Rick A. Bright

Incidence of adamantane resistance among influenza A (H3N2) viruses isolated worldwide from 1994 to 2005: a cause for concern.

BACKGROUND Adamantanes have been used to treat influenza A virus infections for many years. Studies have shown a low incidence of resistance to these drugs among circulating influenza viruses; however, their use is rising worldwide and drug resistance has been reported among influenza A (H5N1) viruses isolated from poultry and human beings in Asia. We sought to assess adamantane resistance among influenza A viruses isolated during the past decade from countries participating in WHOs global influenza surveillance network. METHODS We analysed data for influenza field isolates that were obtained worldwide and submitted to the WHO Collaborating Center for Influenza at the US Centers for Disease Control and Prevention between Oct 1, 1994, and Mar 31, 2005. We used pyrosequencing, confirmatory sequence analysis, and phenotypic testing to detect drug resistance among circulating influenza A H3N2 (n=6524), H1N1 (n=589), and H1N2 (n=83) viruses. FINDINGS More than 7000 influenza A field isolates were screened for specific aminoacid substitutions in the M2 gene known to confer drug resistance. During the decade of surveillance a significant increase in drug resistance was noted, from 0.4% in 1994-1995 to 12.3% in 2003-2004. This increase in the proportion of resistant viruses was weighted heavily by those obtained from Asia with 61% of resistant viruses isolated since 2003 being from people in Asia. INTERPRETATION Our data raise concerns about the appropriate use of adamantanes and draw attention to the importance of tracking the emergence and spread of drug-resistant influenza A viruses.

Development of adenoviral-vector-based pandemic influenza vaccine against antigenically distinct human H5N1 strains in mice

INTRODUCTION Avian H5N1 influenza viruses currently circulating in southeast Asia could potentially cause the next pandemic. However, currently licensed human vaccines are subtype-specific and do not protect against these H5N1 viruses. We aimed to develop an influenza vaccine and assessed its immunogenicity and efficacy to confer protection in BALB/c mice. METHODS We developed an egg-independent strategy to combat the avian influenza virus, because the virus is highly lethal to chickens and the maintenance of a constant supply of embryonated eggs would be difficult in a pandemic. We used a replication-incompetent, human adenoviral-vector-based, haemagglutinin subtype 5 influenza vaccine (HAd-H5HA), which induces both humoral and cell-mediated immune responses against avian H5N1 influenza viruses isolated from people. FINDINGS Immunisation of mice with HAd-H5HA provided effective protection from H5N1 disease, death, and primary viral replication (p<0.0001) against antigenically distinct strains of H5N1 influenza viruses. Unlike the recombinant H5HA vaccine, which is based on a traditional subunit vaccine approach, HAd-H5HA vaccine induced a three-fold to eight-fold increase in HA-518-epitope-specific interferon-gamma-secreting CD8 T cells (p=0.01). INTERPRETATION Our findings highlight the potential of an Ad-vector-based delivery system, which is both egg-independent and adjuvant-independent and offers stockpiling options for the development of a pandemic influenza vaccine.

Induction of heterosubtypic immunity to influenza A virus using a DNA vaccine expressing hemagglutinin-C3d fusion proteins

Cross-protection between different subtypes of influenza A virus has been attributed to heterosubtypic immunity (HSI). Although, HSI can occur in the absence of anti-HA or anti-NA antibodies, HSI seems to be mediated, in part, by cross-reactive antibodies. In this study, we examined the effects of a DNA vaccine expressing an influenza HA fused to three copies of murine C3d of complement (HA-mC3d(3)). HA-mC3d(3) elicited heterosubtypic immunity more efficiently than non-fused forms of HA and protected mice from lethal challenge of influenza with different subtypes. Plasmid encoding for various forms of HA were constructed from two influenza strains, A/Puerto Rico/8/34 (H1N1) or A/Aichi/2/68-x31 (H3N2). Vaccinated mice were analyzed for enhancement of anti-HA titers, affinity maturation of antibody, hemagglutinin-inhibition activity, and altered cytokine profiles. The HA-mC3d(3)-DNA vaccinated mice were protected from heterologous influenza challenge, even though sera from these mice had no viral-neutralizing activity against heterologous virus.

Impact of glycosylation on the immunogenicity of a DNA-based influenza H5 HA vaccine.

Avian H5N1 influenza viruses isolated from humans in Hong Kong in 1997 were divided into two antigenic groups based on the presence or absence of a potential glycosylation site at amino acid residues 154-156 in the HA1 region of the viral hemagglutinin (HA) surface glycoprotein. To assess the impact of glycosylation on the immunogenicity of an HA-expressing DNA vaccine, a series of plasmid vaccine constructs that differed in the presence of potential glycosylation sites at amino acid residues 154-156, 165-167, and 286-288 were used to immunize BALB/c mice. Postvaccination serum IgG, hemagglutination inhibition, and neutralizing antibody titers as well as the morbidity and mortality following a lethal H5N1 viral challenge did not vary significantly among any of the experimental groups. We conclude that the glycosylation pattern of the influenza virus HA1 domain has little impact on the murine antibody response raised to a DNA vaccine encoding the H5 HA, thereby minimizing the concern that the pattern of glycosylation sites encoded by the vaccine match those of closely related H5 viruses.

