Information

Influenza infections and drug design


Why is the neuraminidase used as a target for drugs against influenza virus instead of haemagglutinin? Is there some basic reason that this will make a more effective drug?


It's not that people didn't want to use hemagglutinin as a target for antivirals, it's that they haven't been able to get the antivirals through the approval process yet. There are a number of experimental inhibitors (see for example Progress of small molecular inhibitors in the development of anti-influenza virus agents) but the approval and licensing process is slow and difficult.


National Institute of Allergy and Infectious Diseases (NIAID)

The National Institute of Allergy and Infectious Diseases (NIAID) conducts and supports basic and applied research to better understand, treat, and ultimately prevent infectious, immunologic, and allergic diseases.

Following is a brief description of the major areas of investigation.

  • Acquired Immunodeficiency Syndrome (AIDS). NIAID conducts and supports research on all areas of HIV infection, including developing and testing preventive HIV vaccines, biomedical prevention strategies, and innovative strategies for treating or curing HIV infection and related co-infections and co-morbidities. Since the beginning of the epidemic, NIAID's comprehensive research program has been at the forefront in the fight against HIV/AIDS. NIAID supports a broad array of domestic and international HIV/AIDS research programs and collaborates with more than 7 0 countries through investigator-initiated research grants and multicenter vaccine, therapeutics, microbicide, and prevention clinical research networks. With a number of research programs and initiatives, NIAID is poised to tackle new global research challenges as well as the changing demographics of the HIV/AIDS epidemic.
  • Asthma and Allergic Diseases. NIAID supports programs to examine the causes, pathogenesis, diagnosis, treatment, and prevention of asthma and allergic diseases. Examples of such programs include the Inner-City Asthma Consortium, the Consortium of Food Allergy Research, the Atopic Dermatitis Research Network and the Asthma and Allergic Diseases Cooperative Research Centers. NIAID operates a pediatric allergy clinic at the NIH Clinical Center that serves as a focal point for translational research conducted in collaboration with NIAID intramural laboratories and clinical trials of novel therapies. In addition, NIAID is the lead agency within HHS for research on food allergies.
  • Radiation and Nuclear Countermeasures. NIAID has developed a robust program to accelerate the research and develop­ment of radiation/nuclear medical countermeasures (MCMs) for the Strategic National Stockpile. The NIAID program supports early- to mid-stage research and development to develop medical prod­ucts that can diagnose, mitigate, or treat injuries that can result from radiation exposure from a public health emergency incident. NIAID-sponsored activities focus on MCMs and biodosimetry devices to be used in mass casu­alty radiation/nuclear. The research priority areas of the program are to develop the following: drugs or biologics that can mitigate and/or treat radiation injury when administered at least 24 hours after radiation exposure, drugs that can remove internally contaminated radioactive materials from the body, and biodosimetry methods or devices that can rapidly and accurately distinguish people who have been exposed to radiation.
  • Biodefense and Emerging and Re-emerging Infectious Diseases. NIAID research provides the foundation for developing medical products and strategies to diagnose, treat, and prevent a wide range of infectious diseases, whether those diseases emerge naturally or are deliberately introduced as an act of bioterrorism. Since the 2001 anthrax attacks, NIAID has vastly expanded its portfolio in biodefense and emerging and re-emerging infectious diseases. This research targets pathogens that pose high risks to public health and national security. NIAID conducts and supports research on basic microbiology of and host response to these pathogens as well as development of medical countermeasures. These countermeasures include (1) rapid, accurate diagnostics for natural and bioengineered microbes (2) effective treatments such as antimicrobials, antitoxins, and immunotherapeutics and (3) prophylactic and post-exposure vaccines. NIAID also supports biodefense and emerging infectious disease research through training programs and enhancement of research infrastructure and capacity, and by providing needed research resources and reagents to the scientific community.
  • Enteric Diseases. The global burden of enteric disease is second only to respiratory infection as a cause of sickness and death. Enteric diseases range from persistent, low-grade infections to severe, acute epidemic cholera. An additional burden of disease occurs because enteric infection greatly exacerbates the pathogenicity of diseases such as malaria and HIV/AIDS. Multi-drug resistance is a major problem, making Salmonella, Clostridium difficile, and cholera particularly difficult to treat in the settings where it is most likely to develop a fatal outcome. One of the most severe enteric infections is cholera, the most rapidly killing bacterial disease. NIAID has been involved in many of the most important advances against cholera and other enteric diseases, including supporting the development of oral rehydration therapy, considered to be one of the most important medical advances of the 20th century. Presently, NIAID supports a robust research program of basic and applied research investigating how enteric pathogens cause illness, and developing appropriate diagnostics, vaccines, and therapeutics to prevent infection and to treat patients.
  • Fundamental Immunology. Through both a robust intramural program and investigator-initiated grants and solicited research programs, NIAID supports a strong program to understand basic immune mechanisms, conduct immune profiling, or identify/characterize novel immune cell subsets, pathways, phenomenon, or mechanisms. Examples of NIAID-supported programs include the Human Immunology Profiling Consortium, the Immune Epitope Database, the Immune Mechanisms of Virus Control Program, and Modeling Immunity for Biodefense. NIAID-supported research has yielded a wealth of new information leading to extraordinary growth in the conceptual understanding of the immune system.
  • Transplantation. NIAID supports research that focuses on understanding the role the immune system plays in the success or failure of transplanted cells, tissues, and organs. Researchers are studying ways to selectively control or eliminate unwanted immune responses with the ultimate goal of enhancing long-term transplant survival. Examples of NIAID-supported programs in transplantation include Clinical Trials in Organ Transplantation, Clinical Trials in Organ Transplantation in Children, Clinical Trials in Islet Transplantation, and the Immune Tolerance Network.
  • Immune-Mediated Diseases. NIAID conducts and supports basic, preclinical, and clinical research on immune-mediated diseases, autoimmune disorders, primary immunodeficiency diseases, and the rejection of transplanted organs, tissues, and cells. Efforts are underway to evaluate the safety and efficacy of disease-modifying and tolerance induction strategies for treating immune-mediated diseases, as well as clinical trials to assess the efficacy of hematopoietic stem cell transplantation for treating severe autoimmune disorders. Programs include the Autoimmunity Centers of Excellence, the Immune Tolerance Network (http://immunetolerance.org), Autoimmune Diseases Prevention Centers, Clinical Trials in Organ Transplantation, the Primary Immune Deficiency Treatment Consortium (http://www.rarediseasesnetwork.org/pidtc/), the Primary Immunodeficiency Deficiency Clinic (http://www.niaid.nih.gov/topics/immunedeficiency/pidclinic), the Clinical Islet Transplantation Consortium and the U.S. Immunodeficiency Network (http://www.usidnet.org). NIAID chairs the NIH Autoimmune Diseases Coordinating Committee (ADCC).
  • Malaria and Other Tropical Diseases. Each year, millions of people worldwide are disabled or killed by tropical diseases such as malaria, filariasis, schistosomiasis, leishmaniasis, trypanosomiasis (e.g., Chagas disease and African sleeping sickness), leprosy, and dengue. NIAID supports and conducts basic research on the microbes and parasites that cause tropical diseases, as well as the interactions of these organisms with their human hosts and with animal/invertebrate vectors involved in disease transmission. NIAID also supports and conducts translational and clinical research to develop new and improved diagnostics, drugs, vaccines, and vector management strategies for tropical diseases. These efforts are conducted by U.S. and international investigators receiving Institute support and by NIAID intramural scientists and their collaborators around the world. In addition, the International Centers for Excellence in Research (ICER) program promotes and sustains research programs in developing countries through partnerships with local scientists. The current ICER sites are located in Mali, India, and Uganda. While the ICER program is focused on clinical research in infectious diseases such as malaria and filariasis, each center has the capability to address the research and training needs of greatest relevance to the local population. Clinical research on tropical diseases is largely dependent upon access to populations of patients, vectors, and pathogens/parasites in countries where these diseases are endemic thus, an important complementary objective of NIAID's program is to strengthen international research capacity through research resources and support, scientific collaborations, and research training. In addition, NIAID supports the International Centers of Excellence for Malaria Research (ICEMRs). This program establishes a global network of independent research centers in malaria-endemic settings to provide knowledge, tools, and evidence-based strategies to support researchers working in a variety of settings, especially within governments and healthcare institutions.
  • Influenza. NIAID has supported a comprehensive research program on influenza infections for many years. In response to the emergence and spread of highly pathogenic avian influenza H5N1 and the persistent threat of pandemic influenza, NIAID greatly expanded its influenza program. A broad range of research activities are supported through the intramural program, individual grants and contracts, collaborations with industry partners and investigators in several research networks, including the Vaccine and Treatment Evaluation Units (VTEUs) for the clinical evaluation of candidate products . NIAID intramural researchers conduct cutting edge, comprehensive research on influenza, including its pathogenesis, immunogenicity, transmissibility and genetic variability investigating host immune responses to the virus in animal models and in humans developing vaccines to prevent influenza, especially strains with pandemic potential and studying influenza epidemiology. NIAID investigators recently completed the first human volunteer influenza virus challenge study performed in the U.S. in over a decade. This work provides a critical foundation for vaccine and therapeutics development. NIAID also supports the Centers of Excellence in Influenza Research and Surveillance (CEIRS) network. This program conducts animal influenza surveillance domestically and internationally and focuses on basic research to enhance our understanding of influenza pathogenesis, transmission, evolution, and host response. NIAID also supports activities to develop the next generation of diagnostics, vaccines, and therapeutics and antivirals. NIAID resources and services are available to support early stage development of new vaccine and therapeutic candidates to help advance them through the product development pipeline. Ongoing projects include research to develop a "common epitope" influenza vaccine and therapeutics that protect against all medically important influenza strains systems biology approaches to identify host factors required for influenza infection to expand the number of potential targets for new drug development and clinical research.
  • Genomics and Advanced Technologies. Research fields such as genomics, proteomics, and bioinformatics hold great promise for developing new diagnostics, therapeutics, and vaccines to treat and prevent infectious and immune-mediated diseases. NIAID has made a significant commitment to support and encourage advanced technologies research in Institute labs and in the scientific community. Sophisticated tools are being used to determine the genetic make-up of disease-causing pathogens, to analyze discrepancies among pathogen strains, and to evaluate how immune system responses differ. In addition, data generated through NIAID-supported initiatives is being made rapidly available to the research community. The ultimate goal of the NIAID genomics and advanced technologies program is to allow researchers to use these data to further pursue new discoveries about the causes, treatment, and ultimate prevention of infectious and immune-mediated diseases.
  • Sexually Transmitted Diseases (STDs). More than 15 million Americans each year acquire infectious diseases other than AIDS through sexual contact. STDs such as gonorrhea, syphilis, chlamydia, genital herpes, and human papillomavirus can have devastating consequences, particularly for young adults, pregnant women, and newborn babies. NIAID-supported scientists in STD Cooperative Research Centers, NIAID intramural laboratories, and other research institutions are developing better diagnostic tests, improved treatments, and effective vaccines for STDs.
  • Vaccine Development. Effective vaccines have contributed enormously to improvements in public health in the United States and worldwide during the last century. Research conducted and supported by NIAID has led to new or improved vaccines for a variety of serious diseases, including rabies, meningitis, whooping cough, hepatitis A and B, chickenpox, and pneumococcal pneumonia, to name a few. NIAID supports the Vaccine and Treatment Evaluation Units (VTEUs) for the clinical testing of new vaccines and vaccine technologies at a number of U.S. medical centers. Many vaccines are currently under development in NIAID intramural labs, including vaccines to prevent AIDS, pandemic influenza, childhood respiratory diseases, dengue, and malaria.
  • Adjuvant Discovery and Development. There is a critical need for the identification and characterization of novel adjuvants to boost immunity and increase the efficacy of new or existing vaccines. NIAID supports a robust adjuvant program in both discovery and development with the ultimate goal of advancing candidate adjuvants towards licensure for human use.
  • Drug Research and Development. The development of therapies to treat infectious and immunologic diseases is a key component of NIAID's mission. In collaboration with industry, academia, non-profits, and other government agencies, NIAID has established research programs to facilitate drug development, including screening programs to identify compounds with potential for use as therapeutic agents, facilities to conduct preclinical testing of promising drugs, and clinical trials networks to evaluate the safety and efficacy of drugs and therapeutic strategies in humans.
  • Antimicrobial Resistance. NIAID funds and conducts comprehensive research to study antimicrobial resistance in major viral, bacterial, fungal, and parasitic pathogens. Projects include basic research on the disease-causing mechanisms of pathogens, host-pathogen interactions, and the molecular mechanisms responsible for drug resistance, as well as translational research to develop and evaluate new or improved products for disease diagnosis, intervention, and prevention. NIAID supports clinical trials that assess new and existing antimicrobials and new vaccines relevant to drug-resistant infections through cutting edge intramural research and clinical trial facilities, NIAID-targeted initiatives and clinical trial networks, which include the Adult AIDS Clinical Trials Groups, the Vaccine and Treatment Evaluation Units, and the Antibacterial Resistance Leadership Group (ARLG). Established in 2013, the ARLG develops, designs, implements, and manages a clinical research agenda to increase knowledge of antibacterial resistance. The ARLG aims to advance research by building transformational trials that will change clinical practice and reduce the impact of antibacterial resistance.
  • Minority and Women's Health. Some of the diseases studied by NIAID disproportionately affect women and minority populations. The Institute remains committed to the inclusion of minorities and women in every aspect of its scientific agenda, from recruitment of special populations into clinical studies to the conduct of biomedical research by minority and women researchers. NIAID's Division of Extramural Activities sponsors activities aimed at eliminating the continuing health disparities among these populations. Through the Division's efforts, activities are developed to encourage scientific advances in sex and gender differences research, and to encourage research training of investigators who focus on the health of women and girls, and to stimulate the training and development of researchers from populations that are historically underrepresented in biomedical research in the U.S. The Division also develops innovative training initiatives to increase the number of scientists from diverse backgrounds by supporting undergraduate, graduate, and postgraduate research training in immunologic and infectious diseases. NIAID research results are disseminated to diverse underserved communities through the Institute's outreach activities, which have focused on HIV/AIDS, asthma, sexually transmitted diseases , and autoimmune diseases.

