WO2010009465A2 - Htlv-ii vector and methods of use - Google Patents

Abstract

The disclosure provides compositions and methods of use of an HTLV-II viral vector. In embodiments, the disclosure provides an isolated viral vector comprising at least a portion of the HTLV-II genome encoding the gag, pro, and pol genes and lacking all or a portion of the pX region and a polynucleotide encoding all or a portion of a protein that corresponds to a viral protein from a heterologous virus located within the deletion in the pX region. The viral vectors are useful for inducing an immune response to the viral protein form the heterologous virus.

Description

HTLV-II VECTOR AND METHODS OF USE

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0001] The work performed during the development of this disclosure utilized intramural support from the National Institutes of Health. The United States government has certain rights in the disclosure.

TECHNICAL FIELD

[0002] This disclosure relates to novel viral vectors, particularly for the transfer and expression of genes for vaccines.

BACKGROUND

[0003] Most of the T-cell vaccines developed for HIV are based on microbial vectors having limited replication capacity and do not persist in the host. Such vaccine vectors do not protect macaques from SIV infection and their ability to protect against high virus load is transient (approximately six months). An emerging notion is that these vaccine vectors elicit "too small T-cell responses" that expand "too late" upon virus encounter to sufficiently contain the seeding of the virus in tissues. In addition, few of these vectors target mucosal sites, the first portal of entry for HIV.

[0004] HTLV-II is a human retrovirus that does not cause disease in healthy or in

HIV-I infected individuals. Both HTLV-I and HTLV-II are members of the oncovirinae subfamily of retroviruses and have recently been reclassified as members of the group of viruses which includes bovine leukemia virus (BLV). HTLV-I and HTLV-II share 66% sequence homology. HTLV also shares high sequence homologies with other retroviruses such as bovine leukemia virus (BLV) and the simian T cell leukemia virus (STLV). The discovery and characterization of HTLV-I and II facilitated the characterization of human immunodeficiency virus types 1 and 2 (HIV-I and HIV-2) members of the subfamily of lentiviruses. HTLV shares many biological and molecular characteristics of HIV including routes of transmission, a general T-cell tropism, syncytia induction, and the presence of viral- encoded core proteins that function at both the transcriptional and posttranscriptional level. [0005] The HTLV-II proviral genome has about 8952 nucleotides and includes regions coding for the structural proteins (gag and env) and the viral protease and polymerase (pol), similar to HIV-I. The genome also contains an additional sequence designated pX adjacent to the envelope (env) gene. Four open reading frames (ORFs), designated pX-I, pX- II, pX-III, and pX-IV, were initially identified within the HTLV-I pX. The second coding exon of the transacting viral proteins Tax and Rex are expressed from the overlapping ORFs pX-IV and pX-III, respectively, and are translated from a doubly spliced, bicistronic mRNA. Tax increases the rate of viral transcription by activating the viral promoter in the viral long terminal repeat, while Rex acts posttranscriptionally to increase the ratio of incompletely spliced mRNAs to doubly spliced mRNA. ORFs x-I and x-II contained in the proximal portion of pX have 655 base pairs (bp) and 573 bp for HTLV-I and HTLV-II, respectively, and have been referred to as the nontranslated or untranslated (UT) region. [0006] Recent reports concerning HIV vaccine trials have indicated that current vaccine vectors may not effectively provide for immunity. Thus, there remains a need to develop vaccine vectors useful for providing viral immunity. There further remains a need to develop vaccine vectors useful for treating cancer.

SUMMARY

[0007] One aspect the disclosure provides an isolated viral vector comprising at least a portion of the HTLV-II genome encoding two HTLV-II retroviral long terminal repeat (LTR) sequences. The two LTR sequences encompass at least one to five polynucleotides each independently encoding all or a portion of a protein that corresponds to a gene of interest such as a viral protein from a heterologous virus or a tumor antigen for a target tumor. In some embodiments, the polynucleotides each independently encoding a gene of interest are located near the 3'LTR and positioned in reverse orientation, and thus expressed from the 3'LTR. Also positioned in the reverse orientation can be the HTLV-II sequence (required to protect the 3'LTR from methylation), an internal ribosome entry site (IRES) and multiple closing site (MCS), and a polyadenylation (polyA) signal. In some embodiments, the viral protein is from a heterologous virus such as SIV, HIV, HCV, HPV, EBV, HCMV, and other like chronic infection-based viruses. In still other embodiments the gene of interest encodes all or part of a tumor antigen, for example CA- 125, MUC-I, epithelial tumors antigen (ETA), Tyrosinase, and/or melanoma associated antigen (MAGE). [0008] In another aspect of the disclosure provides an isolated viral vector comprising at least a portion of the HTLV-II genome encoding the gag, pro, and pol genes and lacking all or a portion of the pX region; and a polynucleotide encoding all or a portion of a protein that corresponds to a gene of interest, such as a viral protein from a heterologous virus, located within the deletion in the pX region. In some embodiments, the HTLV-II genome encodes gag, pro, pol, env, tax, and rex. In other embodiments, the HTLV-II genome comprising the polynucleotide sequence of Table 3 has a deletion of nucleotides 6645 to 7153. In some embodiments, the viral protein is from a heterologous virus like SIV, HIV, Hepatitis C, HPV, EBV, HCMV and the like. In yet other embodiments, the viral protein corresponds to any one of the viral proteins selected from the group consisting of Vif, Tat, Gag, Env, Rev, gpl20, gp41, p24, p7, pi 7, tev, and combinations thereof or immunogenic fragments thereof. In still other embodiments the gene of interest encodes all or part of a tumor antigen, for example CA- 125, MUC-I, epithelial tumors antigen (ETA), Tyrosinase, and/or melanoma associated antigen (MAGE). [0009] The disclosure provides methods of use of the viral vector and kits.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 shows HTLV-II infection in the immortalized T cell line, RhM304, as measured by intracellular pi 9Gag staining (Figure IA). Phenotypic analysis conducted by flow cytometry demonstrated that RhM304 cell line is CD3+ CD8+ unlike the Mo-T cell line which is CD3 + CD4 + as shown in Figure IB. Karyotypic analysis of RhM304 cells clearly confirmed the non-human primate origin of the cell line with a diploid set of 21 chromosomes of female gender (Figure IC). Analysis of proviral DNA copies by real-time PCR using primers specific for the gag region demonstrated a higher level of proviral DNA in the Mo-T cells than RhM304 (Figure ID).

[0011] Figure 2 shows that animals M304 and M214 mounted weak antibody responses to HTLV-II by 12 months post infection whereas animal L900 remained seronegative as measured by western blot. Figure 2A. Animals M893, M897, M905, and M906 mounted a stronger antibody response by one month post infection as shown in Figure 2B. [0012] Figure 3 shows PCR analysis of CD4+ or CD8+ purified T-cells from blood or lymph nodes of M304, M214, and L900 revealing the presence of proviral DNA in blood and lymph nodes of M304 and M214 but not L900 (Figure 3A and data not shown for L900). Infection was further confirmed by Gag PCR analysis of all tissues obtained by surgery or at sacrifice, eighteen months after inoculation. Virus was found in the jejunum, ileum and blood and mesenteric lymph nodes of macaques M214 and L304 but not in tissues of macaque L900 (Figure 3B). [0013] Figure 4 shows validation of the presence of viral sequence obtained by amplifying Tax2 in PBMCs, lymph nodes and bone marrow of macaques M214 and M304 but not L900.

[0014] Figure 5 shows induction of HTLV-II viral expression in ex vivo bone marrow samples from M214, M304, and L900. Bone marrow samples were obtained and separated into 3 populations. One population was not further separated. The other populations were separated each into CD34+ enriched or CD34+ depleted cells. Flow cytometry of each of the populations is shown in Figure 5A. The cells in each population were stained with anti-Tax2 antibodies and analyzed by flow cytometry. Animals M214, M304, and L900 all showed the presence of Tax2 in CD34+ cells. Dendritic cells were isolated from peripheral blood from animal L900 and analyzed for the presence of Tax 2. The results are shown in Figure 5B. Tax2 expression is found in both plasmocitoid (pDC) and myeloid derived dendritic cells (mDC). Both subsets of dendritic cells showed expression of pl9 gag. (Figure 5D). The presence of mature viral particles in macaque dendritic cells was found by electron microscopy as shown in Figure 5C. Both sets of dendritic cells were able to transmit virus to SupTl cells as shown in Figure 5E.

[0015] Figure 6 shows the analysis of virus- specific CD4+ and CD8+ T-cell responses in blood and tissues of the infected macaques by intracellular staining following in vitro stimulation with Tax2 and Gag2 overlapping peptides. No detectable responses were found in the blood of macaque L900, consistent with the low/undetectable provirus load in the PBMCs of this animal (Figure 6A). Another animal with a low antibody response to HTLV-II antigens as measured using western blot as shown in Figure 2B is animal M906. Consistent with the results of animal L900, little or no immune responses were found in the blood of M906. Stronger responses were found in the gut tissue. (Figures 6D and 6E). In contrast, low but specific, although sporadic, immune responses were found in blood of animals M304, M214, M893, M897, and M905. (Figures 6B, 6C, 6G, and 6H). Analysis of HTLV-II specific immune responses in the gut demonstrated convincing positive responses to Gag(colon), Tax and Gag (ileum) and Gag (rectum) of animal M214 (Figure 61). Analysis of HTLV-II specific immune responses in the gut demonstrated convincing positive responses to Tax and Gag (jejunum) of animal M893 (Figure 6F). [0016] Figure 7 shows the animals, the relative date of HTLV-II infection and delivery of their progeny. [0017] Figure 8 shows that antibody responses to HTLV antigens were not detectable by western blot in both the mothers and babies when measured within a few months of infection or delivery.

[0018] Figure 9 shows that the antibody response to HTLV-II antigens as measured by ELISA continues to rise in the mothers after delivery. (Figures 9A-D) Analysis of virus- specific CD4+ and CD8+ T-cell responses in the mothers was measured longitudinally in blood of the infected macaques by intracellular staining following in vitro stimulation with Tax2 and Gag2 overlapping peptides as described in Example 3. The results show that most of the mothers have very low virus-specific CD4+ and CD8+ T-cell responses in peripheral blood up to 8 weeks post delivery. One animal P047 showed a strong response to tax and gag at delivery. (Figure 9E-L).

[0019] Figure 10 shows the cloning strategies for cloning heterologous SIV genes into a HTLV-II viral vector.

[0020] Figure 11 shows an illustrative embodiment of a vector comprising two

HTLV-II retroviral long terminal repeats (LTR) encompassing, in reverse orientation and expressed from the 3'LTR, two genes of interest.

[0021] Figure 12 shows a portion of one vector embodiment including the two LTR' s and the material encompassed there between.

[0022] Figure 13: HTLV-II establishes an infection in rhesus macaques, replicating in lymphoid and mucosal tissues, a) Histograms showing the level of HTLV-II gag p 19 protein expressed by Mo-T and RhM304 cells, b) Number of HTLV-II genome copies per million Mo-T and RhM304 cells c) Rh304 karyotype showing that the cell line RhM304 is of macaque origin, d) Serum antibodies to p24 measured by ELISA 1-3 months post HTLV-II infection e) HTLV-II viral burden measured as gag copies per 10⁶ cells isolated from the spleen, ileum, and jejunum in animals M214 and M304. f) Tax2 protein in HTLV-II infected dendritic cells and controls.

[0023] Figure 14: HTLV-II infection stimulates humoral and T cell responses a)

Serum antibodies specific to several HTLV-II proteins measured by western blot in HTLV-II infected macaques, b) Stable CD4+ and CD8+ T-cell counts in the blood of HTLV-II infected macaques c) Representative flow cyometric pseudocolor plots showing the frequency of IFNγ and or TNFα production by CD8+ T-cells following stimulation with HTLV-II tax and gag overlapping peptides or in unstimulated cells, d) - g) Mean HTLV-II specific CD4+ and CD8+ T-cell responses 10 weeks post HTLV-II infection in the blood and the gastrointestinal tract. The frequency of gag or tax specific cells was measured by intracellular cytokine staining for IFNγ/ TNFα or IL2.

[0024] Figure 15: HTLV-II preinfection does not affect SIV_mac25i viral load or the frequency of T-cells a) Study design: Four macaques were infected with HTLV-II; ten months post infection these 4 macaques along with 4 naϊve animals were challenged with SrV_maC25i. Blood, lymphoid, and mucosal tissues were sampled pre and for up to 90 days post SrV_maC25i infection, b) Serum antibodies to HTLV-II measured by western blots pre and post- SIV_maC25i infection, c) SIV_maC25i viral load of HTLV-II co-infected macaques shown in black and controls shown in read measured by the number of SIV_maC25i RNA copies/ml of plasma. d) and e) Absolute CD8+ (left) and CD4+ (right) T-cell count in the blood following SrVmacssi infection.

[0025] Figure 16: Phenotype of CD4+ and CD8+ T-cells in blood is not significantly different between HTLV-II/SIVmac25i co-infected and SIV_mac25i mono- infected monkeys, a) Gating strategy for differentiation of T-cell subsets based on analysis of cell surface co-expression of CD28 and/or CD98 and CCR5. b) Percent of baseline CD4+ memory T-cell count in blood, determined by expression of CD28 and CD95 following SIV mac25i infection, c) Percent of baseline CD4+ CCR5+ T-cell count following SIV_maC25i infection, d) Percent of baseline CD8+ memory T-cell count in blood, determined by expression of CD28 following SIV _mac25i infection . e) Percent of baseline CD8+ effector T- cell count in blood, determined by expression of CD95 and lack of CD28 expression after SIV mac25i infection.

[0026] Figure 17: a) No significant difference between viral load or %CD4+ T-cells in tissues of HTLV-II/SIV _mac25i co-infected and SIV _mac25i mono-infected animals. Number of SIV _mac25i DNA copies per 10⁶ cells in lymph nodes, jejunum, and rectum of HTLV-II co- infected (white) and control (red) animals at 10 and 30 days post-SIV_maC25i infection, b) Percent of CD4+ CD3+ T-cells in the bone marrow and lymph nodes of HTLV-II co-infected and control animals at baseline and 10 and 30 days post-SIV _mac25i infection, c) Percent of CD4+ CD3+ T-cells in jejunum and rectum of HTLV-II co-infected and control animals at baseline and 10 and 30 days post-SIV_maC25i infection, d) and e) Immunohistochemistry showing CD4+ cells and CD8+ cells in tissues of HTLV-II co-infected versus control animals.

[0027] Figure 18: No significant difference in MIPa expression by CD4+ or

CD8+ T-cells in blood of HTLV-II/SIV _mac25i co-infected and SIV_mac25i mono-infected animals, a) Mean fluorescence intensity of MIP lα in CD4+ CD3+ T-cells in the blood of HTLV-II co-infected (black) and control (red) animals from baseline to 60 days post- SIV_maC25i infection, b) Mean fluorescence intensity of MIPIa in CD8+ CD3+ T-cells in the blood of HTLV-II co-infected (black) and control (red) animals from baseline to 60 days post-SIV _mac₂5i infection.

