Research

1918 Influenza Pandemic Caused by Highly Conserved Viruses with Two Receptor-Binding Variants

Ann H. Reid,* Thomas A. Janczewski,* Raina M. Lourens,* Alex J. Elliot,† Rod S. Daniels,† Colin L. Berry,‡ John S. Oxford,‡§ and Jeffery K. Taubenberger*
*Armed Forces Institute of Pathology, Rockville, Maryland, USA; †National Institute for Medical Research, London, United Kingdom; ‡Queen Mary’s School of Medicine and Dentistry, London, United Kingdom; and §Retroscreen Virology, Ltd., London, United Kingdom

Suggested citation for this article: Reid AH, Janczewski TA, Raina M. Lourens RM, Elliot AJ, Rod S, et al. 1918 Influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis [serial online] 2003 Oct [date cited]. Available from: URL: http://www.cdc.gov/ncidod/EID/vol9no10/02-0789.htm

The Spanish influenza pandemic swept the globe in the autumn and winter of 1918-19, and resulted in the deaths of approximately 40 million people. Clinically, epidemiologically, and pathologically, the disease was remarkably uniform, which suggests that similar viruses were causing disease around the world. To assess the homogeneity of the 1918 pandemic influenza virus, partial hemagglutinin gene sequences have been determined for five cases, including two newly identified samples from London, United Kingdom. The strains show 98.9% to 99.8% nucleotide sequence identity. One of the few differences between the strains maps to the receptor-binding site of hemagglutinin, suggesting that two receptor-binding configurations were co-circulating during the pandemic. The results suggest that in the early stages of an influenza A pandemic, mutations that occur during replication do not become fixed so that a uniform “consensus” strain circulates for some time.

The 1918–19 influenza pandemic began, in some parts of the world, with mild outbreaks in the spring of 1918. In the fall of that year, a lethal wave swept the globe. Outbreaks occurred in early September in North America, Europe, and Africa and spread rapidly, so that the disease had peaked and declined worldwide by the end of December (1–4). Many areas had an additional wave of the disease in the early months of 1919. In most communities, the fall wave of the pandemic lasted approximately l month, with 25% to 30% of the population experiencing symptomatic disease. Clinically, epidemiologically, and pathologically, the disease was remarkably uniform, suggesting that similar viruses were causing disease worldwide (5). To assess the homogeneity of the 1918 pandemic influenza virus, partial hemagglutinin (HA) gene sequences were determined for strains from five cases, including two newly identified samples from London, United Kingdom. The strains show 98.9% to 99.8% nucleotide sequence identity. One of the few differences between the strains maps to the receptor-binding site of HA, which suggests that two receptor-binding configurations were co-circulating during the pandemic.

Influenza A virus is capable of rapid genetic change in mammals (6–8). Its polymerase complex lacks proofreading capability, such that one in five virus particles produced is likely to contain a change at one of its approximately 13,500 nt (9). If such a change provides the virus with a competitive advantage, that strain quickly replaces its predecessor. In humans, the need to escape preexisting immunity exerts positive selection pressure on changes in amino acids comprising the antigenic sites of the surface glycoproteins, HA and neuraminidase (NA) (6,10). The process of progressive change in the antigenic properties of the virus is called antigenic drift and results in the emergence of an antigenically distinct variant strain every 2–3 years. Between drift epidemics, the influenza virus appears to be antigenically uniform (11), but the degree of genetic uniformity has not been studied extensively.

In pandemic influenza, one or both of the virus’s surface proteins are replaced with proteins to which the human population has no preexisting immunity (6,12). The virus then spreads explosively, producing symptomatic infection in up to one third of most populations. During the rapid initial spread of a pandemic strain, little antigenic pressure on the virus exists. One might expect the genetic structure under these circumstances to be relatively constant. However, the degree of genetic identity among viral isolates during a pandemic is not known. Very few full-length HA sequences of viruses from the peaks of the 1957 and 1968 pandemics are available, and all of these viruses had been grown at least once in eggs before sequencing—a process that can select for an unpredictable number of sequence changes (13,14). Therefore, this study represents an initial attempt to measure the degree of genetic homogeneity of a pandemic virus. Since the sequences have been obtained directly from clinical material, they contain no sequence changes attributable to culture.