Influenza Antiviral Drug Resistance

When an antiviral drug is fully effective against a virus, that virus is said to be susceptible to that antiviral drug. Influenza viruses are constantly changing, and can sometimes change in ways that might make antiviral drugs work less well or not work at all against these viruses. When an influenza virus changes in the active site where an antiviral drug works, that virus shows reduced susceptibility to that antiviral drug. Reduced susceptibility can be a sign of potential antiviral drug resistance. Antiviral drugs may not work as well in viruses with reduced susceptibility. Influenza viruses can show reduced susceptibility to one or more influenza antiviral drugs.

In the United States, there are four FDA-approved antiviral drugs recommended by CDC this season. Three are neuraminidase inhibitor antiviral drugs: oseltamivir (available as a generic version or under the trade name Tamiflu®) for oral administration, zanamivir (trade name Relenza®) for oral inhalation using an inhaler device, and peramivir (trade name Rapivab®) for intravenous administration. The fourth is a cap-dependent endonuclease (CEN) inhibitor, baloxavir marboxil (trade name Xofluza®) for oral administration, approved for use in the United States during the 2018-2019 season by FDA in October of 2018.

There is another class of influenza antiviral drugs (amantadine and rimantadine) called the adamantanes (which have activity against only influenza A viruses) that are not recommended for use in the United States at this time because of widespread antiviral resistance in circulating influenza A viruses.

How widespread are reduced susceptibility and antiviral resistance in the United States?

In the United States, the majority of the recently circulating influenza viruses have been fully susceptible to the neuraminidase inhibitor antiviral medications and to baloxavir. On the other hand, many flu A viruses are resistant to the adamantane drugs which is why they are not recommended for use at this time.

How does reduced susceptibility and antiviral resistance happen?

Influenza viruses are constantly changing they can change from one season to the next and can even change within the course of one flu season. As a flu virus replicates (i.e., make copies of itself), the genetic makeup may change in a way that results in the virus becoming less susceptible to one or more of the antiviral drugs used to treat or prevent influenza. Influenza viruses can become less susceptible to antiviral drugs spontaneously or emerge during the course of antiviral treatment. Viruses that are less susceptible or resistant vary in their ability to transmit to other people.

How are reduced susceptibility and antiviral resistance detected?

CDC routinely tests flu viruses collected through domestic and global surveillance to see if they have indications of reduced susceptibility to any of the FDA-approved flu antiviral drugs, as this can suggest the potential for antiviral resistance. This data informs public health policy recommendations about the use of flu antiviral medications.

Detection of reduced susceptibility and antiviral resistance involves several laboratory tests, including specific functional assays and molecular techniques (sequencing and pyrosequencing) to look for genetic changes that are associated with reduced antiviral susceptibility.

How has CDC prepared to test for reduced susceptibility and antiviral resistance to the new flu antiviral baloxavir?

CDC&rsquos Influenza Division has taken specific laboratory actions to incorporate the new antiviral drug baloxavir into routine virologic surveillance. This includes the creation and validation of new assays to determine baloxavir susceptibility, and training of laboratorians to conduct baloxavir susceptibility testing.

Seasonal influenza A and B viruses in humans as well as several influenza A viruses that circulate in animals were tested to established baseline susceptibility to baloxavir. In addition, the susceptibility of other distantly related influenza viruses to baloxavir was tested. CDC also is collaborating with the Association of Public Health Laboratories (APHL) and the Wadsworth Center NYSDOH, a National Influenza Reference Center (NIRC), to establish laboratory-testing capacity for baloxavir susceptibility. CDC has trained staff within these partner organizations to use CDC&rsquos new method for assessing baloxavir susceptibility.

What is oseltamivir resistance and what causes it?

Flu viruses are constantly changing (for more information, see How the Flu Virus Can Change. Changes that occur in circulating flu viruses typically involve the structures of the viruses&rsquo two primary surface proteins: neuraminidase (NA) and hemagglutinin (HA). (See image below for a visualization of a flu virus and its HA and NA surface proteins.)

Oseltamivir is the most commonly prescribed of the recommended antiviral drugs in the United States that is used to treat flu illness. Oseltamivir is known as a &ldquoNA inhibitor&rdquo because this antiviral drug binds to NA proteins of a flu virus and inhibits the enzymatic activity of these proteins. By inhibiting NA activity, oseltamivir prevents flu viruses from spreading from infected cells to other healthy cells.

If the NA proteins of a flu virus change, oseltamivir can lose its ability to bind to and inhibit the function of the virus&rsquos NA proteins. This results in &ldquooseltamivir resistance&rdquo (non-susceptibility). A particular genetic change known as the &ldquoH275Y&rdquo mutation is the only known mutation to confer oseltamivir resistance in 2009 H1N1 flu viruses. The &ldquoH275Y&rsquo mutation makes oseltamivir ineffective in treating illnesses with that flu virus by preventing oseltamivir from inhibiting NA activity, which then allows the virus to spread to healthy cells. The H275Y mutation also reduces the effectiveness of peramivir to treat influenza virus infections with this mutation.

How does CDC improve monitoring of influenza viruses for reduced susceptibility and antiviral resistance?

CDC continually improves the ability to rapidly detect influenza viruses with antiviral reduced susceptibility and antiviral resistance through improvements in laboratory methods increasing the number of surveillance sites domestically and globally and increasing the number of laboratories that can test for reduced susceptibility and antiviral resistance. Enhanced surveillance efforts have provided CDC with the capability to detect resistant viruses more quickly, and enabled CDC to monitor for changing trends over time.

How did influenza antiviral susceptibility patterns change during the previous (2019-2020) influenza season?

Antiviral susceptibility patterns changed very little in 2019-2020 compared with the previous season (2018-2019). During the 2018-2019 and 2019-2020 seasons, only a small number of viruses were resistant to oseltamivir. Most of the influenza viruses tested during 2019-2020 continued to be susceptible to the antiviral drugs recommended for influenza by the Centers for Disease Control and Prevention (CDC) and the Advisory Committee on Immunization Practices (ACIP) (oseltamivir, zanamivir, peramivir and baloxavir). Resistance to the adamantane class of antiviral drugs among A/H3N2 and A/H1N1 viruses remained widespread (influenza B viruses are not susceptible to adamantane drugs).

CDC conducts ongoing surveillance and testing of influenza viruses for antiviral reduced susceptibility and resistance among seasonal and novel influenza viruses, and guidance is updated as needed.

Because there were no dramatic changes in antiviral susceptibility patterns during the 2019-2020 flu season, the guidance for the 2020-2021 flu season on the use of influenza antiviral drugs remains unchanged. The latest guidance for clinicians on the use of antiviral drugs for influenza is available on the CDC web site at Antiviral Drugs: Information for Health Professionals.

What can people do to protect themselves against flu viruses with reduced susceptibility and antiviral resistance?

Getting a yearly seasonal flu vaccination is best way to reduce the risk of flu and its potentially serious complications. Flu vaccines protect against an influenza A(H1N1) virus, an influenza A(H3N2) virus, and one or two influenza B viruses (depending on the vaccine). CDC recommends that everyone 6 months of age and older get vaccinated each year. If you are in a group at high risk of serious flu-related complications and become ill with flu symptoms, call your doctor right away, you may benefit from early treatment with an influenza antiviral drug. If you are not at high risk, if possible, stay home from work, school and errands when you are sick. This will help prevent you from spreading your illness to others. See Important Information For People Sick With Flu for more information.


