neu5Ac2en, the amino analog of zanamivir. The mutant enzyme had weaker affinity for the fluorogenic substrate 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid and lower enzymatic activity compared to the wild-type enzyme. The viral variant containing the mutant neuraminidase did not replicate as well as the wild-type virus in culture and was 10,000-fold less infectious than the wild-type virus in a mouse model. These results suggest that although the R292K neuraminidase mutation confers high-level resistance to GS 4071 in vitro, its effect on viral virulence is likely to render this mutation of limited clinical significance.Influenza virus infections remain a serious seasonal health concern and the potential of severe pandemics due to the emergence of new influenza strains, such as the H5N1 “bird flu” recently identified in Hong Kong (39), provides additional impetus to develop potent and effective antiviral agents (24). At present, only amantadine and, in some countries, rimantadine are approved for the treatment and prophylaxis of influenza A infections. However, the usefulness of these two compounds is limited by their lack of activity against influenza B viruses and their rapid selection of drug-resistant mutants which remain transmissible and pathogenic (10, 25).The influenza neuraminidase, which is expressed on the virus surface, has long been considered a valid target for antiviral therapy. This enzyme, which cleaves terminal sialic acid residue from glycoconjugates, is essential for virus proliferation and infectivity (3, 17, 19, 27, 28). The observation that the structural and catalytic amino acids which line the enzyme active site are highly conserved among different influenza neuraminidase types and subtypes (reviewed in reference 6) suggests that inhibitors of this enzyme would be active against a broad range of influenza viruses.Based on information gained from crystallographic studies of influenza neuraminidases complexed with sialic acid or the transition state analog Neu5Ac2en (2, 41, 43), several potent and selective inhibitors of the influenza neuraminidases have been synthesized (15, 16, 43). Two of these, GS 4071 ([3R,4R,5S]-4-acetamido-5-amino-3-[1-ethylpropoxy]1-cyclohexene-1-carboxylic acid), in the form of its oral prodrug GS 4104, and zanamivir (GG167, 4-guanidino-Neu5Ac2en) are currently under clinical development for the prophylaxis and treatment of influenza virus infections. Both compounds have demonstrated efficacy against influenza A and B viruses in vitro (13, 23, 40, 43, 45), in animal models of influenza virus infection (23, 31, 32, 34), and in experimental influenza virus infection in humans (11, 12, 14) when GS 4104 is taken orally and zanamivir is delivered topically to the respiratory tract as an inhalant.Although the development of resistance to zanamivir in animals or people treated with the drug has not been reported, influenza variants resistant to zanamivir due to mutations within their hemagglutinin or neuraminidase genes have been selected in vitro (1, 7, 8, 20, 38). In general, zanamivir-resistant hemagglutinin mutants have been easier to generate than neuraminidase mutations. These hemagglutinin mutants commonly contain amino acid substitutions in or near the sialic acid binding site and are believed to make the virus replication less dependent on neuraminidase activity (7, 20, 33). However, these mutations likely only affect the in vitro, not the in vivo, susceptibility to zanamivir (29).The most common neuraminidase mutation which arises in vitro under selective pressure of zanamivir has been that of the conserved Glu119 residue in the neuraminidase active site (1, 7, 38). Mutations of Glu119, which interacts with the guanidino side chain of zanamivir but not with the natural substrate (43), cause a 100-fold reduction in the sensitivity of the enzyme to zanamivir (1, 7, 38). Viruses containing mutations at this position remain infectious (8) and capable of inducing a febrile response in ferrets (5). Recently, a Lys substitution for the conserved Arg at position 292 has also been reported for a variant selected in the presence of zanamivir (8). The neuraminidase containing this mutation exhibited only a 10-fold reduction in sensitivity to zanamivir. A reassortant virus containing the mutant neuraminidase was 500-fold less infectious than wild-type virus in mice (8).In this report, we describe the first in vitro isolation and characterization of variants of a human influenza virus, A/Victoria/3/75 (H3N2), selected in the presence of GS 4071. In contrast with the experience with amantadine and rimantadine, with which drug-resistant variants can be selected after one or two passages in culture (26), variants with decreased susceptibility to GS 4071 did not readily occur. In the eighth passage, a variant containing two mutations in the stalk region of the hemagglutinin was isolated. This variant exhibited a minor decrease in susceptibility to neuraminidase inhibitors in general. A second variant, containing a conservative substitution of a Lys for an Arg at amino acid 292 of the neuraminidase enzyme active site, was isolated later in the selection process. This mutation caused a marked decrease in the susceptibility of the virus and the sensitivity of the enzyme to GS 4071. However, this mutation also adversely affected neuraminidase enzyme activity, compromised the ability of the virus to replicate in tissue culture, and reduced the infectivity of the virus 10,000-fold in mice.     

