AU753566B2 - Method of inducing an immune response with a live Venezuelan equine encephalitis virus expressing heterologous immunogen - Google Patents

Description

la METHOD OF INDUCING AN IMMUNE RESPONSE WITH A LIVE VENEZUELAN EQUINE ENCEPHALITIS VIRUS EXPRESSING A HETEROLOGOUS IMMUNOGEN Field of the Invention The present invention relates to live attenuated vaccines in general, and particularly relates to attenuated vaccine produced from Venezuelan Equine Encephalitis (VEE) virus.

Background of the Invention All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

Live, attenuated viral vaccines are among the most successful means of controlling viral disease.

However, for some virus pathogens, immunisation with a live virus strain may be either impractical or unsafe.

One alternative strategy is the insertion of genes encoding immunising antigens of such agents into the vaccine strain of another virus. However, relatively few such systems are currently available.

Hahn et al., Proc. Natl. Acad. Sci. USA 89, 2679 (1992), describes Sindbis virus constructs which express a truncated form of the influenza hemagglutinin protein.

The constructs are used to study antigen processing and presentation in vitro and in mice. Although no infectious challenge dose is tested, it is also suggested that such constructs might be used to produce protective B- and Tcell mediated immunity. The final paragraph of the discussion section states: H \cintae\Keep\speci\61757.99 .doc 24/01/02 -2- Although SIN is not likely to be approved for use as a human vaccine, a parallel approach to the one used here for SIN may be applicable for developing live-attenuated vaccine strains using viruses with :imilar replication strategies, such as attenuated strains of Venezuelan equine encephalitis virus.... (citing Davis et al.)" Insofar as applicant is aware, a problem with the Sindbis vector is that the heterologous insert is unstable therein and is "kicked out" of the vector, with the practical limit for stable inserts being about 1 kb.

Davis et al., U.S. Patent No. 5,185,440, describes cDNAs encoding the VEE virus and attenuated mutations which may be incorporated therein for use in making a vaccine. The use of a subgenomic expression 15 system is neither suggested nor disclosed.

Summary of the Invention A first aspect of the present invention is a method of protecting a subject against a disease. The method comprises administering a recombinant Venezuelan 20 Equine Encephalitis (VEE) virus to the subject in. an effective immunogenic amount, with the VEE virus containing at least one attenuating mutation (and typically two or even three different attenuating mutations) and with the VEE virus containing a heterologous RNA segment. The heterologous RNA segment comprises a promoter operable in the subject operatively associated with a RNA encoding an immunogenic protein or peptide effective for protecting the subject from the disease. The heterologous insert may, optionally, itself serve as an attenuating mutation.

In one preferred embodiment, the heterologous RNA segment of the VEE virus is derived from the genome of a pathogenic organism. According t-o this embodiment, the heterologous RNA segment comprising the promoter operatively associated with the RNA encoding the immunogenic protein or peptide is effective for 3 protecting the subject from the disease caused by the pathogenic organism.

A second aspect of the invention is a DNA comprising a cDNA clone coding for an infectious Venezuelan Equine Encephalitis (VEE) virus RNA transcript, said DNA comprising a promoter positioned upstream from said cDNA clone and operatively associated therewith, wherein said infectious virus VEE transcript comprises a heterologous RNA segment encoding an immunogenic protein or peptide and at least one attenuating mutation, for use in the manufacture of a medicament comprising an infectious VEE virus particle.

A third aspect of the present invention is a DNA comprising a cDNA clone coding for an infectious Venezuelan Equine Encephalitis (VEE) virus RNA transcript and a heterologous RNA sequence comprising a promoter positioned upstream from the cDNA clone and operatively associated therewith wherein said infectious VEE virus transcript comprises an RNA encoding an immunogenic protein and further comprises at least one attenuating mutation selected from the group consisting of codons at Elamino acid 272 which specify an attenuating mutation; codons at El amino acid 81 which specify an attenuating mutation; and codons at El amino acid 253 which specify an attenuating mutation.