[0028] Figure 19: No significant difference in Ki67 expression in blood and tissues between HTLV-II/SIV_mac₂5i co-infected and SIV _mac25i mono-infected animals, a) Percent of CD4+ CD3+ T-cells expressing Ki67 in the blood of HTLV-II co-infected and control macaques from baseline through day 90 post-SIV_maC25i infection, b) Percent of CD8+ CD3+ T-cells expressing Ki67 in the blood of HTLV-II co-infected and control macaques from baseline through day 90 post-SIV _mac25i infection, c) Percent of CD4+CD3+ T-cells expressing Ki67 in the lymphoid tissues (blood and bone marrow) of HTLV-II co-infected and control animals at baseline and 10 and 30 days post-SIV _mac25i infection, d) Percent of CD8+CD3+ T-cells expressing Ki67 in the lymphoid tissues (blood and bone marrow) of HTLV-II co-infected and control animals at baseline and 10 and 30 days post-SIV_maC25i infection, e) Percent of CD4+CD3+ T-cells expressing Ki67 in the mucosal tissues (jejunum and rectum) of HTLV-II co-infected and control animals at baseline and 10 and 30 days post- SIV_maC25i infection, f) Percent of CD8+CD3+ T-cells expressing Ki67 in the mucosal tissues (jejunum and rectum) of HTLV-II co-infected and control animals at baseline and 10 and 30 days post-SIV_maC25i infection, g) Immunohistochemistry showing Ki67 expression in the GI tract of HTLV-H/SIV_maC25i co-infected and SIV_maC25i mono-infected animals. [0029] Figure 20: No significant difference in cytokine production by CD8+ T- cells stimulated with SIV _mac25i peptides between HTLV-II/SIV _mac25i co-infected and SIV _mac25i mono-infected macaques, a) Representative flow cytometric plots of CD8+ CD3+ T-cells expressing IFNγ and/or TNFα in the blood both unstimulated and after stimulation with overlapping pools of SIVgag and SIVenv peptides, b) Polyfunctional cytokine response to overlapping SIV_maC25i peptides by CD8+ CD3+ T-cells in the blood of HTLV-II co-infected and control macaques at 7, 30, 60, and 90 days post-SIV_maC25i infection. Green wedge represents cells producing IL2 only; blue represents cells producing IFNγ and/or TNFα; red portion represents cells producing IL2 along with IFNγ and/or TNFα. c) Average total cytokine (IFNγ and/or TNFα, IL2, IL17) production in CD8+ CD3+ T-cells in blood in response to stimulation with SIVenv overlapping peptide pool, d) Average total cytokine (IFNγ and/or TNFα, IL2, IL17) production in CD8+ CD3+ T-cells in blood in response to stimulation with SIVgag overlapping peptide pool. [0030] Figure 21: No significant difference in cytokine production by CD4+ T- cells stimulated with SIV _mac₂5i peptides between HTLV-II/SIV _mac₂5i co-infected and SIV mac25i mono-infected macaques, a) Polyfunctional cytokine response to overlapping SIV_mac25i peptides by CD4+ CD3+ T-cells in the blood of HTLV-II co-infected and control macaques at 7, 30, 60 and 90 days post-SIV_maC25i infection. Green wedge represents cells producing IL2 only; blue represents cells producing IFNγ and/or TNFα; red portion represents cells producing IL2 along with IFNγ and/or TNFα. b) Average total cytokine (IFNγ and/or TNFα, IL2, IL 17) production by CD4+ CD3+ T-cells in blood in response to stimulation with SIVenv overlapping peptide pool, c) Total average cytokine (IFNγ and/or TNFα, IL2, IL17) (IFNγ and/or TNFα, IL2, IL17) production by CD4+ CD3+ T-cells in blood in response to stimulation with SIVenv overlapping peptide pool. [0031] Figure 22 shows the magnitude of the mean S IV_maC25i- specific response in

CD4+ T-cells. Again, there were no significant differences between groups. In all, this data demonstrated that pre-infection with HTLV-II neither enhances nor limits the SIV_maC25i- specific immune responses.

[0032] Figure 23: Illustrative transcription from one embodiment of a vector of a present invention. All transcripts are made using the cellular transcription machinery. A. Initiation of transcription in the R region of the 5' LTR transcript finishes in the R region of the 3' LTR and uses the polyadenylation site of the 3'LTR. This RNA will not express polynucleotide sequences, provides template to be encapsidated and will reconstitute the vector following infection of target cells. B. Expression from the transcriptional start site within the CMV promoter. RNA is polycistronic and encompasses copGFP and first insert (15). Expression of the insert is achieve by reinitiation of translation through the presence of the IRES sequence. The SV40 poly A signal provides polyadenylation. C. Expression from the R region of the 3' LTR in reverse orientation. Insert 16 and 17 are expressed as a polycistronic mRNA. Expression of 16 uses reinitiation of translation through the IRES sequence. Polyadenylation is provided by the SV40 poly A signal.

DETAILED DESCRIPTION

Definitions

[0033] The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure. [0034] The phrase "amino acid" refers to any of the twenty naturally occurring amino acids as well as any modified amino acids. Amino acid sequences can be modified through natural processes such as posttranslational processing, or can include chemical modifications which are known to those skilled in the art. Modifications include, but are not limited to: phosphorylation, ubiquitination, acetylation, amidation, glycosylation, covalent attachment of flavin, ADP-ribosylation, cross-linking, iodination, methylation, etc. [0035] "Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers, which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations, employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0036] "Expression" refers to transcription and translation occurring within a host cell. The level of expression of a DNA molecule in a host cell can be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of DNA molecule encoded protein produced by the host cell (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88).

[0037] The terms "fusion protein" and a "fusion polypeptide" refer to a polypeptide having two portions covalently linked together, where each of the portions is a polypeptide having a different property. The property may be a biological property, such as activity in vitro or in vivo. The property may also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, etc. The two portions may be linked directly by a single peptide bond or through a peptide linker containing one or more amino acid residues. Generally, the two portions and the linker will be in reading frame with each other.

[0038] The phrase "gene of interest" refers to all or a portion of viral antigens such as, for example, HIV, HCV, HBV, HCMV, EBV, etc., and tumor antigens such as, for example, mutated Ras, inosine monophosphate dehydrogenase II (IMPDH2), melanoma antigens, etc. [0039] The phrase "genetically engineered" refers to any recombinant DNA or RNA method used to create a eukaryotic host cell that expresses a target protein at elevated levels, at lowered levels, or in a mutated form. In other words, the host cell has been transfected, transformed, or transduced with a recombinant polynucleotide molecule and thereby altered so as to cause the cell to alter expression of the desired protein. Methods and vectors for genetically engineering host cells are well known in the art; for example, various techniques are illustrated in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons,

New York, 1988, and quarterly updates). Genetically engineering techniques include but are not limited to expression vectors, targeted homologous recombination, and gene activation

(see, for example, U.S. Patent No. 5,272,071 to Chappel) and trans activation by engineered transcription factors (see, for example, Segal et al., 1999, Proc. Natl. Acad. Sci. USA 96(6):

2758-2763).

[0040] "Homology" refers to a degree of complementarity between polynucleotides.

Complementarity affects the efficiency and strength of hybridization between polynucleotide molecules.

[0041] The term "heterologous" as used herein refers to a viral protein that corresponds to a viral protein from a different source than the HTLV-II genome used in the viral vector. For example, the HTLV-II viral vector have the sequence of NC_001488; gl

9626726, and the sequence encoding the viral protein may correspond to that of another virus such as a different strain or isolate of HTLV-II, from a HTLV-I virus, from SIV, and/or from

HIV.

[0042] The phrases "host cell" and "host cells" refer to cells expressing a heterologous polynucleotide molecule. Host cells of the present invention are transfected, transformed, or transduced with the vectors described herein. Examples of suitable host cells useful herein include, but are not limited to, insect cells, mammalian cells, bacterial cells, fungal cells, plant cells, etc.

[0043] The phrase "immunogenic composition" refers to a composition comprising viral vector embodiments or tumor antigen embodiments as described herein that include a polypeptide to elicit an immune response in a subject.

[0044] "Isolated" refers to a polynucleotide or polypeptide separated from at least one contaminant (polypeptide or polynucleotide) with which it is normally associated.

Illustratively, an isolated polynucleotide or polypeptide is in a context or a form different from that in which it is found in nature. [0045] The term "immunogenic effective amount" of a HTLV-II viral vector refers to an amount of the vector that induces an immune response in an animal. The immune response may be determined by measuring a T or B cell response. Typically, the induction of an immune response is determined by the detection of antibodies specific for heterologous viral protein encoded by the HTLV-II viral vector.

[0046] The phrase "nucleic acid sequence" refers to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along a polypeptide chain. The deoxyribonucleotides sequence thus codes for the amino acid sequence. [0047] "Polynucleotide" refers to a linear sequence of nucleotides. The nucleotides may be ribonucleotides, or deoxyribonucleotides, or a mixture of both. Examples of polynucleotides in the context of the present invention include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. The polynucleotides of the present invention can contain one or more modified nucleotides.

[0048] The words "protein", "peptide", and "polypeptide" are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers.

[0049] "Percent (%) nucleic acid sequence identity" with respect to the nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. In some embodiments, the reference HTLV-II nucleic acid sequence is that of NC_001488; gl 9626726. In other embodiments, the HTLV-II nucleic acid sequence is produced synthetically. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. [0050] Several illustrative sequences are included throughout the specification, claims, and Figures. It is understood by those skilled in the art that conservative changes can be made to any one of these sequences without altering the function of the polynucleotide sequence or the polypeptide encoded by the polynucleotide sequence. Thus, a polynucleotide sequence useful as described herein may be represented wholly or in part by the sequences provided herein. Further, it is understood that a polynucleotide sequence useful as described herein can, in some embodiments, have at least about 65% to about 100% sequence identity, including all numbers in between 65% and 100%, to its respective sequence provided herein. For example, an IRES sequence in any particular vector can have at least about 80% sequence identity to the IRES sequence provided herein. In some embodiments, a sequence has at least 90% sequence identity, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater sequence identity to that of the reference nucleic acid sequence.

[0051] For purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence A to, with, or against a given nucleic acid sequence B (which can alternatively be phrased as a given nucleic acid sequence A that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence B) is calculated as follows:

100 times the fraction W/Z where W is the number of nucleotides scored as identical matches by the sequence alignment program in that program's alignment of A and B, and where Z is the total number of nucleotides in B. It will be appreciated that where the length of nucleic acid sequence A is not equal to the length of nucleic acid sequence B, the % nucleic acid sequence identity of A to B will not equal the % nucleic acid sequence identity of B to A.

[0052] "Purify" or "purified" refers to a target protein that is free form at least about

5% to 10% of contaminating proteins. Purification of a protein from contaminating proteins can be accomplished using known techniques, including ammonium sulfate or ethanol precipitation, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Various protein purification techniques are illustrated in Current Protocols in Molecular Biology, Ausubel et al. eds. (Wiley & Sons, New York, 1988, and quarterly updates).

[0053] "Recombinant" refers to a polynucleotide that has been isolated and/or altered by the hand of man such as a HTLV-II viral vector as described herein. A DNA sequence encoding all or a portion of a HTLV-II viral genome may be isolated and combined with a polynucleotide encoding a viral protein from a heterologous virus to form a viral vector. The vector provides for introduction into host cells and amplification of the polynucleotide. [0054] The term "replicable vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked into a cell and providing for amplification of the nucleic acid. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0055] "Variants" of the HTLV-II viral genome refer to a sequence of a viral genome that differs from a reference sequence and includes "naturally occurring" variants as well as variants that are prepared by alteration of one or more nucleotides. In some embodiments, when the viral genome has the sequence of a naturally occurring isolate, the reference sequence may be human HTLV-II (GenBank accession number NC_001488; gl 9626726), and the variant has at least 90% sequence identity to the reference sequence. In other cases, a variant may be prepared by altering or modifying the nucleic acid sequence of the viral genome including by addition, substitution, and deletion of nucleotides. In some embodiments, the variant sequence has a deletion of all or a part of the untranslated region in the pX region of the HTLV-II genome. In some embodiments, a HTLV-II genome has at least 90% sequence identity, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% or greater sequence identity to that of a HTLV-II genome comprising a nucleic acid sequence of GenBank: accession number NC_001488; gl 9626726. Table 3.

I. VIRAL VECTORS AND COMPOSITIONS

[0056] A first aspect of the disclosure relates to recombinant viral vectors and host cells containing the recombinant vectors, as well as methods of making such vectors and host cells by recombinant methods. The viral vectors encoding one or more viral proteins from a heterologous virus are useful as immunogenic compositions to inhibit viral infection and/or to limit the effects of a viral infection. The viral vectors can also be used to encode one or more tumor antigens for use in treatment of various tumor lesions.

[0057] The disclosure, in accordance with embodiments herein, describes an isolated viral vector comprising at least a portion of the HTLV-II genome encoding two HTLV-II retroviral long terminal repeat (LTR) sequences. The 5' and 3' LTR sequences as disclosed herein can include the full LTR sequence or any functional portion of either or both sequences. In some embodiments, a 5' or 3' LTR sequence has at least 90% sequence identity, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater sequence identity to that of the HTLV-II 5' and 3' LTR, respectively, nucleic acid sequence.

[0058] The two LTR sequences encompass one or more polynucleotides, each independently encoding all or a portion of a protein that corresponds to a gene of interest such as a viral protein from a heterologous virus. The LTR sequences drive gene expression and allow integration of the sequence into the host DNA. In some embodiments, one or more of the polynucleotides each independently encoding a gene of interest are located near the 3'LTR and positioned in reverse orientation, and thus expressed from the 3'LTR. Also positioned in the reverse orientation can be an internal ribosome entry site (IRES) and a polyadenylation (polyA) signal.

[0059] Further encompassed by the two LTR sequences and positioned in the forward direction can be one or more of the following: HTLV-II gag ecapsidation signal (and PPT), a DNA FLAP for efficient nuclear import, a promoter such as a CMV promoter, a marker such as green fluorescent protein gene (for example, Chiridius poppei cpGFP mRNA for GFP gene) or other like marker gene, an IRES (internal ribosome entry site) sequence, a polyA signal, a constitutive transport element (CTE) from any simian type D retrovirus, and an HTLV-II sequence (to protect the 3'LTR from methylation).

[0060] In some embodiments, a functional vector comprises an HTLV-II 5'LTR,

HTLV-II gag encapsidation signal, one or more IRES sequences, one or more SV40 polyA signal sequences, CTE, and an HTLV-II 3'LTR. Other embodiments include one or more of the following: DNA FLAP, CMV promoter, CopGFP, and 3' HTLV-II sequence (to protect the 3'LTR from methylation).

[0061] The recipient vector can be a synthetic nucleotide sequence containing an origin of replication (ORI), for example, a bacterial ORI, and a selection marker, for example, a bacterial selection marker such as Amp. SEQ ID NO: 205 is an exemplary plasmid sequence containing an ORI, an ampicillin resistance marker, and a bacterial promoter. In some embodiments, a signal to reduce plasmid copy number to improve vector stability can be inserted into the recipient vector.

[0062] The HTLV-II gag signal is an exemplary encapsidation signal. Additional encapsidation signals can include, for example, HIV encapsidation signal, HTLV-I encapsidation signal, and other retroviral encapsidation signals. Additional encapsidation signals can include other human or animal retrovirus used with a corresponding reverse transcriptase and gag proteins provided in trans by a helper virus (of the same origin) to ensure encapsidation and transcription of the vector.

[0063] The DNA FLAP sequence can be derived from an animal or human lentivirus and allows for integration of the genetic material into the genome of non-dividing cells, increasing nuclear import of the vector.

[0064] Exemplary promoters include, but are not limited to, CMV promoters, as well as other cell specific viral and cellular promoters. Illustrative cell specific promoters include

CD 19 for B -cells and CD 14 for macrophages. An inducible promoter such as tetracycline, ecdysone, or other promoter can be used instead of a CMV promoter to control timing and expression levels of genes placed in the forward orientation.

[0065] Exemplary markers include, but are not limited to, luciferin, green fluorescent protein, yellow fluorescent protein, red fluorescent protein, blue fluorescent protein, etc.

[0066] An IRES sequence allows for translation initiation in the middle of an mRNA sequence.

[0067] The CTE allows incompletely spliced RNA to be transported out of the nucleus to the cytoplasm. Other RNA transfer sequences are contemplated herein, including

RNA transfer sequences encoded by other retroviral genomes such as HTLV-I and HIV.