A ��Hot�� Topic in Dyslipidemia Management����How to Beat a Flush��: Optimizing Niacin Tolerability to Promote Long-term Treatment Adherence and Coronary Disease Prevention

Niacin is the most effective lipid-modifying agent for raising high-density lipoprotein cholesterol levels, but it also causes cutaneous vasodilation with flushing. To determine the frequency of flushing in clinical trials, as well as to delineate counseling and treatment approaches to prevent or manage flushing, a MEDLINE search was conducted of English-language literature from January 1, 1985, through April 7, 2009. This search used the title keywords niacin or nicotinic acid crossed with the Medical Subject Headings adverse effects and human. Niacin flushing is a receptor-mediated, mainly prostaglandin D₂–driven phenomenon, the frequency, onset, and duration of which are largely determined by the distinct pharmacological and metabolic profiles of different niacin formulations. Subjective assessments include ratings of redness, warmth, itching, and tingling. In clinical trials, most (>60%) niacin users experienced mild or moderate flushing, which tended to decrease in frequency and severity with continued niacin treatment, even with advancing doses. Approximately 5% to 20% of patients discontinued treatment because of flushing. Flushing may be minimized by taking niacin with meals (or at bedtime with a low-fat snack), avoiding exacerbating factors (alcohol or hot beverages), and taking 325 mg of aspirin 30 minutes before niacin dosing. The current review advocates an initially slow niacin dose escalation from 0.5 to 1.0 g/d during 8 weeks and then from 1.0 to 2.0 g in a single titration step (if tolerated). Through effective counseling, treatment prophylaxis with aspirin, and careful dose escalation, adherence to niacin treatment can be improved significantly. Wider implementation of these measures should enable higher proportions of patients to reach sufficient niacin doses over time to prevent cardiovascular events.GPR = G_i protein–coupled receptor; HDL-C = high-density lipoprotein cholesterol; NSAID = nonsteroidal anti-inflammatory drug; PG = prostaglandin; RCT = randomized controlled trialReducing low-density lipoprotein cholesterol levels is the primary treatment for preventing cardiovascular disease, largely because of robust evidence from randomized controlled trials (RCTs) involving statins.^1,2 However, many patients optimally treated with statins have residual cardiovascular risk³ associated with low levels of high-density lipoprotein cholesterol (HDL-C), elevated levels of triglycerides, and a preponderance of small, dense (atherogenic) low-density lipoprotein particles.Niacin (3-pyridine-carboxylic acid; C₆H₅N0₂) is the most effective medication to raise HDL-C levels. A water-soluble B-complex (B₃) vitamin used to treat pellagra, niacin was discovered to lower cholesterol levels at higher (gram) doses in 1955.⁴ It was also the first lipid-lowering therapy proven in RCTs to significantly lower (by 27%) the risk of myocardial infarction and the risk of all-cause mortality in the long term (by 11%),^5,6 as well as confer significant angiographic benefits (7,10-15

TABLE 1.

Efficacy and Tolerability Findings From Major Randomized Controlled Trials of Niacin-Containing Therapies^aOpen in a separate windowNiacin improves the entire lipid panel in patients with dyslipidemia, lowering apolipoprotein B–containing lipoproteins and raising apolipoprotein A–containing lipoproteins (eg, high-density lipoproteins).¹⁶ Putative mechanisms for these multidimensional lipid benefits involve interactions of niacin with its G_i protein–coupled receptor (GPR109A) in adipose tissue, reducing free fatty acid release.^17,18One potential obstacle to effective use of lipid therapies is suboptimal treatment adherence and long-term persistence.^19-27 In recent studies, approximately 53% of niacin users did not reach recommended daily maintenance doses of 1.0 g or higher, 92% did not reach doses of 2 g, and flushing severity significantly predicted niacin treatment discontinuation.^28,29 This systematic review (1) explores the pathophysiology of niacin flushing, (2) characterizes its typical clinical presentation, (3) assesses the approximate frequency of niacin flushing and of discontinuations ascribed to flushing in clinical trials, (4) explores potential strategies to prevent or minimize flushing, and (5) surveys ongoing controversies.     