Reconstruction of the 1918 Influenza Pandemic Virus

CDC researchers and their colleagues successfully reconstructed the influenza virus that caused the 1918-19 flu pandemic, which killed as many as 50 million people worldwide. A report of their work, &ldquoCharacterization of the Reconstructed 1918 Spanish Influenza Pandemic Virus external icon ,&rdquo was published in the October 7, 2005 issue of Science. The work was a collaboration among scientists from CDC, Mount Sinai School of Medicine external icon , the Armed Forces Institute of Pathology, and Southeast Poultry Research Laboratory external icon . The following questions and answers describe this important research and related issues.

Note: For a detailed historical summary of this work, including how it was conducted, the people involved, and the lessons learned from it, see The Deadliest Flu: The Complete Story of the Discovery and Reconstruction of the 1918 Pandemic Virus.

Background on the Research

What research does the Science article describe? Why is it important?

Read more on how an expert group of researchers and virus hunters located the lost 1918 virus, sequenced its genome, and reconstructed the virus in a highly safe and regulated laboratory setting at CDC to study its secrets and better prepare for future pandemics.

This report describes the successful reconstruction of the influenza A(H1N1) virus responsible for the 1918 &ldquoSpanish flu&rdquo pandemic and provides new information about the properties that contributed to its exceptional virulence. This information is critical to evaluating the effectiveness of current and future public health interventions, which could be used in the event that a 1918-like virus reemerges. The knowledge from this work may also shed light on the pathogenesis of contemporary human influenza viruses with pandemic potential. The natural emergence of another pandemic virus is considered highly likely by many experts, and therefore insights into pathogenic mechanisms can and are contributing to the development of prophylactic and therapeutic interventions needed to prepare for future pandemic viruses.

What are the reasons for doing these experiments?

The influenza pandemic of 1918-19 killed an estimated 50 million people worldwide, many more than the subsequent pandemics of the 20th century. The biological properties that confer virulence to pandemic influenza viruses have not traditionally been well understood and warranted further study. Research to better understand how the individual genes of the1918 pandemic influenza virus contribute to the disease process provide important insights into the basis of virulence. This kind of information has helped health officials to devise appropriate strategies for early diagnosis, treatment, and prevention, should a similar pandemic virus emerge. Additionally, such research informs the development of general principles with which we can better design antiviral drugs and other interventions against all influenza viruses with enhanced virulence.

Who funded the work described in this article?

Work with the reconstructed 1918 virus was conducted at and supported by CDC. The U.S. Department of Agriculture (USDA), the National Institutes of Health (NIH), and the Armed Forces Institute of Pathology (AFIP) all provided support for many other aspects of this research.

When did CDC begin research on the 1918 virus?

CDC studies of the 1918 influenza virus were begun in 2004 with the initiation of testing of viruses containing subsets of the eight genes of the 1918 virus. Previous articles describing the properties of such viruses were published before 2005. Reconstruction of the entire 1918 virus was begun in August 2005.

Could a 1918-like H1N1 virus re-emerge and cause a pandemic again?

It is impossible to predict with certainty the emergence of a future pandemic, including a 1918-like virus. Pandemics occur when an influenza virus emerges to which there is little, or no, preexisting immunity in the human population. However, it is generally thought that a 1918-like pandemic would be less severe due to the advent of vaccines to prevent flu, current FDA-approved antiviral influenza drugs, and the existing global influenza surveillance system that the World Health Organization maintains.

Are current antivirals and vaccines effective against the 1918 H1N1 virus?

Yes. Oseltamivir (Tamiflu® or generic), has been shown to be effective against similar influenza A(H1N1) viruses and is expected to be effective against the 1918 H1N1 virus. Other antivirals (zanamivir, peramivir and baloxavir) have not been tested against this specific virus but are expected to also be effective. Vaccines containing the 1918 HA or other subtype H1 HA proteins were effective in protecting mice against the 1918 H1N1 virus. Vaccination with current seasonal influenza vaccines is expected to provide some protection in humans since seasonal influenza vaccines provided some level of protection against the 1918 H1N1 virus in mice.

Are new prophylactics and therapeutics that could be effective against the 1918 virus under way?

Scientists continue to work on development of new antivirals which may be effective against a 1918-like virus. The reconstruction of the 1918 H1N1 pandemic virus and subsequent studies that followed showed that the 1918 polymerase genes contribute to efficient replication of the pandemic virus. This insight identified an important virulence factor in the study of influenza that is now targeted for antiviral compound development. Therefore, new polymerase inhibitors promise to add to the clinical management options against influenza virus infections in the future.

Biosafety Precautions

Was the public at risk from the experiments being done on this virus?

The work described in this report was done using stringent biosafety and biosecurity precautions that are designed to protect workers and the public from possible exposure to this virus (for example, from accidental release of the virus into the environment). The 1918 virus used in these experiments has since been destroyed at CDC and does not pose any ongoing risk to the public.

What biosafety and biosecurity precautions for protecting laboratory workers and the public were in place while this work was being done?

Before the experiments were begun, two tiers of internal CDC approval were conducted: an Institutional Biosafety Committee review and an Animal Care and Use Committee review. All viruses containing one or more gene segments from the 1918 influenza virus were generated and handled in accordance with biosafety guidelines of the Interim CDC-NIH Recommendation for Raising the Biosafety Level Laboratory Work Involving Noncontemporary Human Influenza Viruses. Although the 1918 virus was not designated as a select agent at the time this work was performed, all procedures were carried out using the heightened biosecurity elements mandated by CDC&rsquos Select Agent program. The Intra-governmental Select Agents and Toxins Technical Advisory Committee recommended that the reconstructed 1918 influenza virus be added to the list of HHS select agents on September 30, 2005. Following this recommendation, CDC amended its regulations and designated all reconstructed replication competent forms of the 1918 pandemic influenza virus containing any portion of the coding regions of all eight gene segments (reconstructed 1918 Influenza virus) as a select agent.

What are the appropriate biosafety practices and containment conditions for work with the 1918 strain of influenza?

Biosafety Level 3 or Animal Biosafety Level 3 practices, procedures and facilities, plus enhancements that include special procedures (discussed in the next question below), are recommended for work with the 1918 strain. There are four biosafety levels that correspond to the degree of risk posed by the research and involve graded levels of protection for personnel, the environment, and the community. Biosafety Level 4 provides the most stringent containment conditions, Biosafety Level 1 the least stringent. These biosafety levels consist of a combination of laboratory practices and techniques, safety equipment, and laboratory facilities that are appropriate for the operations being performed. The specific criteria for each biosafety level are detailed in the CDC/NIH publication Biosafety in Microbiological and Biomedical Laboratories.

What is Biosafety Level 3 &ldquoenhanced&rdquo? What are the specific enhancements used for work with the 1918 strain of influenza?

A Biosafety Level 3 facility with specific enhancements includes primary (safety cabinets, isolation chambers, gloves and gowns) and secondary (facility construction, HEPA filtration treatment of exhaust air) barriers to protect laboratory workers and the public from accidental exposure. The specific additional (&ldquoenhanced&rdquo) procedures used for work with the 1918 strain include:

  • Rigorous adherence to additional respiratory protection and clothing change protocols
  • Use of negative pressure, HEPA-filtered respirators or positive air-purifying respirators (PAPRs)
  • Use of HEPA filtration for treatment of exhaust air and
  • Amendment of personnel practices to include personal showers prior to exiting the laboratory.

Further details of the biosafety recommendations for work with various human and animal influenza viruses, including 1918 virus, can be found in the interim CDC/NIH guidance for such work at Interim CDC-NIH Recommendation for Raising the Biosafety Level Laboratory Work Involving Noncontemporary Human Influenza Viruses.

How were these experiments conducted safely using containment provided by BSL-3 with enhancements?

Highly trained laboratorians worked with the 1918 influenza virus strain safely using BSL-3-enhanced containment. Researchers at CDC receive specialized training and go through a rigorous biosafety (and security) clearance process. For the work reported in the Science article, the lead CDC researcher provided routine weekly written reports to CDC management officials, including the agency&rsquos Chief Science Officer, and was instructed to notify agency officials immediately of any concerns related to biosafety or biosecurity.

A BSL-3 facility with specific enhancements includes primary (safety cabinets, isolation cabinets, gloves, gowns) and secondary (facility construction) barriers to protect laboratory workers and the public from accidental exposure. Specific enhancements include change-of-clothing and shower-out requirements, and the use of a powered air purifying respirator (PAPR half body suits). The primary and secondary barriers plus additional personal safety practices provide appropriate containment for conducting such influenza research. CDC evaluated the specific studies to be conducted as well as the highly experienced scientific team conducting the research and concluded that this work could proceed under BSL-3 containment with enhancements.

Why was BSL-3-enhanced containment used for work on the 1918 H1N1 virus when most human influenza viruses of the H1N1 subtype are handled under much less stringent containment?

The appropriate biosafety measures for working a given pathogen depend upon a number of factors, including previous experience with the pathogen or similar pathogens, the virulence and transmissibility of the pathogen, the type of experiment, and the availability of vaccines and/or antimicrobial drugs effective against the pathogen. Prior to reconstruction of the 1918 virus, CDC carefully evaluated the specific studies to be conducted and concluded that this research could safely and securely be done under BSL-3-enhanced containment. All viruses containing one or more gene segments from the 1918 influenza virus were generated and handled under high-containment (BSL 3-enhanced) laboratory conditions in accordance with guidelines of NIH and CDC. The recommendations for biosafety levels are made by a panel of experts and are followed in a stringent manner.

A higher level of containment (biosafety level 4) is utilized for work on novel or exotic pathogens for which there is no treatment or vaccine. This is not the case for the 1918 virus. Descendants of the 1918 influenza virus still circulate today, and current seasonal influenza vaccines provide some protection against the 1918 virus. In addition, two types of antiviral drugs, rimantadine (Flumadine) and oseltamivir (Tamiflu® or generic) have been shown to be effective against similar influenza A(H1N1) viruses and are expected to be effective against the 1918 H1N1 virus. Other antivirals (zanamivir, peramivir and baloxavir) have not been tested against this specific virus but also are expected to be effective.

The physical and engineering design of BSL-3-enhanced containment is very similar to that used in BSL-4 laboratories. The BSL-3 laboratory also has state-of-the-art directional airflow control which filters outgoing air, and all waste is autoclaved or decontaminated before it leaves the work area, preventing escape of infectious agents.

Biosecurity Issues

Did the generation of the 1918 Spanish influenza pandemic virus containing the complete coding sequence of the eight viral gene segments violate the Biological Weapons Convention?

No. Article I of the Biological Weapons Convention (BWC) specifically allows for microbiological research for prophylactic, protective, or other peaceful purposes. Article X of the BWC encourages the &ldquofullest possible exchange of&hellipscientific and technological information&rdquo for the use of biological agents for the prevention of disease and other peaceful purposes. Further, Article X of the BWC provides that the BWC should not hamper technological development in the field of peaceful bacteriological activities. Because the emergence of another pandemic virus is considered likely, if not inevitable, characterization of the 1918 virus may enable us to recognize the potential threat posed by new influenza virus strains, and it will shed light on the prophylactic and therapeutic countermeasures that will be needed to control pandemic viruses.