[0068] SV40 polyA signal sequence can be added after the gene of interest resulting in addition of a polyA tail to the 3' end of the mRNA. The polyA tail is involved in nuclear transport, translation, and stability of the mRNA. Thus, when the gene of interest is read from the 5'LTR, the SV40 polyA signal sequence is in the forward direction in the top strand of the DNA. Likewise, when the gene of interest is read from the 3'LTR, the SV40 polyA signal sequence is in the reverse direction in the bottom strand of the DNA.

[0069] Just before the HTLV-II 3'LTR is an HTLV sequence used to protect the 3'

LTR from methylation, thus preserving functionality of the LTR. This HTLV sequence can be of any length useful in minimizing or preventing methylation, from as few as 1 nucleic acid to as many as 500 nucleic acids. In some embodiments, a BLV sequence or an STLV sequence can be used in place of the HTLV sequence. [0070] Both LTRs are orientated 5' to 3' in the forward direction of the upper DNA strand. Both LTRs are required for encapsidation. The 5' LTR has the ability to drive expression of polynucleotides located in the forward orientation of the upper DNA strand. However, the 3'LTR sequence is able to drive expression of polynucleotides in the reverse orientation form the lower DNA strand.

[0071] Transcription from the vector is depicted as shown in Figure 23. All transcripts in this embodiment are made using the cellular transcription machinery. A. Initiation of transcription in the R region of the 5' LTR transcript finishes in the R region of the 3' LTR and uses the polyadenylation site of the 3'LTR. This RNA will not express polynucleotide sequences, provides template to be encapsidated and will reconstitute the vector following infection of target cells. B. Expression from the transcriptional start site within the CMV promoter. RNA is polycistronic and encompasses copGFP and first insert (15). Expression of the insert is achieve by reinitiation of translation through the presence of the IRES sequence. The SV40 poly A signal provides polyadenylation. C. Expression from the R region of the 3' LTR in reverse orientation. Insert 16 and 17 are expressed as a polycistronic mRNA. Expression of 16 uses reinitiation of translation through the IRES sequence. Polyadenylation is provided by the SV40 poly A signal.

[0072] In some aspects, it is desirable to place a gene of interest near the 5'LTR or to place a gene of interest near the 3'LTR. Genes at the 5'LTR are oriented in a forward direction and genes at the 3'LTR end are oriented in the reverse direction. 5'LTR driven expression is limited to about several weeks to about 4 or 5 months, while the 3'LTR driven expression can last the life time of the host cell. Thus, for example, it can be desirable to place a gene of interest such as a viral antigen or tumor antigen near the 3'LTR and on the lower DNA strand for expression in the reverse orientation. It can also be desirable, for example, to place an adjuvant cassette near the 5'LTR and on the top strand DNA for expression in the forward direction. However, in some embodiments, it can be desirable to place certain adjuvants such as cytokines near the 3'LTR for longer term expression. [0073] An exemplary embodiment of a vector is shown in Figure 11. The vector map illustrates the order in which the vector components in one embodiment can be assembled: (1) HTLV-II 5' LTR; (2) HTLV-II gag Ψ signal for encapsidation; (3) DNA FLAP; (4) CMV promoter; (5) Chiridius poppei cpGFP mRNA for green fluorescent protein (GFP) gene; (6) internal ribosome entry site (IRES) sequence and multiple cloning site 1 (MCS 1); (7) SV40 Poly A signal; (8) constitutive transport element (CTE) for RNA nuclear export; (9) SV40 Poly A signal (reverse orientation); (10) vaccine gene or sequence of interest to be expressed from 3' LTR; (11) IRES and MCS 2 in the reverse orientation; (12) vaccine gene or sequence of interest to be expressed from 3' LTR; (13) HTLV sequence required to protect 3' LTR from methylation and MCS 3; and (14) HTLV-II 3' LTR (See Table 6).

[0074] The MCS sequences are segments of DNA that contain restriction sites as described herein or any other cloning sites allowing insertion of the gene of interest into the vector. In the embodiment illustrated by Figure 11 and Table 6, there are three MCS with the following restriction sites: MCS 1: BamHl-Xhol-EcoRl-Hindlll; MCS 2: Sall-Notl- Mlul-BstEII; and MCS 3: BstXl-EcoRV- SphI-TthIII-1. The MCS sequences are read from the top strand (referring to Table 6 and Figure 11), however, in this embodiment, the genes of interest are inserted into the MCS 2 and MCS 3 sites in the reverse orientation. Thus, these two genes of interest are read from the bottom strand and in the reverse direction. [0075] In some embodiments, it is contemplated that a cassette capable of expressing biological adjuvant can be included in the vector. Such adjuvants can be selected from toll receptor molecules, cytokines, anti-apoptotic genes, RNAs, and other modulators of immune response such as viral proteins that affect TCR or MHC class 1. In one embodiment, the cassette is encompassed within the HTLV-II 5'LTR and HTLV-II 3'LTR. [0076] It is important to note that it is within the knowledge of one skilled in the art to reorder vector components in such a manner that the vector is functional. [0077] The disclosure in accordance with embodiments herein also describe an isolated viral vector comprising at least a portion of the HTLV-II genome encoding the gag, pro, and pol genes and lacking all or a portion of the pX region; and a polynucleotide encoding all or a portion of a protein that corresponds to a viral protein from a heterologous virus. To limit redundancy the following disclosure is focused on viral proteins, however, tumor antigens can be inserted in embodiments herein in a similar matter. In some embodiments, the polynucleotide encoding the viral protein from the heterologous virus is located within the deletion in the pX region. The HTLV-II proviral genome has about 8952 nucleotides and includes regions coding for the structural proteins (gag and env) and the viral protease and polymerase (pol), similar to HIV-I. The genome also contains an additional sequence designated pX adjacent to the envelope (env) gene. Four open reading frames (ORFs), designated pX-I, pX-II, pX-III, and pX-IV, were initially identified within the HTLV-I pX. The second coding exon of the transacting viral proteins Tax and Rex are expressed from the overlapping ORFs pX-IV and pX-III, respectively, and are translated from a doubly spliced, bicistronic mRNA. ORFs x-I and x-II contained in the proximal portion of pX have 655 base pairs (bp) and 573 bp for HTLV-I and HTLV-II, respectively, and have been referred to as the nontranslated or untranslated (UT) region. The location of the pX region can be determined by aligning the HTLV-II sequence to that of a reference sequence such as provided in Table 3.

[0078] The HTLV-II genome sequence can be obtained from a naturally occurring isolate. Infected individuals can be identified by the detection of antibodies to HTLV-II polypeptides, or by PCR. The virus may be isolated from lymphoid tissue. Several isolates are known including those that have the sequences as provided in NC_001488, M10060, L20734, Y14365, Af326583, and AO26584. Covas et al, AIDS Research and Human Retroviruses 19:689 (2003) incorporated herein by reference for all purposes. The HTLV genome sequence from a naturally occurring isolate or strain can be modified to form a variant sequence. These HTLV-II genomic variants are useful as HTLV-II viral vectors. [0079] In some embodiments, when the viral genome has the sequence of a naturally occurring isolate, the reference sequence may be human HTLV-II (GenBank accession number NC_001488; gl 9626726), and the variant has at least 90% sequence identity to the reference sequence. In other cases, a variant may be prepared by altering or modifying the nucleic acid sequence of the viral genome including by addition, substitution, and deletion of nucleotides. In some embodiments, the variant sequence has a deletion of all or a part of the pX region of the HTLV-II genome. In some embodiments, a HTLV-II genome has at least about 65% to about 100% sequence identity, including all numbers in between 65 and 100%. For example, if up to 3000 nucleotides are deleted and replaced, the HTLV-II variant genome would have 67% identity to the reference sequence. In some embodiments, a HTLV-II genome has at least 90% sequence identity, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater sequence identity to that of a HTLV-II genome comprising a nucleic acid sequence of GenBank accession number NC_001488; gl 9626726. Table 3.

[0080] In some embodiments, the HTLV-II viral vector is formed with a deletion of all or a portion of the pX region. In some embodiments, this can involve a deletion of all or a portion of nucleotides starting at about nucleotide position 6000 to about 8500, 6000 to 7500, or 6000-7000. In some embodiments, all or a portion of the untranslated region of pX is deleted. In some embodiments, at least 30 to 2500 nucleotides are deleted in the pX region including every number in between 30 and 2500. In other embodiments, at least 30, 100, 200, 300, 400, 500 and up to 1500 nucleotides are deleted in the pX region. [0081] In a specific embodiment, the nucleotide sequence between Pst sites at nucleotide 6661 and 6984 or 6645 and 7153, sequence on Table 3, is deleted. In other embodiments, the 3' region after the tax coding sequence or the pi 1 coding sequence is deleted as shown in Figure 10. In other embodiments, the polynucleotide sequence 3' to the Tax coding sequence and the internal ribosome entry site (IRES) is deleted. [0082] In some embodiments, the HTLV-II variant genome encodes at least the gag, pro, pol proteins. In other embodiments, the HTLV-II viral vector genome comprises polynucleotide sequence encoding gag, pro, pol, and one or more of env, tax, and rex. In some embodiments, the HTLV-II viral vector does not encode any one of p28, plO and/or pi 1. In embodiments, the HTLV-II viral vector can replicate in a human and/or macaque cell, however at a reduced level as compared to a wild type HTLV-II. In some embodiments, the viral vector is attenuated at least about 2 to 10 fold as measured by the production of viral protein. Another criterion for selection is that the HTLV-II viral vector can establish a low level of infection, especially in the gut tissue.

[0083] The HTLV-II viral vector comprises a polynucleotide encoding all or a portion of a gene of interest. In some aspects, and as discussed previously, the polynucleotide encodes all or a portion of a protein that corresponds to a viral protein from a heterologous virus. In other aspects, the polynucleotide encodes a tumor antigen to enhance immunity against a tumor, for example MAGE for melanoma. Further, in some embodiments the viral vector comprises a tumor antigen that will be introduced into dendritic cells and then administered to a subject in need.

[0084] Illustratively, the HTLV-II viral vector comprises a polynucleotide encoding all or a portion of a protein that corresponds to a viral protein from a heterologous virus. A heterologous virus is a virus that is a different source virus. In some embodiments, the heterologous virus is HTLV-I, SIV or HIV. In some embodiments the protein corresponds to a viral protein from a heterologous virus. In such cases, the protein may be artificially constructed based upon a consensus sequence for the viral protein from a number of strains or isolates. The protein may be a consensus sequence from several clades for any one of the viral proteins selected from the group consisting of Vif, Tat, Gag, Env, Rev, gpl20, gp41, p24, p7, p 17, tev, and combinations thereof or immunogenic fragments thereof. In other embodiments, the viral protein is selected from the group consisting of Vif, Tat, Gag, Env, Rev, gpl20, gp41, p24, p7, pl7, tev, and combinations thereof or immunogenic fragments thereof. In some embodiments, the size of the polynucleotide encoding the viral protein will be such that it does not interfere with the ability of the viral vector to replicate in human or macaque cells. In some embodiments, a fragment of viral protein of about 100 amino acids or less will be inserted within the site of the deletion.

[0085] With regard to immunogenic fragments of the viral protein, some are known to those of skill in the art or can be readily identified by binding to neutralizing antibodies, or to immune sera. Those peptides that elicit a CD4 CD8 T cell response can be determined by standard methods as described herein. Typically an immunogenic fragment includes at least 8 amino acids up to the full length protein. Epitopic regions can also be determined using software that can predict epitopes such as is available through Epivax.

[0086] In addition, other modifications may be made to the HTLV-II viral vector. For example, the viral protein may be modified to contain a heterologous protein sequence useful for purification or identification of the viral protein. Examples of additional amino acids include peptide tags that may be added to the polypeptide to facilitate detection and/or purification. Such peptide tags include, but are not limited to, His, HA, Avi, biotin, c-Myc, VSV-G, HSV, V5, or FLAG™. Examples of a polypeptide that can enhance immunogenicity include bovine serum albumin, and/or keyhole lymphocyte hemocyanin (KLH). In addition the polynucleotide sequence may be altered in order to provide unique restriction sites to facilitate cloning of polynucleotides encoding heterologous sequences or to serve to identify the HTLV-II viral vector from other viruses such as naturally occurring variants.

[0087] The polynucleotides encoding a viral protein from a heterologous virus can be produced by standard recombinant methods known in the art, such as polymerase chain reaction (PCR) or reverse transcriptase PCR (Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual, VoIs. 1-3, Cold Spring Harbor Press, Cold Spring Harbor, NY), or the DNA can be synthesized and optimized for expression in bacteria or eukaryotic cells. Primers can be prepared using the polynucleotide sequences that are available in publicly available databases. The polynucleotide constructs may be assembled from polymerase chain reaction cassettes sequentially cloned into a vector containing a selectable marker for propagation in a host. Such markers include but are not limited to dihydrofolate reductase, puromycin, hygromycin or neomycin resistance for eukaryotic cell culture and tetracycline, ampicillin, or kanamycin resistance genes for culturing in host cells.

[0088] Representative examples of appropriate hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces and Salmonella typherium, fungal cells such as yeast; insect cells such as Drosophilia S2 and Spodoptera Sf9, animal cells such as CHO, COS, lymphoid cells and cells lines, and plant cells. Appropriate culture medium and conditions for the above-described host cells are known in the art. [0089] The polynucleotide should be operably linked to an appropriate promoter, such as CMV. Other suitable promoters are known in the art. The expression constructs may further contain sites for transcription initiation, transcription termination, and a ribosome binding site for translation. The coding portion of the mature polypeptide expressed by the constructs preferably includes a translation initiating codon at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0090] HTLV-II viral vectors can be prepared using standard methods. The genomic sequences can be obtained from naturally occurring isolates or can be obtained by PCR amplification using primers as described in Covas et al., cited supra and incorporated by reference herein for all purposes. HTLV-II viral vectors can also be prepared synthetically. Deletion mutants can be constructed according to standard methods using restriction enzymes to remove polynucleotides followed by ligation as described in Green et al., J Vir 69:387 (1995), incorporated by reference herein for all purposes.

[0091] The polynucleotide sequence encoding the gene of interest, for example, a viral protein from a heterologous virus, is inserted into the region of the deletion in the HTLV viral vector. The insertion may be conducted via standard methods such as ligation of a restriction fragment, cassette mutagenesis or other like methods. Replication of the modified viral vector in lymphoid tissue can be determined by introducing the vector into a host cells and detecting production of viral proteins as described herein. Lymphoid cells, such as Jurkat T cells or B cells such as 729 or 293 cells, may be useful in the large scale preparation of the HTLV-II viral vectors described herein.

[0092] Introduction of the recombinant vector into the host cell can be effected by injection, by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid- mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in standard laboratory manuals such as Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual, VoIs. 1-3, Cold Spring Harbor Press, Cold Spring Harbor, NY or Davis et al., 1986, Basic Methods in Molecular Biology. Commercial transfection reagents, such as Lipofectamine (Invitrogen, Carlsbad, CA), Effectene (Qiagen, Valencia, CA) and FuGENE 6™ (Roche Diagnostics, Indianapolis, IN), are also available. [0093] In some embodiments, the disclosure includes a composition comprising an

HTLV-II vector comprising at least a portion of the HTLV-II genome encoding the gag, pro, and pol genes and lacking all or a portion of the pX region and a polynucleotide encoding all or a portion of a protein that corresponds to a viral protein from a heterologous virus located within the deletion in the pX region, and a carrier.

[0094] In some embodiments, the disclosure includes a composition comprising an

HTLV-II vector comprising two HTLV-II retroviral LTR encompassing, in reverse orientation and expressed from the 3'LTR, HTLV-II sequence and one or more genes of interest, and a carrier.

[0095] In some embodiments, the composition includes at least two different viral vectors, wherein each of the viral vectors comprises a polynucleotide encoding a different viral protein. For example, one HTLV-II vector may encode a fragment of the HIV gag and another, a fragment of HIV env from the same or different clades. The different viral proteins may correspond to the viral proteins from the same source virus or different viruses. In embodiments, the composition comprises a plurality of different HTLV-II viral vector each encoding a different viral protein.

