Influenza gain-of-function experiments: their role in vaccine virus recommendation and pandemic preparedness.

In recent years, controversy has arisen regarding the risks and benefits of certain types of gain-of-function (GOF) studies involving avian influenza viruses. In this article, we provide specific examples of how different types of data, including information garnered from GOF studies, have helped to shape the influenza vaccine production process-from selection of candidate vaccine viruses (CVVs) to the manufacture and stockpiling of safe, high-yield prepandemic vaccines for the global community. The article is not written to support a specific pro- or anti-GOF stance but rather to inform the scientific community about factors involved in vaccine virus selection and the preparation of prepandemic influenza vaccines and the impact that some GOF information has had on this process.

Antibody landscapes after influenza virus infection or vaccination.

We introduce the antibody landscape, a method for the quantitative analysis of antibody-mediated immunity to antigenically variable pathogens, achieved by accounting for antigenic variation among pathogen strains. We generated antibody landscapes to study immune profiles covering 43 years of influenza A/H3N2 virus evolution for 69 individuals monitored for infection over 6 years and for 225 individuals pre- and postvaccination. Upon infection and vaccination, titers increased broadly, including previously encountered viruses far beyond the extent of cross-reactivity observed after a primary infection. We explored implications for vaccination and found that the use of an antigenically advanced virus had the dual benefit of inducing antibodies against both advanced and previous antigenic clusters. These results indicate that preemptive vaccine updates may improve influenza vaccine efficacy in previously exposed individuals.

Characteristics of a widespread community cluster of H275Y oseltamivir-resistant A(H1N1)pdm09 influenza in Australia.

BACKGROUND: Oseltamivir resistance in A(H1N1)pdm09 influenza is rare, particularly in untreated community cases. Sustained community transmission has not previously been reported. METHODS: Influenza specimens from the Asia-Pacific region were collected through sentinel surveillance, hospital, and general practitioner networks. Clinical and epidemiological information was collected on patients infected with oseltamivir-resistant viruses. RESULTS: Twenty-nine (15%) of 191 A(H1N1)pdm09 viruses collected between May and September 2011 from Hunter New England (HNE), Australia, contained the H275Y neuraminidase substitution responsible for oseltamivir resistance. Only 1 patient had received oseltamivir before specimen collection. The resistant strains were genetically very closely related, suggesting the spread of a single variant. Ninety percent of cases lived within 50 kilometers. Three genetically similar oseltamivir-resistant variants were detected outside of HNE, including 1 strain from Perth, approximately 4000 kilometers away. Computational analysis predicted that neuraminidase substitutions V241I, N369K, and N386S in these viruses may offset the destabilizing effect of the H275Y substitution. CONCLUSIONS: This cluster represents the first widespread community transmission of H275Y oseltamivir-resistant A(H1N1)pdm09 influenza. These cases and data on potential permissive mutations suggest that currently circulating A(H1N1)pdm09 viruses retain viral fitness in the presence of the H275Y mutation and that widespread emergence of oseltamivir-resistant strains may now be more likely.

Increased detection in Australia and Singapore of a novel influenza A(H1N1)2009 variant with reduced oseltamivir and zanamivir sensitivity due to a S247N neuraminidase mutation.

A novel influenza A(H1N1)2009 variant with mildly reduced oseltamivir and zanamivir sensitivity has been detected in more than 10% of community specimens in Singapore and more than 30% of samples from northern Australia during the early months of 2011. The variant, which has also been detected in other regions of the Asia-Pacific, contains a S247N neuraminidase mutation. When combined with the H275Y mutation, as detected in an oseltamivir-treated patient, the dual S247N+H275Y mutant had extremely high oseltamivir resistance.

Oseltamivir-resistant influenza viruses circulating during the first year of the influenza A(H1N1) 2009 pandemic in the Asia-Pacific region, March 2009 to March 2010.

