Development and approval of live attenuated influenza vaccines based on Russian master donor viruses: Process challenges and success stories

Influenza is a viral infection that affects much of the global population each year. Vaccination remains the most effective tool for preventing the disease. Live attenuated influenza vaccine (LAIV) has been used since the 1950s to protect humans against seasonal influenza. LAIVs developed by the Institute of Experimental Medicine (IEM), Saint Petersburg, Russia, have been successfully used in Russia since 1987.In 2006, the World Health Organization (WHO) announced a Global action plan for influenza vaccines (GAP). WHO, recognizing potential advantages of LAIV over the inactivated influenza vaccine in a pandemic situation, included LAIV in the GAP.BioDiem Ltd., a vaccine development company based in Melbourne, Australia which held the rights for the Russian LAIV, licensed this technology to WHO in 2009. WHO was permitted to grant sub-licenses to vaccine manufacturers in newly industrialized and developing countries to use the Russian LAIV for the development, manufacture, use and sale of pandemic and seasonal LAIVs. To date, WHO has granted sub-licenses to vaccine manufacturers in China (Changchun BCHT Biotechnology Co., Ltd.), India (Serum Institute of India Pvt. Ltd.) and Thailand (Government Pharmaceutical Organization). In parallel, in 2009, IEM signed an agreement with WHO, under which IEM committed to supply pandemic and seasonal candidate vaccine viruses to the sub-licensees.This paper describes the progress made by collaborators from China, India, Russia and Thailand in developing preventive measures, including LAIV against pandemic influenza.     

A pandemic influenza vaccine in India: from strain to sale within 12 months

In the event of a highly pathogenic influenza pandemic, the Indian subcontinent would need 1.2 billion doses of vaccine to immunize its entire population, double if two doses were required to assure immunity. Serum Institute of India Limited (SII) thus became one of six initial grantees of the World Health Organization (WHO) technology transfer initiative to create capacity in developing countries to manufacture H5N1 pandemic influenza vaccine. At the outbreak of the A(H1N1) 2009 influenza pandemic, experience gained from the H5N1 project was used to develop a live attenuated influenza vaccine (LAIV), since this was the only option for the level of surge capacity required for a large-scale immunization campaign in India. SII took <12 months to develop and market its LAIV intranasal vaccine from receipt of the seed strain from WHO. As of November 2010, over 2.5 million persons have been vaccinated with Nasovac(?) with no serious adverse reactions or vaccine failure after 3 months' post-marketing surveillance. The product has been submitted for prequalification by WHO for purchase by United Nations agencies. In parallel, SII also developed an inactivated influenza vaccine, and is currently looking to ensure the sustainability of its influenza vaccine manufacturing capacity.     

Efficacy of live attenuated vaccines against 2009 pandemic H1N1 influenza in ferrets

The advent of the H1N1 influenza pandemic (pH1N1) in 2009 triggered the rapid production of pandemic influenza vaccines, since seasonal influenza vaccines were expected and demonstrated not to provide significant cross-protection against the newly emerged pandemic virus. To increase vaccine production capacity and further evaluate the effectiveness of different candidate pandemic influenza vaccines, the World Health Organization stimulated the evaluation of different vaccination concepts including the use of live attenuated influenza vaccines (LAIVs). Therefore, we have immunized ferrets intranasally with a single dose of pH1N1-LAIV from different manufacturers. They all induced adequate serum HI antibody titers in the ferrets and protected them against intratracheal wild-type pH1N1 virus challenge: pH1N1 virus replication in the upper respiratory tract and lungs was reduced and no disease signs or severe broncho-interstitial pneumonia were observed in any of the vaccinated ferrets. These data together with the relatively efficient production process emphasize the potential of the LAIV concept for pandemic preparedness.     

The development of vaccine viruses against pandemic A(H1N1) influenza

Wild type human influenza viruses do not usually grow well in embryonated hens’ eggs, the substrate of choice for the production of inactivated influenza vaccine, and vaccine viruses need to be developed specifically for this purpose. In the event of a pandemic of influenza, vaccine viruses need to be created with utmost speed. At the onset of the current A(H1N1) pandemic in April 2009, a network of laboratories began a race against time to develop suitable candidate vaccine viruses. Two approaches were followed, the classical reassortment approach and the more recent reverse genetics approach. This report describes the development and the characteristics of current pandemic H1N1 candidate vaccine viruses.     

