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BIT's 6th Annual Congress of International Drug Discovery - Science and Technology
October 18-22, 2008
Beijing, China

Keynote Address
October 18, 2008

Predicting Seasonal and Pandemic Influenza Evolution Via Homologous Recombination
Niman HL
Recombinomics, Inc., Pittsburgh, Pennsylvania, USA

Introduction:  The recent explosion in influenza virus genomic sequence data has led to new insight in mechanisms of rapid genetic evolution.  Homologous recombination has emerged as a mechanism for the exchange of single nucleotide polymorphisms between closely related influenza genomes.  These exchanges can be used in polymorphism tracing experiments, which involves movement onto multiple influenza genetic backgrounds.  These movements can also be used to predict emerging seasonal and pandemic vaccine targets as well as anti-viral resistance.

Methods and materials:  Public influenza sequences were used to identify polymorphism exchanges in seasonal and pandemic influenza, including neuraminidase polymorphism, H274Y, associated with oseltamivir (Tamiflu) resistance, and Q136K, associated with zamanivir (Relenza) resistance in H1N1 seasonal influenza, or hemagglutinin receptor binding domain changes such as M223I, S227N, or M230I in H5N1 clade 2.2 pandemic influenza.

Results:  Polymorphism tracing identified the movement of polymorphisms onto multiple genetic backgrounds which included H1N1 osletamivir resistance marker H274Y which was initially found in clade  2C (Hong Kong/2652 – like) isolates in China (Zhejiang and Gansu) as well as clade 1 (New Caledonia/20 – like) in multiple locations in the United States during the 2006/2007 season.  In the 2007/2008 season this polymorphism moved onto multiple clade 2B (Brisbane/59 – like) backgrounds in the United States, including early isolates in Hawaii, followed by Florida.  The most common sub-clade in the United States was also present throughout Europe, including Norway and France which had the highest reported incidents of H274Y resistance.  Similar genetic background jumps were identified for H1N1 zanamivir resistance marker Q136K which was initially identified on clade 2A (Solomon Island/3 – like) in the Philippines and Australia in 2006, followed by clade 2B in Australia, New Zealand, and Thailand in 2007/2008.  Moreover, in 2008 a mixture containing Q136K was identified in the United States in a Pennsylvania patient as well as the related change, Q136R in a New Jersey patient.  Similar tracings in H5N1 clade 2.2 were also noted, which included concurrent acquisitions of the same polymorphism, G743A, onto multiple genetic backgrounds, clade 2.2.1 and 2.2.3, in multiple countries, including Russia, Egypt, Kuwait, Ghana, and Nigeria.  Similarly, receptor binding domain changes such as V223I were traced from Mongolia to Egypt to Ghana, or M230I from Germany to Egypt.  In addition to these clade 2.2 polymorphism tracings, M230I has also been identified in clade 2.3.2 isolates which were initially identified in Vietnam and Hong Kong in 2006/2007 and have migrated to South Korea, Japan, and Russia in 2008.  These tracings also identify the aggregation of multiple polymorphisms onto the same genetic background, such as the first human H5N1 isolate reported in Nigeria in 2007. 

Conclusion:  Polymorphism tracing identifies predictable acquisitions in seasonal and pandemic influenza genomes.  These acquisitions are acquired via homologous recombination and are applicable to prediction of vaccine targets as well as the emergence of anti-viral resistance.  The data mining from influenza sequences serves as a model for similar analysis in other rapidly evolving genomes.