[0096] In some embodiments, the viral vector is administered in a host cell. For example, a viral vector may be introduced into lymphoid lineage stem cells, or dendritic cells, preferably autologous dendritic cells. In some cases, the host cells are irradiated before administration. The HTLV-II viral vector can be introduced into the dendritic cells and the presence of viral infection in the absence of any viral replication detected before the cells are administered to the subject. Protocols for administration of dendritic cells are known in the art for the treatment of cancer.

[0097] In some embodiments, the compositions of the disclosure comprise an immunogenic effective amount of the HTLV-II viral vector. An immunogenic effective amount is an amount of polynucleotide that induces an immune response to the encoded polypeptide when administered to a host, for example an animal. The actual amount of the immunogenic composition may vary depending on the animal to be immunized, the route of administration and adjuvants. Immunogenic dosages can be determined by those of skill in the art, for example, based on other viral vectors that have been used in clinical trials. The immune response can be humoral, cellular, or both.

[0098] Immune responses can be measured as antibody responses and/or T cell responses. Antibody responses can be measured using ELISA, western blot or other like assay. In addition, antibody responses to the heterologous viral protein can be measured for the capability to participate in ADCC. An exemplary ADCC procedure involves PHA stimulated primary human CD4 blasts and primary human monocytes effectors at a ratio of E:T of 1:1. Plasma samples are added at a final dilution of 1:100. The CD4⁺ lymphocytes are infected with a HIV or SIV virus isolate at a multiplicity of infection of 0.5. The infected cells are then incubated with plasma from immunized and control animals. The cytotoxicity of the cells can then be determined. The production of cytokines and/or chemokines can also be determined when lymphoid cells from animals inoculated with HTLV-II vectors as described herein are assayed as described herein using standard methods. [0099] An embodiment provides an immunogenic composition according to the present disclosure also including immunomodulators such as cytokines or chemokines. In some embodiments, a nucleic acid encodes the immunomodulator or adjuvant. Immunomodulators refers to substances that potentiate an immune response including, but not limited to cytokines and chemokines. Examples of cytokines include but are not limited to IL-2, IL-15, IL-12, or GM-CSF.

[00100] An embodiment provides an immunogenic composition further comprising an adjuvant. Such adjuvants may include ganglioside receptor-binding toxins (cholera toxin, LT enterotoxin, their B subunits and mutants); surface immunoglobulin binding complex CTAl- DD; TLR4 binding lipopolysaccharide; TLR2 -binding muramyl dipeptide; mannose receptor-binding mannan; dectin-1 -binding ss 1,3/1,6 glucans; TLR9-binding CpG- oligodeoxynucleotides; cytokines and chemokines; antigen-presenting cell targeting ISCOMATRIX and ISCOM. Adjuvants such as lipids (fatty acids, phospholipids, Freund's incomplete adjuvant in particular), Vaxfectin, polaxomer, anionic copolymers, CpG units, etc. may be added to the composition. In addition, adjuvants able to prime the mucosal immune system following a systemic immunization, include 25(OH)2D3, cholera toxin, CTAl-DD alone or in combination with ISCOM. In some embodiments, the adjuvant may be encoded or expressed by the expression vector used herein.

[00101] In some embodiments, the compositions of the disclosure comprise an amount that is effective to inhibit viral infection and/or replication of the heterologous virus. An animal model may be available to determine the dosage that provides for a decrease in viral replication and/or infection. For example, a HTLV-II viral vector encoding a SIV gag polypeptide can be tested for the ability to inhibit SIV viral infection and/or replication in the macaque.

[00102] The compositions of the invention can also include a carrier. Carriers include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or animal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™ polyethylene glycol (PEG), and PLURONICS™. [00103] The compositions of the invention can be in the form of sterile injectable preparations, such as sterile injectable aqueous or oleagenous suspensions. For administration as injectable solutions or suspensions, the immunogenic compositions can be formulated according to techniques well-known in the art, using suitable dispersing or wetting and suspending agents, such as sterile oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

[00104] In one embodiment, packaging of HTLV-II vectors of the invention will be accomplished by transient transfection along with a helper virus (providing in trans structural and enzymatic protein required for encapsidation, reverse transcription and integration of HTLV-II origin or any other compatible helper vector), and an envelope expressing vector (that can but not necessarily be of HTLV-II origin. HTLV-II envelope may be substituted to that of VSV-G or any other virus in order to achieve delivery into a specific cell type.). [00105] Transfection could be accomplished in human fibroblasts 239T cell line or any other transfectable cell line able to produce high titer infectious pseudotype particles. Pseudotype virus particles will be purified by ultracentifugation or the use of PEG-based resin or any other means to concentrate virions and used to infect ex- vivo cells that could be reimplanted (transferred) in the patients. Alternatively purified particles could be injected directly in vivo into the peripheral blood or directly to specific immunological sites.

II. USES AND METHODS

[00106] The present disclosure is also directed to uses and methods for administering a composition in accordance with embodiments of the present invention to an animal, including a human or other mammal, birds (including but not limited to chickens, ducks, geese, etc.), fish, etc. As described herein, an animal that requests or requires administration of a viral vector or composition in accordance with the present invention is referred to herein as a subject. In some embodiments, the composition is an immunogenic composition designed to induce an immune response to the viral protein from the heterologous virus. In other embodiments, the composition is used to inhibit or decrease viral infection and/or replication of the heterologous virus in the subject. In still other embodiments, the composition is an immunogenic composition designed to induce an immune response to a tumor antigen for the treatment of cancer in a subject in need thereof.

[00107] In an embodiment, the method comprises administering to a subject an immunogenic effective amount of an immunogenic composition. An immunogenic effective amount is an amount of polynucleotide or other vector that induces an immune response to the encoded polypeptide when administered to the subject. In another embodiment, the polynucleotides are incorporated into the subject's host cells in vivo, and an immunogenic effective amount of the encoded polypeptide or fragment thereof is produced in vivo. In some cases, the HTLV-II viral vector will establish an infection in lymphoid tissue, and particularly in gut associated lymphoid tissue. The actual amount of the immunogenic composition may vary depending on the subject to be immunized, the route of administration, adjuvants, and other like concerns.

[00108] Immunogenic dosages can be determined by those of skill in the art. The immune response may be indicated by T and/or B cell responses. Typically, the immune response is detected by the presence of antibodies that specifically bind to the viral polypeptide. Methods of detecting antibodies are known to those of skill in the art and include such assays as enzyme-linked immunosorbent assays (ELISA), western blot assays, and competition assays. Methods of detecting T cell responses include ELISPOT assays, ICS assays, CD4 CD8 flow cytometry assays, and cytotoxicity assays. [00109] In yet another embodiment, a subject is immunized with an immunogenic composition of the invention and then boosted one or more times with the immunogenic composition. In an embodiment, the subject is boosted about 2 to about 4 weeks after the initial administration of the immunogenic composition. If the subject is to be boosted more than once, there is typically about a 2 to 12 week interval between boosts. In an embodiment, the subject is boosted at about 12 weeks and about 36 weeks after the initial administration of the immunogenic composition. In another embodiment, the subject is a mouse and the mouse is boosted 3 times at 2 week intervals. In yet another embodiment, the subject is a primate and the primate is boosted 1 month and 6 months after the initial administration of the immunogenic composition. The dose used to boost the immune response can include one or more cytokines, chemokines, or immunomodulators not present in the priming dose of the immunogenic composition.

[00110] The methods of the invention also include prime-boost immunization methods utilizing the immunogenic compositions of the invention. The subject may be primed 1 to 8 times. Typically there is a 1, 2, or 3 week interval between administrations. In an embodiment, the subject is primed 3 times at 2 week intervals. The primed subject may then be boosted with the corresponding polypeptide or polypeptides. In an embodiment, the subject is boosted with the polypeptide at least 2 weeks after the last dose of the first viral vector. The dose used to boost the immune response can include one or more cytokines, chemokines, immunomodulators, or other antigens not present in the priming dose. [00111] The methods of the invention also include methods for inhibiting virus infection and/or replication. In some embodiments, the method of the disclosure provides for protective immunity against an infection with the virus that corresponds to the heterologous viral protein that is encoded in the HTLV-II vector embodiments described herein. Inhibition of viral infection and/or replication of the heterologous virus can be determined using detection methods for pro viral DNA or viral antigens (such as by PCR). [00112] Any mode of administration can be used in the methods so long as the mode results in the expression of the desired peptide or protein, in the desired tissue, in an amount sufficient to generate an immune response and/or to inhibit viral infection and/or replication of the heterologous virus. The immunogenic compositions of the invention can be administered by intramuscular (Lm.), subcutaneous (s.c), sublingual, oral, or intrapulmonary route in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, or vehicles. Other suitable routes of administration include, but are not limited to intratracheal, transdermal, intraocular, intranasal, inhalation, intracavity, and intravenous (i.v.) administration. Transdermal delivery includes, but is not limited to intradermal, transdermal, and transmucosal administration. Intracavity administration includes, but is not limited to administration into oral or nasal cavities. The immunogenic compositions can be coated onto particles or nanofibers for delivery or formulated in liposomes.

[00113] Administration modes of the present invention include needle injection; catheter infusion; biolistic injectors; particle accelerators such as, for example, "gene guns" or pneumatic "needleless" injectors such as Med-E-Jet (Vahlsing et al., 1994, J. Immunol. Methods, 171:11-22), Pigjet (Schrijver et al., 1997, Vaccine, 15:1908-1916), Biojector (Davis et al., 1994, Vaccine, 12: 1503-1509; Gramzinski et al., 1998, MoI. Med., 4:109-118), AdvantaJet (Linmayer et al., 1986, Diabetes Care, 9:294-297), or Medi-jector (Martins and Roedl, 1979, Occup. Med., 21:821-824 ); gelfoam sponge depots; biodegradable nanoparticles slowly releasing immunogenic compositions as they degrade in vivo; other commercially available depot materials such as, for example, hydrogels, osmotic pumps, oral or suppositorial solid (tablet or pill) pharmaceutical formulations, topical skin creams, and decanting, polynucleotide coated suture (Qin, Y. et al., 1999, Life ScL, 65:2193-2203), or topical applications during surgery. Certain modes of administration are intramuscular needle-based injection and pulmonary application via catheter infusion. Energy-assisted plasmid delivery (EAPD) methods may also be employed to administer the compositions of the invention. One such method involves the application of brief electrical pulses to injected tissues, a procedure commonly known as electroporation. See generally Mir et al., 1999, Proc. Natl. Acad. Sci USA, 96:4262-7; Hartikka et al., 2001, MoI. Ther., 4:407-15; Mathiesen, 1999, Gene Ther., 6:508-14; Rizzuto et al., 2000, Hum. Gen. Ther. 11:1891-900. [00114] In some embodiments, the subject may be screened for the presence of, for example, HTLV-II infection or proviral DNA prior to administration of the compositions of the disclosure using methods as described herein. Sera from the subjects can be screened using western blot strips or other methods for detecting antibodies to HTLV-II protein. Proviral DNA can be detected using PCR. In some cases, if the subject is positive for HTLV- II infection, it may be desirable to not administer certain compositions described herein. [00115] In some embodiments, the subject may require treatment for a cancerous lesion, for example the subject has ovarian cancer, breast cancer, melanoma or other like disease. The subject, in such case, can be treated using embodiments described herein, where the immunogenic composition induces an immune response against a tumor antigen associated with the tumor. For example, a subject with ovarian cancer may be treated with an immunogenic composition having a vaccine gene or sequence for expression of CA- 125, or a subject with melanoma may be treated with an immunogenic composition having a vaccine gene or sequence for melanoma- associated antigen (MAGE) or tyrosinase. Embodiments herein are directed toward induction of tumor specific cytotoxicity. Note that therapeutic immunity provided to treated subjects can be combined with other conventional cancer treatments, for example chemotherapy, radiation therapy, immunotherapy and the like. [00116] In aspects of the cancerous lesion treatments, tumor antigens can first be expressed in a subject's dendritic cells, (or T cells), and the tumor antigen expressing dendritic cells administered back to the subject. In some cases dendritic cells are harvested from the subject, as is known in the art, and grown in vitro in the presence of tumor antigens derived from the subject's tumor (or expressed or input from recombinant sources). In some embodiments, the tumor antigens are introduced into the autologous dendritic cells using vectors as described herein. The cultured dendritic cells are then administered back to the subject to induce a specific cell-mediated antitumoral cytotoxicity. The combined use of dendritic cells and vector expressed tumor antigens, including known tumor antigens provides an unexpected improvement in tumor cell cytotoxicity. As above, the dendritic cell treatments can be combined with other conventional cancer treatments, for example, chemotherapy, radiation therapy, other immunotherapy methods, and the like. Information related to the manipulation of dendritic cells is located in Sallusto et al., 2002, Arthritis Res., 4 Suppl 3:5127-32 doi 10.1186/ar567. PMID 12110131, incorporated by reference herein for all purposes.

[00117] The present disclosure is also directed to kits for practicing the methods of the invention. In some embodiments, the kit includes a viral vector of the invention, and instructions for administering to a subject (including human) and boosting the subject with a polypeptide. The kit may also include any of the compositions as described herein as well as the heterologous viral protein encoded by the viral vector. In some embodiments, for example, the kit may include reagents useful for screening subjects for prior infection with HTLV-II such as one or more of PCR primers and a probe for detecting HTLV-II proviral DNA in a sample from the subject; an antibody that specifically binds to a HTLV-II protein; or one or more HTLV-II proteins attached to a solid substrate. In some embodiments, the kit may further comprise at least one adjuvant or immunomodulator. The adjuvant or immunomodulator can be encoded by a polynucleotide. In a specific embodiment, the adjuvant is CTAl-DD alone or in combination with ISCOM.

[00118] All publications, patents, and patent applications cited herein are hereby incorporated in their entirety by reference.

EXAMPLES

[00119] The following examples are provided for illustrative purposes only, and are in no way intended to limit the scope of the present invention.

Example 1:

[00120] We have evidence that HTLV-II infects macaques and replicates at very low level in lymphoid tissue and particularly in the gut. We developed a macaque T cell line useful for testing the infectivity of HTLV-II vectors. MATERIALS AND METHODS Immortalization of Rhesus T Cell Line

[00121] Rhesus PBMCs obtained from a female animal (M304) were co-cultivated in a

1 : 1 ratio with the gamma- irradiated HTLV-II producing Mo-T cell line that was originated by a male patient with hairy cell leukemia (this cell line (Mo-T) was immortalized from a patient who had hairy cell leukemia). The patient was HTLV-II infected. (Kalyanaraman VJ Science 1982 p571-3) HTLV-II infection was documented by measuring pl9Gag production by antigen capture assay in the cell culture supernatant and by intracellular pi 9Gag staining. [00122] pl9Gag production was determined using a Zeptomatrix (Buffalo, NY) antigen capture ELISA according to the manufacturer's instructions. Briefly, micro wells coated with high affinity polyclonal antibodies form the capture phase of the assay. These antibodies react strongly with the major gag gene products of HTLV-I and HTLV-II. Viral antigen from the RhM304 cell line was captured by the antibody during the sample incubation step. Captured antigen reacts with an antibody which recognizes pl9 core protein of HTLV-I and HTLV-II. Specifically bound antibody was detected with peroxidase conjugated IgG and color is developed with 3, 31, 5, 51, tetra-methylbenzidine (TMB) as substrate. Resultant absorbance values were proportional to the amount of viral core antigen present in the test specimens.