Did the report provide a &ldquoblueprint&rdquo for bioterrorists to develop and unleash a devastating pandemic on the world?

No. This report does not provide the blueprint for bioterrorist to develop a pandemic influenza strain. The reverse genetics system that was used to generate the 1918 virus is a widely used laboratory technique. While there are concerns that this approach could potentially be misused for purposes of bioterrorism, there are also clear and significant potential benefits of sharing this information with the scientific community: namely, facilitating the development of effective interventions, thereby strengthening public health and national security.

Is the 1918 influenza virus a select agent?

The Intra-governmental Select Agents and Toxins Technical Advisory Committee convened on September 30, 2005, and recommended that the reconstructed 1918 influenza virus be added to the list of HHS select agents. Following this recommendation, CDC amended its regulations and designated all reconstructed replication competent forms of the 1918 pandemic influenza virus containing any portion of the coding regions of all eight gene segments (reconstructed 1918 Influenza virus) as a select agent.

What is the Select Agent Program?

The Centers for Disease Control and Prevention (CDC) regulates the possession, use and transfer of select agents and toxins that have the potential to pose a severe threat to public health and safety. The CDC Select Agent Program oversees these activities and registers all laboratories and other entities in the United States of America that possess, use or transfer a select agent or toxin.


General Overview

Influenza viruses are estimated to cause symptomatic infections in 3-11% of the U.S. population annually and severe disease in about 1.5% of those infected. Although several drugs now available can limit the severity of an influenza infection, yearly vaccination remains the most effective approach to reduce the disease burden caused by influenza viruses.

Current influenza vaccines include split inactivated influenza viruses, live attenuated influenza viruses, and recombinant hemagglutinin (HA) antigens. Each vaccine type has advantages and all of them protect against the two influenza A subtypes (H1N1 and H3N2) and at least one of the influenza B lineages (Yamagata and Victoria) that are responsible for seasonal infections in humans.

Manufacturing split inactivated influenza vaccines generally involves propagating candidate vaccine viruses (CVVs) in eggs or mammalian cells, whereas recombinant HA vaccines are produced using insect cells. Despite these differences, both products are standardized based on the HA antigen content, as responses against HA correlate well with protection.

Each season, several inter-connected challenges can affect the influenza vaccine efficacy: 1) Influenza viruses are constantly evolving, which can cause antigenic drift and occasional antigenic shift in type A viruses 2) vaccine strains must be selected months in advance to meet manufacturing deadlines 3) viral propagation in eggs or cells can lead to unexpected adaptations that can alter important antigens in the vaccine.

Although influenza vaccines have mainly been developed to generate an optimal immune response against HA, influenza viruses do possess a second, less abundant surface antigen, neuraminidase (NA). Like HA, antibodies that recognize NA can provide both matched and cross-protection against influenza virus strains. NA also evolves and drifts independently of HA. These properties imply that by improving the NA response, it might be possible to increase the breadth of the vaccine coverage and mitigate many of the yearly challenges that influenza vaccines face.

In the split inactivated and the live attenuated influenza virus vaccines, NA is present. However, many technical issues must first be solved before the NA component of the annual vaccines can be regulated. Our laboratory is systematically addressing several of these issues to establish a framework for improving the ability of NA to increase the breadth and efficacy of the annual vaccine.


The war against influenza: discovery and development of sialidase inhibitors

The threat of a major human influenza pandemic, in particular from highly aggressive strains such as avian H5N1, has emphasized the need for therapeutic strategies to combat these pathogens. At present, two inhibitors of sialidase (also known as neuraminidase), a viral enzyme that has a key role in the life cycle of influenza viruses, would be the mainstay of pharmacological strategies in the event of such a pandemic. This article provides a historical perspective on the discovery and development of these drugs — zanamivir and oseltamivir — and highlights the value of structure-based drug design in this process.

The emergence of the extremely aggressive avian H5N1 influenza virus, in particular in Asia, has made the likelihood of a human influenza pandemic and the possible socio-economic impact a major worldwide concern 1,2,3 . The appearance of H5N1, and the human fatalities it has already caused, has heightened the awareness of both the general population and governments to the threat of influenza virus to the extent that many governments have implemented preparedness plans, and available anti-influenza drugs are being stockpiled.

Historically, the first drugs available for the treatment of influenza were the adamantane-based M2 ion channel protein inhibitors, rimantidine and amantadine 4,5 . These compounds have only been useful in the treatment of influenza A infection, because only the A strains of the virus have M2 ion channel proteins 4,5,6,7 . Although both drugs can be effective against influenza virus A infection, they have been reported to cause CNS side effects 5,6 , and have given rise to the rapid emergence of drug-resistant viral strains 7 .

Given such issues, there has been considerable effort worldwide to discover novel therapeutic agents against all types of influenza, and several valuable reviews concerned with aspects of influenza virus have been published (for example, Refs 8–11). Here, after providing some background on the influenza virus and its key surface glycoproteins, this article describes the discovery and development of influenza virus sialidase (also known as neuraminidase/exo-α-sialidase EC 3.2.1.18) inhibitors, which are now at the forefront of defences against a flu pandemic. Focused efforts to develop such drugs using structural information began in the 1980s, and provide one of the earliest examples of the application of structure-based drug design.

Influenza virus belongs to the orthomyxoviridae family, which is subdivided into three serologically distinct types: A, B and C. Only influenza virus A and B appear to be of concern as human pathogens as influenza C virus does not seem to cause significant disease 12,13 . Further classification of influenza virus is based on the antigenic properties of its surface glycoproteins haemagglutinin and sialidase 14 (Fig. 1), both of which are essential for infection to proceed (Fig. 2). These surface glycoproteins are carbohydrate-recognizing proteins and in humans are known to recognize the sialic acid N-acetylneuraminic acid (Neu5Ac which has α and β forms when not conjugated compound 1, Fig. 3), which is typically associated as the a-linked terminal carbohydrate unit of upper respiratory tract and lung-associated glycoconjugates 13,15 .

a | A view of the influenza virus haemagglutinin trimer complexed with N-acetylneuraminic acid (Neu5Ac in CPK form). b | A monomeric subunit of influenza A virus sialidase complexed with Neu5Ac (in CPK form). The catalytic site is located near the pseudo-symmetry axis.

The surface of influenza virus A is decorated with three proteins: an M2 ion channel protein, the lectin haemagglutinin and the enzyme sialidase. Typically, the influenza virus adheres to the target host cell by using its surface glycoprotein haemagglutinin to recognize glycoconjugates such as GD1a that display terminal α-linked N-acetylneuraminic acid (α-Neu5Ac, compound 1a, Fig. 3) residues. The virus is then endocytosed, fusion occurs and the host-cell machinery is engaged to produce the necessary viral components. Subsequent viral protein synthesis and particle assembly in the host cell prepares the virion progeny for the budding process to exit the host cell. The enzyme sialidase cleaves the terminal α-Neu5Ac residues from both the newly synthesized virion progeny glycoproteins as well as from the host-cell surface. The action of sialidase enables the host-cell-surface aggregated virion progeny to elute away from the infected cell and seek new host cells to infect. Both haemagglutinin and sialidase have been proposed as potential anti-influenza drug discovery targets. As described in the main text, zanamivir and oseltamivir efficiently block the action of sialidase and significantly inhibit the release mechanism. The M2 ion channel protein of influenza virus A has also been targeted by a class of drugs referred to as the adamantanes, which include amantadine and rimantadine. Last, ribavirin has also been demonstrated to inhibit virus replication by acting on the RNA polymerase function.

1a, α-N-acetylneuraminic acid (α-Neu5Ac) 1b, β-anomer of Neu5Ac 2a, 2-deoxy 2,3-didehydro Neu5Ac (Neu5Ac2en) 2b, N-trifluoroacetylated derivative of Neu5Ac 3, 2-deoxy-α-Neu5Ac 4, 4-amino-4-deoxy-NeuAc2en 5, 4-deoxy-4-guanidino-Neu5Ac2en, now known as zanamivir 6, azide derivative of 4 and 5 7, uronic acid 8, oseltamivir carboxylate (GS 4071) 9, peramivir 10, a pyrrolidine, A-315675 (Ref. 54) 11, oseltamivir (GS 4104) 12, divalent zanamivir.

Haemagglutinin is made up of three identical subunits (Fig. 1a) and is anchored to the lipid membrane of the virus 13 . This glycoprotein seems to have two significant roles. The first is to provide an initial point of contact for the virus to the target host cell-surface glycoconjugates by α-ketosidically linked terminal Neu5Ac residues 10,16,17 . The second is to trigger the internalization process of the virus through fusion of the viral envelope with the host cell 10,18 . A multitude of influenza virus haemagglutinin structures have been determined, as well as the structures of several haemagglutinin–ligand complexes 19,20,21,22 .

Influenza virus sialidase is an enzyme made up of four identical subunits that is also anchored to the viral membrane 23 . The enzyme is an exoglycohydrolase and cleaves α-ketosidically linked Neu5Ac residues that cap the ends of various glycoconjugates 11 . The significance of the sialidase action is that it assists in the movement of virus particles through the upper respiratory tract as well as in the release of virion progeny from infected cells 24,25 . Several influenza virus sialidase crystal structures, including a number of complexes of influenza virus sialidase with Neu5Ac (Fig. 1b) and its derivatives, have been determined 26,27,28 .

The essential roles, although controversial at times, played by both of these surface glycoproteins in the infectious life cycle of the virus (Fig. 2), coupled with a significant body of available structural information, have provided exciting opportunities for rational structure-based discovery of anti-influenza agents 11,29 . The most successful structure-based anti-influenza drug discovery programme has arisen from targeting the sialidase function. This article describes aspects of the discovery of the first potent designed influenza virus sialidase inhibitor — and now commercially available inhaled anti-influenza drug — zanamivir (Relenza GlaxoSmithKline) 30 , and the discovery of the subsequently approved orally bioavailable drug oseltamivir (Tamiflu Gilead/Roche) 31 . More recent approaches to discover next-generation sialidase inhibitors to combat influenza infection, particularly pandemic influenza, are also discussed.

Influenza virus sialidase

Functional and structural information about influenza virus sialidase has been vital in the discovery of potent inhibitors of this enzyme, and its biochemistry has been the subject of intense study 32,33,34 . As noted above, sialidase is believed to play at least two critical roles in the life cycle of the virus, including the facilitation of virion progeny release (Fig. 2) and general mobility of the virus in the respiratory tract. Influenza virus sialidase seems only to cleave terminal α-ketosidically linked Neu5Ac residues 35 . The enzyme mechanism (Box 1) appears to proceed via the formation of a putative sialosyl cation intermediate 33,36 that adopts a distorted half-chair arrangement. This intermediate may be covalently trapped by the enzyme's active site through nucleophilic attack of a strain-independent highly conserved tyrosine residue. This catalytic intermediate is subsequently hydrolytically released as α-Neu5Ac 33 (compound 1a, Fig. 3).

