Molecular clones with mutated HIV gag/pol, SIV gag and SIV env genes

Abstract

Nucleic acid constructs containing HIV-1 gag/pol and SIV gag or SIV env genes which have been mutated to remove or reduce inhibitory/instability sequences are disclosed. Viral particles and host cells containing these constructs and/or viral particles are also disclosed. The exemplified constructs and viral particles of the invention may be useful in gene therapy for numerous disorders, including HIV infection, or as a vaccine for HIV-1 immunotherapy and immunoprophylaxis.

Description

I. TECHNICAL FIELD

The invention relates to nucleic acids comprising mutated HIV-1 gag/pol and SIV gag gene sequences which are capable of being expressed independently of any SIV or HIV regulatory factors. The invention also relates to nucleic acids comprising a mutated SIV env gene sequence, which is capable of being expressed independently of any SIV or HIV regulatory factors. The preferred nucleic acids of the invention are capable of producing infectious viral particles.

The invention also relates to vectors, vector systems and host cells comprising the mutated HIV-1 gag, HIV-1 pol, SIV gag and/or SIV env gene sequences. The invention also relates host cells comprising these nucleic acids and/or vectors or vector systems. The invention also relates to the use of these nucleic acids, vectors, vector systems and/or host cells for use in gene therapy or as vaccines.

II. BACKGROUND

Until recently, gene therapy protocols have often relied on vectors derived from retroviruses, such as murine leukemia virus (MLV). These vectors are useful because the genes they transduce are integrated into the genome of the target cells, a desirable feature for long-term expression. However, these retroviral vectors can only transduce dividing cells, which limits their use for in vivo gene transfer in nonproliferating cells, such as hepatocytes, myofibers, hematopoietic stem cells, and neurons.

Lentiviruses are a type of retrovirus that can infect both dividing and nondividing cells. They have proven extremely efficient at providing long-term gene expression (for up to 6 months) in a variety of nondividing cells (such as, neurons and macrophages) in animal models. See, e.g., Amado et al., Science 285:674-676 (July 1999). It has been proposed that the optimal gene transfer system would include a vector based on HIV, or other lentivirus, that can integrate into the genome of nonproliferating cells. Because retroviruses integrate in the genome of the target cells, repeated transduction is unnecessary. Therefore, in contrast to an adenoviral vector capable of in vivo gene delivery, problems linked to the humoral response to injected viral antigens can be avoided. See, e.g., Naldini et al., Science, 272:263-267 (1996), p. 263.

HIV and other lentiviruses have a complex genome that, in addition to the essential structural genes (env, gag, and pol), contains regulatory (tat and rev) and accessory genes (vpr, vif vpu, and nef). HIV has evolved to efficiently infect and express its genes in human cells, and is able to infect nondividing cells such as macrophages because its preintegration complex can traverse the intact membrane of the nucleus in the target cell. This complex contains, in addition to the viral DNA, the enzyme integrase, the product of the vpr gene, and a protein encoded by the gag gene called matrix. The matrix protein enables the preintegration complex to pass into the nucleus to access the host DNA. Lentiviruses cannot efficiently transduce truly quiescent cells (cells in the G 0 state). However, unlike murine retroviral vectors, in addition to being able to infect dividing cells, HIV-based vectors can achieve effective and sustained transduction and expression of therapeutic genes in nondividing cells, such as hematopoietic stem cells and in terminally differentiated cells such as neurons, retinal photoreceptors, muscle, and liver cells. See, e.g., Amado et al. (July 1999) and Klimatcheva et al., Frontiers in Bioscience 4:d481-496 (June 1999), and the references cited therein.

Although lentiviral vectors can be efficient gene delivery vehicles, there are safety concerns due to their origin. Therefore, the field has turned its attention to the development of vectors and production systems with built-in safety features to prevent the emergence of replication competent lentivirus (RCL). For example, in most laboratory applications, lentiviral vectors are generally created in a transient system in which a cell line is transfected with three separate constructs: a packaging construct, a transfer construct, and an envelope encoding construct. The packaging construct contains the elements necessary for vector packaging (except for env) and the enzymes required to generate vector particles. The transfer construct contains genetic cis-acting sequences necessary for the vector to infect the target cell and for transfer of the therapeutic (or reporter) gene. The lentivirus env gene is generally deleted from the packaging construct and instead the envelope gene of a different virus is supplied in a third vector “the env-coding vector”, although the lentiviruses env gene may be used if it is desired that the vector be intended to infect CD4 + T cells. A commonly used envelope gene is that encoding the G glycoprotein of the vesicular stomatitis virus (VSV-G), which can infect a wide variety of cells and in addition confers stability to the particle and permits the vector to be concentrated to high titers (see, e.g., Naldini et al., Science 272:263-267 (1996) and Akkina et al. J. Virol. 70:2581 (1996). The use of three separate constructs and the absence of overlapping sequences between them minimizes the possibility of recombination during lentivirus (transfer) vector production. In addition, because no viral proteins are expressed by the lentiviral (transfer) vector itself, they do not trigger an effective immune response against cells expressing vector in animal models (a particular problem with vectors based on adenovirus). See, e.g., Amado et al., Science 285:674-676 (July 1999) and the references cited therein. See also Naldini et al. Science 272:263-267 (1996).

The initial packaging plasmids contained most HIV genes except for env. In an effort to improve safety, subsequent HIV vectors have been produced in which the packaging plasmid is devoid of all accessory genes. This process does not interfere with efficient vector production and significantly increases the safety of the system because potential RCLs lack the accessory genes necessary for efficient replication of HIV in humans. Although these vectors can transduce growth-arrested cell lines and neurons in vivo, they have been reported to not efficiently transduce macrophages. The accessory gene vpr is believed to be necessary for HIV infection of these cells using these HIV vectors. See, Zufferey et al., Nature Biotechnol. 15:871-875 (1997). In contrast, as discussed later herein, the HIV-based lentiviral vectors of the present invention do not need any HIV accessory genes in order to be able to infect human macrophages and the other cells tested.

The requirement of vpr or vif for efficient transduction of liver cells has also been reported. See, e.g., Kafri et al., Nature Genet. 17:314 (1997). These results indicate that the requirement of accessory genes for efficient lentivirus-mediated gene transfer is dependent on the type of cell chosen as target, suggesting that future applications of lentiviral vectors may involve vector constructs with different accessory genes, as needed.

Zufferey et al., (1997) describe an HIV vector system in which the virulence genes, env, vif, vpr, vpu, and nef have been deleted. This multiply attenuated vector conserved the ability to transduce growth-arrested cells and monocyte-derived macrophages in culture, and could efficiently deliver genes in vivo into adult neurons. The packaging plasmids described Zufferey et al. (1 997) and Naldini et al. (1996) encode Rev and Tat, in addition to Gag and Pol.

Lentiviral vectors engineered to become packaged into virions in the absence of the regulatory gene tat have also been described. See, e.g., Kim et al., J. Virol. 72:811-816 (1998) and Miyoshi et al. J. Virol. 72:8150-8157 (1998). In these vectors the tat gene has been removed from the packaging plasmid. Kim et al. state that tat is not necessary as long as the serial 5′ LTR promoter is replaced with a strong constitutive promoter. It also has other advantages for HIV therapy. Replacement of the HIV-1 LTR with a constitutive HCMV promoter permits the use of anti-Tat molecules such as Tat transdominant mutants or Tat activation response element decoys as therapeutic agents, since they will not affect vector production. (see p. 814, col. 2). The removal of the tat gene eliminates an essential virulence factor that could contribute to a possible RCL. Kim et al. (1998) describe a vector system which does not contain tat, vf vpr, vpu and nef The preferred vector system includes the rev gene which, the authors state “with RRE, is required for efficient RNA handling in this system.” (p. 811, col. 2). However, Kim et al. also constructed Rev independent constructs using CTE. Kim et al. state that the rev/RRE components could be removed by using a sequence such as the Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE), thereby eliminating all accessory proteins, but this leads to a significant reduction in titer.

Srinivasakumar et al., J. Virol. 71:5841-5848 (1997) describes the generation of stable HIV-1 packaging lines that constitutively express high levels of HIV-1 structural proteins in either a Rev-dependent or a Rev-independent fashion. These cell lines were used to assess gene transfer by using a HIV-1 vector expressing the hygromycin B resistance gene and to study the effects of Rev, Tat, and Nef on the vector titer. The Rev-independent cell lines were created by using gag-pol and env expression vectors that contain the MPMV CTE. This article describes the construction of four plasmids, among others: CMV gagpol-RRE and pCMVenv, which require Rev coexpression for HIV-1 structural gene expression, and pCMV gagpol-CTE and pCMVenv-CTE, which do not. To create Rev-containing and Rev-independent packaging, cell lines, CMT3 cells were transfected with vectors expressing Gag, Gag-Pol, and Env, using a calcium phosphate transfection procedure.

By creating an HIV vector which contained the MPMV CTE (pTR167-CTE) and a packaging cell line which expressed the HIV structural proteins in a Rev-independent fashion, the authors were able to obtain a HIV vector system that functions completely without Rev. The titer of the vector obtained from this system was essentially the same as that obtained from a parallel system which contained Rev. The authors state that, in this context, the CTE seemed to substitute completely for Rev-RRE functions, similar to what was previously observed in transient-expression assays with Rev-dependent constructs. This is in contrast to situations where several rounds of HIV replication were measured. In those cases, titers from CTE-containing viruses were always reduced by at least 1 log unit compared to viruses utilizing Rev and the RRE. (See, Srinivasakumar et al., p. 5847).

The authors state that the advantages of having a HIV vector system that works in the absence of Rev opens the possibility of using it as a delivery vehicle for intracellular immunization against Rev function. Genes encoding Rev antagonists that have dramatic inhibitory effects on HIV replication, such as Rev M10 or RRE decoys, could be introduced into an HIV vector and put into cells normally injectable by HIV. Expression of the “anti-Rev” gene would be expected to dampen HIV infection. Any residual HIV replication should lead to activation of the vector LTR (by Tat) and create a vector-derived RNA that would be packaged by proteins derived from the infectious virus. In this scenario, the wild-type virus would act as a helper that may allow the spread of vector particles to previously nonimmunized cells. Because of the additional vector spread, it is likely that this type of scheme will be more effective in modulating HIV infection in vivo than one based on traditional retrovirus vectors. The authors state that they are currently testing this approach in model systems. (See, Srinivasakumar et al., p. 5847).

Another development in the quest for a safe system is the so-called self-inactivating (SIN) vector. See, e.g., Yu et al., Proc Natl Acad Sci USA 83:3194-8 (1986) and Miyoshi et al., J. Virol. 72:8150 (1998). In Yu et al., a retrovirus-derived vector SIN vector was designed for the transduction of whole genes into mammalian cells. The SIN vector of Yu et al. contains a deletion of 299 base pairs in the 3′ long terminal repeat (LTR), which includes sequences encoding the enhancer and promoter functions. When viruses derived from such vectors were used to infect NIH 3 T3 cells, the deletion was transferred to the 5′ LTR, resulting in the transcriptional inactivation of the provirus in the infected cell. Introduction of a hybrid gene (human metallothionein-promoted c-fos) into cells via a SIN vector was not associated with rearrangements and led to the formation of an authentic mRNA transcript, which in some cases was induced by cadmium. The vector described in Miyoshi et al. also contains a deletion the 3′ (downstream) LTR. A sequence within the upstream LTR serves as a promoter under which the viral genome is expressed. The deletion introduced in the downstream LTR is transferred to the upstream LTR during reverse transcription. This deletion inactivates the LTR promoter and eliminates the production of vector RNA. The gene (or genes) to be transferred (e.g., a reporter or therapeutic gene) is expressed from an exogenous viral or cellular promoter that is inserted into the lentivirus vector. An important safety feature of SIN vectors is that inactivation of the promoter activity of the LTR reduces the possibility of insertional mutagenesis (of the transfer vector) into the host genome. In addition, because the expression of the (transfer) vector RNA is eliminated, the potential for RCL production in the target cell is further minimized. SIN vectors should be particularly useful in gene transfer experiments designed to study the regulated expression of genes in mammalian cells. Absence of enhancer and promoter sequences in both LTRs of the integrated provirus should also minimize the possibility of activating cellular oncogenes and may provide a safer alternative to be used in human gene therapy. Other modifications to enhance safety and specificity include the use of specific internal promoters that regulate gene expression, either temporally or with tissue or cell specificity.

Other strategies to improve safety in human studies would be to use nonhuman lentiviruses such as simian immunodeficiency virus, bovine immunodeficiency virus, or equine infectious anemia virus. Of these, vectors derived from the feline immunodeficiency virus have been engineered to efficiently transduce nondividing human cells. See, e.g., Poeschla et al., Nature Med. 4:354-357 (1998) and WO 99/15641. In addition, White et al., J. Virol. 73:2832-2840 (April 1999) described lentiviral vectors using human and simian immunodeficient virus elements in attempt to improve safety by reducing the likelihood of recombination between packaging constructs and transfer constructs.

The development of efficient packaging lines has proven challenging because expression of the VSV-G envelope and a number of HIV proteins is toxic to cells. Recently, a producer line has been designed in which the expression of packaging genes and VSV-G, and therefore the production of vector, can be turned on at will. Kafri et al., J. Virol. 73-576-584 (1999). The cell line can be expanded for scale-up vector production when the expression of toxic genes is turned off. This cell line produces high titer vector without generating RCL. Hematopoietic stem cells transduced with an HIV vector were transplanted into rhesus macaques as described by Donahue et al. Blood 92 (suppl. 1), abstract 4648.5 (1998) with at least a 14-month follow-up. At that time the procedure proved to be safe; all animals in the study have remained healthy without evidence of circulating HIV or vector. See, Amado et al., Science 285:674-676 (July 1999).

Many gene therapy protocols have been designed to correct a number of inherited metabolic, infectious, or malignant diseases using the hematopoietic stem cell. This cell has the capacity to self-renew and to differentiate into all of the mature cells of the blood and immune systems. Many diseases that affect these systems could potentially be treated by the stable introduction of therapeutic genes into stem cells. Recently, lentiviral vectors were shown to bypass the need for ex vivo stem cell stimulation (which is necessary when using murine retroviral vectors), by mediating efficient gene transfer into very primitive human stem cells that contributed to stable, long-term reconstitution of SCID mouse bone marrow with many hematopoietic lineages. See, e.g., Miyoshi et al., Science 283:682 (1999). Similarly, in a rhesus macaque model of autologous transplantation with lentivirus-transduced stem cells, multilineage gene expression was found, suggesting transduction of an early blood cell progenitor under conditions of minimal stem cell stimulation, ordinarily insufficient for transduction with murine retroviruses. See, Donahue et al., Blood 92 (suppl. 1), abstract 4648.5 (1999) and Amado et al., Science 285:674-676 (July 1999).

In HIV infection, another advantage of lentiviral vectors designed against HIV is their potential to be mobilized by HIV in the infected patient, because the virus supplies all of the necessary elements for packaging of the vector. If these mobilized vectors contained the HIV envelope, they could efficiently transfer their genes (for example, genes custom-designed to confer resistance against HIV) into CD4 + T cells, protecting them from subsequent HIV infection. Lentiviral vectors can also be designed to efficiently express their genes only in CD4 + T cells that are infected with HIV (so called tat-inducible vectors). In these vectors, all HIV genes, including tat and rev, are ablated; cis-acting sequences required for integration, expression, and packaging are retained, and expression is dependent on the activity of the HIV LTR (which requires transactivation by Tat). It has been shown that in this system, vector expression is induced efficiently upon HIV infection. Moreover, in the absence of genes that confer resistance against HIV, stable integration of this vector in permissive cell lines resulted in inhibition of HIV replication. Although the mechanism of HIV inhibition has not been completely elucidated, preliminary results suggest that this vector competes with HIV at the level of reverse transcription. See, An et al., J. Virol., in press, and Amado et al., Science 285:674-676 (1999).

A number of other potential medical applications, where the modification of the genetic material of quiescent cells could result in the prevention or reversal of a disease process, are beginning to be explored. For example, the finding that lentiviral vectors can mediate stable and long-term gene transfer by direct injection of vector into the rat and mouse retina has lent support to the notion of gene therapy for the treatment of retinitis pigmentosa. This degenerative disease of the retina is characterized by photoreceptor cell death, resulting in a slow progression to blindness. Mutations in the CGMP phosphodiesterase β subunit (PDEβ) gene of rod photoreceptors lead to an autosomal recessive form of retinitis pigmentosa in humans, and in the rd mouse model of the disease. Previous studies have shown that adenovirus and adeno-associated virus-mediated PDEP subretinal gene transfer results in a delay in photoreceptor cell death. Using the rd mouse model, a recent study demonstrated that photoreceptors could be rescued in up to 50% of eyes injected with a lentivirus vector containing the murine PDEβ gene. In contrast with the short-term expression previously obtained with adenovirus vectors, PDEβ expression in this study persisted for at least 24 weeks. This finding points to the potential success of gene therapy in a disease that currently lacks effective treatment. See, Takahashi et al., J. Virol., 73:7812-7816 (September 1999) and Amado et al. Science, 285:674-676 (1999).

In nature, the expression of gag, pol, and env of HIV-1 depends on the presence of the viral Rev protein. This dependence is, at least in part, due to the presence of negatively acting sequences (inhibitory or instability elements [INS]) located within unspliced and partially spliced mRNAs. The positive interaction of Rev with the Rev-responsive element [RME] in these mRNAs counteracts the negative effects of the inhibitory sequences.

None of the above references teach or suggest that the gag and/or pol genes described therein may be replaced with the gag and/or pol genes in which the inhibitory/instability have been mutated to render their expression Rev-idependent. Furthermore, there is no disclosure of the specific HIV-1 gag/pol or SIV gag mutated genes described herein.

The gag/pol clone of the invention was made using the method for eliminating inhibitory/instability regions from a gene as first described in U.S. patent application Ser. No. 07/858,747, filed Mar. 27, 1992 (which issued as U.S. Pat. No. 6,174,666) entitled “Method of Eliminating Inhibitory/Instability Regions from mRNA” and later described in a Continuation-in-Part (“CIP”) application, filed as PCT application PCT/US93/02908 on Mar. 29, 1993 and U.S. Pat. Nos. 5,972,596 and 5,965,726. The disclosure of the CIP application was published as International Publication No. WO 93/20212 on Oct. 14, 1993. (The disclosures of these patents and patent applications are specifically incorporated by reference herein in their entirety.) The method was also described in Schwartz et al., J. Virol. 66:7176-7182 (1992).

Schneider et al., J. Virol. 71:4892-4903 (1997), extend the work described in the patent applications and in Schwartz et al. by identifying and characterizing additional INS within gag, protease and pol genes and mutating them in a similar manner. Schneider et al. disclose nucleic acid constructs which contain completely mutated HIV-1 gag genes, but only partially mutated HIV-1 pol genes.

Schneider et al. demonstrate that expression vectors containing an intact or nearly intact p55 gag region allow the production of immature viral particles in mammalian cells in the absence of any other HIV proteins. The introduction of additional mutations in the protease region allowed efficient production of Gag/protease, which resulted in processing of the Pr55 gag precursor and production of mature Gag particles with a lentivirus-like conical-core structure.

Schneider et al. disclose that Rev-independent expression vectors allow the efficient expression of Gag proteins in many cell lines that are not able to support efficient Rev-RRE-dependent rescue of these RNAs. Schneider et al. also disclose that gag/pol expression vectors may be important for vaccination approaches against HIV-1, since the gag/pol region is more conserved than is the env region and may be important for an effective immune response against HIV and for protection against infection. They also state that efficient HIV gene expression in many cells is also of interest for possible gene transfer experiments using lentiviral vectors in nondividing or slowly dividing cells, since HIV and the other lentiviruses are able to infect quiescent cells.

Pavlakis et al., Natl Conf Hum Retroviruses Relat Infect (2nd). (1995), 91, state that Rev-independent Gag expression vectors were able to produce viral particles in human and mouse cells in the absence of any other HIV proteins, and that additional mutations in the pol region allowed the expression of the protease and the processing of the p55 gag precursor. Direct DNA injection of TAT and Rev independent Gag expression vectors in mouse muscle resulted in Gag expression detected by ELISA and in anti-gag antibody response. Several Rev-and Tat-independent Gag expression cassettes were inserted into retroviral vectors and cell lines expressing Gag or Gag fragments that are dominant negative inhibitors of HIV-1 were constructed.

Shiver et al. (1 996) describe the results of DNA vaccination of mice and non-human primates with mutated plasmid DNA encoding either mutated genes encoding HIV-1 gag (p55 gag) or env (gp120 or gp160). Both gag and env vaccine recipients exhibited antigen-specific cytotoxic and helper T lymphocyte (CTL, Th) responses. The results are stated to demonstrate that DNA vaccines elicited long-lived T cell responses in both mice and nonhuman primates that were disseminated throughout the lymphatics.

III. SUMMARY OF THE INVENTION

The invention relates to nucleic acids comprising the nucleic acid sequence of the mutated HIV-1 gag/pol gene shown in FIG. 1 (SEQUENCE ID NO: 1) and vectors and vector systems comprising these nucleic acids.

The invention also relates to nucleic acids comprising the nucleic acid sequence of the mutated SIV gag gene shown in FIG. 3 and vectors and vector systems comprising these nucleic acids.

The invention also relates to nucleic acids comprising the mutated SIV env gene shown in FIG. 17 and vectors and vector systems comprising these nucleic acids.

The invention also relates to products produced by the nucleic acids, e.g., mRNA, protein, and infectious viral particles.

The invention also relates to compositions comprising these nucleic acids and/or their expression products.

The invention also relates to host cells comprising these nucleic acids, vector systems or viral particles.

The invention also relates to uses of these nucleic acids, vector systems, host cells, expression products, and/or compositions to produce mRNA, proteins, and/or infectious viral particles, and/or to induce antibodies and/or cytotoxic or helper T lymphocytes.