The 2009 pandemic H1N1 virus induces anti-neuraminidase (NA) antibodies that cross-react with the NA of H5N1 viruses in ferrets

A miniaturized neuraminidase inhibition (NI) assay using HA-mismatched H6 reassortant viruses was performed to examine the neuraminidase (NA)-specific antibody response in ferrets immunized with live-attenuated influenza vaccine (LAIV) strains. The strains tested possessed different NAs derived from seasonal H1N1 and H3N2, 2009 pandemic H1N1, and the highly pathogenic influenza H5N1 virus. The anti-NA antibodies from the 2009 pandemic strain (A/California/7/2009) immunized ferrets cross-reacted with the NA of H5N1 but not with the NA of seasonal H1N1 viruses. The plaque size reduction assay confirmed the cross-reactivity between the NAs of A/California/7/2009 and the H5N1 virus. Sequence and structural analyses of these N1 NA proteins showed that the NA of the 2009 pandemic H1N1 strain shared at least 22 more amino acids in the head domain with the NAs of the avian H5N1 strains than with the NAs of seasonal human H1N1 viruses. Our data demonstrated LAIV-induced NA antibody responses in ferrets and cross-reactive NA antibodies induced by 2009 pandemic H1N1 and H5N1 LAIV viruses.     

Efficacy of seasonal live attenuated influenza vaccine against virus replication and transmission of a pandemic 2009 H1N1 virus in ferrets

In March 2009, a swine origin influenza A (2009 H1N1) virus was introduced into the human population and quickly spread from North America to multiple continents. Human serologic studies suggest that seasonal influenza virus vaccination or infection would provide little cross-reactive serologic immunity to the pandemic 2009 H1N1 virus. However, the efficacy of seasonal influenza infection or vaccination against 2009 H1N1 virus replication and transmission has not been adequately evaluated in vivo. Here, ferrets received one or two doses of the US licensed 2008-2009 live attenuated influenza vaccine (LAIV) intranasally. An additional group of ferrets were inoculated with the A/Brisbane/59/07 (H1N1) virus to model immunity induced by seasonal influenza virus infection. All vaccinated and infected animals possessed high titer homologous hemagglutination-inhibition (HI) and neutralizing antibodies, with no demonstrable cross-reactive antibodies against 2009 H1N1 virus. However, in comparison to non-immune controls, immunized ferrets challenged with pandemic A/Mexico/4482/09 virus displayed a significant reduction in body temperature and virus shedding. The impact of single-dose LAIV inoculation on 2009 H1N1 disease and virus transmission was also measured in vaccinated ferrets that were challenged with pandemic A/Netherlands/1132/09 virus. Although a single dose of LAIV reduced virus shedding and the frequency of transmission following homologous seasonal virus challenge, it failed to reduce respiratory droplet transmission of 2009 H1N1 virus. The results demonstrate that prior immunization with seasonal LAIV or H1N1 virus infection provides some cross-protection against the 2009 H1N1 virus, but had no significant effect on the transmission efficiency of the 2009 H1N1 virus.     

AF03-adjuvanted and non-adjuvanted pandemic influenza A (H1N1) 2009 vaccines induce strong antibody responses in seasonal influenza vaccine-primed and unprimed mice

Pandemic influenza vaccines have been manufactured using the A/California/07/2009 (H1N1) strain as recommended by the World Health Organization. We evaluated in mice the immunogenicity of pandemic (H1N1) 2009 vaccine and the impact of prior vaccination against seasonal trivalent influenza vaccines (TIV) on antibody responses against pandemic (H1N1) 2009. In naïve mice, a single dose of unadjuvanted H1N1 vaccine (3 μg of HA) was shown to elicit hemagglutination inhibition (HI) antibody titers >40, a titer associated with protection in humans against seasonal influenza. A second vaccine dose of pandemic (H1N1) 2009 vaccine strongly increased these titers, which were consistently higher in mice previously primed with TIV than in naïve mice. At a low immunization dose (0.3 μg of HA), the AF03-adjuvanted vaccine elicited higher HI antibody titers than the corresponding unadjuvanted vaccines in both naïve and TIV-primed animals, suggesting a potential for antigen dose-sparing. These results are in accordance with the use in humans of a split-virion inactivated pandemic (H1N1) 2009 vaccine formulated with or without AF03 adjuvant to protect children and young adults against influenza A (H1N1) 2009 infection.     