Detection of Molecular Markers of Antiviral Resistance in Influenza A (H5N1) Viruses Using a Pyrosequencing Method

ABSTRACT Resistance of influenza viruses to antiviral drugs can emerge following medication or may result from natural variation. Two classes of anti-influenza virus drugs targeting either the M2 protein (amantadine and rimantadine) or neuraminidase (NA; oseltamivir and zanamivir) are currently licensed. These drugs are expected to be important in controlling the early stages of a potential pandemic. In the present study, we describe how a pyrosequencing method can be used to rapidly detect established molecular markers of resistance to M2 blockers and NA inhibitors in influenza A (H5N1) viruses. The residues L26, V27, A30, S31, and G34 in the M2 protein were targeted for pyrosequencing. The NA residues for pyrosequencing analysis included the established markers of drug resistance (H274 and N294), as well as residues of less certain relevance (V116, I117, Q136, K150, and I222). A single pair of pyro-reverse transcription (RT)-PCR primers was designed to allow amplification of an approximately 600-nucleotide-long amplicon of the NA genes of H5N1 viruses from various clades/subclades associated with infections in humans. The sensitivity of the assay was demonstrated by the successful pyrosequencing of RNA extracted from samples of serially diluted (10−5 to 10−7) virus stocks with initial concentrations ranging from 105 to 108 PFU/ml. The markers of resistance were detected in samples with threshold cycle values ranging from 32 to 37, as determined by real-time RT-PCR. The pyrosequencing approach may provide a valuable tool for rapid detection of markers of drug resistance in H5N1 viruses and facilitate the elucidation of the role of such changes in natural and acquired drug resistance.

Antivirals in seasonal and pandemic influenza—future perspectives*

Please cite this paper as: Wathen et al. (2012) Antivirals in seasonal and pandemic influenza—future perspectives. Influenza and Other Respiratory Viruses 7(Suppl. 1), 76–80.

Vaccines as a tool to estimate the burden of severe influenza in children of low-resourced areas (November 30–December 1, 2012, Les Pensieres, Veyrier-du-Lac, France)

There is an increasing focus on influenza in low-resourced areas as a vaccine-preventable cause of severe lower respiratory disease in young children, especially among those under two years of age. The extent of the disease burden is unclear: current etiologic studies may underestimate the impact of influenza if recognized or unrecognized infection occurs some time before severe disease manifestations prompt specimen collection for diagnosis. Because of various methodological challenges, a vaccine probe approach was used to estimate vaccine preventable disease incidence (VPDI) for Streptococcus pneumoniae and Haemophilus influenzae type b, particularly for pneumonia outcomes among young children. A similar approach could be used to determine VPDI for influenza. A highly effective vaccine would facilitate this approach; however, with appropriate design, a less than ideal vaccine also could be used to estimate VPDI. Because influenza vaccine efficacy against severe disease may be greater than against all symptomatic influenza disease, a vaccine probe approach could provide a better measure than etiologic studies of the public health utility of influenza vaccine. The first 6 months of life is a time of particularly increased influenza risk among young children, and an age group for which current vaccines are not approved. Previous studies have found that maternal influenza immunization can reduce acute respiratory infection in the infant during this vulnerable period. Additional randomized, controlled trials are currently underway using a vaccine probe approach to estimate VPDI among mothers and their infants following maternal influenza immunization. The World Health Organization now identifies pregnant women as the highest priority target group for influenza vaccination. Should countries implement this strategy, infants age 6-23 months likely would remain at increased risk; vaccine probe approaches could quantify the public health benefit of immunizing this group.

Influenza Vaccine Manufacture: Keeping Up with Change

Influenza virus causes annual epidemics of respiratory disease that affect all age groups. Manifestations of influenza vary by age and underlying risk condition and, on a population basis, result in substantial morbidity, mortality, and lost productivity. Inactivated vaccines against influenza vi rus have been licensed for >60 years. These vaccines have demonstrated efficacy and effectiveness across broad age groups and among different populations and have a strong record of safety [1-3]. Live atten uated influenza vaccines have been li censed in the United States since 2003 and

Adamantane-Resistant Influenza Infection During the 2004–05 Season

Adamantane-resistant influenza A is an emerging problem, but infections caused by resistant and susceptible viruses have not been compared. We identified adamantane resistance in 47% of 152 influenza A virus (H3N2) isolates collected during 2005. Resistant and susceptible viruses caused similar symptoms and illness duration. The prevalence of resistance was highest in children.

PATH Influenza Vaccine Project: accelerating the development of new influenza vaccines for low-resource countries

The 2009 influenza A/H1N1 pandemic demonstrated that a pandemic influenza virus has the potential to spread more rapidly in today’s highly interconnected world than in the past. While pandemic morbidity and mortality are likely to be greatest in low-resource countries, manufacturing capacity and access to influenza vaccines predominantly exist in countries with greater resources and infrastructure. Even with recently expanded manufacturing capacity, the number of doses available within a 6-month timeframe would be inadequate to fully immunize the global population if the decision to implement a global vaccination program were made today. Improved, affordable vaccines are needed to limit the consequences of a global influenza outbreak and protect low-resource populations. PATH’s Influenza Vaccine Project is supporting a range of activities in collaboration with private- and public-sector partners to advance the development of promising influenza vaccines that can be accessible and affordable for people in low-resource countries.