Ian G. Barr

The global circulation of seasonal influenza A (H3N2) viruses.

Antigenic and genetic analysis of the hemagglutinin of ∼13,000 human influenza A (H3N2) viruses from six continents during 2002–2007 revealed that there was continuous circulation in east and Southeast Asia (E-SE Asia) via a region-wide network of temporally overlapping epidemics and that epidemics in the temperate regions were seeded from this network each year. Seed strains generally first reached Oceania, North America, and Europe, and later South America. This evidence suggests that once A (H3N2) viruses leave E-SE Asia, they are unlikely to contribute to long-term viral evolution. If the trends observed during this period are an accurate representation of overall patterns of spread, then the antigenic characteristics of A (H3N2) viruses outside E-SE Asia may be forecast each year based on surveillance within E-SE Asia, with consequent improvements to vaccine strain selection.

Substitutions Near the Receptor Binding Site Determine Major Antigenic Change During Influenza Virus Evolution

Flu Drift Limited Five antigenic sites in the virus surface hemagglutinin protein, which together comprise 131 amino acid positions, are thought to determine the full scope of antigenic drift of influenza A virus. Koel et al. (p. 976) show that major antigenic change can be caused by single amino acid substitutions. These single substitutions substantially skew the way the immune system “sees” the virus. All substitutions of importance are located next to the receptor-binding site in the hemagglutinin. Because there are few positions of importance for antigenic drift, there are strict biophysical limitations to the substitutions at these positions, which restricts the number of new antigenic drift variants at any point in time. Thus, the evolution of influenza virus may be more predictable than previously thought. The major antigenic changes of the influenza virus are primarily caused by a single amino acid near the receptor binding site. The molecular basis of antigenic drift was determined for the hemagglutinin (HA) of human influenza A/H3N2 virus. From 1968 to 2003, antigenic change was caused mainly by single amino acid substitutions, which occurred at only seven positions in HA immediately adjacent to the receptor binding site. Most of these substitutions were involved in antigenic change more than once. Equivalent positions were responsible for the recent antigenic changes of influenza B and A/H1N1 viruses. Substitution of a single amino acid at one of these positions substantially changed the virus-specific antibody response in infected ferrets. These findings have potentially far-reaching consequences for understanding the evolutionary mechanisms that govern influenza viruses.

Emergence and spread of oseltamivir-resistant A(H1N1) influenza viruses in Oceania, South East Asia and South Africa

The neuraminidase inhibitors (NAIs) are an effective class of antiviral drugs for the treatment of influenza A and B infections. Until recently, only a low prevalence of NAI resistance (<1%) had been detected in circulating viruses. However, surveillance in Europe in late 2007 revealed significant numbers of A(H1N1) influenza strains with a H274Y neuraminidase mutation that were highly resistant to the NAI oseltamivir. We examined 264 A(H1N1) viruses collected in 2008 from South Africa, Oceania and SE Asia for their susceptibility to NAIs oseltamivir, zanamivir and peramivir in a fluorescence-based neuraminidase inhibition assay. Viruses with reduced oseltamivir susceptibility were further analysed by pyrosequencing assay. The frequency of the oseltamivir-resistant H274Y mutant increased significantly after May 2008, resulting in an overall proportion of 64% (168/264) resistance among A(H1N1) strains, although this subtype represented only 11.6% of all isolates received during 2008. H274Y mutant viruses demonstrated on average a 1466-fold reduction in oseltamivir susceptibility and 527-fold reduction in peramivir sensitivity compared to wild-type A(H1N1) viruses. The mutation had no impact on zanamivir susceptibility. Ongoing surveillance is essential to monitor how these strains may spread or persist in the future and to evaluate the effectiveness of treatments against them.

ISCOMs and other saponin based adjuvants

Saponins are chemically a heterogeneous group of sterol glycosides and triterpene glycosides which are common constituents of plants. One source of triterpenoid saponins obtained from the bark of Quillaia saponaria Molina (the soap bark tree) have been known to cause substantial enhancement of immune responses since the 1920s. Despite their use in animal vaccines, the development of saponin-based formulations for human vaccines has been impeded by their complexity and concerns about toxicity. This review briefly covers the use of saponins for animal vaccines but focuses mainly on the development of these adjuvants for use in man. Important aspects include preparation of purified or highly defined saponin fractions, improved understanding of the relationships between adjuvant activity, toxicity and structure of saponins and formulation of saponins into structures with reduced toxicity such as ISCOMs. Recent developments in the understanding of cellular interactions, cytokine induction and the in vivo localisation of saponin containing formulations will also be reviewed.

