Title:  Manipulation of negative stranded RNA viruses by rearrangement of their genes and uses thereof

United States Patent:  6,777,220

Issued:  August 17, 2004

Inventors:  Wertz; Gail W. (Birmingham, AL); Ball; Andrew L. (Birmingham, AL)

Assignee:  Research Development Foundation (Carson City, NV)

Appl. No.:  198371

Filed:  July 18, 2002

Abstract

The present invention provides a method of increasing expression of a promoter distal gene in a virus of the order Mononegavirales, and a recombinant virus constructed by such method. Also provided is a method of attenuating a virus of the order Mononegavirales, and of constructing an attenuated virus useful for a vaccine.

SUMMARY OF THE INVENTION

The non-segmented negative-strand RNA viruses (order Mononegavirales) comprise several important human pathogens. The order of their genes, which is highly conserved, is the major determinant of the relative levels of gene expression, since genes that are close to the single promoter site on the viral genome are transcribed at higher levels than those that occupy more distal positions. An infectious cDNA clone of the prototypic vesicular stomatitis virus (VSV) was manipulated to rearrange the order of four of the five viral genes, while leaving all other aspects of the viral nucleotide sequence unaltered. In one set of cDNA clones, the middle three genes (which encode the phosphoprotein P, the matrix protein M, and the glycoprotein G) were rearranged into all six possible orders. In another set, the gene for the nucleocapsid protein N was moved away from its wild-type promoter-proximal position and placed second, third or fourth. In a final rearrangement, the G protein gene, which encodes the major surface antigen and the target for neutralizing antibodies, was put next to the promoter, in the position for maximum expression. Infectious viruses were recovered from each of these rearranged cDNAs and examined for their levels of gene expression and growth potential in cell culture, and their immunogenicity and virulence in mice. Rearrangement changed the expression levels of the encoded proteins and attenuated the viruses to different extents both in cultured cells and in mice. Increasing the expression of the G protein enhanced and accelerated the immune response in inoculated mice. Since the Mononegavirales do not undergo homologous recombination, gene rearrangement should be irreversible and thus provides a rational method for developing securely attenuated live vaccines against this type of virus.

In one embodiment of the, present invention, there is provided a method of increasing expression of a promoter distal gene in a virus of the order Mononegavirales, comprising the step of rearranging gene order of the virus by moving the promoter distal gene toward a wild-type 3' promoter proximal position site.

In another embodiment of the present invention, there is provided a recombinant virus of the order Mononegavirales having a rearranged genome, wherein the genome is rearranged by moving a promoter distal gene of the virus toward a wild type 3' promoter proximal position site. Such recombinant virus can be used for accelerating and enhancing a protective immune response.

In still another embodiment of the present invention, there is provided a method of attenuating a virus of the order Mononegavirales by rearranging gene order of the virus by moving a gene away from its wild type position, or by rearranging gene order of the virus by moving an essential limiting factor gene away from its wild type 3' promoter proximal position site.

In yet another embodiment of the present invention, there is provided a method of constructing an attenuated virus useful for a vaccine, comprising the steps of: rearranging the gene order of the virus by moving a gene away from its wild-type 3' promoter proximal position site, wherein the gene is an essential limiting factor for genome replication; and placing a gene coding for an immune response inducing antigen in the position closest to the 3' end of the gene order of the virus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention illustrates that introduction of specific changes into the genome of a negative strand RNA virus allowed translocation of the gene for the nucleocapsid (N) protein to successive positions on the genome and demonstrated directly that the position of a gene relative to the promoter determined the level of expression. Levels of N protein synthesis control the level of RNA replication. Consistent with this, the present invention demonstrates that as the level of N mRNA and protein synthesis in cells infected with viruses N2, N3 and N4 was reduced, the level of genomic RNA replication was also reduced. Correspondingly, the production of infectious virus in cell culture was reduced in increments up to four orders of magnitude with virus N4. Finally, concomitant with reduced replication potential, the lethality of viruses N2, N3, and N4 for mice following IN inoculation was reduced by approximately one, two or three orders of magnitude, respectively, compared to the wild-type virus.

These data demonstrate that translocating a single gene essential for replication to successive positions down the viral genome lowered the growth potential in cell culture and the lethality of the viruses for mice in a stepwise manner. However, the ability of the viruses to elicit a protective immune response in mice was not altered in correspondence with the reduction in virulence. Therefore, since the viruses all contained the wild-type complement of genes and all were competent to replicate, albeit at reduced levels, the level of replication was sufficient to induce a protective host response. Thus, for some rearranged viruses, the protective dose and the lethal dose were 1,000 fold different, in contrast to the situation with wild-type virus where the lethal dose and protective dose overlap. Taken together, these data suggest a means of attenuating non-segmented negative strand RNA viruses in a predictable, incremental manner that would allow one to determine an optimal level of attenuation to avoid disease production without loss of replication potential to induce a sufficient immune response.