A fourth aspect of the present invention is aDNA comprising a cDNA clone coding for an infectious Venezuelan Equine Encephalitis (VEE) virus RNA transcript and a heterologous RNA sequence comprising a promoter positioned upstream from said cDNA clone and operatively associated with an RNA encoding an immunogenic protein further comprising: a first attenuating mutation which is a codon at Elamino acid 253 which specifies an attenuating mutation; and a second attenuating mutation which is an inactivated E3 amino acid 56 to 59 cleavage recognition site.

xFurther aspects of the present invention include H;\cintae\Keep\speci\61757.99.doc 24/01/02 3a an infectious VEE virus RNA transcript encoded by cDNA clones as given herein; infectious VEE virus particles containing such RNA transcripts; and pharmaceutical formulations comprising such infectious VEE virus particles, in an effective immunogenic amount in a pharmaceutically acceptable carrier.

Frolov et al., Proceedings IXth International Congress of virology, Glasgow, Scotland, August: 8-13, 1993, pg. 67, discusses recombinant VEE viruses which express a Hepatitis B virus antigenic protein. The use of an attenuated VEE virus, strain 230, is described. However, it is not suggested that the attenuated virus itself be administered to humans. To the contrary, these H:\cintae\Keep\speci\61757.99.doc 24/01/02 i -4viruses are used to manufacture the antigenic Hepatitis B virus proteins themselves in tissue culture, which are then harvested and administered to humans. Strain 230 itself replicates poorly, if at all, in humans.

The foregoing and other objects and aspects of the present invention are explained in detail in the specification set forth below.

Brief Description of the Drawings Figure 1 is an illustration of the structure of the shuttle vector containing the structural genes of VEE with three attenuating mutations in the E2 gene, and .a second 26S promoter and multiple cloning site inserted directly downstream from the C-terminus of El, in the 3.2 kb pUC118 plasmid.

*o 15 Figures 2a-2c illustrate double promoter vectors.

Figure 3 is a Northern blot analysis of total intracellular RNA.from baby hamster kidney (BHK) cells infected with an attenuated VEE mutant, with the mutant containing an HA gene inserted downstream of a second subgenomic promoter, or with the mutant containing an •inserted HA gene in a non-coding orientation.

Figures 4a-4d illustrate the immunocytochemical staining of VEE vector-infected BHK cell monolayers.

S* 25 Cells were infected with either influenza PR/8/34, VEE.

vector containing the complete influenza HA gene, VEE vector without insert.

Figures 5a-5c are graphical illustrations of the clinical signs observed in inoculated mice challenged intranasally with influenza. Mice were inoculated with PBS, VEE vector without insert, or VEE vector with the complete influenza HA gene.

Figure 6 is a graphical illustration of previral challenge anti-flu IgG ELISA titers of serum obtained from inoculated mice. Mice were inoculated with PBS, VEE vector without insert, or VEE vector with the complete influenza HA gene.

Figure 7 is a graphical illustration of the titer of viable influenza virus observed in lung tissue of inoculated mice 4 days after challenge with influenza.

Mice were inoculated with PBS, VEE vector without insert, or VEE vector with the complete influenza HA gene.

Detailed Description of the Invention Complementary DNA sequences encoding live Venezuelan Equine Encephalitis (VEE) virus and pharmaceutical formulations containing the same are known. See, N. Davis et al., U.S. Patent No.

5,185,440 (Applicant specifically intends that the disclosures of all patent references cited herein be incorporated herein by reference in their entirety).