[00123] Intracellular staining for viral antigens was performed using a mouse anti-pl9

Gag antibody (Zeptomatrix, Buffalo, NY). Briefly, cell pellets were fixed in 100 μl IC Fixation Buffer (eBioscience). Intracellular staining was performed in 100 μl of Permeabilization Buffer using a 1:250 dilution of the anti-pl9 Gag antibody. After incubation for 30 min in the dark at room temperature, cells were washed twice and stained with anti- mouse IgG Alexa-Fluor488 antibody (Invitrogen) for 30 min in the dark at room temperature. Next, cells were washed and resuspended in 1% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS. Four-parameter flow cytometry analysis was performed using CELLQuest software. List mode data files were analyzed using FlowJo software (Tree Star, Ashland, OR). [00124] A cell line derived from this culture was designated RhM304.

Cell Line Characterization

[00125] The RhM304 cell line was characterized for the presence of T cell surface antigens, karyotype, and the presence of HTLV-II proviral DNA transcripts. [00126] A phenotypic analysis for CD3, CD4, CD8 and CD19 was conducted by flow- cytometry.

[00127] Karyotypic analysis of RhM304 cells was obtained. The cultured cells were treated with colcemid, a drug that disrupts the mitotic spindle apparatus to prevent the completion of mitosis and arrests the cells in metaphase. The cells were fixed, dropped on a microscope slide, dried, and stained with Giemsa.

[00128] Analysis of proviral HTLV-II DNA copies was demonstrated by real-time

PCR using primers specific for the gag region. A brief description of the method follows.

Genomic DNAs from PBMCs or tissue samples were isolated by DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer's protocol except DNA elution step. DNA was eluted with 10 mM Tris (pH8.0). Genomic DNA was quantified by OD₂6o measurements that were obtained using the Spectrophotometer (NanoDrop 1000).

[00129] The TaqMan probe and PCR primers for the real-time PCR were designed within the gag gene of HTLV-II (Shimotohno et al., PNAS; 1985). The TaqMan probe and primer sequences are shown in the Table 1.

Table 1 Probe and primers for HTLV-II detection

[00130] TaqMan probes labeled at the 5'end with the reporter dye FAM (6-carboxy- fluorescein) and at the 3' end with the quencher dye TAMRA (6-carboxytetram ethyl - rhodamine) (Integrated DNA Technologies). Albumin DNA quantification performed in parallel on all samples in order to determine the amount of cellular DNA present and used as an internal reference to normalize variations due to differences DNA extraction. [00131] The 25 ul PCR mixture for HTLV-II and nonhuman primate albumin DNA consisted of 1 ug of DNA extracted from Cells or tissues; 200 nM primers; 100 nM probe; 2X TaqMan Universal PCR Mastermix (Applied Biosystems) which consists of 10 mM Tris- HCl (pH 8.3), 50 mM KCl, 5 mM MgCl₂, 300 uM each of dATP, dCTP, and dGTP, 60OuM dUTP, 0.625 U of AmpliTaq Gold DNA polymerase, and 0.25 U uracil N-glycosylase (UNG). For both HTLV-II and nonhuman primate albumin DNA amplification, 1 cycle at 5O⁰C for 2 min and 1 cycle at 95⁰C for 10 min were followed by a two step-PCR procedure consisting of 15 seconds at 95⁰C and 1 min at 60°c for 50 cycles. The amplification was performed using ABI Prism 7500 Sequence Detector system (Applied Biosystems).

RESULTS

Transformation of Normal Rhesus Peripheral Blood Cells by HTLV-II In Vitro

[00132] HTLV-II infection in the immortalized T cell line, RhM304, was documented by measuring pl9Gag production by antigen capture assay in the cell culture supernatant

(data not shown) and by intracellular pi 9Gag staining (Figure IA). The results of intracellular pi 9Gag staining are shown in Figure IA.

[00133] To further characterize this cell line, a phenotypic analysis was conducted by flow cytometry. Our data demonstrated that RhM304 cell line is CD3+ CD8+ unlike the Mo-

T cell line which is CD3 + CD4 + as shown in Figure IB. Karyotypic analysis of RhM304 cells clearly confirmed the non-human primate origin of the cell line with a diploid set of 21 chromosomes of female gender (Figure 1C).

[00134] Analysis of proviral DNA copies by real-time PCR using primers specific for the gag region demonstrated a higher level of proviral DNA in the Mo-T cells as compared to

RhM304 (Figure ID).

DISCUSSION

[00135] These results show that HTLV-II is able to infect rhesus PBMC in vitro and that we confirm previous study on the CD8+ preferential tropism of this virus in vitro. However, it is desirable that the HTLV-II viral vector be attenuated as compared to wild type. The HTLV-II viral vector can be constructed to eliminate p28, and p 10 and pi 1 (ref Cockerell G.L Blood 1996, 1030; Yamamoto et al, Retrovirology 2008, 5:38). Attenuation will be confirmed by quantitating viral replication in cells in vitro. Any insertional mutagenesis will be minimized as the virus will be attenuated. In addition, it has been reported that in HTLV-II infected individuals also infected with HIV there is a lower progression to AIDS, indicating that the presence of HTLV-II in immunosuppressed individuals provides beneficial effects. (Turci, M. et al., JAIDS, 2006 p:100; Beilke et al., CID 2004:39; Bassani CID 2007:44; Beilke et al., CID, 2007:44). Example 2:

HTLV-II Infects Rhesus Macaques and Replicates in the Gut

[00136] To assess HTLV-II infectivity in rhesus macaques, seven animals were inoculated intravenously.

MATERIALS AND METHODS

Animal Innoculations

[00137] Seven rhesus macaques were inoculated intravenously with 10⁸ lethally irradiated HTLV-II producing cells of human (Mo-T) or rhesus (RhM304) origin. Animal M214 received a heterologous inoculation with Mo-T cells, while macaques M304 and L900 received respectively an autologous or allogenic inoculation with RhM304 cell line. M893, M897, M905, and M906 received heterologous inoculation with Mo-T cell line. Since the allogenic inoculation with RhM304 cell line did not induce a detectable immune response in animal L900, we decided to inoculate the four new animals with the Mo-T cell line.

Detection of Infection

[00138] The presence of antibodies specific for viral antigens in inoculated macaques was evaluated at 1, 6, 12 and 18 months post-inoculation by western-blot. Several tissue samples were also obtained and analyzed by real-time PCR using primers specific for the gag region or tax 2 as described in Example 1.

[00139] For western blot, blot strips were obtained from Zeptomatrix, Inc. (Buffalo,

NY). The blots contain MTA-I, a unique HTLV-I envelope recombinant protein (rgp46-I), K55, and GD21, a common yet specific HTLV-I and HTLV-II epitope of the envelope protein. Each strip also includes an internal sample addition control to minimize the risk of false negatives due to operational errors.

[00140] The assay was carried out according to the manufacturer's instructions.

Briefly, blot strips were incubated at room temperature in Tris buffer with Tween 20. Buffer was removed and blotting buffer added. Sera samples were added and the strips were incubated for 1 hour at room temperature on a rocker platform. The strips were washed and goat anti-human IgG conjugated with alkaline phosphatase was added. The strips were incubated for 1 hour at room temperature. The conjugate was removed and substrate solution was then added. After 15 minutes of incubation, the strips were washed and dried. Induction of Viral Expression

[00141] We obtained bone marrow from animals M214, M304 and L900. After overnight culture in RPMI supplemented with 10% FCS and 1% antibiotic/antimycotic, cells from the bone marrow were divided into 3 populations: not treated, CD34+ depleted, and CD34+ enriched using magnetic sorting technology (Miltenyi). Briefly, cells were frrst stained with anti-CD34-PE antibody (BD, Biosciences) for 15 minutes at room temperature. Cells were then stained with anti-PE Microbeads (Miltenyi) and separated on MACS columns (Miltenyi). The cells were stained intracellularly with antibodies to Tax 2 (kind gift from Mahieux R., France) and sorted by flow cytometry.

Purification and Analysis of Dendritic Cells

[00142] Dendritic cells were purified from the blood of animal L900 by enriching for peripheral blood mononuclear cells (PBMCs) using ficoll-hypaque separation. Blood myeloid dendritic cells were isolated from monkey PBMCs using immunomagnetic separation using a non-primate CDIc (BDCA-I)+ dendritic cell isolation kit (Miltenyi Biotec) . The isolation was performed in a two-step procedure. First, B cells and monocytes are magnetically labeled and depleted using a cocktail of CD20 and CD 14 MicroBeads. Subsequently, the pre- enriched dendritic cells in the non-magnetic flow-through fraction were magnetically labeled and enriched using anti CDIc microbeads. The highly pure enriched cell fraction was CDIc (BDCA-I)⁺ type+1 myeloid dendritic cells (MDCIs). For the isolation of Plasmacytoid dendritic cells, cells were depleted of T cells (CD3 microbeads) B cells (CD20), NK cells (CD16) and monocytes (CD14), and then positively selected using anti CD123. The highly pure enriched cell fraction was plasmacytoid dendritic cells. For evaluation by flow cytometry cells were stained with antibodies to CD123, CDl Ic, CDIc, CD3, CD4, CD8, CD14, CD16, and CD20.

[00143] The presence of Tax 2 and pl9 Gag in these cells was determined by staining these cells with antibodies to Tax2 or pl9 and sorting the cells by flow cytometry. The presence of Tax 2 can also be determined by PCR as described in Example 1. Primers useful for such amplification include those in Table 2.

Table 2 HTLV-II TAX Primers

[00144] The presence of virus particles was determined using standard electron microscopy protocols.

[00145] Transmission of virus to human T cell lymphoma line SupTl cells (ATCC

CRL- 1942) was determined by co culturing 200,000 dendritic cells with 800,000 SupTl cells for 24-48 hrs. Infected SupTl cells were identified by GFP expression.

RESULTS

[00146] Animals M304 and M214 mounted weak antibody responses to HTLV-II by 12 months post infection whereas animal L900 remained sero-negative. Animals M304 and L900 received the RhM304 cell line that we showed was producing less virus than the Mo-T cell line. See Figure 2A. Animals M893, M897, M905, and M906 mounted a strong antibody response at 1 month post infection as shown in Figure 2B. These animals received the HTLV- II infected human Mo-T cell line.

[00147] PCR analysis of CD4+ or CD8+ purified T-cell from blood or lymph nodes of

M304, M214, and L900 revealed the presence of proviral DNA in blood and lymph nodes of M304 and M214 but not L900 (Figure 3A and data not shown for L900). Infection was further confirmed by Gag PCR analysis of all tissues obtained by surgery or at sacrifice, eighteen months after inoculation. Virus was found in the jejunum, ileum and blood and mesenteric lymph nodes of macaques M214 and L304 but not in tissues of macaques L900 (Figure 3B). Proviral DNA was not found in lymph nodes, colon, rectum, vagina, cervical, dorsal, and lumbar spinal cord, bone marrow, cerebrum, cerebellum, brain stem and spinal fluid cells. The results in Figure 3A are obtained with nested- PCR in order to increase the sensitivity of the assay. Data shown in Figure 3B were obtained by real-time PCR. Either 1 microgram or 100 nanograms of DNA were used for the real-time PCR. We cannot exclude that the virus is present in this tissue but at a level that is below the detection level of this technique. Furthermore, validation of the presence of viral sequence was obtained by amplifying Tax2 in PBMCs, lymph nodes and bone marrow of macaques M214 and M304 but not L900 (Figure 4).

DISCUSSION [00148] Most of the T-cell vaccines developed for HIV are based on microbial vectors that have limited replication capacity and do not persist in the host. Such vaccines do not protect macaques from SIV infection and their ability to protect against high virus load is transient (approximately six months). An emerging notion is that these vaccine vectors elicit "too small T-cell responses" that expand "too late" upon virus encounter to sufficiently contain the seeding of the virus in tissues. In addition few of these vectors target mucosal sites, the first portal of entry for HIV.

[00149] A continuous expression of low level of SIV antigen(s) in the gut may maintain a sufficient level of effectors CD8 memory cells able to decrease early seeding of the virus and sufficient level of central memory cells that may limit the broadcasting of the virus at distal sites. A vaccine based on a vector that is able to infect gut tissues and persist at a low level may provide the type of protection that is needed to provide immunity against SIV and HIV infection.

[00150] The antibody response to HTLV-II antigens post infection in some of macaques was weak or non-detectable. For example, animal L900 did not show much of an antibody response. Other animals, however, showed a stronger antibody response when inoculated with Mo-T cells.

[00151] We have obtained evidence that indeed HTLV-II infects macaques and replicates at very low level in lymphoid tissue and particularly in the gut. Significantly, virus was found in gut lymphoid tissues such as the jejunum, ileum and mesenteric lymph nodes of macaques. This is important because it is thought that the first portal of entry of infection with HIV is in the gut.

[00152] Importantly HTLV-II infects dendritic cells both in vivo and in vitro and both

HTLV-II infected myeloid and plasmacytoid dendritic cells have a mature phenotype. Even in animals that showed a poor antibody response, the presence of virus could be detected in CD34+ bone marrow cells. Infection of dendritic cells isolated from peripheral blood was confirmed by detecting the presence of tax 2, pl9Gag, and by electron microscopy. The macaque dendritic cells were also able to transmit HTLV-II infection to a human T lymphoid cell line. HTLV-II can use the dendritic cell antigen presenting cell (APC) function to infect T cells.

[00153] These results indicate that a vaccine vector using a HTLV-II could infect gut tissues and persist at a low level. The ability of HTLV-II to infect dendritic cells is important for antigen presentation and the development of T cell responses including CD8 memory cell responses. Example 3:

HTLV-II Induces Low Level of Virus Specific CD4+ and CD8+ T-CeIl Responses in Blood and Tissues of Infected Macaques

[00154] Analysis of virus-specific CD4+ and CD8+ T-cell responses were measured longitudinally in blood and tissues of the infected macaques by intracellular staining following in vitro stimulation with Tax2 and Gag2 overlapping peptides.

MATERIALS AND METHODS

[00155] Peripheral blood cells were isolated from the macaques and cultured in the presence of Tax-2 or Gag-2 overlapping peptides. The sequences are shown in Tables 4 and 5. SEB was used as a positive control.

[00156] Intracellular cytokine staining (ICC) staining was performed using the anti-

TNF-α, -IFN-γ, and -IL-2 Abs. A total of 10⁶ fresh PBMC in 1 ml of complete medium were incubated for 1 h at 37⁰C in the absence or presence of a pool of Gag, Tax peptides (1 mg/ml) of HTLV-II and in the presence of CD28 and CD49d (1 mg/ml each) in a 5-ml round-bottom tube. After addition of 10 μg/ml brefeldin A (Sigma- Aldrich), cells were incubated for 5 h at 37⁰C and processed for surface and ICC staining. Briefly, cells were washed with 1% FCS in PBS, surface stained for 20 min with CD8β-PE (Immunotech) and CD4-PerCP (BD Biosciences), washed again, and fixed and permeabilized with Cytofix (BD Pharmingen) for 20 min at 4⁰C in the dark. Following two further washes in Perm/Wash buffer (BD Pharmingen), cells were intracellularly stained with FITC-conjugated anti-TNF, anti-IFN-γ and allophycocyanin-conjugated anti-IL-2 (BD Pharmingen), incubated for 20 min at 37⁰C, fixed with 1% paraformaldehyde in PBS, and analyzed on a FACSCalibur. [00157] Four-parameter flow cytometry analysis was performed using CELLQuest software. List mode data files were analyzed using FlowJo software (Tree Star, Ashland, OR). A positive response was defined as follows: the frequency of cytokines-i- T cells observed in the sample that was stimulated with no peptides was used as a cut-off.