The invention also relates to the use of these nucleic acid constructs, vectors, vector systems and or host cells for use in immunotherapy and immunoprophylaxis, e.g., as a vaccine, or in genetic therapy after expression, preferably in humans. The nucleic acid constructs of the invention can include or be incorporated into lentiviral vectors or other expression vectors or they may also be directly injected into tissue cells resulting in efficient expression of the encoded protein or protein fragment. These constructs may also be used for in-vivo or in-vitro gene replacement, e.g., by homologous recombination with a target gene in-situ.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D . DNA sequence of a mutated HIV-1 gag/pol molecular clone (SEQUENCE ID NO: 1). The gagpol terminator is located at positions 4305-4397 of SEQUENCE ID NO: 1.

FIGS. 2A-2F . Comparison of the sequence of the wild—type and mutated poi region in pCMVgagpolBNkan. Position #1 in the figure is position 2641 in plasmid pCMVgagpolBNkan. The comparison starts at position 1872 from the gag initiator ATG.

FIG. 3 . DNA sequence of a mutated SIV gag molecular clone (SIVgagDX).

FIGS. 4A-4D . Comparison of the mutated SIV gag DNA sequence in SIVgagDX with the wild type SIV sequence from Simian (macaque) immunodeficiency virus isolate 239, clone lambda siv 239-1 (GenBank accession No. M33262).

FIG. 5 . Schematic diagram of some components of sample versions of a lentiviral system. BGH poly (A): bovine growth hormone poly (A) signal; MSD: mutated splice donor site; ψ: encapsidation signal; SD, splice donor site; SA, splice acceptor site; “X” indicates that the ATG codon of the partial gag gene sequence is mutated so that translation of this gene does not occur.

FIG. 6 . Schematic diagram of the packaging construct pCMVgagpolBNkan.

FIG. 7 . Schematic diagram of transfer construct 1 : pmBCwCNluci. The packaging signal, the CMV promoter and the coding region for the luciferase gene are flanked by the 5′ and 3 HIV-1 LTRs, which provide promoter and polyadenylation signals, as indicated by the arrows. Three consecutive arrows indicate the U5, R, and U3 regions of the LTR, respectively. The transcribed portions of the LTRs are shown in black. Some restriction endonuclease cleavage sites are also indicated.

FIG. 8 . Schematic diagram of transfer construct 1 : pmBCmCNluci. Symbols are as above.

FIGS. 9A-9D . DNA sequence of packaging construct pCMVgagpolBNkan.

FIGS. 10A-10E . DNA sequence of transfer construct 1 : pmBCwCNluci.

FIGS. 11A-11E . DNA sequence of transfer construct 1 : pmBCmCNluci.

FIG. 12 . Nucleotide sequence of the region BssHII (711) to ClaI (830) in wild-type HIV-1 molecular clones HXB2 and NL4-3, and in the transfer constructs. The translation initiator signal for Gag protein (ATG) is underlined. pmBCwCNluci and pmBCmCNluci (transfer constructs 1 and 2 ) contain the sequence mBCwCN. Transfer construct 3 contains the sequence m2BCwCN. In contrast to the sequence mBCwCN, m2BCwCN has different mutations at the 5′ splice site region and has an intact Gag ATG.

FIG. 13 . Bar graph showing levels of gag protein that is released from cells upon transient transfection with pCMVgagpolBNkan (labeled pCMVBNKan in the figure).

FIG. 14 . Bar graph showing reverse transcriptase activity from the Rev-independent gag-pol HIV-1 vector pCMVgagpolBNkan (labeled pCMVBNKan in the figure).

FIGS. 15A-15D . Bar graphs showing the amount of luciferase per nanogram of p24 Gag protein detected in cells transducted with PCMVgagpolBNkan Rev-independent gag-HIV-1 based retroviral vectors. The results show that with PCMVgagpolBNkan Rev-independent gag-HIV-1 based retroviral vectors display high transduction efficiency in (A) 293 cells, (B) human lymphoid cells, (C) human myeloid cells (U937), as well as (D) non-dividing cells such as primary human macrophages.

FIG. 16 . Schematic diagram of the SIV envelope encoding vector CMVkan/R-R-SIVgp160CTE.

FIGS. 17A-17D . DNA sequence of the SIV envelope encoding vector CMVkan/R-R-SIVgp160CTE containing a mutated SIV env gene.

V. MODES FOR CARRYING OUT THE INVENTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only, and are not restrictive of the invention, as claimed. The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention and, together with the description, serve to explain the principles of the invention.

One aspect of the invention comprises vectors that encode the Gag and/or Pol of HIV-1 in a Rev-independent manner. An example of such a vector which is described herein is the plasmid pCMVgagpolBNkan, which encodes the complete Gag and Pol of HIV-1 in a Rev-independent manner, and also contains a gene conferring kanamycin resistance. This plasmid is Tat and Rev-independent and was generated by eliminating the inhibitory/instability sequences present in the gag/pol mRNA without altering the amino acid sequence of the proteins coded by the genes.

The gag/pol clone of the invention is a DNA construct of the gag/pol region of HIV which has had the inhibitory/instability regions removed. The construct is expected to be useful as a component a new type of lentivirus vector for use in gene therapy or as a vaccine.

The gag, pol or gag/pol sequences of the invention can be highly expressed in human and other mammalian cells in the absence of any other regulatory and structural protein of HIV, including Rev. When the gag/pol sequences are combined with a sequence encoding an envelope protein, such as the VSV G protein or the HIV envelope protein (e.g., in the same vector or in another expression vector), infectious virus is produced after transfection into human cells. When a gene encoding a non-HIV envelope protein is used, for example, in the presence of the HIV gag/pol gene, the virus particles produced would contains only the HIV proteins Gag and Pol.

Lentiviral vectors or vector systems based on the gag, pol or gag/pol sequences of this invention, as exemplified by the Rev-independent pCMVgagpol BNkan construct described herein, may be used for gene therapy in vivo (e.g., parenteral inoculation of high titer vector) or ex vivo (e.g., in vitro transduction of patient's cells followed by reinfusion into the patient of the transduced cells). These procedures are been already used in different approved gene therapy protocols.

The HIV gag/pol clone and SIV gag clone of the invention were made using the method for eliminating inhibitory/instability regions from a gene as described in U.S. Pat. No. 6,174,666, and also in related U.S. Pat. Nos. 5,972,596 and 5,965,726, which are incorporated by reference herein. This method does not require the identification of the exact location or knowledge of the mechanism of function of the INS. Generally, the mutations are such that the amino acid sequence encoded by the mRNA is unchanged, although conservative and non-conservative amino acid substitutions are also envisioned where the protein encoded by the mutated gene is substantially similar to the protein encoded by the non-mutated gene. The mutated genes can be synthetic (e.g., synthesized by chemical synthesis), semi-synthetic (e.g., a combination of genomic DNA, cDNA, or PCR amplified DNA and synthetic DNA), or recombinantly produced. The genes also may optionally not contain introns. The nucleic acids of the invention may also contain Rev-independent fragments of these genes which retain the desired function (e.g., for antigenicity of Gag or Pol, particle formation (Gag) or enzymatic activity (Pol)), or they may also contain Rev-independent variants which have been mutated so that the encoded protein loses a function that is unwanted in certain circumstances. In the latter case, for example, the gene may be modified to encode mutations (at the amino acid level) in the active site of reverse transcriptase or integrase proteins to prevent reverse transcription or integration. Rev-independent fragments of the gag gene are described in U.S. patent application Ser. No. 07/858,747, filed Mar. 27, 1992, and also in related U.S. Pat. Nos. 5,972,596 and 5,965,726, which are incorporated by reference herein.

In addition to being capable of producing HIV Gag and Pol proteins in the absence of Rev regulatory protein in a cell in vivo, the HIV gag/pol clone and SIV gag clone of the invention are also capable of producing HIV Gag and Pol proteins in the absence of any added cis acting transport element, such as CTE or CTE-like elements (collectively referred herein as RNA Transport Elements (RTE)). Experiments indicate that the mutated vectors of the invention for SIV gag are far superior to those adding CTE (see Qiu et al., J. Virol. 73:9145-52 (1999)).

The expression of the proteins encoded by these vectors after transfection into human cells may be monitored at both the level of RNA and protein production. RNA levels are quantitated by methods known in the art, e.g., Northern blots, S1 mapping or PCR methods. Protein levels may also be quantitated by methods known in the art, e.g., western blot or ELISA or fluorescent detection methods. A fast non-radioactive ELISA protocol can be used to detect gag protein (DUPONT or COULTER gag antigen capture assay).

At least three types of lentiviral vectors based on the gag/pol genes of the invention for use in gene therapy and/or as a vaccine are envisioned, i.e., lentiviral vectors having

a) no round of replication (i.e., a zero replication system)

b) one round of replication

c) a fully replicating system

For a system with no round of replication, a gag/pol gene, or separate gag and pol genes, or fragments of these genes, expressed using appropriate transcription units, e.g., a CMV promoter and a BGH poly (A) site. This will allow expression of the gag/pol unit (or gag or pol or fragment(s) thereof) for vaccine purposes. This expression can be accomplished without the production of any functional retroviral enzymes, provided that the appropriate mutation(s), e.g., a missense mutation, are introduced. In a zero replication system, a virus stock will be administered to the cells or animals of interest. For example, if one creates and uses a virus stock with the exemplified system using the packaging vector PCMVgagpolBNkan, the transfer construct pmBCwCNluci or pmBCmCNluci, and the envelope containing vector pHCMV-G, one obtains a zero replication system. The virus particles produced by such system can infect cells, and the reverse transcribed transfer construct DNA will go into the nucleus but, because the coding regions for viral structural proteins are not present, there will be no virus expression and replication (0 rounds). If one transfects cells in vivo with the same 3 DNAs, they will go to the nucleus, express viral proteins, make infectious virus particles and go out and infect another cell or cells (1 round). Since in vivo delivery of three plasmids may result in lower expression, at least two different embodiments are envisioned. In the first, two plasmids may be used, e.g., MV1 shown in FIG. 5 and an envelope expression plasmid such as pHCMV-G. Other plasmids encoding functional envelopes from HIV, SIV, or other retroviruses can also be used. Transfection by the two plasmids results in infectious virus that can infect and integrate into new cells (1 round). The infected cells produce gagpol but virus propagation is not possible in the absence of env.

For a system with one round of replication, at least two additional embodiments are envisioned. In the first method, a combination of the genes, e.g., a gag/pol gene, an env encoding gene and, preferably, a gene encoding a reporter protein or other polynucleotide or protein of interest, are delivered into the cells of interest in vivo. As discussed above for the exemplified system, if one transfects cells in vivo with the same 3 DNAs, they will go to the nucleus, express viral proteins, make infectious virus particles, be released and infect another cell or cells (1 round).

In another embodiment, the same result (i.e., only one round of replication) can be obtained by using transfer vectors that have deletions in the 3′ LTR and in which a heterologous-promoter (e.g., the CMV-promoter, or inducible promoter, or tissue-specific promoter), is used in place of the ‘3’ LTR promoter. The mutations in the 3′ LTR making it inactive upon reverse transcription and integration. This is because the integrated provirus derives both its 5′ LTR and its 3′ LTR from the 3′ LTR of the starting (transfer) construct. (This is a well-known property of all retroviruses and has been used to make self-inactivating vectors (SIN)). There are several reasons one may want to inactivate the incoming LTR promoter, one of which is to use a different tissue specific or regulated promoter for expression of a gene of interest in the integrated provirus. Note that, with SIN vectors, if one uses a viral stock made in vitro after transfection into cells and collection of infectious virus, there will be no round of replication. If one transfects cells with the DNAs in vivo, there will be one round of replication. If functional gag, pol, or env are not included in the DNA mix, there will not be any infection at all (i.e., infectious viruses will not be made).

A fully replicating Rev-independent system has not been constructed yet, although it is expected that a functional system can be constructed using Rev-independent gag/pol and env sequences. If desired, extra posttranscriptional control elements such as the CTE element, which can replace Rev and give infectious virus (see e.g., Zolotukhin et al., J. Virol.68:944-7952 (1994)) are included. The fully replicating system should be in one piece, containing the LTR, packaging signal, gag/pol, splice site, env, tat, one or more CTE or CTE-like elements (if desired for optimal results), and LTR. Tat is thought to be required in this construct, at least in non-permissive cells. Such a system is depicted in FIG. 5 , (construct MV 2 ). In this system, a cell or animal of interest (preferably human) would be infected with virus stock that then propagates. CTE or CTE-like elements (depicted in construct MV 2 as RTE (RNA Transport Elements)) are desirable since they have been shown to improve expression, and since many retroviruses require the presence of posttranscriptional control elements. There are several types of CTE and CTE-like elements, and these elements appear to work via a different pathway from the Rev-RRE pathway. See, e.g., Tabernero et al., J. Virol. 71:95-101 (1997). See also, Pavlakis and Nappi, PCT/US99/11082, filed May 22, 1999, published as WO 99/61596 on Dec. 2, 1999 (and incorporated herein by reference), which describes a new type of post-transcriptional control element that is able to replace CTE and HIV RRE/Rev. The Pavlakis-Nappi element does not work in the same way as CTE and does not have any sequence or structure homology.

In a preferred embodiment, a lentiviral system of the invention comprises the following three components:

1. a packaging vector containing nucleic acid sequences encoding the elements necessary for vector packaging such as structural proteins (except for HIV env) and the enzymes required to generate vector particles, the packaging vector comprising at least a mutated HIV or SIV gag/pol gene of the invention;

2. a transfer vector containing genetic cis-acting sequences necessary for the vector to infect the target cell and for transfer of the therapeutic or reporter or other gene(s) of interest, the transfer vector comprising the encapsidation signal and the gene(s) of interest or a cloning site for inserting the gene(s) of interest; and

3. a vector containing sequences encoding an element necessary for targeting the viral particle to the intended recipient cell, preferably the gene encoding the G glycoprotein of the vesicular stomatis virus (VSV-G) or amphotrophic MuLV or lentiviral envs.

Using the CMV promoter or other strong, high efficiency, promoter instead of the HIV-1 LTR promoter in the packaging vector, high expression of gag, pol or gag/pol can be achieved in the total absence of any other viral protein. The exchange of the HIV-1 LTR promoter with other promoters is beneficial in the packaging vector or other vectors if constitutive expression is desirable and also for expression in other mammalian cells, such as mouse cells, in which the HIV-1 promoter is weak. Vectors containing the sequences of the invention can be used for the Rev independent production of HIV-1 Gag/Pol, HIV-1 Gag, HIV-1 Pol, and SIV Gag proteins. In certain embodiments, the presence of heterologous promoters will also be desired in the transfer vector and the envelope encoding vector, when such vectors are used.

The gene(s) of interest are chosen according to the effect sought to be achieved. For gene therapy purposes there will be at least one therapeutic gene encoding a gene product which is active against the condition it is desired to treat or prevent. Alternatively or additionally, there may be a gene which acts as a marker by encoding a detectable product. Therapeutic genes may encode, for example, an anti-sense RNA, a ribozyme, a transdominant negative mutant of a target protein, a toxin, a conditional toxin, an antigen that induces antibodies or helper T-cells or cytotoxic T-cells, a single chain antibody or a tumor suppresser protein. See, e.g., WO 98/17816.

An even more extensive list of genes of interest for use in lentiviral vectors is described, e.g., in WO 99/04026 on page 10, line 20 to page 12, line 7. Table 2 of Klimatcheva et al. (1999) also provides a list of disorders and target cells for gene therapy, as well as a number of lentiviral vectors used by others. This list includes genetic/metabolic deficiencies, viral infection and cancer. Inherited genetic defects such as adenosine deaminase deficiency, familial hypercholesterolemia, cystic fibrosis, mucopolysaccharidosis type VII, types I and II diabetes, classical phenylketonuria and Gaucher disease are diseases which are listed as being possible to overcome by lentiviral vector-mediated gene therapy because they constitute single-gene deficiencies for which the involved genes are known. Viral diseases are also listed as constituting appropriate targets for lentiviral gene delivery. In particular, a number of gene therapy approaches have been proposed for the treatment of HIV infection and, for some of these strategies, phase I studies have recently begun in humans. The article states that preliminary studies have dealt with defective murine oncoviruses for delivery of anti-sense RNAs, ribozymes and trans-dominant proteins against HIV replication.

In any of the vectors, but preferably in the transfer vector, an inserted gene could have an internal ribosomal entry site (IRES), e.g., from picornaviral RNA. An IRES will be used in circumstances that one wants to express two proteins from the same promoter. For example one protein of interest and a marker gene, e.g., green fluorescent protein (GFP) or a marker gene and a drug resistance gene (e.g. the firefly luciferase gene and neomycin phosphotransferase gene) as described on p. 58 of WO 99/04026, for example. Using an IRES the expression of the two proteins is coordinated. A further gene or genes may also be present under the control of a separate promoter. Such a gene may encode for example a selectable marker, or a further therapeutic agent which may be among the therapeutic agents listed above. Expression of this gene may be constitutive; in the case of a selectable marker this may be useful for selecting successfully transfected packaging cells, or packaging cells which are producing particularly high titers of the retroviral vector particles. Alternatively or additionally, the selectable marker may be useful for selecting cells which have been successfully infected with the lentiviral vector and have the provirus integrated into their own genome.

One way of performing gene therapy is to extract cells from a patient, infect the extracted cells with a lentiviral vector and reintroduce the cells back into the patient. A selectable marker may be used to provide a means for enriching for infected or transduced cells or positively selecting for only those cells which have been infected or transduced, before reintroducing the cells into the patient. This procedure may increase the chances of success of the therapy. Selectable markers may be for instance drug resistance genes, metabolic enzyme genes, or any other selectable markers known in the art. Typical selection genes encode proteins that confer resistance to antibiotics and other toxic substances, e.g., histidinol, puromycin, hygromycin, neomycin, methotrexate etc. and cell surface markers.

However, it will be evident that for many gene therapy applications of lentiviral vectors, selection for expression of a marker gene may not be possible or necessary. Indeed expression of a selection marker, while convenient for in vitro studies, could be deleterious in vivo because of the inappropriate induction of cytotoxic T lymphocytes (CTLs) directed against the foreign marker protein. Also, it is possible that for in vivo applications, vectors without any internal promoters will be preferable. The presence of internal promoters can affect for example the transduction titres obtainable from a packaging cell line and the stability of the integrated vector. Thus, single transcription unit vectors, which may be bi-cistronic or poly-cistronic, coding for one or two or more therapeutic genes, may be the preferred vector designed for use in vivo. See, e.g., WO 98/17816.

Suitable host or producer cells for use in the invention are well known in the art. May lentiviruses have already been split into replication defective genomes and packaging components. For those which have not the technology is available for doing so. The producer cell encodes the viral components not encoded by the vector genome such as the Gag, Pol and Env proteins. The gag, pol and env genes may be introduced into the producer cell transiently, or may be stably integrated into the cell genome to give a packaging cell line. The lentiviral vector genome is then introduced into the packaging cell line by transfection or transduction to create a stable cell line that has all of the DNA sequences required to produce a lentiviral vector particle. Another approach is to introduce the different DNA sequences that are required to produce lentiviral vector particle, e.g., the env coding constrict, the gag-pol coding construct and the transfer construct into the cell simultaneously by transient triple transfection.

Target cells identified by Klimatcheva et al. (1999), and the references cited therein, include airway epithelial cells for cystic fibrosis; retinal photoreceptor cells for retinitis pigmentosa; progenitors for red blood cells, macrophages, and lymphocytes for hematopoietic disorders, sickle cell anemia, β-thalassemia, lysosomal storage disorders, mucopolysaccharidoses, and severe combined immunodeficiency syndrome; bone marrow cells and macrophages for Gaucher's disease; liver cells for familial hypercholesterolaemia; T-lymphocytes and macrophages for HIV infection; brain tissue, neurons, and glial cells for neurodegenerative diseases such as Parkinson's and Alzheimer's diseases; endothelial cells and cardiac myocytes for cardiovascular diseases; and cancer cells in various tissues (e.g. liver or brain) for cancer. Target cells for other diseases would be apparent to one of skill in the art.

Vaccines and pharmaceutical compositions comprising at least one of the nucleic acid sequences, vectors, vector systems, or transduced or transfected host cells of the invention and a physiologically acceptable carrier are also part of the invention.

As used herein, the term “transduction” generally refers to the transfer of genetic material into the host via infection, e.g., in this case by the lentiviral vector. The term “transfection” generally refers to the transfer of isolated genetic material into cells via the use of specific transfection agents (e.g., calcium phosphate, DEAE Dextran, lipid formulations, gold particles, and other microparticles) that cross the cytoplasmic membrane and deliver some of the genetic material into the cell nucleus.

Systems similar to those described herein can be produced using elements of lentiviruses in addition to the HIV and/or SIV genes described herein.

Pharmaceutical Compositions

The pharmaceutical compositions of the invention contain a pharmaceutically and/or therapeutically effective amount of at least one nucleic acid construct, vector, vector system, viral particle/virus stock, or host cell (i.e., agents) of the invention. In one embodiment of the invention, the effective amount of an agent of the invention per unit dose is an amount sufficient to cause the detectable expression of the gene of interest. In another embodiment of the invention, the effective amount of agent per unit dose is an amount sufficient to prevent, treat or protect against deleterious effects (including severity, duration, or extent of symptoms) of the condition being treated. The effective amount of agent per unit dose depends, among other things, on the species of mammal inoculated, the body weight of the mammal and the chosen inoculation regimen, as is well known in the art. The dosage of the therapeutic agents which will be most suitable for prophylaxis or treatment will also vary with the form of administration, the particular agent chosen and the physiological characteristics of the particular patient under treatment. The dose is administered at least once. Subsequent doses may be administered as indicated.

To monitor the response of individuals administered the compositions of the invention, mRNA or protein expression levels may be determined. In many instances it will be sufficient to assess the expression level in serum or plasma obtained from such an individual. Decisions as to whether to administer another dose or to change the amount of the composition administered to the individual may be at least partially based on the expression levels.

