4
WORKING GROUPS, DAY 1

WORKING GROUP 1 INFLUENZA VIRULENCE AND ANTIGENIC CHANGE

Chairperson—Robert Webster

Briefer—Peter Palese

Rapporteur—Robert Lamb


Charge: The charge of this working group is to define research priorities associated with understanding influenza virulence, with tracking and predicting antigenic change, and with defining which antigenic changes may be associated with increased virulence, risk of transmission, and pandemic potential.


Potential issues to consider:

  1. What studies are needed to define the genetic loci for pathogenicity in avian and human influenza virus strains?

  2. What studies are needed to affirm the hypothesis that incremental acquisition of genetic changes can lead to influenza pandemics as compared with the sudden emergence of previous pandemics?

  3. What studies are needed to track the rate of antigenic change in avian and human influenza virus strains and to predict changes that may occur?

  4. What studies are needed to determine whether pandemic risk can be predicted by virulence factors and/or antigenic characteristics?

REPORT TO PLENARY

Rapporteur—Dr. Robert Lamb

One question concerned how we would define the loci for pathogenicity with influenza virus. We know at the molecular level the importance of the hemagglutinin’s cleavage site. It is required to becleaved to HA1 and HA2 which activates the molecule for fusion..

We know that if we have a multi-basic residue at the HA cleavage site, it has the potential to be cleaved in the trans-Golgi network, and that correlates directly with high pathogenicity of viruses in many cases. If that cleavage site has only a single basic residue, it very often correlates with low pathogenicity. We know that the ability of neuraminidase to bind the protease plasminogen is a determinant of pathogenicity, because binding plasminogen then causes cleavage of the hemagglutinin, such as found for the WSN strain.



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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings 4 WORKING GROUPS, DAY 1 WORKING GROUP 1 INFLUENZA VIRULENCE AND ANTIGENIC CHANGE Chairperson—Robert Webster Briefer—Peter Palese Rapporteur—Robert Lamb Charge: The charge of this working group is to define research priorities associated with understanding influenza virulence, with tracking and predicting antigenic change, and with defining which antigenic changes may be associated with increased virulence, risk of transmission, and pandemic potential. Potential issues to consider: What studies are needed to define the genetic loci for pathogenicity in avian and human influenza virus strains? What studies are needed to affirm the hypothesis that incremental acquisition of genetic changes can lead to influenza pandemics as compared with the sudden emergence of previous pandemics? What studies are needed to track the rate of antigenic change in avian and human influenza virus strains and to predict changes that may occur? What studies are needed to determine whether pandemic risk can be predicted by virulence factors and/or antigenic characteristics? REPORT TO PLENARY Rapporteur—Dr. Robert Lamb One question concerned how we would define the loci for pathogenicity with influenza virus. We know at the molecular level the importance of the hemagglutinin’s cleavage site. It is required to becleaved to HA1 and HA2 which activates the molecule for fusion.. We know that if we have a multi-basic residue at the HA cleavage site, it has the potential to be cleaved in the trans-Golgi network, and that correlates directly with high pathogenicity of viruses in many cases. If that cleavage site has only a single basic residue, it very often correlates with low pathogenicity. We know that the ability of neuraminidase to bind the protease plasminogen is a determinant of pathogenicity, because binding plasminogen then causes cleavage of the hemagglutinin, such as found for the WSN strain.