The phi. se5 "attenuating mutation" and "attenuating amino acid" as used herein mean a nucleotide mutation or an amino acid coded for in view of such mutation which result in a decreased probability of 20 causing disease in its host a loss of virulence), in accordance with standard terminology in the art (See, B. Davis et al., Microbiology, 132 (3d ed. 1980), whether the mutation be a substitution mutation or an inframe deletion mutation. The phrase "attenuating mutation" excludes mutations which would be lethal to the virus. Examples of known VEE attenuating mutations include codons at E2 amino acid position 76 which specify an attenuating mutation, codons at E2 amino acid position 209 which specify an attenuating mutation, and codons at E2 amino acid 120 which specify an attenuating mutation (see, U.S. Patent No. 5,185,440 to N. Davis et a G to C mutation at viral RNA nucleotide 3.

Novel attenuating mutations disclosed herein which may be used to carry out the present invention include codons at El amino acid 272 which specify an attenuating mutation (preferably a substitution mutation, such as a threonine or serine (most preferably threonine)); codons at El amino acid 81 which specify an attenuating mutation (preferably a substitution mutation, such as an isoleucine or leucine (most preferably isoleucine)) and codons at El amino acid 253 which specify an attenuating mutation (preferably a substitution mutation such as a serine or threonine (most preferably serine)).

A novel pair of attenuating mutations which may be inserted together in a cDNA clone encoding an attenuated VEE virus is a first attenuating mutation which is a codon at El amino acid 253 which specifies an attenuating mutation; and a second mutation which is an inactivated E3 amino acid 56 to 59 cleavage recognition site. An advantage of this combination of attenuating mutations is that the inactivated cleavage s.te, of ite, is letha to th- vi rus. Thu, if attenuating mutation at El amino acid 253 reverts to the •virulent wild-type, the remaining mutation kills the virus. The E3 amino acid 56 to 59 cleavage recognition site may be inactivated by any suitable means: the cleavage recognition site may be deleted, in whole or in part; a substitution mutation may be made therein an arginine to aspartic acid substitution mutation at 25 amino acid 59).

Attenuating mutations may be introduced into cDNAs encoding live VEE by any suitable means, such as site-directed mutagenesis (see, U.S. Patent No.

4,873,192 to Kunkel).

The immunogenic protein or peptide, or "immunogen" may be any immunogen suitable for protecting the subject against a disease, including but not limited to microbial, bacterial, protozoal, parasitic, and viral diseases. For example, the immunogen may be an influenza virus immunogen an influenza virus hemagglutinin (HA) surface protein, or an equine influenza virus immunogen), or a lentivirus immunogen an equine 7 infectious anaemia virus immunogen, a Human Immunodeficiency Virus (HIV) immunogen, such as an HIV-1 immunogen, or an HIV-2 immunogen. The immunogen may also be a coronavirus immunogen a transmissible gastroenteritis virus immunogen for pigs, or an infectious bronchitis virus immunogen for chickens) or a flavivirus immunogen a yellow fever virus immunogen or a Japanese encephalitis virus immunogen). If desired, an advantage of the instant invention is that the heterologous insert containing the DNA encoding the immunogen as given above may be a relatively large insert, at least 1 kilobase in length.

The promoter is preferably a Venezuelan equine encephalitis virus 26S subgenomic promoter. This definition is intended to include derivatives of this promoter such as deletion mutants thereof, so long as activity as a promoter is retained. Subjects which may be administered the live attenuated viruses and vaccine formulations disclosed herein include both human and animal horse, donkey, 20 pigs, mouse, hamster, monkey, chicken) subjects.

Vaccine formulations of the present invention comprise an immunogenic amount of a live attenuated virus as disclosed herein in combination with a pharmaceutically acceptable carrier. An "immunogenic amount" is an amount of 25 the attenuated virus sufficient to evoke an immune response, particularly an immune response to the protein or peptide encoded by the heterologous RNA carried by the virus, in the subject to which the virus is administered.

An amount of from about 101 to 10 5 plaque forming units of the live virus per dose is suitable, depending upon the age and species of the subject being treated. Exemplary pharmaceutically acceptable carriers include, but are not limited to sterile pyrogen-free water and sterile pyrogenfree physiological saline solution.