Several high-resolution X-ray crystal structures of sialidase complexed with various small-molecule inhibitors have been determined, including Neu5Ac (Fig. 4a). Most strikingly, the active site consists of a number of distinct adjoining pockets that are lined by eight highly conserved amino-acid residues that make direct contact with Neu5Ac and its derivatives 27 (Fig. 4b). In addition, there are a further ten amino-acid residues invariant in all strains of influenza virus within the vicinity of the active site that appear to be important primarily in the stabilization of the architecture of the active site 27,37 .

a | α-N-acetylneuraminic acid (α-Neu5Ac) bound in a boat-like conformation to the influenza virus sialidase active site. b | Eight invariant amino-acid residues make direct contact with Neu5Ac. c | Neu5Ac interactions with the influenza A virus sialidase active site (derived from LIGPLOT software 67 ).

Upon binding, Neu5Ac-containing glycoconjugates are oriented in the active site through interaction with a cluster of three arginine residues and the Neu5Ac moiety makes a number of significant contacts with active-site residues 38,39 (Fig. 4c). Specifically, further orientation of the Neu5Ac moiety is facilitated by several additional interactions within the active site, including hydrogen bonding of the C-5 acetamido group carbonyl oxygen to Arg152 and its N–H to a buried water molecule. Favourable hydrophobic contacts to residues Trp178 and Ile222 are also made by the methyl of the C-5 acetamido group. Additional hydrogen-bond networks are formed by the C-8 and C-9 hydroxyl groups of the glycerol side chain to the carboxylate oxygens of residue Glu276, while the C-4 hydroxyl group associates with the carboxylate oxygen of Glu119. All of the amino-acid residues mentioned above are fully conserved across the natural strains of influenza virus known so far 23,37 .

Structure-based inhibitor design

Although several influenza virus sialidase inhibitors had been reported in the literature before structural information was available, none had shown efficacy in an in vivo model (for reviews, see Refs 11,29,40). From these studies, the unsaturated Neu5Ac derivative 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en compound 2a, Fig. 3), a micromolar inhibitor of influenza virus sialidase, has provided the most potent inhibitor core template.

The first sialidase crystals were successfully grown from purified protein and subsequently, more suitable crystals were obtained for diffraction purposes and resulted in the X-ray crystal structure determination of influenza virus sialidase 26,27,37 . This structural information proved valuable in the discovery and development of zanamivir and oseltamavir, as described below.

The discovery and development of zanamivir. The initial crystal structure 26,27 of influenza virus sialidase had a resolution of ∼ 3.0 Å, and consequently had limited value in structure-based drug design. Therefore, an initial focus in the discovery of influenza virus sialidase inhibitors was on substrate-like Neu5Ac derivatives, particularly 2-deoxy-α- D -N-acetylneuraminic acid (2-deoxy-α-Neu5Ac) derivatives (compound 3, Fig. 3). Based on an understanding of the enzyme's catalytic mechanism 33,40 , it was thought that such compounds might not be rapidly metabolized and should be recognized by the enzyme as a result of the compound's substrate/product-like characteristics 41 .

The parent unsubstituted template of this series, 2-deoxy-α-Neu5Ac (compound 3, Fig. 3), was the first compound produced by the von Itzstein group to be evaluated in vivo in a mouse model of influenza infection by Glaxo researchers led by Charles Penn and Janet Cameron. This compound showed weak, but measurable, effects in animals infected with influenza virus. It was intriguing that 2-deoxy-α-Neu5Ac, but not the unsaturated N-trifluoroacetylated derivative of Neu5Ac (compound 2b, Fig. 3 the most potent influenza virus sialidase inhibitor up until the late 1980s 42,43,44,45 ) had in vivo, albeit weak, activity. The general view was that compound 2b may have suffered from either metabolism 44 or rapid clearance, as demonstrated by others for the parent template, Neu5Ac2en (compound 2a, Fig. 3) 45 . To better understand this interesting observation, Penn, Cameron and colleagues re-evaluated Neu5Ac2en in their established mouse model under identical conditions to those used for 2-deoxy-α-Neu5Ac, and it demonstrated good activity in the mouse model experiments.

On the basis of these experiments, the choice of carbohydrate template was changed. Concomitantly, further refinement of a number of sialidase crystal structures (Box 1) in complex with Neu5Ac and the now confirmed in vivo inhibitor, Neu5Ac2en, were successfully completed. This improvement in structural resolution enabled the commencement of a fully fledged structure-based drug design effort based on these X-ray crystal structures. Computational chemistry techniques were used to probe the active site of influenza virus sialidase in an attempt to design structurally modified Neu5Ac2en derivatives that might be more potent inhibitors 30,39,46 . Moreover, these X-ray structural studies identified residues within the active site that are conserved in sialidases across all influenza A and B viruses and provided an exciting opportunity for the development of compounds that should target all influenza A and B virus strains. Energetically favourable interactions between various functional groups and the residues within the binding pocket were revealed through the application of GRID software 47 . Most importantly, the significance of the Neu5Ac2en C-4 hydroxyl group-binding domain within the sialidase active site was realized. This realization, with further considerations, directed attention to the replacement of the Neu5Ac2en C-4 hydroxyl group by a basic group such as, in the first instance, an amino group. The 4-substituted Neu5Ac2en derivative, 4-amino-4-deoxy-Neu5Ac2en (compound 4, Fig. 3), was predicted 39,46 to have higher affinity for the enzyme than the parent compound Neu5Ac2en as a result of salt-bridge formation with the conserved amino acid Glu119.

Importantly, with further analysis, it was found that the conserved Neu5Ac2en C-4 hydroxyl group-binding domain could accommodate a larger basic functional group. This analysis, together with chemical intuition, led to the conclusion that incorporation of a larger, more basic functionality in place of the Neu5Ac2en C-4 hydroxyl group was of value. Thus, substitution of the C-4 hydroxyl group with a guanidinyl functionality to provide 4-deoxy-4-guanidino-Neu5Ac2en (compound 5, Fig. 3) was predicted to significantly improve affinity for the enzyme. This improvement in affinity was believed to be driven by interactions between two conserved C-4 binding domain amino acids (Glu119 and Glu227) and the larger basic C-4 guanidinyl moiety 30,39,46 4-deoxy-4-guanidino-Neu5Ac2en engages most of the volume available within the sialidase active site (Fig. 5).

a | The inhibitor zanamivir is shown in CPK model in complex with influenza virus A sialidase. b | Oseltamivir carboxylate engages the sialidase active site via an induced fit mechanism. The crystal structure of the influenza virus sialidase–oseltamivir carboxylate complex has provided an excellent rationale of why this compound can efficiently engage the sialidase active site 11,31,55 . Two superimposed structures are shown. In green is the X-ray crystal structure of influenza virus sialidase selected active-site amino-acid residues in complex with 2-deoxy 2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en compound 2a, Fig. 3) In white is the X-ray crystal structure of influenza virus sialidase with the same selected active-site amino-acid residues in complex with oseltamivir carboxylate (compound 8, Fig. 3). The cyclohexene ring of oseltamivir carboxylate (white) is oriented within the sialidase active site such that the carboxylate, acetamide and C-4 substituent interact with the sialidase active site similar to those observed for Neu5Ac2en (green) and derivatives, as expected. However, complexation of oseltamivir carboxylate induces Glu276 to adopt an alternative conformation that is stabilized by a charge–charge interaction to Arg224. This induced fit establishes a hydrophobic area in which the two ethyl groups of oseltamivir carboxylate are then well accommodated. c | Surface display of N-acetylneuraminic acid (atomic colours) and oseltamivir carboxylate (carbon atoms in magenta) influenza virus A sialidase structures superimposed. The key amino-acid residue Glu276 that undergoes reorientation upon binding of oseltamivir carboxylate (see main text) and Arg224 are shown. The His110 (atomic colours) to Tyr110 (cyan) mutation results in oseltamivir resistance, as it seems to prevent the essential rearrangement of Glu276. These mutants remain sensitive to zanamivir as this inhibitor engages Glu276 in an identical manner to that of the substrates recognized by the enzyme (Fig. 4).

The two target molecules — 4-amino-4-deoxy-Neu5Ac2en and 4-deoxy-4-guanidino-Neu5Ac2en (Fig. 3) — were synthesized using the key C-4 azide intermediate 48 (compound 6, Fig. 3). Evaluation of these C-4 derivatives as influenza virus sialidase inhibitors confirmed that both were competitive inhibitors 30,49 . Moreover, both compounds were found to be highly potent inhibitors of virus replication for all influenza A and B virus strains evaluated in vitro 30,49,50 and in vivo 30 . In the case of influenza A (N2) virus sialidase, 4-amino-4-deoxy-Neu5Ac2en inhibited the enzyme by a factor of 100-times greater than the parent compound Neu5Ac2en (4-amino-4-deoxy-Neu5Ac2en: Ki 4 × 10 −8 M Neu5Ac2en: Ki 4 × 10 −6 M) 49 . As expected, the derivative 4-deoxy-4-guanidino-Neu5Ac2en was found to be more potent, with an improved affinity as much as 10,000-fold compared with Neu5Ac2en (4-deoxy-4-guanidino-Neu5Ac2en: Ki 2 × 10 −10 M) 30,49,50 .

X-ray crystallographic structure determination of influenza virus sialidase–inhibitor complexes for 4-amino-4-deoxy-Neu5Ac2en and 4-deoxy-4-guanidino-Neu5Ac2en confirmed, in general, that these inhibitors engaged the enzyme's active site in the predicted binding modes 30 . Specifically, the 4-amino group of 4-amino-4-deoxy-Neu5Ac2en was shown 30 to establish a salt bridge with Glu119, and although 4-deoxy-4-guanidino-Neu5Ac2en displayed the predicted 30,46 lateral binding between the terminal guanidinyl nitrogens and the carboxylate of Glu227, Glu119 was found to be slightly further removed 30 than proposed and stacked parallel to the guanidinyl group 38 . However, Glu119 is still within a distance close enough for electrostatic interaction with the guanidinyl group 30,38 .

Fortuitously, 4-deoxy-4-guanidino-Neu5Ac2en was found to be highly selective for influenza virus sialidase and displayed considerably lower affinity for other sialidases from different sources 49 . The selectivity of influenza virus sialidase inhibitors has become of increasing interest as a result of potential side effects in patients as a result of, in part, the possible inhibition of endogenous human sialidases 51 .

The more potent inhibitor, 4-deoxy-4-guanidino-Neu5Ac2en (compound 5, Fig. 3), was selected as the lead drug candidate by Glaxo under the generic name zanamivir. Owing to its limited oral bioavailability (due to its highly polar nature and rapid excretion of the compound), it was developed as an inhaled formulation, which delivers the drug directly to the primary site of infection. As zanamivir was a first-in-class drug, significant effort in the establishment of appropriate end-point measurements of clinical benefit, in cooperation with regulatory agencies such as the US Food and Drug Administration (FDA), was needed. Following its success in clinical trials, zanamivir was approved in 1999 as the first sialidase-targeting anti-influenza drug, with the tradename Relenza.