Materials and Methods

Patients and Samples

The genetic sequences encoding the HA1 domains of three 1918 influenza strains have been determined (15,16). Two of the strains came from U.S. soldiers who died on September 26, 1918: one in Camp Upton, New York, and one in Fort Jackson, South Carolina. The third came from an Inuit woman who died in mid-November 1918 in a remote village on the Seward Peninsula of Alaska.

To obtain further samples for analysis, we examined autopsy material of 14 patients who died in the fall and winter of 1918 to 1919. The material consisted of formalin-fixed, paraffin-embedded tissues, stained slides, and clinical records from the files of the Morbid Anatomy Department of the Royal London Hospital. The cases were preselected by histologic criteria for further analysis, and samples were taken from patients who died from acute influenza after clinical courses of <1 week (16–18). Of these 14 lung samples, 4 were positive for influenza RNA on subsequent molecular genetic analysis, but only 2 had sufficient material for HA1 sequencing. The first patient was a 50-year-old woman admitted to the hospital on November 12, 1918, with influenza and pneumonia. She died on November 13. The postmortem diagnosis was bronchopneumonia. The second patient was a 25-year-old man admitted to the hospital on February 13, 1919. He died on February 15 of influenza. The postmortem diagnosis was lobar pneumonia with toxemia.

Methods

Sample preparation, reverse transcription, polymerase chain reaction (PCR), and sequencing were performed as described previously (15). (Primers used are available upon request.) PCR was performed from at least two separate reverse transcription reactions, and products from at least two PCR reactions were sequenced in each case to ensure accuracy and exclude amplification artifacts. Sequences used to evaluate the complexity of pandemic and epidemic influenza strains were obtained from the Influenza Sequence Database (available from: URL: http://www.flu.lanl.gov/).

Results

	Figure
	Click to view enlarged image Figure. Partial HA1 domain cDNA sequences from five 1918-19 cases...

The 563-bp fragments sequenced for this study, encoding the antigenic and receptor-binding sites of the HA1 domain (19–21), represent the most variable portion of the influenza genome (Figure). The London cases were designated A/London/1/1918 (H1N1) and A/London/1/1919 (H1N1). These two sequences, when compared to the three previously sequenced North American strains (15), differ from each other by 1 nt to 3 nt, showing a sequence identity of 98.9% to 99.8%.

A/London/1/1918 shows 2 nt differences, compared to A/Brevig Mission/1/1918, one of which would change the amino acid at codon 188 from G to S (amino acid numbering is aligned to the H3 influenza HA). This residue is near several of the residues that have been shown experimentally to affect receptor-binding specificity of H1 HAs (21–23) and next to one of the mapped Sb antigenic site residues (19,20). A/London/1/1919 shows 3 nt differences from A/Brevig Mission/1/1918, 2 of which are nonsynonymous, resulting in changes of V223I and D225G. The V223I change is near Ca antigenic site residues, and the D225G change is at a residue that functions both in receptor-binding and as a Ca antigenic site residue. Amino acid 225 also varies among North American strains; A/New York/1/1918, like A/London/1/1919, has a glycine at position 225, as do most avian influenza strains. A/South Carolina/1/1918 and A/Brevig Mission/1/1918, like A/London/1/1918 and most subsequent human H1 strains, have aspartic acid at this position (Figure) (15). The relative genetic homogeneity of the 1918–19 isolates encouraged us to analyze sequences from the 1957 and 1968 pandemics.

GenBank contains complete HA1 domain–encoding sequences for eight 1957 H2N2 strains. As noted in previous studies of receptor-binding specificity (22,24), the 1957 strains have undergone varying passage histories, but all have been passed at least once. Three of the strains have been sequenced more than once and differ by as many as 8 nt within the same strain. Between sequences, the number of nucleotide differences ranges from only 1 nt difference between A/Chile/6/1957 and A/Davis/1/1957 to 12 differences between one of the A/Japan/305/1957 sequences and one of the A/Singapore/1/1957 sequences. Overall, the sequences show 98.9% to 99.9% identity at the nucleotide level, and 98.5% to 100% identity at the amino acid level.

More limited sequence data are available for the 1968 H3N2 pandemic strains. The complete HA1 domain sequence is available for only three strains, two of which have been sequenced twice each. The two A/NT/60/68/29C sequences differ by 4 nt. The most divergent sequences differ by 24 nt (A/NT/60/68/29C vs. A/Hong Kong/1/68), thus showing 97.6% to 100% identity between sequences at the nucleotide level, and 96.0% to 100% identity at the amino acid level.