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2 69 23 .doc 23/11/99 8 Administration of the live attenuated viruses disclosed herein may be carried out by any suitable means, including both parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection), by in ovo injection in birds and by topical application of the virus (typically carried in the pharmaceutical formulation) to an airway surface. Topical application of the virus to an airway surface can be carried out by intranasal administration by use of a dropper, swab, or inhaler which deposits a pharmaceutical formulation intranasally). Topical application of the virus to an airway surface can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid particles and liquid particles) containing the virus as an aerosol suspension, and then causing the subject to inhale the respirable particles. Methods and apparatus for administering respirable particles of pharmaceutical formulations are well known, and any conventional technique 20 can be employed. See, U.S. Patent No. 5,304,125 to D. Leith; U.S. Patent No. 5,299,566 to C. Davis and R.

Snyder; U.S Patent No. 5,290,550 to R. Fisher and W.

Metzger; and U.S. Patent No. 5,292,498 to R. Boucher.

For the purposes of this specification it will be 25 clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

The following examples are provided to illustrate the present invention, and should not be construed as limiting thereof.

EXAMPLE 1 Construction of a "Second Promoter" Expression Vector A full-length cDNA clone derived from the TRD strain of VEE, pV3507, and containing three attenuating mutations in the E2 gene was employed in this study. The \\.elbfiles\homeS\Helen\Keep\speci\P36153(div.26 9 2 3 .doc 23/11/99 8a mutations occur at E2 76 lys, E2 120 lys and E2 209 lys, as reported in Davis et al., Virology 183:20 (1991). The cDNA clone was digested with TthlllT, converted to blunt ends with Klenow fragment of E. coli DNA polymerase, and \\melb-files\hone$\Helefl\Keep\SpeCi\P36153{div.26923) .doc 23/1 1/99 -9then digested with EcoRI. The 3.9 kb fragment was isolated and ligated with M12 mpl9 RF DNA that had been digested with HindIII, treated with Klenow fragment and then digested with EcoRI. The resulting M13 phage, TE3, contained the structural gene region of pV3507, but not the 26S promoter region. The HindIII site was regenerated in the ligation. Single-stranded DNA from phage was produced following transformation of E. coli CJ236 (dut-ung-) with TE3 and used in the procedure outlined by Kunkel, Proc. Natl. Acad. Sci. USA 82:488 (1985), with a synthetic oligonucleotide designed to give the following insertion (in bold): 3: i 3' end of El gene -34/+14 26S promoter-ClaI site the 5' end of 3' untranslated region Sntll,315-ntll,326 ntll,327-ntll,338

AAACATAATTGA/GAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATCG/ATACAGCAGCAA

(SEQ ID NO: 1) The regions flanking the inserted cnerT are identified Vs nrleide nimbr-c -aed on *g o• go o the full-length VEE sequence, as identified by Kinney et al., Virol. 170:19 (1989).

The correct insertion was identified initially by screening with Clal digestion of phage RF DNA, and confirmed by sequencing across the junction between the El gene and the 3'-untranslated region on single-stranded phage DNA.

Thereafter, the DNA fragment containing the pV3507 structural genes and the inserted second 26S promoter was subcloned into pUC118 using HindIII and EcoRI. The HindIII and EcoRI sites were removed by digestion followed by conversion to blunt ends with Klenow fragment and religation to form the pUC118 second promoter clone.

A 1.5 kb SalI-SalI "stuffer" fragment lacking an initiator AUG codon was isolated from the glycoprotein gene region of the TR5000 full-length Sindbis clone (identified by Schoepp et al., Virology 193:149 (1993)), and inserted into -the SalI site of the Clal2 adaptor plasmid. The Clal2 plasmid containing a multiple cloning site (mcs) flanked by Clal sites was identified by Hughes et al., J. Virol. 61:3004 (1987).

Using Clal, the multiple cloning site containing the inserted TR5000 sequence was cloned into the unique Clal site of the pUC118 second promoter clone.