Session 54 - New Preventive and Therapeutic Vaccine Discovery and Development

October 21
The Evolution of Influenza via Recombination, with Emphasis on H5N1 and Vaccine Target Development

The acquisition of single nucleotide polymorphisms via homologous recombination represents a paradigm shift in influenza genetics.  This novel mechanism is applied to influenza sequence data from seasonal and pandemic (H5N1) isolates to map distribution patterns of single nucleotide polymorphisms.  These distribution patterns are used to identify polymorphisms which are appended onto diverse genetic backgrounds to generate antigen drift, which is predictable.  This approach has been applied primarily to clade 2.2 (Qinghai strain) to identify acquisition patterns, including dramatic examples in isolates from Egypt.  These isolates acquire polymorphisms from diverse locations outside of Egypt, as well as from local isolates, which are associated with vaccine resistance in poultry populations.  The applications of clade 2.2 evolution extends to clade 2.3 (Fujian strain), including clade 2.3.2 / 2.3.4 reassortants in South Korea, Japan, and southeastern Russia, as well as clade 2.1 in Indonesia as well as seasonal flu.  These acquisitions are predictable and reliability is increased as the sequence database grows, which can be used to create emerging genomes prior to emergence.  The application of these predictions to vaccine target creation and selection will be discussed.


Options for the Control of Influenza VI
June 17-23, 2007
Toronto, Ontario, Canada

H5N1 Clade 2.2 Polymorphism Tracing Identifies Influenza Recombination and Potential Vaccine Targets
Niman HL, Saad, MD, Boynton, BR, Monteville MR
Recombinomics, Inc., Pittsburgh, Pennsylvania, USA, U.S. Naval Medical Research Unit 3 (NAMRU-3), Cairo, EGY

Avian influenza virus H5N1 Clade 2.2 was first detected at Qinghai Lake in May 2005 in long range migratory birds.  Subsequently, Clade 2.2 has been detected in wild birds, domestic poultry, and patients in 47 countries in Asia, Europe, and Africa Polymorphism tracing was used to track and monitor emerging polymorphisms.
Newly emerging polymorphisms in H5N1 clade 2.2 sequences in public sequence databases at Genbank and Los Alamos were analyzed and compared to each other and sequences from other H5N1 sub-clades or serotypes.
The newly emerging polymorphisms define regional markers and linking markers between countries that correlate with known migratory bird flyways. The newly emerging polymorphism could be traced to both low and high path avian influenza isolates. Sequences in Egypt and Nigeria from the current season contained regional markers from last season, with new markers appended onto the established genetic background defined by isolates from the 2005/2006 season. HA from the first reported human H5N1 isolate from Nigeria, A/Nigeria/6e/07, had two western Africa region markers (identified in isolates from Nigeria, Ivory Coast, Burkina Faso, and Sudan) combined with one marker form northern Germany/ Switzerland, and three others from Egypt/Djibouti. One of these three was also present in recent H5N1 isolates from chickens in Hunan and geese from Shantou in China. Although the isolates from China were not Clade 2.2, the isolates also contained additional polymorphisms in other recent Clade 2.2 isolates from Egypt. Isolates from the familial cluster in Gharbiya contained polymorphisms in HA, NA, and PB2 that matched the geese from Shantou. Two additional isolates from adjacent regions in Beni Suef and Fayoum governorates had a three BP deletion in HA which was also present in the chicken isolates from Hunan.
Newly emerging Clade 2.2 polymorphisms in Africa define migratory pathways and parental strains of H5N1, which create novel combinations of polymorphisms via recombination.
Use of these acquisition patterns to predict novel vaccine targets will be discussed.

NA Phylogram

HA Phylogram

Emergence and Convergence of H5N1 Clade 2.2 Hemagglutinin M230I in Egypt
Niman HL, Saad MD, Boynton BR, Aly MM, Arasa A-SA, Monteville, MR
Recombinomics, Inc., Pittsburgh, Pennsylvania, USA, U.S. Naval Medical Research Unit 3 (NAMRU-3), Cairo, EGY, Central Laboratory for Veterinary Quality Control, Giza, EGY