Studies from epidemic years have yielded similar results. A 2001 study (25) examined variation in the HA gene of human H3N2 viruses in Spain from 1996 to 2000. During this time, strains antigenically similar to A/Wuhan/359/1995 were replaced by strains similar to A/Sydney/5/1997 and then by strains similar to A/Panama/2007/1999. Within the groups of viruses belonging to each antigenic group, sequence variation was minimal. For example, among the viruses that reacted antigenically with Sydney, but not Panama and Wuhan, 2–10 nt differences occurred over the 591 nt sequenced (98.3% to 99.7% identity).

An unpublished study provides sequences of the HA1 domain of the H3-subtype HA of 16 A/Sydney/05/1997-like (H3N2) influenza virus isolates circulating in Canada during the 1997/98 influenza epidemic season (GenBank no. AF087700–AF087702, AF087707, AF087708, AF096306–AF096316) (26). Two of the isolates had identical sequences, while the others varied by 1 nt to 14 nt over 981 nt (98.6% to 100% identity).

Discussion

The three North American 1918 influenza strains sequenced previously were isolated from patients separated by nearly 2 months in time and almost 4,000 miles in distance (27). Two nucleotide differences were found among these three strains, one of which resulted in an amino acid substitution in the receptor-binding site (15). All three cases likely derived from the initial introduction of the fall wave into the United States, believed to have occurred in Boston in early September 1918. The virus then spread rapidly from Camp Devens, Massachusetts, the first U.S. army base to experience the epidemic, which then reached army bases throughout the eastern United States within 2 weeks (2). Influenza probably reached Brevig Mission, Alaska, via Seattle, Washington. The pandemic reached Camp Lewis, Washington, in mid-September, following the arrival of a troop ship from Philadelphia, Pennsylvania (1,2), and spread to Seattle by late September. After careful screening to exclude sick passengers, a ship left Seattle for Nome, Alaska, in mid-October, but days after its arrival local residents began falling ill (1). An account of the pandemic as it occurred in Brevig Mission reports that visitors from Nome brought the disease to the village in November (28). This chain of events suggests that the Alaskan outbreak was not the result of a separate introduction of the 1918 influenza from Asia to the West Coast of the United States.

The spring wave of the 1918 epidemic was widespread in France and Spain during April and May but did not reach England until June. The fall wave also arrived somewhat later in England than in continental Europe and the United States; peak mortality in London occurred during the first 2 weeks of November (2). A second peak occurred in the third week of February 1919. One strain from each of these peaks was sequenced for this study.

Our results show that strains separated by over 7,500 miles (Brevig Mission, Alaska, to London, United Kingdom) and several months (September 26, 1918, to February 15, 1919) share a sequence identity of 99%. This level of genetic homogeneity is slightly higher than that seen for the available 1957 and 1968 pandemic strains, but the 1957 and 1968 strains were not sequenced directly from clinical material. Sequences from different passages of the same strain were sometimes as different from each other as they were from other strains (29), suggesting that sequence heterogeneity observed was the result of culture adaptation, making it impossible to determine how homogeneous the pandemic viruses actually were. Even so, the 1957 and 1968 pandemic strains show >97% identity between strains. Similar levels of genetic homogeneity were seen in strains from case-patients isolated from a drift epidemic in 1997. Thus, influenza viruses circulating during a single outbreak, whether epidemic or pandemic, show levels of sequence identity consistent with the uniformity of the 1918 cases.

Despite the uniformity of the 1918 strains, one of the variable sites is an amino acid known to be important in receptor binding (21). At a subset of amino acids critical for receptor binding, avian strains differ from swine H1s at only one amino acid, E190D (15). At these amino acids, two of the cases (A/New York/1/1918 and A/London/1/1919) are identical to that of A/sw/Iowa/1976/31 (a classical swine strain). The other 1918 cases have an additional change from the avian consensus at amino acid 225. Since swine viruses with the same receptor site as A/sw/Iowa/1976/31 bind both SAa2,3Gal and SAa2,6Gal (14), A/New York/1/1918 and A/London/1/1919 probably also had the capacity to bind both receptors. Because two of five 1918-19 analyzed fall wave strains from case-patients have the swine-like receptor-binding pattern, the E190D change alone is apparently sufficient to allow viral replication in the human respiratory tract. However, the existence of three strains with the additional G225D change shows that both receptor-binding variants were co-circulating throughout the pandemic. The current evidence does not suggest progression from one receptor-binding pattern to the other during the pandemic, since the two variants are present, on both continents, both early and late in the pandemic. Since residue 225 has also been identified as part of the Ca antigenic site (19), the co-circulating strains possibly differed in antigenic reactivity as well as receptor-binding characteristics.