Digestion with SalI and self-ligation produced the shuttle vector with the structure shown in Figure 1.

Thereafter, a viable full-length VEE second promoter expression vector including three attenuating mutations was prepared. The 3.4 kb AflII-NotI fragment isolated from the shuttle vector and containing a portion of the viral structural genes, the inserted downstream second promoter and the 3'-untranslated region, was used 15 to replace the homologous AflII-NotI region of the pV3507 full-length clone. Transformants were screened with Clal. Plasmids containing a Clal site were linearized at the unique NotI site and transcribed in vitro with T7 RNA polymerase. Transcripts were quantitated by 20 incorporation of alpha- 3 P-labeled UTP. Thereafter, monolayers were transfected with transcripts using cationic liposomes (Lipofectin, BRL) and then overlaid with agarose for assay of plaque formation. The transcripts had specific infectivities comparable to those produced from the virulent pV3000 parent clone, indicating that they were fully infectious. Figures 2a- 2c illustrate double promoter vectors. Figure 2b is an illustration of pV4002, one of the downstream second promoter expression vectors.

EXAMPLE 2 Construction of an Expression Vector Containing the Influenza HA gene The complete coding sequence of the HA gene from influenza strain PR/8 cloned into the BamHI site of pGem4 was obtained from Dr. Andy Caton at the Wistar Institute. The 1.7, kb BamHI fragment containing the HA -11sequence was ligated to BamHI digested, dephosphorylated shuttle vector DNA. V4002 DNA was cut with Clal and dephosphorylated. Thereafter, the Clal fragment containing the HA sequence was purified from the shuttle vector and ligated to prepare V4002 DNA. Clones containing the HA gene in the multiple cloning site downstream of the second 26S promoter were identified using digestion with HpaI. Clones including both coding (pV4002HA) and noncoding (pV4002AH) orientations were identified. Tests for the specific infectivity of the transcripts from these clones showed that they were as infectious as parental transcripts, but that these HAcontaining genomes made smaller plaques on baby hamster kidney (BHK) cell monolayers. From these results, we 15 determined that the VEE expression vector containing the three attenuating mutations in E2 and an inserted 1.7 kb gene was st relcation competent, ut appeared to grow more slowly than the vector without an insert.

EXAMPLE 3 20 Stability Test of HA Containing Vectors During Replication in Tissue Culture Total RNA from vector-infected cells was analyzed for HA sequence-containing subgenomic RNA transcripts using Northern blots probed with VEE-specific or HA-specific probes as described in Sambrook et al-, Molecular Cloning pp 7.39-7.52. Purified cytoplasmic RNA was glyoxylated, electrophoresed on agarose gels containing 0.01 M sodium phosphate (pH and transferred to Biotrans nylon membranes (ICN) with 7.5 M sodium hydroxide. A VEE-specific "P-labeled riboprobe was made using a subclone of the VEE glycoprotein gene region (nt 9493 to 10486) in the pGEM3 transcription vector, pGEM19. The pGEM4 HA clone obtained from Dr.

Andy Caton at the Wistar Institute was used to generate an HA-specific 3 2 P-labeled riboprobe. (The HA clone used contained a single nucleotide deletion which resulted in -12translation of a truncated protein. This mutation affected our ability to detect any expression of protein from this vector, although subgenomic HA-containing viral RNAs were detected.) Duplicate membranes were hybridized to either probe, dried and exposed to x-ray film. The results are illustrated in Figure 3. Lanes A and D contain total cytoplasmic RNA from cells infected with a VEE strain containing three attenuating mutations in E2.