A Phase 3, Double-Blind, Randomized, Placebo-Controlled Study of Armodafinil for Excessive Sleepiness Associated With Jet Lag Disorder

OBJECTIVE: To assess the effect of armodafinil, the longer-lasting isomer of modafinil, on jet lag disorder.PARTICIPANTS AND METHODS: This double-blind, randomized, parallel-group, multicenter study was conducted between September 18, 2008, and February 9, 2009. Adults with a history of jet lag symptoms on previous flights through multiple time zones flew from the United States to France (a 6-hour time zone change) for a 3-day laboratory-based study period. Participants received armodafinil (50 or 150 mg/d) or placebo each morning. Wakefulness was assessed by the coprimary outcomes, mean sleep latency on the Multiple Sleep Latency Test (MSLT) (average of all MSLT sessions across days 1 and 2) and Patient Global Impression of Severity in relation to jet lag symptoms (averaged across days 1 and 2).RESULTS: A total of 427 participants received armodafinil at 50 mg/d (n=142), armodafinil at 150 mg/d (n=143), or placebo (n=142). Armodafinil at 150 mg/d provided a significant benefit in sleep latency on the MSLT (days 1-2: mean, 11.7 minutes vs 4.8 minutes for placebo; P<.001) and participants'' perception of their overall condition in relation to jet lag symptoms (Patient Global Impression of Severity, days 1-2: mean, 1.6 vs 1.9 for placebo; P<.05). The most frequently reported adverse events for armodafinil at 150 mg/d were headache (27%), nausea (13%), diarrhea (5%), circadian rhythm sleep disorder (5%), and palpitations (5%).CONCLUSION: Armodafinil increased wakefulness after eastward travel through 6 time zones.Trial Registration: clinicaltrials.gov identifier: {"type":"clinical-trial","attrs":{"text":"NCT00758498","term_id":"NCT00758498"}}NCT00758498ANCOVA = analysis of covariance; KSS = Karolinska Sleepiness Scale; MSLT = Multiple Sleep Latency Test; NPSG = nocturnal polysomnography; PGI-S = Patient Global Impression of Severity; STAI = State and Trait Anxiety Inventory; SWD = shift work disorderJet lag disorder is a circadian rhythm sleep disorder that occurs as a consequence of rapid travel through multiple time zones.¹ The traveler may experience excessive sleepiness, fatigue, insomnia, irritability, gastrointestinal disturbance, or other symptoms after arrival at the destination.^2-4 Jet lag symptoms arise from the desynchronization between the body''s circadian rhythm, which is synchronous with the location of departure, and the new sleep/wake cycle required at the destination.^1-4 Effects tend to be more severe when a greater number of time zones are traversed, and following eastbound travel.^2,5,6 Although the percentage of people flying across multiple time zones who develop jet lag disorder is unclear, it is estimated that possibly up to two-thirds of all travelers experience jet lag and may experience symptoms such as excessive sleepiness during the day or insomnia.² A treatment for excessive sleepiness that promotes daytime wakefulness may be especially beneficial to travelers who have a limited amount of time at their destination, precluding a circadian readjustment.Armodafinil, the longer-lasting R-isomer of racemic modafinil,⁷ is a wakefulness-promoting medication. The terminal half-life of the R-isomer is approximately 15 hours, compared with 3 to 4 hours for the S-isomer.^8,9 In a study of patients with shift work disorder (SWD), another circadian rhythm disorder, armodafinil 150 mg/d significantly improved wakefulness and clinicians'' perception of patients'' overall condition, compared with placebo.¹⁰ The primary objective of this study was to evaluate armodafinil (50 mg/d and 150 mg/d) for treatment of excessive sleepiness associated with jet lag disorder due to eastbound travel in a population of travelers with a history of jet lag symptoms.     