Avian influenza virus H5N1 Clade 2.2 was first detected at Qinghai Lake in May 2005 in long range migratory birds.  Clade 2.2 has been detected in wild birds, domestic poultry, and patients in Egypt.  Polymorphism tracing was used to track and monitor emerging polymorphisms, including HA M230I, encoded by two previously detected codons, ATA and ATT.
Between October 2006 and March 2007, NAMRU-3, a World Health Organization Regional Influenza Reference Laboratory in Cairo, Egypt, sequenced 13 poultry and 9 human samples which were H5N1 RT-PCR positive.
All H5N1 hemagglutinin sequences were Clade 2.2 containing an HA cleavage site of GERRRKKR, and a number of regional polymorphisms detected in human and poultry samples collected in 2006 between March and June.  This season, eight of the thirteen poultry hemagglutinin sequences had M230I.  Six were encoded by ATT and two was encoded by ATA.  Similarly, four of the nine human sequences had M230I. Three were encoded by ATA and one was encoded by ATT.  The four M230I positive infections were fatal. Although M230I has been detected in H5N1 in Asia previously, the detection M230I in the twelve samples collected in the 2006/2007 season represents the first report of M230I in Clade 2.2.   Two of the human samples were from a cluster of three patients from the same extended family, who died in December, 2006.  In addition to M230I, which is adjacent to the receptor binding domain, the two samples also had V223I located within the receptor binding domain, as well as NA N294S, an alteration linked to oseltamivir resistance.  Although these emerging polymorphisms also have not been reported in human Clade 2.2, all polymorphisms have been reported previously in H5N1 in Asia. 
Sequencing of H5N1 in Egypt has defined a number of newly emerging regional specific polymorphisms on a Clade 2.2 genetic background.  These polymorphisms can be used to identify parental strains, which contribute polymorphisms which are placed onto new genetic backgrounds via recombination.  The monitoring of these polymorphisms can be used to develop new vaccine targets prior to the emergence of the novel combinations of polymorphisms.     

Canadian Swine H1N1 and H1N2 Evolution Via Recombination
Niman HL
Recombinomics, Inc., Pittsburgh, Pennsylvania, USA

Several constellations of influenza genes have emerged in swine in recent years.
Classical swine H1N1 was present in North America from the first influenza isolates in 1931 through 1998. In 1998 triple reassortants were identified which had human H3, N2, and PB1 genes, swine NP, MP, and NS genes, and avian PA and PB2 genes Subsequent isolates replaced the human H3 with swine H1or the N2 with N1. Recent H1N1 and H1N2 swine isolates in Canada contained a constellation of five or seven swine genes and one or three human genes. All had a human PB1 gene.
Analysis of the sequences from the 2003/2004 swine isolates identified recombination with earlier swine sequences. Most striking was the recombination in swine PB2 and PA genes. Most had regions of identity with two 1977 H1N1 swine isolates, A/swine/Tennessee/26/77(H1N1) and A/swine/Tennessee/24/77(H1N1). One had regions of identity in PA with a 1931 H1N1 swine isolate, A/swine/Iowa/1976/31(H1N1). Additional regions of identity in PB2 in A/swine/North Carolina/35922/98(H3N2) and A/swine/Korea/CY02/02(H1N2) were found.
The regions of identity were extensive, covering as much as 80% of the gene segments. Nested regions of recombination were also seen.. The regions of identity, which were maintained with absolute fidelity for over 25 years, raise questions about the role of random mutations in the seasonal variation in swine and human influenza.
Although the PB1 sequence was human, the 2003/2004 swine sequences were most closely related to human sequences from the mid 90’s. This slower rate of evolutionary change in swine provides an alternate gene pool for human influenza evolution.
The conservation of these changes and the tracing of the changes to earlier isolates provides a novel approach for the analysis of the 1918 pandemic strain, which shares polymorphisms with human and swine isolates of the early 1930’s. The relationship between the 1918 pandemic strain and human and swine isolates is maintained across all eight gene segments.
The role of recombination in the evolution of human and swine influenza will be discussed. 