The term “unit dose” as it pertains to the inocula refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined quantity of active material (e.g., nucleic acid, virus stock or host cell) calculated to produce the desired effect in association with the required diluent. The titers of the virus stocks to be administered to a cell or animal will depend on the application and on type of delivery (e.g., in vivo or ex vivo). The virus stocks can be concentrated using methods such as centrifugation. The titers to be administered ex vivo are preferably in the range of 0.001 to 1 infectious unit/cell. Another method of generating viral stocks is to cocultivate stable cell lines expressing the virus with the target cells. This method has been used to achieve better results when using traditional retroviral vectors because the cells can be infected over a longer period of time and they have the chance to be infected with multiple copies of the vector.

For in vivo administration of nucleic acid constructs, vectors, vector systems, virus stocks, or cells which have been transduced or transfected ex vivo, the dose is to be determined by dose escalation, with the upper dose being limited by the onset of unacceptable adverse effects. Preliminary starting doses may be extrapolated from experiments using lentiviral vectors in animal models, by methods known in the art, or may be extrapolated from comparisons with known retroviral (e.g., adenoviral) doses. Generally, small dosages will be used initially and, if necessary, will be increased by small increments until the optimum effect under the circumstances is reached. Exemplary dosages are within the range of 10 8 up to approximately 5×10 15 particles.

Inocula are typically prepared as a solution in a physiologically acceptable carrier such as saline, phosphate-buffered saline and the like to form an aqueous pharmaceutical composition.

The agents of the invention are generally administered with a physiologically acceptable carrier or vehicle therefor. A physiologically acceptable carrier is one that does not cause an adverse physical reaction upon administration and one in which the nucleic acids are sufficiently soluble to retain their activity to deliver a pharmaceutically or therapeutically effective amount of the compound. The pharmaceutically or therapeutically effective amount and method of administration of an agent of the invention may vary based on the individual patient, the indication being treated and other criteria evident to one of ordinary skill in the art. A therapeutically effective amount of a nucleic acid of the invention is one sufficient to prevent, or attenuate the severity, extent or duration of the deleterious effects of the condition being treated without causing significant adverse side effects. The route(s) of administration useful in a particular application are apparent to one or ordinary skill in the art.

Routes of administration of the agents of the invention include, but are not limited to, parenteral, and direct injection into an affected site. Parenteral routes of administration include but are not limited to intravenous, intramuscular, intraperitoneal and subcutaneous. The route of administration of the agents of the invention is typically parenteral and is preferably into the bone marrow, into the CSF intramuscular, subcutaneous, intradermal, intraocular, intracranial, intranasal, and the like. See, e.g., WO 99/04026 for examples of formulations and routes of administration.

The present invention includes compositions of the agents described above, suitable for parenteral administration including, but not limited to, pharmaceutically acceptable sterile isotonic solutions. Such solutions include, but are not limited to, saline and phosphate buffered saline for nasal, intravenous, intramuscular, intraperitoneal, subcutaneous or direct injection into a joint or other area.

In providing the agents of the present invention to a recipient mammal, preferably a human, the dosage administered will vary depending upon such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history and the like.

The administration of the pharmaceutical compositions of the invention may be for either “prophylactic” or “therapeutic” purpose. When provided prophylactically, the compositions are provided in advance of any symptom. The prophylactic administration of the composition serves to prevent or ameliorate any subsequent deleterious effects (including severity, duration, or extent of symptoms) of the condition being treated. When provided therapeutically, the composition is provided at (or shortly after) the onset of a symptom of the condition being treated.

For all therapeutic, prophylactic and diagnostic uses, one or more of the agents of the invention, as well as antibodies and other necessary reagents and appropriate devices and accessories, may be provided in kit form so as to be readily available and easily used.

Where immunoassays are involved, such kits may contain a solid support, such as a membrane (e.g., nitrocellulose), a bead, sphere, test tube, rod, and so forth, to which a receptor such as an antibody specific for the target molecule will bind. Such kits can also include a second receptor, such as a labeled antibody. Such kits can be used for sandwich assays to detect toxins. Kits for competitive assays are also envisioned.

VI. INDUSTRIAL APPLICABILITY

Mutated genes of this invention can be expressed in the native host cell or organism or in a different cell or organism. The mutated genes can be introduced into a vector such as a plasmid, cosmid, phage, virus or mini-chromosome and inserted into a host cell or organism by methods well known in the art. In general, the mutated genes or constructs containing these mutated genes can be utilized in any cell, either eukaryotic or prokaryotic, including mammalian cells (e.g., human (e.g., HeLa), monkey (e.g., Cos), rabbit (e.g., rabbit reticulocytes), rat, hamster (e.g., CHO and baby hamster kidney cells) or mouse cells (e.g., L cells), plant cells, yeast cells, insect cells or bacterial cells (e.g., E. coli. The vectors which can be utilized to clone and/or express these mutated genes are the vectors which are capable of replicating and/or expressing the mutated genes in the host cell in which the mutated genes are desired to be replicated and/or expressed. See, e.g., F. Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience (1992) and Sambrook et al. (1989) for examples of appropriate vectors for various types of host cells. The native promoters for such genes can be replaced with strong promoters compatible with the host into which the gene is inserted. These promoters may be inducible. The host cells containing these mutated genes can be used to express large amounts of the protein useful in enzyme preparations, pharmaceuticals, diagnostic reagents, vaccines and therapeutics.

Mutated genes or constructs containing the mutated genes may also be used for in-vivo or in-vitro gene therapy. For example, a mutated gene of the invention will produce an mRNA in situ to ultimately increase the amount of protein expressed. Such gene include viral genes and/or cellular genes. Such a mutated gene is expected to be useful, for example, in the development of a vaccine and/or genetic therapy.

The constructs and/or proteins made by using constructs encoding the mutated gag, env, and pol genes could be used, for example, in the production of diagnostic reagents, vaccines and therapies for AIDS and AIDS related diseases. The inhibitory/instability elements in the HIV-1 gag gene may be involved in the establishment of a state of low virus production in the host. HIV-1 and the other lentiviruses cause chronic active infections that are not cleared by the immune system. It is possible that complete removal of the inhibitory/instability sequence elements from the lentiviral genome would result in constitutive expression. This could prevent the virus from establishing a latent infection and escaping immune system surveillance. The success in increasing expression of the entire gag/pol gene by eliminating the inhibitory sequence element suggests that one could produce lentiviruses without any negative elements. Such lentiviruses could provide a novel approach towards attenuated vaccines.

For example, vectors expressing high levels of Gag can be used in immunotherapy and immunoprophylaxis, after expression in humans. Such vectors include retroviral vectors and also include direct injection of DNA into muscle cells or other receptive cells, resulting in the efficient expression of gag, using the technology described, for example, in Wolff et al., Science 247:1465-1468 (1990), Wolff et al., Human Molecular Genetics 1(6):363-369 (1992) and Ulmer et al., Science 259:1745-1749 (1993). Further, the gag constructs could be used in transdominant inhibition of HIV expression after the introduction into humans. For this application, for example, appropriate vectors or DNA molecules expressing high levels of p55 gag or p37 gag would be modified to generate transdominant gag mutants, as described, for example, in Trono et al., Cell 59:113-120 (1989). The vectors would be introduced into humans, resulting in the inhibition of HIV production due to the combined mechanisms of gag transdominant inhibition and of immunostimulation by the produced gag protein. In addition, the gag constructs of the invention could be used in the generation of new retroviral vectors based on the expression of lentiviral gag proteins. Lentiviruses have unique characteristics that may allow the targeting and efficient infection of non-dividing cells. Similar applications are expected for vectors expressing high levels of env.

Identification of similar inhibitory/instability elements in SIV indicates that this virus is a convenient model to test these hypotheses. SIV similarly modified could be used in place of HIV in an effort to further minimize the possibility of rearrangement events that would lead to the generation of infectious HIV.

The following examples illustrate certain embodiments of the present invention, but should not be construed as limiting its scope in any way. Certain modifications and variations will be apparent to those skilled in the art from the teachings of the foregoing disclosure and the following examples, and these are intended to be encompassed by the spirit and scope of the invention.

EXAMPLE 1

Rev-Independent HIV-1 Gag/Pol Molecular Clone

FIG. 1 shows the DNA sequence of a Rev-independent HIV-1 gag/pol molecular clone. This DNA sequence shown encodes the complete Gag and Pol of HIV-1 and can be expressed in a Rev-independent manner when operably linked to a promoter. The Rev-independent gag sequence was described in U.S. Pat. Nos. 6,174,666, 5,972,596 and 5,965,726 and the Rev-independent pol sequence was generated by eliminating the inhibitory/instability sequences using the methods described in those,patents. Others have reportedly made Rev independent gag sequences by optimizing codon usage for human cells (see, e.g., WO 98/34640).

FIG. 2 shows an alignment of the sequence of the wild-type and mutated pol region in pCMVgagpolBNkan. Position #1 in the figure is position 2641 in plasmid pCMVgagpolBNkan.

The elimination of INS in gag, pol and env regions allows the expression of high levels of authentic HIV-1 structural proteins in the absence of the Rev regulatory factor of HIV-1.

EXAMPLE 2

Rev-Independent SIV Gag Molecular Clone

FIG. 3 shows the DNA sequence of a Rev-independent SIV gag molecular clone, SIVgagDX. FIG. 4 shows the comparison of wild type (WT) and mutant (SIVgagDX) sequences. The wild type SIV sequence is from Simian (macaque) immunodeficiency virus isolate 239, clone lambda siv 239-1 (GenBank accession No. M33262).

EXAMPLE 3

Rev-Independent SIV Env Molecular Clone

FIG. 16 shows a schematic diagram, and FIG. 17 shows the DNA sequence, of the “env-coding” vector CMVkan/R-R-SIVgp160CTE, which is an example of a vector comprising a mutated lentiviral env gene sequence which is capable of being expressed independently of any SIV or HIV regulatory factors. “CMV” denotes the cytomegalovirus promoter, “SRV-CTE” denotes the constitutive transport element (CTE) of Simian Retrovirus Type 1; “all-STOP” denotes a sequence providing translational stops in all three reading frames; “BGH terminator” denotes the bovine growth hormone polyadenylation signal. Other posttranscriptional control elements can be used instead of the indicated SRV-CTE, for example the one described by Pavlakis and Nappi, PCT/US99/11082, filed May 22, 1999, which was published as WO 99/61596 on Dec. 2, 1999 (and which is incorporated herein by reference).

As mentioned previously above, such a vector encoding a lentiviral env gene may be used if it is desired that the vector infect CD4 + T cells. Also as mentioned previously above, the CTE element (i.e., the SRV-CTE element in the case of vector CMVkan/R-R-SIVgp160CTE), can be replaced with another post-transcriptional control element, such as the Pavlakis-Nappi element, that is able to replace CTE and HIV RRE/Rev. See Pavlakis and Nappi, PCT/US99/11082, filed May 22, 1999, which was published as WO 99/61596 on Dec. 2, 1999 (and which is incorporated herein by reference).

EXAMPLE 4

Lentivirial Vector System

FIG. 5 is a schematic of some of the components of a preliminary version of the Rev-independent lentiviral vector system exemplified herein, including a packaging construct and three different transfer vectors which may be used. In the lentiviral system exemplified herein, the packaging construct also contains the gene for kanamycin resistance. The lentiviral system exemplified herein also contains the vector pHCMV-G, which is shown in FIG. 5 .

In the packaging construct shown in FIG. 5 , “CMV” denotes the cytomegalovirus promoter, “Gag” denotes the gag gene, which generates components of the virion core, “Pro” denotes “protease” “RT” denotes “reverse transcriptase,” “Int” denotes “integrase” and “BGH poly (A)” denotes the bovine growth hormone polyadenylation signal. The protease, reverse transcriptase, and integrase genes comprise the “pol” gene. In transfer construct 1 , “LTR” denotes the HIV “long terminal repeat”, which contains a HIV promoter; “mSD” denotes “mutated splice donor site,” which is present in the construct so that splicing of the RNA transcript does not occur; “ψ” denotes the encapsidation signal; “wGA” denotes part of the wild-type gag gene which contains sequences believed to be necessary for encapsidation; “X” indicates that the ATG codon of the partial gag gene sequence is mutated so that translation of this gene does not occur; “CMV” denotes the cytomegalovirus promoter and luciferase is used as a reporter gene. Luciferase can be replaced with any gene of interest. Another HIV LTR is present at the 3′ end of transfer construct 1 . Replacement of this LTR in constructs such as the transfer construct 1 , 2 , or 3 with a promoter-enhancer deleted HIV LTR leads to inactivation of LTR after integration. Transfer construct 2 is similar to transfer construct 1 , the difference being that a mutated part of the gag gene (denoted “mGa”) is used instead of the wild-type part of the gag gene. Transfer construct 3 (pm2BCwCNluci) has different mutations at the 5′ splice site and has an intact ATG codon so that translation of part of the mutated gag gene occurs. Transfer construct 3 also has a 5′ CMV promoter instead of a 5′ LTR promoter. This construct is expressed independent of the presence of HIV Tat protein. The transfer constructs expressed from the LTR promoter are partially dependent on Tat protein. In 293 cells significant expression can be achieved in the absence of Tat. See, e.g., Valentin et al., Proc. Natl Acad. Sci. U S A. 95:8886-91 (1988).

EXAMPLE 5

Generation of Packaging Construct pCMVgagpol BNkan

FIG. 6 shows a schematic map of the packaging construct pCMV gagpolBNKan. The nucleotide numbering is that of the HXB2R sequence (Genbank accession number K03455 and M38432), where +1 is the start of transcription.

The sequence in HIV-1 gag/pol region was mutated in order to eliminate all the INS. The fragment from the beginning of gag to BsrGI site in pol and the fragment KE [KpnI(3700)-EcoRI(4194)] were previously mutated described in Schneider et al., J. Virol. 71:4892-4903 (1997) and in U.S. Pat. Nos. 6,174,666, 5,972,596 and 5,965,726.

To generate pCMVgagpolBNkan, three fragments within HIV-1 pol region were mutated. They are fragment BP [BsrGI(2207)PflMI(3032)], fragment PK [PflMI(3032)-KpnI(3700)] and fragment EN [EcoRI(4194)-NdeI(4668)]. Mutagenesis was performed using a modified version of the method described by Ho et al., Gene 77:51-59 (1989) and DNA shuffling (Zhao and Arnold, Nucl. Acid Res. 25(6), 1307-1308 (1997). Sixteen oligonucleotides extending over the complete sequence of the three fragments were designed. Six oligos corresponded to fragment BP, six to fragment PK, and four to fragment EN (the oligonucleotides ranged from 130 to 195 bases in length; adjacent oligos overlapped by twenty nucleotides). Each fragment was assembled in two steps:

1) PCR; the reaction was carried out in standard pfu buffer with 10 pmol of each purified big oligo, 0.2 mM of each dNTPs and 2.5 u pfu DNA polymerase enzyme (Stratagene) in a 50 μl final volume. The PCR program was: 3 min 96° C. followed by 50 cycles of 1 min 94° C., 1 min 55° C., and 1 min +5 s/cycle 72° C., ended by 7 min at 72° C. After PCR, the big oligonucleotides were removed from the assembled mutated fragment.

2) The second step was to specifically amplify the assembled products with 30 mer primers located at the 5′ and 3′ end of each mutated fragment. One microliter of the assembled PCR product was used as template in a 25-cycle PCR reaction with 50 pmol of each primer, 1×pfu buffer, 0.2 mM of each dNTP and 2.5 u pfu DNA polymerase in a 50 μl final volume. The PCR program was: 3 min 96° C., 10 cycles of 30 s 94° C., 30 s 55° C., 45 s 72° C., followed by another 14 new line cycles of 30 s 94° C., 30 s 55° C., 45 s +20 s/cycle 72° C., and finally 7 min 72° C. This program gave a single PCR product of the correct size. The amplified BP, PK and EN fragments were individually cloned into PCR-script™ vector using PCR-script™ Amp SK(+) Cloning Kit (Stratagene). Clones were randomly selected and sequenced. The correct BP, PK and EN fragments together with fragment KE previously mutated by Schneider et al. were ligated between BsrGI and KpnI site of p55AM1-R5 (which was previously described in Schneider et al., J. Virol. 71: 4892-4903 (1997)) to produce a completely mutated gagpol ORF. The new plasmid containing the completely mutated gag/pol was named pLTRgagpolBN. BN stands for the modification of the fragment between B srGI and N deI. The mutated gag/pol was then cloned into a CMVkan vector containing the cytomegalovirus major late promoter (GenBank accession no. X17403) and the kanamycin resistance gene, resulting in pCMVgagpolBNkan. The plasmid backbone comes from pVR1332 provided by Vical Inc., and described in Hartikka et al., Hum Gene Ther. 7:1205-17 (1996).

It is understood that different plasmid backbones can be used, e.g., to provide good expression in vivo, in the case of DNA injection, for example.

EXAMPLE 6

Construction of Transfer Vectors pmBCwCNluci and pmBCmCNluci

The HIV-1 sequence BC, between BssHII (257) and ClaI (376), contains the major splice donor site and the encapsidation signal. Six oligos (33 to 46 bases) were designed to introduce mutations on the splice donor site and the AUG start codon of gag. The BC fragment was assembled, amplified and sequenced as described in the section concerning the construction of pCMVgagpolBN.

The mutated BC fragment and a fragment of wild type gag between ClaI (376) and Nsi (793) were placed between the BssHII and Nsi sites of p55RRE (Schneider et al., J. Virol. 71:4892-4903 (1997)) to generate pmBCwCN. In parallel, the fragment between ClaI (376) and NsiI sites of mutated gag from p55BM1-10SD+ was used to generate pmBCmCN. (p55BM1-10OSD+ is similar to p55BM1-10, which is described in Schneider et al. (1997), but contains in addition the intact splice donor and encapsidation site upstream of gag). The region between NsiI and XhoI containing 3′ part of gag and RRE in pmBCwCN and pmBCmCN was replaced by a ClaI-XhoI fragment containing CMV promoter and luciferase gene from pHR′-CMVluci (vector from D. Trono) to generate pmBCwCNluci and pmBCmCNluci (which are shown as transfer constructs 1 and 2 in FIG. 5 , and schematically depicted in FIGS. 7 and 8 , respectively). The sequences of these plasmids are shown in FIGS. 10 and 11 , respectively. Different versions of these plasmids have also been created, by standard procedures, with variations in the region of the encapsidation site, the first splice donor site, and the initiator gag AUG. For example, the transfer construct pm2BcwCNluci (which is shown as transfer construct 3 in FIG. 5 ) has different mutations in the 5′ splice site region and has an intact ATG. A comparison of the sequences in the BssHII-Cla I region of transfer constructs 1 and 2 (mBCwCN frag), transfer construct 3 (m2BCwCN frag), HXB2 and NL43 is shown in FIG. 12 .

EXAMPLE 7

Preparation of Viral Particles

Lentiviral particles were generated by transient cotransfection of 293 human kidney cells with a combination of three plasmids: pCMVgagpolBNkan, pmBCwCNluci or pmBCmCNluci (transfer vector) and pHCMV-G (Yee et al., Proc. Natl. Acad. Sci., USA, 91:9564-9568 (1994) a plasmid coding for the envelope VSV-G (glycoprotein of vesicular stomatitis virus).

The day before the transfection, 293 cells were plated at a density of 10 6 cells/plate on a 60 mm plate. Plasmid DNA was transfected by the Ca-phosphate precipitation method in the following proportions: 3 μg packaging construct, 6 μg transfer construct and 100 ng VSV-G encoding construct, pHCMV-G. [Note that the LTR promoter can be expressed in 293 cells in the absence of Tat with a moderate decrease in efficiency. The transfer constructs can be fully Tat independent after replacement of the LTR promoter with a CMV (see, e.g., transfer construct 3 in FIG. 5 ) or other promoter in such a way that the mRNA start site is at the beginning of the LTR R region.] In the present experiments for preparation of viral particles 500 ng of a Tat expression plasmid was included in the transfection.

Cells were washed the day after transfection and were kept in DMEM medium for another 48 hours before the supernatants were harvested. Supernatants were spun at 1,200 rpm for 7 mins to eliminate any floating cells. pCMVgagpolBNkan produces high levels of Gag protein that is efficiently released from the cells (FIG. 13 ), and also produces high levels of functional Pol as judged by levels of reverse transcriptase activity similar to those found upon expression of complete HIV-1 (FIG. 14 ).

Supernatants from 293 transfected cells were used to transduce several human cell lines (293, Jurkat, U937) and non-dividing human primary macrophages.

EXAMPLE 8

Cell Transduction

Transduction was performed by incubating for 3-4 hours at 37° C. the target cells with 1-2 ml of supernatant containing the retroviral vectors. The amount of retroviral vector present in the supernatant was normalized by p24 content (measured by ELISA). Equal amounts of p24 gag protein were used for infection of cells. This way, differences in production of the different preparations was minimized.

The macrophages used for transduction were isolated from the peripheral blood of healthy donors by adherence to plastic. Cells were cultured in RPMI +20% fetal calf serum (FCS) +10% human serum (HS). After 1 week, non-adherent cells were washed off with PBS and the macrophages were kept in culture for another 1-2 weeks in the absence of human serum. The cells were washed 2-4 times with PBS before transduction.

Cells were harvested 48 hours after transduction (seven days for primary macrophages) and the transduction efficiency was determined by measuring luciferase activity in cell extracts from the cultures. The results of the transduction experiments in 293 Jurkat, U937 and primary macrophages are shown in FIGS. 15A-D . These results demonstrate that Rev-independent gag-HIV-1 based retroviral vectors display high transduction efficiency in (A) 293 cells, (B) human lymphoid cells, (C) human myeloid cells (U937), as well as (D) non-dividing cells such as primary human macrophages.

EXAMPLE 9

Use Of Nucleic Acids of the Invention In Immunoprophylaxis or Immunotherapy

In postnatal gene therapy, new genetic information has been introduced into tissues by indirect means such as removing target cells from the body, infecting them with viral vectors carrying the new genetic information, and then reimplanting them into the body; or by direct means such as encapsulating formulations of DNA in liposomes; entrapping DNA in proteoliposomes containing viral envelope receptor proteins; calcium phosphate co-precipitating DNA; and coupling DNA to a polylysine-glycoprotein carrier complex. In addition, in vivo infectivity of cloned viral DNA sequences after direct intrahepatic injection with or without formation of calcium phosphate coprecipitates has also been described. mRNA sequences containing elements that enhance stability have also been shown to be efficiently translated in Xenopus laevis embryos, with the use of cationic lipid vesicles. See, e.g., J. A. Wolff, et al., Science 247:1465-1468 (1990) and references cited therein.