Discovery and development of oseltamivir. The discovery of zanamivir provided a platform for further sialidase-targeted anti-influenza drugs. Significant work has been undertaken in structure–activity relationship studies with Neu5Ac-based derivatives and uronic acid carbohydrate-based templates derived from N-acetylglucosamine (compound 7, Fig. 3) 52 . Moreover, substantial effort has been applied towards the development of influenza virus sialidase inhibitors that are based on non-carbohydrate templates (for a review, see Ref. 11). For example, potent and selective inhibitors of influenza virus sialidase and of influenza virus infection in vivo have been developed based on a range of core templates including cyclohexenes such as oseltamivir carboxylate (compound 8, Fig. 3 originally known as GS 4071) 31 cyclopentanes such as peramivir 53 (compound 9, Fig. 3) and pyrrolidines such as A-315675 (compound 10, Fig. 3) 54 . Most noteworthy has been the development of the cyclohexene derivative GS 4071, and its prodrug, oseltamivir, the first orally active sialidase inhibitor to be approved for treating influenza.

Significant synthetic organic and medicinal chemistry based on the functionalized cyclohexene and cyclohexane shikimic acid and quinic acid core templates, respectively, were undertaken in the discovery of GS 4071. On the basis of interactions of the unsaturated Neu5Ac derivatives 4 and 5, three key concepts were used in the discovery of GS 4071 and oseltamivir. First, based on previous mechanistic studies 33,36,39 , positioning the double bond in the inhibitor to more closely mimic the putative transition state sialosyl cation (Box 1) was investigated.

Second, replacing the glycerol moiety of 4-amino-4-deoxy-Neu5Ac2en by a lipophilic group was explored on the basis that the hydrophobic backbone of the glycerol side chain makes contact with the protein, even though the C-8 and C-9 hydroxyl groups make a bidentate interaction with Glu276. This replacement was attempted in the hope that optimization of the hydrophobic character would lead to new sialidase inhibitors with improved lipophilicity while maintaining inhibitor activity. Wholesale replacement of the glycerol side chain by a lipophilic moiety was counter-intuitive, as it had been demonstrated (see previous section) that the glycerol-binding domain engaged the C-8 and C-9 hydroxyl groups through specific interactions (Fig. 4c).

Extensive manipulation of the alkyl side chain, in an elegant structure–activity investigation, led to the development of the optimized 3-pentyl ether side chain. An X-ray crystallographic study of an influenza virus sialidase–GS 4071 complex clearly showed that the architecture of the active site had been altered on binding of GS 4071 (Refs 31,55). Specifically, Glu276 reorients outwards from the glycerol side-chain binding domain to interact with Arg224 and in doing so generates a considerable hydrophobic area within this domain. This rearrangement, which was not predictable, provided exciting new opportunities for further sialidase inhibitor-based anti-influenza drug discovery. Although GS 4071 binds in an identical fashion to zanamivir and other Neu5Ac2en derivatives, this induced fit is essential for the inhibitor to successfully engage the active site to provide the inhibitor's potent efficacy (Fig. 5c).

Third, although it had been hoped that GS 4071 (which is more lipophilic than zanamivir) might have had sufficient oral bioavailability, this was found not to be the case, and so a prodrug strategy was used. Oseltamivir (GS 4104 compound 11, Fig. 3), the ethyl ester prodrug of GS 4071, is readily converted to the active form in vivo by the action of endogenous esterases. Following its success in clinical trials as an orally administered treatment for influenza virus infection, oseltamivir (developed by Gilead) was approved in late 1999, and is now marketed by Roche under the trade name Tamiflu.

Experience with sialidase inhibitors

As zanamivir and then oseltamivir came to market, the questions of clinically significant resistance development and possible unknown side effects remained to be answered. It was hoped that zanamivir, a drug derived from the naturally occurring sialic acid Neu5Ac with minimal additional functionalization, would not result in viable mutants — as functionally important amino-acid residues had been targeted — or unknown side effects. So far, over 8 years of clinical experience with zanamivir has not provided evidence to suggest otherwise, although this may simply be the result of the limited use of this drug.

In the case of oseltamivir, which has been much more widely used, a viable resistant influenza virus mutant has emerged 56 . Interestingly, this mutation targets and effectively blocks the essential rearrangement of Glu276 within the sialidase active site and as a consequence the drug has significantly reduced affinity (Fig. 5b). This oseltamivir-resistant virus remains sensitive to zanamivir 56 . These experiences may provide some evidence that maintaining a strong resemblance to the natural substrate, Neu5Ac, might reduce the prospect of the development of viable drug-resistant mutants. This is because when drug binding depends on either active-site amino-acid reorientation or interactions with non-essential active-site amino acids, the possibility of escape mutants might increase. With regards to adverse effects of sialidase inhibitors in general, the only significant adverse effects that have been reported in the past 2 years have been some speculation about adverse neuropsychiatric effects of oseltamivir in certain age groups 57 .

In light of the current pandemic threat and the emergence of resistance to oseltamivir, the development of next-generation anti-influenza drugs must be a high priority. To this end, the US FDA has established a fast-track programme for the development of such drugs. So far, the FDA has provided fast-track designation for the Biocryst injectable candidate peramivir (compound 9, Fig. 3). Biota and Sankyo have also announced their intention to develop a new influenza virus sialidase inhibitor that is a divalent zanamivir (compound 12, Fig. 3), which is currently in clinical trials.

Both of these developments are important as they could give rise to alternatives to the currently available drugs that may be required in the case of a pandemic or with the appearance of significant drug resistance. An injectable drug such as peramivir might be of great value to patients who cannot readily take tablets or have limited lung capacity. Also, a long-acting sialidase inhibitor, such as divalent zanamivir, that might reduce the number of treatments required to as little as once a week compared with the current twice-daily requirement is appealing.

A further important consideration for the development of next-generation sialidase inhibitors is the cost of drug production. The experiences from the development of both the relatively expensive zanamivir and oseltamivir provide valuable insight towards the design of next-generation potent influenza virus sialidase inhibitors from relatively inexpensive starting materials, such as N-acetylglucosamine 52 .

Recently, Russell and colleagues have described that influenza virus sialidases may be grouped into two distinct families: namely Group 1 and Group 2 influenza virus sialidases 58 . It has been suggested that the Group 1 sialidases such as N1 from the now infamous avian H5N1 strain undergo significant rearrangement around the so-called 150 loop upon binding of substrates and inhibitors. In apo structures of the Group 1 influenza virus sialidases, the 150 loop is in a more open orientation that presents a larger active-site cavity. These enzymes complexed with the inhibitor oseltamivir carboxylate reveal that this 150 loop eventually closes to tightly coordinate the inhibitor 58 . The fact that there is a more open enzyme architecture in the apo and initially inhibitor-complexed structures provides new and exciting opportunities for exploitation in sialidase-targeted anti-influenza drug discovery.

An important remaining question is who will fund the development of such compounds. It is unlikely that pharmaceutical companies would race to bring to market another anti-influenza drug, particularly as there are already two good drugs available. It may be that national governments or not-for-profit organizations will have to support such development, particularly if nations wish to stockpile a variety of anti-influenza drugs.

Alternative drug discovery targets 59,60,61 , such as the RNA polymerase 59 , the haemagglutinin protein 60 or the M2 ion channel protein 61 , that are essential in the virus' life cycle are also under investigation and may, in either a combination therapy approach 61 with the sialidase inhibitors or in their own right, provide new classes of anti-influenza drugs. Combination therapy might also reduce the potential of resistance development 61 . Of course, although anti-influenza drugs will buy time and save lives, the 'Holy Grail' would be the development of a universal vaccine that would protect against all influenza virus strains past, present and future. Much effort is going on in the development of improved vaccines however, there is still a long way to go 62,63 . There is no doubt that we must continue efforts in drug discovery to win the war against this deadly virus, or risk another potential pandemic that may result in mortality similar to that experienced between 1918 and 1919.

Box 1 | Enyzmatic mechanism of influenza virus sialidase

It was originally proposed that the solution-dominant α-sialoside 2 C5 conformer binds to the influenza virus sialidase and is distorted by the active-site environment from this chair conformation into an α-boat conformer (figure). X-ray crystallographic studies 64,65 of sialidase–Neu5Ac (N-acetylneuraminic acid) complexes confirmed both distortion of the substrate upon binding and the formation of a salt bridge between the substrate's negatively charged carboxyl group and highly conserved triarginyl cluster.

The departure of the aglycon residue would appear to be facilitated by the resulting conformational strain through the formation of an oxocarbocation ion intermediate, a sialosyl cation, that has been identified by kinetic isotope effect measurements and molecular modelling studies 33,39 . The negatively charged environment within that region of the sialidase catalytic site is thought to stabilize the charged intermediate 33 . A water molecule then reacts in a stereoselective manner with the sialosyl cation intermediate to afford α-Neu5Ac (compound 1a) as the first product of release that then mutarotates to the thermodynamically more favourable β-anomer (compound 1b) 33,39 .

Alternatively, it has been proposed 11,66 that all sialidases, irrespective of origin, may trap the cation to form a glycosyl-enzyme covalent intermediate, a common feature of retaining glycohydrolases 66 , that is stereospecifically hydrolysed to afford compound 1a.


Results

Efficacy of antiviral treatment

Since the conversion factor of virus from TCID50/mL to infectious virus particles (α) might affect the dynamics of drug resistance, we first assess the impact of α by studying the number of breakthrough infections. We simulated 1000 infections treated with an adamantane or a NAI started at t = 0, and inoculated with NV(0) = αV(0) virions. We determined the fraction of patients who developed a symptomatic infection despite having received preventive antiviral therapy. We defined a symptomatic infection as one in which the viral titer exceeds the symptomatic threshold of 1% of peak untreated viral titer, as defined in [65]. Furthermore, we assumed that both the wild-type and drug-resistant virions are identical (same fitness), differing only in their susceptibility to the antiviral (mi or ni). Studies suggest that circulating drug-resistant strains tend to have fitness equivalent to the wild-type strain [21, 66, 67]. The effect of fitness will be examined in later sections. Fig 3 shows the number of breakthrough infections in our 1000 simulated treated infections as a function of the conversion factor, α, for increasing antiviral efficacies.

Fractions of simulated infections (out of 1000) resulting in breakthrough symptomatic infections despite treatment initiated at infection onset with either adamantanes (top row) or NAIs (bottom row), assuming the complete (left column) or internal (right column) mutation model as a function of the conversion factor, α. Mutant and wild-type virus are assumed to have equal fitness.

When breakthrough infections occurred, the viral load consisted almost exclusively of drug-resistant mutant virus, with wild-type viral titers typically remaining below the symptomatic threshold. When α is small, i.e., when there are few virions per measured TCID50/mL of nasal wash, the number of breakthrough infections is small. That is because the rate of virion production, αp, is small and provides fewer opportunities for resistance to develop. As α is increased, the number of breakthrough infections increases until it reaches a maximum value. The production rate is no longer a limiting factor here and the asymptotic value reflects the balance of a decreasing infection rate and an increasing viral inoculum.