Genetic stability of live attenuated vaccines against potentially pandemic influenza viruses

BackgroundEnsuring genetic stability is a prerequisite for live attenuated influenza vaccine (LAIV). This study describes the results of virus shedding and clinical isolates’ testing of Phase I clinical trials of Russian LAIVs against potentially pandemic influenza viruses in healthy adults.MethodsThree live attenuated vaccines against potentially pandemic influenza viruses, H2N2 LAIV, H5N2 LAIV and H7N3 LAIV, generated by classical reassortment in eggs, were studied. For each vaccine tested, subjects were randomly distributed into two groups to receive two doses of either LAIV or placebo at a 3:1 vaccine/placebo ratio. Nasal swabs were examined for vaccine virus shedding by culturing in eggs and by PCR. Vaccine isolates were tested for temperature sensitivity and cold-adaptation (ts/ca phenotypes) and for nucleotide sequence.ResultsThe majority of nasal wash positive specimens were detected on the first day following vaccination. PCR method demonstrated higher sensitivity than routine virus isolation in eggs. None of the placebo recipients had detectable vaccine virus replication.All viruses isolated from the immunized subjects retained the ts/ca phenotypic characteristics of the master donor virus (MDV) and were shown to preserve all attenuating mutations described for the MDV. These data suggest high level of vaccine virus genetic stability after replication in humans.During manufacture process, no additional mutations occurred in the genome of H2N2 LAIV. In contrast, one amino acid change in the HA of H7N3 LAIV and two additional mutations in the HA of H5N2 LAIV manufactured vaccine lot were detected, however, they did not affect their ts/ca phenotypes.ConclusionsOur clinical trials revealed phenotypic and genetic stability of the LAIV viruses recovered from the immunized volunteers. In addition, no vaccine virus was detected in the placebo groups indicating the lack of person-to-person transmission.LAIV TRIAL REGISTRATION at ClinicalTrials.gov: H7N3-NCT01511419; H5N2-NCT01719783; H2N2-NCT01982331.     

Development of influenza vaccine production capacity by the Government Pharmaceutical Organization of Thailand: addressing the threat of an influenza pandemic

In 2005, a year after highly pathogenic avian influenza outbreaks in Thailand, the Thai Government issued a National Strategy Plan for Pandemic Influenza Preparedness, a major objective of which was the domestic production of seasonal influenza vaccine. It was considered that sustained influenza vaccine production was the best guarantee of a pandemic vaccine in the event of a future pandemic. The Government decided to provide funds to establish an industrial-scale influenza vaccine production plant, and gave responsibility for this challenging project to the Government Pharmaceutical Organization (GPO). In 2007, with support from the World Health Organization (WHO), the GPO started to develop egg-based, trivalent inactivated influenza vaccine (IIV) in a renovated pilot plant. In early 2009, during the second year of the project, the GPO turned its attention to develop a pandemic live attenuated influenza vaccine (PLAIV) against the influenza A (H1N1) virus. By December 2010, the H1N1 PLAIV had successfully completed Phase II clinical trials and was awaiting registration approval from the Thai Food and Drug Administration (TFDA). The GPO has also started to develop an H5N2 PLAIV, which is expected to enter clinical trials in January 2011. The next step in 2011 will be the development and clinical evaluation of seasonal LAIV. To meet the needs of the national seasonal influenza vaccination programme, the GPO aims to produce 2 million doses of trivalent IIV in 2012 and progressively increase production to the maximum annual capacity of 10 million doses. This article relates how influenza vaccine production capacity was developed and how major challenges are being met in an expeditious manner, with strong local and global commitment.     

