United States Patent:  6,060,308

Assignee:  Connaught Laboratories Limited (North York, CA)

Filed:  September 4, 1997

A vector comprising a first DNA sequence which is complementary to at least part of an alphavirus RNA genome and having the complement of complete alphavirus DNA genome replication regions, a second DNA sequence encoding a paramyxovirus protein, particularly a respiratory syncytial virus fusion (RSV F) protein or a RSV F protein fragment that generates antibodies that specifically react with RSV F protein, the first and second DNA sequences being under the transcriptional control of a promoter is described. Such vector may be used to produce an RNA transcript which may be used to immunize a host, including a human host, to protect the host against disease caused by paramyxovirus, particularly respiratory syncytial virus, by administration to the host.

SUMMARY OF THE INVENTION

The present invention provides novel immunogenic materials and immunization procedures based on such novel materials for immunizing against disease caused by paramyxoviridae, including respiratory syncytial virus and parainfluenza virus. In particular, the present invention is directed towards the provision of RNA vaccines against disease caused by infection with paramyxoviridae.

In accordance with one aspect of the present invention, there is provided a vector, comprising a first DNA sequence which is complementary to at least part of an alphavirus RNA genome and having the complement of complete alphavirus RNA genome replication regions; a second DNA sequence encoding a paramyxovirus protein or a protein fragment that generates antibodies that specifically react with the paramyxovirus protein; the second DNA sequence being inserted into a region of the first DNA sequence which is non-essential for replication; the first and second DNA sequences being under transcriptional control of a promoter.

The paramyxovirus protein may be selected from the group consisting of a parainfluenza virus (PIV) and a respiratory syncytial virus (RSV). The PIV protein may be PIV-1, PIV-2, PIV-3 or PIV-4, particularly the HN or F glycoproteins of PIV-3. The RSV protein particularly may be the F or G glycoprotein of RSV.

The second DNA sequence may encode a full length RSV F protein, or may encode a RSV F protein lacking the transmembrane anchor and cytoplasmic tail. The lack of the coding region for the transmembrane anchor and cytoplasmic tail results in a secreted form of the RSV F protein.

The second DNA sequence preferably encodes a RSV F protein and lacks a SpeI restriction site, and optionally, also lacking the transmembrane anchor and cytoplasmic tail encoding region. The absence of the SpeI restriction site may be carried out by mutating nucleotide 194 (T) of the RSV F gene to a C, which eliminates the SpeI without altering the amino acid sequence. The nucleotide sequence (SEQ ID No: 1) and encoded amino acid sequence (SEQ ID No: 2) of the mutated truncated RSV F gene is shown in FIG. 2.

The alphavirus preferably is a Semliki Forest virus and the first DNA sequence is the Semliki Forest viral sequence contained in plasmid pSFVl. The promoter used preferably is the SP6 promoter.

The vector preferably is one having the identifying characteristics of plasmid pMP37 (ATCC 97905) as shown in FIG. 1C and, more preferably, is the plasmid pMP37.

The mutant DNA sequence encoding an RSV F protein or a fragment thereof capable of inducing antibodies that specifically react with RSV F protein constitutes another aspect of the present invention, and preferably is that shown in FIG. 2 (SEQ ID No: 1).

The novel vector provided herein may be linearized and transcribed to an RNA transcript. In accordance with a further aspect of the invention, there is provided an RNA transcript of a vector as provided herein.

The RNA transcripts provided herein may be provided in the form of an immunogenic composition for in vivo administration to a host for the generation in the host of antibodies to paramyxovirus protein, such immunogenic compositions comprising, as the active component thereof, an RNA transcript as provided herein. Such immunogenic compositions, which are provided in accordance with another aspect of the invention, may be formulated with any suitable pharmaceutically-acceptable carrier for the in vivo administration and may produce a protective immune response.

In a yet further aspect of the present invention, there is provided a method of immunizing a host against disease caused by infection with paramyxovirus, which comprises administering to the host an effective amount of an RNA transcript as provided herein.

The present invention also includes a novel method of using a gene encoding an RSV F protein or an fragment of an RSV F protein capable of generating antibodies which specifically react with RSV F protein to protect a host against disease caused by infection with respiratory syncytial virus, which comprises isolating said gene; operatively linking said gene to a DNA sequence which is complementary to at least part of an alphavirus RNA genome and having the complement of complete alphavirus RNA genome replication regions in a region of said DNA sequence which is non-essential for replication to form a vector wherein said gene and DNA sequence are under transcriptional control of a promoter; linearizing the vector while maintaining said gene and DNA sequence under said transcriptional control of the promoter; forming an RNA transcript of said linearized vector; and introducing said RNA transcript to said host.

The vector employed preferably is plasmid pMP37 and the linearizing step is effected by cleavage at the SpeI site.

In addition, the present invention includes a method of producing a vaccine for protection of a host against disease caused by infection with respiratory syncytial virus (RSV), which comprises isolating a first DNA sequence encoding an RSV F protein from which the transmembrane anchor and cytoplasmic tail are absent and lacking any SpeI restriction site; operatively linking said first DNA sequence to a second DNA sequence which is complementary to at least part of an alphavirus RNA genome and having the complete alphavirus genome replication regions in a region of said second DNA sequence which is non-essential for replication to form a vector wherein said first and second DNA sequences are under transcriptional control of a promoter; linearizing the vector while maintaining said first and second DNA sequences under said transcriptional control of the promoter; forming a RNA transcript of said linearized vector; and formulating said RNA transcript as a vaccine for in vivo administration. The vector employed preferably is plasmid pMP37 and the linearizing step is effected by cleavage at the SpeI site.

Advantages of the present invention include the provision of RNA transcripts which are useful in generating an immune response by in vivo administration.

Claim 1 of 28 Claims

1. A vector, comprising:

a first DNA sequence which is complementary to at least part of an alphavirus RNA genome and having the complement of complete alphavirus RNA genome replication regions,

a second DNA sequence encoding a respiratory syncytial virus (RSV) F protein and lacking a SpeI restriction site or an RSV F protein fragment lacking a SpeI restriction site that provokes the generation of antibodies that specifically react with the RSV F protein,

said second DNA sequence being inserted into a region of said first DNA sequence which is non-essential for replication thereof,

said first and second DNA sequences being under transcriptional control of a promoter.

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