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings We know that the presence of carbohydrate sites around the cleavage site of HA are important for pathogenicity. We know that certain mutations in the NS1 protein affect cytokine production in some viruses. And we know that mutations that affect replication and decrease replication—such as in the cold-adapted virus and the so-called PB2 627 mutation found in many of the H5 viruses—lead to a difference in pathogenicity in the mouse. One important consideration is that the very same residue makes no difference when the studies are done in ferrets. Researchers are now using mice, ferrets, and chickens as animal models. The use of non-human primates for influenza virus studies is somewhat questionable. Although mice are obviously the smallest and cheapest animal, there are major questions about the use of inbred mice, particularly the popular strains that lack the MX1 gene, as those do not produce the induction of a normal antiviral state. The best model animal system is probably the ferret: it requires no adaptation for human viruses. However, we do need to agree internationally on one strain of ferret, because British ferrets and Japanese ferrets and American ferrets are not all the same. Another problem is that very few immunological reagents exist for the ferret. How would we study a cytokine response? We need a ferret DNA sequence genome project, like those under way for another 11 or so organisms, from zebra fish to mouse, and of course humans before that. We also need to increase the number of ferrets. That doesn’t just mean ordering a few more. We need a very large breeding colony—the approach taken with the breeding of woodchucks for hepatitis-B studies. We should also determine if other small animal models would work, such as hamsters and mini-pigs. An underlying need is for biological containment facilities for both tissue-culture work and animal work. We need to determine the genes needed for both transmission and pathogenicity, which we would do by making reassortments using reverse genetics—the so-called 6 plus 2, 5 plus 3, etc combinations of 8 genes. We would then study the genetic basis underlying transmission, virulence, and pathogenicity in terms of the function of proteins, the structure of proteins at the atomic level, and how proteins go together to make complexes such as polymerase. We do not completely understand the components of what can be reassorted from one strain versus another. The suggestion is there are incompatibilities among proteins—that not all P proteins can go together. We were asked whether incremental changes in the genome lead to pandemics. We know that from 1997 to 2005 the H5N1 virus gained the ability to kill ducks, and that it has transferred to cats and killed them. We need studies where a series of viruses have changed in virulence pathogenicity. We need to obtain the genome sequences of all these viruses and post them on public databases. The new NIAID-NIH–sponsored public database with The Institute of Genome Research (TIGR) sequencing genomes of human viruses is a start, but we need such an approach for other viruses as well. We also need reverse genetics working for all of these viruses. And when we have all those components in place, we can then show that mutations are sufficient and necessary for the property of pathogenicity being examined. The third question we were asked was whether studies are needed to track the rate of antigenic change in avian and human strains, and to predict the changes that occur. As background, note that only three hemagglutinin subtypes—H1, H2 and H3—cause major human disease

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings We know, as Dr. Palese has often pointed out, that the 1918 virus caused a W-shaped death rate. That is, if we plot the number of deaths on the Y-axis and the age groups that died on the X-axis, we find that young people died early on, and then there was a gap, and older people died much later. People between 30 and 60 years of age presumably had prior exposure to an H1-like virus. We should add that it is thought that H3 and H1 both circulated before 1918, and that there is no evidence that H5, H7, H9, or any of the highly pathogenic viruses in avian species were found in humans in those earlier times. A question about viral archeology inevitably comes up—the sort of studies that Dr. Jeff Taubenberger has done. Such investigation is very difficult because tissues were not preserved before 1900. His work was done on formalin fixed tissues. The question was raised whether we could examine tombs in churches for examples of antibodies, but we felt that would be extremely difficult. At the theoretical level we can continue to predict changes and the effect on antigenic epitopes. An example is the paper in the Proceedings of the National Academies of Science by evolutionary biologist Dr. Simon Levin and his group, who predicted HA clusters and antigenic evolution. Such studies should continue. Surveillance of influenza viruses—now occurring throughout the world—should also continue, as should nucleotide sequencing of viral genomes. Ideally, we need better immunological markers. We point out that hemagglutinin inhibition tests are insensitive, and that they don't work on H5 viruses. Question 4 addressed the studies needed to determine pandemic risk associated with antigenic characteristics. We need to determine the extent of antigenic variation that yields a pandemic strain. We need both human and animal studies to follow how much variation can occur in a strain and still lead to disease and pandemic potential. We would like to know whether preexisting antibody to one subtype can have an effect on infection with another subtype. That is, we can look at the ability of viruses of different subtypes to infect animals such as ferret that already have antibody levels to common human viruses. If we already have some antigenic determinants to a component of any one of those viruses, might that provide some benefit against infection with another influenza virus? Lastly, Dr. Purnell Choppin and others asked whether we need studies that ask whether human genetic changes increase or decrease susceptibility to influenza virus infection. Could such studies be coupled to the NCI cancer genome project? For the mouse, such work could easily be coupled with the huge forward genetics projects occurring at five centers in the United States for mutagenizing the mouse on a total random basis and mapping any genetic changes. Could we use those leftover mice to determine if their susceptibility to influenza had increased or decreased? Although the mouse may not be a very good model, those projects provide an avenue of research. Our workshop’s first priority is to determine the sequences of human, animal, and avian isolates within an epidemiological framework. We want to stress the latter, because just having the sequences doesn’t help. We need to know the clinical data from human cases to accompany the sequences. Our second major recommendation is to determine the genes and their function for transmission and pathogenicity in ferrets using qualified reagents. That one sentence