Zanamivir-Resistant Influenza Viruses with a Novel Neuraminidase Mutation

ABSTRACT The neuraminidase inhibitors zanamivir and oseltamivir are marketed for the treatment and prophylaxis of influenza and have been stockpiled by many countries for use in a pandemic. Although recent surveillance has identified a striking increase in the frequency of oseltamivir-resistant seasonal influenza A (H1N1) viruses in Europe, the United States, Oceania, and South Africa, to date there have been no reports of significant zanamivir resistance among influenza A (H1N1) viruses or any other human influenza viruses. We investigated the frequency of oseltamivir and zanamivir resistance in circulating seasonal influenza A (H1N1) viruses in Australasia and Southeast Asia. Analysis of 391 influenza A (H1N1) viruses isolated between 2006 and early 2008 from Australasia and Southeast Asia revealed nine viruses (2.3%) that demonstrated markedly reduced zanamivir susceptibility and contained a previously undescribed Gln136Lys (Q136K) neuraminidase mutation. The mutation had no effect on oseltamivir susceptibility but caused approximately a 300-fold and a 70-fold reduction in zanamivir and peramivir susceptibility, respectively. The role of the Q136K mutation in conferring zanamivir resistance was confirmed using reverse genetics. Interestingly, the mutation was not detected in the primary clinical specimens from which these mutant isolates were grown, suggesting that the resistant viruses either occurred in very low proportions in the primary clinical specimens or arose during MDCK cell culture passage. Compared to susceptible influenza A (H1N1) viruses, the Q136K mutant strains displayed greater viral fitness than the wild-type virus in MDCK cells but equivalent infectivity and transmissibility in a ferret model.

2009 Influenza A(H1N1) Seroconversion Rates and Risk Factors Among Distinct Adult Cohorts in Singapore

CONTEXT Singapore experienced a single epidemic wave of 2009 influenza A(H1N1) with epidemic activity starting in late June 2009 and peaking in early August before subsiding within a month. OBJECTIVE To compare the risk and factors associated with H1N1 seroconversion in different adult cohorts. DESIGN, SETTING, AND PARTICIPANTS A study with serial serological samples from 4 distinct cohorts: general population (n = 838), military personnel (n = 1213), staff from an acute care hospital (n = 558), and staff as well as residents from long-term care facilities (n = 300) from June 22, 2009, to October 15, 2009. Hemagglutination inhibition results of serum samples taken before, during, and after the epidemic and data from symptom questionnaires are presented. MAIN OUTCOME MEASURES A 4-fold or greater increase in titer between any of the 3 serological samples was defined as evidence of H1N1 seroconversion. RESULTS Baseline titers of 40 or more were observed in 22 members (2.6%; 95% confidence interval [CI], 1.7%-3.9%) of the community, 114 military personnel (9.4%; 95% CI, 7.9%-11.2%), 37 hospital staff (6.6%; 95% CI, 4.8%-9.0%), and 20 participants from long-term care facilities (6.7%; 95% CI, 4.4%-10.1%). In participants with 1 or more follow-up serum samples, 312 military personnel (29.4%; 95% CI, 26.8%-32.2%) seroconverted compared with 98 community members (13.5%; 95% CI, 11.2%-16.2%), 35 hospital staff (6.5%; 95% CI, 4.7%-8.9%), and only 3 long-term care participants (1.2%; 95% CI, 0.4%-3.5%). Increased frequency of seroconversion was observed for community participants from households in which 1 other member seroconverted (adjusted odds ratio [OR], 3.32; 95% CI, 1.50-7.33), whereas older age was associated with reduced odds of seroconversion (adjusted OR, 0.77 per 10 years; 95% CI, 0.64-0.93). Higher baseline titers were associated with decreased frequency of seroconversion in community (adjusted OR for every doubling of baseline titer, 0.48; 95% CI, 0.27-0.85), military (adjusted OR, 0.71; 95% CI, 0.61-0.81), and hospital staff cohorts (adjusted OR, 0.50; 95% CI, 0.26-0.93). CONCLUSION Following the June-September 2009 wave of 2009 influenza A(H1N1), 13% of the community participants seroconverted, and most of the adult population likely remained susceptible.

