Interferon inducing genetically engineered attenuated viruses

Details Interferon inducing genetically engineered attenuated viruses Interferon inducing genetically engineered attenuated viruses

1999-06-11

2002-10-22

Housel, James (Department: 1648)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Virus or component thereof

C424S199100, C435S069100, C435S320100

Reexamination Certificate

active

06468544

ABSTRACT:

1. INTRODUCTION
The present invention relates to engineering attenuated viruses by altering a non-coding region or the coding sequence of a viral nonstructural (NS) gene. In particular, the present invention relates to engineering live attenuated influenza viruses which induce interferon and related pathways. The present invention further relates to the use of the attenuated viruses and viral vectors against a broad range of pathogens and/or antigens, including tumor specific antigens. The present invention also relates to a host-restriction based selection system for the identification of genetically manipulated influenza viruses. In particular, the present invention relates to a selection system to identify influenza viruses which contain modified NS gene segments.
2. BACKGROUND OF THE INVENTION
2.1. ATTENUATED VIRUSES
Inactivated virus vaccines are prepared by “killing” the viral pathogen, e.g., by heat or formalin treatment, so that it is not capable of replication. Inactivated vaccines have limited utility because they do not provide long lasting immunity and, therefore, afford limited protection. An alternative approach for producing virus vaccines involves the use of attenuated live virus vaccines. Attenuated viruses are capable of replication but are not pathogenic, and, therefore, provide for longer lasting immunity and afford greater protection. However, the conventional methods for producing attenuated viruses involve the chance isolation of host range mutants, many of which are temperature sensitive; e.g., the virus is passaged through unnatural hosts, and progeny viruses which are immunogenic, yet not pathogenic, are selected.
Recombinant DNA technology and genetic engineering techniques, in theory, would afford a superior approach to producing an attenuated virus since specific mutations could be deliberately engineered into the viral genome. However, the genetic alterations required for attenuation of viruses are not known or predictable. In general, the attempts to use recombinant DNA technology to engineer viral vaccines have mostly been directed to the production of subunit vaccines which contain only the protein subunits of the pathogen involved in the immune response, expressed in recombinant viral vectors such as vaccinia virus or baculovirus. More recently, recombinant DNA techniques have been utilized in an attempt to produce herpes virus deletion mutants or polioviruses which mimic attenuated viruses found in nature or known host range mutants. Until very recently, the negative strand RNA viruses were not amenable to site-specific manipulation at all, and thus could not be genetically engineered.
2.2. THE INFLUENZA VIRUS
Virus families containing enveloped single-stranded RNA of the negative-sense genome are classified into groups having non-segmented genomes (Paramyxoviridae, Rhabdoviridae) or those having segmented genomes (Orthomyxoviridae, Bunyaviridae and Arenaviridae). The Orthomyxoviridae family, described in detail below, and used in the examples herein, contains only the viruses of influenza, types A, B and C.
The influenza virions consist of an internal ribonucleoprotein core (a helical nucleocapsid) containing the single-stranded RNA genome, and an outer lipoprotein envelope lined inside by a matrix protein (M). The segmented genome of influenza A consists of eight molecules (seven for influenza C) of linear, negative polarity, single-stranded RNAs which encode ten polypeptides, including: the RNA-directed RNA polymerase proteins (PB2, PB1 and PA) and nucleoprotein (NP) which form the nucleocapsid; the matrix proteins (M1, M2); two surface glycoproteins which project from the lipoprotein envelope: hemagglutinin (HA) and neuraminidase (NA); and nonstructural proteins whose function is unknown (NS1 and NS2). Transcription and replication of the genome takes place in the nucleus and assembly occurs via budding on the plasma membrane. The viruses can reassort genes during mixed infections.