RESULTS

[00158] No detectable responses were found in the blood of macaque L900, consistent with the low/undetectable pro virus load in the PBMCs of this animal (Figure 6A). Another animal with a low antibody response to HTLV-II antigens as shown in Figure 2B is animal M906. Consistent with the results of animal L900, little or no immune responses were found in the blood. Higher level responses were found in the gut tissue. See Figures 6D and 6E. [00159] In contrast, low but specific, although sporadic, immune responses were found in blood of animals M304, M214, M893, M897, and M905 (Figure 6B, 6C, 6G, and 6H). Analysis of HTLV-II specific immune responses in the gut demonstrated convincing positive responses to Gag(colon), Tax and Gag (ileum) and Gag (rectum) of animal M214 (Figure 61). Analysis of HTLV-II specific immune responses in the gut demonstrated convincing positive responses to Tax and Gag (jejunum) of animal M893 (Figure 6F).

DISCUSSION

[00160] HTLV-II induces a "smoldering" infection in macaques that mirrors HTLV-II infection of humans. Its localization to and persistence in the gut renders this virus an interesting candidate as a delivery system for an HIV vaccine. The immune responses elicited by this virus are low but very effective in controlling HTLV-II replication. It is therefore anticipated that the expression of HIV or other viral antigens by this vector may confer an adequate control of HIV replication as well.

Example 4:

Transmission of HTLV-II and development of the immunity to HTLV-II in babies born of mothers infected with HTLV-II.

[00161] The animals, date of infection and their progeny are shown in Figure 7.

RESULTS

[00162] Antibody responses to HTLV antigens were determined using western blot strips as described in Example 2. The results are shown in Figures 8 and 9. Antibody responses to HTLV-II antigens are not detectable in both the mothers and babies when measured at within a few months of infection or delivery using western blot. (Figure 8)

When measured using ELISA, the antibody response to HTLV-II antigens was detected and continues to rise in the mothers after delivery. (Figures 9A-D)

[00163] Analysis of virus-specific CD4+ and CD8+ T-cell responses in the mothers were measured longitudinally in blood of the infected macaques by intracellular staining following in vitro stimulation with Tax2 and Gag2 overlapping peptides as described in

Example 3. The results are shown in Figure 9E-L. [00164] The results show that most of the mothers have very low virus-specific CD4+ and CD8+ T-cell responses in peripheral blood up to 8 weeks post delivery. One animal P047 showed a strong response to tax and gag at delivery.

DISCUSSION

[00165] The results from the mothers infected with HTLV-II are consistent with those reported in Example 3. The majority of animals exhibit a very low antibody response to infection with HTLV-II. Virus-specific CD4+ and CD8+ T-cell responses in peripheral blood cells are also very low. Responses in gut tissue in the infected mothers have not yet been measured. The babies will be studied as well to determine whether they are infected with HTLV-II and whether they develop immunity in a similar way.

Example 5:

HTLV-II Deletion Mutant

[00166] One challenge in developing a viral vector is to identify a virus that can be modified by the addition of heterologous DNA without losing the capacity to infect and/or replicate in cells. In our initial studies with HTLV-II we deleted gag, pol, and gag and pol, and cloned HIV gag or pol genes in the location of the deleted genes. However, we found that these constructs were not able to produce infectious virus particles.

[00167] A deletion mutant of HTLV-II with a deletion of about 324 base pairs at the 3' end of ORF pX-I is able to infect and replicate in rabbits. This mutant has been described by Cockerell et al., Blood 87:1030 (1996). Four open reading frames (ORFs), designated pX-I, pX-II, pX-III, and pX-IV, were initially identified within the HTLV-I pX. The second coding exon of the transacting viral proteins Tax and Rex are expressed from the overlapping ORFs pX-IV and pX-III, respectively, and are translated from a doubly spliced, bicistronic mRNA. Tax increases the rate of viral transcription by activating the viral promoter in the viral long terminal repeat, while Rex acts posttranscriptionally to increase the ratio of incompletely spliced mRNAs to doubly spliced mRNA. ORFs x-I and x-II contained in the proximal portion of pX have 655 base pairs (bp) and 573 bp for HTLV-I and HTLV-II, respectively, and have been referred to as the nontranslated or untranslated (UT) region. They are not known to be expressed, and are nonconserved between the two viruses. Alternatively spliced mRNAs and protein isoforms encoded in the pX region have been identified. However, analysis of HTLV-I and -II UT deletion mutants have shown that these sequences are not necessary for successful induction, transmission, or maintenance of the transformed cell type or in vitro expression of other viral polypeptides. The UT region represents an area of the HTLV-II genome that can be deleted in order to provide a location for heterologous polynucleotide sequence. A representative genomic sequence for HTLV-II can be found in GenBank at NC_001488, gl 9626726 or as described in Shimotohno, PNAS 82:3101 (1985).

Cloning

[00168] Heterologous DNA encoding a fragment of anyone of HIV and/or SIV proteins are cloned into the Pstl site (position 6661) of the HTLV-II mutant. In some cases, an artificial DNA maybe constructed that encodes a consensus sequence of any one of the genes of HIV or SIV. In some cases, the DNA fragment is no more than 400 base pairs.

Different cloning strategies are shown in Figure 10.

[00169] The DNA sequences encoding the genes from HIV or SIV strains or isolates are known to those of skill in the art and are obtainable at the NCBI database or at the Los

Alamos database. Fragments may be selected based on ability of the polypeptide to react with immune serum, neutralizing antibodies whether monoclonal or polyclonal, and/or to stimulate

T cell responses.

[00170] Cloning of the heterologous viral sequences may be conducted by methods known in the art including PCR amplification or synthetic production of HIV fragments followed by ligation in the HTLV-II vector.

[00171] A number of different HTLV-II based vectors (Swarms) will be produced each having a heterologous DNA sequence encoding a different gene or fragment thereof. For example, one vector may be constructed with the consensus sequence for the env and another with a sequence encoding all or a portion of gag from clade B of HIV. Several constructs covering all HIV clades can also be constructed. A number of these viral vectors can be combined into one immunogenic composition.

Infectivity

[00172] Infectivity of the HTLV-II viral vector will first be screened in the macaque or human T cells as described in Example 1. We can transfect 729 B-cells and make stable cell lines or we can transfect 293 cells or other easily transfectable cells. Cells are irradiated before administration. An alternative strategy is to electrophorate directly the construct DNA in the skin. If the viral vector exhibits infectivity in the cell line, animal model test for infectivity will be done in rabbits or macaques as described in Example 2. Immune Response

[00173] Immune responses in the animals to the heterologous viral protein encoded by the heterologous DNA present in the HTLV-II viral vector will be determined as described in Examples 2 and 3. ELISA and PCR kits are commercially available for detecting HIV and SIV viral antigens. Western blot strips are also commercially available for the detection of antibody responses to HIV and/or SIV antigens. Virus-specific CD4+ and CD8+ T-cell responses in various tissues can be determined as described in Example 3. The immune response will also be evaluated for the capacity to participate in ADCC of cells infected with the virus corresponding to the heterologous viral protein.

Immunogenic Compositions

[00174] Immunogenic compositions will be formed from one or more of the HTLV-II viral vectors. The HTLV-II viral vector selected will express a heterologous gene or portion thereof. Another criterion for selection is that the HTLV-II viral vector can establish a low level of infection, especially in the gut tissue. Another criterion for selection is that the HTLV-II viral vector induces a low level antibody response to the heterologous gene or fragment thereof and a higher virus- specific CD4+ and CD8+ T-cell response, especially in the gut tissue. Desirably, the HTLV-II viral vector induces antibodies that can participate in ADCC. As discussed above, such compositions may include more than one HTLV-II viral vector, each encoding a different polypeptide or fragment thereof.

Example 6:

Pre-existing Infection with the Human T-cell Lymphtropic Virus Type 2 does neither Exacerbate nor Attenuate SIVm_ar75i Infection in macaques

[00175] HIV- 1 vaccine strategies have focused on viral vectors delivering HIV-I antigens. These vectors stimulate strong, systemic antigen-specific responses, but are unable to protect from infection since they generate only limited mucosal responses. The only vaccine approach that has conferred protection in the SrV_m3C2S1 macaque model is a live attenuated virus (Desrosiers, R. C. 2004. Nat. Med. 10:221-223), suggesting that persistent expression of viral antigens in mucosal and lymphoid tissues may be necessary. An HTLV-II vector expressing HIV-I antigens at mucosal sites that stimulates and maintain T-cell responses in the gut may confer protection from infection by quickly eliminating cells infected by the founder virus. This study establishes that the Indian Rhesus macaque model for HTLV-II infection suitable to test this hypothesis as it demonstrates that HTLV-II targets systemic, lymphoid as well as has mucosal tissues of rhesus macaques, stimulates humoral immune responses, and systemic and mucosal T-cell responses. Importantly, it is demonstrated here that macaques dually infected with HTLV-II and SIV_maC25i have similar viral and T-cell dynamics as SIV_maC25i singly infected macaques.

MATERIALS AND METHODS Experimental HTLV-II and SIV_mac₂5i infection

[00176] The eleven Indian rhesus macaques (RM) used in this study were housed and cared for under the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care International (IACUC). RMs were housed at Advanced Biosciences Laboratory in Rockville, MD. Seven RMs were inoculated with 10⁸ HTLV-II- infected lethally irradiated cells, of human (Mo-T) or rhesus (M304) origin. Mo-T-cells are an HTLV- II producing cell line that was derived from a male patient with hairy cell leukemia (Kalyanaraman, V. S., M. G. Sarngadharan, M. Robert-Guroff, I. Miyoshi, D. Golde, and R. C. Gallo. 1982. Science 218:571-573). To create an HTLV-II expressing rhesus macaque cell line, we co-cultured cells from macaque M304 with gamma irradiated Mo-T-cells at a 1:1 ratio. Cells were maintained in RPMI- 1640 complete medium supplemented with 10% heat inactivated FCS, 1% Penicillin/Streptomycin and 20U/ml recombinant human IL-2. HTLV-II infection was documented by measuring extracellular p 19 Gag production by antigen capture assay and by intracellular pl9 Gag staining. The cell line derived from this culture was designated RhM304. Animals M304 and L900 were inoculated with HTLV-II infected cells from M304, i.e. an autologous or allogenic infection respectively, while animal M214 received a heterologous infection with Mo-T-cells. Both heterologous and autologous infections produced a persistent HTLV-II infection; therefore, the remaining HTLV-II infections were done using irradiated Mo-T-cells. Four additional animals (M893, M897, M905, M906) were HTLV-II infected; ten months post HTLV-II infection, these four animals and four naϊve macaques were infected intra-rectally with SIV_maC25i (10⁵ TCID 50).

Blood and tissue collection

[00177] Mononuclear cells were isolated from blood, rectal, jejunal pinch biopsies, lymph node biopsies, and bone marrow aspirates. Mononuclear cells were separated from whole blood and bone marrow aspirates by density gradient centrifugation (Ficoll). Lymph nodes were homogenized and passed through a 100 μm cell strainer and mononuclear cells purified by density gradient centrifugation (Ficoll, Rectal, and jejunal pinch biopsies were treated with 1 mM of ultra pure Dithiothreitol (Invitrogen, Carlsbad, CA) for 20 min followed by incubation in 0.1M EDTA solution in calcium/magnesium free HBSS with penicillin- streptomycin for 60 minutes to remove the epithelial layer. Lamina propria lymphocytes were separated, following the removal of the intraepithelial lymphocytes, by incubating with collagenase D (400 U/ml; Boehringer Mannheim, Mannheim, Germany) and DNase (1 μg/ml; Invitrogen) for 2 h at 37⁰C in Iscove's modified Dulbecco's medium (GIBCO BRL) supplemented with 10% FBS and penicillin-streptomycin. The dissociated mononuclear cells were then placed over 42% Percoll (General Electric Healthcare, Piscataway, NJ) and centrifuged at 2000 rpm for 30 min at 4⁰C. Immunophenotypic studies were performed by polychromatic flow cytometry (as detailed below) on mononuclear cells derived from all tissues. All cells were stained the day of sampling, except for cells from the jejunum and rectum, which were allowed to rest overnight and then stained.

Immunophenotyping and intracellular cytokine assay

[00178] Four and eight-color flow cytometric analysis was performed on mononuclear cells from blood and tissues. Surface staining was performed for 30 minutes at room temperature with antibodies to CD3 (cloneSP34-2), CD4+ (L200), CCR5 (clone 3A9), and CD95 (clone DX2) all obtained from BD Biosciences (CITY STATE). Anti-CD28 (clone CD28.2) was obtained from eBiosciences and anti-CD8 (clone 3B5) was obtained from Invitrogen (CITYSTATE). Following surface staining, cells were permeabilized and stained with anti-Ki67 (clone B56 from BD Biosciences). To monitor SIV and HTLV-II specific immune responses, lymphocytes were resuspended at 10⁶ cells per ml in RPMI- 1640 complete medium with 10% heat- inactivated FCS in combination with anti-CD28 (Biosource International, Camarillo, CA), anti-CD49d (Becton Dickinson, San Jose, CA), and monensin (BD Biosciences). Cells were incubated with media only or withl μg/ml of 15mers overlapping by 11 amino acids peptide pools derived from the entire HTLV-II Tax, HTLV-II Gag, SIV_maC25i Gag and SIV_maC25i Env proteins sequence. Stimulation with either phorbol myristate acid (PMA) and Ionomycin A23187 (Sigma CITY STATE) or staphylococcal endotoxin B (SEB, Toxin Technologies, Sarasota, FL) was used as a positive control. Cells were stimulated for 5 hours, washed, and stained for CD3, CD4+, and CD8+ for 30 minutes at room temperature. Following surface staining, lymphocytes were permeabilized with FACS Perm/Wash solution (BD Biosciences), and stained intracellularly for IFN γ (clone B27), TNFα (clone MABI l), MIPl α (clone 11A3), and IL2 (clone MQ1-17H12) all from BD Biosciences, in addition to IL17 (clone eBio64DEC17) from e-Bioscience. All cells were fixed with 1% paraformaldehyde and at least 100,000 events acquired on either a FACS Calibur or LSRII. Data analysis was performed with Flowjo (Treestar CA) and graphical and statistical analysis was performed using Graph Pad Prizm.

Infection of primary dendritic cells

[00179] Plasmacytoid dendritic cells (pDC) were isolated from PBMCs from uninfected rhesus macaques. Non-pDC lineages were first removed using a commercially available kit (Miltenyi Biotec) followed by positive selection of BDCA-4 (NP-I) cells. A portion of the cells were immediately fixed, permeabilized, and stained for the presence of Tax-2. The remaining pDCs were infected with HTLV-II harvested from the supernatant of MoT-cells by incubating the virus with pDC for 3 hours, washing, and replating in six well plates at 5 xlO /well. These pDC were cultured in RPMI- 1640 medium supplemented with 10% human AB serum and IL- 3 (lOng/ml), and the cells harvested, permeabilized, and stained for Tax-2 either 2 or 6 days later.

Karyotyping

[00180] Karyotypic analysis of RhM304 cells was obtained by treating the cultured cells with colcemid, a drug that disrupts the mitotic spindle apparatus to prevent the completion of mitosis and arrests the cells in metaphase. The cells were fixed, dropped on a microscope slide, dried, and stained with Giemsa.

Serological analysis and Intracellular staining for HTLV-II antigens

[00181] The presence of antibodies to viral antigens was determined by Western blot on the sera collected from infected animals. Blot strips were obtained from ZeptoMetrix Corporation (Buffalo, NY). The blots contain the Gag proteins as well as MTA-I, a unique HTLV-I envelope recombinant protein rgp46-I, and GD21, a common, yet specific HTLV-I and HTLV-II epitope of the envelope protein. Each strip also includes an internal sample control to minimize the risk of false negatives due to operational errors. The assay was carried out according to the manufacturer's instructions. [00182] Intracellular staining for viral antigens was performed using a mouse anti-pl9 gag antibody (Zeptomatrix, Buffalo, NY) or a rabbit anti-tax2 antibody (kindly provided by Mahieux R., Pateur Institute ,France). Briefly, cell pellets were fixed in 100 μl IC Fixation Buffer (eBioscience). Intracellular staining was performed in 100 μl of Permeabilization Buffer using a 1:250 dilution of the anti-pl9 gag antibody or 1:100 dilution of the anti-tax2 antibody and incubated for 30 min in the dark at room temperature. Cells were washed twice and stained with anti-mouse IgG Alexa-Fluor488 antibody or anti-rabbit Alexa-Fluor488 (Invitrogen) for 30 min in the dark at room temperature. Next, cells were washed and resuspended in 1% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS. Four-parameter flow cytometric analysis was performed using a FACS Calibur with CELLQuest software.

