EP1226238A2

EP1226238A2 - Method for creating divergent populations of nucleic acid molecules and proteins

Info

Publication number: EP1226238A2
Application number: EP00969738A
Authority: EP
Inventors: Wolfgang A. Renner; Lars Nieba
Original assignee: Cytos Biotechnology AG
Current assignee: Cytos Biotechnology AG
Priority date: 1999-10-27
Filing date: 2000-10-26
Publication date: 2002-07-31
Also published as: WO2001030989A3; AU7938900A; WO2001030989A2

Abstract

The present invention is related to the fields of molecular biology, virology, immunology and medicine. The invention provides methods for creating divergent populations of nucleic acid molecules and proteins, as well as nucleic acid molecules and proteins produced by these methods. The invention also provides processes for producing populations of antigens and antigenic determinants useful in the production of vaccines for the treatment of infectious diseases and as pharmaccine to prevent cancer.

Description

METHOD FOR CREATING DIVERGENT POPULATIONS OF NUCLEIC ACID MOLECULES AND PROTEINS

Background of the Invention

Field of the Invention

The present invention is related to the fields of molecular biology, virology, immunology and medicine. The invention provides methods for creating divergent populations of nucleic acid molecules and proteins, as well as nucleic acid molecules and proteins produced by these methods. The invention also provides processes for producing populations of nucleic acid molecules which encode antigens and antigenic determinants, as well as the antigens and antigenic determinants themselves, useful in the production of vaccines for the treatment of infectious diseases and as pharmaccine for the treatment and prevention of cancer.

Related Art

New vaccines, which take the naturally occurring variability into account the rapidly evolving nature of infectious agents, have to mimic viral, bacterial, and parasitic evolution. This sequence-design process, used in nature, has yielded results far superior to those obtained so far by in vitro rational approaches. Patten, P. et al, Curr. Opinion Biotechnol. 8:724 (1997). Recent technological advances have demonstrated that it is now possible to create large pools of quasispecies of a single protein. However, unlike with natural selection, where multiple environmental forces select organisms with genomes that allow them to meet a variety of challenges, in vitro evolution exerts highly focused selection pressure on organisms in isolation that does not usually reflect the type of pressure exerted by natural selection. The most popular methods to create such in vitro libraries are error-prone PCR or repeated oligonucleotide directed mutagenesis. Caldwell, R.C. et al., PCR Method Applic. 2:28 (1992); You, L. et al, Protein Eng. 9:77 (1994); Reidhaar-Olson, J. et al, Methods Enzymol. 208: 564 ( 1991 ). More recently, DNA shuffling has been shown to be a powerful method for creating large libraries in vitro. Stemmer, W.P.C., Nature 370:389 (1994). These methods have been used to improve several enzymes, such as beta-lactamase (Stemmer, W., Nature 570:389 (1994)), GFP (Crameri, A. etal, Nat. Biotech. 14:315 (1996)), fucosidase (Zhang J. et al, Proc. Natl. Acad. Sci.

94:4504 (1997)), TK (Christians etal, Nat. Biotech. 17:259 (1999)) and esterase (Giver et al, Proc. Natl. Acad. Sci. 95:12809 (1998)). However, all of the methods described above are limited to use in prokaryotic amplification systems. This is a severe limitation, because prokaryotic cells lack the glycosylation machinery present in eukaryotic cells. A further limitation of the in vitro mutagenic methods is the necessity of transformation followed by a transfection, if eukaryotic expression is necessary, resulting in a severe decrease in library size. A direct in vivo mutagenesis method would broaden the current window significantly. Further, many of the highly immunogenic viral proteins, such as the HIV-1 envelope protein gpl60 or hemagglutinin (HA) of Influenza virus, are heavily glycosylated. Thus, it is advantageous to produce such proteins in eukaryotic cells for use in vaccine compositions.

Normally, the immune system is capable of mounting potent attacks on invading viruses, as well as other pathogens, and eliminates many of them by either pathogen-specific T cells or neutralizing antibody responses. Mims, CA,

Pathogenesis of Infectious Disease, Academic, London (1982). However, some viruses may persist despite an immune response by changing CTL-epitopes. McMichael A. et al, Ann. Rev. Immunol. 15:271 (1997); Webster R. et al, Nature 296: 115 (1982); Weiner A. et al., Proc. Natl. Acad. Sci. 92:2755 (1995). Pircher et al, Nature 346:629 (1990). A number of groups have shown, for example, that Lymphocyte Choriomeningitis Virus (LCMV), a negative stranded RNA virus with high natural mutation rate, may escape MHC-dependent immune surveillance in an individual by in vivo selection of viral mutants that are resistant to recognition by cytotoxic T cells. The same phenomenon has been observed with other viruses having a high natural mutation rate, such as HIV, Hepatitis B virus (HBV), and Hepatitis C virus (HCV). McMichael A. et al, Ann. Rev. Immunol. 15:271 (1997); Webster, R.G. et al, Nature 296:1 15 (1982); Weiner, A. et al., Proc. Natl. Acad. Sci. 92:2755 (1995).

Moreover, viruses may form antibody escape mutants (serotype) on a population level, e.g., Influenza virus and Rhinovirus.

HIV

The rapid replication and large population size of HIV in vivo (Ho, D.D. et al, Nature 373:123-126 (1995); Wei, X. et al, Nature 373:1 7-122 (1995)) imply that this virus can be considered an ideal Darwinian population for the purposes of modeling genetic variation. Coffin, J. et al, Curr. Top. Microbiol. Immunol. 176: 143 (1992). Although the single-cycle mutation rate for HIV is not known, precedent from other retroviruses suggest that it probably lies around 10^"5 per base per replication cycle. Klarmann, G. et al, J. Biol. Chem. 268:9793 (1993); Ricchetti, M. et al, EMBO J. 9:1583 (1990). Given in vivo replication kinetics with more than 10⁹ new cells infected every day and an HIV-1 genome size of approximately 10⁴ bases, the probabilities indicate that each and every possible single point mutation could occur between 10⁴ and 10⁵ times per day in an HIV-infected individual.

The influence of small selective forces in population dynamics is so strong that no mutation can be assumed to be truly neutral. Coffin, J. et al., Curr. Top. Microbiol. Immunol. 176: 43 (1992). The pattern in which mutations accumulate also renders unlikely models of pathogenesis that rely on the appearance of some specific mutations in HIV for the progression to late stage disease. Nowak, M. etal, Science 254:963 (1991); Tersmette, M. etal, J. Virol. 63 (1989); McMichael, A. et al, Ann. Rev. Immunol. 15:27 (1997). This is so because even extremely complex patterns of mutations will appear in the HIV population quite early in the infection process. Thus, the failure of such viruses to become dominant in the population early in the infection presumably reflects their selective disadvantage at that time. By the same token, the appearance of such mutants later in disease stages presumably reflects the changing selective environment in the infected host. Further, these viruses could not appear to contribute to pathogenesis and are more likely a symptom of the end stage than its cause. Coffin, J., Science 2(57:483 (1995).

To date, HIV resistant mutants have been identified which have partial or complete resistance to all compounds used or seriously considered for HIV therapy. Schinazi, R. et al, Int. Antiviral News 2:72 (1994); Tantillo, J. et al, J.

Mol. Biol. 243:364 (1994). Further, such mutations may be an inevitable consequence of antiviral therapy. The pattern of disappearance of wild-type virus and appearance of mutant virus, after treatment, combined with the coincident decline in CD4 cell numbers, strongly imply that, at least with some compounds, the appearance of mutant virus is the major cause underlying failure of therapy.

Coffin, J., Science 267:483 (1995). The virtually inevitable and rapid occurrence of mutations which confer resistance to therapeutic agents has strong implications for drug design and application.

Because the wild-type virus remains for only a short time after the onset of treatment, no matter how effective the compound is at inhibiting replication of the virus clinical benefit of this compound is likely to be highly transitory. Coffin, J., Science 267:483 (1995). This consideration implies that sound strategies for design of the next generation of antiviral therapies or antiviral vaccines will include the screening of promising compounds effective against all potential mutants of the particular virus.

Influenza Virus

Influenza A viruses periodically cause epidemics in humans, horses, pigs, birds and occasionally in other animals, such as mink, whales and seals. A hallmark of these viruses is their variability both in antigenicity and pathogenicity. The so-called antigenic drift occurs by mutations in the immunodominant genes, leading to an accumulation of amino acid sequence changes which alter the antigenic sites so that they are no longer recognized by the host's immune system. For example, the changes observed in hemagglutinin (HA) are distributed over the entire protein. Webster, R. et al, Nature 296:115-121 (1982); Webster, R. et al, Cell 50:665-666 (1987). The single-cycle mutation rate for Influenza virus is estimated 10^"5 per base per replication cycle. Variants of the HA molecule which have changes in their amino acid sequence can have sufficient selective survival advantages in a population having immunity to Influenza A virus to produce an epidemiological impact. As a result of the ever occurring genetic changes in Influenza A virus, in order to protect populations against this virus, current vaccines must be given annually. Kennedy, M., Nurse Pract. 17:21 (1998). To facilitate world wide vaccination, the next generation vaccine should include a range of possible mutations of Influenza A virus which can be generated by genetic drift.

Summary of the Invention

The present invention provides methods for creating divergent populations of nucleic acid molecules and proteins, as well as products derived from these methods. More specifically, the invention relates to increasing the error rates which occur during the replication of nucleic acids to obtain mixed populations of divergent nucleic acid molecules and expression products.

In one general aspect, the present invention provides methods for producing modified nucleic acid molecules which result from the replication or production of RΝA in the presence of one or more agents (e.g., nucleoside analogs) or under conditions (e.g. , exposure to ionizing radiation) which increase mutation rates.

In one specific aspect, the invention provides methods for preparing populations of viral vectors comprising:

(a) inserting a gene of interest into an alphaviral vector;

(b) replicating the alphaviral vector in the presence of an alphaviral replicase and one or more (e.g., two, three, four, five, six, or more) nucleoside analogs to produce modified genes of interest; and

(c) repeating step (b) for a sufficient number of times that the nucleotide sequences of the modified genes of interest in at least 90% of the members of the population which contain at least one mutation in the gene of interest are 90-95%, 90-99%, 95-99%, 97-99%, or 95-97% identical to the nucleotide sequence of the gene of interest.

In another aspect, the invention provides methods for preparing populations of viral vectors comprising: (a) inserting a gene of interest into an alphaviral vector;

(b) replicating the alphaviral vector in the presence of an alphaviral replicase and one or more nucleoside analogs to produce modified genes of interest; and

(c) repeating step (b) for a sufficient number of times that the modified genes of interest encode polypeptides which are 90-95%, 90-99%,

95-99%, 97-99%, 90-99.5%, 95-99.5%, 97-99.5%, or 95-97% identical to the polypeptide encoded by the gene of interest.

Further, the populations of viral vectors generated by the methods described above, may be produced by any number of means. For example, replication can be catalyzed by alphaviral replicases having varying functional properties. Examples of such replicases include wild-type and temperature sensitive, non-cytopathic alphaviral replicases. In most instances, however, the vectors are replicated in the presence of a temperature sensitive, non-cytopathic, alphaviral replicase and one or more nucleoside analogs. These vectors may be produced, for example, in prokaryotic (e. g. , bacterial) or eukaryotic (e. g. , human) cells. Specific eukaryotic cells suitable for use with the present invention include baby hamster kidney (BHK), Chinese hamster ovary (CHO), and COS cells.

In one specific aspect, a nucleic acid molecule which encodes the alphaviral replicase (e.g., a temperature sensitive, non-cytopathic, alphaviral replicase) is chromosomally integrated into the genome of a cell line used to produce the population of viral vectors.

The alphaviral replicase may also be encoded either by the members of the population of viral vectors or by a separate vector (e.g., a plasmid or double minute). In another specific aspect, the populations of viral vectors may be prepared in the presence of one or more specific nucleoside analogs, such as 5-azacytidine (AZT), 5-fluorouridine (5-FU), 5-hydroxy-2'deoxycytidine, Mitomycin C, furyl-furamide, 4-nitroquinolinel -oxide, N-methyl-N'-nitro-N- n i tro s o gu ani d i n e , N 4 - am ino cyti d ine , 8 - o xo - g uano s i ne , N l -methyl-N4-aminocytidine, 3-methylcytidine, 5-bromocytidine, 5-nitrosocytidine, 3-methyluridine, O4-isobutyluridine, 3-methyladenosine,

8-hydroxylguanosine, and N6-methyladenosine.

The invention further provides populations of alphaviral vectors which encode an alphaviral replicase, wherein at least 90% of the members of the population which contain at least one mutation in the gene of interest comprise modified genes of interest having nucleotide sequences which are 90-95%,

90-99%, 95-99%, 97-99%, or 95-97% identical to the nucleotide sequence of a gene of interest.

The invention also provides populations of alphaviral vectors which encode alphaviral replicases and comprise modified genes of interest which encode polypeptides 90-95%, 90-99%, 95-99%, 97-99%, 90-99.5%, 95-99.5%,

97-99.5%, or 95-97% identical to the polypeptide encoded by a gene of interest.

In a related aspect, the alphaviral vectors of the invention do not encode an alphaviral replicase. In this aspect, replicase expression, when needed, is provided by a separate nucleic acid molecule. The invention also provides populations of vectors prepared by a method comprising:

(a) inserting a gene of interest into an alphaviral vector;

(c) repeating step (b) for a sufficient number of times that the nucleotide sequences of the modified genes of interest in at least 90% of the members of the populations which contain at least one mutation in the gene of interest are 90-95%, 90-99%, 95-99%, 97-99%, or 95-97% identical to the nucleotide sequence of the gene of interest. In one related aspect, the gene of interest encodes a polypeptide. In another related aspect, the gene of interest encodes an untranslated RNA.

The population of alphaviral vectors may further comprise portions of the Semliki Forest Virus, Sindbis virus, Venezuelan equine encephalomyelitis virus, or Ross River Virus genomes.

In another related aspect, the members of the population of alphaviral vectors are packaged.

In one specific aspect, the population of alphaviral vectors contain a gene of interest derived from Lymphocyte Choriomeningitis Virus (LCMV), Influenza virus, Human Immunodeficiency Virus Type 1 (HIV-1), Human

Immunodeficiency Virus Type 2 (HIV-2), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Hepatitis E Virus (HEV), Hepatitis G Virus (HGV), Rhinovirus, or a species of Trypanosoma (e.g. , Trypanosoma congolense, Trypanosoma vivax, Trypanosoma cruzi, Trypanosoma brucei) or Plasmodium (e.g., Plasmodium falciparum, Plasmodium vivax).

In a further aspect, the population of alphaviral vectors is administered to an individual in admixture with a pharmaceutically acceptable diluent, adjuvant, or carrier as part of a pharmaceutical composition. In a related aspect, this pharmaceutical composition is suitable for use as a vaccine. In yet another aspect, the invention provides methods for vaccinating individuals comprising administering to these individuals pharmaceutically effective amounts of populations of alphaviral vectors which encode alphaviral replicases, wherein the nucleotide sequences of the modified genes of interest in at least 90% of the members of the populations which contain at least one mutation in the gene of interest are 90-95%, 90-99%, 95-99%, 97-99%, or

95-97%) identical to the nucleotide sequence of the gene of interest.

In another aspect, the invention provides methods for vaccinating individuals comprising administering to these individuals pharmaceutically effective amounts of populations of alphaviral vectors which encode alphaviral replicases and comprise modified genes of interest which encode polypeptides 90-95%, 90-99%, 95-99%, 97-99%, 90-99.5%, 95-99.5%, 97-99.5%, or 95-97% identical to the polypeptide encoded by a gene of interest.

Further, the population of alphaviral vectors which is administered to individuals may further comprise portions of the Semliki Forest Virus, Sindbis virus, Venezuelan equine encephalomyelitis virus, or Ross River Virus genomes.