Immunogenicity and efficacy of the monovalent,trivalent and quadrivalent intranasal live attenuated influenza vaccines containing different pdmH1N1 strains

A ferret challenge study was conducted to address the efficacy of the egg-based and Madin-Darby canine kidney (MDCK)-based live attenuated influenza vaccine (LAIV) strains. Vaccines derived as 6:2 reassortants from the A/Leningrad/134/17/57 master donor strain and the HA and NA components from the A/California/07/2009 (A/Cal)- and A/Michigan/45/2015 (A/Mich)-like strains of type A H1N1 influenza virus were used in the study. Monovalent, trivalent and quadrivalent formulations of the LAIV containing either of the two H1N1 strains were analysed. A total of ten groups of six animals each were immunised intranasally (i.n.) with a single dose of 0.5-ml vaccine formulation or placebo and challenged on day 28 with the homologous wild-type A/Cal or A/Mich strain. Immune response post immunisation and virus replication post challenge were studied.Both the strains derived from embryonated eggs or MDCK cells, irrespective of the vaccine valency, were capable of rendering complete protection from virus replication in the lung. The A/Mich vaccine strain showed higher immune titres and efficacy than the A/Cal vaccine strain in all the vaccine formulations. The haemagglutination inhibition and virus neutralisation antibody titres were induced, and the reduction in the virus load in the respiratory tract was observed to be higher in animals treated with the monovalent formulation compared to the trivalent and quadrivalent formulations. Overall, it appears that the monovalent formulations render better protection from infection and would therefore be the best candidate during a pandemic.     

Lessons from pandemic influenza A(H1N1): the research-based vaccine industry's perspective

As A(H1N1) influenza enters the post-pandemic phase, health authorities around the world are reviewing the response to the pandemic. To ensure this process enhances future preparations, it is essential that perspectives are included from all relevant stakeholders, including vaccine manufacturers. This paper outlines the contribution of R&D-based influenza vaccine producers to the pandemic response, and explores lessons that can be learned to improve future preparedness.The emergence of 2009 A(H1N1) influenza led to unprecedented collaboration between global health authorities, scientists and manufacturers, resulting in the most comprehensive pandemic response ever undertaken, with a number of vaccines approved for use three months after the pandemic declaration. This response was only possible because of the extensive preparations undertaken during the last decade.During this period, manufacturers greatly increased influenza vaccine production capacity, and estimates suggest a further doubling of capacity by 2014. Producers also introduced cell-culture technology, while adjuvant and whole virion technologies significantly reduced pandemic vaccine antigen content. This substantially increased pandemic vaccine production capacity, which in July 2009 WHO estimated reached 4.9 billion doses per annum. Manufacturers also worked with health authorities to establish risk management plans for robust vaccine surveillance during the pandemic. Individual producers pledged significant donations of vaccine doses and tiered-pricing approaches for developing country supply.Based on the pandemic experience, a number of improvements would strengthen future preparedness. Technical improvements to rapidly select optimal vaccine viruses, and processes to speed up vaccine standardization, could accelerate and extend vaccine availability. Establishing vaccine supply agreements beforehand would avoid the need for complex discussions during a period of intense time pressure.Enhancing international regulatory co-operation and mutual recognition of approvals could accelerate vaccine supply, while maintaining safety standards. Strengthening communications with the public and healthcare workers using new approaches and new channels could help improve vaccine uptake. Finally, increasing seasonal vaccine coverage will be particularly important to extend and sustain pandemic vaccine production capacity.     

Control of pandemic (H1N1) 2009 influenza virus infection of ferret lungs by non-adjuvant-containing pandemic and seasonal vaccines

The pandemic H1N1 2009 influenza virus caused relatively mild disease in most infected people but some suffered extensively from primary lung infection, many more than would have occurred with seasonal influenza infection. Early commercially available pandemic H1N1 vaccines did not contain adjuvant, as did many of the subsequent vaccines, and could not stop infection with the pandemic virus in vaccinated ferrets. Nevertheless, we showed that virus loads in the lungs were greatly diminished in ferrets vaccinated once with an unadjuvanted pandemic vaccine and challenged with 10(6)EID(50) wildtype A/California/07/2009 (H1N1). In addition, a single inoculation with seasonal vaccine showed beneficial reduction in pandemic pulmonary virus loads in the absence of any detectable cross-reactive serological responses. Ferrets primed with either seasonal or pandemic vaccine and then boosted with pandemic vaccine also showed less extensive lung infection when challenged with a tenfold higher dose of pandemic virus. These results implicate non-classical protective mechanisms that prevent severe pulmonary disease but not viral shedding and imply that particular non-adjuvanted vaccines may have retained the ability to induce these responses.     