Lanes B and E contain total cytoplasmic RNA from cells infected with the same mutant strain with a second subgenomic promoter followed by the influenza HA gene in the coding orientation (V4002HA). Lanes C and F contain Stotal cytoplasmic RNA from cells infected with the VEE •vector containing the HA gene in the noncoding 15 orientation (V4002AH). Lanes A, B and C were probed with a "P-labeled riboprobe complementary to a portion of the S* influenza HA gene. Lanes D, E and F were probed with a 3 2 P-labeled riboprobe complementary to a portion of the VEE glycoprotein genes. The positions of ribosomal RNA 20 markers were determined in a parallel lane stained with ethidium bromide. VEE genome length RNA (40S) was not detectable in this experiment.

SThe results indicate that deletion mutants of both the V4002HA and V4002AH vectors were arising during replication in tissue culture. However, some subgenomic RNAs in V4002HA-infected cells still contained RA sequences, and a significant fraction of these were of-a size to accommodate the complete HA gene. The results revealed some instability of the inserted sequence.

-13- EXAMPLE 4 Expression in Cultured BHK cells of Influenza PR/8 HA Gene from a Downstream Promoter Expression Vector with Two Attenuating Mutations The complete coding sequence of the HA gene from influenza PR/8 cloned into the HindIII site of pBR322 was obtained from Dr. P. Palese at the Mt. Sinai School of Medicine. The HA-containing HindIII fragment was ligated to the HindIII-cut and dephosphorylated shuttle vector (see Figure Clal was then used to insert these HA sequences in both coding (V4036a) and noncoding (V4036e) orientations into a VEE second promoter expression vector that carried two attenuating mutations, E2 lys 209 and El thr 272. RNA transcribed in vitro from these clones showed comparable specific infectivities to RNA from the parental clone without the HA gene. Virus-conLaining supernatants obtained following transfection of BHK cells with cationic liposomes contained both small and large plaques, with 20 the proportion of large plaques increasing with time. A greater proportion of large plaque variants were seen with V4036a, carrying HA in the coding orientation, than with V4036e.

BHK cell monolayers were infected at a multiplicity of 1 with either egg-grown influenza virus (PR/8 strain), the second promoter expression vector with the HA gene in the coding orientation, or the vector without the insert. At 6 hr post-infection, the monolayers were fixed with methanol:acetone at 20 OC and air dried. Using the horseradish peroxidase, biotin-avidin detection system (Vector labs), cells were tested for the presence of viral antigens using an HAspecific monoclonal antibody, or VEE-specific hyperimmune mouse ascites fluid, as primary antibodies. Cells were then counterstained with Meyer's hematoxylin. The monolayers infected with influenza PR/8 or with the coding HA vector showed positive staining for HA. The -14results are illustrated in Figures 4a-4d. Figure 4a illustrates cells infected with influenza PR/8 stained with anti-HA antibody. Figure 4b illustrates VEE HAvector infected cells stained with anti-HA antibody.

Figure 4c illustrates cells infected with VEE vector without insert stained with anti-VEE antibody. Figure 4d illustrates cells infected with VEE vector without insert stained with anti-HA antibody.

The HA stain in HA vector infected cells was cytoplasmic, and was as intense, under these conditions, *as the staining for HA in the influenza-infected control monolayers. These results indicate that influenza HA can *be expressed at normal levels from the second 26S promoter in a form that is detectable by this anti-HA 15 monoclonal antibody, and that an infectious VEE virus carrying the inserted gene can be produced.

EXAMPLE Protection of Mice Against Influenza Challenge Four-week-old CD-1 mice were inoculated subcutaneously into each rear footpad with 1 x 104 pfu of diluent (PBS) alone, the HA-expressing doubly attenuated vector (V4036a), or with the vaccine vector without insert. Three weeks later, the mice were challenged intranasally with 10 s EIDs 5 (50% egg infectious dose) of influenza virus. The results are reported i± Figures 5a-5c. All 24 control mice suffered severe disease and 50% died. Only one of 12 HA-vectorinoculated mice died, and another exhibited signs of disease for one day and recovered.