In one specific aspect, the population of alphaviral vectors may contain a gene of interest derived from Rhinovirus, Lymphocyte Choriomeningitis Virus

(LCMV), Influenza virus, Human Immunodeficiency Virus Type 1 (HIV-1),

Human Immunodeficiency Virus Type 2 (HIV-2), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Hepatitis E Virus (HEV), Hepatitis G Virus (HGV), or a species of Trypanosoma (e.g., Trypanosoma congolense, Trypanosoma vivax,

Trypanosoma cruzi, Trypanosoma brucei) or Plasmodium (e.g., Plasmodium falciparum, Plasmodium vivax). In an even more specific aspect, the gene of interest encodes glycoprotein 120 (gpl20), glycoprotein 140 (gpl40), or glycoprotein 160 (gpl60) of HIV-1.

In another specific aspect, the members of the population of alphaviral vectors administered to individuals are packaged.

In yet another aspect, the invention provides methods for vaccinating individuals comprising administering to these individuals pharmaceutically effective amounts of polypeptide expression products of populations of viral vectors, wherein these polypeptide expression products are produced by:

(a) inserting a gene of interest into an alphaviral vector;

(b) replicating the alphaviral vector in the presence of an alphaviral replicase and one or more nucleoside analogs to produce modified genes of interest;

(c) repeating step (b) for a sufficient number of times that the modified genes of interest encode polypeptides which are 90-95%, 90-99%, 95-99%, 97-99%, 90-99.5%, 95-99.5%, 97-99.5%, or 95-97% identical to the polypeptide encoded by the gene of interest; (d) inserting the alphaviral vector containing the modified genes of interest into host cells; (e) expressing the modified genes of interest to produce polypeptide expression products; and

(f) recovering the polypeptide expression products. Further, the population of alphaviral vectors which is administered to individuals may further comprise portions of the Semliki Forest Virus, Sindbis virus, Venezuelan equine encephalomyelitis virus, or Ross River Virus genomes.

In one specific aspect, the population of alphaviral vectors may contain a gene of interest derived from Rhino virus, Lymphocyte Choriomeningitis Virus

(LCMV), Influenza virus, Human Immunodeficiency Virus Type 1 (HIV-1), Human Immunodeficiency Virus Type 2 (HIV-2), Hepatitis B Virus (HBV),

Hepatitis C Virus (HCV), Hepatitis E Virus (HEV), Hepatitis G Virus (HGV), or ^' a species of Trypanosoma (e.g., Trypanosoma congolense, Trypanosoma vivax, Trypanosoma cruzi, Trypanosoma brucei) or Plasmodium (e.g., Plasmodium falciparum, Plasmodium vivax). In an even more specific aspect, the gene of interest encodes glycoprotein 120 (gp 120), glycoprotein (gp 140), or glycoprotein

160 (gpl60) of HIV-1. Further, the gene of interest may also encode hemagglutinin protein of Influenza virus or the hemagglutinin HA1 domain.

In each embodiment of the invention, the gene of interest and the alphavirus replicase may be encoded by two separate vectors or polynucleotides.

Brief Description of the Figures

FIG. 1. The DNA of pCYTts (1) is inserted into the nucleus. The eukaryotic promoter (solid horizontal arrow) drives transcription (2) into mRNA (3). Translation (4) of the first open reading frame (ORF) of the mRNA results in the production of a temperature-sensitive replicase (ts-replicase protein) (5). The second open ORF encoding the gene of interest is not accessible to ribosomes. Thus no translation (6) of the gene of interest occurs. At low temperature the ts-replicase catalyzes replication (7) of the mRNA (3) into full- length (-) strand RNA (8). The ts-replicase also catalyzes subsequent replications (9, 10) into full-length (+) strand RNA (11) and subgenomic RNA (12). The ≤ubgenomic RNA (12) is then translated (13) into the protein of interest (not shown). The combination of amplification and qualitati\ e change of the RNA results in unprecedented tightness and regulatability of the expression of the gene of interest. Abbreviations in FIG. 1 are as follows: Rous Sarcoma Virus promoter

(RSN pr.), cw-acting sequence elements (CSE), non-structural proteins 1 -4 (nsP 1 , nsP2, nsP3, nsP4), gene of interest (G.O.I.), and subgenomic promoter (S.G.). FIG. 2 is a schematic representation of the pCYTts vector. The pCYTts vector contains, in addition to the elements shown in FIG. 1, an ampicillin resistance marker for selection in bacterial cells and a ColEl sequence which directs high copy number bacterial amplification. The pCYTts vector was prepared as described in Example 1.

FIG. 3A-3D shows the complete cDΝA sequence of pCYTts (SEQ ID

ΝO:l). FIG.4A-4E shows the nucleotide sequence of the pSinRep5 vector (SEQ

ID NO:2).

FIG. 5 is a schematic representation of the pSinRep5 vector. Restriction sites which can be used to insert genes of interest in operable linkage with a subgenomic promoter are indicated. FIG. 6A-6B show agarose gels stained with ethidium bromide. FIG. 6A, lane 1, contained an amplified chloramphenicol acetyltransferase (CAT) gene.

FIG. 6B, lane 1, contained an EcøRI digest of the amplified CAT gene. Lane 2 in each panel of FIG. 6A-6B contained DNA markers.

FIG.7A-7B show agarose gels stained with ethidium bromide. FIG. 7A, lane 1 , contained DNA markers. FIG.7A, lanes 2-4 contained PCR amplification products of the ΕPO gene. The lanes of FIG. 7B contained the following: lanes

1-2: Kpnl/Stul double digests of the amplified ΕPO gene (FIG. 7A, lane 2); lane

3 : undigested control (FIG.7A, lane 2); lanes 4-5 : Kpnl/Stul double digests of the amplified ΕPO gene (FIG. 7A, lane 3); lane 6: undigested control (FIG.7A, lane 3); lanes 7-8: Kpnl/Stul double digests of the amplified ΕPO gene (FIG.7 A, lane

4); lane 9: undigested control (FIG. 7 A, lane 4). Detailed Description of the Preferred Embodiments

The present invention provides a versatile new technology which allows for the production of nucleic acid and protein quasispecies. These quasispecies have identical nucleic acid or protein origins, but differ in their nucleotide or amino acid sequences. The methods of the invention thus allow for the mimicking of the natural evolution of molecules, while being faster in the creation of mutated molecular species than many previously described methods.

Further, when used in combination with alphaviral vaccine technologies, the methods of the present invention allow for the creation of highly efficient vaccines against a vast number of infectious diseases. Representative alphaviral vaccine technologies suitable for use in preparing vaccines of the invention are disclosed below; in PCT publication WO 00/32227; and in U.S. Appl. Nos.

60/1 10,414, 60/142,788, 09/449,631, and 60/202,341, respectively filed on

November 30, 1998, July 8, 1999, November 30, 1999, and May 5, 2000, each of which (including PCT publication WO 00/32227) are incorporated herein by reference.

In brief, the present invention provides methods for creating divergent populations of nucleic acid molecules and proteins, as well as products derived from these methods. More specifically, methods are provided for preparing populations of vectors which contain variant forms of a gene of interest, referred to herein as "modified genes of interest." Further provided are polynucleotides produced by these methods, as well as the expression products of these polynucleotides (e.g., RNAs and polypeptides).

In one general aspect, the present invention is directed to methods for inducing mutations in nucleic acid molecules which employ nucleoside analogs, as well as other agents and conditions, that increase the error rate naturally present during nucleic acid replication and synthesis.

About 30 years ago, nucleoside analogs were used to introduce temperature sensitive mutants in RSV (Linial et al, "RNA tumor viruses," in Weiss et al, eds., Cold Spring Harbor, N.Y. (1982), pp. 649-783), MSV (Linial et al, "RNA tumor viruses," in Weiss et al, eds., Cold Spring Harbor, N.Y. (1982), pp. 649-783), Sin (Burge, B. etal, Virology 30:203 (1966)), SF (Strauss, J. et al, Microbiol. Rev. 58:491 (1994)) and others (Halle, S., J. Virol. 2:1228 (1968)) and as potential chemotherapeutic against leukemia (Karon, M. et al, Blood 42:359 (1963)).

5-Azacytidine (AZT), for example, is incorporated into RNA and induces transitions and transversions. Further, this nucleoside analog can increase the mutation rate during retroviral replication by more than 10-fold. Pathak, V. et al. , J. Virol. 66:3093 (1992). A number of other nucleoside analogs have been described which are potent mutagens. Watanabe et al, Mutat. Res. 314:39

(1994).

The present invention is not directed to the use of nucleoside analogs for the treatment of diseases, but instead takes advantage of the mutagenicity of these compounds. In general, when eukaryotic cells carrying a gene of interest replicate their nucleic acid in the presence of nucleoside analogs (e.g., AZT,

5-FU), the gene of interest will be randomly mutated and the result is a pool of quasispecies. However, due to proofreading activity, the mutation rate of the eukaryotic transcription machinery is very low in general.

Mutation rates in eukaryotic cells can be increased, for example, by the use of alphaviral vectors which use replicases to replicate their RNA. The replication of the gene of interest is therefore controlled by a replicase, for which the spontaneous mutation frequency can be increased at least 200-fold by AZT treatment. Halle, S., J. Virol. 2:1228-1229 (1968). Since the alphaviral replicases, similar to HIV or Influenza virus, do not have any proofreading activity, it is possible to mimic the natural mutation and decrease the time required for creating a large pool of quasispecies. In general, the present invention can be used for any purpose where a pool of quasispecies occurs or is desired. For example, vaccines of the present invention can be used for the treatment and prevention of diseases caused by viral escape mutants. As shown below in Example 3, large differences in the amino acid sequence of a polypeptide can be induced using the methods of the present invention.

One advantage of the present invention is that it provides an in vivo method for mutagenizing nucleic acid molecules wherein polypeptide expression products of the nucleic acid molecules are correctly glycosylated.

Definitions

The following definitions are provided to clarify the subject matter which the inventors consider to be the present invention. Unless otherwise defined herein, the terms used throughout the specification have the meaning commonly associated with them by those practicing in the field of virology and molecular biology.

As used herein, the term "RNA virus" refers to a virus which has packaged RNA. These viruses either (1) convert the nucleic acids of all or part of their s from RNA to DNA during at least one phase of their life cycle or

(2) replicate their nucleic acids as RNA without ever converting all or part of their genomes to DNA. Examples of RNA viruses include retroviruses and alphaviruses. Examples of retroviruses include human immunodeficiency viruses I, II and III, foamy viruses (FVs), human endogenous retroviruses (HERVs), porcine endogenous retrovirus (PERVs), murine endogenous retroviruses

(MERVs), Gibbon Ape Leukemia Virus (GALV), and murine leukemia virus (MLV).

As used herein, the term "alphavirus" refers to any of the RNA viruses included within the genus Alphavirus. Descriptions of the members of this genus are contained in Strauss and Strauss, Microbiol. Rev., 5S:491-562 (1994).

Examples of alphaviruses include Aura virus, Bebaru virus, Cabassou virus, Chikungunya virus, Easter equine encephalomyelitis virus, Fort morgan virus, Getah virus, Kyzylagach virus, Mayoaro virus, Middleburg virus, Mucambo virus, Ndumu virus, Pixuna virus, Tonate virus, Triniti virus, Una virus, Western equine encephalomyelitis virus, Whataroa virus, Sindbis virus (SIN), Semliki forest virus (SFV), Venezuelan equine encephalomyelitis virus

(VEE), and Ross River Virus (RRV).

As used herein, the phrase "gene of interest" refers to a polynucleotide which is inserted in a viral vector (i.e., is heterologous to the viral vector) and then modified at the nucleotide level by the methods of the present invention. In most instances the gene of interest will encode an RNA or polypeptide expression product.

As used herein, the phrase "expression product" refers to the RNA or polypeptide product encoded by a gene of interest. Specific examples of expression products include untranslated RNAs and polypeptides, such as glycoprotein 120 (gpl20), glycoprotein 140 (gpl40), and glycoprotein 160

(gp 160) of HIV-1.

As used herein, the phrase "untranslated RNA" refers to an RNA which either does not encode an open reading frame or encodes an open reading frame, or portion thereof, but in a format in which a polypeptide will not be produced

(e.g., no initiation codon is present). Examples of untranslated RNAs include tRNA molecules, rRNA molecules, antisense RNA molecules, and ribozymes. Antisense RNA is not translated to produce a polypeptide. However, in some instances, antisense RNA can be converted to a translatable sense strand (e.g. , via the action of a replicase) and a polypeptide may then be produced.

As used herein, the term "replicase" refers to a polymerase which catalyzes the production of an RNA molecule from another RNA molecule. This term is used herein synonymously with the phrase "RNA-Dependent RNA polymerase." As used herein, the phrase "temperature-sensitive" refers to a variant form of an enzyme which has altered temperature requirements as compared to the wild-type form. For example, a temperature- sensitive enzyme may have low catalytic activity at a temperature where the wild-type form of the same enzyme has high catalytic activity. An example of a temperature-sensitive enzyme is the replicase protein encoded by the pCYTts vector, which has readily detectable replicase activity at temperatures below 34 °C and has low or undetectable activity at 37°C. The pCYTts vector is described in PCT/IB99/00523, filed March 25, 1999, which is incoφorated herein by reference.

As used herein, the term "recombinant host cell" refers to a host cell into which one or more nucleic acid molecules of the invention have been introduced. As used herein, the phrase "non-infective packaged RNA molecules" refers to packaged RNA molecules which will essentially undergo only one round of host cell infection and are not pathogenic. These molecules are thus "infective" but only for a single infectious entry into a host cell and are not capable of reproducing to form additional infectious particles. As used herein, the term "vector" refers to an agent (e.g., a plasmid or virus) used to transmit genetic material to a host cell. A vector may be composed of either DNA or RNA. Further, the term "alphaviral vector" refers to a vector which contains nucleic acid from an alphavirus and can replicate its nucleic acid as RNA. The alphaviral vector itself, however, may in one form comprise DNA. As used herein, the term "population" refers to a group of nucleic acid molecules (e.g., viral vectors or particles) or polypeptides, wherein members of the group have related but differing sequences. Of course, two or more specific members of the population may comprise nucleic acid or polypeptides which are identical to each other. The members of a "population" will generally be variants derived from one original nucleic acid molecule or polypeptide. One example of a population of nucleic acid molecules is a group of alphaviral vectors which have undergone one or more rounds of replication catalyzed by an RNA replicase in the presence of a sufficient quantity of one or more nucleoside analogs to induce mutations in members of the group.

As used herein, the term "quasispecies" refers to a population of viral vectors, nucleic acids, or polypeptides produced by the methods of the present invention.

As used herein, the term "mutation" refers to an alteration in the nucleotide sequence (e.g., substitution, deletion, or insertion) of a nucleic acid molecule. When the nucleic acid which is the subject of the mutation encodes a polypeptide, the alteration can result in a silent change, the introduction of a frame shift, the insertion of a stop codon, or the replacement of one codon with another codon that encodes the same or different amino acid residue. As explained below, depending on the actual circumstances, it will generally be advantageous when practicing the present invention to introduce mutations which do not result in the introduction of a stop codons or frame shifts in the coding region of the gene of interest.

As used herein, the term "mutation rate" refers to the overall rate in which mutations are introduced into a nucleic acid molecule. This rate may be defined by a number of parameters but is generally defined in terms of alterations per a specified number of bases per replication cycle. For example, as already noted, current data suggests that the mutation rate for HIV and Influenza virus is about 10^"5 per base per replication cycle. Mutation rate may also be measured by determining the number of alterations which occur during each replication cycle. As used herein, the term "nucleoside analog" refers to a molecule which sufficiently resembles a naturally occurring nucleoside (/. e. , adenosine, cytidine, guanosine, thymidine, uridine) to be incorporated into a polynucleotide chain during nucleic acid replication. This term also includes compounds which resemble nucleoside analogs structurally and are modified in vivo to a form which allows for their incoφorated into a polynucleotide chain during nucleic acid replication, as well as compounds which can be used a building blocks for either direct synthesis of RNA or synthesis of RNA after in vivo modification by a viral replicase. Specific examples of nucleoside analogs are set out below.

As used herein, the term "individual" refers to all animals which have immune systems. These animals include vertebrates, humans, and domesticated animals (e.g., horses, camels, pigs, cattle, mice, rats , rabbits, birds, reptiles, and fish). Methods for Producing Divergent Alphaviral Vectors

In general, the present invention is directed in part to methods for preparing populations of viral vectors. These methods involve the replication of viral vectors in the presence of one or more agents, or under conditions, which increase mutation rate during replication of nucleic acids. Examples of suitable agents for increasing mutation rates include nucleoside analogs and other compounds which bind to nucleic acid molecules (e.g. , ethidium bromide, S YBR Green I stain). See Singer et al, Mutat. Res. 439:37-47 (1999).