Influenza virus adsorbs via HA to sialyloligosaccharides in cell membrane glycoproteins and glycolipids. Following endocytosis of the virion, a conformational change in the HA molecule occurs within the cellular endosome which facilitates membrane fusion, thus triggering uncoating. The nucleocapsid migrates to the nucleus where viral mRNA is transcribed as the essential initial event in infection. Viral mRNA is transcribed by a unique mechanism in which viral endonuclease cleaves the capped 5′-terminus from cellular heterologous mRNAs which then serve as primers for transcription of viral RNA templates by the viral transcriptase. Transcripts terminate at sites 15 to 22 bases from the ends of their templates, where oligo(U) sequences act as signals for the template-independent addition of poly(A) tracts. Of the eight viral mRNA molecules so produced, six are monocistronic messages that are translated directly into the proteins representing HA, NA, NP and the viral polymerase proteins, PB2, PB1 and PA. The other two transcripts undergo splicing, each yielding two mRNAs which are translated in different reading frames to produce M1, M2, NS1 and NS2. In other words, the eight viral mRNAs code for ten proteins: eight structural and two nonstructural. A summary of the genes of the influenza virus and their protein products is shown in Table I below.
TABLE I
INFLUENZA VIRUS GENOME RNA SEGMENTS AND CODING
ASSIGNMENTS
a
Length
d
Molecules
Length
b
Encoded
(Amino
Per
Segment
(Nucleotides)
Polypeptide
c
Acids)
Virion
Comments
1
2341
PB2
759
30-60
RNA transcriptase component;
host cell RNA cap binding
2
2341
PB1
757
30-60
RNA transcriptase component;
initiation of transcription;
endonuclease activity?
3
2233
PA
716
30-60
RNA transcriptase component;
elongation of mRNA chains?
4
1778
HA
566
 500
Hemagglutinin; trimer;
envelope glycoprotein;
mediates attachment to cells
5
1565
NP
498
1000
Nucleoprotein; associated with
RNA; structural component of
RNA transcriptase
6
1413
NA
454
 100
Neuraminidase; tetramer;
envelope glycoprotein
7
1027
M
1
252
3000
Matrix protein; lines inside of
envelope
M
2
 96
Structural protein in plasma
membrane; spliced mRNA
?
 ?9
Unidentified protein
8
 890
NS
1
230
Nonstructural protein; function
unknown
NS
2
121
Nonstructural protein: function
unknown; spliced mRNA
a
Adapted from R. A. Lamb and P. W. Choppin (1983), Reproduced from the Annual Review of Biochemistry, Volume 52, 467-506.
b
For A/PR/8/34 strain
c
Determined by biochemical and genetic approaches
d
Determined by nucleotide sequence analysis and protein sequencing
The Influenza A genome contains eight segments of single-stranded RNA of negative polarity, coding for nine structural and one nonstructural proteins. The nonsructural protein NS1 is abundant in influenza virus infected cells, but has not been detected in virions. NS1 is a phosphoprotein found in the nucleus early during infection and also in the cytoplasm at later times of the viral cycle (King et al., 1975, Virology 64: 378). Studies with temperature-sensitive (ts) influenza mutants carrying lesions in the NS gene suggested that the NS1 protein is a transcriptional and post-transcriptional regulator of mechanisms by which the virus is able to inhibit host cell gene expression and to stimulate viral protein synthesis. Like many other proteins that regulate post-transcriptional processes, the NS1 protein interacts with specific RNA sequences and structures. The NS1 protein has been reported to bind to different RNA species including: vRNA, poly-A, U6,
sn
RNA, 5′ untranslated region as of viral mRNAs and ds RNA (Qiu et al., 1995, Rna 1:304; Qiu et al., 1994, J. Virol. 68:2425). Expression of the NS1 protein from cDNA in transfected cells has been associated with several effects: inhibition of nucleo-cytoplasmic transport of mRNA, inhibition of pre-mRNA splicing, inhibition of host mRNA polyadenylation and stimulation of translation of viral mRNA (Fortes, et al., 1994, Embo J. 13:704; Enami, K. et al, 1994, J. Virol. 68: 1432 de la Luna, et al., 1995, J. Virol. 69

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