United States Patent:  6,045,790

Appl. No.:  939556

It is known that the human influenza virus strain A/Puerto Rico/8/34 grows particularly well in eggs and that reasserted viruses having it as a parent may also grow well in eggs. It has now been found that certain reassortants of A/PR/8/34 and equine influenza viruses, namely those which comprise the RNA7 segment from A/PR/8/34, will grow in cell culture, even though the parent equine influenza virus will not. Thus the specification describes and claims: reassorted viruses comprising genes for surface antigens of equine influenza viruses and the RNA7 segment from A/PR/8/34; methods of obtaining such viruses by reassortment; methods of propagating such reasserted viruses in cell culture, especially Vero cells; vaccines against equine influenza comprising such reassorted viruses; and methods of vaccinating equines against influenza.

BRIEF SUMMARY OF THE INVENTION

It has now surprisingly been found that if an equine influenza virus is genetically reasserted to produce a virus containing certain RNA derived from A/Puerto Rico/8/34, the genetically reasserted virus is able to grow in cell culture. There is no clear correlation between having a high yield in eggs and the ability to grow in cell culture, and there is no teaching in the prior art to suggest that a virus which grows well in eggs is likely to grow in cell culture.

Therefore a first aspect of the present invention provides a genetically reasserted virus derived from an equine influenza virus and the human influenza virus A/Puerto Rico/8/34, which reasserted virus comprises RNA derived from the equine influenza virus coding for at least one surface antigen and an RNA segment derived from A/Puerto Rico/8/34 which codes for matrix protein.

It is to be understood that it is not necessary for one of the parental viruses to be A/PR/8/34, only for the said RNA segment in the reasserted virus to be ultimately derived from A/PR/8/34. Thus A/PR/8/34 could be a "grandparent" or "great-grandparent" etc of the virus in accordance with the invention. It has been established that the said RNA segment which codes for matrix protein is that which is termed RNA 7.

A genetically reasserted virus in accordance with the invention has at least some of the antigenic characteristics of the parent equine influenza virus but is endowed with the ability to grow in certain cell cultures, especially cultures of Ineteroploid monkey cells, for example Vero cells.

Human influenza virus strain A/Puerto Rico/8/34 is deposited with the American Type Culture Collection under accession no. VR 95. Strains of equine influenza virus are available from The Animal Health Trust, Equine Research Station, P.O. Box 5, Balaton Lodge, Snailwell Road, Newmarket, Suffolk CB8 7DW and from WHO Collaborating Centre, Holly Hill, London.

Whereas it has been established by the applicants that the RNA segment which codes for matrix protein enables growth in cell culture to take place, it is not clear as to whether the matrix protein itself confers this ability: it may be some other gene product coded for by the same RNA segment.

The involvement of matrix protein in general in virus growth in cell culture was suggested by Bosch et al, (in Negative Strand Viruses and the Host Cell (1978), Academic Press, edited by B. W. J. Mahy and others, page 465). However, this paper relates to the growth of fowl plague virus (FPV) and no mention is made of equine influenza virus. In view of the well known difficulty in making predictions about the behaviour of one type of influenza virus based on observations of another, this distinction is by no means trivial. See, for example Scholtissek et al, Virology (1977) 81 74-80, which illustrates the proposition that apparently small changes between influenza viruses have profound effects. This is amplified by Sweet and Smith (Microb. Revs. (1980) 44(22), 303-30). Furthermore, there has been no suggestion that the segment in A/PR/8/34 coding for matrix protein is effective in conferring the ability to grow in cell culture.

In a preferred genetically reasserted virus in accordance with the present invention, the RNA which is derived from the equine influenza virus codes for a haemagglutinin antigen. A further preferred genetically reassorted virus contains, in addition, RNA derived from the equine influenza virus which codes for a neuraminidase surface protein. Suitable strains of equine influenza virus are A/Eq 1/Newmarket/77 and A/Eq 2/Brentwood/79. Other strains of equine influenza virus from which a genetically reasserted virus according to the present invention may be derived include A/Eq 1/Cornell/74, A/Eq 2/Columbus/2/78 and A/Eq 2/Cambridge/80.

A second aspect of the present invention provides a process for the preparation of a genetically reasserted virus in accordance with the first aspect of the invention, which process comprises (a) allowing (i) equine influenza virus (termed herein the equine virus) and (ii) the human influenza virus strain A/Puerto Rico/8/34 or a virus comprising the RNA 7 segment thereof (termed herein the PR8 virus) to grow under conditions in which genetic reassortment can take place, (b) selecting for genetically reasserted viruses having surface antigens from only the equine influenza virus but other RNA from the human influenza virus strain, and (c) selecting those reassortants from step (b) which will grow in cell culture.