Pre-challenge anti-flu serum IgG ELISA titers were measured, and the results are illustrated in Figure 6. The geometric mean ELISA titer of anti-HA serum IgG in the HA-vector inoculated mice was 246, while sera from only 3 of 24 control mice gave a detectable titer, and they were positive only at the lowest dilution tested The two HA-vector-inoculated mice affected by the influenza challenge showed no detectable anti-HA IgG.

Therefore, in 10 of 12 mice, the HA-vector elicited a detectable level of anti-HA serum IgG, and 11 of 12 mice were protected against lethal influenza challenge.

EXAMPLE 6 Protection of Mice Against Influenza Challenge Four-week-old CD-1 mice were inoculated subcutaneously into each rear footpad with 1 x 104 pfu of diluent (PBS) alone, the HA-expressing doubly 10 attenuated vector, or with the vaccine vector without insert. Three weeks later, the mice were challenged intranasally with 1 0 S EIDso (50% egg infectious dose) of influenza virus. The lungs were removed 4 days after challenge. Lung tissue was homogenized in PBS 0.1% BSA to give a 20% suspension, centrifuged, aliquoted and frozen at -70^C. For each animal, two aliquots were assayed for pfu on MDCK cells under agarose containing 0.1% trypsin. No influenza infectivity was detected in the lungs of mice previously immunized with the HA-vector 20 at a detection level of 1.25 x 102 pfu/gm tissue pfu/average lung). The geometric mean titers (represented by solid dots) calculated for the animals in the control groups with measurable virus titers, were 3.04 x 106 pfu/gm for PBS-inoculated mice, and 1.93 x 106 pfu/gm for mice inoculated with VEE vector alone. The results are reported in Figure 7. The results suggest a very low level of challenge virus replication in the vaccinated animals.

The foregoing is illustrative of the present invention and is not to be construed as limiting thereof.

The invention is defined by the following claims, with equivalents of the claims to be included therein.

The entire disclosure in the complete specification of our Australian Patent Application No. 26923/95 is by this cross-reference incorporated into the present specification.

-16- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Johnston. Robert E.

Davis. Nancy L.

Smith. Jonathon F.

Grieder. Franziska 8.

(ii) TITLE OF INVENTION: METHOD OF INDUCING AN IMMUNE RESPONSE WITH A LIVE VENEZUELAN EQUINE ENCEPHALITIS VIRUS EXPRESSING A HETEROLOGOUS IMMUNOGEN (iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Kenneth D. Sibley STREET: Post Office Drawer 34009 CITY: Charlotte STATE: North Carolina COUNTRY: USA ZIP: 28234 COMPUTER READABLE FORM: vj ,U R M•t- MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: US 08/250.445 FILING DATE: 27-MAY-1994

CLASSIFICATION:

*(viii) ATTORNEY/AGENT INFORMATION: NAME: Sibley. Kenneth D.

REGISTRATION NUMBER: 31.665 REFERENCE/DOCKET NUMBER: 5470-79 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (919) 420-2200 TELEFAX: (919) 881-3175 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 74 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear -17- (i1) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION; SEQ ID NO:1: AAACATAATT GAGAGGGGCC CCTATAACTC TCTACGGCTA ACCTGAATGG ACTAGGACAT CGATACAGCA GCAA 74

Claims (30)

1. A DNA comprising a cDNA clone coding for an infectious Venezuelan Equine Encephalitis (VEE) virus RNA transcript, said DNA comprising a promoter positioned upstream from said cDNA clone and operatively associated therewith, wherein said infectious virus VEE transcript comprises a heterologous RNA segment encoding an immunogenic protein or peptide and at least one attenuating mutation, when used in the manufacture of a medicament comprising an infectious VEE virus particle.

2. A DNA according to claim 1, wherein said medicament is a vaccine.

3. A DNA according to claim 1 or 2, wherein said heterologous RNA segment contains a promoter.

4. A DNA according to any one of claims 1 to 3, 20 wherein said promoter is a VEE 26S subgenomic promoter.

5. A DNA according to any one of claims 1 to 4, wherein said heterologous RNA segment is at least 1 e. kilobase in length.