ISCOMs: an adjuvant with multiple functions

Aluminum salts are currently the only widely used adjuvant for human vaccines. Over the past 10–15 years, a large research effort has attempted to find novel adjuvants with ability to induce abroad range of immune responses, including cell‐mediated immunity. The immunostimulating complex or ISCOM is one adjuvant with multiple adjuvant properties. ISCOMs are open cage‐like complexes typically with a diameter of about 40 nm that are built up by cholesterol, lipid, immunogen, and saponins from the bark of the tree Quillaia saponaria Molina. ISCOMs have been demonstrated to promote antibody responses and induce T helper cell as well as cytotoxic T lymphocyte responses in a variety of experimental animal models, and have now progressed to phase I and II human trials. This review describes recent developments in the understanding of the structure, composition, and preparation of ISCOMs and will cover important aspects of the understanding of the adjuvant functions of ISCOMs and how they act on the immune system. J. Leukoc. Biol. 64: 713–723; 1998.

Efficacy of Soap and Water and Alcohol-Based Hand-Rub Preparations against Live H1N1 Influenza Virus on the Hands of Human Volunteers

BACKGROUND Although pandemic and avian influenza are known to be transmitted via human hands, there are minimal data regarding the effectiveness of routine hand hygiene (HH) protocols against pandemic and avian influenza. METHODS Twenty vaccinated, antibody-positive health care workers had their hands contaminated with 1 mL of 10(7) tissue culture infectious dose (TCID)(50)/0.1 mL live human influenza A virus (H1N1; A/New Caledonia/20/99) before undertaking 1 of 5 HH protocols (no HH [control], soap and water hand washing [SW], or use of 1 of 3 alcohol-based hand rubs [61.5% ethanol gel, 70% ethanol plus 0.5% chlorhexidine solution, or 70% isopropanol plus 0.5% chlorhexidine solution]). H1N1 concentrations were assessed before and after each intervention by viral culture and real-time reverse-transcriptase polymerase chain reaction (PCR). The natural viability of H1N1 on hands for >60 min without HH was also assessed. RESULTS There was an immediate reduction in culture-detectable and PCR-detectable H1N1 after brief cutaneous air drying--14 of 20 health care workers had H1N1 detected by means of culture (mean reduction, 10(3-4) TCID(50)/0.1 mL), whereas 6 of 20 had no viable H1N1 recovered; all 20 health care workers had similar changes in PCR test results. Marked antiviral efficacy was noted for all 4 HH protocols, on the basis of culture results (14 of 14 had no culturable H1N1; (P< .002) and PCR results (P< .001; cycle threshold value range, 33.3-39.4), with SW statistically superior (P< .001) to all 3 alcohol-based hand rubs, although the actual difference was only 1-100 virus copies/microL. There was minimal reduction in H1N1 after 60 min without HH. CONCLUSIONS HH with SW or alcohol-based hand rub is highly effective in reducing influenza A virus on human hands, although SW is the most effective intervention. Appropriate HH may be an important public health initiative to reduce pandemic and avian influenza transmission.

Identification of vaccine candidate antigens from a genomic analysis of Porphyromonas gingivalis.

Porphyromonas gingivalis is a key periodontal pathogen which has been implicated in the etiology of chronic adult periodontitis. Our aim was to develop a protein based vaccine for the prevention and or treatment of this disease. We used a whole genome sequencing approach to identify potential vaccine candidates. From a genomic sequence, we selected 120 genes using a series of bioinformatics methods. The selected genes were cloned for expression in Escherichia coli and screened with P. gingivalis antisera before purification and testing in an animal model. Two of these recombinant proteins (PG32 and PG33) demonstrated significant protection in the animal model, while a number were reactive with various antisera. This process allows the rapid identification of vaccine candidates from genomic data.

Epidemiological, antigenic and genetic characteristics of seasonal influenza A(H1N1), A(H3N2) and B influenza viruses: Basis for the WHO recommendation on the composition of influenza vaccines for use in the 2009–2010 Northern Hemisphere season

Influenza vaccines form an important component of the global response against infections and subsequent illness caused in man by influenza viruses. Twice a year, in February and September, the World Health Organisation through its Global Influenza Surveillance Network (GISN), recommends appropriate influenza viruses to be included in the seasonal influenza vaccine for the upcoming Northern and Southern Hemisphere winters. This recommendation is based on the latest data generated from many sources and the availability of viruses that are suitable for vaccine manufacture. This article gives a summary of the data and background to the recommendations for the 2009-2010 Northern Hemisphere influenza vaccine formulation.