This study is the first to examine the genetic homogeneity of a pandemic influenza virus directly from clinical material. The results suggest that in the early stages of a pandemic, mutations that occur during replication do not become fixed so that a uniform consensus strain circulates for some time. Studies of influenza strains circulating after 1919 should provide insight into how pandemic viruses evolve after the initial waves through immunologically vulnerable populations. In terms of pandemic planning, our results indicate that a specific antiviral drug or vaccine would have a uniform effect during the important and often lethal first wave of a pandemic (30,31).

This work was supported by a grant from the National Institute of Allergy and Infectious Diseases (R01 AI50619-01) to J.K.T. and by the intramural funds of the Armed Forces Institute of Pathology. J.S.O., C.L.B., and R.S.D. gratefully acknowledge financial support from the Wellcome Trust and the Ian Heap fund.

Ms. Reid is a research biologist in the Molecular Pathology Division at the Armed Forces Institute of Pathology. Her principal research interest is pandemic influenza.

http://www.cdc.gov/ncidod/EID/vol9no10/02-0789.htm

 

Patent Application Suggests Influenza Vaccines Likely Contaminated and Can Induce Inadvertent Infections

Abstract:

Influenza viruses for use in preparing human vaccines have traditionally been grown on embryonated hen eggs, although more modern techniques grow the virus in mammalian cell culture e.g. on Vero, MDCK or PER.C6 cell lines. The inventor has realised that the conditions used for influenza virus 5 culture can increase the risk that pathogens other than influenza virus may grow in the cell lines and have identified specific contamination risks. Suitable tests can thus be performed during manufacture in order to ensure safety and avoid iatrogenic infections.

Claims:

1. A process for preparing an influenza vaccine from influenza virus that has been grown in a culture of a mammalian cell line, comprising a step in which the vaccine and/or the culture is tested for the presence of an infectious agent that can grow in said cell line but that does not grow in embryonated hen eggs.

2. A process for preparing an influenza vaccine from influenza virus that has been grown in a culture of a mammalian cell line, comprising a step in which the vaccine and/or the culture is treated to remove and/or inactivate an infectious agent that can grow in the cell line but does not grow in embryonated hen eggs.

3. The process of claim 1 or claim 2, wherein the mammalian cell line is a MDCK cell line, a Vero cell line, or a PER.C6 cell line.

4. The process of any preceding claim, wherein the infectious agent is selected from the group consisting of: Pneumovirinae; Morbilliiviruses of the Paramyxoviridae family; Enteroviruses of the Picornaviridae family; mammalian Reoviridae; and Birnaviridae.

5. The process of claim 4, wherein the infectious agent is selected from the group consisting of: respiratory syncytial virus; measles virus; Coxsackie viruses; echoviruses; enteroviruses; orthoreoviruses; rotaviruses; and infectious bursal disease virus.

6. The process of any preceding claim, wherein the mammalian cell line is a Vero cell line, and wherein the infectious agent is selected from the group consisting of: Metapneumoviruses of the Paramyxoviridae family; Rubulaviruses of the Paramyxoviridae family; Togaviridae; Coronaviridae; Rhinoviruses of the Picornaviridae family; varicella zoster virus; Polyomaviridae; porcine circoviruses; porcine picornaviruses; Chlamydia bacteria; and Parvoviruses.

7. The process of claim 6, wherein the infectious agent is selected from the group consisting of: human metapneumovirus; mumps virus; Rubellavirus; SARS coronavirus; M-strains of Rhinovirus; SV-40 polyomavirus; BK polyomavirus; JC polyomavirus; swine vesicular disease virus; Teschen-Talfan virus; C. trachomatis; C. pneumoniae; C. psittaci; canine parvovirus; and porcine parvoviruses.