Recently, it has also been shown that injection of pure RNA or DNA directly into skeletal muscle results in significant expression of genes within the muscle cells. J. A. Wolff, et al., Science 247:1465-1468 (1990). Forcing RNA or DNA introduced into muscle cells by other means such as by particle-acceleration (N. -S. Yang, et al. Proc. Natl. Acad. Sci. USA 87:9568-9572 (1990); S. R. Williams et al., Proc. Natl. Acad. Sci. USA 88:2726-2730 (1991)) or by viral transduction should also allow the DNA or RNA to be stably maintained and expressed. In the experiments reported in Wolff et al., RNA or DNA vectors were used to express reporter genes in mouse skeletal muscle cells, specifically cells of the quadriceps muscles. Protein expression was readily detected and no special delivery system was required for these effects. Polynucleotide expression was also obtained when the composition and volume of the injection fluid and the method of injection were modified from the described protocol. For example, reporter enzyme activity was reported to have been observed with 10 to 100 μl of hypotonic, isotonic, and hypertonic sucrose solutions, Opti-MEM, or sucrose solutions containing 2 mM CaCl 2 and also to have been observed when the 10- to 100-μl injections were performed over 20 min. with a pump instead of within 1 min.

Enzymatic activity from the protein encoded by the reporter gene was also detected in abdominal muscle injected with the RNA or DNA vectors, indicating that other muscles can take up and express polynucleotides. Low amounts of reporter enzyme were also detected in other tissues (liver, spleen, skin, lung, brain and blood) injected with the RNA and DNA vectors. Intramuscularly injected plasmid DNA has also been demonstrated to be stably expressed in non-human primate muscle. S. Jiao et al., Hum. Gene Therapy 3:21-33 (1992).

It has been proposed that the direct transfer of genes into human muscle in situ may have several potential clinical applications. Muscle is potentially a suitable tissue for the heterologous expression of a transgene that would modify disease states in which muscle is not primarily involved, in addition to those in which it is. For example, muscle tissue could be used for the heterologous expression of proteins that can immunize, be secreted in the blood, or clear a circulating toxic metabolite. The use of RNA and a tissue that can be repetitively accessed might be useful for a reversible type of gene transfer, administered much like conventional pharmaceutical treatments. See J. A. Wolff, et al., Science 247:1465-1468 (1990) and S. Jiao et al., Hum. Gene Therapy 3:21-33 (1992).

It had been proposed by J. A. Wolff et al., supra, that the intracellular expression of genes encoding antigens might provide alternative approaches to vaccine development. This hypothesis has been supported by a recent report that plasmid DNA encoding influenza A nucleoprotein injected into the quadriceps of BALB/c mice resulted in the generation of influenza A nucleoprotein-specific cytotoxic T lymphocytes (CTLs) and protection from a subsequent challenge with a heterologous strain of influenza A virus, as measured by decreased viral lung titers, inhibition of mass loss, and increased survival. J. B. Ulmer et al., Science 259:1745-1749 (1993).

Therefore, it appears that the direct injection of RNA or DNA vectors encoding the viral antigen can be used for endogenous expression of the antigen to generate the viral antigen for presentation to the immune system without the need for self-replicating agents or adjuvants, resulting in the generation of antigen-specific CTLs and protection from a subsequent challenge with a homologous or heterologous strain of virus.

CTLs in both mice and humans are capable of recognizing epitopes derived from conserved internal viral proteins and are thought to be important in the immune response against viruses. By recognition of epitopes from conserved viral proteins, CTLs may provide cross-strain protection. CTLs specific for conserved viral antigens can respond to different strains of virus, in contrast to antibodies, which are generally strain-specific.

Thus, direct injection of RNA or DNA encoding the viral antigen has the advantage of being without some of the limitations of direct peptide delivery or viral vectors. See J. A. Ulmer et al., supra, and the discussions and references therein). Furthermore, the generation of high-titer antibodies to expressed proteins after injection of DNA indicates that this may be a facile and effective means of making antibody-based vaccines targeted towards conserved or non-conserved antigens, either separately or in combination with CTL vaccines targeted towards conserved antigens. These may also be used with traditional peptide vaccines, for the generation of combination vaccines. Furthermore, because protein expression is maintained after DNA injection, the persistence of B and T cell memory may be enhanced, thereby engendering long-lived humoral and cell-mediated immunity.

1. Vectors for the Immunoprophylaxis or Immunotherapy Against HIV-1

The mutated gag, pol or gag/pol sequences will be inserted in expression vectors using a strong constitutive promoter such as CMV or RSV, or an inducible promoter such as HIV-1.

The vector will be introduced into animals or humans in a pharmaceutically acceptable carrier using one of several techniques such as injection of DNA directly into human tissues; electroporation or transfection of the DNA into primary human cells in culture (ex vivo), selection of cells for desired properties and reintroduction of such cells into the body, (said selection can be for the successful homologous recombination of the incoming DNA to an appropriate preselected genomic region); generation of infectious particles containing the gag gene, infection of cells ex vivo and reintroduction of such cells into the body; or direct infection by said particles in vivo.

Substantial levels of protein will be produced leading to an efficient stimulation of the immune system.

In another embodiment of the invention, the described constructs will be modified to express mutated Gag proteins that are unable to participate in virus particle formation. It is expected that such Gag proteins will stimulate the immune system to the same extent as the wild-type Gag protein, but be unable to contribute to increased HIV-1 production. This modification should result in safer vectors for immunotherapy and immunophrophylaxis.

EXAMPLE 10

Inhibition of HIV-1 Expression Using Transdominant (TD)-TD-Gag-TD Rev or Td Gap-Pro-TD Rev Genes

Direct injection of DNA or use of vectors other than retroviral vectors will allow the constitutive high level of trans-dominant Gag (TDgag) in cells. In addition, the approach taken by B. K. Felber et al., Science 239:184-187 (1988) will allow the generation of retroviral vectors, e.g. mouse-derived retroviral vectors, encoding HIV-1 TDgag, which will not interfere with the infection of human cells by the retroviral vectors. In the approach of Felber, et al., supra, it was shown that fragments of the HIV-1 LTR containing the promoter and part of the polyA signal can be incorporated without detrimental effects within mouse retroviral vectors and remain transcriptionally silent. The presence of Tat protein stimulated transcription from the HIV-1 LTR and resulted in the high level expression of genes linked to the HIV-1 LTR.

The generation of hybrid TDgag-TDRev or TDgag-pro-TDRev genes and the introduction of expression vectors in human cells will allow the efficient production of two proteins that will inhibit HIV-1 expression. The incorporation of two TD proteins in the same vector is expected to amplify the effects of each one on viral replication. The use of the HIV-1 promoter in a matter similar to one described in B. K. Felber, et al., supra, will allow high level Gag and Rev expression in infected cells. In the absence of infection, expression will be substantially lower. Alternatively, the use of other strong promoters will allow the constitutive expression of such proteins. This approach could be highly beneficial, because of the production of a highly immunogenic gag, which is not able to participate in the production of infectious virus, but which, in fact, antagonizes such production. This can be used as an efficient immuniprophylactic or immunotherapeutic approach against AIDS.

Examples of trans-dominant mutants are described in Trono et al., Cell 59:112-120 (1989).

1. Generation of Constructs Encoding Transdominant Gag Mutant Proteins

Gag mutant proteins that can act as trans-dominant mutants, as described, for example, in Trono et al., supra, will be generated by modifying vector p37M1-10OD or p55M1-13P0 to produce transdominant Gag proteins at high constitutive levels.

The transdominant Gag protein will stimulate the immune system and will inhibit the production of infectious virus, but will not contribute to the production of infectious virus.

The added safety of this approach makes it more acceptable for human application.

VII. REFERENCES

U.S. Pat. No. 6,174,666 issued Jan. 16, 2001 (Pavlakis and Felber)

U.S. Pat. No. 5,972,596 issued Oct. 26, 1999 (Pavlakis and Felber)

U.S. Pat. No. 5,965,726 issued Oct. 12, 1999 (Pavlakis and Felber)

WO 98/17816 Lentiviral Vectors (Kingsman & Kingsman) (Oxford Biomedica Ltd)

WO 98/34640 (Shiver, J. W., Davies, M-E M., Freed, D. C., Liu, M. A. and Perry, H. C. -Merck & Co., Inc.)

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WO 99/04026 Lentiviral Vectors (Chen, Gasmi, Yee and Jolly) (Chiron)

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WO 99/30742 Therapeutic Use of Lentiviral Vectors (Naldini and Song)

WO 99/51754 Infectious Pseudotyped Lentiviral Vectors Lacking Matrix Protein and Uses Thereof (Goettlinger, Reil and Bukovsky) (Dana Farber Cancer Inst Inc)

PCT/US99/11082 Post-Transcriptional Regulatory Elements and Uses Thereof (Pavlakis and Nappi), filed May 22, 1999, published as WO 99/61596 on Dec. 2, 1999

Akkina, R. K., Walton, R. W., Chen, M. L., Li, Q-X, Planelles, V and Chen, I. S. Y., “High-efficiency gene transfer into CD34 + cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G,” J. Virol. 70:2581-2585 (1996)

Amado, R. G. & Chen, I. S. Y., “Letinviral vectors-the promise of gene therapy within reach?,” Science 285:674-676 (July 1999)

Donahue, R. E., An, D. S., Wersto, R. P., Agricola, B. A., Metzger, M. E. and Chen, I. S. Y., “Transplantation of immunoselected CD34 + cells transduced with a EGFP-expressing lentiviral vector in non-human primates,” Blood 92(suppl. 1):383b, Abstract #4648.5 (1998)

Fox, J. L., “Researchers wary of fear-based ban on lentivirus gene therapy,” Nature Biotechnology 16:407-408 (1998)

Goldman, M. J., Lee, P. S., Yang, J. S. & Wilson, J. M., “Lentiviral vectors for gene therapy of cystic fibrosis,” Hum Gene Ther. 8, 2261-2268 (1997)

Hartikka J, Sawdey M, Cornefert-Jensen F, Margalith M, Barnhart K, Nolasco M, Vahlsing H L, Meek J, Marquet M, Hobart P, Norman J, and Manthorpe M., “An improved plasmid DNA expression vector for direct injection into skeletal muscle,” Hum Gene Ther. 7:1205-17 (1996)

Kafri, T., Blomer, U., Peterson, D. A., Gage, F. H. & Verma, I. M., “Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors,” Nat Genet. 17, 314-317 (1997)

Kafri, T., van Praag, H., Ouyang, L., Gage, F. G. and Verma, I. M., “A packaging cell line for lentivirus vectors,” J. Virol. 73:576-584 (1999)

Kim, V. N., Mitrophanous, K., Kingsman, S. M., and Kingsman, A. J., “Minimal Requirement for a Lentivirus Vector Based on Human Immunodeficiency Virus Type 1”, J. Virol. 72:811-816 (1998)

Klimatcheva, E., Rosenblatt, J. D. and Planelles, V., “Lentiviral vectors and gene therapy,” Frontiers in Bioscience 4:d481-496 (June 1999)

Miyoshi, H., Takahashi, M., Gage, F. H. & Verma, I. M., “Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector,” Proc Natl Acad Sci USA. 94: 10319-10323 (1997)

Miyoshi, H., Blomer,U., Takahashi, M., Gage, F. H., and Verma, I. M., “Development of self-inactivating lentivirus vector,” J. Virol. 72:8150-8157 (1998)

Miyoshi, H., Smith, K. A., Mosier, D. E., Verma, I. M. and Torbett, B. E., “Transduction of human CD34 + cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors,” Science 283:682-686 (1999)

Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H., Verma, I. M. & Trono, D., “In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector,” Science. 272, 263-267 (1996)

Naviaux, R. K, Costanzi, E., Haas, M. and Verma, I., “The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses,” J Virol. 70:5701-5705 (1996)

Pavlakis, G. N., Schneider, R.; Song, S., Nasioulas, G., Zolotukhin, A., Felber, B. K., Trauger, R., Cox, J., and Manthorpe, M., “Use of simple Rev-independent HIV-1 gag expression vectors in gene therapy and gene vaccine applications,” Natl Conf Hum Retroviruses Relat Infect (2nd), Jan. 29-Feb. 2,(1995); 91.

Poeschla, E. M., Wong-Staal, F. & Looney, D. J., “Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors,” Nature Med. 4:354-357 (1998)

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Those skilled in the art will recognize that any gene encoding a mRNA containing an inhibitory/instability sequence or sequences can be modified in accordance with the exemplified methods of this invention or their functional equivalents.

Modifications of the above described modes for carrying out the invention that are obvious to those of skill in the fields of genetic engineering, virology, immunology, medicine, and related fields are intended to be within the scope of the following claims.

Every reference cited hereinbefore throughout the application is hereby incorporated by reference in its entirety.

