Fusion proteins

Details Fusion proteins Fusion proteins

1997-04-30

2001-02-27

Guzo, David (Department: 1636)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C530S395000

Reexamination Certificate

active

06194546

ABSTRACT:

This invention relates to the construction of fusion proteins.
In previous attempts to express immunogenic epitopes of foot-and-mouth disease virus (FMDV) using vaccinia virus protein, fusions have been made to beta-galactosidase. High levels of fusion protein were synthesised. However, animals vaccinated with the recombinant vaccinia virus failed to produce neutralizing antibody (Newton et al, 1986). The reason for this poor response may be because beta-galactosidase is expressed in the cytoplasm. We have therefore investigated whether an antigen which is expressed on the surface of a cell would elicit a better response.
DNA sequences were constructed, each encoding a fusion protein comprising influenza virus haemagglutinin protein (HA) and, at antigenic site A of the HA, an epitope from part of the major FMDV antigenic site. The DNA sequences were incorporated into the vaccinia virus genome. Cells infected with the recombinant viruses not only expressed the fusion protein but did so such that the FMDV epitopes could be detected using anti-FMDV serum. Further, the infected cells reacted with polyclonal anti-HA antibody. It is therefore possible to change the sequence of influenza HA yet still retain all its properties of membrane insertion.
This approach for presenting antigenic epitopes to the immune system has general applicability. Accordingly, the present invention provides a DNA sequence encoding a fusion protein comprising influenza virus HA at a site of which normally occupied by a natural antigenic epitope thereof a different antigenic epitope is provided. In other words all or part of a natural antigenic epitope of HA may be replaced by a different antigenic epitope.
For expression of the fusion protein, the DNA sequence is incorporated in an expression vector. The DNA sequence is incorporated in a vector such that the vector, when provided in a eucaryotic host, is capable of expressing the fusion protein. A eucaryotic host is required for correct glycosylation of the HA. The vector may be a viral vector which incorporates the DNA sequence such that the fusion protein is expressed in cells infected with the vector. A vaccine may comprise such a viral vector and a physiologically acceptable carrier or diluent. In such an instance, the viral vector preferably is a recombinant vaccinia virus which incorporates the DNA sequence. Alternatively, a vaccine may comprise the fusion protein and a physiologically acceptable carrier or diluent.
Influenza virus HA is the most thoroughly studied integral membrane protein with detailed information available on its three-dimensional structure (Wilson et al, 1981) and its antigenicity (Wiley et al, 1981). The HA genes from several influenza subtypes have been expressed in a number of eukaryotic cells using various specific vectors (eg Smith et al, 1983; Gething and Sambrook, 1982). When expressed in recombinant vaccinia virus infected cells the HA is glycosylated normally and transported to the cell surface where it can be detected by serological methods.
Antigenic site A “loops out” from the surface of HA. This site can be determined by epitope mapping. Typically, it corresponds to HA amino acid residues 140 to 146. The HA may correspond to any influenza virus type or subtype. Consequently, it is at HA antigenic site A for example at which the antigenic epitope for the fusion protein is provided. All or part of site A may be replaced by the antigenic epitope. Typically, though, the epitope is inserted between HA residues 142 or 143 and residue 146 with the loss of the intervening HA residues. However, the antigenic epitope may be provided at any of the other natural antigenic epitopes of HA, sites B, C, D or E.
Any antigenic epitope may be provided at the natural antigenic epitope of HA. The epitope may be a corresponding epitope from a different type or subtype of influenza virus as that to which the HA corresponds. Alternatively or additionally, one or more of the other natural antigenic epitopes of HA of the same or of a different type or subtype of influenza virus may be provided. In this way a polyvalent influenza vaccine may be formed.
More usually, however, a heterologous antigenic epitope is provided. By “heterologous” is meant an epitope which is not an epitope of an influenza virus. The size of the heterologous epitope usually is larger than the portion of the HA site A which it replaces. The heterologous epitope may form part of a longer amino acid sequence. The insert may have from 4 to 26 amino acid residues. A heterologous antigenic epitope may be provided with an influenza virus epitope. Two or more heterologous antigenic epitopes may be provided. In this way, polyvalent vaccines can be presented.
The heterologous epitope may be that of a virus, bacterium or protozoan. As examples of viral epitopes, there may be mentioned those of FMDV, poliovirus human rhinovirus and hepatitis B virus. Protozoan whose epitopes may be provided include the malaria parasite
Plasmodium falciparum.
The major FMDV antigenic sites correspond to amino acid residues 141 to 160 and 200 to 213 of the VP1 capsid protein. Either or both of these amino acid residues may therefore be provided, for example at site A. Alternatively parts of these sequences may be provided e.g. residues 142 to 145, 146 to 151 or 142 to 151. Suitable DNA constructs incorporating parts of the major antigenic site of FMDV type O
1
Kaufbeuren, and their corresponding amino acid sequences as denoted by the one-letter code, are shown below together with the DNA and amino acid sequences for HA.
140                                     150
HA
AAA AGG GGA CCT GGT AGC GGT TTT TTC AGT AGA
 K   R   G   P   G   S   G   F   F   S   R

HA-FMDV
AAA AGG GGA CCG AAC CTG CGT TTT TTC AGT AGA
142-145
 K   R   G   P   N   L   R   F   F   S   R
           [142  FMDV   145]

HA-FMDV
AAA AGG GGA CCG GGT GAC CTG CAG GTT CTG TTT
146-151
 K   R   G   P   G   D   L   Q   V   L   F
               [146       FMDV      151]

TTC AGT AGA
 F   S   R

HA-FMDV
AAA AGG GGA CCG AAC CTG CGT GGT GAC CTG CAG
142-151
 K   R   G   P   N   L   R   G   D   L   Q
           [142             FMDV

GTT CGT GGT TTT TTC AGT
 V   L   G   F   F   S
     151]
The DNA sequence encoding the fusion protein can be prepared by providing a DNA sequence encoding HA, for example as a plasmid, and modifying this sequence by providing in the correct frame at the site of the DNA encoding a natural antigenic epitope of HA a DNA sequence corresponding to the desired antigenic epitope it is wished to incorporate in the fusion protein. A vector capable of expressing the fusion protein may be prepared by incorporating the DNA sequence encoding the fusion protein between translational start and stop signals in a vector suitable for use in

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