The internal mutation model predicts fewer breakthrough infections for a given drug efficacy than the complete mutation model. This is because an internal mutation will only be carried by the core protein, not the surface protein, giving the antiviral a second chance to prevent its spread through the drug-sensitive surface protein. Both the internal and complete mutation models predict adamantanes are better than NAIs at suppressing breakthrough infections due to resistance emergence. This is because adamantanes act before viral replication, unlike NAIs which act afterwards, leaving little opportunity for a mutation to occur. Interestingly, the fraction of breakthrough infections under adamantane therapy given a late mutation is approximately equivalent to that under NAI therapy for an early mutation.

To evaluate the effect of the drug-resistant strain’s fitness relative to its wild-type counterpart, we varied the relative fitness while fixing α = 10 4 virions/[TCID50/mL], the value at which the fraction of breakthrough infections has reached its asymptotic value and consistent with an estimate of this conversion factor used in [38]. The results are shown in Fig 4. Perhaps the most prominent feature of these plots is the clear fitness threshold needed to produce any breakthrough infections. This threshold is determined by the basic reproductive number of the model, which is the number of secondary infections produced by a single infected cell (see Methods). Essentially, the basic reproductive number must be greater than one in order for the infection to grow and when the fitness of the drug resistant mutant is small, this threshold is not met. When the threshold is surpassed, the number of breakthrough infections rises, sometimes quite dramatically. We again see that for a given drug efficacy, the IM assumption produces fewer breakthrough infections than the CM assumption for both adamantanes and NAIs. Under a particular mutation assumption and for a given fitness and drug efficacy, adamantanes are more effective at suppressing infections than NAIs, but adamantane CM is equivalent to NAI IM.

Number of breakthrough infections during treatment with adamantanes (top row) and NAIs (bottom row) under the complete mutation (left column) and internal mutation (right column) assumptions as a function of the relative fitness of the drug-resistant mutant. The conversion factor is fixed to α = 10 4 virions/[TCID50/mL].

Drug resistance in the absence of treatment

Drug resistance has been known to emerge even in the absence of treatment [68]. This is because a mutation which confers resistance against an antiviral almost always emerges over the course of an infection as a result of random mutations [43], but that mutation will only grow to sufficient levels to be detectable in a patient’s viral shedding if its presence does not affect the fitness of the virus significantly. Thus, we set out to determine what conditions will enable a drug-resistant strain to emerge even in the absence of drug pressure.

Fig 5 shows the three measures of drug resistance described in Methods as a function of the relative fitness of the mutant in the absence of drug treatment. For the fraction of mutants in the cumulative viral titer (left) and the total number of mutants (center), we also investigated the effect of infection initiation with different initial virus mixtures consisting of entirely wild-type, entirely mutant, or a mixture containing 50% of each.

Fraction (left), total number (center) and time of detection (right) of drug resistant mutants produced in an influenza infection in the absence of drug treatment. Adamantane CM mutants are in red, adamantane IM mutants are in maroon, NAI CM mutants in blue, and NAI IM mutants are in green. We show the mean of 1000 simulations with error bars indicating the standard deviation (error bars are too small to be visible in left two graphs).

In the absence of drug treatment, the fraction of drug-resistant mutants (Fig 5, left) is negligible when their relative fitness is low (<0.2)—even if the initial viral inoculum consists entirely of drug-resistant mutant. When the viral inoculum consists entirely of wild-type virus, the fraction of drug-resistant mutants remains negligible unless the drug resistant mutants have a high relative fitness. When the initial viral inoculum consists of both wild-type and mutant virus, the infection switches from predominantly wild-type virus to predominantly drug-resistant mutant near a relative fitness of 1. There are, however, some dynamical differences caused by the mutation assumptions and mechanism of drug action. When the initial viral inoculum consists of 100% mutant virus, IM adamantane-resistant mutants become dominant at slightly lower fitness than the other models and the CM NAI-resistant mutants become dominant at slightly higher fitness. The order is reversed when the initial viral inoculum is 100% wild-type virus. In the case of a 100% mutant inoculum, the IM models, which we’ve seen lower the effective rates of mutations, also lower the rate of reverse mutations from mutant to wild-type such that when there are already large numbers of mutant virus, the IM models prevent growth of the wild-type virus. For a particular mutation assumption, adamantanes require a lower fitness to dominate the infection than NAIs when the initial inoculum consists of mutant virus. This is because the fitness cost for adamantane resistance is assumed to affect the infection rate β which reduces the chances of infecting a cell, whereas the fitness cost for NAIs is assumed to affect the infection rate p which means that the few virions that escape the cell can easily infect new cells and continue the infection.

Even in the absence of drug treatment, we still see the total number of drug resistant mutants produced during the infection (Fig 5, center) rising to very high levels at relative fitness less than 1. The total number of mutants also shows some interesting dynamical differences between the two drug treatments and mutation assumptions. This can be seen particularly at the extremes of viral inoculum (initial inoculum consisting entirely of mutant or entirely of wild-type), where the adamantane-resistant mutants are a slightly larger fraction of the total viral titer than the NAI-resistant mutants at low relative fitness (<0.5), and a slightly smaller fraction of the total viral titer at high relative fitness (>1.5). This difference is due to the small (or large) value of pμ of NAI-resistant mutants. At low relative fitness, the production of NAI-resistant mutants is suppressed and so NAI-resistant mutants are not detected. As the relative fitness increases, pμ increases without bound, as does the number of NAI-resistant mutants produced and released over the course of the infection, so the mutants become a large fraction of the population at lower relative fitness than the adamantanes. We also finally see a difference in the dynamics of adamantane CM and NAI IM models. The NAI IM behaves like the adamantane CM model at low relative fitness where the internal mutation incurs an extra fitness cost. At high fitness, however, the few virions with mismatched RNA and surface proteins are far outnumbered by conventionally packed virions, so their effect on infection dynamics is minimal and we see little difference between the IM and CM models for both adamantanes and NAIs.

The time of detection (Fig 5, right) of drug resistant mutants ranges between 1.7–2.3 days post-infection (dpi) at low relative fitness and 1.3–1.5 dpi at high relative fitness. Not unexpectedly, there is a steady decrease in the time of detection as mutant fitness increases because the mutants can spread more easily as their fitness increases and they are therefore detected earlier in the infection. Interestingly, there are differences in the time of detection of NAI-resistant and adamantane-resistant mutants. When the relative fitness is less than 1, the NAI CM mutants are the slowest to emerge because of the drastically reduced production rate of these mutants. NAI IM mutants will not have production suppressed when they are initially produced as they are packed in with the wild-type surface proteins and so emerge a little sooner. The adamantane IM mutants are the fastest to emerge because when initially produced, they have the wild-type surface proteins and so can easily infect cells which will then produce drug-resistant mutants. The situation is reversed once the relative fitness is greater than 1. The NAI CM mutants now have increased production causing them to emerge rapidly and the adamantane AP mutants have a reduced infection rate causing them to emerge later.

Drug resistance in the presence of treatment

In the presence of drug treatment, the drug-resistant mutants have a competitive advantage and are expected to emerge sooner and at lower relative fitness than in the absence of drug treatment. We examine our three measures of drug resistance during treatment initiated at t = 0 in order to understand the nature of this competitive advantage. The results are shown in Fig 6, which shows the fraction of mutants in the viral titer (left column), total number of mutants (center column), and time of detection (right column) for infections treated at 60% (top row), 70% (second row), 80% (third row), and 90% (bottom row) efficacy.

Fraction (left column), total number (center column) and time of detection (right column) of drug resistant mutants produced in an influenza infection given drug treatment at 60% (top row), 70% (second row), 80% (third row), and 90% (bottom row) efficacy. Adamantane CM mutants are in red, adamantane IM mutants are in maroon, NAI CM mutants in blue, and NAI IM mutants are in green. We show the mean of 1000 simulations with error bars indicating the standard deviation.

In the presence of drug treatment, the relative fitness at which drug-resistant mutants begin to dominate the infection shifts to lower relative fitness. Not surprisingly, drug resistant mutants rise to high levels at low fitness in the presence of drug treatment. We still see dynamical differences between the two drug treatments and the two mutation models, particularly for infections initiated with an inoculum consisting entirely of wild-type virus. These differences, however, diminish as the drug efficacy increases. It seems that the competitive advantage conferred by high efficacy drug treatment trumps any small additional competitive advantage incurred by drug mechanism or viral surface proteins.

Perhaps more important than the number of mutants produced is the time at which the mutants will become detectable during the infection. As the drug efficacy increases, the time to detection of mutants also increases. While this seems somewhat contradictory in the face of the competitive advantage of the drug-resistant mutants, we must remember that we are considering infections initiated entirely with wild-type virus. As the drug efficacy increases, the growth rate of the wild-type infection slows and it takes longer to produce that first drug-resistant mutant. Once that first mutant appears, it has the competitive edge and will grow quickly, but it is the long wait for that first mutant virus that increases the mean time to detection. Unfortunately, this highlights a potential problem with our model. Slow-growing infections that fester for 5–10 d before producing detectable levels of mutant virus are unlikely to occur in most humans. The human immune response will likely clear a slow-growing infection before it has the opportunity to produce a drug-resistant mutant.

Delayed treatment

Another short-coming of the previous section is the assumption of treatment initiated at the onset of infection. While both adamantanes and NAIs are occasionally used prophylactically to prevent influenza outbreaks from spreading, they are more often given to patients who are not only already infected, but are most likely experiencing symptoms [69]. The delay in treatment allows the wild-type virus to grow unimpeded for some time affording the opportunity for a drug-resistant mutant to arise stochastically in the absence of antiviral pressure. Initially, this drug resistant mutant will not have a competitive edge, but once antiviral therapy begins, whatever drug-resistant mutants have been produced suddenly have a competitive advantage and will begin to grow. In order to determine what effect delayed treatment might have on the emergence of drug-resistant mutants, we simulated infections with treatment at 98% efficacy initiated at 0, 12 and 48 h post-infection. The results are shown in Fig 7.

Wild-type and mutant virus for treatment initiated at t = 0 (left column), at 12 h (center column) and at 48 h right column. The top row shows dynamics for the adamantane CM model the second row shows the adamantane IM model the third row shows the NAI CM model and the fourth row shows the NAI IM model. In each plot, we present 10 simulations. Treatment efficacy is assumed to be 98% and the two strains are assumed to have equal fitness. The dashed line denotes the threshold of detection.