1918 Pandemic Evolution Via Recombination Between Human and Swine H1N1
Niman HL
Recombinomics, Inc., Pittsburgh, Pennsylvania, USA

Although complete sequences of all eight gene segments of the 1918 pandemic strain of H1N1 have been published, the origin and evolution of the strain remain unclear. 10 polymorphisms in the four internal genes distinguished the 1918 strain from avian isolates, but pandemic polymorphisms match both human and swine H1N1 sequences. Similarly, phylogenetic analysis indicates the pandemic strain is most closely related to human and swine H1N1 from the early 1930’s. Recent H1N1 and H1N2 sequences from swine in Canada demonstrate absolute fidelity with extended regions of PA and PB2 from 1977 swine isolates from 1977, as well as regions of PA identity with a swine isolate from 1931. These identities strongly suggested that the swine evolution was via recombination and support use of polymorphism tracing to determine parental strains.
Although human and swine sequences from isolates collected prior to the 1918 pandemic are not available, full sequences have been generated on human and swine H1N1 isolates from the 1930’s.  Representative human, A/WSN/33, or swine, A/swine/Iowa/15/30 or A/swine/Iowa/1976/31, contain over 90% of the polymorphisms in six of the eight gene segments of A/Brevig Mission/1/1918 or A/South Carolina/1/18. The matches in the remaining two genes, NA and NP, are 84% and 86%, respectively. In six of the eight genes, the percentage of human polymorphisms are higher than the swine polymorphisms, PB2(51/40), PB1(55/40), PA(52/39), HA(53/49), MP(52/40), NS(55/41) while the swine polymorphisms are greater in the remaining two gene segments, NP(42/47) and NA(34/51). Moreover, although the matching regions alternate, the regions are defined by clustered polymorphisms. These acquisition patterns provide additional support for generation of the pandemic strain via recombination between human and swine H1N1.
The differences between the 1918 pandemic strain containing polymorphisms that are found in two mammalian sources, swine and human, and a pandemic strain such as avian H5N1 which has acquired the ability to efficiently transmit in a mammalian host, will be discussed.

Detection of Oseltamivir Resistance Mutation N294S in Humans with Influenza A H5N1

Saad MD, Boynton BR, Earhart KC, Mansour MM, Niman HL, Elsayed NM, Nayel AI, Abdelghani AS, Essmat HM, Labib EM, Ayoub EA, Monteville MR

U.S. Naval Medical Research Unit 3 (NAMRU-3), Cairo, EGY; Recombinomics, Inc., Pittsburgh, Pennsylvania, USA; Ministry of Health, Arabic Republic of Egypt, Cairo, EGY.

Introduction: From Mar 2006 to Feb 2007 Egypt had 22 human cases of Influenza A H5N1 with 13 deaths. All patients were treated with oseltamivir using WHO guidelines.
Subjects and Methods: In Dec 2006, NAMRU-3 received 3 human specimens from the Ministry of Health  that were reportedly positive for influenza H5N1. The cases included 2 females (age 35 and 16) and one male (age 26) from an extended family living in the Nile Delta. All patients participated in the slaughter of household ducks on Dec 13 and developed influenza like illness between Dec 15 - 19.  On Dec 21, a throat swab was collected from each patient before beginning oseltamivir treatment.  The patients were transferred to Abbassia Chest hospital in Cairo on Dec 23 for respiratory deterioration, and additional throat swabs were collected. All 3 patients died from pneumonia complicated by adult respiratory distress syndrome and multi-organ failure.  The specimens were tested at NAMRU-3 using RT-PCR. Those testing positive for H5N1 were further analyzed by genomic sequencing. 
Results: H5N1 was confirmed in 2 patients (16F and 26M ), while the third remains under study. The NA gene sequence from specimens collected before and after antiviral therapy revealed a mutation (N294S) known to confer resistance to oseltamivir.  The sequences were confirmed by the Influenza Division at the Center for Disease Control.  Phenotypic analysis, using the chemiluminescent NA inhibition assay, revealed a 12-15 fold reduction in oseltamivir susceptibility, but sensitivity to zanaimivir.
Conclusions: N294S is reported frequently in patients treated with oseltamivir for seasonal influenza., but has been reported in only one patient with H5N1. This patient was treated with a prophylactic dose of oseltamivir followed by a full course of therapy. N294S has not previously been described in humans before oseltamivir treatment.  Although N294S has been detected in H5N1 infected birds in Asia, this is the first demonstration of N294S in Clade 2.2. This is also the first report of a H5N1 virus containing the N294S mutation being transmitted directly from an avian species to a human as judged by the mutation being present before the administration of antiviral therapy. 