#             SEQUENCE LISTING
<160> NUMBER OF SEQ ID NOS: 19
<210> SEQ ID NO 1
<211> LENGTH: 4338
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
#Sequence:  Mutated
Human Immunodeficiency Virus - 1
#Gag/Pol gene
<400> SEQUENCE: 1
atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgatggga aa
#aaattcgg     60
ttaaggccag ggggaaagaa gtacaagcta aagcacatcg tatgggcaag ca
#gggagcta    120
gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag ac
#aaatactg    180
ggacagctac aaccatccct tcagacagga tcagaggagc ttcgatcact at
#acaacaca    240
gtagcaaccc tctattgtgt gcaccagcgg atcgagatca aggacaccaa gg
#aagcttta    300
gacaagatag aggaagagca aaacaagtcc aagaagaagg cccagcaggc ag
#cagctgac    360
acaggacaca gcaatcaggt cagccaaaat taccctatag tgcagaacat cc
#aggggcaa    420
atggtacatc aggccatatc acctagaact ttaaatgcat gggtaaaagt ag
#tagaagag    480
aaggctttca gcccagaagt gatacccatg ttttcagcat tatcagaagg ag
#ccacccca    540
caggacctga acacgatgtt gaacaccgtg gggggacatc aagcagccat gc
#aaatgtta    600
aaagagacca tcaatgagga agctgcagaa tgggatagag tgcatccagt gc
#atgcaggg    660
cctattgcac caggccagat gagagaacca aggggaagtg acatagcagg aa
#ctactagt    720
acccttcagg aacaaatagg atggatgaca aataatccac ctatcccagt ag
#gagagatc    780
tacaagaggt ggataatcct gggattgaac aagatcgtga ggatgtatag cc
#ctaccagc    840
attctggaca taagacaagg accaaaggaa ccctttagag actatgtaga cc
#ggttctat    900
aaaactctaa gagctgagca agcttcacag gaggtaaaaa attggatgac ag
#aaaccttg    960
ttggtccaaa atgcgaaccc agattgtaag accatcctga aggctctcgg cc
#cagcggct   1020
acactagaag aaatgatgac agcatgtcag ggagtaggag gacccggcca ta
#aggcaaga   1080
gttttggccg aggcgatgag ccaggtgacg aactcggcga ccataatgat gc
#agagaggc   1140
aacttccgga accagcggaa gatcgtcaag tgcttcaatt gtggcaaaga ag
#ggcacacc   1200
gccaggaact gccgggcccc ccggaagaag ggctgttgga aatgtggaaa gg
#aaggacac   1260
caaatgaaag attgtactga gagacaggct aattttttag ggaagatctg gc
#cttcctac   1320
aagggaaggc cagggaattt tcttcagagc agaccagagc caacagcccc ac
#cagaagag   1380
agcttcaggt ctggggtaga gacaacaact ccccctcaga agcaggagcc ga
#tagacaag   1440
gaactgtatc ctttaacttc cctcagatca ctctttggca acgacccctc gt
#cacagtaa   1500
ggatcggggg gcaactcaag gaagcgctgc tcgatacagg agcagatgat ac
#agtattag   1560
aagaaatgag tttgccagga agatggaaac caaaaatgat aggggggatc gg
#gggcttca   1620
tcaaggtgag gcagtacgac cagatactca tagaaatctg tggacataaa gc
#tataggta   1680
cagtattagt aggacctacc tacacctgtc aacataattg gaagaaatct gt
#tgacccag   1740
atcggctgca ccttgaactt ccccatcagc cctattgaga cggtgcccgt ga
#agttgaag   1800
ccggggatgg acggccccaa ggtcaagcaa tggccattga cgaaagagaa ga
#tcaaggcc   1860
ttagtcgaaa tctgtacaga gatggagaag gaagggaaga tcagcaagat cg
#ggcctgag   1920
aacccctaca acactccagt cttcgcaatc aagaagaagg acagtaccaa gt
#ggagaaag   1980
ctggtggact tcagagagct gaacaagaga actcaggact tctgggaagt tc
#agctgggc   2040
atcccacatc ccgctgggtt gaagaagaag aagtcagtga cagtgctgga tg
#tgggtgat   2100
gcctacttct ccgttccctt ggacgaggac ttcaggaagt acactgcctt ca
#cgatacct   2160
agcatcaaca acgagacacc aggcatccgc taccagtaca acgtgctgcc ac
#agggatgg   2220
aagggatcac cagccatctt tcaaagcagc atgaccaaga tcctggagcc ct
#tccgcaag   2280
caaaacccag acatcgtgat ctatcagtac atggacgacc tctacgtagg aa
#gtgacctg   2340
gagatcgggg cagcacagga ccaagatcga ggagctgaga cagcatctgt tg
#aggtgggg   2400
actgaccaca ccagacaaga agcaccagaa ggaacctccc ttcctgtgga tg
#ggctacga   2460
actgcatcct gacaagtgga cagtgcagcc catcgtgctg cctgagaagg ac
#agctggac   2520
tgtgaacgac atacagaagc tcgtgggcaa gttgaactgg gcaagccaga tc
#tacccagg   2580
catcaaagtt aggcagctgt gcaagctgct tcgaggaacc aaggcactga ca
#gaagtgat   2640
cccactgaca gaggaagcag agctagaact ggcagagaac cgagagatcc tg
#aaggagcc   2700
agtacatgga gtgtactacg acccaagcaa ggacctgatc gcagagatcc ag
#aagcaggg   2760
gcaaggccaa tggacctacc aaatctacca ggagcccttc aagaacctga ag
#acaggcaa   2820
gtacgcaagg atgaggggtg cccacaccaa cgatgtgaag cagctgacag ag
#gcagtgca   2880
gaagatcacc acagagagca tcgtgatctg gggcaagact cccaagttca ag
#ctgcccat   2940
acagaaggag acatgggaga catggtggac cgagtactgg caagccacct gg
#atccctga   3000
gtgggagttc gtgaacaccc ctcccttggt gaaactgtgg tatcagctgg ag
#aaggaacc   3060
catcgtggga gcagagacct tctacgtgga tggggcagcc aacagggaga cc
#aagctggg   3120
caaggcaggc tacgtgacca accgaggacg acagaaagtg gtgaccctga ct
#gacaccac   3180
caaccagaag actgagctgc aagccatcta cctagctctg caagacagcg ga
#ctggaagt   3240
gaacatcgtg acagactcac agtacgcatg ggcatcatcc aagcacaacc ag
#accaatcc   3300
gagtcagagc tggtgaacca gatcatcgag cagctgatca agaaggagaa ag
#tgtacctg   3360
gcatgggtac cagcacacaa aggaattgga ggaaatgaac aagtagataa at
#tagtcagt   3420
gctgggatcc ggaaggtgct gttcctggac gggatcgata aggcccaaga tg
#aacatgag   3480
aagtaccact ccaactggcg cgctatggcc agcgacttca acctgccacc tg
#tagtagca   3540
aaagaaatag tagccagctg tgataaatgt cagctaaaag gagaagccat gc
#atggacaa   3600
gtagactgta gtccaggaat atggcagctg gactgcacgc acctggaggg ga
#aggtgatc   3660
ctggtagcag ttcatgtagc cagtggatat atagaagcag aagttatccc tg
#ctgaaact   3720
gggcaggaaa cagcatattt tcttttaaaa ttagcaggaa gatggccagt aa
#aaacaata   3780
cacacggaca acggaagcaa cttcactggt gctacggtta aggccgcctg tt
#ggtgggcg   3840
ggaatcaagc aggaatttgg aattccctac aatccccaat cgcaaggagt cg
#tggagagc   3900
atgaacaagg agctgaagaa gatcatcgga cagtgaggga tcaggctgag ca
#cctgaaga   3960
cagcagtgca gatggcagtg ttcatccaca acttcaaaag aaaagggggg at
#tggggggt   4020
acagtgcagg ggaaaggatc gtggacatca tcgccaccga catccaaacc aa
#ggagctgc   4080
agaagcagat caccaagatc cagaacttcc gggtgtacta ccgcgacagc cg
#caacccac   4140
tgtggaaggg accagcaaag ctcctctgga agggagaggg ggcagtggtg at
#ccaggaca   4200
acagtgacat caaagtggtg ccaaggcgca aggccaagat catccgcgac ta
#tggaaaac   4260
agatggcagg tgatgattgt gtggcaagta gacaggatga ggattagaac ct
#ggaagagc   4320
ctggtgaagc accatatg
#
#
#4338
<210> SEQ ID NO 2
<211> LENGTH: 2507
<212> TYPE: DNA
<213> ORGANISM: Human immunodeficiency virus type
#1
<400> SEQUENCE: 2
tgtacagaga tggaaaagga agggaaaatt tcaaaaattg ggcctgaaaa tc
#catacaat     60
actccagtat ttgccataaa gaaaaaagac agtactaaat ggagaaaatt ag
#tagatttc    120
agagaactta ataagagaac tcaagacttc tgggaagttc aattaggaat ac
#cacatccc    180
gcagggttaa aaaagaaaaa atcagtaaca gtactggatg tgggtgatgc at
#atttttca    240
gttcccttag atgaagactt caggaaatat actgcattta ccatacctag ta
#taaacaat    300
gagacaccag ggattagata ccatacctag tataaacaat gagacaccag gg
#atttgata    360
tcagtacaat gtgcttccac agggatggaa aggatcacca gcaatattcc aa
#agtagcat    420
gacaaaaatc ttagagcctt ttagaaaaca aaatccagac atagttatct at
#caatacat    480
ggatgatttg tatgtaggat ctgacttaga aatagggcag catagaacaa aa
#atagagga    540
gctgagacaa catctgttga ggtggggact taccacacca gacaaaaaac at
#cagaaaga    600
acctccattc ctttggatgg gttatgaact ccatcctgat aaatggacag ta
#cagcctat    660
agtgctgcca gaaaaagaca gctggactgt caatgacata cagaagttag tg
#gggaaatt    720
gaattgggca agtcagattt acccagggat taaagtaagg caattatgta aa
#ctccttag    780
aggaaccaaa gcactaacag aagtaatacc actaacagaa gaagcagagc ta
#gaactggc    840
agaaaacaga gagattctaa aagaaccagt acatggagtg tattatgacc ca
#tcaaaaga    900
cttaatagca gaaatacaga agcaggggca aggccaatgg acatatcaaa tt
#tatcaaga    960
gccatttaaa aatctgaaaa caggaaaata tgcaagaatg aggggtgccc ac
#actaatga   1020
tgtaaaacaa ttaacagagg cagtgcaaaa aataaccaca gaaagcatag ta
#atatgggg   1080
aaagactcct aaatttaaac tgcccataca aaaggaaaca tgggaaacat gg
#tggacaga   1140
gtattggcaa gccacctgga ttcctgagtg ggagtttgtt aatacccctc ct
#ttagtgaa   1200
attatggtac cagttagaga aagaacccat agtaggagca gaaaccttct at
#gtagatgg   1260
ggcagctaac agggagacta aattaggaaa agcaggatat gttactaata ga
#ggaagaca   1320
aaaagttgtc accctaactg acacaacaaa tcagaagact gagttacaag ca
#atttatct   1380
agctttgcag gattcgggat tagaagtaaa catagtaaca gactcacaat at
#gcattagg   1440
aatcattcaa gcacaaccag atcaaagtga atcagagtta gtcaatcaaa ta
#atagagca   1500
gttaataaaa aaggaaaagg tctatctggc atgggtacca gcacacaaag ga
#attggagg   1560
aaatgaacaa gtagataaat tagtcagtgc tggaatcagg aaagtactat tt
#ttagatgg   1620
aatagataag gcccaagatg aacatgagaa atatcacagt aattggagag ca
#atggctag   1680
tgattttaac ctgccacctg tagtagcaaa agaaatagta gccagctgtg at
#aaatgtca   1740
gctaaaagga gaagccatgc atggacaagt agactgtagt ccaggaatat gg
#caactaga   1800
ttgtacacat ttagaaggaa aagttatcct ggtagcagtt catgtagcca gt
#ggatatat   1860
agaagcagaa gttattccag cagaaacagg gcaggaaaca gcatattttc tt
#ttaaaatt   1920
agcaggaaga tggccagtaa aaacaataca tacagacaat ggcagcaatt tc
#accagtgc   1980
tacggttaag gccgcctgtt ggtgggcggg aatcaagcag gaatttggaa tt
#ccctacaa   2040
tccccaaagt caaggagtag tagaatctat gaataaagaa ttaaagaaaa tt
#ataggaca   2100
ggtaagagat caggctgaac atcttaagac agcagtacaa atggcagtat tc
#atccacaa   2160
ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag ta
#gacataat   2220
agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aa
#aattttcg   2280
ggtttattac agggacagca gaaatccact ttggaaagga ccagcaaagc tc
#ctctggaa   2340
aggtgaaggg gcagtagtaa tacaagataa tagtgacata aaagtagtgc ca
#agaagaaa   2400
agcaaagatc attagggatt atggaaaaca gatggcaggt gatgattgtg tg
#gcaagtag   2460
acaggatgag gattagaaca tggaaaagtt tagtaaaaca ccatatg
#              2507
<210> SEQ ID NO 3
<211> LENGTH: 2467
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
#Sequence:  Mutated
Human Immunodeficiency Virus - 1
#Pol gene
<400> SEQUENCE: 3
tgtacagaga tggagaagga agggaagatc agcaagatcg ggcctgagaa cc
#cctacaac     60
actccagtct tcgcaatcaa gaagaaggac agtaccaagt ggagaaagct gg
#tggacttc    120
agagagctga acaagagaac tcaggacttc tgggaagttc agctgggcat cc
#cacatccc    180
gctgggttga agaagaagaa gtcagtgaca gtgctggatg tgggtgatgc ct
#acttctcc    240
gttcccttgg acgaggactt caggaagtac actgccttca cgatacctag ca
#tcaacaac    300
gagacaccag gcatccgcta ccagtacaac gtgctgccac agggatggaa gg
#gatcacca    360
gccatctttc aaagcagcat gaccaagatc ctggagccct tccgcaagca aa
#acccagac    420
atcgtgatct atcagtacat ggacgacctc tacgtaggaa gtgacctgga ga
#tcgggcag    480
cacaggacca agatcgagga gctgagacag catctgttga ggtggggact ga
#ccacacca    540
gacaagaagc accagaagga acctcccttc ctgtggatgg gctacgaact gc
#atcctgac    600
aagtggacag tgcagcccat cgtgctgcct gagaaggaca gctggactgt ga
#acgacata    660
cagaagctcg tgggcaagtt gaactgggca agccagatct acccaggcat ca
#aagttagg    720
cagctgtgca agctgcttcg aggaaccaag gcactgacag aagtgatccc ac
#tgacagag    780
gaagcagagc tagaactggc agagaaccga gagatcctga aggagccagt ac
#atggagtg    840
tactacgacc caagcaagga cctgatcgca gagatccaga agcaggggca ag
#gccaatgg    900
acctaccaaa tctaccagga gcccttcaag aacctgaaga caggcaagta cg
#caaggatg    960
aggggtgccc acaccaacga tgtgaagcag ctgacagagg cagtgcagaa ga
#tcaccaca   1020
gagagcatcg tgatctgggg caagactccc aagttcaagc tgcccataca ga
#aggagaca   1080
tgggagacat ggtggaccga gtactggcaa gccacctgga tccctgagtg gg
#agttcgtg   1140
aacacccctc ccttggtgaa actgtggtat cagctggaga aggaacccat cg
#tgggagca   1200
gagaccttct acgtggatgg ggcagccaac agggagacca agctgggcaa gg
#caggctac   1260
gtgaccaacc gaggacgaca gaaagtggtg accctgactg acaccaccaa cc
#agaagact   1320
gagctgcaag ccatctacct agctctgcaa gacagcggac tggaagtgaa ca
#tcgtgaca   1380
gactcacagt acgcactggg catcatccaa gcacaaccag accaatccga gt
#cagagctg   1440
gtgaaccaga tcatcgagca gctgatcaag aaggagaaag tgtacctggc at
#gggtacca   1500
gcacacaaag gaattggagg aaatgaacaa gtagataaat tagtcagtgc tg
#ggatccgg   1560
aaggtgctgt tcctggacgg gatcgataag gcccaagatg aacatgagaa gt
#accactcc   1620
aactggcgcg ctatggccag cgacttcaac ctgccacctg tagtagcaaa ag
#aaatagta   1680
gccagctgtg ataaatgtca gctaaaagga gaagccatgc atggacaagt ag
#actgtagt   1740
ccaggaatat ggcagctgga ctgcacgcac ctggagggga aggtgatcct gg
#tagcagtt   1800
catgtagcca gtggatatat agaagcagaa gttatccctg ctgaaactgg gc
#aggaaaca   1860
gcatattttc ttttaaaatt agcaggaaga tggccagtaa aaacaataca ca
#cggacaac   1920
ggaagcaact tcactggtgc tacggttaag gccgcctgtt ggtgggcggg aa
#tcaagcag   1980
gaatttggaa ttccctacaa tccccaatcg caaggagtcg tggagagcat ga
#acaaggag   2040
ctgaagaaga tcatcggaca agtgagggat caggctgagc acctgaagac ag
#cagtgcag   2100
atggcagtgt tcatccacaa cttcaaaaga aaagggggga ttggggggta ca
#gtgcaggg   2160
gaaaggatcg tggacatcat cgccaccgac atccaaacca aggagctgca ga
#agcagatc   2220
accaagatcc agaacttccg ggtgtactac cgcgacagcc gcaacccact gt
#ggaaggga   2280
ccagcaaagc tcctctggaa gggagagggg gcagtggtga tccaggacaa ca
#gtgacatc   2340
aaagtggtgc caaggcgcaa ggccaagatc atccgcgact atggaaaaca ga
#tggcaggt   2400
gatgattgtg tggcaagtag acaggatgag gattagaacc tggaagagcc tg
#gtgaagca   2460
ccatatg
#
#
#        2467
<210> SEQ ID NO 4
<211> LENGTH: 1533
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
#Sequence:  Mutated
Simian Immunodeficiency Virus Gag ge
#ne
<400> SEQUENCE: 4
atgggcgtga gaaactccgt cttgtcaggg aagaaagcag atgaattaga aa
#aaattagg     60
ctacgaccca acggaaagaa aaagtacatg ttgaagcatg tagtatgggc ag
#caaatgaa    120
ttagatagat ttggattagc agaaagcctg ttggagaaca aagaaggatg tc
#aaaaaata    180
ctttcggtct tagctccatt agtgccaaca ggctcagaaa atttaaaaag cc
#tttataat    240
actgtctgcg tcatctggtg cattcacgca gaagagaaag tgaaacacac tg
#aggaagca    300
aaacagatag tgcagagaca cctagtggtg gaaacaggaa ccaccgaaac ca
#tgccgaag    360
acctctcgac caacagcacc atctagcggc agaggaggaa actacccagt ac
#agcagatc    420
ggtggtaact acgtccacct gccactgtcc ccgagaaccc tgaacgcttg gg
#tcaagctg    480
atcgaggaga agaagttcgg agcagaagta gtgccaggat tccaggcact gt
#cagaaggt    540
tgcaccccct acgacatcaa ccagatgctg aactgcgttg gagaccatca gg
#cggctatg    600
cagatcatcc gtgacatcat caacgaggag gctgcagatt gggacttgca gc
#acccacaa    660
ccagctccac aacaaggaca acttagggag ccgtcaggat cagacatcgc ag
#gaaccacc    720
tcctcagttg acgaacagat ccagtggatg taccgtcagc agaacccgat cc
#cagtaggc    780
aacatctacc gtcgatggat ccagctgggt ctgcagaagt gcgtccgtat gt
#acaacccg    840
accaacattc tagatgtaaa acaagggcca aaagagccat ttcagagcta tg
#tagacagg    900
ttctacaaaa gtttaagagc agaacagaca gatgcagcag taaagaattg ga
#tgactcaa    960
acactgctga ttcaaaatgc taacccagat tgcaagctag tgctgaaggg gc
#tgggtgtg   1020
aatcccaccc tagaagaaat gctgacggct tgtcaaggag taggggggcc gg
#gacagaag   1080
gctagattaa tggcagaagc cctgaaagag gccctcgcac cagtgccaat cc
#cttttgca   1140
gcagcccaac agaggggacc aagaaagcca attaagtgtt ggaattgtgg ga
#aagaggga   1200
cactctgcaa ggcaatgcag agccccaaga agacagggat gctggaaatg tg
#gaaaaatg   1260
gaccatgtta tggccaaatg cccagacaga caggcgggtt ttttaggcct tg
#gtccatgg   1320
ggaaagaagc cccgcaattt ccccatggct caagtgcatc aggggctgat gc
#caactgct   1380
cccccagagg acccagctgt ggatctgcta aagaactaca tgcagttggg ca
#agcagcag   1440
agagaaaagc agagagaaag cagagagaag ccttacaagg aggtgacaga gg
#atttgctg   1500
cacctcaatt ctctctttgg aggagaccag tag
#
#       1533
<210> SEQ ID NO 5
<211> LENGTH: 1532
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
#Sequence:  Consensus
sequence of mutated Simian Immunodef
#iciency Virus
Gag gene (SIVgagDX) with wild-type
#SIV 239 Gag
gene
<400> SEQUENCE: 5
atgggcgtga gaaactccgt cttgtcaggg aagaaagcag atgaattaga aa
#aaattagg     60
ctacgaccca acggaaagaa aaagtacatg ttgaagcatg tagtatgggc ag
#caaatgaa    120
ttagatagat ttggattagc agaaagcctg ttggagaaca aagaaggatg tc
#aaaaaata    180
ctttcggtct tagctccatt agtgccaaca ggctcagaaa atttaaaaag cc
#tttataat    240
actgtctgcg tcatctggtg cattcacgca gaagagaaag tgaaacacac tg
#aggaagca    300
aaacagatag tgcagagaca cctagtggtg gaaacaggaa cmacmgaaac ya
#tgccraar    360
acmwstmgac caacagcacc atctagcggc agaggaggaa aytacccagt ac
#arcaratm    420
ggtggtaact aygtccacct gccaytrwsc ccgagaacmy traaygcytg gg
#tmaarytg    480
atmgaggara agaarttygg agcagaagta gtgccaggat tycaggcact gt
#cagaaggt    540
tgcaccccct aygacatyaa ycagatgytr aaytgygtkg gagaccatca rg
#cggctatg    600
cagatyatcm gwgayatyat maacgaggag gctgcagatg ggacttgcag ca
#cccacaac    660
cagctccaca acaaggacaa cttagggagc cgtcaggatc agayatygca gg
#aacmacyw    720
sytcagtwga ygaacaratc cagtggatgt acmgwcarca gaacccsatm cc
#agtaggca    780
acatytacmg kmgatggatc carctgggky tgcaraartg ygtymgwatg ta
#yaacccra    840
cmaacattct agatgtaaaa caagggccaa aagagccatt tcagagctat gt
#agacaggt    900
tctacaaaag tttaagagca gaacagacag atgcagcagt aaagaattgg at
#gactcaaa    960
cactgctgat tcaaaatgct aacccagatt gcaagctagt gctgaagggg ct
#gggtgtga   1020
atcccaccct agaagaaatg ctgacggctt gtcaaggagt aggggggccg gg
#acagaagg   1080
ctagattaat ggcagaagcc ctgaaagagg ccctcgcacc agtgccaatc cc
#ttttgcag   1140
cagcccaaca gaggggacca agaaagccaa ttaagtgttg gaattgtggg aa
#agagggac   1200
actctgcaag gcaatgcaga gccccaagaa gacagggatg ctggaaatgt gg
#aaaaatgg   1260
accatgttat ggccaaatgc ccagacagac aggcgggttt tttaggcctt gg
#tccatggg   1320
gaaagaagcc ccgcaatttc cccatggctc aagtgcatca ggggctgatg cc
#aactgctc   1380
ccccagagga cccagctgtg gatctgctaa agaactacat gcagttgggc aa
#gcagcaga   1440
gagaaaagca gagagaaagc agagagaagc cttacaagga ggtgacagag ga
#tttgctgc   1500
acctcaattc tctctttgga ggagaccagt ag
#
#        1532
<210> SEQ ID NO 6
<211> LENGTH: 8366
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
#Sequence:  DNA
sequence of the construct pCMVgagpol
#BNKan containing a CMV
promoter, a HIV gag/pol gene and
#a kanamycin
resistance gene
<400> SEQUENCE: 6
cctggccatt gcatacgttg tatccatatc ataatatgta catttatatt gg
#ctcatgtc     60
caacattacc gccatgttga cattgattat tgactagtta ttaatagtaa tc
#aattacgg    120
ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gt
#aaatggcc    180
cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg ta
#tgttccca    240
tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cg
#gtaaactg    300
cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt ga
#cgtcaatg    360
acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tt
#tcctactt    420
ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tg
#gcagtaca    480
tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cc
#cattgacg    540
tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cg
#taacaact    600
ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat at
#aagcagag    660
ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt ga
#cctccata    720
gaagacaccg ggaccgatcc agcctccgcg ggcgcgcgtc gacagagaga tg
#ggtgcgag    780
agcgtcagta ttaagcgggg gagaattaga tcgatgggaa aaaattcggt ta
#aggccagg    840
gggaaagaag aagtacaagc taaagcacat cgtatgggca agcagggagc ta
#gaacgatt    900
cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac tg
#ggacagct    960
acaaccatcc cttcagacag gatcagagga gcttcgatca ctatacaaca ca
#gtagcaac   1020
cctctattgt gtgcaccagc ggatcgagat caaggacacc aaggaagctt ta
#gacaagat   1080
agaggaagag caaaacaagt ccaagaagaa ggcccagcag gcagcagctg ac
#acaggaca   1140
cagcaatcag gtcagccaaa attaccctat agtgcagaac atccaggggc aa
#atggtaca   1200
tcaggccata tcacctagaa ctttaaatgc atgggtaaaa gtagtagaag ag
#aaggcttt   1260
cagcccagaa gtgataccca tgttttcagc attatcagaa ggagccaccc ca
#caggacct   1320
gaacacgatg ttgaacaccg tggggggaca tcaagcagcc atgcaaatgt ta
#aaagagac   1380
catcaatgag gaagctgcag aatgggatag agtgcatcca gtgcatgcag gg
#cctattgc   1440
accaggccag atgagagaac caaggggaag tgacatagca ggaactacta gt
#acccttca   1500
ggaacaaata ggatggatga caaataatcc acctatccca gtaggagaga tc
#tacaagag   1560
gtggataatc ctgggattga acaagatcgt gaggatgtat agccctacca gc
#attctgga   1620
cataagacaa ggaccaaagg aaccctttag agactatgta gaccggttct at
#aaaactct   1680
aagagctgag caagcttcac aggaggtaaa aaattggatg acagaaacct tg
#ttggtcca   1740
aaatgcgaac ccagattgta agaccatcct gaaggctctc ggcccagcgg ct
#acactaga   1800
agaaatgatg acagcatgtc agggagtagg aggacccggc cataaggcaa ga
#gttttggc   1860
cgaggcgatg agccaggtga cgaactcggc gaccataatg atgcagagag gc
#aacttccg   1920
gaaccagcgg aagatcgtca agtgcttcaa ttgtggcaaa gaagggcaca cc
#gccaggaa   1980
ctgccgggcc ccccggaaga agggctgttg gaaatgtgga aaggaaggac ac
#caaatgaa   2040
agattgtact gagagacagg ctaatttttt agggaagatc tggccttcct ac
#aagggaag   2100
gccagggaat tttcttcaga gcagaccaga gccaacagcc ccaccagaag ag
#agcttcag   2160
gtctggggta gagacaacaa ctccccctca gaagcaggag ccgatagaca ag
#gaactgta   2220
tcctttaact tccctcagat cactctttgg caacgacccc tcgtcacagt aa
#ggatcggg   2280
gggcaactca aggaagcgct gctcgataca ggagcagatg atacagtatt ag
#aagaaatg   2340
agtttgccag gaagatggaa accaaaaatg atagggggga tcgggggctt ca
#tcaaggtg   2400
aggcagtacg accagatact catagaaatc tgtggacata aagctatagg ta
#cagtatta   2460
gtaggaccta cacctgtcaa cataattgga agaaatctgt tgacccagat cg
#gctgcacc   2520
ttgaacttcc ccatcagccc tattgagacg gtgcccgtga agttgaagcc gg
#ggatggac   2580
ggccccaagg tcaagcaatg gccattgacg aaagagaaga tcaaggcctt ag
#tcgaaatc   2640
tgtacagaga tggagaagga agggaagatc agcaagatcg ggcctgagaa cc
#cctacaac   2700
actccagtct tcgcaatcaa gaagaaggac agtaccaagt ggagaaagct gg
#tggacttc   2760
agagagctga acaagagaac tcaggacttc tgggaagttc agctgggcat cc
#cacatccc   2820
gctgggttga agaagaagaa gtcagtgaca gtgctggatg tgggtgatgc ct
#acttctcc   2880
gttcccttgg acgaggactt caggaagtac actgccttca cgatacctag ca
#tcaacaac   2940
gagacaccag gcatccgcta ccagtacaac gtgctgccac agggatggaa gg
#gatcacca   3000
gccatctttc aaagcagcat gaccaagatc ctggagccct tccgcaagca aa
#acccagac   3060
atcgtgatct atcagtacat ggacgacctc tacgtaggaa gtgacctgga ga
#tcgggcag   3120
cacaggacca agatcgagga gctgagacag catctgttga ggtggggact ga
#ccacacca   3180
gacaagaagc accagaagga acctcccttc ctgtggatgg gctacgaact gc
#atcctgac   3240
aagtggacag tgcagcccat cgtgctgcct gagaaggaca gctggactgt ga
#acgacata   3300
cagaagctcg tgggcaagtt gaactgggca agccagatct acccaggcat ca
#aagttagg   3360
cagctgtgca agctgcttcg aggaaccaag gcactgacag aagtgatccc ac
#tgacagag   3420
gaagcagagc tagaactggc agagaaccga gagatcctga aggagccagt ac
#atggagtg   3480
tactacgacc caagcaagga cctgatcgca gagatccaga agcaggggca ag
#gccaatgg   3540
acctaccaaa tctaccagga gcccttcaag aacctgaaga caggcaagta cg
#caaggatg   3600
aggggtgccc acaccaacga tgtgaagcag ctgacagagg cagtgcagaa ga
#tcaccaca   3660
gagagcatcg tgatctgggg caagactccc aagttcaagc tgcccataca ga
#aggagaca   3720
tgggagacat ggtggaccga gtactggcaa gccacctgga tccctgagtg gg
#agttcgtg   3780
aacacccctc ccttggtgaa actgtggtat cagctggaga aggaacccat cg
#tgggagca   3840
gagaccttct acgtggatgg ggcagccaac agggagacca agctgggcaa gg
#caggctac   3900
gtgaccaacc gaggacgaca gaaagtggtg accctgactg acaccaccaa cc
#agaagact   3960
gagctgcaag ccatctacct agctctgcaa gacagcggac tggaagtgaa ca
#tcgtgaca   4020
gactcacagt acgcactggg catcatccaa gcacaaccag accaatccga gt
#cagagctg   4080
gtgaaccaga tcatcgagca gctgatcaag aaggagaaag tgtacctggc at
#gggtacca   4140
gcacacaaag gaattggagg aaatgaacaa gtagataaat tagtcagtgc tg
#ggatccgg   4200
aaggtgctgt tcctggacgg gatcgataag gcccaagatg aacatgagaa gt
#accactcc   4260
aactggcgcg ctatggccag cgacttcaac ctgccacctg tagtagcaaa ag
#aaatagta   4320
gccagctgtg ataaatgtca gctaaaagga gaagccatgc atggacaagt ag
#actgtagt   4380
ccaggaatat ggcagctgga ctgcacgcac ctggagggga aggtgatcct gg
#tagcagtt   4440
catgtagcca gtggatatat agaagcagaa gttatccctg ctgaaactgg gc
#aggaaaca   4500
gcatattttc ttttaaaatt agcaggaaga tggccagtaa aaacaataca ca
#cggacaac   4560
ggaagcaact tcactggtgc tacggttaag gccgcctgtt ggtgggcggg aa
#tcaagcag   4620
gaatttggaa ttccctacaa tccccaatcg caaggagtcg tggagagcat ga
#acaaggag   4680
ctgaagaaga tcatcggaca agtgagggat caggctgagc acctgaagac ag
#cagtgcag   4740
atggcagtgt tcatccacaa cttcaaaaga aaagggggga ttggggggta ca
#gtgcaggg   4800
gaaaggatcg tggacatcat cgccaccgac atccaaacca aggagctgca ga
#agcagatc   4860
accaagatcc agaacttccg ggtgtactac cgcgacagcc gcaacccact gt
#ggaaggga   4920
ccagcaaagc tcctctggaa gggagagggg gcagtggtga tccaggacaa ca
#gtgacatc   4980
aaagtggtgc caaggcgcaa ggccaagatc atccgcgact atggaaaaca ga
#tggcaggt   5040
gatgattgtg tggcaagtag acaggatgag gattagaacc tggaagagcc tg
#gtgaagca   5100
ccatatggcg ttcgaagcta gcctcgagat ccagatctgc tgtgccttct ag
#ttgccagc   5160
catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc ac
#tcccactg   5220
tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt ca
#ttctattc   5280
tggggggtgg ggtggggcag cacagcaagg gggaggattg ggaagacaat ag
#caggcatg   5340
ctggggatgc ggtgggctct atgggtaccc aggtgctgaa gaattgaccc gg
#ttcctcct   5400
gggccagaaa gaagcaggca catccccttc tctgtgacac accctgtcca cg
#cccctggt   5460
tcttagttcc agccccactc ataggacact catagctcag gagggctccg cc
#ttcaatcc   5520
cacccgctaa agtacttgga gcggtctctc cctccctcat cagcccacca aa
#ccaaacct   5580
agcctccaag agtgggaaga aattaaagca agataggcta ttaagtgcag ag
#ggagagaa   5640
aatgcctcca acatgtgagg aagtaatgag agaaatcata gaatttcttc cg
#cttcctcg   5700
ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tc
#actcaaag   5760
gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gt
#gagcaaaa   5820
ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt cc
#ataggctc   5880
cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aa
#acccgaca   5940
ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tc
#ctgttccg   6000
accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt gg
#cgctttct   6060
caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gc
#tgggctgt   6120
gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tc
#gtcttgag   6180
tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa ca
#ggattagc   6240
agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ct
#acggctac   6300
actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cg
#gaaaaaga   6360
gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt tt
#ttgtttgc   6420
aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat ct
#tttctacg   6480
gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat ga
#gattatca   6540
aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aa
#tctaaagt   6600
atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc ac
#ctatctca   6660
gcgatctgtc tatttcgttc atccatagtt gcctgactcc gggggggggg gg
#cgctgagg   6720
tctgcctcgt gaagaaggtg ttgctgactc ataccaggcc tgaatcgccc ca
#tcatccag   6780
ccagaaagtg agggagccac ggttgatgag agctttgttg taggtggacc ag
#ttggtgat   6840
tttgaacttt tgctttgcca cggaacggtc tgcgttgtcg ggaagatgcg tg
#atctgatc   6900
cttcaactca gcaaaagttc gatttattca acaaagccgc cgtcccgtca ag
#tcagcgta   6960
atgctctgcc agtgttacaa ccaattaacc aattctgatt agaaaaactc at
#cgagcatc   7020
aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aa
#aaagccgt   7080
ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag at
#cctggtat   7140
cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ct
#cgtcaaaa   7200
ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga ga
#atggcaaa   7260
agcttatgca tttctttcca gacttgttca acaggccagc cattacgctc gt
#catcaaaa   7320
tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag ac
#gaaatacg   7380
cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg ca
#ggaacact   7440
gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ct
#ggaatgct   7500
gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg ga
#taaaatgc   7560
ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ct
#catctgta   7620
acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc at
#cgggcttc   7680
ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc cc
#atttatac   7740
ccatataaat cagcatccat gttggaattt aatcgcggcc tcgagcaaga cg
#tttcccgt   7800
tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag tt
#ttattgtt   7860
catgatgata tatttttatc ttgtgcaatg taacatcaga gattttgaga ca
#caacgtgg   7920
ctttcccccc ccccccatta ttgaagcatt tatcagggtt attgtctcat ga
#gcggatac   7980
atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tc
#cccgaaaa   8040
gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aa
#ataggcgt   8100
atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ct
#gacacatg   8160
cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag ac
#aagcccgt   8220
cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc gg
#catcagag   8280
cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cg
#taaggaga   8340
aaataccgca tcagattggc tattgg
#
#            8366
<210> SEQ ID NO 7
<211> LENGTH: 271
<212> TYPE: PRT
<213> ORGANISM: Escherichia coli
<400> SEQUENCE: 7
Met Ser His Ile Gln Arg Glu Thr Ser Cys Se
#r Arg Pro Arg Leu Asn
1               5
#                 10
#                 15
Ser Asn Met Asp Ala Asp Leu Tyr Gly Tyr Ly
#s Trp Ala Arg Asp Asn
20
#             25
#             30
Val Gly Gln Ser Gly Ala Thr Ile Tyr Arg Le
#u Tyr Gly Lys Pro Asp
35
#         40
#         45
Ala Pro Glu Leu Phe Leu Lys His Gly Lys Gl
#y Ser Val Ala Asn Asp
50
#     55
#     60
Val Thr Asp Glu Met Val Arg Leu Asn Trp Le
#u Thr Glu Phe Met Pro
65
# 70
# 75
# 80
Leu Pro Thr Ile Lys His Phe Ile Arg Thr Pr
#o Asp Asp Ala Trp Leu
85
#                 90
#                 95
Leu Thr Thr Ala Ile Pro Gly Lys Thr Ala Ph
#e Gln Val Leu Glu Glu
100
#           105
#           110
Tyr Pro Asp Ser Gly Glu Asn Ile Val Asp Al
#a Leu Ala Val Phe Leu
115
#       120
#       125
Arg Arg Leu His Ser Ile Pro Val Cys Asn Cy
#s Pro Phe Asn Ser Asp
130
#   135
#   140
Arg Val Phe Arg Leu Ala Gln Ala Gln Ser Ar
#g Met Asn Asn Gly Leu
145                 1
#50                 1
#55                 1
#60
Val Asp Ala Ser Asp Phe Asp Asp Glu Arg As
#n Gly Trp Pro Val Glu
165
#               170
#               175
Gln Val Trp Lys Glu Met His Lys Leu Leu Pr
#o Phe Ser Pro Asp Ser
180
#           185
#           190
Val Val Thr His Gly Asp Phe Ser Leu Asp As
#n Leu Ile Phe Asp Glu
195
#       200
#       205
Gly Lys Leu Ile Gly Cys Ile Asp Val Gly Ar
#g Val Gly Ile Ala Asp
210
#   215
#   220
Arg Tyr Gln Asp Leu Ala Ile Leu Trp Asn Cy
#s Leu Gly Glu Phe Ser
225                 2
#30                 2
#35                 2
#40
Pro Ser Leu Gln Lys Arg Leu Phe Gln Lys Ty
#r Gly Ile Asp Asn Pro
245
#               250
#               255
Asp Met Asn Lys Leu Gln Phe His Leu Met Le
#u Asp Glu Phe Phe
260
#           265
#           270
<210> SEQ ID NO 8
<211> LENGTH: 8937
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
#Sequence:  DNA
sequence of transfer construc pmBCwC
#Nluci
<400> SEQUENCE: 8
tggaagggct aatttggtcc caaaaaagac aagagatcct tgatctgtgg at
#ctaccaca     60
cacaaggcta cttccctgat tggcagaact acacaccagg gccagggatc ag
#atatccac    120
tgacctttgg atggtgcttc aagttagtac cagttgaacc agagcaagta ga
#agaggcca    180
aataaggaga gaagaacagc ttgttacacc ctatgagcca gcatgggatg ga
#ggacccgg    240
agggagaagt attagtgtgg aagtttgaca gcctcctagc atttcgtcac at
#ggcccgag    300
agctgcatcc ggagtactac aaagactgct gacatcgagc tttctacaag gg
#actttccg    360
ctggggactt tccagggagg tgtggcctgg gcgggactgg ggagtggcga gc
#cctcagat    420
gctacatata agcagctgct ttttgcctgt actgggtctc tctggttaga cc
#agatctga    480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aa
#gcttgcct    540
tgagtgctca aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta ga
#gatccctc    600
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg ga
#cttgaaag    660
cgaaagtaaa gccagaggag atctctcgac gcaggactcg gcttgctgaa gc
#gcgcacgg    720
caagaggcga ggggcggcgc ctgacgagga cgccaaaaat tttgactagc gg
#aggctaga    780
aggagagagc tcggtgcgag agcgtcagta ttaagcgggg gagaattaga tc
#gatgggaa    840
aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat ag
#tatgggca    900
agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc ag
#aaggctgt    960
agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga ac
#ttagatca   1020
ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat aa
#aagacacc   1080
aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaaaaa ag
#cacagcaa   1140
gcagcagctg acacaggaca cagcaatcag gtcagccaaa attaccctat ag
#tgcagaac   1200
atccaggggc aaatggtaca tcaggccata tcacctagaa ctttaaacga ta
#agcttggg   1260
agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aa
#cgaccccc   1320
gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg ac
#tttccatt   1380
gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat ca
#agtgtatc   1440
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tg
#gcattatg   1500
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta tt
#agtcatcg   1560
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cg
#gtttgact   1620
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tg
#gcaccaaa   1680
atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa at
#gggcggta   1740
ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt ca
#gatcgcct   1800
ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgactctag ag
#gatccatc   1860
taagtaagct tggcattccg gtactgttgg taaaatggaa gacgccaaaa ac
#ataaagaa   1920
aggcccggcg ccattctatc ctctagagga tggaaccgct ggagagcaac tg
#cataaggc   1980
tatgaagaga tacgccctgg ttcctggaac aattgctttt acagatgcac at
#atcgaggt   2040
gaacatcacg tacgcggaat acttcgaaat gtccgttcgg ttggcagaag ct
#atgaaacg   2100
atatgggctg aatacaaatc acagaatcgt cgtatgcagt gaaaactctc tt
#caattctt   2160
tatgccggtg ttgggcgcgt tatttatcgg agttgcagtt gcgcccgcga ac
#gacattta   2220
taatgaacgt gaattgctca acagtatgaa catttcgcag cctaccgtag tg
#tttgtttc   2280
caaaaagggg ttgcaaaaaa ttttgaacgt gcaaaaaaaa ttaccaataa tc
#cagaaaat   2340
tattatcatg gattctaaaa cggattacca gggatttcag tcgatgtaca cg
#ttcgtcac   2400
atctcatcta cctcccggtt ttaatgaata cgattttgta ccagagtcct tt
#gatcgtga   2460
caaaacaatt gcactgataa tgaattcctc tggatctact gggttaccta ag
#ggtgtggc   2520
ccttccgcat agaactgcct gcgtcagatt ctcgcatgcc agagatccta tt
#tttggcaa   2580
tcaaatcatt ccggatactg cgattttaag tgttgttcca ttccatcacg gt
#tttggaat   2640
gtttactaca ctcggatatt tgatatgtgg atttcgagtc gtcttaatgt at
#agatttga   2700
agaagagctg tttttacgat cccttcagga ttacaaaatt caaagtgcgt tg
#ctagtacc   2760
aaccctattt tcattcttcg ccaaaagcac tctgattgac aaatacgatt ta
#tctaattt   2820
acacgaaatt gcttctgggg gcgcacctct ttcgaaagaa gtcggggaag cg
#gttgcaaa   2880
acgcttccat cttccaggga tacgacaagg atatgggctc actgagacta ca
#tcagctat   2940
tctgattaca cccgaggggg atgataaacc gggcgcggtc ggtaaagttg tt
#ccattttt   3000
tgaagcgaag gttgtggatc tggataccgg gaaaacgctg ggcgttaatc ag
#agaggcga   3060
attatgtgtc agaggaccta tgattatgtc cggttatgta aacaatccgg aa
#gcgaccaa   3120
cgccttgatt gacaaggatg gatggctaca ttctggagac atagcttact gg
#gacgaaga   3180
cgaacacttc ttcatagttg accgcttgaa gtctttaatt aaatacaaag ga
#tatcaggt   3240
ggcccccgct gaattggaat cgatattgtt acaacacccc aacatcttcg ac
#gcgggcgt   3300
ggcaggtctt cccgacgatg acgccggtga acttcccgcc gccgttgttg tt
#ttggagca   3360
cggaaagacg atgacggaaa aagagatcgt ggattacgtc gccagtcaag ta
#acaaccgc   3420
gaaaaagttg cgcggaggag ttgtgtttgt ggacgaagta ccgaaaggtc tt
#accggaaa   3480
actcgacgca agaaaaatca gagagatcct cataaaggcc aagaagggcg ga
#aagtccaa   3540
attgtaactc gagggggggc ccggtacctt taagaccaat gacttacaag gc
#agctgtag   3600
atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tc
#ccaaagaa   3660
gacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct ga
#ttggcaga   3720
actacacacc agggccaggg gtcagatatc cactgacctt tggatggtgc ta
#caagctag   3780
taccagttga gccagataag gtagaagagg ccaataaagg agagaacacc ag
#cttgttac   3840
accctgtgag cctgcatgga atggatgacc ctgagagaga agtgttagag tg
#gaggtttg   3900
acagccgcct agcatttcat cacgtggccc gagagctgca tccggagtac tt
#caagaact   3960
gctgacatcg agcttgctac aagggacttt ccgctgggga ctttccaggg ag
#gcgtggcc   4020
tgggcgggac tggggagtgg cgagccctca gatgctgcat ataagcagct gc
#tttttgcc   4080
tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ct
#aactaggg   4140
aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gt
#gtgcccgt   4200
ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gt
#ggaaaatc   4260
tctagcaccc cccaggaggt agaggttgca gtgagccaag atcgcgccac tg
#cattccag   4320
cctgggcaag aaaacaagac tgtctaaaat aataataata agttaagggt at
#taaatata   4380
tttatacatg gaggtcataa aaatatatat atttgggctg ggcgcagtgg ct
#cacacctg   4440
cgcccggccc tttgggaggc cgaggcaggt ggatcacctg agtttgggag tt
#ccagacca   4500
gcctgaccaa catggagaaa ccccttctct gtgtattttt agtagatttt at
#tttatgtg   4560
tattttattc acaggtattt ctggaaaact gaaactgttt ttcctctact ct
#gataccac   4620
aagaatcatc agcacagagg aagacttctg tgatcaaatg tggtgggaga gg
#gaggtttt   4680
caccagcaca tgagcagtca gttctgccgc agactcggcg ggtgtccttc gg
#ttcagttc   4740
caacaccgcc tgcctggaga gaggtcagac cacagggtga gggctcagtc cc
#caagacat   4800
aaacacccaa gacataaaca cccaacaggt ccaccccgcc tgctgcccag gc
#agagccga   4860
ttcaccaaga cgggaattag gatagagaaa gagtaagtca cacagagccg gc
#tgtgcggg   4920
agaacggagt tctattatga ctcaaatcag tctccccaag cattcgggga tc
#agagtttt   4980
taaggataac ttagtgtgta gggggccagt gagttggaga tgaaagcgta gg
#gagtcgaa   5040
ggtgtccttt tgcgccgagt cagttcctgg gtgggggcca caagatcgga tg
#agccagtt   5100
tatcaatccg ggggtgccag ctgatccatg gagtgcaggg tctgcaaaat at
#ctcaagca   5160
ctgattgatc ttaggtttta caatagtgat gttaccccag gaacaatttg gg
#gaaggtca   5220
gaatcttgta gcctgtagct gcatgactcc taaaccataa tttctttttt gt
#tttttttt   5280
ttttattttt gagacagggt ctcactctgt cacctaggct ggagtgcagt gg
#tgcaatca   5340
cagctcactg cagcccctag agcggccgcc accgcggtgg agctccaatt cg
#ccctatag   5400
tgagtcgtat tacaattcac tggccgtcgt tttacaacgt cgtgactggg aa
#aaccctgg   5460
cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gt
#aatagcga   5520
agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aa
#tggcgcga   5580
aattgtaaac gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tc
#agctcatt   5640
ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat ag
#accgagat   5700
agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tg
#gactccaa   5760
cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac ca
#tcacccta   5820
atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aa
#gggagccc   5880
ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag gg
#aagaaagc   5940
gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg ta
#accaccac   6000
acccgccgcg cttaatgcgc cgctacaggg cgcgtcccag gtggcacttt tc
#ggggaaat   6060
gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tc
#cgctcatg   6120
agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat ga
#gtattcaa   6180
catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt tt
#ttgctcac   6240
ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg ag
#tgggttac   6300
atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga ag
#aacgtttt   6360
ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg ta
#ttgacgcc   6420
gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tg
#agtactca   6480
ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg ca
#gtgctgcc   6540
ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg ag
#gaccgaag   6600
gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tc
#gttgggaa   6660
ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tg
#tagcaatg   6720
gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc cc
#ggcaacaa   6780
ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc gg
#cccttccg   6840
gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cg
#gtatcatt   6900
gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac ga
#cggggagt   6960
caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc ac
#tgattaag   7020
cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aa
#aacttcat   7080
ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac ca
#aaatccct   7140
taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa ag
#gatcttct   7200
tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc ac
#cgctacca   7260
gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aa
#ctggcttc   7320
agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg cc
#accacttc   7380
aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc ag
#tggctgct   7440
gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt ac
#cggataag   7500
gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gc
#gaacgacc   7560
tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tc
#ccgaaggg   7620
agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg ca
#cgagggag   7680
cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cc
#tctgactt   7740
gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cg
#ccagcaac   7800
gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ct
#ttcctgcg   7860
ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga ta
#ccgctcgc   7920
cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gc
#gcccaata   7980
cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cg
#acaggttt   8040
cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct ca
#ctcattag   8100
gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tg
#tgagcgga   8160
taacaatttc acacaggaaa cagctatgac catgattacg ccaagctcgg aa
#ttaaccct   8220
cactaaaggg aacaaaagct gctgcagggt ccctaactgc caagccccac ag
#tgtgccct   8280
gaggctgccc cttccttcta gcggctgccc ccactcggct ttgctttccc ta
#gtttcagt   8340
tacttgcgtt cagccaaggt ctgaaactag gtgcgcacag agcggtaaga ct
#gcgagaga   8400
aagagaccag ctttacaggg ggtttatcac agtgcaccct gacagtcgtc ag
#cctcacag   8460
ggggtttatc acattgcacc ctgacagtcg tcagcctcac agggggttta tc
#acagtgca   8520
cccttacaat cattccattt gattcacaat ttttttagtc tctactgtgc ct
#aacttgta   8580
agttaaattt gatcagaggt gtgttcccag aggggaaaac agtatataca gg
#gttcagta   8640
ctatcgcatt tcaggcctcc acctgggtct tggaatgtgt cccccgaggg gt
#gatgacta   8700
cctcagttgg atctccacag gtcacagtga cacaagataa ccaagacacc tc
#ccaaggct   8760
accacaatgg gccgccctcc acgtgcacat ggccggagga actgccatgt cg
#gaggtgca   8820
agcacacctg cgcatcagag tccttggtgt ggagggaggg accagcgcag ct
#tccagcca   8880
tccacctgat gaacagaacc tagggaaagc cccagttcta cttacaccag ga
#aaggc      8937
<210> SEQ ID NO 9
<211> LENGTH: 8937
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
#Sequence:  DNA
sequence from transfer construct pmB
#CmCNluci
<400> SEQUENCE: 9
tggaagggct aatttggtcc caaaaaagac aagagatcct tgatctgtgg at
#ctaccaca     60
cacaaggcta cttccctgat tggcagaact acacaccagg gccagggatc ag
#atatccac    120
tgacctttgg atggtgcttc aagttagtac cagttgaacc agagcaagta ga
#agaggcca    180
aataaggaga gaagaacagc ttgttacacc ctatgagcca gcatgggatg ga
#ggacccgg    240
agggagaagt attagtgtgg aagtttgaca gcctcctagc atttcgtcac at
#ggcccgag    300
agctgcatcc ggagtactac aaagactgct gacatcgagc tttctacaag gg
#actttccg    360
ctggggactt tccagggagg tgtggcctgg gcgggactgg ggagtggcga gc
#cctcagat    420
gctacatata agcagctgct ttttgcctgt actgggtctc tctggttaga cc
#agatctga    480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aa
#gcttgcct    540
tgagtgctca aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta ga
#gatccctc    600
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg ga
#cttgaaag    660
cgaaagtaaa gccagaggag atctctcgac gcaggactcg gcttgctgaa gc
#gcgcacgg    720
caagaggcga ggggcggcgc ctgacgagga cgccaaaaat tttgactagc gg
#aggctaga    780
aggagagagc tcggtgcgag agcgtcagta ttaagcgggg gagaattaga tc
#gatgggaa    840
aaaattcggt taaggccagg gggaaagaag aagtacaagc taaagcacat cg
#tatgggca    900
agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc ag
#aaggctgt    960
agacaaatac tgggacagct acaaccatcc cttcagacag gatcagagga gc
#ttcgatca   1020
ctatacaaca cagtagcaac cctctattgt gtgcaccagc ggatcgagat ca
#aggacacc   1080
aaggaagctt tagacaagat agaggaagag caaaacaagt ccaagaagaa gg
#cccagcag   1140
gcagcagctg acacaggaca cagcaatcag gtcagccaaa attaccctat ag
#tgcagaac   1200
atccaggggc aaatggtaca tcaggccata tcacctagaa ctttaaacga ta
#agcttggg   1260
agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aa
#cgaccccc   1320
gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg ac
#tttccatt   1380
gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat ca
#agtgtatc   1440
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tg
#gcattatg   1500
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta tt
#agtcatcg   1560
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cg
#gtttgact   1620
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tg
#gcaccaaa   1680
atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa at
#gggcggta   1740
ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt ca
#gatcgcct   1800
ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgactctag ag
#gatccatc   1860
taagtaagct tggcattccg gtactgttgg taaaatggaa gacgccaaaa ac
#ataaagaa   1920
aggcccggcg ccattctatc ctctagagga tggaaccgct ggagagcaac tg
#cataaggc   1980
tatgaagaga tacgccctgg ttcctggaac aattgctttt acagatgcac at
#atcgaggt   2040
gaacatcacg tacgcggaat acttcgaaat gtccgttcgg ttggcagaag ct
#atgaaacg   2100
atatgggctg aatacaaatc acagaatcgt cgtatgcagt gaaaactctc tt
#caattctt   2160
tatgccggtg ttgggcgcgt tatttatcgg agttgcagtt gcgcccgcga ac
#gacattta   2220
taatgaacgt gaattgctca acagtatgaa catttcgcag cctaccgtag tg
#tttgtttc   2280
caaaaagggg ttgcaaaaaa ttttgaacgt gcaaaaaaaa ttaccaataa tc
#cagaaaat   2340
tattatcatg gattctaaaa cggattacca gggatttcag tcgatgtaca cg
#ttcgtcac   2400
atctcatcta cctcccggtt ttaatgaata cgattttgta ccagagtcct tt
#gatcgtga   2460
caaaacaatt gcactgataa tgaattcctc tggatctact gggttaccta ag
#ggtgtggc   2520
ccttccgcat agaactgcct gcgtcagatt ctcgcatgcc agagatccta tt
#tttggcaa   2580
tcaaatcatt ccggatactg cgattttaag tgttgttcca ttccatcacg gt
#tttggaat   2640
gtttactaca ctcggatatt tgatatgtgg atttcgagtc gtcttaatgt at
#agatttga   2700
agaagagctg tttttacgat cccttcagga ttacaaaatt caaagtgcgt tg
#ctagtacc   2760
aaccctattt tcattcttcg ccaaaagcac tctgattgac aaatacgatt ta
#tctaattt   2820
acacgaaatt gcttctgggg gcgcacctct ttcgaaagaa gtcggggaag cg
#gttgcaaa   2880
acgcttccat cttccaggga tacgacaagg atatgggctc actgagacta ca
#tcagctat   2940
tctgattaca cccgaggggg atgataaacc gggcgcggtc ggtaaagttg tt
#ccattttt   3000
tgaagcgaag gttgtggatc tggataccgg gaaaacgctg ggcgttaatc ag
#agaggcga   3060
attatgtgtc agaggaccta tgattatgtc cggttatgta aacaatccgg aa
#gcgaccaa   3120
cgccttgatt gacaaggatg gatggctaca ttctggagac atagcttact gg
#gacgaaga   3180
cgaacacttc ttcatagttg accgcttgaa gtctttaatt aaatacaaag ga
#tatcaggt   3240
ggcccccgct gaattggaat cgatattgtt acaacacccc aacatcttcg ac
#gcgggcgt   3300
ggcaggtctt cccgacgatg acgccggtga acttcccgcc gccgttgttg tt
#ttggagca   3360
cggaaagacg atgacggaaa aagagatcgt ggattacgtc gccagtcaag ta
#acaaccgc   3420
gaaaaagttg cgcggaggag ttgtgtttgt ggacgaagta ccgaaaggtc tt
#accggaaa   3480
actcgacgca agaaaaatca gagagatcct cataaaggcc aagaagggcg ga
#aagtccaa   3540
attgtaactc gagggggggc ccggtacctt taagaccaat gacttacaag gc
#agctgtag   3600
atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tc
#ccaaagaa   3660
gacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct ga
#ttggcaga   3720
actacacacc agggccaggg gtcagatatc cactgacctt tggatggtgc ta
#caagctag   3780
taccagttga gccagataag gtagaagagg ccaataaagg agagaacacc ag
#cttgttac   3840
accctgtgag cctgcatgga atggatgacc ctgagagaga agtgttagag tg
#gaggtttg   3900
acagccgcct agcatttcat cacgtggccc gagagctgca tccggagtac tt
#caagaact   3960
gctgacatcg agcttgctac aagggacttt ccgctgggga ctttccaggg ag
#gcgtggcc   4020
tgggcgggac tggggagtgg cgagccctca gatgctgcat ataagcagct gc
#tttttgcc   4080
tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ct
#aactaggg   4140
aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gt
#gtgcccgt   4200
ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gt
#ggaaaatc   4260
tctagcaccc cccaggaggt agaggttgca gtgagccaag atcgcgccac tg
#cattccag   4320
cctgggcaag aaaacaagac tgtctaaaat aataataata agttaagggt at
#taaatata   4380
tttatacatg gaggtcataa aaatatatat atttgggctg ggcgcagtgg ct
#cacacctg   4440
cgcccggccc tttgggaggc cgaggcaggt ggatcacctg agtttgggag tt
#ccagacca   4500
gcctgaccaa catggagaaa ccccttctct gtgtattttt agtagatttt at
#tttatgtg   4560
tattttattc acaggtattt ctggaaaact gaaactgttt ttcctctact ct
#gataccac   4620
aagaatcatc agcacagagg aagacttctg tgatcaaatg tggtgggaga gg
#gaggtttt   4680
caccagcaca tgagcagtca gttctgccgc agactcggcg ggtgtccttc gg
#ttcagttc   4740
caacaccgcc tgcctggaga gaggtcagac cacagggtga gggctcagtc cc
#caagacat   4800
aaacacccaa gacataaaca cccaacaggt ccaccccgcc tgctgcccag gc
#agagccga   4860
ttcaccaaga cgggaattag gatagagaaa gagtaagtca cacagagccg gc
#tgtgcggg   4920
agaacggagt tctattatga ctcaaatcag tctccccaag cattcgggga tc
#agagtttt   4980
taaggataac ttagtgtgta gggggccagt gagttggaga tgaaagcgta gg
#gagtcgaa   5040
ggtgtccttt tgcgccgagt cagttcctgg gtgggggcca caagatcgga tg
#agccagtt   5100
tatcaatccg ggggtgccag ctgatccatg gagtgcaggg tctgcaaaat at
#ctcaagca   5160
ctgattgatc ttaggtttta caatagtgat gttaccccag gaacaatttg gg
#gaaggtca   5220
gaatcttgta gcctgtagct gcatgactcc taaaccataa tttctttttt gt
#tttttttt   5280
ttttattttt gagacagggt ctcactctgt cacctaggct ggagtgcagt gg
#tgcaatca   5340
cagctcactg cagcccctag agcggccgcc accgcggtgg agctccaatt cg
#ccctatag   5400
tgagtcgtat tacaattcac tggccgtcgt tttacaacgt cgtgactggg aa
#aaccctgg   5460
cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gt
#aatagcga   5520
agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aa
#tggcgcga   5580
aattgtaaac gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tc
#agctcatt   5640
ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat ag
#accgagat   5700
agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tg
#gactccaa   5760
cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac ca
#tcacccta   5820
atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aa
#gggagccc   5880
ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag gg
#aagaaagc   5940
gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg ta
#accaccac   6000
acccgccgcg cttaatgcgc cgctacaggg cgcgtcccag gtggcacttt tc
#ggggaaat   6060
gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tc
#cgctcatg   6120
agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat ga
#gtattcaa   6180
catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt tt
#ttgctcac   6240
ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg ag
#tgggttac   6300
atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga ag
#aacgtttt   6360
ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg ta
#ttgacgcc   6420
gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tg
#agtactca   6480
ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg ca
#gtgctgcc   6540
ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg ag
#gaccgaag   6600
gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tc
#gttgggaa   6660
ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tg
#tagcaatg   6720
gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc cc
#ggcaacaa   6780
ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc gg
#cccttccg   6840
gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cg
#gtatcatt   6900
gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac ga
#cggggagt   6960
caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc ac
#tgattaag   7020
cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aa
#aacttcat   7080
ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac ca
#aaatccct   7140
taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa ag
#gatcttct   7200
tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc ac
#cgctacca   7260
gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aa
#ctggcttc   7320
agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg cc
#accacttc   7380
aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc ag
#tggctgct   7440
gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt ac
#cggataag   7500
gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gc
#gaacgacc   7560
tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tc
#ccgaaggg   7620
agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg ca
#cgagggag   7680
cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cc
#tctgactt   7740
gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cg
#ccagcaac   7800
gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ct
#ttcctgcg   7860
ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga ta
#ccgctcgc   7920
cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gc
#gcccaata   7980
cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cg
#acaggttt   8040
cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct ca
#ctcattag   8100
gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tg
#tgagcgga   8160
taacaatttc acacaggaaa cagctatgac catgattacg ccaagctcgg aa
#ttaaccct   8220
cactaaaggg aacaaaagct gctgcagggt ccctaactgc caagccccac ag
#tgtgccct   8280
gaggctgccc cttccttcta gcggctgccc ccactcggct ttgctttccc ta
#gtttcagt   8340
tacttgcgtt cagccaaggt ctgaaactag gtgcgcacag agcggtaaga ct
#gcgagaga   8400
aagagaccag ctttacaggg ggtttatcac agtgcaccct gacagtcgtc ag
#cctcacag   8460
ggggtttatc acattgcacc ctgacagtcg tcagcctcac agggggttta tc
#acagtgca   8520
cccttacaat cattccattt gattcacaat ttttttagtc tctactgtgc ct
#aacttgta   8580
agttaaattt gatcagaggt gtgttcccag aggggaaaac agtatataca gg
#gttcagta   8640
ctatcgcatt tcaggcctcc acctgggtct tggaatgtgt cccccgaggg gt
#gatgacta   8700
cctcagttgg atctccacag gtcacagtga cacaagataa ccaagacacc tc
#ccaaggct   8760
accacaatgg gccgccctcc acgtgcacat ggccggagga actgccatgt cg
#gaggtgca   8820
agcacacctg cgcatcagag tccttggtgt ggagggaggg accagcgcag ct
#tccagcca   8880
tccacctgat gaacagaacc tagggaaagc cccagttcta cttacaccag ga
#aaggc      8937
<210> SEQ ID NO 10
<211> LENGTH: 122
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
#Sequence:  DNA
sequence of the BSSHII to ClaI
#fragment in transfer
construct pmBCwCNluci and pmBCmCNluci
<400> SEQUENCE: 10
cgcgcacggc aagaggcgag gggcggcgcc tgacgaggac gccaaaaatt tt
#gactagcg     60
gaggctagaa ggagagagct cggtgcgaga gcgtcagtat taagcggggg ag
#aattagat    120
cg
#
#
#             122
<210> SEQ ID NO 11
<211> LENGTH: 122
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
#Sequence:  DNA
sequence of the BSSHII to ClaI
#fragment in transfer
construct 3
<400> SEQUENCE: 11
cgcgcacggc aagaggcgag gggcggcgcc tggggaggac gccaaaaatt tt
#gactagcg     60
gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg ag
#aattagat    120
cg
#
#
#             122
<210> SEQ ID NO 12
<211> LENGTH: 122
<212> TYPE: DNA
<213> ORGANISM: Human immunodeficiency virus type
#1
<400> SEQUENCE: 12
cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt tt
#gactatcg     60
gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg ag
#aattagat    120
cg
#
#
#             122
<210> SEQ ID NO 13
<211> LENGTH: 122
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
#Sequence:  Plurality
Consensus sequence of DNA sequence
#of the BSSHII
to CLaI fragment in HIV-1 and
#transfer constructs
<400> SEQUENCE: 13
cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt tt
#gactagcg     60
gaggctagaa ggagagagat gggtgcgaga gcgtcggtat taagcggggg ag
#aattagat    120
aa
#
#
#             122
<210> SEQ ID NO 14
<211> LENGTH: 122
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
#Sequence:  DNA
sequence of construct CMVkan/R-R-SIVgp1
#60 CTE
<400> SEQUENCE: 14
cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt tt
#gactagcg     60
gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg ag
#aattagat    120
cg
#
#
#             122
<210> SEQ ID NO 15
<211> LENGTH: 6978
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
#Sequence:  DNA
sequence of construct CMVkan/R-R-SIVgp1
#60 CTE
<400> SEQUENCE: 15
cctggccatt gcatacgttg tatccatatc ataatatgta catttatatt gg
#ctcatgtc     60
caacattacc gccatgttga cattgattat tgactagtta ttaatagtaa tc
#aattacgg    120
ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gt
#aaatggcc    180
cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg ta
#tgttccca    240
tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cg
#gtaaactg    300
cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt ga
#cgtcaatg    360
acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tt
#tcctactt    420
ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tg
#gcagtaca    480
tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cc
#cattgacg    540
tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cg
#taacaact    600
ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat at
#aagcagag    660
ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt ga
#cctccata    720
gaagacaccg ggaccgatcc agcctccgcg ggccgcgcta agtatgggat gt
#cttgggaa    780
tcagctgctt atcgccatct tgcttttaag tgtctatggg atctattgta ct
#ctatatgt    840
cacagtcttt tatggtgtac cagcttggag gaatgcgaca attcccctct tt
#tgtgcaac    900
caagaatagg gatacttggg gaacaactca gtgcctacca gataatggtg at
#tattcaga    960
agtggccctt aatgttacag aaagctttga tgcctggaat aatacagtca ca
#gaacaggc   1020
aatagaggat gtatggcaac tctttgagac ctcaataaag ccttgtgtaa aa
#ttatcccc   1080
attatgcatt actatgagat gcaataaaag tgagacagat agatggggat tg
#acaaaatc   1140
aataacaaca acagcatcaa caacatcaac gacagcatca gcaaaagtag ac
#atggtcaa   1200
tgagactagt tcttgtatag cccaggataa ttgcacaggc ttggaacaag ag
#caaatgat   1260
aagctgtaaa ttcaacatga cagggttaaa aagagacaag aaaaaagagt ac
#aatgaaac   1320
ttggtactct gcagatttgg tatgtgaaca agggaataac actggtaatg aa
#agtagatg   1380
ttacatgaac cactgtaaca cttctgttat ccaagagtct tgtgacaaac at
#tattggga   1440
tgctattaga tttaggtatt gtgcacctcc aggttatgct ttgcttagat gt
#aatgacac   1500
aaattattca ggctttatgc ctaaatgttc taaggtggtg gtctcttcat gc
#acaaggat   1560
gatggagaca cagacttcta cttggtttgg ctttaatgga actagagcag aa
#aatagaac   1620
ttatatttac tggcatggta gggataatag gactataatt agtttaaata ag
#tattataa   1680
tctaacaatg aaatgtagaa gaccaggaaa taagacagtt ttaccagtca cc
#attatgtc   1740
tggattggtt ttccactcac aaccaatcaa tgataggcca aagcaggcat gg
#tgttggtt   1800
tggaggaaaa tggaaggatg caataaaaga ggtgaagcag accattgtca aa
#catcccag   1860
gtatactgga actaacaata ctgataaaat caatttgacg gctcctggag ga
#ggagatcc   1920
ggaagttacc ttcatgtgga caaattgcag aggagagttc ctctactgta aa
#atgaattg   1980
gtttctaaat tgggtagaag ataggaatac agctaaccag aagccaaagg aa
#cagcataa   2040
aaggaattac gtgccatgtc atattagaca aataatcaac acttggcata aa
#gtaggcaa   2100
aaatgtttat ttgcctccaa gagagggaga cctcacgtgt aactccacag tg
#accagtct   2160
catagcaaac atagattgga ttgatggaaa ccaaactaat atcaccatga gt
#gcagaggt   2220
ggcagaactg tatcgattgg aattgggaga ttataaatta gtagagatca ct
#ccaattgg   2280
cttggccccc acagatgtga agaggtacac tactggtggc acctcaagaa at
#aaaagagg   2340
ggtctttgtg ctagggttct tgggttttct cgcaacggca ggttctgcaa tg
#ggagccgc   2400
cagcctgacc ctcacggcac agtcccgaac tttattggct gggatagtcc aa
#cagcagca   2460
acagctgttg gacgtggtca agagacaaca agaattgttg cgactgaccg tc
#tggggaac   2520
aaagaacctc cagactaggg tcactgccat cgagaagtac ttaaaggacc ag
#gcgcagct   2580
gaatgcttgg ggatgtgcgt ttagacaagt ctgccacact actgtaccat gg
#ccaaatgc   2640
aagtctaaca ccaaagtgga acaatgagac ttggcaagag tgggagcgaa ag
#gttgactt   2700
cttggaagaa aatataacag ccctcctaga ggaggcacaa attcaacaag ag
#aagaacat   2760
gtatgaatta caaaagttga atagctggga tgtgtttggc aattggtttg ac
#cttgcttc   2820
ttggataaag tatatacaat atggagttta tatagttgta ggagtaatac tg
#ttaagaat   2880
agtgatctat atagtacaaa tgctagctaa gttaaggcag gggtataggc ca
#gtgttctc   2940
ttccccaccc tcttatttcc agcagaccca tatccaacag gacccggcac tg
#ccaaccag   3000
agaaggcaaa gaaagagacg gtggagaagg cggtggcaac agctcctggc ct
#tggcagat   3060
agaatatatc cactttctta ttcgtcagct tattagactc ttgacttggc ta
#ttcagtaa   3120
ctgtaggact ttgctatcga gagtatacca gatcctccaa ccaatactcc ag
#aggctctc   3180
tgcgacccta cagaggattc gagaagtcct caggactgaa ctgacctacc ta
#caatatgg   3240
gtggagctat ttccatgagg cggtccaggc cgtctggaga tctgcgacag ag
#actcttgc   3300
gggcgcgtgg ggagacttat gggagactct taggagaggt ggaagatgga ta
#ctcgcaat   3360
ccccaggagg attagacaag ggcttgagct cactctcttg tgagggacag ag
#aattcgga   3420
tccactagtt ctagactcga gggggggccc ggtacgagcg cttagctagc ta
#gagaccac   3480
ctcccctgcg agctaagctg gacagccaat gacgggtaag agagtgacat tt
#ttcactaa   3540
cctaagacag gagggccgtc agagctactg cctaatccaa agacgggtaa aa
#gtgataaa   3600
aatgtatcac tccaacctaa gacaggcgca gcttccgagg gatttgtcgt ct
#gttttata   3660
tatatttaaa agggtgacct gtccggagcc gtgctgcccg gatgatgtct tg
#gtctagac   3720
tcgagggggg gcccggtacg atccagatct gctgtgcctt ctagttgcca gc
#catctgtt   3780
gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tg
#tcctttcc   3840
taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tc
#tggggggt   3900
ggggtggggc agcacagcaa gggggaggat tgggaagaca atagcaggca tg
#ctggggat   3960
gcggtgggct ctatgggtac ccaggtgctg aagaattgac ccggttcctc ct
#gggccaga   4020
aagaagcagg cacatcccct tctctgtgac acaccctgtc cacgcccctg gt
#tcttagtt   4080
ccagccccac tcataggaca ctcatagctc aggagggctc cgccttcaat cc
#cacccgct   4140
aaagtacttg gagcggtctc tccctccctc atcagcccac caaaccaaac ct
#agcctcca   4200
agagtgggaa gaaattaaag caagataggc tattaagtgc agagggagag aa
#aatgcctc   4260
caacatgtga ggaagtaatg agagaaatca tagaatttct tccgcttcct cg
#ctcactga   4320
ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa ag
#gcggtaat   4380
acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aa
#ggccagca   4440
aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tc
#cgcccccc   4500
tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga ca
#ggactata   4560
aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cg
#accctgcc   4620
gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ct
#caatgctc   4680
acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gt
#gtgcacga   4740
accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg ag
#tccaaccc   4800
ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gc
#agagcgag   4860
gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct ac
#actagaag   4920
gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa ga
#gttggtag   4980
ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gc
#aagcagca   5040
gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cg
#gggtctga   5100
cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat ca
#aaaaggat   5160
cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gt
#atatatga   5220
gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct ca
#gcgatctg   5280
tctatttcgt tcatccatag ttgcctgact ccgggggggg ggggcgctga gg
#tctgcctc   5340
gtgaagaagg tgttgctgac tcataccagg cctgaatcgc cccatcatcc ag
#ccagaaag   5400
tgagggagcc acggttgatg agagctttgt tgtaggtgga ccagttggtg at
#tttgaact   5460
tttgctttgc cacggaacgg tctgcgttgt cgggaagatg cgtgatctga tc
#cttcaact   5520
cagcaaaagt tcgatttatt caacaaagcc gccgtcccgt caagtcagcg ta
#atgctctg   5580
ccagtgttac aaccaattaa ccaattctga ttagaaaaac tcatcgagca tc
#aaatgaaa   5640
ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gt
#ttctgtaa   5700
tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt at
#cggtctgc   5760
gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aa
#ataaggtt   5820
atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aa
#agcttatg   5880
catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aa
#tcactcgc   5940
atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cg
#cgatcgct   6000
gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ct
#gccagcgc   6060
atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ct
#gttttccc   6120
ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gc
#ttgatggt   6180
cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg ta
#acatcatt   6240
ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tc
#ccatacaa   6300
tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat ac
#ccatataa   6360
atcagcatcc atgttggaat ttaatcgcgg cctcgagcaa gacgtttccc gt
#tgaatatg   6420
gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg tt
#catgatga   6480
tatattttta tcttgtgcaa tgtaacatca gagattttga gacacaacgt gg
#ctttcccc   6540
ccccccccat tattgaagca tttatcaggg ttattgtctc atgagcggat ac
#atatttga   6600
atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aa
#gtgccacc   6660
tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gt
#atcacgag   6720
gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tg
#cagctccc   6780
ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gt
#cagggcgc   6840
gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag ag
#cagattgt   6900
actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga ga
#aaataccg   6960
catcagattg gctattgg
#
#
#6978
<210> SEQ ID NO 16
<211> LENGTH: 879
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
#Sequence:  SIV
gp160env IN PLASMID CMVkan/R-R-SIVgp160
# CTE
<400> SEQUENCE: 16
Met Gly Cys Leu Gly Asn Gln Leu Leu Ile Al
#a Ile Leu Leu Leu Ser
1               5
#                 10
#                 15
Val Tyr Gly Ile Tyr Cys Thr Leu Tyr Val Th
#r Val Phe Tyr Gly Val
20
#             25
#             30
Pro Ala Trp Arg Asn Ala Thr Ile Pro Leu Ph
#e Cys Ala Thr Lys Asn
35
#         40
#         45
Arg Asp Thr Trp Gly Thr Thr Gln Cys Leu Pr
#o Asp Asn Gly Asp Tyr
50
#     55
#     60
Ser Glu Val Ala Leu Asn Val Thr Glu Ser Ph
#e Asp Ala Trp Asn Asn
65
# 70
# 75
# 80
Thr Val Thr Glu Gln Ala Ile Glu Asp Val Tr
#p Gln Leu Phe Glu Thr
85
#                 90
#                 95
Ser Ile Lys Pro Cys Val Lys Leu Ser Pro Le
#u Cys Ile Thr Met Arg
100
#           105
#           110
Cys Asn Lys Ser Glu Thr Asp Arg Trp Gly Le
#u Thr Lys Ser Ile Thr
115
#       120
#       125
Thr Thr Ala Ser Thr Thr Ser Thr Thr Ala Se
#r Ala Lys Val Asp Met
130
#   135
#   140
Val Asn Glu Thr Ser Ser Cys Ile Ala Gln As
#p Asn Cys Thr Gly Leu
145                 1
#50                 1
#55                 1
#60
Glu Gln Glu Gln Met Ile Ser Cys Lys Phe As
#n Met Thr Gly Leu Lys
165
#               170
#               175
Arg Asp Lys Lys Lys Glu Tyr Asn Glu Thr Tr
#p Tyr Ser Ala Asp Leu
180
#           185
#           190
Val Cys Glu Gln Gly Asn Asn Thr Gly Asn Gl
#u Ser Arg Cys Tyr Met
195
#       200
#       205
Asn His Cys Asn Thr Ser Val Ile Gln Glu Se
#r Cys Asp Lys His Tyr
210
#   215
#   220
Trp Asp Ala Ile Arg Phe Arg Tyr Cys Ala Pr
#o Pro Gly Tyr Ala Leu
225                 2
#30                 2
#35                 2
#40
Leu Arg Cys Asn Asp Thr Asn Tyr Ser Gly Ph
#e Met Pro Lys Cys Ser
245
#               250
#               255
Lys Val Val Val Ser Ser Cys Thr Arg Met Me
#t Glu Thr Gln Thr Ser
260
#           265
#           270
Thr Trp Phe Gly Phe Asn Gly Thr Arg Ala Gl
#u Asn Arg Thr Tyr Ile
275
#       280
#       285
Tyr Trp His Gly Arg Asp Asn Arg Thr Ile Il
#e Ser Leu Asn Lys Tyr
290
#   295
#   300
Tyr Asn Leu Thr Met Lys Cys Arg Arg Pro Gl
#y Asn Lys Thr Val Leu
305                 3
#10                 3
#15                 3
#20
Pro Val Thr Ile Met Ser Gly Leu Val Phe Hi
#s Ser Gln Pro Ile Asn
325
#               330
#               335
Asp Arg Pro Lys Gln Ala Trp Cys Trp Phe Gl
#y Gly Lys Trp Lys Asp
340
#           345
#           350
Ala Ile Lys Glu Val Lys Gln Thr Ile Val Ly
#s His Pro Arg Tyr Thr
355
#       360
#       365
Gly Thr Asn Asn Thr Asp Lys Ile Asn Leu Th
#r Ala Pro Gly Gly Gly
370
#   375
#   380
Asp Pro Glu Val Thr Phe Met Trp Thr Asn Cy
#s Arg Gly Glu Phe Leu
385                 3
#90                 3
#95                 4
#00
Tyr Cys Lys Met Asn Trp Phe Leu Asn Trp Va
#l Glu Asp Arg Asn Thr
405
#               410
#               415
Ala Asn Gln Lys Pro Lys Glu Gln His Lys Ar
#g Asn Tyr Val Pro Cys
420
#           425
#           430
His Ile Arg Gln Ile Ile Asn Thr Trp His Ly
#s Val Gly Lys Asn Val
435
#       440
#       445
Tyr Leu Pro Pro Arg Glu Gly Asp Leu Thr Cy
#s Asn Ser Thr Val Thr
450
#   455
#   460
Ser Leu Ile Ala Asn Ile Asp Trp Ile Asp Gl
#y Asn Gln Thr Asn Ile
465                 4
#70                 4
#75                 4
#80
Thr Met Ser Ala Glu Val Ala Glu Leu Tyr Ar
#g Leu Glu Leu Gly Asp
485
#               490
#               495
Tyr Lys Leu Val Glu Ile Thr Pro Ile Gly Le
#u Ala Pro Thr Asp Val
500
#           505
#           510
Lys Arg Tyr Thr Thr Gly Gly Thr Ser Arg As
#n Lys Arg Gly Val Phe
515
#       520
#       525
Val Leu Gly Phe Leu Gly Phe Leu Ala Thr Al
#a Gly Ser Ala Met Gly
530
#   535
#   540
Ala Ala Ser Leu Thr Leu Thr Ala Gln Ser Ar
#g Thr Leu Leu Ala Gly
545                 5
#50                 5
#55                 5
#60
Ile Val Gln Gln Gln Gln Gln Leu Leu Asp Va
#l Val Lys Arg Gln Gln
565
#               570
#               575
Glu Leu Leu Arg Leu Thr Val Trp Gly Thr Ly
#s Asn Leu Gln Thr Arg
580
#           585
#           590
Val Thr Ala Ile Glu Lys Tyr Leu Lys Asp Gl
#n Ala Gln Leu Asn Ala
595
#       600
#       605
Trp Gly Cys Ala Phe Arg Gln Val Cys His Th
#r Thr Val Pro Trp Pro
610
#   615
#   620
Asn Ala Ser Leu Thr Pro Lys Trp Asn Asn Gl
#u Thr Trp Gln Glu Trp
625                 6
#30                 6
#35                 6
#40
Glu Arg Lys Val Asp Phe Leu Glu Glu Asn Il
#e Thr Ala Leu Leu Glu
645
#               650
#               655
Glu Ala Gln Ile Gln Gln Glu Lys Asn Met Ty
#r Glu Leu Gln Lys Leu
660
#           665
#           670
Asn Ser Trp Asp Val Phe Gly Asn Trp Phe As
#p Leu Ala Ser Trp Ile
675
#       680
#       685
Lys Tyr Ile Gln Tyr Gly Val Tyr Ile Val Va
#l Gly Val Ile Leu Leu
690
#   695
#   700
Arg Ile Val Ile Tyr Ile Val Gln Met Leu Al
#a Lys Leu Arg Gln Gly
705                 7
#10                 7
#15                 7
#20
Tyr Arg Pro Val Phe Ser Ser Pro Pro Ser Ty
#r Phe Gln Gln Thr His
725
#               730
#               735
Ile Gln Gln Asp Pro Ala Leu Pro Thr Arg Gl
#u Gly Lys Glu Arg Asp
740
#           745
#           750
Gly Gly Glu Gly Gly Gly Asn Ser Ser Trp Pr
#o Trp Gln Ile Glu Tyr
755
#       760
#       765
Ile His Phe Leu Ile Arg Gln Leu Ile Arg Le
#u Leu Thr Trp Leu Phe
770
#   775
#   780
Ser Asn Cys Arg Thr Leu Leu Ser Arg Val Ty
#r Gln Ile Leu Gln Pro
785                 7
#90                 7
#95                 8
#00
Ile Leu Gln Arg Leu Ser Ala Thr Leu Gln Ar
#g Ile Arg Glu Val Leu
805
#               810
#               815
Arg Thr Glu Leu Thr Tyr Leu Gln Tyr Gly Tr
#p Ser Tyr Phe His Glu
820
#           825
#           830
Ala Val Gln Ala Val Trp Arg Ser Ala Thr Gl
#u Thr Leu Ala Gly Ala
835
#       840
#       845
Trp Gly Asp Leu Trp Glu Thr Leu Arg Arg Gl
#y Gly Arg Trp Ile Leu
850
#   855
#   860
Ala Ile Pro Arg Arg Ile Arg Gln Gly Leu Gl
#u Leu Thr Leu Leu
865                 8
#70                 8
#75
<210> SEQ ID NO 17
<211> LENGTH: 271
<212> TYPE: PRT
<213> ORGANISM: Escherichia coli
<400> SEQUENCE: 17
Met Ser His Ile Gln Arg Glu Thr Ser Cys Se
#r Arg Pro Arg Leu Asn
1               5
#                 10
#                 15
Ser Asn Met Asp Ala Asp Leu Tyr Gly Tyr Ly
#s Trp Ala Arg Asp Asn
20
#             25
#             30
Val Gly Gln Ser Gly Ala Thr Ile Tyr Arg Le
#u Tyr Gly Lys Pro Asp
35
#         40
#         45
Ala Pro Glu Leu Phe Leu Lys His Gly Lys Gl
#y Ser Val Ala Asn Asp
50
#     55
#     60
Val Thr Asp Glu Met Val Arg Leu Asn Trp Le
#u Thr Glu Phe Met Pro
65
# 70
# 75
# 80
Leu Pro Thr Ile Lys His Phe Ile Arg Thr Pr
#o Asp Asp Ala Trp Leu
85
#                 90
#                 95
Leu Thr Thr Ala Ile Pro Gly Lys Thr Ala Ph
#e Gln Val Leu Glu Glu
100
#           105
#           110
Tyr Pro Asp Ser Gly Glu Asn Ile Val Asp Al
#a Leu Ala Val Phe Leu
115
#       120
#       125
Arg Arg Leu His Ser Ile Pro Val Cys Asn Cy
#s Pro Phe Asn Ser Asp
130
#   135
#   140
Arg Val Phe Arg Leu Ala Gln Ala Gln Ser Ar
#g Met Asn Asn Gly Leu
145                 1
#50                 1
#55                 1
#60
Val Asp Ala Ser Asp Phe Asp Asp Glu Arg As
#n Gly Trp Pro Val Glu
165
#               170
#               175
Gln Val Trp Lys Glu Met His Lys Leu Leu Pr
#o Phe Ser Pro Asp Ser
180
#           185
#           190
Val Val Thr His Gly Asp Phe Ser Leu Asp As
#n Leu Ile Phe Asp Glu
195
#       200
#       205
Gly Lys Leu Ile Gly Cys Ile Asp Val Gly Ar
#g Val Gly Ile Ala Asp
210
#   215
#   220
Arg Tyr Gln Asp Leu Ala Ile Leu Trp Asn Cy
#s Leu Gly Glu Phe Ser
225                 2
#30                 2
#35                 2
#40
Pro Ser Leu Gln Lys Arg Leu Phe Gln Lys Ty
#r Gly Ile Asp Asn Pro
245
#               250
#               255
Asp Met Asn Lys Leu Gln Phe His Leu Met Le
#u Asp Glu Phe Phe
260
#           265
#           270
<210> SEQ ID NO 18
<211> LENGTH: 2640
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial
#Sequence:  DNA
sequence of mutated SIV gene in
#construct
CMVkan/R-R-SIVgp160 CTE
<400> SEQUENCE: 18
atgggatgtc ttgggaatca gctgcttatc gccatcttgc ttttaagtgt ct
#atgggatc     60
tattgtactc tatatgtcac agtcttttat ggtgtaccag cttggaggaa tg
#cgacaatt    120
cccctctttt gtgcaaccaa gaatagggat acttggggaa caactcagtg cc
#taccagat    180
aatggtgatt attcagaagt ggcccttaat gttacagaaa gctttgatgc ct
#ggaataat    240
acagtcacag aacaggcaat agaggatgta tggcaactct ttgagacctc aa
#taaagcct    300
tgtgtaaaat tatccccatt atgcattact atgagatgca ataaaagtga ga
#cagataga    360
tggggattga caaaatcaat aacaacaaca gcatcaacaa catcaacgac ag
#catcagca    420
aaagtagaca tggtcaatga gactagttct tgtatagccc aggataattg ca
#caggcttg    480
gaacaagagc aaatgataag ctgtaaattc aacatgacag ggttaaaaag ag
#acaagaaa    540
aaagagtaca atgaaacttg gtactctgca gatttggtat gtgaacaagg ga
#ataacact    600
ggtaatgaaa gtagatgtta catgaaccac tgtaacactt ctgttatcca ag
#agtcttgt    660
gacaaacatt attgggatgc tattagattt aggtattgtg cacctccagg tt
#atgctttg    720
cttagatgta atgacacaaa ttattcaggc tttatgccta aatgttctaa gg
#tggtggtc    780
tcttcatgca caaggatgat ggagacacag acttctactt ggtttggctt ta
#atggaact    840
agagcagaaa atagaactta tatttactgg catggtaggg ataataggac ta
#taattagt    900
ttaaataagt attataatct aacaatgaaa tgtagaagac caggaaataa ga
#cagtttta    960
ccagtcacca ttatgtctgg attggttttc cactcacaac caatcaatga ta
#ggccaaag   1020
caggcatggt gttggtttgg aggaaaatgg aaggatgcaa taaaagaggt ga
#agcagacc   1080
attgtcaaac atcccaggta tactggaact aacaatactg ataaaatcaa tt
#tgacggct   1140
cctggaggag gagatccgga agttaccttc atgtggacaa attgcagagg ag
#agttcctc   1200
tactgtaaaa tgaattggtt tctaaattgg gtagaagata ggaatacagc ta
#accagaag   1260
ccaaaggaac agcataaaag gaattacgtg ccatgtcata ttagacaaat aa
#tcaacact   1320
tggcataaag taggcaaaaa tgtttatttg cctccaagag agggagacct ca
#cgtgtaac   1380
tccacagtga ccagtctcat agcaaacata gattggattg atggaaacca aa
#ctaatatc   1440
accatgagtg cagaggtggc agaactgtat cgattggaat tgggagatta ta
#aattagta   1500
gagatcactc caattggctt ggcccccaca gatgtgaaga ggtacactac tg
#gtggcacc   1560
tcaagaaata aaagaggggt ctttgtgcta gggttcttgg gttttctcgc aa
#cggcaggt   1620
tctgcaatgg gagccgccag cctgaccctc acggcacagt cccgaacttt at
#tggctggg   1680
atagtccaac agcagcaaca gctgttggac gtggtcaaga gacaacaaga at
#tgttgcga   1740
ctgaccgtct ggggaacaaa gaacctccag actagggtca ctgccatcga ga
#agtactta   1800
aaggaccagg cgcagctgaa tgcttgggga tgtgcgttta gacaagtctg cc
#acactact   1860
gtaccatggc caaatgcaag tctaacacca aagtggaaca atgagacttg gc
#aagagtgg   1920
gagcgaaagg ttgacttctt ggaagaaaat ataacagccc tcctagagga gg
#cacaaatt   1980
caacaagaga agaacatgta tgaattacaa aagttgaata gctgggatgt gt
#ttggcaat   2040
tggtttgacc ttgcttcttg gataaagtat atacaatatg gagtttatat ag
#ttgtagga   2100
gtaatactgt taagaatagt gatctatata gtacaaatgc tagctaagtt aa
#ggcagggg   2160
tataggccag tgttctcttc cccaccctct tatttccagc agacccatat cc
#aacaggac   2220
ccggcactgc caaccagaga aggcaaagaa agagacggtg gagaaggcgg tg
#gcaacagc   2280
tcctggcctt ggcagataga atatatccac tttcttattc gtcagcttat ta
#gactcttg   2340
acttggctat tcagtaactg taggactttg ctatcgagag tataccagat cc
#tccaacca   2400
atactccaga ggctctctgc gaccctacag aggattcgag aagtcctcag ga
#ctgaactg   2460
acctacctac aatatgggtg gagctatttc catgaggcgg tccaggccgt ct
#ggagatct   2520
gcgacagaga ctcttgcggg cgcgtgggga gacttatggg agactcttag ga
#gaggtgga   2580
agatggatac tcgcaatccc caggaggatt agacaagggc ttgagctcac tc
#tcttgtga   2640
<210> SEQ ID NO 19
<211> LENGTH: 813
<212> TYPE: DNA
<213> ORGANISM: Escherichia coli
<400> SEQUENCE: 19
atgagccata ttcaacggga aacgtcttgc tcgaggccgc gattaaattc ca
#acatggat     60
gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg tg
#cgacaatc    120
tatcgattgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg ca
#aaggtagc    180
gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga at
#ttatgcct    240
cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact ca
#ccactgcg    300
atccccggga aaacagcatt ccaggtatta gaagaatatc ctgattcagg tg
#aaaatatt    360
gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg ta
#attgtcct    420
tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa ta
#acggtttg    480
gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca ag
#tctggaaa    540
gaaatgcata agcttttgcc attctcaccg gattcagtcg tcactcatgg tg
#atttctca    600
cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt tg
#gacgagtc    660
ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg tg
#agttttct    720
ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga ta
#tgaataaa    780
ttgcagtttc atttgatgct cgatgagttt ttc
#
#        813

