Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or... – Inactivation or attenuation; producing viral subunits
Reexamination Certificate
1999-06-16
2002-07-23
Wortman, Donna C (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
Inactivation or attenuation; producing viral subunits
C435S235100
Reexamination Certificate
active
06423528
ABSTRACT:
This invention relates to variants of herpes simplex virus type 1 (HSV-1) which lack neurovirulence. Such variants are of value in the preparation of live attenuated vaccines for the prevention of HSV infections in humans.
Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are important human pathogens which infect more than 80% of the general population and cause recurrent mucocutaneous lesions. Following replication HSV enters the peripheral nervous system where active replication is turned off by an unknown mechanism. Thereafter a latent infection in neurons is established which persists for the life of the host. HSV can reactivate from the latent state to produce infectious lesions. HSV is responsible for a broad spectrum of clinical diseases ranging from relatively benign cutaneous lesions to fatal viral encephalitis.
A considerable amount of research has already been devoted to elucidation of the genetic organisation of both HSV-1 and HSV-2. The HSV-1 genome is a linear double stranded DNA molecule of approximately 152 kilobase pairs consisting of two components L and S. Each component consists of unique sequences U
1
and U
s
, flanked by inverted repeats. The organisation of the HSV-2 genome is similar but not identical. For a detailed description of the genetic organisation of HSV-1 and HSV-2 (see McGeoch, 1987).
The identification of genes involved in viral pathogenicity and the elucidation of their precise functions is of fundamental importance to the understanding of the biology of herpes simplex virus (HSV). A number of variants of both HSV type 1 (strain 17) and HSV type 2 (strain HG52) with defined deletions in the unique and repeat sequences of both the long and short regions of the viral genome have already been isolated and characterised (Brown et al 1984, Harland and Brown 1985, Brown and Harland 1987, MacLean and Brown, 1987a and b, Harland and Brown 1989). Little is known, however, about the molecular mechanisms which regulate the neurovirulence of HSV. It has been shown that a deletion variant of HSV-2 strain HG52, termed JG2604, is avirulent on intracerebral inoculation of mice (Taha et al, 1989a). JH2604 has
—
1488 base pair deletion within both copies of the long repeat region of the genome [i.e. terminal long repeat (TR
L
) and internal inverted long repeat (IR
L
) regions].
An HSV-1 strain 17/HSV-2 strain HG52 recombinant (initially isolated in the Institute of Virology, Glasgow by Marsden et al, 1978), termed RE6, has also been reported to be avirulent in mice (Thompson et al 1989).
In HSV-1, inverted repeats of the L component designated ab and b′a′ are each approximately 9 Kbp whereas those of the S component c′a′ and ca, are each approximately 6.5 Kbp. A sequence shared by the inverted repeats of the L and S components is designed the ‘a’ sequence. This sequence has been reported (Chou and Roizman 1986) to contain the promoter-regulatory sequence and the transcription initiation sites for a diploid gene located in the b sequence of the inverted repeats of the L component. Working with HSV strain F these authors reported that there was a transcribed open reading frame (ORF) between the ‘a’ sequence and an immediate early gene designated IE1. By the use of antipeptide sera they were able to show that the ORF specified a protein designated ICP 34.5 (Ackermann et al 1986). Recently Chou and Roizman (1990) have reported that their now predicted ORF is conserved in 2 other HSV-1 strains analysed but not in Glasgow strain HSV-1 (17) syn+. It has been suggested by Chou et al (1990) that the neurovirulence locus of HSV-1 comaps with, and requires the expression of, ICP 34.5.
Surprisingly it has now been found that HSV-1 Glasgow strain 17 variants modified in the terminal portion of R
L
lack neurovirulence.
Such variants are incapable of replicating in CNS neurons, but are able, in mice to elicit a good immunological and cell mediated response since they are capable of replication in the peripheral tissue. This ability emphasises the vaccine potential of these strains.