Viral load

[00183] SIV RNA was quantified in the plasma and SIV DNA was quantified in the blood and tissues of macaques by NASB as previously described (Romano, J. W., K. G. Williams, R. N. Shurtliff, C. Ginocchio, and M. Kaplan. 1997. Immunol. Invest. 26:15-28). Quantification of HTLV-II DNA in the blood and tissues was performed by extracting genomic DNA with the DNeasy tissue kit according to the manufacturer's protocol except DNA elution step QIAGEN). DNA was eluted with 10 mM Tris (pH8.0). Five hundred ng of genomic DNA was subjected to real-time PCR. The TaqMan probe and PCR primers for the real-time PCR were designed within the gag gene of HTLV-II (Shimotohno, K., Y. Takahashi, N. Shimizu, T. Gojobori, D. W. Golde, I. S. Chen, M. Miwa, and T. Sugimura. 1985. Proc. Natl. Acad. Sci. U. S. A 82:3101-3105). The TaqMan probe and primer sequences are follows: probe, 5'-FAM- AGGCGTGG AC ACCCAAGG AC AAAAC - TAMRASp-3' SEQ ID NO:; forward primer, 5'-GGGAGATGCTCCGGACATG -3' SEQ ID NO:; reverse primer, 5'-CGTGGTTGGACCACAAG GA -3' SEQ ID NO:. Reaction conditions were as follows: The 25 μl PCR mixture for HTLV-II and macaque albumin DNA consisted of 500 ng of DNA extracted from tissues or PBMCs, 200 nM primers; 100 nM probe; 2X TaqMan Universal PCR Mastermix (Applied Biosystems). For both HTLV-II and macaque albumin DNA amplification, 1 cycle at 50⁰C for 2 min and 1 cycle at 95 ⁰C for 10 min were followed by a two step-PCR procedure consisting of 15 seconds at 95 ⁰C and 1 min at 60 ⁰C for 50 cycles. The amplification was performed using ABI Prism 7500 Sequence Detector system (Applied Biosystems). The normalized value of the HTLV-II proviral DNA load was calculated as HTLV-II DNA copy number/macaque albumin gene copy number x 2 x 10⁶ and expressed as the number of HTLV-II pro viral DNA copies per 10⁶ Cells (Chung, A., E. Rollman, S. Johansson, S. J. Kent, and I. Stratov. 2008. Curr. HIV. Res. 6:515-519). (Chung et al. J. Virol Methods 152 (l-2):91-7, 2008). Nested semi-quantitative PCR reaction was used also used to amplify a 128-nucleotide sequence in the HTLV-II Tax gene. DNA was extracted from blood and lymph nodes. DNA extraction was performed using the DNeasy Blood and Tissue Kit (Quiagen, Valencia, CA), according to the manufacturer's instructions. The PCR reaction included 250 ng of DNA, the Platinum High Fidelity Supermix (Invitrogen, Carlsbad, CA) and primers specific for either β-actin (forward 5'- CGGTTGGCCTTGGGGTTCAGGGGG-3' SEQ ID NO:; reverse 5'- ATCGTGGGGCGCCCC AGGCACCA-3' SEQ ID NO:) or the HTLV-II tax gene (forward 5'-TGGATACCCCGTCTACGTGT-S' SEQ ID NO:; reverse 5'- GAGCTGACAACGCGTCCATC-3' SEQ ID NO:; nested forward 5'- GTGTTTGGCGATTGTGTAC A-3' SEQ ID NO:; nested reverse 5'- CCATCGATGGGGTCCCA-3' SEQ ID NO:). The primers were designed using Primer3 software (Whitehead Institute for Biochemical Research).

Statistical analyses

Two way ANOVAs with Dunn's multiple comparison post tests were performed to assess the statistical significance of the biological data.

RESULTS

HTLV-II establishes a persistent infection in rhesus macaques and replicates in lymphoid and mucosal tissues

[00184] To determine the optimum method for establishing an HTLV-II infection in rhesus macaques, we inoculated three monkeys with 10⁸ lethally irradiated, HTLV-II producing cells of either human (Mo-T) or rhesus (RhM304) origin. We performed a heterologus infection in one animal (M214), which was given the HTLV-II cell line Mo-T (Table 6). This cell line was originally generated from cells obtained from a male patient with a T-cell variant of hairy cell leukemia (Kalyanaraman, V. S., M. G. Sarngadharan, M. Robert-Guroff, I. Miyoshi, D. Golde, and R. C. GaUo. 1982. Science 218:571-573). It contains a replication competent genome of HTLV-II and two defective HTLV-II genomes. To perform an autologus infection, we cultured PBMCs from one macaque (M304) with lethally irradiated Mo-T-cells and generated a HTLV-II expressing macaque cell line, named RhM304. HTLV-II infection and gene expression in RhM304 and Mo-T-cells was confirmed by PCR and flow cytometric staining for Gag (Figure 13A and 13B). RhM304 was confirmed to be a female of non-human primate origin with a diploid set of 22 chromosomes (Figure 13C). RhM304 cells were lethally irradiated and used to infect animal M304 and a third macaque, L900. Two of the animals, M214 and M304, became persistently infected and seroconverted; antibodies to HTLV-II p24 were detected by ELISA (Figure 13D). Eighteen months after inoculation, the animals were sacrificed and blood, bone marrow, spleen, lymph nodes, intestinal, vaginal, and brain tissue was collected along with spinal fluid. HTLV-II proviral DNA was amplified from the spleen, ileum, and jejunum (Figure 13E) whereas, proviral DNA was not detected in the brain or in the spinal cord of any of the animals (data not shown). HTLV-II was also demonstrated to infect primary rhesus macaque dendritic cells and Figure 13F shows Tax staining in macaque myeloid derived dendritic cells after 6 days in culture. Altogether these findings demonstrate that HTLV-II infects rhesus macaques and replicates in lymphoid and mucosal tissues. Interestingly, similar to HTLV-II infected humans, low levels of virus replication were observed.

HTLV-II -specific immune responses in rhesus macaques

[00185] As both autologous and heterologus HTLV-II expressing cells were infectious in rhesus macaques, we inoculated 4 additional animals (M893, M897, M905, and M906) with the irradiated Mo-T-cell line (Table 6) and followed T-cell dynamics, and humoral and cellular immune responses to HTLV-II in these animals. The presence of antibodies specific for viral antigens was evaluated by Western blot in all the HTLV-II infected macaques. All four animals mounted robust antibody responses against HTLV-II antigens within 30 days of infection, which persisted for over 6 months (Figure 14A). HTLV-II tax DNA was amplified by nested PCR from the lymph nodes of all four animals at 3 months post infection (Figure 14B) but not in the DNA of uninfected macaques used as a control (data not shown). Interestingly, both CD8+ and CD8+ T- cells contained HTLV-II tax, suggesting that similar to infection in humans (Casoli, C, E. Pilotti, and U. Bertazzoni. 2007. AIDS Rev. 9:140- 149). ^u HTLV-II can replicate in both T-cell types in macaques. Importantly, HTLV-II infection did not induce a loss of CD4+ or CD8+ T-cells in vivo and only minor fluctuations in the number of CD4+ and CD8+ T-cells were observed (Figure 14C). [00186] HTLV-II- specific T-cell responses in the blood and gastrointestinal were measured by intracellular cytokine staining for IFN -γ, TNF-α, and IL-2 in response to peptide pools of 2 major antigenic proteins of HTLV-II Gag and Tax. Flow cytometric analysis of the frequency of CD8+ T-cells producing cytokines demonstrated low levels of antigen specific responses in the blood and the gut (Figure 14D-14G). HTLV-II specific responses peaked 3- 4 months post infection and very low-level responses were detectable up to 10 months postinfection (data not shown).

Comparable viral and cellular dynamics in the blood of dually HTLV-II/ SIV_2Si infected or SIV₂₅i infected macaques.

[00187] The effects of pre-existing HTLV-II infection in the acute phases of HIV-I infection in humans are unknown. We therefore studied the effect of HTLV-II infection on SIV replication and immune response in macaques co-infected with HTLV-II and SIV_maC25i to address these questions. As summarized in the study design (Figure 15A), 10 months post HTLV-II infection we challenged macaques with SIV_maC25i intra-rectally. The viral burden and T-cell dynamics in HTLV-II-infected macaques super-infected with SIV_maC25i was then characterized and compared to SIV_maC25i singly infected controls. Western blot and nested PCR confirmed the persistence of antibodies to HTLV-II antigens and HTLV-II Tax DNA in blood before and after SIV_maC25i infection in the HTLV-II pre-infected macaques (Figure 15B and C). The quantity of SIV RNA in the plasma was quantified by RT PCR (Figure 15D). Peak viremia occurred at 14 days post-infection, with an average of 1.1 X 10⁸ copies/ml for the HTLV-II /SIV_maC25i group and 1.2 X 10⁸ copies/ml in the controls. Set point occurred by 60 days post infection with an average of 8.4 X 10⁶ copies/ml in the HTLV- H/SIV_maC25i group and 7.2 X 10⁶ copies/ml in the controls. Neither peak viral load nor set point viremia was significantly different between the two groups. SIV_maC25i infection caused a sharp decline in the number of CD4+ T-cells in the blood of both groups; this decline stabilized by 30 days post infection (Figure 15E). Absolute CD4+ T-cell counts were not significantly different between the groups. These data support the clinical findings in HTLV- II infected patients where HIV- 1 infection was not found to influence HTLV-II viral load (Turci, M., E. Pilotti, P. Ronzi, G. Magnani, A. Boschini, S. G. Parisi, D. Zipeto, A. Lisa, C. Casoli, and U. Bertazzoni. 2006. J Acquir. Immune. Defic. Syndr. 41:100-106).

Memory CD4+ and CD8+ T-cells in the blood of singly or dually infected macaques [00188] Memory CD4+T cells, particularly those that express CCR5, are the primary targets of HIV-I and SIV_maC25i infection. These target cells are rapidly lost in the acute phase of the infection. HTLV-II infected CD8+ T-cells secrete MIPl α, a natural ligand for CCR5. The production of MIP lα by HTLV-II infected CD8+ T-cells isolated from co-infected individuals have been shown to inhibit HIV-I replication (Casoli, C, E. Vicenzi, A. Cimarelli, G. Magnani, P. Ciancianaini, E. Cattaneo, P. Dall'Aglio, G. PoIi, and U. Bertazzoni. 2000. Blood 95:2760-2769). Thus, MIPIa secretion by HTLV-II infected cells and its binding to CCR5 on circulating or mucosal CD4+ T-cells may theoretically, protect this cell type from the typical rapid depletion observed during SIV_maC25i/HIV-l infections. To examine whether this is the case, we determined the frequency of CD4+ CCR5+ T-cells (pre- gated on CD3+ lymphocytes) using the gating strategy shown in Figure 16A. To control for animal-to-animal variation, the frequency of CD4+CCR5+ T-cells is displayed as a percent of baseline value over the course of the infection. A severe depletion of CD4+CCR5+ cells was observed in both groups by 21 days post infection (Figure 16B) and no difference in the rate of decline of CD4+ CCR5+ was observed between the co-infected animals and controls. Activation and proliferation drive T-cell differentiation from naϊve to memory and effector cells. In order to compare the phenotype of T-cells in HTLV-II/SIV_maC25i co-infected animals to SIV _maC25i singly-infected controls, we characterized the differentiation status of PBMCs using polychromatic flow cytometry. Figure 16A shows the gating strategy, where CD4+ and CD8+ T-cells are first pregated as CD3+ lymphocytes. T-cells that express only CD28 were considered naϊve (N), cells dually positive for CD28 and CD95 were characterized as central memory cells (CM); and cells positive for CD95 only were categorized as effector/ effector memory cells (EM)(Pitcher, C. J., S. I. Hagen, J. M. Walker, R. Lum, B. L. Mitchell, V. C. Maino, M. K. Axthelm, and L. J. Picker. 2002. J. Immunol. 168:29-43). In CD4+ PBMCs, similar to CCR5+ cells, a sharp decline in the fraction of memory cells was observed in both groups, which then stabilized between day 30 and day 60 (Figure 16C). An increase in the absolute number of CD8+ T-cell was observed following SIV_maC25i infection in both groups (Figure 16D) this increase was mainly due to an expansion of CD8+ effector cells (graphed as a fraction of baseline 16E). The CD8+ T-cell expansion coincides with the post-peak decline in viremia, suggesting a contribution of CD 8+ T-cells to the partial control of viremia, as observed in several other studies during SIV infection. The acute increase in the percentage of CD8+ effectors peaked at 21 days post infection and then declined transiently, only to continue to increase in the chronic phase; this is likely due to the continued generalized immune activation observed during chronic HIV-I and SIV_maC25i infections. Altogether, there were no significant differences between the two groups of CD8+ T-cells (Figure 16D and 16E). CD4+ T-cell loss in the lymphoid and gastrointestinal tract of HTLV-II/SIV _mar.7.si infected macaques.

[00189] The tropism of HTLV-II for T-cells and its potential ability to activate T-cells in lymphoid and mucosal sites raised the possibility that HTLV-II infection may increase SIV_maC25i replication by creating more viral targets. We therefore monitored the level of virus replication and CD4+ T-cell depletion in lymphoid and mucosal sites in HTLV-II/SIV_maC25i co-infected macaques and controls. Bone marrow aspirates, along with biopsies of lymph nodes, jejunum, and rectum were collected before infection and at 10 and 30 days post SIV_maC25i infection and SIV_maC25i DNA was quantified by real time PCR on the DNA from lymph nodes and the gastrointestinal tract. SIV DNA was amplified in all tissues tested at both 10 and 30 days post infection (Figure 17A). The level of SIV_maC25i proviral DNA in the tissues including the gastrointestinal tract was not significantly different between the two groups. Similar to the measurements in blood, we monitored the frequency of CD4+ T-cells in the lymphoid organs (i.e. bone marrow and lymph nodes) (Figure 17B) and the gastrointestinal tract (Figure 17C). Both HTLV-II and SIV_maC25i co-infected and SIV_maC25i singly infected macaques showed a decline in the frequency of CD4+ T-cells in both lymphoid organs at 30 days post infection, but there was no significant difference between the groups. A significant depletion of lamina propria CD4+ T-cells was observed in the jejunum and rectum (Figure 17C). However, the extent of the depletion was severe in both groups; these observations were confirmed by immunohistochemical staining which enumerated the absolute number of CD4+ expressing cells in the rectum (Figure 17D). Interestingly, immunohistochemical staining demonstrated a greater number of CD4+ cells in the rectum before SIV infection in HTLV-II pre-infected animals. However, it is unclear as to whether this increase in number was due to HTLV-II induced proliferation. We observed no difference in the number of CD8+ expressing cells in the rectum (Figure 17E) or the frequency of CD8+ T-cells (data not shown) in the singly or dually infected animals before or after SIV infection. Thus, at least in rhesus macaques, the HTLV-H/SIV_mac₂5i co-infection did neither attenuate SIV_maC25i replication nor the loss of CD4+ T-cells at mucosal sites (Figure 17).