Preferred growth conditions involve simultaneous infection of hen eggs with both viruses and incubation for 24 to 48 (preferably 36) hours at 37^oC. Genetically reasserted viruses can subsequently be selected by antibody pressure and susceptibility to an antiviral agent that acts specifically against equine influenza viruses, such as amantadine and rimantadine. Thus the viruses can be incubated and grown in the presence of antibody to both surface antigens of the PR8 virus parent. Such antibodies suppress the growth of the PR8 virus parent and genetically reasserted viruses bearing the PR8 virus surface antigens. At the same stage the viruses may be grown in the presence of for example amantadine, to which all known equine influenza viruses are sensitive, and to which A/Puerto Rico/8/8/34 is resistant. The only viruses which can grow under the combination of these conditions will be genetically reasserted viruses which do not bear surface antigens from A/PR/8/34 but which comprise the genetic segment (namely RNA 7) from A/PR/8/34 which confers amantadine resistance and which has replaced the genetic segment from equine influenza virus which confers amantadine sensitivity. See Lubeck et al J. Virol (1978) 28 (3) 710-16.

The antigenic identity of genetically reasserted viruses thus produced can then be determined, for example by haemagglutination-inhibition and neuraminidase-inhibition assays to establish the type of equine influenza antigens on the resultant viruses. Genetically reasserted viruses with desired surface antigens are then be tested for their ability to grow in, for example, Vero cells, and further adapted to the cells by serial passage, for example six times.

A genetically reassorted virus in accordance with the first aspect of the present invention can be grown in cell culture, for example Vero cells, preferably with the addition of trypsin. Growth would suitably be allowed to take place for 2 to 5 days at 37^oC. in the presence of from 0.1 to 20 .mu.g/ml, preferably 2.5 .mu.g/ml trypsin. A greater concentration of trypsin can be used if it has been partially inactivated by gamma-irradiation sterilisation. Subsequently, harvested virus can then be attenuated by serial passage or inactivated before incorporation into a vaccine. Inactivation may be achieved by formalin (at a concentration of from 1 part in 500 to 1 part in 3000, preferably 1 part in 1000 to 1 part in 2000 of a 40% w/w aqueous stock solution) or acetylethyleneimine or ethyleneimine or beta-propiolactone.

A third aspect of the present invention provides a vaccine comprising inactivated or attenuated genetically reasserted virus in accordance with the first aspect of the present invention or obtained by a method in accordance with the second aspect of the invention in association with a veterinarily acceptable carrier. For broader protection the vaccine preferably comprises two genetically reasserted viruses derived from different parent strains of equine influenza virus, i.e. one genetically reasserted virus derived from a serotype 1 equine influenza virus and a second one derived from a serotype 2 equine influenza virus. In addition a third element may be added to the vaccine such as tetanus toxoid.

Veterinarily acceptable carriers, in this instance, are liquid media suitable for use as vehicles to introduce the virus particles into the animals. Examples of such a carrier are saline solution and phosphate buffered saline.

Where the vaccine comprises inactivated genetically reasserted virus(es) an adjuvant may be added for stimulating the immune response and thereby enhancing the effect of the vaccine. Convenient adjuvants for use in the present invention include Freund's complete adjuvant and, more particularly, saponin, Corynebacterium parvum (Coparvax), aluminium phosphate and aluminium hydroxide or a mixture of these or other known adjuvants. (The word "Coparvax" is a Trade Mark). Alternatively the vaccine may be formulated into an oil-in-water emulsion using oils such as Marcol and/or Arlacel.

Conveniently the vaccines may be so formulated as to contain a final concentration of a single genetically reassorted virus in the range of from 1 to 50, preferably 2 to 15, most preferably 10, .mu.g/dose. After formulation the vaccine may be incorporated into a sterile container which is then sealed and stored at a low temperature, for example 4^oC., or may be freeze dried.

In order to induce immunity in horses to equine influenza one or more doses of the vaccine, formulated as described above, may be administered. It is recommended that each dose is 0.5 to 5 ml, preferably 1 to 3 ml, most preferably 2 ml of vaccine.

When the vaccine contains two genetically reasserted viruses derived from serotype 1 and serotype 2 equine influenza virus respectively, the concentration for each virus is in the range of from 1 to 50, preferably 2 to 15, most preferably 10, .mu.g/dose. Each dose is 0.5 to 5 ml, preferably 1 to 3 ml, most preferably 2 ml of vaccine.

A fourth aspect of the present invention provides a method of immunising horses against influenza, which method comprises administering to a horse a non-toxic, effective immunising amount of a vaccine in accordance with the third aspect of the present invention.

A vaccine of the present invention is desirably administered by subcutaneous or intramuscular injection. The treatment may consist of a single dose of vaccine or a plurality of doses over a period of time. An advantageous treatment schedule requires administration of two doses of vaccine with an interval of 3 to 7, preferably 4 to 6, weeks between doses. If longer protection is required, booster doses may be administered after longer intervals, for instance after 6 months or annually.

Claim 1 of 6 Claims

1. A genetically reassorted virus grown in cell culture in sufficient quantities to be useful for vaccine preparation without need of further multiplication, said virus being derived from an equine influenza virus and the human influenza virus A/Puerto Rico/ 8/34 which reassorted virus comprises RNA derived from the equine influenza virus coding for at least one neuraminidase or haemagglutinin surface antigen and the RNA segment derived from A/Puerto Rico/8/34 which codes for matrix protein.

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