8. The process of any preceding claim, further comprising a step in which the vaccine and/or the culture is tested for the presence of a pathogen selected from the group consisting of: parainfluenza viruses; Herpesviridae; Adenoviridae; Mycoplasma; avian circoviruses; and avian Reoviridae.

9. A process for preparing an influenza vaccine from influenza virus that has been grown in a culture of a first mammalian cell line, comprising a step in which the vaccine and/or the culture is tested for the presence of an infectious agent that can grow in said first cell line but that does not grow in a second mammalian cell line.

10. A process for preparing an influenza vaccine from influenza virus that has been grown in a culture of a first mammalian cell line, comprising a step in which the vaccine and/or the culture is treated to remove and/or inactivate an infectious agent that can grow in said first cell line but does not grow in a second mammalian cell line.

11. The process of claim 9 or claim 10, wherein (a) the first mammalian cell line is selected from the group consisting of: a MDCK cell line, a Vero cell line, or a PER.C6 cell line; (b) the second mammalian cell line is selected from the group consisting of: a MDCK cell line, a Vero cell line, or a PER.C6 cell line; and (c) the first and second mammalian cell lines are different.

12. The process of any one of claims 9 to 11, wherein the first cell line is a Vero cell line, and wherein the infectious agent is selected from the group consisting of: Metapneumoviruses of the Paramyxoviridae family; Rubulaviruses of the Paramyxoviridae family; Togaviridae; Coronaviridae; Rhinoviruses of the Picornaviridae family; varicella zoster virus; Polyomaviridae; porcine circoviruses; porcine picornaviruses; Chlamydia bacteria; Parvoviruses; and Birnaviridae.

13. The process of claim 12, wherein the infectious agent is selected from the group consisting of: human metapneumovirus; mumps virus; Rubellavirus; SARS coronavirus; M-strains of Rhinovirus; SV-40 polyomavirus; BK polyomavirus; JC polyomavirus; swine vesicular disease virus; Teschen-Talfan virus; C. trachomatis; C. pneumoniae; C. psittaci; canine parvovirus; porcine parvoviruses; and infectious bursal disease virus.

14. The process of any one of claims 9 to 13, further comprising a step in which the vaccine and/or the culture is tested for the presence of a pathogen selected from the group consisting of: parainfluenza viruses; Herpesviridae; Adenoviridae; Mycoplasma; avian circoviruses; and avian Reoviridae.

15. The process of claim 2 or claim 10, comprising a further step wherein, after said removal and/or inactivation, the vaccine and/or culture is tested for the presence of said infectious agent.

16. A process for preparing an influenza vaccine from influenza virus that has been grown in a culture of a mammalian cell line or in hens eggs, comprising a step in which the vaccine and/or the culture is tested for the presence of a pathogen selected from the group consisting of: parainfluenza viruses; Herpesviridae; Adenoviridae; Mycoplasma; avian circoviruses; avian Reoviridae; and Birnaviridae.

17. The process of claim 16, wherein the pathogen is selected from the group consisting of: PIV-1; PIV-2; PIV-3; herpes simplex virus 1; herpes simplex virus 2; human adenovirus; simian adenovirus; orthoreoviruses; and infectious bursal disease virus.

18. The process of any preceding claim, wherein the culture is tested by immunochemical detection and/or nucleic acid detection.

19. The process of claim 18, wherein detection is by ELISA and/or PCR (including RT-PCR).

20. An influenza vaccine that has been grown in a culture of a mammalian cell line, wherein the vaccine has been confirmed as free from the presence of an infectious agent that can grow in said cell line but that does not grow in embryonated hen eggs.

21. An influenza vaccine that has been grown in a culture of a first mammalian cell line, wherein the vaccine has been confirmed as free from the presence of an infectious agent that can grow in said first cell line but that does not grow in a second mammalian cell line.

22. The vaccine of claim 18 or claim 19, wherein vaccine was grown in a culture of a MDCK cell line, of a Vero cell line, or of a PER.C6 cell line.