An efficacy of 98% is sufficient to suppress all breakthrough infections in three of the four models when applied at the onset of infection. Even with a 12 h delay, the adamantane IM model predicts suppression of the infection, while the remaining models predict that there will be breakthrough infections. The only model for which there is no risk to treatment is the adamantane IM model, since even treatment delayed by 48 h does not result in mutant virus rising to detectable levels. For the remaining models, treatment, while potentially beneficial to the patient, presents the risk of encouraging a drug-resistant infection. This is particularly evident for the adamantane CM and NAI IM models which predict that treatment initiated at t = 0 will suppress infections, but when treatment is delayed by even as little as 12 h, which is still before symptoms usually appear, many patients will develop drug-resistant infections. Another potential risk is seen in the predictions made by the two NAI models, which show long-lasting drug-resistant infections when treatment is initiated at 48 h.

Effect of an immune response

To evaluate the effect of an immune response, we first examine the number of breakthrough infections (Fig 8). When we use the same drug efficacies as for the model without an immune response, we see that for all models except the NAI CP, the number of breakthrough infections falls to less than 10%. Even for NAI CP, the number of breakthrough infections is reduced in the presence of an immune response, although it still remains quite high—about 50% of treated patients will become symptomatically infected when the mutant fitness is equal to the wild-type fitness. We again see that there is a minimum fitness needed to produce breakthrough infections. Since the immune response has changed the basic reproductive number of our model, the minimum fitness threshold for breakthrough infections to occur is slightly higher than in the absence of an immune response (∼15% with the immune response compared to ∼10% without).

Number of breakthrough infections in the presence of an immune response during treatment started at t = 0 with adamantanes (top row) and NAIs (bottom row) under the complete mutation (left column) and internal mutation (right column) assumptions as a function of the relative fitness of the drug-resistant mutant. The conversion factor, α is fixed to 10 4 .

The addition of an immune response also alters the infection dynamics both in the presence and absence of drug treatment. Fig 9 shows the fraction of mutants, the total number of mutants and the time of detection for infections in the absence of treatment (top row) and in the presence of treatment at 60% (center row) and 70% (bottom row) drug efficacy. In the absence of drug treatment, the fraction of mutants, total number of mutants and time of detection in the presence of an immune response look quite similar to those found without the immune response (Fig 5). Careful inspection, however shows that the mutants need a slightly higher fitness to dominate the infection and will take slightly longer to reach detection levels when an immune response is present. When there is an immune response, the mutants need to not only successfully compete for resources with the wild-type virus, but they also need to evade the immune response making it even harder for them to multiply. In the presence of drug treatment, the effect of the immune response is more evident. With 60% drug efficacy, the mutants must have a relative fitness of at least 0.2 in order to produce an infection with a 70% drug efficacy, the mutants need to have a relative fitness of at least 0.5 in order to produce an infection. This minimum threshold was not observed in the absence of an immune response because mutant virus could linger and slowly grow over long periods of time, so that even mutant virus with very low fitness could eventually multiply to high numbers. The immune response puts an end to these slow-growing infections so that if a mutant virus is not fit enough to multiply to large numbers before the immune response kicks in, there will not be an infection. Since these slow-growing infections have been eliminated, the time of detection is substantially reduced in the presence of drug treatment, with the average time of detection remaining below 4 dpi.

Fraction (left column), total number (center column) and time of detection (right column) of drug resistant mutants produced in an influenza infection in the presence of an immune response given no drug treatment (top row) and drug treatment at 60% (second row) or 70% (bottom row). Adamantane CM mutants are in red, adamantane IM mutants are in maroon, NAI CM mutants in blue, and NAI IM mutants are in green. We show the mean of 1000 simulations with error bars indicating the standard deviation.


6. Lab-on-a-Chip/Microchip Devices

A more versatile and powerful technology, lab-on-a-chip/microchip (LoC), provides a new route to develop a new generation of POC influenza tests. LoC technology originates from microelectromechanical system (MEMS) technology, with a focus on chemical and biological applications. It has many fascinating advantages, such as high reaction efficiency, low reagent/energy consumption, low waste generation, and a small footprint. LoC technology has been utilized to develop assays targeting multiple pathogens. With respect to influenza tests, Soh’s group reported a disposable microfluidic chip for sample-to-answer genetic analysis of H1N1 virus [36]. Bhattacharyya and Klapperich fabricated a plastic microfluidic solid phase extraction device to isolate viral RNA from mammalian cells infected with the influenza A (H1N1) virus [37]. In a recent report, Klapperich’s group described a disposable microchip integrated with solid-phase extraction and RT-PCR modalities capable of extracting and amplifying influenza A RNA directly from clinical specimens in less than three hours [38]. A continuous-flow microfluidic RT-PCR chip and disposable electrical printed (DEP) chips have been employed for rapid amplification and sensing of a swine-origin influenza virus. Using the RT-PCR chip method, the assay could be completed in 15 min and signals were detected with the DEP chip [39]. A miniaturized all-in-one, real-time RT-PCR instrument was developed, allowing automated sample preparation and diagnosis within 2.5 h. Typing and sub-typing of seasonal influenza A H1N1 was demonstrated using this system [40]. Another portable, user-friendly microchip NAT system was reported for POC diagnosis of influenza, with sensitivity close to that of a benchtop RT-PCR instrument. A microchip electrophoretic immunoassay coupled with laser-induced fluorescence detector was developed to detect swine influenza virus [41]. This system allows rapid and simultaneous concentration of viral particles and further separation of the virus-antibody complexes from the unbound antibody [42]. These influenza assays can be implemented using LoC technology and the microchip influenza assays exhibit impressive results. However, more efforts in further system improvements and assay validation are necessary to adapt the new LoC tests to real POC settings.


Influenza infections and drug design - Biology

In the last 100 years there have been three major influenza pandemics: Spanish Flu in 1918, Asian Flu in 1957 and Hong Kong Flu in 1968. These claimed the lives of approximately 50 million, 2 million and 1 million people respectively. Added to this is the annual death toll of 250,000 to 500,000 people worldwide with a further 3 to 4 million people suffering severe illness. These statistics make influenza an extremely important pathogen. In 1997 the alarming emergence of a new, highly pathogenic subtype, H5N1, which has a 50% mortality rate, provided a major impetus for renewed influenza research. However the battle against influenza is going to be difficult. Recently another subtype, H1N1, has emerged. This subtype causes a relatively mild infection in humans, however is highly transmittable between people and a new influenza pandemic was declared by the World Health Organization. If this virus were to acquire some of the lethal capabilities of H5N1, then the ensuing pandemic could be devastating.

In this timely book, internationally renowned scientists critically review the current research and the most important discoveries in this highly topical field. Subjects covered include the NS1 protein of influenza A virus, the structure of influenza NS1, influenza B hemagglutinin, influenza A nucleoprotein, influenza A hemagglutinin glycoproteins, the M2 channel, virulence genes of the 1918 pandemic influenza, influenza virus polymerase, gene diagnostic microarrays, and computer-assisted vaccine design.

Highly informative and well referenced, this book is essential reading for all influenza specialists and is recommended reading for all virologists, immunologists, molecular biologists, public health scientists and research scientists in pharmaceutical companies.

"This is a good quality, concise book on the basic nature of influenza viruses that comprehensively covers the current work on influenza." from Doodys

"a series of excellent and high-powered articles . an up-to-date review of the advancements in molecular influenza virology . of interest to a range of readers including post-graduate and basic science researchers, virologists and those involved with drug design and development." from Expert Rev. Vaccines (2010) 9: 719-720.

"I particularly enjoyed a very thorough account of the influenza A haemagglutinin . it's a nicely put together book that summarizes recent developments on the structural side of influenza replication. Appropriate audiences for the book would be final-year virology students and influenza researchers." from Microbiol. Today (2010)

(EAN: 9781904455578 9781912530816 Subjects: [virology] [microbiology] [medical microbiology] [molecular microbiology] )


Influenza infections and drug design - Biology

Three times in the last century, influenza viruses have undergone major genetic changes resulting in global pandemics that had devastating effects. The most infamous pandemic was the Spanish Flu which affected up to 25% of the world population and is thought to have killed at least 40 million people in 1918-1919. More recently, two other influenza pandemics, the Asian Flu in 1957 and the Hong Kong Flu in 1968, killed millions of people worldwide. These caused severe disease, not only in the young and the elderly, who are usually very susceptible to influenza, but also among healthy younger persons. In 1997 and 2003 a new influenza A virus of H5N1 subtype emerged in Asia and was transmitted directly from birds to humans with lethal outcomes. Despite monumental efforts to contain them, the H5N1 viruses expanded their territory and caused a major outbreak in wild waterfowl in China in 2005. Indeed, they have even been transmitted to Siberia and Kazakhstan.

Despite extensive, coordinated efforts by various agencies and disciplines, both national and international, we are ill-equipped for a new influenza pandemic. In fact it is highly unlikely that adequate supplies of vaccine for the H5N1 viruses will be prepared prior to the occurrence of the next pandemic. Many countries are stockpiling influenza drugs, with the hope that the inevitable emergence of drug-resistant viruses will not nullify those efforts immediately. To combat the outbreaks that will undoubtedly occur in the near future a better understanding of influenza virus itself, the virus-host interaction, and mechanisms of drug resistance is urgently needed.

In this timely book world renowned scientists (including the 1996 Nobel Prize Winner, Peter Doherty) critically review the most important issues in this rapidly expanding field. Topics covered include analysis of influenza RNP, viral entry and intracellular transport, epidemiology, host range and pathogenicity, antivirals, vaccines, H5 viruses, and much more. Essential reading for all influenza virologists, molecular biologists, public health scientists and research scientists in pharmaceutical companies.

"This book is highly recommended for infectious diseases specialists, virologists, vaccinologists, vaccine and drug development professionals, and anyone who wishes to learn more about influenza" from Clin. Inf. Diseases (2006) 43: 802-804.

". interesting and readable . lucidly written . an important resource . " from Clin. Inf. Diseases (2006) 43: 802-804.

"This is a wonderful book . I have seldom read such a high-powered series of articles on molecular virology . I do not think that the book will be easily outdated or even outclassed . " from J. Antimicrobial Chemother. (2006) 58: 909.

". excellent value for money and strongly recommended." from Microbiology Today (2006).

"should be on the shelf of every flu virologist, public health scientist, and vaccinologist . I recommend it for all virologists and public health scientists who want to have the most updated picture on influenza and why a flu pandemic may occur in the near future. " from ASM Microbe (March 2007).

"This is one of the best books currently available on Influenza virus research." from Sai Vikram Vemula, Purdue University.

(EAN: 9781904455066 9781913652319 Subjects: [virology] [microbiology] [medical microbiology] )


2017 Summer School on Modeling Immunology

The summer school will be taught by the following individuals:


Trevor Bedford
Assistant Member, Fred Hutch Center
Vaccine & Infectious Disease Division
Andreas Handel
Associate Professor, Department of Epidemiology & Biostatistics
University of Georgia School of Public Health
Frederick “Erick” Matsen
Associate Member, Fred Hutch Center
Computational Biology Program
Paul Thomas
Associate Member, St. Jude Faculty
Immunology Department


Watch the video: Influenza - The Flu Virus (November 2021).