Pandemic and seasonal H1N1 influenza hemagglutinin-specific T cell responses elicited by seasonal influenza vaccination

Understanding whether seasonal influenza vaccines can elicit antibody and T cell responses against the 2009 pandemic H1N1 strain is important. We compared T cell and antibody responses elicited by trivalent inactivated influenza vaccine (TIV) and live attenuated influenza vaccine (LAIV) in healthy adults. Both vaccines boosted pre-existing T cells to the seasonal and pandemic hemagglutinin (HA) but responses were significantly greater following immunization with LAIV. Antibody titers were significantly boosted only by TIV. The relationship between antibody and T cell responses and the effect of the magnitude of pre-existing immunity on vaccine-induced responses were also evaluated. Cross reactive T cell responses to the pandemic H1N1 HA existed among the cohort before the circulation of the virus to varying degrees and these responses were boosted by seasonal vaccination.     

Vaccination against pandemic influenza A/H1N1 among healthcare workers and reasons for refusing vaccination in Istanbul in last pandemic alert phase

Coverage of the HCWs as target population is one of the important determinants for the impact of vaccination. To determine the vaccination against the pandemic influenza A/H1N1 among HCWs, we conducted a cross-sectional questionnaire survey in a public hospital in Istanbul from December 7 to December 22, 2009. Out of total 941 HCWs 718 (76.3%) completed the questionnaires. Nearly one-fourth (23.1%) of the participants were vaccinated against pandemic influenza A/H1N1. Occupation (being a doctor), receiving seasonal influenza vaccine in 2009, agreement with safety of pandemic influenza A/H1N1 vaccine and being comprehend that HCWs have a professional responsibility for getting vaccinated was the strongest independent predictive factor for accepting the pandemic influenza A/H1N1 vaccine (p < .0001). The most frequent reasons for refusing pandemic vaccine were fear of side effects and doubts about vaccine efficacy. Among HCWs 59.6% were recommending pandemic influenza vaccination to a patient even if indicated. In conclusion vaccination against pandemic influenza A/H1N1 is insufficient among HCWs. Misinformed or inadequately informed HCWs are important barrier to pandemic influenza vaccine coverage of the general public also. Educational campaigns concerning HCWs should include evidence based and comprehensible information about possible adverse effects and their incidence besides the advantages of vaccine.     

Improvement of H5N1 influenza vaccine viruses: influence of internal gene segments of avian and human origin on production and hemagglutinin content

The H5N1-clade 1 influenza vaccine strain NIBRG-14 produces exceptionally low amounts of antigen, a problem recently encountered also for initial pandemic H1N1-2009 vaccine seeds. Here, we report on a strategy that may contribute to overcome this obstacle. Influenza vaccine viruses usually consist of two segments coding for the antigenic HA and NA proteins of a wild-type strain and the six residual internal gene segments of the vaccine donor strain A/PR/8/34 (PR8). To enhance the antigen yield from H5N1 vaccine virus we generated by reverse genetics a set of PR8-based reassortant viruses expressing the HA and NA segments of the prototypic strain A/Vietnam/1203/2004 and additional replacements of the internal M or PB1 genes of PR8. The reassortants were compared to the parental PR8 and H5N1 viruses in terms of growth in embryonated chicken eggs and the amount of incorporated antigenic HA protein. Compared to NIBRG-14, three out of six viruses displayed an increased replication in embryonated chicken eggs and higher HA content that was also maintained after ether/detergent extraction of virions. Electron microscopic analysis showed that the reassortment hardly affected particle shape and size. Two selected H5N1 reassortant viruses were investigated concerning their pathogenicity in ferrets and found to behave as low pathogenic as the PR8 donor strain. In conclusion, this study shows that replication and antigen content of PR8-derived H5N1 influenza vaccine viruses can be improved by incorporation of heterologous internal gene segments without compromising their attenuated character.     