As shown below in Example 3, nucleoside analogs can be used to induce relatively high levels of mutations in a gene of interest during nucleic acid replication. The mutation rate found using the alphaviral vector system disclosed in Example 3 is approximately 20 times higher than when replication proceeds in the absence of the AZT.

Examples of nucleoside analogs suitable for use in the methods of the present invention include 5-azacytidine (AZT), 5-fluorouridine (5-FU),

5-hydroxy-2'-deoxycytidine, 4-nitroquinolinel -oxide, furyl-furamide, mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, N4-aminocytidine, 2', 3'- dideoxycytidine (ddC), 2', 3'-dideoxyinosine (ddl), Videx), and 2', 3'-didehydro- 2', 3'-dideoxythymine, 8-oxo-guanosine, Nl-methyl-N4-aminocytidine, 3-methylcytidine, 5-bromocytidine, 5-nitrosocytidine, 3-methyluridine,

O4-isobutyluridine, 3-methyladenosine, 8-hydroxylguanosine, and N6-methyladenosine. Additional nucleoside analogs suitable for use in the practice of the present invention are disclosed in Arimilli et al, U.S. Patent No. 5,886,179, Cook et al, U.S. Patent No. 5,914,396, United States Patent 5,902,881 , Cheruvallath et al, U.S. Patent No. 5,902,881 , Hosteller, U.S. Patent

No. 5,879,700, Marquez et al, U.S. Patent No. 5,869,666, Beauchamp, U.S. Patent No. 5,043,339, and Harnden et al, U.S. Patent No. 5,166,198, each of which are incoφorated herein by reference.

Another method for increasing mutation rates during nucleic acid replication involves the induction of deoxyribonucleoside triphosphate (dNTP) pool imbalances. Julias et al, J. Virol. 72:7941-79419 (1998), for example, report that dNTP pool imbalances are associated with increase rates of misincorporation and hypermutation during in vitro reverse transcription reactions. Further, it is known that treatment of cells with thymidine and hydroxyurea induces significant dNTP pool imbalances. Thus, the present invention includes the use of compounds which alter dNTP pools and increase error rates during nucleic acid replication. In particular, the present invention includes the use of thymine, hydroxyurea, and other agents which alter dNTP pools to increase mutation rates during nucleic acid replication.

Nucleoside pool imbalances can also be used to induce to increase error rates which normally occur during the replication of RNA.

It is also known that exposure of nucleic acid molecules to radiation (e.g. , gamma radiation, ultraviolet light) can increase mutation rates. Colussi et al, Mutat. Res. 401:89-97 (1998), Amundson et al, Int. J. Radiat. Biol (59:555-563 (1996). The methods of the invention thus include the use of radiation to increase the mutation rate normally associated with the replication of nucleic acid molecules.

Free radicals produced by ionizing radiation or generated by chemical systems are known in the art to damage nucleic acid molecules and can result in the induction of mutations. Another method for increasing mutation rates involves the exposure of nucleic acid molecules to free radicals. While Hirst,

Br. Dent. J. 170:115-117 (1991) obtained negative data regarding mutagenesis using a Salmonella typhimurium assay, Hirst suggested that free radicals produced by ultrasonic emissions could increase mutation rates. The methods of the invention thus include the use of free radicals to increase the mutation rate normally associated with the replication of nucleic acid molecules.

Alkylating agents, such as N-methyl-N-nitrosourea, have been known for some time to induce mutations in nucleic acid molecules. Hince et al, Mutat. Res. 46: 1-10 (1977). The methods of the invention thus include the use of alkylating agents to increase the mutation rate normally associated with the replication of nucleic acid molecules. Intercalating agents, such as ethidium bromide, as well as other agents which bind to nucleic acid molecules, have been shown to have mutagenic activity. For example, S YBR Green I stain, a non- intercalating nucleic acid stain, has been shown using the Ames test to induce mutations. Singer et al, Mutat. Res. 439:37-47 (1999). Thus, the methods of the invention also include the use of nucleic acid binding agents (e.g., intercalating agents and other agents which bind to nucleic acids) to increase the mutation rate normally associated with the replication of nucleic acid molecules.

The methods of the present invention can be practiced with virtually any agent or under any conditions which increase the mutation rate of nucleic acid molecules during replication. Thus, in one aspect, the invention is directed to methods for inducing mutations in nucleic acid molecules involving the replication of these molecules in the presence of an agent or under conditions which increase the rate at which mutations occur during replication. One skilled in the art would understand how to adjust the concentrations of the mutagenic agent or the particular conditions to achieve a desired mutation rate. For example, when ionizing radiation is used to produce mutagenized populations of vectors, the intensity of the radiation or duration of exposure can be adjusted to induce a particular number of mutations per base per replication cycle (e.g., 10^"4). Similarly, when nucleoside analogs are used to induce mutations in replicating vectors, the concentrations of these analogs or the exposure time can be adjusted to induce a particular number of mutations per base per replication cycle. One example of the use of nucleoside analogs to induce mutations in vectors is disclosed in Example 3. As one skilled in the art would appreciate, an inevitable consequence of mutating the viral vectors of the invention is that some of these vectors will cease to function in a manner required for their propagation and/or expression of the gene of interest. For example, essential components required for either vector replication or transcription of the gene of interest can be mutated and become non-functional. The end result in either instance is that the expression product of the particular modified gene of interest will not be produced. Various methods, examples of which are set out below, can be employed to lessen the deleterious effects of induced mutations on the vectors containing the gene of interest.

In one embodiment, the number of essential elements of the viral vectors required for vector replication and transcription of the gene of interest or the modified genes of interest are kept to a minimum. Depending on the particular applications of the vector, the enzyme which catalyzes replication of the nucleic acid (e.g., replicase) may be encoded by nucleic acid other than that of the vectors itself (e.g. , integrated into the host cell genome, contained on a separate vector). This strategy exposes fewer vector components to the mutagenic process.

In a preferred embodiment, the viral vector used with the methods of the invention is an alphaviral vector, such as the pCYTts vector shown in FIGs. 1-3. As shown in FIG. 1, the RNA form of this vector is encoded by DNA. The pCYTts vector encodes a temperature sensitive, non-cytopathic replicase which both catalyzes the production of full-length genomic RNA and mRNA of the gene of interest (or modified genes of interest) driven by the subgenomic promoter.

Mutations are known in the art which render the replicase protein non-cytopathic. See Weiss et al, J. Virol. 33:463-474 (1980); Dryga et al, Virology 225:74-83 (1997). One such mutation results from the exchange of the proline residue at position 726 to another of the 20 natural occurring amino acids, such as a serine (abbreviated as "Pro 726 Ser"). These mutations may be introduced by a number of means, including site directed mutagenesis.

The creation and the identification of mutations which render the Sindbis replicase non-cytopathic are described in more detail elsewhere. Weiss et al, J.

Virol. 33:463-474 (1980); Dryga et al., Virology 228:74-83 (1997); patent application WO 97/38087. Further, methods for inducing such mutations are known in the art. See, e.g., Sambrook, J. et al, eds., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel, F. et al, eds., CURRENT PROTOCOLS IN

MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997). Temperature sensitivity (ts) may be conferred by the introduction of a mutation in the nsP4 gene of the replicase. Preferably, mutations which confer a temperature-sensitive phenotype upon replicase activities are in a protein in complementation group F. Lemm et al, J. Virol. (5*^:3001-3011 (1990). For example, a temperature-sensitive phenotype may be conferred by changing Gly

153 ofnsP4 to Glu.

Additionally, any other mutation which renders replicase activity temperature-sensitive can be used in the practice of the invention. Methods for creating and identifying new temperature-sensitive mutants are described by Burge and Pfefferkorn. Burge and Pfefferkorn, Virol. 30:204-213 (1966); Burge and Pfefferkorn, Virol 30:214-223 (1966).

The modified genes of interest may be excised from the viral vectors following one or more rounds of mutagenesis and reinserted into a viral vector which has not undergone mutagenesis according to the methods of the invention. In other words, a population of modified genes of interest can be excised from viral vectors which has undergone mutagenesis and inserted into a vector which has not undergone mutagenesis. Depending on the use of the modified genes of interest, the second vector may be a vector having components derived from an RNA virus. In most instances, when a population of modified genes of interest is to be excised from a viral vector subsequent to mutagenesis, the viral vector will be reverse transcribed to cDNA prior to excision. Further, once the population of modified genes of interest is reinserted into another vector, it may be desirable to maintain these vectors as DNA. Maintaining these vectors in a DNA format allows for the replication of the vector, and the population of modified genes of interest, in the absence of considerable numbers of additional mutations. The lack of considerable numbers of additional mutations may be advantageous when batch consistency is desired, for example, when vaccine compositions are produced comprising populations of modified genes of interest or their expression products. In general, the conditions for producing the vectors of the invention will be adjusted such that the population of divergent vectors will contain modified genes of interest having, on average, a specified number of mutations per unit number of bases. The specific level of mutations desired can be achieved during one or more nucleic acid replication cycles.

More specifically, conditions can be adjusted such that a specific percentage of the viral vectors will contain modified genes of interest in which a certain percentage of nucleotides of this gene have been altered by insertion, deletion or substitution. In one embodiment, for example, at least 90% of the members of the total population of alphaviral vectors will contain modified genes of interest having nucleotide sequences which are 95-99% identical to the nucleotide sequence of the gene of interest.

In another embodiment, at least 90% of the members of the population of alphaviral vectors which contain at least one mutation (e.g., one, two three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, or thirty) in the gene of interest will contain modified genes of interest having nucleotide sequences which are 95-99% identical to the nucleotide sequence of the gene of interest.

As one skilled in the art would appreciate, the percentage of the members of the population of viral vectors (e.g. , alphaviral vectors) which contain modified genes of interest having the specified identity to the nucleotide sequence of the gene of interest can vary with the particular application for which the vectors are used. In general, however, the percentage of the total viral vector population having the specified identity to the nucleotide sequence of the gene of interest will be at least 70%, 73%, 75%, 78%, 80%, 83%, 85%, 88%, 90%, 93%, 95%,

97%) or 99%, as well as intervening whole number and fraction numbers. Similarly, in general, the percentage of the viral vector population which contains at least one mutation in the gene of interest having the specified identity to the nucleotide sequence of the gene of interest will be at least 70%, 73%), 75%, 78%, 80%, 83%, 85%, 88%, 90%, 93%, 95%, 97% or 99%, as well as intervening whole number and fraction numbers. Further, conditions can be adjusted to obtain vectors wherein the percent identity between a specified percentage of the modified genes of interest and the gene of interest is between 80-99%, 90-95%, 90-99%, 95-99%, 95-97%, 97-99%, or 98-99%. Conditions can also be adjusted to obtain vectors wherein the percent identity between a specified percentage of the modified genes of interest and the gene of interest varies between lower and upper ranges having the following values: 70%, 73%, 75%, 78%, 80%, 83%, 85%, 88%, 90%, 93%, 95%, 97% or 99%. Of course, the value of the lower range must be lower than the value of the upper range. One example of such a range would be 80-93%) and, thus, the percent identity between a specified percentage of the modified genes of interest and the gene of interest would be between 80-93%.

Similarly, conditions can be adjusted to obtain vectors wherein the percent identity between the expression product the modified genes of interest, or a specified percentage thereof, and the expression product of the gene of interest (e.g., a polypeptide) is between 80-99%, 90-95%, 90-99%, 95-99%, 95-97%,

97-99%, 98-99%, 80-99.5%, 90-99.5%, 95-99.5%, 97-99.5%, or 98-99.5%. Conditions can also be adjusted to obtain vectors wherein the percent identity between the expression products of the modified genes of interest, or a specified percentage thereof, and the expression product of the gene of interest varies between lower and upper ranges having the following values: 70%, 73%, 75%),

78%, 80%, 83%, 85%, 88%, 90%, 93%, 95%, 97%, 99%, or 99.5%. Of course, as above, the lower range must be lower than the value of the upper range. Thus, one example of such a range would again be 80-93%) and the percent identity between the expression products, or a specified percentage thereof, of the modified genes of interest and the expression product of the gene of interest would be between 80-93%.

As used herein, the phrase "% identity" or "%> identical" refers to a relationship between two polynucleotide sequences or two polypeptides determined by comparing the sequences of these molecules to each other. More specifically, these phrases refer to the degree of sequence relatedness between the two polynucleotide or two polypeptides as determined by the nucleotide or amino acid sequence match between their sequences.

As one skilled in the art would recognize, in order to determine whether a particular percentage of the modified genes of interest of the members of a population of the invention, or the polypeptides encoded by these modified genes, share, for example, 95-99% identity with either a gene of interest, or a polypeptide encoded by a gene of interest, it will generally be necessary to determine the nucleotide or amino acid sequence of a representative number of members (e.g., 50, 75, 100, 150, 200 or 250) of the population. Statistical analysis can then be used to determine whether the nucleotide sequences of the modified genes of interest of a particular percentage of the members of the population (e.g., 90%), or the encoded polypeptide, fall within a specified range of identity (e.g., 90-99%) with either the nucleotide sequence of the gene of interest or the amino acid sequence encoded by this gene. Numerous publications are available which review methods for performing statistical analyses. (See, e.g., Daniel, W., BIOSTATISTICS: A FOUNDATION FOR ANALYSIS IN THE HEALTH SCIENCES, John Wiley & Sons, 1974 and 1978, the entire disclosure of which is incoφorated herein by reference.)

One skilled in the art would recognize that the use of statistics to show that at least 95% of the vectors or polypeptides in a population, for example, requires that certain assumptions about the population be made. The populations of the present invention are believed to either comprise or closely approximate a normal distribution. Thus, as explained by the example below, whether "at least" a particular percentage of the modified genes of interest, for example, of a population share a particular range of identity with a gene of interest can be determined by sequencing a representative number of the modified genes of interest in the population, followed by a determination of whether the average number of members of the population sharing a particular range of identity with a gene of interest comprise, within one standard deviation, "at least" the specified percentage. Statistical analyses can also be used to determine whether the members of the population fall within a specified range of identity as compare to the nucleotide sequence of the gene of interest or the amino acid sequence of the encoded polypeptide. For example, a population wherein a mean of 97.3% (+/-1.3%=one standard deviation) of the modified genes of interest share, on average (i.e., mean), 95.4% (+/-3.2%=one standard deviation) identity with the gene of interest would be said to comprise a population wherein at least 95% of the members of the population are 90-99% identical to the nucleotide sequence of the gene of interest.

One skilled in the art would recognize that when determining %> identity between two populations of nucleic acids or polypeptides, any one of several frames of reference could be used. For example, one could determine the nucleic acid or amino acid sequences of a representative number of molecules (e.g., the modified genes of interest) in the total population and then calculate the average

% identity that these molecules share with a reference molecule (e.g., the gene of interest). Alternatively, one could (1) determine the sequence of a representative number of molecules in a population, (2) identify those molecules which contain at least one mutation (e.g., the modified genes of interest), and (3) calculate the average % identity that these molecules share with a reference molecule (e.g., the gene of interest). The first method set out above is referred to herein as the "total population" method for assessing percent identity, and the second method set out above is referred to herein as the "mutant population" method for assessing percent identity. The % identity between two polynucleotide or two polypeptides can be determined by a number of art known methods. Several of these methods are described in COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G., eds.,

Humana Press, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, 1987; SEQUENCE ANALYSIS PRIMER, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ; Carillo etal., SIAMJ. AppliedMαth. 48:1073 (1988); Altschul, S.,J Mol. Biol. 279:555- 65 (1991); Altschul, J. Mol. Evol. 3<5:290-300 (1993); Altschul, S. et αl, J. Mol. Biol. 275:403-410 (1990); Claverie et αl, Computers in Chemistry 17:191-201

(1993); Henikoff et αl, Proc. Nαtl. Acαd. Sci. USA 59: 10915-10919 (1992); Karlin et αl., Proc. Nαtl. Acαd. Sci. USA 57:2264-2268 (1990); Karlin et αl., Proc. Nαtl. Acαd. Sci. USA 90:5873-5877 (1993); States et αl., J. Comput. Biol. 7:39-50 (1994). Methods for determining % identity have been codified in publicly available computer programs. Examples of computer programs useful for determining identity and similarity between two sequences include the GCG program package (Devereux et αl., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, and FASTA (Altschul et al, J. Mol. Biol. 275:403-410 (1990). Another example is the BLASTX program, which is publicly available from NCBI and other sources. See BLAST Manual, Altschul et al. , NCBI NLM NIH Bethesda, Md. 20894; Altschul et al, J. Mol. Biol. 275:403-410 (1990).