6. A DNA according to any one of claims 1 to wherein said heterologous RNA segment contains a sequence derived from the genome of a pathogenic organism.

7. A DNA according to any one of claims 1 to 6, wherein said immunogenic protein or peptide is selected from the group consisting of influenza immunogens, lentivirus immunogens, coronavirus immunogens, and flavivirus immunogens.

8. A DNA according to any one of claims 1 to 7, s wherein said heterologous RNA segment encodes an influenza H:\Pcabral\Keep\speci\61757.99.doc 22/08/02 I I 19 immunogen.

9. A DNA according to any one of claims 1 to 8, wherein said heterologous RNA segment encodes an influenza virus hemagglutinin surface protein. A DNA according to any one of claims 1 to 7, wherein said heterologous RNA segment encodes a lentivirus immunogen.

11. A DNA according to any one of claims 1 to 7, wherein said heterologous RNA segment encodes a coronavirus immunogen.

12. A DNA according to any one of claims 1 to 7, wherein said heterologous RNA segment encodes a flavivirus immunogen.

13. A DNA according to any one of claims 1 to 6, 20 wherein said heterologous RNA segment encodes an equine infectious anaemia virus immunogen.

14. An infectious VEE virus RNA transcript encoded by a cDNA of any of Claims 1 to 14. An infectious VEE virus particle containing an infectious VEE virus RNA transcript of claim 14.

16. A pharmaceutical formulation comprising an infectious VEE virus particle according to claim 15 in a Spharmaceutically acceptable carrier.

17. A DNA comprising the cDNA clone of any one of claims 1 to 15, further comprising a second attenuating mutation which is an inactivated E3 amino acid 56 to 59 cleavage recognition site. H;\Pcabral\Keep\speci\61757.99.doc 22/08/02 20

18. A DNA according to claim 17, wherein said E3 amino acid 56 to 59 cleavage recognition site is deleted.

19. A DNA according to claim 17, wherein said E3 amino acid 56 to 59 cleavage recognition site contains an inactivating attenuating mutation. A DNA according to claim 17, wherein said inactivating amino acid 56 to 59 cleavage recognition site contains an arginine to aspartic acid substitution mutation at amino acid 59.

21. An infectious VEE virus RNA transcript encoded by a cDNA clone of any of claims 17 to

22. An infectious VEE virus particle containing a RNA transcript of claim 21.

23. A pharmaceutical formulation comprising an 20 infectious VEE virus particle according to claim 22 in an effective immunogenic amount in a pharmaceutically acceptable carrier.

24. A method of protecting a subject against a disease, comprising administering a pharmaceutical formulation according to claim 16 or claim 23, to said subject in an effective immunogenic amount, said immunogenic protein or peptide encoded by said heterologous RNA segment is effective for protecting said subject from said disease.

25. A method according to claim 24, wherein said administering step is a parenteral administration step.

26. A method according to claim 24, wherein said administering step is carried out by topically applying ST said virus to an airway surface of said subject. H:\Pcabral\Keep\speci\61757.99.doc 22/08/02 21

27. A method according to claim 24, wherein said administering step is an intranasal administration step.

28. A method according to claim 24, wherein said administering step is an inhalation administration step.

29. Use of a DNA according to claim 1, for the manufacture of a medicament for protecting a subject against a disease. Use of an infectious VEE virus transcript according to claim 14, for the manufacture of a medicament for protecting a subject against a disease.

31. A DNA according to claim 1, substantially as herein described with reference to the Examples.

32. An infectious VEE virus RNA transcript according 20 to claim 14, substantially as herein described with reference to the Examples.

33. A pharmaceutical formulation according to claim 23, substantially as herein described with reference to the Examples.

34. A method according to claim 24, substantially as herein described with reference to the Examples. Dated this 22nd day of August 2002 THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H:\Pcabral\Keep\speci\61757.99.doc 22/08/02