23. An influenza vaccine in which mammalian reovirus is undetectable by RT-PCR.

24. The vaccine of claim 23, which is free from ovalbumin and from chicken DNA.

25. An influenza vaccine obtained or obtainable by the process of any one of claims 1 to 17.

26. The vaccine of any one of claims 20 to 25, which is a live virus vaccine.

27. The vaccine of any one of claims 20 to 25, which is an inactivated virus vaccine.

28. The vaccine of claim 27, which is a whole virus vaccine, a split virus vaccine, or a viral subunit vaccine.

29. The vaccine of any one of claims 20 to 28, which is a trivalent influenza vaccine.

30. The vaccine of any one of claims 20 to 29, which includes a pandemic influenza virus strain.

31. The vaccine of claim 30, which includes a H5 or H7 influenza virus strain.

32. The vaccine of any one of claims 20 to 31, which is for administration to a patient by injection, by an intranasal route, by an oral route, by an intradermal route, by a transcutaneous route, or by a transdermal route.

33. The vaccine of any one of claims 20 to 32, which is for pediatric immunisation.

34. The vaccine of any one of claims 20 to 33, which includes 1-20 μg of influenza virus haemagglutinin per strain.

35. The vaccine of any one of claims 20 to 34, which includes an adjuvant.

Description:

[0001]All documents cited herein are incorporated by reference in their entirety.

TECHNICAL FIELD

[0002]This invention concerns the production and quality control of influenza virus vaccines.

BACKGROUND ART

[0003]Influenza viruses for use in preparing human vaccines have traditionally been grown on embryonated hen eggs, although more modern techniques grow the virus in mammalian cell culture e.g. on Vero cells, MDCK cells or PER.C6 cells. The change in virus growth substrate has provided an opportunity for regulatory re-assessment of influenza vaccine safety. For example, contamination with host cell DNA has been a regulatory concern for the cell-derived vaccines [1], but has not been of concern in the past for vaccines grown in eggs.

[0004]The safety issues surrounding egg-derived influenza vaccines are thus different from those surrounding vaccines grown in cell culture, with cell-derived vaccines being under closer scrutiny. It is an object of the invention to address these different safety issues, and in particular to provide methods for enhancing the safety of influenza vaccines grown on cell culture.

DISCLOSURE OF THE INVENTION

[0005]By definition, the use of mammalian cell substrates for influenza vaccine production involves culturing the cells under conditions that are well suited to viral growth and replication. The inventor has realised that these conditions increase the risk that pathogens other than influenza virus may grow in the cell culture, thereby leading to potential contamination of the final vaccine product. Tests for contamination are generally not difficult to perform, but a manufacturer first has to know what tests to perform. The inventor has identified specific contamination risks, and their work means that suitable tests can be performed during manufacture in order to ensure the safety and quality of influenza vaccines grown on cell culture. Some of the contaminants may be harmless in a final vaccine product, but their presence can interfere with influenza virus propagation and downstream purification, and so their removal is primarily of concern for quality and reproducibility, other contaminants would be harmful in a final vaccine, and so their removal is primarily a safety concern.

[0006]The risk of contamination arising from viral co-culture is not without precedent (e.g. certain early poliovirus vaccine batches were contaminated by simian virus 40 (`SV40`), a polyomavirus), but there have not been any previous disclosures on identifying specific risks associated with cell culture for human influenza vaccine production. every new year brings a new risk of contamination, particularly as multiple passages are involved during preparation of seed viruses for manufacturers, thereby increasing the risk of parallel growth of adventitious pathogenic agents.

[0007]The inventor has identified infectious agents that can grow in the conditions used for growing influenza viruses in cell culture but that do not grow in hen eggs. These infectious agents represent a new contamination risk for influenza vaccines that was never of concern for traditional influenza vaccines. Thus the invention provides a process for preparing an influenza vaccine from influenza virus that has been grown in a culture of a mammalian cell line, comprising a step in which the vaccine and/or the culture is tested for the presence of an infectious agent that can grow in said cell line but that does not grow in embryonated hen eggs.

[0008]The inventor has also identified infectious agents that grow in some cell substrates used for influenza vaccine production but do not grow in others. These infectious agents are thus a contamination risk only for certain influenza vaccines. Thus the invention also provides a process for preparing an influenza vaccine from influenza virus that has been grown in a culture of a first mammalian cell line, comprising a step in which the vaccine and/or the culture is tested for the presence of an infectious agent that can grow in said first cell line but that does not grow in a second mammalian cell line.