During the first year of the influenza A(H1N1) 2009 pandemic, unprecedented amounts of the neuraminidase inhibitors, predominantly oseltamivir, were used in economically developed countries for the treatment and prophylaxis of patients prior to the availability of a pandemic vaccine. Due to concerns about the development of resistance, over 1,400 influenza A(H1N1) 2009 viruses isolated from the Asia-Pacific region during the first year of the pandemic (March 2009 to March 2010) were analysed by phenotypic and genotypic assays to determine their susceptibility to the neuraminidase inhibitors. Amongst viruses submitted to the World Health Organization Collaborating Centre for Reference and Research in Melbourne, Australia,oseltamivir resistance was detected in 1.3% of influenza A(H1N1) 2009 strains from Australia and 3.1% of strains from Singapore, but none was detected in specimens received from other countries in Oceania or south-east Asia, or in east Asia. The overall frequency of oseltamivir resistance in the Asia-Pacific region was 16 of 1,488 (1.1%). No zanamivir-resistant viruses were detected. Of the 16 oseltamivir-resistant isolates detected, nine were from immunocompromised individuals undergoing oseltamivir treatment and three were from immunocompetent individuals undergoing oseltamivir treatment. Importantly, four oseltamivir-resistant strains were from immunocompetent individuals who had not been treated with oseltamivir, demonstrating limited low-level community transmission of oseltamivir-resistant strains. Even with increased use of oseltamivir during the pandemic, the frequency of resistance has been low, with little evidence of community-wide spread of the resistant strains. Nevertheless, prudent use of the neuraminidase inhibitors remains necessary, as does continued monitoring for drug-resistant influenza viruses.

A new pandemic influenza A(H1N1) genetic variant predominated in the winter 2010 influenza season in Australia, New Zealand and Singapore.

Pandemic H1N1 influenza virus is of global health concern and is currently the predominant influenza virus subtype circulating in the southern hemisphere 2010 winter. The virus has changed little since it emerged in 2009, however, in this report we describe several genetically distinct changes in the pandemic H1N1 influenza virus. These variants were first detected in Singapore in early 2010 and have subsequently spread through Australia and New Zealand. At this stage, these signature changes in the haemagglutinin and neuraminidase proteins have not resulted in significant antigenic changes which might make the current vaccine less effective, but such adaptive mutations should be carefully monitored as the northern hemisphere approaches its winter influenza season.

Adamantane resistance in influenza A(H1) viruses increased in 2007 in South East Asia but decreased in Australia and some other countries.

The adamantanes (amantadine and rimantadine) were the initial antivirals licensed for use against influenza A viruses and have been used in some countries to control seasonal influenza and have also been stockpiled for potential pandemic use. While high rates of resistance have been observed in recent years with A(H3) viruses, the rates of resistance with A(H1) viruses has varied widely. In this study we analysed 281 human influenza A viruses isolated in 2007 that were referred to the WHO Collaborating Centre for Reference and Research in Melbourne, mainly from Australia and the surrounding regions, for evidence of resistance to adamantanes and a subset of these was examined for resistance to the neuraminidase inhibitors (NIs). We found that the rates of adamantane resistance in A(H3) viruses continued to increase in most countries in 2007 but a distinct variation was seen with A(H1) resistance levels. A(H1) viruses from Australia, New Zealand and Europe had low rates of resistance (2-9%) whereas viruses from a number of South East (SE) Asian countries had high rates of resistance (33-100%). This difference can be attributed to the spread of A/Brisbane/59/2007-like viruses to many parts of the world with the exception of SE Asia where A/Hong Kong/2652/2006-like viruses continue to predominate. When these two A(H1) subgroups were compared for their in vitro sensitivity to the other class of influenza antiviral drugs, the neuraminidase inhibitors, no difference was seen between the groups with both showing normal levels of sensitivity to these drugs, The finding of reducing A(H1) resistance rates in Australia and rising levels in SE Asia in 2007, reverses the trend seen in 2006 when A(H1) resistance levels were rising in Australia and elsewhere but remained low in most of SE Asia.

Genetic analysis of two influenza A (H1) swine viruses isolated from humans in Thailand and the Philippines.