Parameters of many of the publicly available computer programs for determining %> identity between the nucleotide or amino acid sequences of two molecules can be set by the user. In many instances where such software is used in conjunction with the present invention, the default parameters of the particular computer program employed for determining % identity will be used.

Generally, the method used to determine % identity will be designed to take into account insertions, deletions and substitutions in the sequences of the nucleic acid molecules being compared. As an illustration, by a polynucleotide of a modified gene of interest having a nucleotide sequence having, for example, 95-99% "identity" to a reference gene of interest it is intended that the nucleotide sequence of the modified gene of interest is identical to the reference gene of interest except that the nucleotide sequence of the modified gene of interest may include 1 to 5 point mutations per 100 nucleotides of the reference gene of interest nucleotide sequence. In other words, in a modified gene of interest having a nucleotide sequence 95-99% identical to the nucleotide sequence of a reference gene of interest, 1-5% of the nucleotides in the reference gene of interest may be deleted or substituted with other nucleotides in the modified gene of interest, or a number of nucleotides between 1-5% of the total nucleotides in the reference gene of interest sequence may be inserted into the modified gene of interest. These insertions, substitutions, or deletions may occur at the 5' or 3' terminal positions of the reference gene of interest nucleotide sequence or anywhere between those terminal positions, either interspersed individually among nucleotides in the reference gene of interest sequence or in one or more contiguous groups within the reference gene of interest sequence.

Whether the nucleotide sequences of a modified gene of interest and a gene of interest (i. e. , a reference nucleotide sequence) are between, for examples, 80-99%, 90-99%, 95-99%, 95-97%, 97-99%, or 98-99% identical can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 5371 1). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, between 95-99%> identical to a reference nucleotide sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

Once the % identity shared between the nucleotide sequences of a representative number of members of either the total population of modified genes of interest or the population of molecules which contain at least one mutation in the gene of interest and the gene of interest are individually determined, as discussed immediately above, statistical analyses can then be performed to determine whether the nucleotide sequences of the members of the population fall within a specified range of identity as compare to the reference nucleotide sequence.

Analogously, by a polypeptide encoded by a modified gene of interest having an amino acid sequence having at least, for example, 95-99%> identity to a reference amino acid sequence encoded by a gene of interest (reference polypeptide) is intended that the amino acid sequence of the polypeptide encoded by the modified gene of interest is identical to the amino acid sequence of the reference polypeptide except that the polypeptide encoded by the modified gene of interest sequence may include up to 1 to 5 amino acid alterations per 100 amino acids of the reference polypeptide sequence. In other words, in a polypeptide encoded by a modified gene of interest having an amino acid sequence 95-99% identical to a reference polypeptide sequence, 1-5% of the amino acid residues in the reference polypeptide sequence may be deleted or substituted with other amino acids in the polypeptide encoded by a modified gene of interest, or a number of amino acids between 1-5% of the total amino acid residues in the reference polypeptide sequence may be inserted into the polypeptide encoded by a modified gene of interest. These insertions, substitutions, or deletions may occur at the amine or carboxy terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, either interspersed individually among amino acid residues in the reference polypeptide or in one or more contiguous groups within the reference polypeptide.

Whether the amino acid sequence of a polypeptide encoded by a modified gene of interest and the amino acid sequence of a polypeptide encoded by a gene of interest (i.e., a reference nucleotide sequence) are between, for examples, 80-99%, 90-99%, 95-99%, 95-97%, 97-99%, 98-99%, 80-99.5%, 90-99.5%, 95-99.5%), 97-99.5%, or 98-99.5% identical can also be determined conventionally using known computer programs such the Bestfit program. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, between 95-99% identical to a reference amino acid sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed. Once the % identity shared between the amino acid sequences of a representative number of members of either (1) the total population of polypeptides encoded by the modified genes of interest or (2) the population of polypeptides encoded by the modified genes of interest which contain at least one mutation in the gene of interest and the polypeptide encoded by the gene of interest are individually determined, as discussed immediately above, statistical analyses may then be performed to determine whether the amino acid sequences of the members of the population fall within a specified range of identity as compare to the reference amino acid sequence.

When the vectors of the invention are intended for use in vaccine compositions, the introduction of mutations which result in substitutions, as compared to deletions and insertions will generally be preferred. This is so because insertions and deletions will often introduce frame shifts in the coding sequence of the polypeptide encoded by the gene of interest.

Further, when the vectors of the invention are intended for use in vaccine compositions, it will generally be advantageous to mutagenize the gene of interest using a method which minimizes the introduction of stop codons.

Vectors of the Invention

Vectors can be used in the practice of the present invention in a number of ways. One of these ways involves the insertion of a gene of interest into a vector followed by mutagenesis using the methods described above to produce a population of vectors which contain modified genes of interest. The vectors which undergo mutagenesis will generally contain mutations in nucleic acid sequences other than just those of the gene of interest.

Another way that vectors can be used with the invention involves the excision of modified genes of interest from the vector in which mutagenesis was performed followed by insertion of these genes into separate vectors. The vector in which mutagenesis was performed and that in which the modified genes of interest are inserted, depending on the application of the modified genes of interest, can be the same or different vector. The vectors of the invention may be composed of either DNA or RNA.

In general, when mutagenesis of the gene of interest is performed, the vector will be in an RNA form. However, the mutagenized vectors containing the modified genes of interest can be reverse transcribed into DNA and, if desired, maintained in this format to prevent the introduction of considerable numbers of additional mutations.

When RNA vectors are used in the practice of the present invention, these vectors will generally contain essential c s-acting genetic elements necessary for replication. Further, additional elements required for replication (e.g. , replicase coding sequences) which function in trans may be encoded by one or more separate nucleic acid molecules (e.g. , plasmid DNA, chromosomal DNA, vector

RNA, helper virus DNA or RNA).

The vectors of the invention may also contain genetic elements which allow for chromosomal integration of vector nucleic acid. Such elements are useful for the stable maintenance of heterologous nucleic acid sequences and include nucleic acid sequences which confer both site-specific and site-independent integration. Site-specific integration (e.g., homologous integration) and site-independent integration, sometimes referred to as "random integration," can be used to introduce heterologous nucleic acid (e.g., a gene of interest, modified genes of interest) into eukaryotic chromosomes. Methods for inserting genetic material into eukaryotic chromosomes are available from numerous sources including Sambrook, J. et al, eds. (MOLECULAR CLONING, A LABORATORY MANUAL, 2nd. edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)).

One example of an RNA viral vector suitable for use with the present invention is the pCYTts vector shown in FIGs. 2 and 3. This vector can be converted between RNA and DNA formats and can replicate when present in the RNA format. Further, this vector contains the following components: a Rous Sarcoma Virus (RSV) promoter, c/s-acting sequence elements (CSE), non-structural proteins 1-4 (nsPl, nsP2, nsP3, nsP4), a gene of interest, and a subgenomic promoter (S.G.).

5 The pCYTts vector shown in FIGs. 2 and 3 is described in

PCT/IB99/00523, filed March 25, 1999, which is incorporated herein by reference in its entirety.

Another example of an RNA viral vector suitable for use with the present invention is the pSinRep5 vector shown in FIGs.4A-4E and 5. (InvitroGen BV,

0 P.O. Box 2312, 9704 CH Groningen, The Netherlands; Bredenbeek^' et al, J.

Virol 77:6439-6446 (1993).) pSinRep5 contains nucleic acid which encodes a wild-type alphaviral replicase. Thus, this replicase in cytopathic and eukaryotic cells which express it will generally become non-viable within 24 hours after initiation of expression.

5 An additional example of an RNA viral vector suitable for use with the present invention is the pTE vector (Hahn et al, Proc. Natl. Acad. Sci. (USA) 89:2679-2683 (1992)). The pTE vector encodes a wild-type replicase and viral structural proteins, the coding sequences of which are located 3' to a subgenomic promoter. Further, the gene of interest in the pTE vector is located 3' to a second

.0 subgenomic promoter. Expression of the wild-type replicase from this vector leads to the production of infectious viral particles and relatively rapid cell death.

Additional features of the pSinRepS vectors are described in "Sindbis

Expression System", Version C, (InvitroGen Catalog No. K750-1), the entire disclosure of which incorporated herein by reference. t5 One skilled in the art would recognize that expression of a wild-type replicase, such as that encoded by pSinRep5, in host cells will result in relatively rapid host cell death. Expression of such a replicase, however, may be advantageous when rapid production of expression products, replicons, or viral particles is desired.

\0 Inducible or constitutive promoters can be used to both confer expression of RNA forms of vectors of the invention and the production of expression products of modified genes of interests. One example of a promoter which can confer expression of nucleic acid in a DNA format is the RSV promoter of pCYTts. Numerous additional promoters suitable for inducing expression of RNA from DNA molecules are known and include cytomegalovirus (CMV) promoters, simian virus 40 (SV40) promoters, myeloproliferative sarcoma virus

(MPSV) promoters, glucocorticoid promoters, metallothionein promoters, Herpes simplex virus thymidine kinase (HSVTK) promoters, human immuno deficiency (HIV) promoters, mouse mammary tumor virus (MMTV) promoters, human polyomavirus BK (BKV) promoters, and Moloney murine leukemia virus (MuLV) promoters.

Vectors suitable for use with the present invention will generally contain a cloning site for the insertion of a gene of interest. Depending on the particular application, this cloning site may or may not be operably linked to a promoter for the expression of mRNA from the RNA or DNA form of the vector. Examples of promoters which confer the expression of mRNA from an

RNA molecule include the subgenomic promoters of alphaviruses (e.g., the subgenomic promoter of pCYTts).

When the mutagenized vectors containing the modified genes of interest are reverse transcribed into a DNA format or the modified genes of interest are excised and inserted into separate DNA vectors, it may not be necessary to express the modified genes of interest from vectors in an RNA format. Instances where it could be desirable to express modified genes of interest in an RNA format are where these RNA molecules are screened to identify those which encode polypeptides having specific characteristics and when RNA vectors containing the modified genes of interest are administered to an individual.

DNA molecules of the invention can also contain packaging signals which direct the packaging of RNA molecules into viral particles. These RNA molecules can be packaged in the presence of wild-type virus or defective helper virus RNA. With respect to alphaviral vectors, a significant improvement was made with the development of defective helper RNA molecules. See Bredenbeek, P. et al, J. Virol. 67:6439-6446 (1993). These defective helper RNA molecules contain c.s-acting sequences, required for replication of the full-length transcription product, and subgenomic RNA promoter sequences which drive the expression of the structural protein genes. For example, in cells containing both RNA molecules with packaging signals and the defective helper virus RNA, alphaviral non-structural proteins allow for replication and amplification of the defective helper virus RNA sequences which are translated to produce virion structural proteins. Since the defective helper virus RNA lacks packaging signals, these molecules are not packaged into assembled virions. Thus, viral particles produced in this way contain essentially only RNA sequences encoding the gene of interest and, when necessary for the particular application, other sequences required for temperature-sensitive regulation of gene expression. These non-infective packaged RNA molecules do not contain sequences encoding virion structural proteins and, thus, undergo only one round of host cell infection and are non-pathogenic.

Non-infective packaged RNA molecules can be used to "infect" a culture of suitable host cells simply by addition of the particles to culture medium containing these cells. The preparation of non-infective alphaviral particles is described in a number of sources, including "Sindbis Expression System", Version C, (InvitroGen Catalog No. K750-1).

Further, when vectors are prepared in a DNA format, these vectors may also be packaged. Graham et al, U.S. Patent No. 5, 919,676 and Spooner et al. , U.S. Patent No. 5,885,808, for example, describe the preparation of packaged adenoviral particles which contain inserted genes. Only three regions of the adenoviral genome (the left inverted terminal repeat, the packaging signals, and the right inverted terminal repeat) are known to be required in cis for viral DNA replication and packaging of viral DNA into virion particles. All other regions of the viral genome appear to be required only to produce viral products that act in trans to allow viral replication and production of infectious viruses. Thus, when all essential viral proteins and RNA are provided by a helper virus, vectors can be designed and constructed wherein the majority of the viral DNA is deleted and essentially only those viral sequences mentioned above that are required in cis for viral DNA replication and packaging remain. The invention is thus directed to packaged adenoviral particles, as well as packaged viral particles of other viruses, which contain modified genes of interest.

The vectors of the invention can also contain genetic elements which confer additional functional characteristics such as selection markers, sequences which result in high copy number host cell amplification, and sequences which allow for chromosomal integration of vector nucleic acid. Markers for the selection of prokaryotic and eukaryotic cells containing vectors of the present invention are well known in the art and include tetracycline, ampicillin, puromycin, neomycin, and kanamycin resistance. DNA molecules containing nucleic acid which confer phenotypes suitable for selection are available from numerous sources including Stratagene (11011 North Torrey Pines Road, La Jolla, CA 92037, USA) and Promega (2800 Woods Hollow Road,

Madison, WI 53711, USA). Nucleic acid elements which result in high copy number amplification are also known in the art and include the ColEl sequence contained in the pCYTts vector.

As noted supra, the vectors of the invention may also contain genetic elements which allow for chromosomal integration of vector nucleic acid.

The vectors of the invention may also be designed to confer expression of the modified genes of interest as fusion proteins. These polypeptides may include not only secretion signals but also additional heterologous functional regions. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage. A region may also be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art. For example, the nucleic acid encoding the modified genes of interest may be linked to a signal peptide which will allow secretion of the protein or its compartmentalization in a subcellular compartment. Such signal peptides maybe designed with or without specific protease sites such that the signal peptide is amenable to subsequent removal. Alternatively, the native signal peptide for this protein may be used.

The nucleic acid encoding the modified genes of interest may also be fused to nucleic acid encoding at least a portion of the Fc region of an immunoglobulin. For example, the fusion protein expression products of such constructs may contain the amino-terminal portion of polypeptides encoded by the modified genes of interest and the Fc region forms the carboxy terminal portion. In these fusion proteins, the Fc region will often be limited to the hinge region and the C_H2 and C_H3 domains.

The nucleic acid encoding the modified genes of interest may also be fused to a hexa-histidine (HIS) peptide, such as the tag provided in the pQE vector (Qiagen, Inc.), among others, many of which are commercially available.

This HIS peptide provides for convenient purification of the fusion protein.

Recombinant Host Cells

A variety of different recombinant host cells can be produced which generate and contain the viral vectors of the invention. The particular host cell selected for use will vary with the characteristics of the viral vectors and with the application for which the vectors are to be used. For example, when the host cells are used to produce the expression products of the modified genes of interest, it may be advantageous to have these genes chromosomally integrated into the host cell's genome. Further, various components of the particular expression system can be encoded by the host cell genome or other nucleic acid molecule (e.g., plasmid, helper virus) within the host cell. For example, when an alphavirus is used to produce modified genes of interest, the replicase activity can be encoded by the host cell genome. Similarly, when packaged, infectious or non-infectious viral particles are desired, the structural proteins required for packaging can be encoded by the host cell genome. Polo, M. et al, Proc. Natl. Acad. Sci. U.S.A. 9(5:4598-4603 (1999), for example, describes cell lines stably transformed with expression cassettes which constitutively produced RNA transcripts encoding the Sindbis virus structural proteins under the regulation of their subgenomic promoter. Cells of this type are suitable for use in the production of packaged alphaviral vectors.

Alphaviral vectors have the advantage of having a wide host range and thus can be used with a considerable number of cell types. Sindbis virus, for instance, infects cultured mammalian, reptilian, and amphibian cells, as well as some insect cells. Clark, H., J Natl. Cancer Inst. 57:645 (1973); Leake, C, J.