[0009]The invention also provides a process for preparing an influenza vaccine from influenza virus that has been grown in a culture of a mammalian cell line, comprising a step in which the vaccine and/or the culture is treated to remove and/or inactivate an infectious agent that can grow in the cell line but does not grow in embryonated hen eggs. Similarly, the invention provides a process for preparing an influenza vaccine from influenza virus that has been grown in a culture of a first mammalian cell line, comprising a step in which the vaccine and/or the culture is treated to remove and/or inactivate an infectious agent that can grow in said first cell line but does not grow in a second mammalian cell line. After removal and/or inactivation, the vaccine/culture may be tested for the presence of the infectious agent e.g. to verify that it is has been removed/inactiv Influenza viruses grown on cell culture are at particular risk from contamination because the strains used for vaccine production are changed every year, and so new cultures have to be established every year. This annual change in production materials means that ated.

[0010]The invention also provides an influenza vaccine that has been obtained by a process of the invention. The invention also provides an influenza vaccine that is obtainable by a process of the invention.

[0011]The invention also provides an influenza vaccine that has been grown in a culture of a mammalian cell line, wherein the vaccine has been confirmed as free from the presence of an infectious agent that can grow in said cell line but that does not grow in embryonated hen eggs. Similarly, the invention provides an influenza vaccine that has been grown in a culture of a first mammalian cell line, wherein the vaccine has been confirmed as free from the presence of an infectious agent that can grow in said first cell line but that does not grow in a second mammalian cell line.

[0012]The invention also provides an influenza vaccine in which mammalian reovirus is undetectable by RT-PCR (e.g. using the L1-based RT-PCR technique disclosed in reference 16, using primers L1.rv5, L1.rv6, L1.rv7 and LV1.rv8 as taught). Not having been grown on eggs, the vaccine will be free from ovalbumin and from chicken DNA.

The Mammalian Cell Line

[0013]The influenza vaccines of the invention are grown in mammalian cell lines, rather than being grown in embryonated eggs. Typical mammalian cell lines used in production of biologicals include: MDCK; CHO; BHK; Vero; MRC-5; PER.C6; WI-38; etc. Preferred mammalian cell lines for growing influenza viruses include: MDCK cells [2-5], derived from Madin Darby canine kidney; Vero cells [6-8], derived from African green monkey (Cercopithecus aethiops) kidney; or PER.C6 cells [9], derived from human embryonic retinoblasts.

[0014]These cell lines are widely available e.g. from the American Type Cell Culture (ATCC) collection [10], or from the Coriell Cell Repositories [11]. For example, the ATCC supplies various different Vero cells under catalog numbers CCL-81, CCL-81.2, CRL-1586 and CRL-1587, and it supplies MDCK cells under catalog number CCL-34.

for more check patent app

Demographic information for Ukraine fatalities linked to D225G receptor binding in H1N1 mutation

November 23, 8:00 AMLA Health Technology ExaminerVictoria Nicks

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Fatal H1N1 Flu Virus Mutation in Ukraine and Norway
NAID

The receptor binding domain (RBD) for the Ukraine H1N1 mutation, D225G, has been linked to fatal cases of swine flu infection. Demographic and genetic analysis for the H1N1 mutation taking place in the Ukraine virus has been published by Mill Hill, the London laboratory that performed testing on samples from the Ukraine. D225G is the same RBD that was found in the virus that caused the 1918 Spanish flu outbreak.

H1N1 mutation fatalities

According to this analysis of the Mill Hill lab information, each of the fatal cases of flu from the Ukraine virus contained this marker. D225G is the name for the receptor binding domain of this particular virus. A virus attaches to charged molecules on host cells, but each type of virus attaches to a different type of molecule, in a different way. The H1N1 mutation found in the Ukraine attaches in the same place, in the same way, as the Spanish flu virus from 1918.

Detection of the H1N1 mutation

While positive results for the H1N1 virus with D225G are not widespread, this may be the result of the methods of testing. Tests for swine flu typically consist of a nasal swab, which is unlikely to pick up this mutated virus, due to its location. The H1N1 mutation that is causing fatalities in the Ukraine and Norway affects the respiratory system, and is found deep in the lungs.

Ukraine Health Ministry confirms high death rate

The Ukraine virus has caused 381 deaths since October 29, according to a statement from the Ukraine Health Ministry on November 21. The death rate increases daily, despite quarantine measures, and may affect the upcoming elections.

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