Claims

1. A nucleic acid construct comprising a HIV-1 gag/pol gene having the coding sequence of the gag/pol gene set forth in FIG. 1 (SEQUENCE ID NO: 1).

2. A nucleic acid construct comprising a HIV-1 pol gene having the coding sequence of the pol gene set forth in FIG. 2 (SEQUENCE ID NO: 3).

3. A nucleic acid construct comprising an HIV or SIV 5′ LTR, a packaging signal, a gag/pol gene comprising the sequence set forth in FIG. 1 (SEQUENCE ID NO: 1), a 5′ splice site, a 3′ splice site, an env gene, a tat gene, a functional RNA transport element and a 3′ HIV or SIV LTR, said nucleic acid construct being able to produce functional Gag. Pol and Env virion components.

4. A vector comprising the nucleic acid construct of claim 1, 2 or 3.

5. An isolated host cell comprising the nucleic acid construct of claim 1, 2 or 3.

6. The host cell of claim 5 wherein said cell is a eukaryote.

7. The host cell of claim 6 wherein said cell is a human cell.

8. The host cell of claim 5 wherein said cell is a prokaryote.

9. The host cell of claim 8 wherein said cell is E. coli.

10. A composition comprising the nucleic acid A construct of claim 1, 2, or 3 and a pharmaceutically acceptable carrier.

11. A lentiviral expression system comprising the following:(a) a packaging vector comprising a HIV-1 gag/pol gene having the nucleotide sequence set forth in FIG. 1 (SEQUENCE ID NO: 1); (b) a transfer vector; and (c) an envelope encoding vector.

12. An isolated host cell comprising the lentiviral expression system of claim 11.

13. The host cell of claim 12, wherein said cell is a eukaryote.

14. The host cell of claim 13 wherein said cell is a human cell.

15. A process for making a lentiviral particle comprising expressing, in a host cell, HIV Gag and HIV Pol from a vector comprising the nucleotide sequences encoding HIV Gag and HIV Pol set forth in FIG. 1 (SEQUENCE ID NO: 1) and expressing a gene encoding an envelope protein by growing the host cell under conditions suitable to cause expression of HIV Gag, HIV Pol and envelope protein so that a lentiviral particle is formed.

16. A lentiviral expression system which is capable of functioning in the absence of Rev, Tat, and any viral RNA transport element comprising the following:(a) a packaging vector comprising a HIV-1 gag/pol gene which is capable of functioning in the absence of Rev, Tat, and any viral RNA transport element; (b) a transfer vector; and (c) an envelope encoding vector wherein the HIV-1 gag/pol gene has the coding sequence of the HIV-1 gag/pol gene set forth in FIG. 1 (SEQUENCE ID NO: 1).

17. A process for making a lentiviral particle in the absence of Rev, Tat, or any viral RNA transport element comprising expressing HIV Gag and HIV Pol in a host cell from a HIV-1 gag/pol gene which is capable of functioning in the absence of Rev, Tat, and any viral RNA transport element and expressing an Envelope protein from a envelope encoding gene whose expression is independent of Rev, Tat, or any viral RNA transport element wherein the HIV-1 gag/pol gene has the coding sequence of the HIV-1 gag/pol gene set forth in FIG. 1 (SEQUENCE ID NO: 1).

18. The lentiviral expression system of claim 16 wherein the packaging vector has the DNA sequence of packaging construct pCMVgag/polBNKan set forth in FIG. 9 (SEQUENCE ID NO: 6).

19. The lentiviral expression system of claim 16 wherein the transfer vector has the DNA sequence of pmBCwCNluci set forth in FIG. 10 (SEQUENCE ID NO: 8) or pmBCmCNluci set forth in FIG. 11 (SEQUENCE ID NO: 9).

20. An isolated host cell comprising the lentiviral expression system of claim 16.

21. The host cell of claim 20 wherein said cell is a eukaryote.

22. The host cell of claim 21 wherein said cell is a human cell.