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings encompasses several needs. Such an effort will require ferrets—lots of them. We are talking about determining not just which genes are involved but also their function. That will require a lot of basic research. Research Recommendations Priorities Determine sequences of human, animal and avian isolates within an epidemiological framework. Need for clinical data from human cases. Determine the genes and their function for transmission and pathogenicity in ferrets using qualified reagents

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings Working Group 1 Presentation Slides: Influenza Virulence and Antigenic Change-Dr. Lamb, Rapporteur

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings (Slides available on accompanying CD)

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings Working Group 1 Briefing Slides: Influenza Virulence and Antigenic Change-Dr. Palese, Briefer

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings (Slides available on accompanying CD)

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings WORKING GROUP 2 CONTROLLING ANIMAL INFLUENZA AND DECREASING ANIMAL-TO-HUMAN TRANSMISSION Chairperson—David Halvorson Briefer—David Swayne Rapporteur—Bruce Innis Charge: The charge to this group will be to define research needs related to strategies for controlling the spread of influenza among animals and decreasing the risk of spread from animals to people. Specific research areas may include assessing the role and impacts of vaccine in animals; environmental modifications and precautions to decrease the spread of infection within and between farms; optimal approaches to culling animal populations; and prevention or management of exposure to infected animals by people. Specific Questions: What studies are needed to determine whether the circulating avian influenza H5N1 virus in Southeast Asia is likely to cause a human pandemic? What studies are needed to assess the effectiveness of strategies to control animal influenza? What studies are needed to assess optimal approaches to use and to assess impact of avian influenza vaccines? What effective cross-cutting technologies for control of animal influenza are translatable to human pandemic scenarios? Report to Plenary Rapporteur: Dr. Bruce Innis We selected several objectives for the intermediate 5-to-10-year horizon. The highest priority is to focus resources on country-specific epidemiological studies targeted at understanding both disease and transmission in animals and in humans. We focused principally on avian influenza. The countries now affected in Southeast Asia do not have a unitary system of poultry production. Thus it is important to look not only at industrial settings but also at village farms, where much bird raising occurs, and also at live markets, and to remember that animals are raised for recreational uses and captivity. It is also important to investigate the epidemiology of humans who care for these animals—we are dismayed that more is not being done on that front. We would like to know where transmission is taking place. Why are people who apparently are exposed not becoming sick? And in many cases, why are they not becoming infected?

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings WORKING GROUP 4 ANTIVIRALS AND NON-SPECIFIC APPROACHES, TREATMENTS AND IMMUNOTHERAPIES Chairperson – Richard Whitley Briefer – Fred Hayden Rapporteur – Charles Hackett Charge: This working group will identify research priorities related to the development, evaluation, and use of antiviral drugs and immunotherapies. Issues to consider include studying the impacts of currently available antiviral drugs and strategies to increase their effectiveness or efficiency; studies of other licensed products that may have benefit in treatment of prevention of influenza; studies of drugs in the development pipeline; and studies of immune therapies and biologicals. Research needed to predict, identify, evaluate, and decrease antiviral resistance also is relevant Specific Questions: What studies are needed to identify the optimal strategies for use of currently available antiviral drugs? What studies are needed to evaluate licensed agents that may moderate the occurrence or severity of influenza? What new antiviral agents are being developed and what research is needed to evaluate these agents or to identify new drug treatment or prevention options? What potential immunotherapeutics could be developed and what research is needed for their evaluation? What studies can help in prediction, identification, assessment, and prevention of antiviral drug resistance? What studies on the pathophysiology of influenza can offer insights for prevention? Report to Plenary Rapporteur: Dr. Charles Hackett At the top of our list are clinical trials for antivirals and immunotherapeutics. One of the main priorities is to develop pandemic protocols now. We reflected on experience with SARS, where public health authorities faced difficult decisions on which of many different possible courses of action they could take unless they had determined ahead of time which protocols to put in place. We need to obtain data on the virologic course and immune responses following pandemic influenza infection, as well as response to therapy following human H5 infections and other potential pandemic infections. A major priority is safety and tolerability of available drugs—oseltamivir pharmacokinetics (PK)—especially in infants less than 1 year old. We also need to determine the PK and tolerability of parenteral drug use, and to assess the long-term (12–20 weeks) tolerability of oseltamivir in inhaled zanamivir prophylaxis.