Gen. Virol. 35:335 (1977); Stollar, V. in THE TOGA VIRUSES, R.W. Schlesinger, Ed., Academic Press, (1980), pp. 583-621. Thus, numerous recombinant host cells can be used in the practice of the invention.

The selection of a host cell suited for a particular application will vary with a number of factors including the polypeptide and RNA expression products of the modified genes of interest which is expressed. For example, when a glycoprotein is produced, it is generally desirable to express this protein in a cell type which will glycosylate the protein in a manner similar to that of the native protein. BHK, COS, Vero, HeLa and CHO cells are particularly suitable for the production of heterologous proteins because they have the potential to glycosylate heterologous proteins in a manner similar to human cells (Watson, E. et al., Glycobiology 4:227, (1994)) and can be selected (Zang, M. et al, Bio/Technology 13:389 (1995)) or genetically engineered (Renner, W. et al, Biotech. Bioeng. 47:476 (1995); Lee K. et al, Biotech. Bioeng. 50:336 (1996)) to grow in serum-free medium, as well as in suspension.

One significant advantage of the methods of the present invention is that mammalian host cells can be readily used to produce the expression products of the invention. Mammalian cells are especially useful when polypeptide expression products are desired in glycosylated form. One example of a situation where it is advantageous to produce polypeptides having glycosylation patterns similar to those expressed in an individual (e.g., a human) is where the polypeptide expression products of the modified genes of interest are to be used in vaccination protocols against agents which alter their epitopes (e.g., HIV-1, HIV-2, Influenza virus, Hepatitis C virus, and cancers such as liver carcinomas, stomach carcinomas, skin carcinomas, and ovarian tumors).

One vector which is particularly suitable for preparing populations of modified genes of interest is the pCYTts vector shown in FIGs. 2 and 3.

As noted above, replicase activity may be expressed from nucleic acid of the vector containing the modified gene of interest, nucleic acid of a separate vector, or nucleic acid which is integrated into host cell chromosomes.

When the stable transfer of vector nucleic acid to recombinant host cells is desired, this nucleic acid may be chromosomally integrated into the host cell genome. In such instances, vector nucleic acid will be maintained in the host cell and transferred to cellular progeny. For example, the inclusion of long terminal repeats of retroviruses in gene transfer vectors has been found to confer stable maintenance of vector nucleic acid in recombinant host cells. Peng, L. et al, J. Surg. Res. 69: 193-198 (1997); Qing, K. et al, J. Virol. 77:5663-5667 (1997). Thus, chromosomal integration of vector nucleic acid is one mechanism by which such nucleic acids can be stably maintained in recombinant host cells. These nucleic acids can integrate into host cell chromosomes either without regard to chromosomal location or at one or more specific chromosomal loci (e.g., via homologous recombination). These recombinant host cells may then be cultured in vitro or introduced into an individual.

When polypeptide expression products are intended for administration to individuals of a particular species, these polypeptides can be produced in host cells of that species. Further, when polypeptides are intended for administration to mammals, these polypeptides will generally be produced in cells of mammalian origin (e.g., COS, CHO, BHK, MCF-7, MCF-10A, ISO-HAS, HaCaT, HeLa, Hepa 6, Hep3B, and Hep G2 cells). Introduction of the polynucleotide vectors into host cells can be effected by methods described in standard laboratory manuals (see, e.g., Sambrook, J. et al, eds., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd. edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), Chapter 9; Ausubel, F. et al, eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997), Chapter 16), including methods such as electroporation, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, and infection. Methods for the introduction of exogenous DNAs into host cells are discussed in Feigner, P. et al, U.S. Patent No. 5,580,859. Non-infective or infective, packaged RNA or DNA can also be used to infect host cells. Packaged RNA and DNA molecules can be introduced into host cells by contacting them with host cells in culture media.

In one aspect, the invention is directed to modified genes of interest, vectors, and viral particles produced according to the methods of the invention which are isolated from recombinant host cells. These modified genes of interest, vectors, and viral particles will generally be amplified in the recombinant host cells prior to recovery from either the culture medium or the cells themselves.

As suggested above, one disadvantage to the use of prokaryotic cells for the production of mammalian proteins is that prokaryotic cells lack the glycosylation machinery present in eukaryotes. The methods of the present invention have the advantage of being useful for the production of polypeptide expression products of modified genes of interest in higher eukaryotic cells such as mammalian cells. This is especially advantageous when the goal is to mimic the three-dimensional structure of viral epitopes. Thus, when the expression products of the invention are to be used in vaccine compositions designed to elicit protective immunity against viruses which alter their antigenic determinants, these expression products will generally be produced in mammalian cells.

When mammalian cells are used as recombinant host cells for the production of viral-based core particles, these cells will generally be grown in tissue culture. Methods for growing cells in culture are well known in the art

(see, e.g., Celis, J., ed., CELL BIOLOGY, Academic Press, 2^nd edition, (1998); Sambrook, J. et al. , eds., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd. edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel, F. et al, eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997); Freshney, R., CULTURE OF ANIMAL CELLS, Alan R. Liss, Inc. (1983)).

Expression Products of the Invention

The vectors and recombinant host cells of the invention may be used for the production of polypeptide and RNA molecules. Thus, the invention provides methods for producing polypeptide and RNA expression products of the modified genes of interest in host cells, comprising the step of introducing nucleic acid molecules of the invention into host cells and recovering the expression products from either the culture medium or the host cells themselves.

Recombinant host cells which express a gene of interest will generally be used to either express this gene in individuals or in in vitro cultures. In one aspect, the present invention provides methods for producing polypeptide and RNA expression products of the modified genes of interest comprising introducing nucleic acid molecules of the invention into recombinant host cells and recovering the expression products from either the culture medium or the host cells themselves. In a related aspect, the invention provides isolated polypeptide and RNA expression products of the modified genes of interest produced according to the methods of the present invention.

Depending on the molecule which is expressed, the molecule may be obtained either from the culture supernatant or by lysing the recombinant host cells. When the expression product is a protein, it will often be possible to obtain the expression product from the culture supernatant. This will be so even when the protein does not have a naturally associated secretory signal. Codons encoding such a signal can be added to the vector nucleic acids of the invention and will result in the expression of a fusion protein which will be secreted from the recombinant host cell. Nucleic acids encoding such signal peptides are known in the art and are publically available. See, e.g., pPbac and pMbac vectors, STRATAGENE 1997/1998 CATALOG, Catalog #211503 and #211504, Stratagene, 1 1011 North Torrey Pines Road, La Jolla, CA 92037, USA.

Host cells may also be infected with packaged or unpackaged nucleic acid molecules. The gene product of interest may then be recovered and purified by any suitable means.

The protein expressed from the modified genes of interest can be recovered and purified from recombinant cell cultures by methods known in the art including ammonium sulfate precipitation, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and high performance liquid chromatography. Methods for purifying proteins are described in numerous sources. See, e.g. , Celis, J., ed., CELL BIOLOGY, Academic Press, 2^nd edition, (1998).

Methods for purifying RNA molecules are also known in the art. See, e.g., Celis, J., ed., CELL BIOLOGY, Academic Press, 2^nd edition, (1998). These methods include phenol/chloroform extraction, digestion with DNAses followed by precipitation of the undigested RNA molecules, and column chromatography (e.g., oligo dT column chromatography). Further, RNA molecules can be separated from other cellular material using the single-step guanidinium- thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi,

Anal. Biochem. 162:156-159 (1987).

A number of different bioprocess parameters can be varied in order to increase the amount of expression product produced during the cell culture process. The conditions under which the host cells are grown (e.g., medium composition, pH, oxygen concentration, agitation, and, for the case of anchorage-dependent cells, the surface provided and the carrier of that surface) prior to exposure to the nucleic acid molecules of the invention or induction of gene expression influence both the cell density achieved at a given time and the physiological state of the cells. These culture conditions will thus affect the expected cellular response to vector exposure or the induction signal (e.g., shifting to a permissive temperature). Further, the cell culture process-conditions mentioned above can be varied to maximize the production of expression product and, often, the characteristics (e.g., glycosylation pattern) of that expression product.

The overall cell culture process employing nucleic acid molecules of the invention for the production of expression product can be implemented in a variety of bioreactor configurations (e.g., stirred-tank, perfused, membrane enclosed, encapsulated cell, fluidized bed, and air-lift reactors) and scales (from laboratory T-flasks to thousands of liters), chosen to accommodate the requirements of the host cell line utilized (e.g., anchorage dependency, O₂ concentrations), to maximize the production of expression product, and to facilitate subsequent recovery and purification of expression product.

The invention is also directed to the production of polypeptide or RNA expression products of modified genes of interest using mammalian cells grown in serum-free or protein- free culture media. For example, by long-term culture under conditions restricting serum access or selecting for suspension growth,

CHO cell lines are selected which are able to grow in serum-free medium and/or in suspension. Zang, M. et al, Bio/Technology 13:389 (1995). Further, by genetic modification of CHO KI cells, a modified cell line designated CHO KI :cycE was obtained which grows as suspended single cells in protein-free culture media. Renner, W. etal, Biotech. Bioeng. 47:476 (1995). CHO mutants

(e.g., LEC10 cells) have also been isolated which produce glycoproteins having different glycosylation patterns than those produced in parental CHO cells. Stanley, P., Glycobiology 2:99 (1992). Alternatively, CHO cells capable of synthesizing glycoproteins with correspondingly modified oligosaccharides may be obtained by genetic modifications which alter the activities of enzymes involved in oligosaccharide biosynthesis. Minch etα/., Biotechnol. Prog. 77:348 (1995).

Further, a number of different bioprocess parameters can be varied in order to alter the glycosylation pattern of polypeptide products produced by the recombinant host cells of the invention. These factors include medium composition, pH, oxygen concentration, lack or presence of agitation, and, for the case of anchorage-dependent cells, the surface provided. Thus, the glycosylation pattern of glycoproteins may be altered by choosing the host cell in which these proteins are expressed in and the conditions under which the recombinant host cells are grown.

Uses of Vectors of the Invention

In one aspect, the present invention provides pharmaceutical compositions suitable for use as vaccines wherein the individual members of the population are generally not separated from each other prior to administration to an individual. These compositions comprise one or more populations of vectors of the present invention, or the polypeptide expression products of such populations, in a pharmaceutically acceptable carrier.

The invention thus provides in one aspect multi-component vaccines ( . e. , heterogenous vaccines) composed of either multiple vectors which express divergent polypeptides or the divergent polypeptides themselves. The heterogeneity of the vaccines compositions of the present invention vaccine will be advantageous in many instances because these vaccine compositions are likely to be more effective than single vector or polypeptide vaccines in eliciting broad protective immune responses against viruses which alter their antigenic determinants. Thus, a multi-component vaccine of the present invention may contain a considerable number of permutations of the gene of interest (e.g., 10⁶,

10⁷, or 10⁸ different polypeptide expression products.

In addition, the mutation rate can be adjusted such that the amino acid sequences encoded by the modified genes of interest in a specified percentage of the vector population will have a specified percentage or number of amino acid substitutions, deletions or insertions as compared to the gene of interest.

In a separate embodiment, specific members of the population of modified genes of interest are separated from the other members of the population and screened for usefulness in eliciting the production of antibodies or a protective immune response. Thus, vaccine compositions of the invention also include vaccines wherein the polypeptide expression product of a single modified gene of interest, or modified gene of interest itself, is administered to an individual in admixture with a pharmaceutically acceptable carrier.

Further within the scope of the invention are whole cell and whole viral vaccines. Such vaccines may be produced recombinantly and involve the expression of polypeptide expression products of modified genes of interest. For example, the polypeptide expression products of modified genes of interest may be either secreted or localized intracellular, on the cell surface, or in the periplasmic space. Further, when a recombinant virus is used, the polypeptide expression products of modified genes of interest may, for example, be localized in the viral envelope, on the surface of the capsid, or internally within the capsid.

Whole cell vaccines which employ cells expressing heterologous proteins are known in the art. See, e.g., Robinson, K. et al, Nature Biotech. 75:653-657 (1997); Sirard, J. et al, Infect. Immun. (55:2029-2033 (1997); Chabalgoity, J. et al, Infect Immun. (55:2402-2412 (1997). These cells may be administered live or may be killed prior to administration. Chabalgoity, J. et al, supra, for example, report the successful use in mice of a live attenuated Salmonella vaccine strain which expresses a portion of a platyhelminth fatty acid-binding protein as a fusion protein on its cell surface.

Further included within the scope of the invention are vaccines comprising non-natural molecular scaffold coated with expression products of the modified genes of interest. Examples of such non-natural molecular scaffold are set out below and are disclosed in U.S. Provisional Appl. Nos. 60/110,414 and 60/142,788; respectively filed on November 30, 1998 and July 8, 1999, entitled "Ordered Molecular Presentation of Antigens, Method of Preparation and Use," the entire disclosures of which are incoφorated herein by reference. In part, the inventions disclosed in U.S. Provisional Appl. Nos. 60/110,414 and 60/142,788 are directed to composition comprising (A) a non-natural molecular scaffold and (B) an antigen or antigenic determinant.

The non-natural molecular scaffold comprises (i) a core particle selected from the group consisting of (1) a core particle of non-natural origin and (2) a core particle of natural origin; and (ii) an organizer comprising at least one first attachment site, wherein the organizer is connected to the core particle by at least one covalent bond.

The antigen or antigenic determinant has at least one second attachment site which is selected from the group consisting of (i) an attachment site not naturally occurring with the antigen or antigenic determinant; and (ii) an attachment site naturally occurring with the antigen or antigenic determinant.

The inventions disclosed in U.S. Provisional Appl. Nos. 60/110,414 and 60/142,788, as well as in PCT publication WO 00/32227, provide an ordered and repetitive antigen array through an association of the second attachment site to the first attachment site by way of at least one non-peptide bond. Thus, the antigen or antigenic determinant and the non-natural molecular scaffold are brought together through this association of the first and the second attachment site to form an ordered and repetitive antigen array.

As used herein, the term "organizer" is used to refer to an element bound to a core particle in a non-random fashion that provides a nucleation site for creating an ordered and repetitive antigen array. An organizer is any element comprising at least one first attachment site that is bound to a core particle by at least one covalent bond. An organizer may be a protein, a polypeptide, a peptide, an amino acid (i.e., a residue of a protein, a polypeptide or peptide), a sugar, a polynucleotide, a natural or synthetic polymer, a secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a combination thereof, or a chemically reactive group thereof.

The expression products of the modified genes of interest having a second attachment site form an association with the non-natural molecular scaffold having one or more first attachment sites, by way of a non-peptide bond (e.g., covalent bond, ionic bond, hydrophobic interaction), to form an ordered and repetitive antigen array.

In one embodiment of the invention, the expression products of the modified genes of interest are linked to one or more second attachment sites for preparing an organized and repetitive array associated with the non-natural molecular scaffold. While one skilled in the art would recognize groups which can be used to produce appropriate second attachment sites, representative examples of attachment sites include, the following: an antigen, an antibody or antibody fragment, biotin, avidin, strepavidin, a receptor, a receptor ligand, a ligand, a ligand-binding protein, an interacting leucine zipper polypeptide, an amino group, a chemical group reactive to an amino group, a carboxyl group, chemical group reactive to a carboxyl group, a sulfhydryl group, a chemical group reactive to a sulfhydryl group, or a combination thereof

As used herein, the term "core particle" refers to a rigid structure with an inherent repetitive organization that provides a foundation for attachment of an

"organizer". A core particle as used herein may be the product of a synthetic process or the product of a biological process.

As used herein, the phrase "non-natural molecular scaffold" refers to any product made by the hand of man that may serve to provide a rigid and repetitive array of first attachment sites. Ideally but not necessarily, these first attachment sites are in a geometric order. The non-natural molecular scaffold may be organic or non-organic and may be synthesized chemically or through a biological process, in part or in whole. The non-natural molecular scaffold is comprised of: (a) a core particle, either of natural or non-natural origin; and (b) an organizer, which itself comprises at least one first attachment site and is connected to a core particle by at least one covalent bond. In a particular embodiment, the core particle may be a virus, virus-like particle, a virus capsid particle, a phage, a recombinant form thereof, or synthetic particle.