Influenza viruses A/Philippines/341/2004 (H1N2) and A/Thailand/271/2005 (H1N1) were isolated from two males, with mild influenza providing evidence of sporadic human infection by contemporary swine influenza. Both viruses were antigenically and genetically distinct from influenza A (H1N1 and H1N2) viruses that have circulated in the human population. Genetic analysis of the haemagglutinin genes found these viruses to have the highest degree of similarity to the classical swine H1 viruses circulating in Asia and North America. The neuraminidase gene and the internal genes were found to be more closely related to viruses circulating in European swine, which appear to have undergone multiple reassorting events. Although transmission of swine influenza to humans appears to be a relatively rare event, swine have been proposed as the intermediate host in the generation of potential pandemic influenza virus that may have the capacity to cause human epidemics resulting in high morbidity and mortality.

The emergence of adamantane resistance in influenza A(H1) viruses in Australia and regionally in 2006.

The adamantanes (amantadine and rimantadine) were the first antivirals licensed for use against influenza A viruses and have been used in some countries to control seasonal influenza. While increasing resistance of A(H3) viruses to this class of drug has been reported in recent years, only low levels of resistance were seen with A(H1) viruses until the 2005-2006 influenza season in the USA. In this study we analysed 101 human influenza A viruses isolated in 2006 that were referred to the WHO Collaborating Centre for Reference and Research in Melbourne, from Australia and the surrounding regions, for evidence of resistance to adamantanes. We found that whereas previously A(H1) resistant viruses were rare, 21.8% of the 2006 viruses had a resistant genotype. By comparison, 58.6% of influenza A(H3) viruses isolated in 2006 that were tested at the Centre, had a resistant genotype.

Increased adamantane resistance in influenza A(H3) viruses in Australia and neighbouring countries in 2005.

The prevention and control of disease caused by seasonal and potential pandemic influenza viruses is currently managed by the use influenza vaccines and antivirals. The adamantanes (amantadine and rimantadine) were the first antivirals licensed for use against influenza A viruses and have been used extensively in some countries. Since the early 2000s increased resistance to these drugs has been reported especially in the A(H3) viruses. In this study we analysed recent human influenza A strains isolated in Australia and regionally for evidence of resistance to adamantanes and found evidence of significant resistant emerging during 2005.

Susceptibility of highly pathogenic A(H5N1) avian influenza viruses to the neuraminidase inhibitors and adamantanes.

Since 2003, highly pathogenic A(H5N1) influenza viruses have been the cause of large-scale death in poultry and the subsequent infection and death of over 140 humans. A group of 55 influenza A(H5N1) viruses isolated from various regions of South East Asia between 2004 and 2006 were tested for their susceptibility to the anti-influenza drugs the neuraminidase inhibitors and adamantanes. The majority of strains were found to be fully sensitive to the neuraminidase inhibitors oseltamivir carboxylate, zanamivir and peramivir; however two strains demonstrated increased IC50 values. Sequence analysis of these strains revealed mutations in the normally highly conserved residues 116 and 117 of the N1 neuraminidase. Sequence analysis of the M2 gene showed that all of the A(H5N1) viruses from Vietnam, Malaysia and Cambodia contained mutations (L26I and S31N) associated with resistance to the adamantane drugs (rimantadine and amantadine), while strains from Indonesia were found to be a mix of both adamantane resistant (S31N) and sensitive viruses. None of the A(H5N1) viruses from Myanmar contained mutations known to confer adamantane resistance. These results support the use of neuraminidase inhibitors as the most appropriate class of antiviral drug to prevent or treat human A(H5N1) virus infections.

Outbreak of influenza amongst residential school students in Malaysia.

In the months of July and August 2003, an outbreak of acute respiratory illness caused by influenza A virus occurred among students in seven residential schools situated in the northern part (Perak) of Peninsular Malaysia. Out of 4989 students, aged 13 to 18 years (mean = 15.9), 1419 (28%) were effected by influenza-like illness. All patients were treated as outpatients except for 36 students who required admission for high fever, severe coughing and shortness of breath. Abnormal chest X-ray findings were noted for those that required inpatient management. Influenza A virus was isolated from 37 sputum specimens, 20 throat swabs and three nasal swab specimens from a total of 278 clinical samples obtained from 180 patients. Isolates from each of the outbreaks were sent to WHO Collaborating Centre for Reference and Research on Influenza, Melbourne, Australia for antigenic and genetic analysis. One school outbreak was due to influenza A (H1N1), A/New Caledonia/20/99-like virus while the other six school outbreaks were due to influenza A (H3N2) viruses which were A/Fujian/411/2002-like).