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings We also need to look at high-priority/high-risk populations, such as pregnant women and immuno-compromised hosts. We need data on these populations, because we need to resolve questions about safety and efficacy of treatments before using them in a pandemic. We also need to study emergence of H5N1 resistance in animal models, and strategies for preventing it. Animal models seem to be the best vehicle for obtaining good data. We need to assess the probability of licensure for orally inhaled zanamivir for disease prophylaxis, because oseltamivir resistance was uncovered and because well-controlled studies show that this approach may work. The short-term priority is to accelerate drug development and discovery programs, including assessment of orphan drugs. We want to support operational infrastructure research. We decided that the strength of our group is to recommend research that will go hand-in-hand with policy development. Thus we see the need for clinical trials of drugs administered more than 48 hours after viral infection, and data on differences in sites of care and drug deployment and response time. We need research on physician prescription of antiviral drugs, because many physicians are not geared up to prescribe such drugs. That would have to change if antiviral drugs were a major cornerstone of a pandemic treatment. That effort should start now, and would dovetail with policy development. We need to test systematic approaches to influencing inflammatory expression and disease. We drew that lesson from looking at SARS, where inflammation damages lungs and steroids were used for treatment, even though steroids have unintended and longer-term consequences. We need to look at other ways of controlling inflammation as a priority. In the medium term, we pointed to accelerated clinical trials of antivirals. We need to test oseltamivir monotherapy versus combination with an M2 inhibitor or ribavirin or other novel therapies in high-risk populations. We also need to test therapeutic efficacy of parenteral peramivir in hospitalized influenza patients, and to test prophylactic efficacy and tolerability of topical long-acting neuraminidase inhibitors. We need to develop a contemporary virus challenge pool or pools for studies of experimental human influenza. This is an important piece of the puzzle, but obtaining the appropriate challenge virus that will provide the characteristics of the disease that we need to test takes time. We also need to use those to test candidate immunomodulators and antivirals in healthy challenge patients. We need to develop immune-based therapies such as monoclonal antibodies, polyclonal antibodies, and polyclonal antibodies for therapy and prophylaxis, including monoclonal antibodies that target multiple proteins, so the virus will not escape by mutation of the targeted epitope. We also need to consider polyclonal antibodies. New developments in polyclonal antibodies from animals with humanized immune systems might provide a passive therapy for pandemic influenza. This slide shows examples of potent and specific inhibitors of neuraminidase of various viruses, influenzas A and B. They have different potencies according to the viral subtypes. Some are available and some are investigational. Thus there is a pipeline of long-acting neuraminidase

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings inhibitors, including conjugated sialidase, HA inhibitors, polymerase inhibitors including siRNA, and protease inhibitors. However, the pipeline must be developed. The longer-term goal is to support ongoing small molecule discovery programs. One with promise is siRNA as a systemic or topical antiviral; siRNA holds potential as a novel approach toward new antiviral targets, such as polymerases. We also need to address incentives for industrial partners, as it is very difficult for a drug company to suddenly pursue another antiviral. Over the longer-term, innate immune effector molecules need development both as specific antiviral molecules, and also for general innate immune activation as a strategy for broad prophylaxis. We need to promote the development of innate-immune system based modulators of inflammatory cascades as alternatives to steroids. What should we be doing now? One suggestion was to create a clinical trial infrastructure for therapeutics, including a uniform protocol for development and data capture. Some trials could be done in Southeast Asia. To develop a public health policy, we also need research on transmission and treatment factors. Such an effort would include operational research to define the optimal infrastructure for developing, stockpiling, and distributing efficacious antiviral agents as quickly as possible.

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings Working Group 4 Presentation Slides: Antivirals and Non-Specific Approaches, Treatments and Immunotherapies-Dr. Hackett, Rapporteur

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings (Slides available on accompanying CD)

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings Working Group 4 Briefing Slides: Antivirals and Non-Specific Approaches, Treatments and Immunotherapies-Dr. Hayden, Briefer

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings

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John R. La Montagne Memorial Symposium on Pandemic Influenza Research: Meeting Proceedings (Slides available on accompanying CD)