Virus-like particles (VLPs) are supermolecular structures built in a symmetric manner from many protein molecules of one or more types. They lack the viral genome and, therefore, are noninfectious. VLPs can often be produced in large quantities by heterologous expression and can be easily purified.

Examples of VLPs include the capsid proteins of Hepatitis B virus (Ulrich et al, Virus Res. 50: 141-182 (1998)), measles virus (Warnes et al, Gene 160:173-178 (1995)), Sindbis virus, rotavirus (US Patent Nos. 5,071,651 and

5, 374 ,426), foot-and-mouth-disease virus (Twomey etal., Vaccine 13: 1603-1610, (1995)), Norwalk virus (Jiang, X., etal, Science 250:1580-1583 (1990); Matsui, S.M., et al, J. Clin. Invest. 57: 1456-1461 (1991)), the retroviral GAG protein (PCT Patent Appl. No. WO 96/30523), the retrotransposon Ty protein pi, the surface protein of Hepatitis B virus (WO 92/11291) and human papilloma virus (WO 98/15631). In some instances, recombinant DNA technology may be utilized to fuse a heterologous protein to a VLP protein (Kratz, P.A., et al. , Proc. Natl. Acad. Sci. USA 9(5: 1915-1920 (1999)).

In a related embodiment, the expression products of the modified genes of interest are expressed as fusion proteins linked to a second polypeptide which acts as an attachment site (e. g. , a JUN or EOS leucine zipper domain, strepavidin, biotin). The second polypeptide has binding affinity for a first attachment site (e.g., a JUN or EOS leucine zipper domain, strepavidin, biotin) located on the surface of a core particle. After binding to the surface of the core particle, the fusion proteins and core particle are co-administered to an individual as an organized and repetitive antigen array.

An example of this embodiment is disclosed below in Examples 4-6. Examples 4-6 disclose the construction and mutagenesis of an HIV-1 gpl40-FOS fusion construct. The HIV-1 gpl40-EOS fusion construct is prepared in an alphaviral vector and then mutagenized by replication in the presence of nucleoside analogs. The modified forms of HIV-1 gpl40-EOS then associate with particles having JUN polypeptides on their surfaces. See Example 7.

In a related embodiment, when it is desirable to avoid significant numbers of mutations in the second polypeptide, the modified genes of interest may be generated by the methods described herein, excised from the vectors used for mutagenesis, and reinserted into new vectors where they are linked to the nucleic acid encoding the second polypeptide.

A multi-component vaccine can also be prepared using techniques known in the art by combining populations of vectors of the invention or polypeptide expression products of these populations, with additional antigenic components (e.g. , diphtheria toxin, tetanus toxin, and/or other compounds known to elicit an immune response). Such vaccines are useful for eliciting protective immune responses to organisms which naturally express the gene of interest, or variants thereof, and other pathogenic agents.

As suggested above, the vaccines of the present invention also include DNA vaccines. DNA vaccines are currently being developed for a number of infectious diseases. Anwer, K. etal, PharmRes. 7(5:889-895 (1999); McCluskie,

M. et al, Mol Med. 5:287-300 (1999); Boyer, J. et al, Nat. Med. 3:526-532 (1997); reviewed in Spier, R., Vaccine 74:1285-1288 (1996). Such DNA vaccines contain nucleic acid molecules which express modified genes of interest. DNA vaccines have been shown to display efficacy in treatment or prevention of cancer, allergic diseases and autoimmunity. Kowalczyk, D. et al,

Cell. Mol. Life. Sci. 55:751-770 (1999) provide a review directed to the topic of immune responses to DNA vaccines. Further, attenuated strains of bacteria have been shown to function as suitable delivery vehicle for DNA vaccines. Dietrich, G. etal, Immunol. Today 20:251-253 (1999). In addition, recent studies suggest that DNA vaccines can be used to induce protective immune responses to viral infections. Chen, Y. etal, J. Gen. Virol. 50:1393-1399 (1999); Huang, H. etal, Viral Immunol. 72:1-8 (1999). Also, immunization of mice with Semliki Forest viral vectors encoding the Influenza A virus antigens nucleoprotein and hemagglutinin have recently been shown to induce immune responses that are protective against challenge infection with Influenza virus. Berglund, P. et al,

Vaccine 77:497-507 (1999).

When nucleic acid vectors of the invention are used for vaccination, these vectors may be administered in package or unpackaged forms. Animal studies have recently shown that packaged recombinant replicon particles can elicit durable, antigen-specific, and virus-neutralizing antibody responses. See

Colombage, G. et al, Virology 250:151-163 (1998). Methods for preparing packaged viral vectors have been described above.

When nucleic acid vectors of the invention are used for vaccination, these vectors may be self-replicating or non-self-replicating. Self-replicating RNA vectors, for example, have recently been shown to be potentially useful in vaccination protocols. Ying, H. et al, Nat. Med. 5:823-827 (1999). When the intent is to vaccinate an individual against a pathogenic agent such as a virus, vectors which express the modified genes of interest or the expression products of the genes of interest themselves may be administered to that individual. The vaccine compositions of the invention are especially useful for eliciting immune responses to pathogenic agents which alter their epitopes.

As already noted, examples of such agents include HIV- 1 , HIV-2, Influenza virus and parasites such as those of the genera Trypanosoma (e.g., Trypanosoma congolense, Trypanosoma vivax, Trypanosoma cruzi, Trypanosoma brucei) or Plasmodium (e.g., Plasmodium falciparum, Plasmodium vivax). Additional examples include members of the retrovirus group and viruses which replicate their nucleic acid as RNA.

When a gene of interest is selected for modification by the methods described herein for the preparation of an anti-viral vaccine composition, this gene will often be (1) of viral origin, (2) a gene which is normally altered during the life cycle of the virus, and (3) a gene wherein the elicitation of an immune response in response to the gene product can attenuate or prevent viral infection. Representative examples of viral genes which potentially meet these criteria are as follows: Rhino virus, HIV- 1 gp41 , gp 120, gp 140, and gp 160; Influenza A virus nucleoprotein, neuramidase, and hemagglutinin; Sindbis virus nsP2 and glycoproteins El and E2; Hepatitis C virus NS3, NS4A, NS5 A, NS5B; Hepatitis

G virus (HGV) V37D, V36S, P37R, and C40P. Further, genes derived from a considerable number of additional viruses (e.g. , Ross River Virus (RRV), Semliki Forest Virus (SFV), Hepatitis E virus, Lymphocyte Choriomeningitis Virus (LCMV)) are also useful for the preparation of anti-viral vaccine compositions. As noted above, the present invention also relates to the administration of a vaccine which is co-administered with a molecule capable of modulating immune responses. Kim, J. et al, Vaccine 7(5:1828-1835 (1998) and Kim, J. et al, Nature Biotech. 75:641-646 (1997), for example, report the enhancement of immune responses produced by DNA immunizations when DNAs encoding molecules which stimulate the immune response are co-administered. In a similar fashion, the vaccines of the present invention may be co-administered with either nucleic acids encoding immune modulators or the immune modulators themselves. These immune modulators include proteins such as granulocyte macrophage colony stimulating factor (GM-CSF), CD86, CD80, Interleukin-12, Interleukin-4, Interferon-γ, or a complement degradation product. The vaccines of the present invention may be used to confer resistance to infectious agents by either passive or active immunization. When the vaccines of the present invention are used to confer resistance to an infective agent through active immunization, a vaccine of the present invention is administered to an individual to elicit a protective immune response which either prevents or attenuates the infection caused by the infective agent. When the vaccines of the present invention are used to confer resistance to an infective agent through passive immunization, the vaccine is provided to a host individual (e.g., human, dog, or mouse), and the antisera elicited by this vaccine is recovered and directly provided to a recipient suspected of having an infection caused by the infective agent.

Polypeptides expression products of the invention may be administered in pure form or may be coupled to a macromolecular carrier. Example of such carriers are proteins and carbohydrates. Suitable proteins which may act as macromolecular carrier for enhancing the immunogenicity of the polypeptides of the present invention include keyhole limpet hemacyanin (KLH), tetanus toxoid, pertussis toxin, bovine serum albumin, and ovalbumin. Methods for coupling the polypeptides of the present invention to such macromolecular carriers are disclosed in Harlow et al, Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988), the entire disclosure of which is incoφorated herein by reference.

The present invention thus concerns and provides a means for preventing or attenuating an infection resulting from organisms which express antigens that are recognized and bound by antisera produced in response to the polypeptides of the present invention. As used herein, a vaccine is said to prevent or attenuate a disease if its administration to an individual results either in the total or partial attenuation (i.e., suppression) of a symptom or condition of the disease, or in the total or partial immunity of the individual to the disease.

The administration of the vaccine (or the antisera which it elicits) may be for either a "prophylactic" or "therapeutic" puφose. When provided prophylactically, the compound(s) are provided in advance of any symptoms caused by the infective agent. The prophylactic administration of the compound(s) serves to prevent or attenuate any subsequent infection. When provided therapeutically, the compound(s) is provided upon or after the detection of symptoms which indicate that an individual may be infected with an infective agent. The therapeutic administration of the compound(s) serves to attenuate any actual infection. Thus, the vectors of the present invention, or the expression products of these vectors, may be provided either prior to the onset of infection (so as to prevent or attenuate an anticipated infection) or after the initiation of an actual infection. Further, polypeptide expression products of the invention may be expressed as fusion proteins. Generally, these fusion proteins will be designed to either aid in purification (e.g., will have His tag) or to have increased immunogenicity (e.g., will be fused to KLH).

A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient individual and is otherwise suitable for administration to that individual. Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient. While in all instances the vaccine of the present invention is administered as a pharmacologically acceptable compound, one skilled in the art would recognize that the composition of a pharmacologically acceptable compound varies with the individual to which it is administered. For example, a vaccine intended for human use will generally not be co-administered with Freund's adjuvant. Further, the level of purity of the vectors of the invention, or expression products of these vectors, will normally be higher when administered to a human than when administered to a non-human individual.

As would be understood by one of ordinary skill in the art, when the vaccine of the present invention is administered to an individual, it may be in a composition which may contain salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the composition. Adjuvants are substances that can be used to specifically augment a specific immune response. These substances generally perform two functions: (1 ) they protect the antigen(s) from being rapidly catabolized after administration and (2) they nonspecifically stimulate immune responses.

Normally, the adjuvant and the composition are mixed prior to presentation to the immune system, or presented separately, but into the same site of the individual being immunized. Adjuvants can be loosely divided into several groups based upon their composition. These groups include oil adjuvants (for example, Freund's complete and incomplete), mineral salts (for example,

AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄(SO₄), silica, kaolin, and carbon), polynucleotides (for example, poly IC and poly AU acids), and certain natural substances (for example, wax D from Mycobacterium tuberculosis, as well as substances found in Corynebacterium parvum, or Bordetella pertussis, and members of the genus Brucella). Other substances useful as adjuvants are the saponins such as, for example, Quil A. (Superfos A/S, Denmark). Preferred adjuvants for use in the present invention include aluminum salts, such as AlK(SO₄)₂, AlNa(SO₄)₂, and AlNH₄(SO₄). Examples of materials suitable for use in vaccine compositions are provided in Remington 's Pharmaceutical Sciences (Osol, A, Ed, Mack Publishing Co, Easton, PA, pp. 1324-1341 (1980), which reference is incoφorated herein by reference).

The therapeutic compositions of the present invention can be administered parenterally by injection, rapid infusion, nasopharyngeal absoφtion (intranasopharangeally), dermoabsoφtion, or orally. The compositions may alternatively be administered intramuscularly, or intravenously. Compositions for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absoφtion. Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water. Besides the inert diluents, such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents.

Therapeutic compositions of the present invention can also be administered in encapsulated form. For example, intranasal immunization of mice against Bordetella pertussis infection using vaccines encapsulated in biodegradable microsphere composed of poly(DL-lactide-co-glycolide) has been shown to stimulate protective immune responses. Shahin, R. et al, Infect. Immun. (53: 1 195-1200 (1995). Similarly, orally administered encapsulated Salmonella typhimurium antigens have also been shown to elicit protective immunity in mice. Allaoui-Attarki, K. et al, Infect. Immun. (55:853-857 (1997). Encapsulated vaccines of the present invention can be administered by a variety of routes including those involving contacting the vaccine with mucous membranes (e.g., intranasally, intracolonicly, intraduodenally).

Many different techniques exist for the timing of the immunizations when a multiple administration regimen is utilized. It is possible to administer the compositions of the invention more than once to increase the levels and diversities of expression of the immunoglobulin repertoire expressed by the immunized individual. Typically, if multiple immunizations are given, they will be given one to two months apart.

According to the present invention, an "effective amount" of a therapeutic composition is one which is sufficient to achieve a desired biological effect.

Generally, the dosage needed to provide an effective amount of the composition will vary depending upon such factors as the individual's age, condition, sex, and extent of disease, if any, and other variables which can be adjusted by one of ordinary skill in the art.

The antigenic preparations of the invention can be administered by either single or multiple dosages of an effective amount. Effective amounts of the compositions of the invention can vary from 0.01-1,000 μg/ml per dose, more preferably 0.1-500 μg/ml per dose, and most preferably 10-300 μg/ml per dose.

Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

Examples

The following enzymes and reagents were used in the experiments described in the examples which follow: T4 DNA ligase was obtained from New England Biolabs; Taq DNA Polymerase, QIAprep Spin Plasmid Kit, QIAGEN Plasmid Midi Kit, QIAExII Gel Extraction Kit, QIAquick PCR Purification Kit were obtained from QIAGEN; QuickPrep Micro mRNA Purification Kit was obtained from Pharmacia; Superscript One-step RT PCR Kit, fetal calf serum (FCS), bacto-tryptone and yeast extract were obtained from Gibco BRL; Oligonucleotides were obtained from Microsynth (Switzerland); restriction endonucleases were obtained from Roche Diagnostics, Boehringer Mannheim,

New England Biolabs or MBI Fermentas; Pwo. polymerase and dNTPs were obtained from Boehringer Mannheim. HP-1 medium was obtained from Cell culture Technologies (Glattbrugg, Switzerland). All standard chemicals were obtained from Fluka-Sigma-Aldrich and all cell culture materials were obtained from TPP. Nucleoside analogs were obtained from FLUKA-SIGMA-Aldrich-

Supelco (Buchs, Switzerland).

DNA manipulations were carried out by standard techniques. DNA was prepared either from 2 ml bacterial culture using the QIAprep Spin Plasmid Kit or from 50 ml culture using the QIAGEN Plasmid Midi Kit, both according to the protocols provided by the manufacturer. For restriction digest, DNA was incubated at least 2 hours with the respective restriction enzyme at a concentration of 5-10 units of enzyme per μg DNA under appropriate conditions (buffer and temperature as recommended by the manufacturer). Digests with more than one enzyme were performed simultaneously if reaction conditions were appropriate for all enzymes, otherwise consecutively. DNA fragments to be isolated for further manipulations were separated by electrophoresis in a 0.7 to 1.5% agarose gel, excised from the gel and purified with the QiaExII Gel Extraction Kit according to the protocol provided by the manufacturer. For ligation of DNA fragments, 100 to 200 pg of purified vector DNA were incubated overnight with a threefold molar excess of the insert fragment at 16°C in the presence of 1 units of T4 DNA ligase in the buffer provided by the manufacturer (total volume: 10-20 μl). 1/10 to ^l aliquot of the ligation reaction was used for transformation of E. coli XL 1 -Blue (Stratagene) by electroporation using a Gene Pulser (BioRAD) and 0.1 cm Gene Pulser Cuvettes (BioRAD) at 200 Ω, 25 μF,

1.7 kV. After electroporation, the cells were incubated for 1 hour in 1 ml Luria broth medium (Luria, S. E., and Delbruck, M., Genetics 25:491-511 (1943)) with shaking, before plating on selective LB agar.

Example 1 Expression of Viral Particles

One μg of RNase-free vector (pTE-CAT; Xiong, C. et al, Science

243:1188-1191 (1989)) was linearized by digestion. Subsequently in vitro transcription is carried out using the SP6 in vitro transcription kit

(InvitroscripCAP by InvitroGen, InvitroGen BV, NV Leek, The Netherlands). The resulting 5 '-capped mRNAs was analyzed on reducing agarose-gels.