Distinct germinal center selection at local sites shapes memory B cell response to viral escape

Respiratory influenza virus infection induces cross-reactive memory B cells targeting invariant regions of viral escape mutants. However, cellular events dictating the cross-reactive memory B cell responses remain to be fully defined. Here, we demonstrated that lung-resident memory compartments at the site of infection, rather than those in secondary lymphoid organs, harbor elevated frequencies of cross-reactive B cells that mediate neutralizing antibody responses to viral escape. The elevated cross-reactivity in the lung memory compartments was correlated with high numbers of V_H mutations and was dependent on a developmental pathway involving persistent germinal center (GC) responses. The persistent GC responses were focused in the infected lungs in association with prolonged persistence of the viral antigens. Moreover, the persistent lung GCs supported the exaggerated B cell proliferation and clonal selection for cross-reactive repertoires, which served as the predominant sites for the generation of cross-reactive memory progenitors. Thus, we identified the distinct GC selection at local sites as a key cellular event for cross-reactive memory B cell response to viral escape, a finding with important implications for developing broadly protective influenza vaccines.Protective memory responses provided by parental influenza vaccines primarily depend on neutralizing IgG antibodies (Abs) directed against hemagglutinin (HA), a major glycoprotein on the virus surface (Gerhard, 2001; Plotkin, 2013). The membrane distal region of the HA globular head is highly immunogenic and is the primary target of anti-HA Abs elicited by vaccination (Skehel and Wiley, 2000). However, the HA globular head undergoes continual antigenic evolution (Wiley et al., 1981), making vaccine-induced Abs less effective against drifted viruses. Moreover, new subtypes can emerge rapidly and unexpectedly, as experienced in the 2009 A/H1N1 pandemic virus and sporadic human infection with avian viruses such as H5N1 and H7N9. Thus, the evolving threats of influenza virus underscore the need for influenza vaccines that are more broadly protective.HA conserved regions can be targeted by broadly cross-reactive Abs that exhibit potent virus-neutralizing activity in vitro and in vivo (Okuno et al., 1993; Throsby et al., 2008; Sui et al., 2009; Yoshida et al., 2009; Corti et al., 2010; Krause et al., 2011; Wrammert et al., 2011). Such cross-reactive Abs were observed in IgG and IgA fractions after respiratory exposure of viruses (Tamura et al., 1992; Tumpey et al., 2001; Margine et al., 2013). Of note, cross-reactive IgG Abs were higher in humans infected with influenza virus than in humans parentally boosted with vaccines (Moody et al., 2011; Wrammert et al., 2011; Li et al., 2012; Pica et al., 2012; Margine et al., 2013), suggesting that the cellular pathways for cross-reactive Ab responses are more active after respiratory virus infection.Pulmonary-infected influenza virus initially primes virus-binding B cells in the lung-draining mediastinal LNs (MLNs; Coro et al., 2006). The infected lungs, albeit at delayed kinetics, also participate in the primary immune response, concordant with the ectopic formation of induced bronchus-associated lymphoid tissue (iBALT; Moyron-Quiroz et al., 2004; Halle et al., 2009). iBALTs are able to support germinal center (GC) formation (Moyron-Quiroz et al., 2004), suggesting intraorgan development of long-lived plasma cells and memory B cells, which are crucial cellular components for humoral memory responses (Joo et al., 2008; Onodera et al., 2012; Tarlinton and Good-Jacobson, 2013). Although immediate protection against homologous reinfection is mediated by preexisting neutralizing Abs from long-lived plasma cells, memory B cells serve as a reservoir of cross-reactive Ab repertoires in West Nile virus infection (Purtha et al., 2011). Therefore, it is now postulated that memory B cells are important for the broad protection against escape mutants, against which strain-specific Abs are no longer effective (Baumgarth, 2013). However, the memory B cell subset reserving cross-reactive repertoires and its developmental pathway has not been fully characterized.Here, using two types of fluorochrome-labeled HA probes, we identified the cross-reactive memory B cell subset and dissected its developmental pathway after pulmonary influenza virus infection. Our data revealed a striking heterogeneity in the tissue localization, persistence, and selection for cross-reactivity among virus-specific GC responses. Among such heterogeneous GC responses, persistent GCs in the infected lungs profoundly selected and supplied cross-reactive memory repertoires into local sites, thereby potentiating the cross-protection at the site of infection.     