The rapid identification of human influenza neuraminidase N1 and N2 subtypes by ELISA.

An ELISA assay was developed to allow the rapid and accurate identification of human influenza A N1 and N2 neuraminidases. Initial testing using a fetuin pre-coating of wells correctly identified 81.7% of the neuraminidase type from a series of human A(H1N1), A(H1N2) and A(H3N2) viruses. This result could be improved to detect the neuraminidase subtype of almost all human influenza A viruses from a large panel of viruses isolated from 2000 to 2005, if the fetuin pre-coating was removed and the viruses were coated directly onto wells. This method is simple, rapid and can be used to screen large numbers of currently circulating human influenza A viruses for their neurraminidase subtype and is a good alternative to RT-PCR.

Identification of genetic diversity by cultivating influenza A(H3N2) virus in vitro in the presence of post-infection sera from small children.

Antigenic variants probably arise in the field by escaping herd immunity. We have earlier found that sera from small children are more strain-specific than sera from adults and could therefore, provide favourable conditions for selecting antigenic escape mutants. We had access to small volumes of anonymous sera collected in Norway after the epidemic season 1999/00, which was dominated by the A/Panama/2007/99 (H3N2) variant. The HA gene of the representative strain of that season was genetically identical to A/South Australia/147/99 (H3N2) and was selected for this study. Two sera from children aged 4 and 3 years, respectively, and one adult (64 years old) were used to attempt selecting antigenic escape mutants. Virus was grown in MDCK cells in the presence of human serum and escaped variants were tested by haemagglutination-inhibition tests. Although variant strains were occasionally identified, their HA1 genetic sequence did not identify obvious changes at known antigenic sites. However, by cloning and subsequent sequencing, the genetic diversity of the parent virus was found to be significantly reduced when grown in the presence of human sera. Data also showed that the two children's sera selected additional mutants from those already present in the parent pool and that the two sera selected different mutants. On a community level, it is possible that antigenic changes could be accumulated in a step-wise manner when epidemic virus is transmitted from one small child to the next, each with a restricted and possibly variant antibody repertoire.

Isolation of avian influenza viruses from two different transhemispheric migratory shorebird species in Australia.

Shorebirds on their southerly migration from Siberia to Australia, may pass through Asian regions currently experiencing outbreaks of highly pathogenic H5N1 influenza. To test for the presence of avian influenza viruses in migratory shorebirds arriving in Australia during spring 2004, 173 cloacal swabs were collected from six species. Ten swabs were positive for influenza A, with H4N8 viruses detected in five red-necked stints and H11N9 viruses detected in five sharp-tailed sandpipers. No H5N1 viruses were detected. All isolated viruses were non-pathogenic in domestic chickens. These results further demonstrate the potential for migratory shorebirds to carry and potentially spread influenza viruses.

An influenza A(H3) reassortant was epidemic in Australia and New Zealand in 2003.

During 2003, Australia and New Zealand experienced substantial outbreaks of influenza. The strain responsible was an A(H3N2) influenza virus described as A/Fujian/411/2002-like, which had circulated as a minor variant in the previous Northern Hemisphere (NH) winter, mainly in Korea and Japan. Early in the year the isolates were very similar to those that had been previously isolated in the NH, however, a reassortant strain emerged early in the New Zealand winter, followed by the appearance of similar viruses in Australia and other regional areas. While the hemagglutinin HA1 sequence of these viruses demonstrated only minor differences from the A/Fujian/411/2002 reference strain, the neuraminidase gene was clearly different from that of other recently circulating H3 viruses and most closely matched an earlier reference strain A/Chile/6416/2001. Three internal genes (NS, NP, M) in the reassortant viruses were also more closely related to the A/Chile/6416/2001 lineage. This reassortant A(H3) virus predominated in Australia and New Zealand in 2003 was also seen in Brazil and Malaysia during 2003 and was widespread in the United States and Europe during their 2003-04 winter. Interestingly most of the strains of A(H3) that were isolated at the beginning of the 2004 winter in Australia, did not have this earlier A/Chile/6416/2001-like neuraminidase but had a neuraminidase that was similar to that of the reference strain A/Fujian/411/2002. This was suggestive of the re-introduction of influenza A(H3) from other countries, however, there was still low level circulation of the reassortant virus in 2004 with isolates detected in Australia and Singapore.