Five μg of in vitro transcribed mRNA was electroporated into BHK 21 cells (ATCC: CCL10) according to InvitroGen's manual ("Sindbis Expression System", Version C, (InvitroGen Catalog No. K750-1), InvitroGen BV, The Netherlands). After 12 to 14 hours incubation at 37°C, the cells were harvested and the medium containing the released virus particles was then used in a plaque assay to determine the plaque forming units.

A new BHK cell layer was infected with 10⁷ viral particles (TE virus). HP-1 medium was supplemented with either 5-azacytidine (AZT) (400 μg/ml - 12 μg/ml) or 5-fluorouridine (5-FU) (40 μg/ml and 20 μg/ml). Twelve hours post infection, the medium was changed to RPMI without Cys and Met (RPMI/-S) for Vi hour. The RPMI/-S medium was replaced by RPMI/-S supplemented with ³⁵S-Cys and ³⁵S-Met. After 30 min the radioactive medium was replaced by regular RPMI medium and further incubated for 2 hours at 37°C. The supernatant was harvested and mixed with chloroform/methanol to precipitate the proteins. The protein pellet was analyzed by SDS-gel-electrophoresis, followed by an autoradiography. Due to difficulties in resolving the capsid protein, only El and E2 were visible on the autoradiographs (data not shown). AZT turned out to be the more potent mutagen (data not shown). The maximal concentration of AZT where protein expression and virus budding is still possible was determined to be around 200 μg/ml. The best results, in terms of protein yield, were obtained with concentrations below 100 μg/ml. Concentrations above 100 μg/ml decreased the yield of viral particles significantly, indicating a high mutation rate which might have inactivated the replicase. However, the temperature-sensitive mutations were generated in the presence of 10 μg/ml AZT. Our data clearly showed that the mutagen concentration can be increased much further, without affecting the protein folding and the virus budding.

Example 2

Production of Sindbis Virus Particles Containing Chloramphenicol Acetyltransferase (CA T) RNA

One μg of RNase-free vector (pTE-CAT; Xiong, C. et al, Science 243:1188-1191 (1989)) was linearized by digestion. Subsequently in vitro transcription was carried out using the SP6 in vitro transcription kit (InvitroscripCAP by InvitroGen, InvitroGen BV, The Netherlands). The resulting 5 '-capped mRNAs was analyzed on reducing agarose-gels. Five μg of in vitro transcribed mRNA were electroporated into BHK 21 cells (ATCC: CCL10) according to InvitroGen's manual ("Sindbis Expression System", Version C, (InvitroGen Catalog No. K750-1), InvitroGen BV, The Netherlands, the entire disclosure of which is incoφorated herein by reference). The resulting

5 '-capped mRNAs were analyzed on reducing agarose-gels.

Five μg of in vitro transcribed mRNA were electroporated into BHK 21 cells (ATCC: CCL10) according to InvitroGen's manual ("Sindbis Expression System", Version C, (InvitroGen Catalog No. K750-1), InvitroGen BV, The Netherlands). After 12 to 14 hours incubation at 37°C, the cells were harvested and the medium containing the released virus particles was then used in a plaque assay to determine the plaque-forming units.

A new BHK cell layer was infected with 10⁷ viral particles in the presence of 15 μg/ml - 200 μg/ml AZT. Two hours post infection the cells were washed with HP- 1 medium and further incubated for 10 hours at 37 ° C in the presence of

15 μg/ml - 200 μg/ml AZT. The supernatant was harvested and stored at -20°C. The cells were disrupted and mRNA was isolated using the mRNA purification kit (Pharmacia Biotech, Switzerland). CAT was amplified by RT-PCR using the following oligonucleotides and conditions: 5 '-subgenomic: 5'-CTAATACTACAACACCACCACC-3' (SEQ ID NO:3)

3 '-CAT: 5 '-CCCGCCCTGCCACTCATCGC-3 ' (SEQ ID NO:4)

First-Strand Synthesis

Two hundred ng of mRNA was incubated with 0.5 μg oligo dT primer in a final volume of 33 μl according to manufacture manual (Ready to go First Strand Beads, Pharmacia Biotech, Dϋbendorf, Switzerland).

PCR

For the second-strand synthesis, 8 μl of the first strand reaction were mixed with 40 pmol of each primer (1 and 2), 0.1 mM MgSO₄ and 2.5 U Taq polymerase in a total volume of 100 μl. The temperature cycles were as follows: A single denaturation step at 95°C for 2 minutes; followed by 25 cycles of 92°C for 45 sec; 48°C for 30 sec; and 72 °C for 1 minute, followed by 10 cycles of 92 °C for 45 sec, 55 °C for 30 sec and 72 °C for 1 minute. The PCR product was analyzed by restriction digest and the correct fragments were obtained (FIG.6B). Only in the sample where 15 μg/ml AZT were present in the medium, CAT could be amplified (FIG. 6A). At very high concentration of AZT in the medium, no RT-PCR product was obtained, indicating that a very high mutation rate was achieved and therefore the primers annealing was impossible.

Example 3

Production of Sindbis Virus Particles Containing Erythropoietin RNA

One μg of RNase-free vector (pDH-EB; Bredenbeek et al, J. Virol. 77 :6439-6446 (1993)) was linearized by Ec RI digestion. Subsequently, in vitro transcription was carried out using the SP6 in vitro transcription kit (InvitroscripCAP by InvitroGen, InvitroGen BV, NV Leek, The Netherlands).

The resulting 5 '-capped mRNA was analyzed on reducing agarose-gels.

Five μg of in vitro transcribed mRNA were electroporated into 1 C4 cell line (patent application pCYTts) according to InvitroGen's manual ("Sindbis Expression System", Version C, (InvitroGen Catalog No. K750-1), InvitroGen BV, The Netherlands). After 10 hours ofincubationat37°C,the FCS-containing medium was replaced by HP-1 medium without FCS, followed by an additional incubation at 30 °C for 72 hours. The supernatant was harvested and analyzed for viral particle production.

A new BHK cell layer was infected with 10⁷ viral particles in the presence of 10 μg/ml AZT. Two hours post infection the cells were washed with HP-1 medium and further incubated for 30 hours at 37 °C in the presence of 10 μg/ml AZT. The supernatant was harvested and viral RNA was isolated using the high pure viral RNA kit (Boehringer Mannheim). The following RT-PCR was carried out using the One-Step-RT-PCR System (Gibco BRL). The following primers and conditions were chosen:

Epo-3 ' - Primer: 5 '-CCTGCATGCTCATCTG-3 ' (SEQ ID NO:5)

Epo-5' - Primer: 5'-GGGGTGCACGAATGTC-3' (SEQ ID NO:6)

Twelve μl viral RNA were incubated with 100 pmol of each primer (3 and 4), 0.2 mM dNTPs, 1.2 mM MgSO₄, and 1.0 μl of the superscript II RT/Taq mixture in a total volume of 50 μl.

The temperature cycles were as follows: An initial 30 minute cycle for the reverse transcription at 50 °C; followed by 40 cycles of 92 °C for 30 sec; 55 °C for 30 sec, and 72 °C for 90 sec. The obtained PCR product showed the expected length of 600 bp (FIG. 7A). The RT-PCR fragments were digested with Kpnl and Stul (FIG. 7B). The obtained 430 bp cleavage products were isolated and cloned into pBluescript via Kpnl and EcoRV. The DNA sequence of 10 clone were analyzed by DNA sequencing using IRD 800 labeled primers and a Licor sequencer.

Table 1:

Transitions, transversions, insertions and deletions occurring in the presence of 10 μg/ml AZT in the ΕPO-gene

As shown in Table 1, most frequently deletions were occurring as described earlier for AZT; whereas no other bias could be observed. AZT induced transversions and transitions, also insertions could be observed. The overall mutation rate was determined to be 1 in 100. This was 20 times higher as determined for the control reaction treated exactly as the one described above, the only difference was that no AZT was present in the medium.

Example 4 Construction ofgpl40-FOS

The gpl40 gene (Swiss-Prot:P03375) without the internal protease cleavage site was amplified from the original plasmid pAbT4674 (ATCC 40829) containing the full length gpl60 gene by PCR using the following oligonucleotides:

HIV-1 :

5'-ACTAGTCTAGAatgagagtgaaggagaaatatc-3' (SEQ ID NO:7) HIV-end:

5'-TAGCATGCTAGCACCGAAtttatctaattccaataattcttg-3' (SEQ ID NO: 8) HIV-Cleav::

5'-tgcCAGTTTCTCGAGCTGGGTAGCTTTCAGctttgccttggtgggtgctac-3' (SEQ ID NO:9) HIV-Cleav2

5 ' -CTGAAAGCTACCCAGCTCGAGAAACTGgcagtgggaataggagctttg-3 ' ( SEQ I D NO : 10 )

For PCR I, 100 pmol of oligo HIV-1 and HIV-Cleav2 and 5 ng of the template DNA were used in the 75 μl reaction mixture, containing 4 units of Taq or Pwo polymerase, 0.1 mM dNTPs and 1.5 mM MgSO₄.

PCR parameters: 30 cycles with an annealing temperature of 60 °C and an elongation time of 2 minutes at 72 °C. For PCR II, 100 pmol of oligo HIV-end and HIV-Cleav and 5 ng of the template DNA were used in the 75 μl reaction mixture, containing 4 units of Pwo polymerase, 0.1 mM dNTPs and 1.5 mM MgSO₄.

PCR parameters: 30 cycles with an annealing temperature of 60°C and an elongation time of 50 seconds at 72°C.

Both PCR fragments were purified and isolated and taken for an assembly PCR.

For the assembly PCR, 100 pmol of oligo HIV-1 and HIV-end and 2 ng of each PCR fragment (PCR I and PCR II) were used in the 75 μl reaction mixture, containing 4 units of Taq or Pwo polymerase, 0.1 mM dNTPs, and 1.5 nM MgSO₄.

PCR parameters: 30 cycles with an annealing temperature of 60 °C and an elongation time of 2.5 minutes at 72 °C.

The obtained PCR fragment was digested with Xbal and Nhel. At the C-terminal end of gp 140, the EOS amphiphatic helix was fused in frame.

The DNA sequence coding for the EOS amphiphatic helix domain was PCR-amplified from vector pJuFo (Crameri & Suter, Gene 137:69 (1993)), using the oligonucleotides: EOS-HI V:

5'-ttcggtgctagcggtggcTGCGGTGGTCTGACCGAC-3' (SΕQ ID NΟ:l 1)

EOS-Apa:

5'-gatgctgggcccttaaccGCAACCACCGTGTGCCGCC-3' (SΕQ ID NΟ.12)

For the PCR, 100 pmol of each oligo and 5 ng of the template DNA was used in the 75 μl reaction mixture, containing 4 units of Taq or Pwo polymerase,

0.1 mM dNTPs and 1.5 mM MgSO₄. The temperature cycles were as follows:

Ninety-five °C for 2 minutes, followed by 5 cycles of 95 °C (45 seconds), 60°C (30 seconds), 72°C (25 seconds), and followed by 25 cycles of 95°C (45 seconds), 68°C (30 seconds), 72°C (20 seconds). The obtained PCR fragment was digested with Nhel and Bspl20L. One expression vector for gpl40-EOS was obtained in a 3 -fragment ligation of both PCR fragments into pSinRep5. The obtained vector pSinRep5- gpl40-EOS was controlled by restriction analysis and DNA sequencing.

The other expression vector for gpl40-EOS was obtained by ligating the sequence controlled gpl40 gene into pCYTts via Xbal and Bspl2OL. The obtained vector pCYTts-gpl40-EOS was controlled by restriction analysis.

Example 5 Expression of gpl40-Fos using pCYTts-gpl40-FOS

Twenty μg of pCYT-gpl40-EOS were linearized by restriction digest. The reaction was stopped by phenol-chloroform extraction, followed by an isopropanol precipitation of the linearized DNA. The restriction reaction was checked by agarose-gel electrophoresis. For the transfection, 5.4 μg of linearized pCTYts-gpl40-EOS was mixed with 0.6 μg of linearized pSV2Neo in 30 μl of H₂O, and 30 μl of 1 M CaCl₂ solution were added. After addition of 60 μl phosphate buffer (50 mM HΕPΕS, 280 mM NaCl, 1.5 mM Na₂HPO₄, pH 7.05), the solution was vortexed for 5 seconds, followed by an incubation at room temperature for 25 seconds. The solution was immediately added to 2 ml HP-1 medium containing 2% FCS (2% FCS medium). The medium of a 80% confluent BHK21 cell culture in a 6-well plate was then replaced by the DNA- containing medium. After an incubation for 5 hours at 37 °C in CO₂ incubator, the DNA-containing medium was removed and replaced by 2 ml of 15% glycerol in 2% FCS medium. The glycerol-containing medium was removed after a 30- second incubation phase and the cells were washed by rinsing with 5 ml of HP-1 medium containing 10% FCS. Finally, 2 ml of fresh HP-1 medium containing 10% FCS was added.

Stably transfected cells were selected and grown in selection medium (HP-1 medium, supplemented with G418) at 37 °C in a CO₂ incubator. Single cells were sorted in a cell sorter and grown in confluency. The cultures were split, followed by a 12 hour growth period at 37°C. One part of the cells was shifted to 30°C to induce the expression of soluble gpl40-EOS. The other part was kept at 37 °C. All 100 clones were tested for gpl40Fos expression by Western-blot.

The expression of soluble gpl40-EOS was determined by Western- blotting. After SDS-PAGΕ, proteins were transferred to Protan nitrocellulose membranes (Schleicher & Schuell, Germany). The membrane was blocked with 1 % bovine serum albumin (Sigma) in TBS (1 OxTBS per liter: 87.7 g NaCl, 66.1 g Trizma hydrochloride (Sigma), and 9.7 g Trizma base (Sigma), pH 7.4) for 1 hour at room temperature, followed by an incubation with an anti-GP120 antibody (Fitzgerald Industries International, USA) for 1 hour. The blot was washed 3 times for 10 minutes with TBS-T, and incubated for 1 hour with an alkaline- phosphatase-anti-mouse IgG conjugate. After washing 2 times for 10 minutes with TBS-T and 2 times for 10 minutes with TBS, the development reaction was carried out using alkaline phosphatase detection reagents (10 ml AP buffer (100 mM Tris/HCl, 100 mM NaCl, pH 9.5) with 50 μl NBT solution (7.7% Nitro Blue

Tetrazolium (Sigma) in 70%) dimethylformamide) and 37 μl of X-Phosphate solution (5% of 5-bromo-4-chloro-3-indolyl phosphate in dimethylformamide). The best expressing cell line was further analyzed by ΕLISA and silver stained 2-D gel electrophoresis.

Example 6

Expression of a gpl40 quasispecies-pool

The best gpl40-EOS expressing cell line is grown at 37 °C to 70% confluency (8 T- 150 flasks). HP- 1 medium is exchanged by HP- 1 supplemented with nucleoside analogs (AZT, 5-FU, 5-hydroxy-2'deoxycytidine, 4- Nitroquinolinel -oxide, furyl-furamide, mitomycin C, N-methyl-N'-nitro-N- nitrosoguanidine and N4-aminocytidine). The cells are incubated at 30°C to induce gp 140-EOS expression. Seventy-two hours after induction, a first sample is taken and analyzed by Western Blot, exactly as described in Example 5, for heterogeneity. One hundred-twenty hours after induction, a second sample is taken and analyzed by Western Blot and 2D gel-electrophoresis for heterogeneity of the protein pool. The supernatant is harvested approximately 200 hours after induction and the gpl40-EOS protein pool is purified by ion exchange chromatography, followed by gel filtration. The protein pool is also analyzed for its glycosylation pattern. The above- described procedure is carried out for all 8 nucleoside analogs to determine the most effective mutagen for gpl40-EOS.

The most powerful mutagen is taken at different concentration and the expression is carried out exactly as described above, followed by the protein analyzation. The obtained quasispecies pool is purified by ion exchange chromatography, followed by gel filtration in the presence of 2 mM β-mercaptoethanol or 5 mM DTT.

Example 7 Preparation of the Alpha Vaccine particles

Purified Virus-like particles fused to a JUN helix (Renner et al, U.S.