A prospective study of fasting plasma glucose and risk of stroke in asymptomatic men

OBJECTIVE: To examine the association between levels of fasting plasma glucose (FPG) and incidence of stroke outcomes in a large cohort of asymptomatic men.PATIENTS AND METHODS: Participants were 43,933 men (mean ± SD age, 44.3±9.9 years) who were free of known cardiovascular disease at baseline and whose FPG levels were assessed during a preventive medical examination at the Cooper Clinic, Dallas, TX, between January 7, 1971, and March 11, 2002. Patients with diagnosed diabetes mellitus (DM) or low FPG (<80 mg/dL [to convert to mmol/L, multiply by 0.0555]) were excluded. Fatal stroke was identified through the National Death Index, and nonfatal stroke was ascertained from mail-back surveys.RESULTS: A total of 595 stroke events (156 fatal and 456 nonfatal strokes; 17 men reported a nonfatal stroke before they died of stroke) occurred during 702,928 person-years of exposure. Age-adjusted fatal, nonfatal, and total stroke event rates per 10,000 person-years for normal FPG (80-109 mg/dL), impaired fasting glucose (110-125 mg/dL), and undiagnosed DM (≥126 mg/dL) were 2.1, 3.4, and 4.0 (P_trend=.002); 10.3, 11.8, and 18.0 (P_trend=.008); and 8.2, 9.6, and 12.4 (P_trend=.008), respectively. After further adjusting for potential confounders, the direct association between FPG and fatal, nonfatal, or total stroke events remained significant (P_trend=.02, .03, and .01, respectively). For FPG levels of 110 mg/dL or greater, each 10-unit increment of FPG was associated with a 6% higher risk of total stroke events (P=.05).CONCLUSION: Hyperglycemia (FPG, ≥110 mg/dL), even below the DM threshold (such as with impaired fasting glucose), was associated with a higher risk of fatal, nonfatal, or total stroke events in asymptomatic men.ACLS = Aerobics Center Longitudinal Study; ADA = American Diabetes Association; BMI = body mass index; CI = confidence interval; CVD = cardiovascular disease; DM = diabetes mellitus; ECG = electrocardiography; FPG = fasting plasma glucose; ICD = International Classification of Diseases; IFG = impaired fasting glucose; MetS = metabolic syndrome; NDI = National Death IndexThe primary causes of death and disability in patients with diabetes mellitus (DM) are cardiovascular and cerebrovascular complications.^1-5 Previous studies have reported an independent and direct association of clinically diagnosed DM and stroke^2,5-7; however, about half of all patients with type 2 DM are undiagnosed because they remain asymptomatic for long periods.⁸ Those with undiagnosed DM often have elevated levels of fasting plasma glucose (FPG) but no symptoms of DM. To date, few studies have examined the effect of FPG on stroke events, and the findings are inconclusive.^6,9-13 Some have reported a positive association between elevated FPG levels and stroke,^9,10 whereas others failed to identify impaired glucose levels as a significant risk predictor for stroke.^6,11-13 The inconsistent findings may be due to differences in study populations, length of follow-up, stroke outcome definition (such as fatal, nonfatal, or a combination), confounders selection, or a combination of these factors.For editorial comment, see page 1038In 2003, the American Diabetes Association (ADA) recommended changing the lower limit for the diagnosis of impaired fasting glucose (IFG) from 110 to 100 mg/dL (to convert to mmol/L, multiply by 0.0555).¹⁴ However, the need for this change has since been questioned, and concerns have been raised regarding the potential public health implications.^15-18 Therefore, we used the widely accepted 1997 ADA guidelines¹⁹ to define the IFG (FPG, 110-125 mg/dL) and DM (FPG, ≥126 mg/dL). We evaluated the association between FPG (including undiagnosed DM and IFG, as well as lower levels of FPG [100-109 mg/dL]) and fatal, nonfatal, and fatal/nonfatal combined stroke in a large cohort of men while controlling for cardiorespiratory fitness, an independent predictor of mortality and morbidity.^20,21