Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-02-25
2003-08-12
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069300, C435S183000, C424S186100
Reexamination Certificate
active
06605457
ABSTRACT:
The present invention relates to a recombinant influenza neuraminidase, an expression vector with which the recombinant neuraminidase can be expressed in host cells, methods for producing and purifying recombinant neuraminidase, vaccines against influenza and the use of recombinant neuraminidase according to the invention.
Influenza A and B virus epidemics cause considerable discomfort to those affected and have a great influence on social and economic lie. They cause a significant mortality rate in older people and in patients with chronic illnesses. Since their introduction during the 1940s, inactivated vaccines based on virus material cultured in chicken eggs have been found to be clearly effective against influenza infection and have resulted in a significant fall in the mortality rate of high-risk populations.
The influenza viruses are unique among the viruses of the bronchial tubes because they undergo a significant antigenic variation (so-called “drift”) in their two surface antigens, that is, the hemagglutinin (HA) and the neuramindase (NA).
In addition, influenza A in particular can escape the prevalent immunity due to the phenomenon of “shift”. Appearing herein in the human virus is an NA gene which comes from the animal reservoir of influenza genes. In 1957 the NA1-type virus prevalent up to that time was thus replaced by a new NA2-type virus. Since 1977 the NA1-type viruses have also returned to the human population. The present vaccines must therefore preferably be aimed against both NA1 and NA2-type viruses.
NA catalyses the removal of terminal sialic acid residues of glycosyl groups whereby potential receptors for HA are destroyed (Gottschalk, 1957; Burnet and Stone, 1974). It is assumed that NA is essential in preventing virus aggregation and in an efficient spreading from cell to cell (Colman and Ward, 1985).
Each NA molecule (M
r
=240,000) has a toadstool-like structure which consists of four identical polypeptide chains built up of two dimers which are linked to disulphide bridges and in turn held together by non-covalent bonds (Bucher and Kilbourne, 1972; Laver and Valentine, 1969; Varghese et al., 1983; Ward et al., 1983). Otherwise than HA, NA is anchored in the lipid membrane by a non-spliced, NA-terminal, lipophilic sequence (Fields et al., 1983; Block et al., 1982), the so-called membrane anchor. The greatest part of the total structure protrudes above the membrane and forms there a distal, box-shaped “head” area localised on top of an elongate “stalk” region (Wrigley et al., 1973′). Inside the head each monomer has its own catalytic site and contains at least four NA-linked glycosyl groups (Colman et al., 1983; Ward et al., 1982). The presence of O-glycosylation has not yet been demonstrated up to the present time.
On account of their external localization the HA and NA antigens represent the most important viral target structures for the host immune system. Of antibodies which bind specifically to HA it is thought that they neutralise the viral infectivity, probably by blocking the early steps of infection (Hirst, 1942; Kida et al., 1983). NA-specific antibodies normally do not prevent the initial infection of a target cell (Jahiel and Kilbourne, 1966; Kilbourne et al., 1968; Johanssen et al., 1988) but precisely the spread of the virus. In addition, due to competition mechanisms, the immunologic response to NA appears to be partly suppressed in favour of the more frequently occurring HA antigen (Johanssen et al., 1987, Kilbourne, 1976). As net result the effect of NA immunity is generally overshadowed by the neutralising HA antibodies. For this reason the attention of vaccine designers has been focussed for a long time almost exclusively on HA.
A number of experimental observations indicate however that NA is indeed capable of playing a significant part in the build-up of protective immunity to influenza (Schulman et al., 1968; Johansen and Kilbourne, 1990; Johansen et al., 1993). Fundamental studies into the immunogenic potential of NA necessitate the availability of very pure antigens in sufficient quantities and with the correct three-dimensional conformation. Up until now NA has been prepared by treating viral envelopes with detergents (Gallagher et al., 1984; Kilbourne et al., 1968) or by proteolytic cleavage of the protein head, often by means of pronase (Seto et al., 1966; Rott et al., 1974), followed by purifying of the NA. Although to some extent usable, these methods have considerable limitations in respect of yield and purity.
It is therefore the objects of the present invention to provide a recombinant influenza neuraminidase which has antigenic properties corresponding with the naturally occurring neuraminidase and is folded in the correct manner.
Such a recombinant neuraminidase in substantially isolated form can be obtained according to the invention by:
a) culturing in a suitable culture medium host cells which are transformed with a neuraminidase expression vector or infected with a virus which is transformed with a neuraminidase expression vector, wherein the expression vector comprises at least a part of the coding region of a neuraminidase gene of an influenza virus minus the region which codes for the membrane anchor, or a modified version thereof, preceded in phase by a signal sequence; and
b) isolating the expression product neuraminidase from the culture medium.
The recombinant neuraminidase according to the invention which is secreted in the culture medium can for instance be used for fundamental studies, wherein the separate vaccination with NA is performed in order to determine the role of NA in a vaccine. In practice recombinant NA will however probably still be used in combination with HA in order to increase the degree of protection (percentage of the inoculated population that is effectively protected against an infection) and the protection persistence (protection against later epidemic strains).
More particularly the invention provides a recombinant influenza NA2 neuraminidase which can be obtained by culturing host cells in a suitable culture medium and isolating the expression product neuraminidase from the culture medium. This entails in practice for instance that a recombinant expression module from pAc2IVNAs is crossed in a wild-type baculovirus or a derivative thereof. Host cells are then infected with this recombinant baculovirus.
The host cells used for the production of the recombinant influenza neuraminidase preferably originate from lower eukaryotic organisms such as insects, preferably the insect cell line sf9, but can also be yeast cells such as Saccharomyces or Pichia.
The present invention further relates to two vectors for expressing a secretable influenza neuraminidase comprising a replication origin, at least a part of the coding region of the influenza neuraminidase gene minus the region which codes for the membrane anchor, or modified versions thereof, a signal sequence located at 5′ from the coded region and coupled in phase thereto, a promoter located at 5′ from the signal sequence and a transcription terminator located at 3′ from the coding region. More particularly the invention provides a vector for use in expression a secretable influenza NA2 neuramindase comprising a replication origin, the coding region of the influenza NA2 neuraminidase gene of the virus strain A/Victoria/3/75 minus the region which codes for the membrane anchor, or modified versions thereof.
For expression in insect cells such a vector is placed in a cell together with a wild-type baculovirus or derivative thereof. A recombinant baculovirus results due to the occurrence of a double homologous recombination, wherein the expression module from the vector is introduced into the viral genome. After plaque purification a stock of recombinant baculoviruses is obtained which can subsequently be used to infect for instance Sf9-cells.
The signal sequence preferably originates from the hemagglutinin gene of the influenza NA2 virus A/Victoria/3/75 (H3N2). The invention preferably comprises the vector
Deroo Tom Maria
Fiers Walter Charles
Min Jou Willy Alfons
Prouty Rebecca E.
Rao Manjunath N.
Vlaams Interuniversitair Instituut Voor Biotechnologie
Webb Ziesenheim & Logsdon Orkin & Hanson, P.C.
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