Prov. Appl. No. 60/110,414, filed November 30, 1998, the entire disclosure of which is incoφorated by reference herein) are concentrated using Millipore Ultrafree Centrifugal Filter Devices with a molecular cut-off of 100 kD according to the protocol supplied by the manufacturer. The pH is adjusted to 8.0 and the virus-like particles (VLPs) are incubated with 5 mM DTT for 5 hours. Purified reduced VLPs are incubated with at least a 200-fold molar excess of the gpl40-EOS protein pool in an appropriate buffer (pH 7.5-8.5). The mixture is incubated for 1 hour at 4°C and dialyzed against an appropriate buffer (pH 7.5- 8.5) supplemented with a redox shuffle system, but without DTT or β-mercaptoethanol, for at least 10 hours at 4°C. After concentration of the particles using a Millipore Ultrafree Centrifugal Filter Device with a molecular weight cut-off of 100 kD, the mixture is passed through a Sephacryl S-300 gel filtration column (Pharmacia). Viral particles are eluted with the void volume. It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

The entire disclosure of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are hereby incorporated by reference.

Claims

What Is Claimed Is:

1. A method of preparing a population of viral vectors comprising:

(a) inserting a gene of interest into an alphaviral vector;

(b) replicating said alphaviral vector in the presence of an alphaviral replicase and one or more nucleoside analogs to produce modified genes of interest; and

(c) repeating step (b) for a sufficient number of times that the nucleotide sequences of the modified genes of interest in at least 90% of the members of said population which contain at least one mutation in the gene of interest are 90-99%) identical to the nucleotide sequence of the gene of interest.

2. The method of claim 1, wherein the nucleotide sequences of the modified genes of interest in at least 90% of the members of said population which contain at least one mutation in the gene of interest are 97-99% identical to the nucleotide sequence of the gene of interest.

3. The method of claim 1, wherein the nucleotide sequences of the modified genes of interest in at least 90% of the members of said population which contain at least one mutation in the gene of interest are 95-97%) identical to the nucleotide sequence of the gene of interest.

4. The method of claim 1, wherein the alphaviral vector is pSinRep5.

5. The method of claim 1, wherein the alphaviral replicase is a temperature-sensitive, non-cytopathic replicase.

6. The method of claim 5, wherein the alphaviral vector is pCYTts.

7. The method of claim 1 , wherein said population is produced in a eukaryotic cell.

8. The method of claim 7, wherein said eukaryotic cell is a baby hamster kidney, Chinese hamster ovary or COS cell.

9. The method of claim 7, wherein said eukaryotic cell is of human origin.

10. The method of claim 7, wherein nucleic acid encoding said temperature sensitive, non-cytopathic alphaviral replicase is chromosomally integrated into the genome of said eukaryotic cell.

11. The method of claim 1 , wherein said alphaviral replicase is encoded by the members of said population.

12. The method of claim 1, wherein said nucleoside analogs are selected from the group consisting of:

(a) 5-azacytidine;

(b) 5-fluorouridine;

(c) 5 -hydroxy-2'deoxycytidine;

(d) Mitomycin C;

(e) furyl-furamide;

(f) 4-nitroquinoline 1 -oxide;

(g) N-methyl-N'-nitro-N-nitrosoguanidine;

(h) N4-aminocytidine;

0) 8-oxo-guanosine;

0⁾ N 1 -methyl-N4-aminocytidine;

(k) 3-methylcytidine;

(1) 5-bromocytidine;

(m) 5-nitrosocytidine;

(n) 3-methyluridine;

(o) O4-isobutyluridine; (p) 3-methyladenosine;

(q) 8-hydroxylguanosine; and

(r) N6-methyladenosine.

13. A method of preparing a population of viral vectors comprising: (a) inserting a gene of interest into an alphaviral vector;

(c) repeating step (b) for a sufficient number of times that the modified genes of interest encode polypeptides which are 90-99.5%) identical to the polypeptide encoded by the gene of interest.

14. The method of claim 13, wherein the modified genes of interest encode polypeptides which are 97-99.5% identical to the polypeptide encoded by the gene of interest.

15. The method of claim 13, wherein the modified genes of interest encode polypeptides which are 95-97% identical to the polypeptide encoded by the gene of interest.

16. The method of claim 13, wherein the alphaviral vector is pSinRep5.

17. The method of claim 13, wherein the alphaviral replicase is a temperature-sensitive, non-cytopathic replicase.

18. The method of claim 17, wherein the alphaviral vector is pCYTts.

19. The method of claim 13, wherein said population is produced in a eukaryotic cell.

20. The method of claim 13, wherein said eukaryotic cell is a baby hamster kidney, Chinese hamster ovary or COS cell.

21. The method of claim 20, wherein said eukaryotic cell is of human origin.

22. The method of claim 21, wherein nucleic acid encoding said alphaviral replicase is chromosomally integrated into the genome of said eukaryotic cell.

23. The method of claim 13, wherein said alphaviral replicase is encoded by the members of said population.

24. The method of claim 13, wherein said nucleoside analogs are selected from the group consisting of:

(a) 5-azacytidine;

(b) 5-fluorouridine;

(c) 5-hydroxy-2'deoxycytidine; (d) Mitomycin C;

(e) furyl-furamide;

(f) 4-nitroquinoline 1 -oxide;

(g) N-methyl-N'-nitro-N-nitrosoguanidine; and (h) N4-aminocytidine; (i) 8-oxo-guanosine;

(j) Nl-methyl-N4-aminocytidine;

(k) 3-methylcytidine;

(1) 5-bromocytidine;

(m) 5-nitrosocytidine; (n) 3-methyluridine;

(o) O4-isobutyluridine; (p) 3-methyladenosine;

(q) 8-hydroxylguanosine; and

(r) N6-methyladenosine.

25. A population of alphaviral vectors which encode an alphaviral replicase and comprise modified genes of interest having nucleotide sequences which are 90-99% identical to the nucleotide sequence of a gene of interest.

26. The population of claim 25, wherein the nucleotide sequences of said modified genes of interest are 97-99% identical to the nucleotide sequence of the gene of interest.

27. The population of claim 25, wherein the nucleotide sequences of said modified genes of interest are 95-97% identical to the nucleotide sequence of the gene of interest.

28. The population of claim 25, wherein the alphaviral replicase is a temperature-sensitive, non-cytopathic replicase.

29. The population of claim 25, wherein said gene of interest encodes a polypeptide.

30. The population of claim 25, wherein said gene of interest encodes an untranslated RNA.

31. The population of claim 25, wherein the gene of interest comprises nucleic acid from Semliki Forest Virus, Sindbis virus, Venezuelan equine encephalomyelitis virus, or Ross River Virus.

32. The population of claim 25, wherein said gene of interest comprises a nucleic acid of a virus or organism selected from the group consisting of:

(a) Influenza virus; (b) Human Immunodeficiency Virus Type 1 (HIV-1);

(c) Human Immunodeficiency Virus Type 2 (HIV-2);

(d) Hepatitis B Virus;

(e) Hepatitis C Virus;

(f) Hepatitis E Virus; (g) Hepatitis G Virus;

(h) Rhinovirus;

(i) Lymphocyte Choriomeningitis Virus;

(j) Trypanosoma cruzi;

(k) Trypanosoma brucei; (1) Plasmodium falciparum; and

(m) Plasmodium vivax.

33. A pharmaceutical composition comprising a population of alphaviral vectors of claim 25 in admixture with a pharmaceutically acceptable diluent, adjuvant, or carrier.

34. The pharmaceutical composition of claim 33 which is suitable for use as a vaccine.

35. A population of alphaviral vectors which encode an alphaviral replicase and comprise modified genes of interest which encode polypeptides 90-99.5% identical to the polypeptide encoded by a gene of interest.

36. The population of claim 35, wherein said modified genes of interest encode polypeptides 95-99.5%) identical to the polypeptide encoded by a gene of interest.

37. The population of claim 35, wherein said modified genes of interest encode polypeptides 97-99.5% identical to the polypeptide encoded by a gene of interest.

38. The population of claim 35 , wherein the gene of interest comprises nucleic acid from Semliki Forest Virus, Sindbis virus, Venezuelan equine encephalomyelitis virus, or Ross River Virus.

39. The population of claim 35, wherein the alphaviral replicase is a temperature-sensitive, non-cytopathic replicase.

40. The population of claim 35 which is produced in a eukaryotic host cell.

41. The population of claim 40, wherein said eukaryotic cell is a baby hamster kidney, Chinese hamster ovary or COS cell.

42. The population of claim 35, wherein the gene of interest comprises a nucleic acid of a virus or organism selected from the group consisting of: (a) Influenza virus;

(b) Human Immunodeficiency Virus Type 1 (HIV-1);

(c) Human Immunodeficiency Virus Type 2 (HIV-2);

(d) Hepatitis B Virus;

(e) Hepatitis C Virus; (f) Hepatitis E Virus;

(g) Hepatitis G Virus;

(h) Rhino virus;

(i) Lymphocyte Choriomeningitis Virus;

(j) Trypanosoma cruzi; (k) Trypanosoma brucei; (1) Plasmodium falciparum; and (m) Plasmodium vivax.

43. A pharmaceutical composition comprising the population of claim 35 in admixture with a pharmaceutically acceptable diluent, adjuvant, or carrier.

44. The pharmaceutical composition of claim 43 which is suitable for use as a vaccine.

45. A population of vectors prepared by a method comprising;

(a) inserting a gene of interest into an alphaviral vector;

(c) repeating step (b) for a sufficient number of times that the nucleotide sequences of the modified genes of interest in at least 90%) of the members of said population which contain at least one mutation in the gene of interest are 90-99% identical to the nucleotide sequence of the gene of interest.

46. The population of claim 45, wherein the nucleotide sequences of the modified genes of interest in at least 90% of the members of said population which contain at least one mutation in the gene of interest are 97-99%) identical to the nucleotide sequence of the gene of interest.

47. The population of claim 45, wherein the nucleotide sequences of the modified genes of interest in at least 90% of the members of said population which contain at least one mutation in the gene of interest are 90-97% identical to the nucleotide sequence of the gene of interest.

48. The population of claim 45, wherein the alphaviral vector is pSinRep5.

49. The population of claim 45, wherein the alphaviral replicase is a temperature-sensitive, non-cytopathic replicase.

50. The population of claim 49, wherein the alphaviral vector is pCYTts.

51. The population of claim 45, wherein the members of said population are packaged.

52. The population of claim 45 which is produced in a eukaryotic host cell.

53. The population of claim 52, wherein said eukaryotic cell is a baby hamster kidney, Chinese hamster ovary or COS cell.

54. The population of claim 45 , wherein the gene of interest comprises a nucleic acid of a virus or organism selected from the group consisting of:

(a) Influenza virus;

(b) Human Immunodeficiency Virus Type 1 (HIV-1); (c) Human Immunodeficiency Virus Type 2 (HIV-2);

(d) Hepatitis B Virus;

(e) Hepatitis C Virus;

(f) Hepatitis E Virus;

(g) Hepatitis G Virus; (h) Rhino virus;

(i) Lymphocyte Choriomeningitis Virus;

(j) Trypanosoma cruzi;

(k) Trypanosoma brucei;

(1) Plasmodium falciparum; and (m) Plasmodium vivax.

55. A pharmaceutical composition comprising the population of claim 45 in admixture with a pharmaceutically acceptable diluent, adjuvant, or carrier.

56. The pharmaceutical composition of claim 55 which is suitable for use as a vaccine.

57. A method of vaccinating an individual comprising administering to said individual a pharmaceutically effective amount of a population of alphaviral vectors which encode an alphaviral replicase, wherein the nucleotide sequences of modified genes of interest in at least 90% of the members of said population which contain at least one mutation in a gene of interest are 90-99%) identical to the nucleotide sequence of the gene of interest.

58. The method of claim 57, wherein the nucleotide sequences of the modified genes of interest in at least 90% of the members of said population which contain at least one mutation in a gene of interest are 97-99% identical to the nucleotide sequence of the gene of interest.

59. The method of claim 57, wherein the nucleotide sequences of the modified genes of interest in at least 90% of the members of said population which contain at least one mutation in a gene of interest are 95-97% identical to the nucleotide sequence of the gene of interest.

60. The method of claim 57, wherein the alphaviral vector is pSinRep5.

61. The method of claim 57, wherein the alphaviral replicase is a temperature-sensitive, non-cytopathic replicase.

62. The method of claim 61 , wherein the alphaviral vector is pCYTts.

63. The method of claim 57, wherein the gene of interest comprises nucleic acid from Semliki Forest Virus, Sindbis virus, Venezuelan equine encephalomyelitis virus, or Ross River Virus.

64. The method of claim 57, wherein the gene of interest comprises nucleic acid from HIV-1.

65. The method of claim 64, wherein the gene of interest is glycoprotein 120, glycoprotein 140, or glycoprotein 160 of HIV-1.

66. The method of claim 57, wherein the gene of interest is encodes the hemagglutinin protein of Influenza virus or the hemagglutinin HA1 domain.

67. The method of claim 57, wherein the members of said population are packaged.

68. A method of vaccinating an individual comprising administering to said individual a pharmaceutically effective amount of a population of alphaviral vectors which encode a temperature sensitive, non-cytopathic alphaviral replicase and comprise modified genes of interest which encode polypeptides 90-99.5% identical to the polypeptide encoded by a gene of interest.

69. The method of claim 68, wherein the population of alphaviral vectors comprises modified genes of interest which encode polypeptides 95-97% identical to the polypeptide encoded by a gene of interest.

70. The method of claim 68, wherein the population of alphaviral vectors comprises modified genes of interest which encode polypeptides 97-99.5% identical to the polypeptide encoded by a gene of interest.

71. The method of claim 68, wherein the alphaviral vector is pSinRep5.

72. The method of claim 68, wherein the alphaviral replicase is a temperature-sensitive, non-cytopathic replicase.

73. The method of claim 72, wherein the alphaviral vector is pCYTts.

74. The method of claim 68, wherein the gene of interest comprises nucleic acid from Semliki Forest Virus, Sindbis virus, Venezuelan equine encephalomyelitis virus, or Ross River Virus.

75. The method of claim 68, wherein the gene of interest comprises nucleic acid from HIV-1.

76. The method of claim 75, wherein the gene of interest is glycoprotein 120, glycoprotein 140, or glycoprotein 160 of HIV-1.

77. The method of claim 68, wherein the gene of interest is encodes the hemagglutinin protein of Influenza virus or the hemagglutinin HA1 domain.

78. The method of claim 68, wherein the members of said population are packaged.

79. A method of vaccinating an individual comprising administering to said individual a pharmaceutically effective amount of polypeptide expression products of a population of viral vectors, wherein said polypeptide expression products are produced by:

(a) inserting a gene of interest into an alphaviral vector; (b) replicating said alphaviral vector in the presence of an alphaviral replicase and one or more nucleoside analogs to produce modified genes of interest;

(c) repeating step (b) for a sufficient number of times that the modified genes of interest encode polypeptides which are 90-99.5%) identical to the polypeptide encoded by the gene of interest;

(d) inserting said alphaviral vector containing the modified genes of interest into a host cells;

(e) expressing the modified genes of interest to produce polypeptide expression products; and

(f) recovering said polypeptide expression products.

80. The method of vaccination of claim 79, wherein the modified genes of interest encode polypeptides 95-97%) identical to the polypeptide encoded by a gene of interest.

81. The method of vaccination of claim 79, wherein the modified genes of interest encode polypeptides 97-99.5% identical to the polypeptide encoded by a gene of interest.

82. The method of claim 79, wherein the alphaviral vector is pSinRep5.

83. The method of claim 79, wherein the alphaviral replicase is a temperature-sensitive, non-cytopathic replicase.

84. The method of claim 83, wherein the alphaviral vector is pCYTts.

85. The method of vaccination of claim 79, wherein the gene of interest comprises nucleic acid from Semliki Forest Virus, Sindbis virus, Venezuelan equine encephalomyelitis virus, or Ross River Virus.

86. The method of vaccination of claim 79, wherein the gene of interest comprises nucleic acid from HIV-1.

87. The method of vaccination of claim 86, wherein the gene of interest is glycoprotein 120, glycoprotein 140, or glycoprotein 160 of HIV-1.