Advancing new vaccines against pandemic influenza in low-resource countries

With the support of the Biomedical Advanced Research and Development Authority (BARDA), PATH is working with governments and vaccine manufacturers to strengthen their influenza vaccine manufacturing capacity and improve their ability to respond to emerging pandemic influenza viruses. Vaccines directed against influenza A/H5N1 and A/H7N9 strains are a particular focus, given the potential for these viruses to acquire properties that may lead to a pandemic. This paper will review influenza vaccine development from a developing country perspective and PATH's support of this effort. Several vaccines are currently in preclinical and clinical development at our partners for seasonal and pandemic influenza in Vietnam (IVAC and VABIOTECH), Serbia (Torlak), China (BCHT), Brazil (Butantan), and India (SII). Products in development include split, whole-virus inactivated and live attenuated influenza vaccines (LAIVs). Additionally, while most manufacturers propagate the virus in eggs, PATH is supporting the development of cell-based processes that could substantially increase global manufacturing capacity and flexibility. We review recent data from clinical trials of pandemic influenza vaccines manufactured in developing countries. An important discussion is on the role of whole virion vaccines for H5N1, given the poor immunogenicity of split vaccines and the complexity involved in developing potent adjuvants.     

Association of vaccine handling conditions with effectiveness of live attenuated influenza vaccine against H1N1pdm09 viruses in the United States

PurposeThis analysis examined potential causes of the lack of vaccine effectiveness (VE) of live attenuated influenza vaccine (LAIV) against A/H1N1pdm09 viruses in the United States (US) during the 2013–2014 season. Laboratory studies have demonstrated reduced thermal stability of A/California/07/2009, the A/H1N1pdm09 strain utilized in LAIV from 2009 through 2013–2014.MethodsPost hoc analyses of a 2013–2014 test-negative case-control (TNCC) effectiveness study investigated associations between vaccine shipping conditions and LAIV lot effectiveness. Investigational sites provided the LAIV lot numbers administered to each LAIV recipient enrolled in the study, and the vaccine distributor used by the site for commercially purchased vaccine. Additionally, a review was conducted of 2009–2014 pediatric observational TNCC effectiveness studies of LAIV, summarizing effectiveness by type/subtype, season, and geographic location.ResultsFrom the 2013 to 2014 TNCC study, the proportion of LAIV recipients who tested positive for H1N1pdm09 was significantly higher among children who received a lot released between August 1 and September 15, 2013, compared with a lot shipped either earlier or later (21% versus 4%; P < 0.01). A linear relationship was observed between the proportion of subjects testing positive for H1N1pdm09 and outdoor temperatures during truck unloading at distributors’ central locations. The review of LAIV VE studies showed that in the 2010–2011 and 2013–2014 influenza seasons, no significant effectiveness of LAIV against H1N1pdm09 was demonstrated for the trivalent or quadrivalent formulations of LAIV in the US, respectively, in contrast to significant effectiveness against A/H3N2 and B strains during 2010–2014.ConclusionsThis study showed that the lack of VE observed with LAIV in the US against H1N1pdm09 viruses was associated with exposure of some LAIV lots to temperatures above recommended storage conditions during US distribution, and is likely explained by the increased susceptibility of the A/California/7/2009 (H1N1pdm09) LAIV strain to thermal degradation.Clinical trial registry: NCT01997450     

Influenza pandemic (H1N1) 2009 activity during summer 2009. Effectiveness of the 2008-9 trivalent vaccine against pandemic influenza in Spain

Introduction

The Spanish influenza surveillance system (SISS) maintained its activity during the summer of 2009 to monitor the influenza pandemic.

Objectives

To describe pandemic influenza activity from May to September 2009 and to estimate the effectiveness of the 2008-9 seasonal influenza vaccine against laboratory-confirmed pandemic (H1N1) 2009 influenza.

Methods

Data from the SISS were used to identify the trend of pandemic (H1N1) 2009 influenza outside the influenza season. For the effectiveness study, we compared the vaccination status of notified cases [influenza-like illnesses (ILI) laboratory confirmed as pandemic influenza] with that of the test-negative controls.

Results

The first laboratory-confirmed case of the pandemic virus was notified in the system in week 20/2009. The ILI rate increased gradually in the study period, exceeding basic activity in week 38. The proportion of pandemic (H1N1) 2009 influenza viruses detected by the system represented 14% in week 20/2009 and rapidly increased to 90% in week 34. The adjusted vaccine effectiveness of the 2008-9 seasonal vaccine against laboratory-confirmed pandemic influenza was 12% (-30; 41).

Conclusions

The SISS became an essential tool for pandemic monitoring in Spain. The improved SISS will provide more accurate information on influenza activity in future seasonal or pandemic waves. Using surveillance data, we could not demonstrate the effectiveness of the seasonal 2008-9 vaccine against laboratory-confirmed pandemic influenza.