Spring Ahead: Microbiology Issues in 2007
San Ramon, California
24th Annual NCASM Spring Meeting

March 31 - Emerging Influenza Issues

1:50 - 2:40 PM

Qinghai H5N1 Evolution Via Recombination

Henry L. Niman, Ph.D., Recombinomics, Inc

Dr. Niman will introduce us to the 3 R's of influenza viral evolution - recombination, reassortment and random mutation.  He will discuss the role of viral recombination in the development of antiviral resistance and outline methodologies being used to select influenza vaccine targets.

HA Polymorphisms Egypt (slide 53)
NA Polymorphisms Egypt (slide 54)
Egypt Collaborators (slide 58)

3:10 - 4:00 PM

Bruce Boynton, M.D. Naval Medical Research Unit #3

Avian Influenza in Egypt: A View from the Trenches

Dr. Boynton will provide an "on the ground" look at the introduction and movement of H5N1 in Egypt.  He will shed light on the more personal aspects of how this epidemic has impacted medical practice, economics, society & politics.


Targeted Immunotherapeutics & Vaccine Summit -
     Novel Vaccines: Bridging Research Development, and Production
Cambridge, Massachusets

August 21 - Overcoming Challenges
9:35 - 10:05 AM

Pandemic and Seasonal Influenza Evolution Via Recombination - Selection of Vaccine Targets

Henry L. Niman, Ph.D., President & Founder, Recombinomics, Inc.

Pandemic H5N1 and seasonal H3N2 influenza rapidly evolve via recombination.Identification of parental strains allows for prediction of sequences of emerging virus prior to emergence. Data mining of sequence databases also allows for prediction of time and location of significant genetic changes, including altered affinity in the receptor binding domain. Recombination rules can be used to identify novel vaccine targets and increase lead time over emerging viruses.

H5N1 Hong Kong PB2 Recombinant (slide 11)
H5N1 Hong Kong PB1 Recombinant (slide 12)
H5N1 Hong Kong PA Recombinants (slide 13)
H5N1 Hong Kong NP Recombinant (slide 14)
H9N2 Korea NA Recombinants (slide 15)
H3N2 Korea HA Recombinants
(slide 17)
Canadian Swine PB2 Recombination (slide 23)
Canadian Swine PA Recombination (slide 24)
Canadian Swine PB1 Haplotypes (slide 26)

August 21 - Panel Discussion -  Funding for Vaccines

4:30 - 5:30 PM

Funding for Vaccines continues to be a contentious subject that often presents considerable frustrations for researchers and developers. Though the public clamors for protection from the threat of pandemics and infectious diseases, there is often a lack of understanding about what is needed to provide adequate facilities and lead time in order to generate ample product. Mutating viruses tend to stay at least a few steps ahead of vaccine production.And funding is scarce for providing vaccines where they are needed most—throughout the developing world. Join us in this discussion as experts from various vaccine pathways address this important issue.

David S. Cho, Ph.D., M.P.H., Influenza Product Development Program Officer, Influenza, SARS, & Related Viral Respiratory Diseases Section, NIAID / NIH

Georges Thiry, Ph.D., Project Management, International AIDS Vaccine Initiative (IAVI)

Daniel Zimmerman, Ph.D., Senior Vice President of Research, Cellular Immunology, CEL-SCI Corporation

Douglas Holtzman, Ph.D., M.P.H., Senior Program Officer, Infectious Diseases Global Health Program, Bill & Melinda Gates Foundation

Henry L. Niman, Ph.D., President & Founder, Recombinomics, Inc.



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