Since the Mononegavirales have not been observed to undergo homologous recombination, gene rearrangement is predicted to be irreversible, and therefore, the present invention provides a rational, alternative method for developing stably attenuated live vaccines against the non-segmented negative strand RNA viruses. Furthermore, based on the close similarity of genome organization and control of gene expression, this approach to generating attenuated viruses should be applicable to the entire family of Mononegavirales, which includes the Rhabdoviridae, such as rabies, the Paramyxoviridae, such as measles, mumps, respiratory syncytial virus, and parainfluenza viruses I-IV, and the Filoviridae such as Ebola and Marburg viruses. These represent some of the most problematic viral pathogens extent.

In one embodiment of the present invention, there is provided a method of increasing expression of a promoter distal gene in a virus of the order Mononegavirales, comprising the step of rearranging gene order of the virus by moving the promoter distal gene toward a wild-type 3' promoter proximal position site. Preferably, the distal gene encodes a surface glycoprotein. For vesicular stomatitis virus, one distal gene that encodes a surface glycoprotein is the gene for the attachment glycoprotein G. For respiratory syncytial virus, one distal gene that encodes a surface glycoprotein is referred to as the attachment glycoprotein (G) gene; another distal gene that encodes a surface glycoprotein is the respiratory syncytial virus fusion (F) protein gene. For the measles virus, the distal gene that encodes a surface glycoprotein is referred to as the H (hemagglutinin) gene. For the mumps and parainfluenza viruses, the distal gene that encodes a surface glycoprotein is referred to as the HN (hemagglutinin/neuraminidase) gene. A person having ordinary skill in this art would readily recognize, for each specific virus of the order Mononegavirales, which distal gene that encodes a surface glycoprotein would be manipulated in order to perform the methods of the present invention.

In another embodiment of the present invention, there is provided a recombinant virus of the order Mononegavirales having a rearranged genome, wherein the genome is rearranged by moving a promoter distal gene of the virus toward a wild type 3' promoter proximal position site. Such recombinant virus can be used for accelerating and enhancing a protective immune response.

In still another embodiment of the present invention, there is provided a method of attenuating a virus of the order Mononegavirales by rearranging gene order of the virus by moving a gene away from its wild type position, or by rearranging gene order of the virus by moving an essential limiting factor gene away from its wild type 3' promoter proximal position site. Preferably, the gene is placed in the next to last position in the gene order of the virus. Furthermore, it is preferable that the gene which is an essential limiting factor for genome replication is the nucleocapsid (N) gene. Representative examples of viruses of the order Mononegavirales are a Rhabdovirus, such as rabies virus or vesicular stomatitis virus, a Paramyxovirus, such as measles, mumps, parainfluenza virus or respiratory syncytial virus (human and bovine), or a Filovirus, such as Ebola virus or Marburg virus. The present invention also includes a virus attenuated according to this method.

In yet another embodiment of the present invention, there is provided a method of constructing an attenuated virus useful for a vaccine, comprising the steps of rearranging gene order of the virus by moving a gene away from its wild-type 3' promoter proximal position site, wherein the gene is an essential limiting factor for genome replication; and placing a gene coding for an immune response inducing antigen in the position closest to the 3' end of the gene order of the virus. Preferably, the essential limiting factor gene is the nucleocapsid (N) gene and the gene is placed in the next to last position in the gene order of the virus. Still preferably, the gene coding for an immune response inducing antigen may be the attachment glycoprotein (G) gene, a fusion gene or the hemagglutinin/neuraminidase gene. A person having ordinary skill in this art would be able to readily substitute suitable immune response-inducing antigens. The present invention also includes a virus attenuated according to this method.

In accordance with the present invention, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual (1982); "DNA Cloning: A Practical Approach," Volumes I and II (D. N. Glover ed. 1985); "Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic Acid Hybridization" [B. D. Hames & S. J. Higgins eds. (1985)]; "Transcription and Translation" [B. D. Hames & S. J. Higgins eds. (1984)]; "Animal Cell Culture" [R. I. Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984). Therefore, if appearing herein, the following terms shall have the definitions set out below.

The present invention also demonstrates that it is possible to increase the expression of a promoter distal gene, e.g., the G gene, which encodes the attachment glycoprotein, by moving it to a promoter proximal site. To show that an increase in the production of the G protein during infection could elicit a greater protective immune response, changes were engineered into an infectious cDNA clone of the VSV genome and two novel viruses were recovered in which the glycoprotein gene was moved from its normal fourth position to the first position in the gene order. One virus had the gene order 3'-G-N-P-M-L-5' (G1N2) and the second 3'-G-P-M-N-L-5' (G1N4). The in vitro and in vivo characteristics of these viruses were assessed and compared to those of viruses having the gene orders 3'-P-M-G-N-L-5' (G3N4) and 3'-N-P-M-G-L-5' (N1G4), the latter being the wild-type gene order. Differences were observed in the replication of these viruses in cell culture, lethality in mice, kinetics and levels of antibody production, and their ability to protect against challenge with a lethal dose of VSV.

Claim 1 of 18 Claims

What is claimed is:

1. A method of increasing expression of a promoter distal gene in a virus of the order Mononegavirales, comprising the step of:

rearranging gene order of said virus by moving said promoter distal gene toward a wild-type 3' promoter proximal position site.

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