Characterization of two outer membrane protein antigens of Porphyromonas gingivalis that are protective in a murine lesion model.

Porphyromonas gingivalis is a key periodontal pathogen that has been implicated in the aetiology of chronic adult periodontitis. The aim of this study was to characterize two potential vaccine candidates (PG32 and PG33) identified from a previous genomic sequence analysis. Gene knockout studies suggested that these proteins play an important role in bacterial growth and are transcriptionally linked. Analysis of 14 laboratory and clinical isolates of P. gingivalis found that in all strains, both genes were present with a high level of conservation and that the two proteins were also expressed in vitro. Truncated recombinant PG32 and PG33 proteins were produced in Escherichia coli in an attempt to increase the solubility of the proteins while retaining their native conformation. While most of the truncated proteins remained insoluble, two truncated proteins showed good solubility and high levels of protection in the P. gingivalis murine lesion model and may be considered as potential vaccine candidates for further testing in models of human periodontal disease.

Reassortants in recent human influenza A and B isolates from South East Asia and Oceania.

From 2000 to 2002, human influenza A and B viruses that were genetic reassortants of contemporary circulating human strains, were isolated in South East Asia and Oceania. Similar to reports from other regions, A(H1N2) isolates were found to be reassortants of circulating A(H3N2) viruses that had acquired only the haemagglutinin gene of an A(H1N1) virus. Some of these reassortants from Thailand and Singapore predate those previously recorded during the winter of 2001-2002 in Europe and the Middle East and may be precursors of these viruses. The B reassortants had a haemagglutinin similar to an earlier B strain, B/Shangdong/7/97 (B/Victoria/2/87-lineage) and a neuraminidase similar to the recently circulating B/Sichuan/379/99 virus (B/Yamagata/16/88-lineage). Despite the early occurrences of A(H1N2) reassortants and the extensive circulation of A(H1) viruses in South East Asia and Oceania during 2000-2001, these reassortant influenza A viruses have to date not been prominent unlike Europe and the Middle East where they were common in the 2001-2002 winter. In contrast the reassortant B viruses, which first emerged in this region in early 2002, rapidly became the predominant strains isolated from patients with influenza B in South East Asia and Oceania.

Characterization and expression of a novel Porphyromonas gingivalis outer membrane protein, Omp28.

We report the characterization of a Porphyromonas gingivalis gene, designated omp28, encoding a protein that we have previously purified and characterized as a 28-kDa outer membrane protein. The deduced amino acid sequence of the omp28 open reading frame displayed an outer membrane leader sequence and lipoprotein attachment site but did not exhibit any significant overall sequence identity with protein sequences in the databases. A small stretch of amino acids (19 residues) exhibits 50% sequence identity with a segment of a fimbrial protein from Dichelobacter nodosus involved in adhesion, suggesting that Omp28 may be a surface adhesin/receptor of P. gingivalis. Using the pET-24 vector we expressed recombinant Omp28 (rOmp28) in Escherichia coli. Western blot analyses of purified rOmp28 with rabbit antisera to a P. gingivalis outer membrane preparation, protective rat anti-whole P. gingivalis antisera and pooled human sera from chronic periodontitis patients showed that the recombinant was recognized by all antisera. Further, anti-rOmp28 antisera exhibited strong reactivity with a panel of four laboratory strains and 10 clinical isolates of P. gingivalis from the United States, Sudan, Romania and Norway. These results suggest that Omp28 is expressed by a wide distribution of P. gingivalis strains.