According to the present invention there is provided an HSV-1 strain the genome of which is modified in the terminal portion of R
L
within Bam HI s (0-0.02 and 0.81-0.83 mu).
By Bam HI s it will be appreciated that what is meant is each copy of the approximately 3 Kb Bam HI s fragment of the HSV R
L
region.
The term ‘modified’ is used herein to denote disruption of the Bam HI s fragment by deletion of one or more nucleotides, insertion of additional nucleotides or any other alteration of the nucleotide sequence such as rearrangement, or substitution.
The HSV-1 strain may be a spontaneously isolated deletion variant of may be a wild type strain into which the desired modification has been introduced.
Such modifications in the HSV strain may be made by genetic manipulation, for example by site-directed mutagenesis, or by excision of a portion of the genome with or without replacement with a pre-prepared DNA cassette incorporating the required modification. Alternatively one may isolate naturally occurring HSV-1 variants, e.g. deletion variants.
Preferably the HSV-1 strain of the invention is a Glasgow strain 17 variant.
In one preferred aspect the HSV-1 strain is a strain in which at least 100 nucleotides in the Bam HI s′ region between the Alu I site at 125074 np and 125972 np within the a sequence and its counterpart sequence in TR
L
have been deleted.
More preferably 0.5 to 3 kb of the Bam HI s′ region and its counterpart in TR
L
is deleted, still more preferably about 0.7-2.5 kb is deleted.
In one specific example the HSV-1 variant is a strain designated 1714 which is a spontaneously occurring deletion variant of variant 1702 and lacks 759 bp within each copy of the Bam HI s fragment of the R
L
region as described hereinbelow, in which the deletion associated with non-neurovirulence is located between nucleotide positions 125213 and 125972.
Such a deletion removes one complete copy of the 18 bp DR
1
element of the ‘a’ sequence and terminates 1105 bp upstream of the 5′ end of the immediate early gene 1.
In another specific example the HSV-1 variant is a variant designated 1716 in which the 759 bp deletion in variant 1714 has been introduced into the wild type Glasgow strain 17
+
.
In order to understand the invention more clearly reference may be made to
FIG. 1
hereinbelow in which:
(a) Shows the HSV-1 genome (with map units marked) in the prototype orientation; and
(b) Shows an expansion of BamHI k(s+g). The BamHI (B) and AluI (A) sites flanking the deletion in 1714/1716 are marked. All coordinates are based on the numbering of McGeoch et al (1988). Also indicated are the positions of the 5′ end of IE1, the ‘a’ sequence, the DR
1
/U
b
boundary in the ‘a’ sequence, a 189 bp conserved open reading frame between HSV-1 and HSV-2 (R
L
ORF) and the end points of the 759 bp deletion in 1714/1716. The deletion extends from the DR
1
/U
b
boundary to remove the 5′ 107 bp of the R
L
ORF.
The present invention further provides a whole virus vaccine comprising an HSV-1 strain according to the invention wherein such vaccine comprises an immunoprotective and non-toxic amount of the strain of the invention. Such vaccine may comprise the strain alone or in conjunction with other antigens and/or adjuvants.
Due to their non-pathogenic nature, the viruses of the present invention are exceptional candidates for further modification. For example they may be further modified so as to carry heterologous antigens. The virus can be engineered so as to express antigens from HSV-2, such as HSV-2 gD. Such a virus, elicits both antibody and CTL responses to both type 1 and type 2 virus and, moreover, enhances the overall immune response. Similarly other antigens from the other pathogens may be presented by the viruses of the present invention. For example, gene products from HCMV, VZV, EBV, HHV6, HHV7, and HIV as well as other envelope viruses may be presented.
Moreover, the virus of the present invention may be modified
Brown Susanne M.
MacLean Alasdair R.
Nixon & Vanderhye P.C.
The University Court of the University of Glasgow
Wortman Donna C
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