[00190] Early in HIV-I infection, viruses that utilize the chemokine receptor CCR5 for entry into T-cells and macrophages dominate (Scarlatti, G., E. Tresoldi, A. Bjorndal, R. Fredriksson, C. Colognesi, H. K. Deng, M. S. Malnati, A. Plebani, A. G. Siccardi, D. R. Littman, E. M. Fenyo, and P. Lusso. 1997. Nat. Med. 3:1259-1265). HIV-I and SIV_mac25i entry can be inhibited by the binding of the natural ligands for CCR5; these CC-chemokines include MIP lα, MIPl β, and RANTES (Cocchi, F., A. L. DeVico, A. Garzino-Demo, S. K. Arya, R. C. GaUo, and P. Lusso. 1995. Science 270:1811-1815)( Lehner, T., Y. Wang, M. Cranage, L. Tao, E. Mitchell, C. Bravery, C. Doyle, K. Pratt, G. Hall, M. Dennis, L. Villinger, and L. Bergmeier. 2000. Immunology 99:569-577). HTLV-II infected T-cells have been shown to spontaneously secrete MIP lα; the induction of MIP lα is likely the result of the viral protein Tax transactivating the chemokine promoter (Lewis, M. J., V. W. Gautier, X. P. Wang, M. H. Kaplan, and W. W. Hall. 2000. J Immunol 165:4127-4132). To characterize the ability of CD4+ and CD8+ T-cells to spontaneously produce MIP lα, we cultured PBMCs for 5 hours in the presence of monensin and performed intracellular cytokine staining for MIPIa. Both the MIPIa expression per cell (MFI) and the frequency of MIPIa production (data not shown) decreased after SIVi_HaC2S1 infection (Figure 18A and 18B). However, no difference was observed in the level of spontaneous MIP lα produced by CD4+ or CD8+ T- cells from co-infected animals and controls at any time point pre- or post-SIV_mac251 infection (Figure 18A and 18B). In addition, no correlation was observed between the levels of spontaneous MIP lα produced and viral load in either HTLV-II/S IV_matasi infected macaques or controls. MIPIa in the plasma of HTLV-II/S IVmac25i co-infected animals or SrVmac25i singly infected animals was undetectable by ELISA.

T-cell proliferation in singly and dually infected macaques

[00191] Increased T-cell proliferation is a hallmark of HTLV infections (Dezzutti, C.

S., D. R. Sasso, D. L. Rudolph, and R. B. LaI. 1998. J Infect. Dis. 177:1489-1496). Thus, we monitored the level of proliferation by staining for the nuclear antigen Ki67 in CD4+ and CD8+ T-cells from blood and tissues following SIVmac25i infection. Before SIVmac25i infection, the levels of Ki67 expression on both CD4+ and CD8+ T-cells were similar in HTLV-II infected and uninfected controls (Figure 19 and 20). An increase in the percentage of CD4+ T-cells expressing Ki67 was observed in the blood following SrVmac25i infection; this peaked at 30 days and continued to be elevated in the chronic phase (Figure 19A). The frequency of CD4+Ki67+ cells increased in the bone marrow with similar kinetics to the blood, the lymph nodes, jejunum, and rectum also demonstrated increases in activating and proliferating CD4+ T-cells. In general, SIV_mac251 singly infected animals had a greater percentage of CD4+ Ki67+ T-cells than those doubly infected with HTLV-II and SIV_maC25i, however, this difference was only significantly different in rectal biopsies (Figure 19D). [00192] Following SIV_maC25i infection there was a dramatic increase in the percentage of CD8+ T-cells expressing Ki67, which peaked 21-30 days post-infection. The frequency of proliferating CD8+ T-cells then declined between 30-60 days, but continued to be significantly elevated in the chronic phase compared to pre-SIV_maC25i infection levels. There were no significant differences in the magnitude of proliferating CD8+ T-cells in the blood of HTLV-II-infected and control macaques (Figure 20A). Lymphoid tissues showed increases in the percent of CD8+ Ki67+ cells post SIV_m3C2S1 infection in both groups, with no significant difference in levels of proliferation between groups (Figure 20B). In mucosal tissues, similar to systemic sites, SIV_maC25i infection caused an increase in CD8+ T-cell proliferation; however, the extent of this increase was similar in both HTLV-II pre-infected animals and controls (Figure 20C). These observations were confirmed by immunohistochemistry for CD8+ and Ki67 in the rectum (Figures 2OD and 20E). Overall SIV_maC25i infection was associated with a significant increase in the level of proliferating T- cells in blood and tissues. HTLV-II pre-infection did however, not affect the magnitude or the duration of this proliferation. Paradoxically our findings indicated somewhat lower levels of proliferation in the HTLV-II infected animals; however, in general these differences were not statistically significant from the level in the SIV_maC25i mono-infected macaques.

Limited polvfunctional SIV_mac25i responses in infected animals

[00193] HIV- 1 infection induces measurable virus-specific T-cell responses; however, these responses are not of sufficient quality and/or quantity to eliminate virus replication (Betts, M. R., M. C. Nason, S. M. West, S. C. De Rosa, S. A. Migueles, J. Abraham, M. M. Lederman, J. M. Benito, P. A. Goepfert, M. Connors, M. Roederer, and R. A. Koup. 2006. Blood 107:4781-4789). The goal of any vaccination strategy for HIV-I is to eliminate or at least reduce HIV-I replication so that both transmission and disease progression are prevented. Live attenuated viruses with low levels of virus replication may prime and continually boost immune responses, creating a protective immune response and successfully immunizing the individual. In order to evaluate HTLV-II as a potential platform for an HIV-I vaccine, we sought to determine if HTLV-II pre-infection impacted either the quantity or the quality of the S IV_maC25i- specific response. Previous studies have indicated that HTLV-II infection and specifically, its transactivating Tax II protein can induce IFNγ production (Betts, M. R., M. C. Nason, S. M. West, S. C. De Rosa, S. A. Migueles, J. Abraham, M. M. Lederman, J. M. Benito, P. A. Goepfert, M. Connors, M. Roederer, and R. A. Koup. 2006. Blood 107:478 l-4789)(Biglione, M., O. Vidan, R. Mahieux, M. de Colombo, de los Angeles de Basualdo, M. Bonnet, G. Pankow, M. A. De Efron, A. Zorrilla, F. Tekaia, E. Murphy, G. de The, and A. Gessain. 1999. AIDS Res. Hum. Retroviruses 15:407-417). The quality of an HIV-I or S IV_maC25i- specific immune response has been determined by monitoring the ability of CD4+ and CD8+ T-cells to respond to viral antigens by secreting multiple cytokines and chemokines. Using polychromatic flow cytometry, we assessed and the ability of CD4+ and CD8+ T-cells to simultaneously produce the cytokines IFNγ and/or TNFα, IL2 and IL17 and evaluated the magnitude of the S IV_maC25i- specific response. Overlapping peptide pools to SIV_maC25i Gag and SIV_maC25i Env were used to stimulate mononuclear cells from the blood. Figure 2OA shows representative flow cytometric plots of CD8+ T-cells either unstimulated, or stimulated with SIV_maC25i Gag or SIV_maC25i Env. The proportion of CD+ and CD4+ T-cells responding to SIV_maC25i Env stimulation by concurrently producing multiple cytokines (3 or 2 functions) was not significantly different in HTLV-H/SIV_maC25i co-infected animals compared to SIV_maC25i singly infected controls (Figures 21B and 22A). Similarly, no difference in polyfunctional responses was observed in CD8+ T-cells stimulated with SIV_maC25i Gag (data not shown). Figures 21C and 21D show the magnitude of the mean SIV_maC25i-specific response following SIV_maC25i infection. Overall, the immune response to SIV_maC25i Env was of greater magnitude than the response to Gag; however, no clear differences in the magnitude of the response to either antigen was observed between HTLV-H/SIV_maC25i co- infected animals and controls. Figure 22B and 22C show the magnitude of the mean SIV_maC25i -specific response in CD4+ T-cells. Again, there were no significant differences between groups. In all, this data demonstrated that pre-infection with HTLV-II neither enhances nor limits the SIV_maC25i- specific immune responses.

DISCUSSION

[00194] HTLV-II infection is prevalent among intravenous drug users (IDU) co- infected with HIV-I (Turci, M., E. Pilotti, P. Ronzi, G. Magnani, A. Boschini, S. G. Parisi, D. Zipeto, A. Lisa, C. Casoli, and U. Bertazzoni. 2006. J Acquir. Immune. Defic. Syndr. 41:100- 106). The effects of HTLV-II infection on progression to AIDS are controversial as multiple studies have suggested a protective role for HTLV-II in HTLV-II/HIV-1 co-infected patients while others have reported no beneficial effect. In the former studies the maintenance of CD4+ T-cells, lower levels of virus replication and immune activation has been reported in dually infected HTLV-II/HIV- 1 individuals, versus patients infected with HIV-I only (Bassani, S., M. Lopez, C. Toro, V. Jimenez, J. M. Sempere, V. Soriano, and J. M. Benito. 2007. Clin. Infect. Dis. 44: 105-110)( Casoli, C, E. Pilotti, and U. Bertazzoni. 2007. AIDS Rev. 9:140-149)( Giacomo, M., E. G. Franco, C. Claudio, C. Carlo, D. A. Anna, D. Anna, and F. Franco. 1995. Eur. J Epidemiol. 11:527-533). Additionally, several IDU long-term non progressors with stable CD4+ counts, in the absence of antiretro viral therapy, have been reported to be also infected with HTLV-II (Turci, M., E. Pilotti, P. Ronzi, G. Magnani, A. Boschini, S. G. Parisi, D. Zipeto, A. Lisa, C. Casoli, and U. Bertazzoni. 2006. J Acquir. Immune. Defic. Syndr. 41: 100-106) (Willy, R. J., C. M. Salas, G. E. Macalino, and J. D. Rich. 1999. Diagn. Microbiol. Infect. Dis. 35:269-270). A possible mechanism underlying the milder disease course has been ascribed to the increased expression of β chemokines in HTLV-II- infected patients (Casoli, C, E. Vicenzi, A. Cimarelli, G. Magnani, P. Ciancianaini, E. Cattaneo, P. Dall'Aglio, G. PoIi, and U. Bertazzoni. 2000. Blood 95:2760-2769)(Lewis, M. J., V. W. Gautier, X. P. Wang, M. H. Kaplan, and W. W. Hall. 2000. J Immunol 165:4127- 4132)(Pilotti, E., L. Elviri, E. Vicenzi, U. Bertazzoni, M. C. Re, S. Allibardi, G. PoIi, and C. Casoli. 2007. Blood 109:1850-1856), particularly of MIPIa, a chemochine that inhibits viral entry by binding to CCR5. Despite many years since the discovery of HTLV-II/HIV- 1 co- infection, little is known about the effects of HTLV-II on the HIV-specific response and on viral and T-cell dynamics in the blood and tissues. Clinical studies in dually infected patients have provided useful information, but lengths of infection, routes of infection, pathogenicity of virus isolates, and drug use, all of which can affect the immune system, are not easily controlled for. Indeed cross-sectional studies demonstrating differences in immunological parameters such as immune activation and cytokine secretion in dually infected patients versus controls (Bassani, S., M. Lopez, C. Toro, V. Jimenez, J. M. Sempere, V. Soriano, and J. M. Benito. 2007. Clin. Infect. Dis. 44: 105-110) may be more reflective of the levels of HIV- 1 replication rather than an effect of HTLV-II infection. Thus, an animal model where the route, dose, and length of each infection can be controlled can provide useful information. Here, we have established a model of HTLV-II infection and SIV_maC25i co-infection in rhesus macaques. HTLV-II infection of macaques was persistent as demonstrated by the durability of serum antibodies to HTLV-II antigens and the detection of proviral DNA in the blood and tissues of the infected macaques. The data demonstrated that HTLV-II persists at low level in the lymphoid and importantly in the mucosal tissues of macaques similar to HTLV-II- infected humans whereby the pro virus DNA levels have been reported to be often undetectable or a few genome copies per million PBMCs (Beilke, M. A., V. L. Traina-Dorge, M. Sirois, A. Bhuiyan, E. L. Murphy, J. M. Walls, R. Fagan, E. L. Winsor, and P. J. Kissinger. 2007. Clin. Infect. Dis. 44:1229-1234)(Turci, M., E. Pilotti, P. Ronzi, G. Magnani, A. Boschini, S. G. Parisi, D. Zipeto, A. Lisa, C. Casoli, and U. Bertazzoni. 2006. J Acquir. Immune. Defic. Syndr. 41:100-106). Repeated exposures to HTLV-II may induce a more robust infection in the blood of rhesus macaques similar to HTLV-I and STLV-I infected animals (Fultz, P. N., T. McGinn, I. C. Davis, J. W. Romano, and Y. Li. 1999. J Infect. Dis. 179:600-611) (Traina-Dorge, V. L., L. N. Martin, R. Lorino, E. L. Winsor, and M. A. Beilke. 2007. J Infect. Dis. 195:562-571). Unlike other co-infection studies with SIV_maC25i and either HTLV-I or STLV-I performed in non-human primates, we observed no indication of pathology associated with HTLV-II infection (Fultz, P. N., T. McGinn, I. C. Davis, J. W. Romano, and Y. Li. 1999. J Infect. Dis. 179:600-611) (Traina-Dorge, V. L., L. N. Martin, R. Lorino, E. L. Winsor, and M. A. Beilke. 2007. J Infect. Dis. 195:562-571). [00195] Recent observations that primary dendritic cells are permissive for HTLV-I, and that infected dendritic cells can efficiently transfer virus to CD4+ T-cells (Jones, K. S., C. Petrow-Sadowski, Y. K. Huang, D. C. Bertolette, and F. W. Ruscetti. 2008. Nat. Med. 14:429-436), together with reports that HTLV- I-infected dendritic cells have been isolated from infected individuals (Hishizawa, M., K. Imada, T. Kitawaki, M. Ueda, N. Kadowaki, and T. Uchiyama. 2004. Br. J Haematol. 125:568-575)(Jones, K. S., C. Petrow-Sadowski, Y. K. Huang, D. C. Bertolette, and F. W. Ruscetti. 2008. Nat. Med. 14:429-436), suggest that dendritic cells may play a role in HTLV-I transmission and/or persistence. In the present work, we show that HTLV-II can also infect rhesus macaques' dendritic cells. HTLV-II infection was not associated with significant changes in CD4+ and CD8+ T-cell counts. Co- infection with SrV_m3C2S1 of HTLV-II infected macaques did not affect the level of SIV_maC25i RNA in tissues or plasma; similarly HTLV-II pre-infection did not affect SIV_maC25i-specific immune responses. Accordingly, the rate of decline of systemic and mucosal CD4+ T-cell and specifically CD4+CCR5+ T-cells, the target for SIV_maC25i infection were indistinguishable in singly and dually infected macaques. These results indicate that at least in this experimental model, HTLV-II pre-infection did affect the ability of SIV_maC251 to infect CD4+ T-cells, replicate to high titers and induce a profound loss of CD4+T-cells. HTLV-II infects also CD8+ T-cells which secrete β chemokines such as MIP lα, a natural ligand for CCR5 that inhibits HIV-I and SIV_maC25i infection. SIV_maC25i infection caused a decline in both the percentage and the mean florescent intensity of MIP Ice; however, on T-cells there was no difference between the levels of MIP lα spontaneously produced in HTLV- H/SIV_maC25i infected animals and in controls. HTLV-I is known to stimulate the proliferation of T-cell and in HTLV-I, the clonal expansion of T-cell can lead to neoplastic transformation (Lairmore, M. D. and G. Franchini. 2007, p. 2071-2106. In D. M. Knipe and P. M. Howley (eds.), Fields Virology. Lippincott Williams & Wilkins, Philadelphia). In the case of HTLV- II, long term infection has been associated with increased T-cell number (Bartman, M. T., Z. Kaidarova, D. Hirschkorn, R. A. Sacher, J. Fridey, G. Garratty, J. Gibble, J. W. Smith, B. Newman, A. E. Yeo, and E. L. Murphy. 2008. Blood 112:3995-4002) but no clear association with hematological diseases is proven. Thus, we evaluated the frequency of proliferating CD4+ and CD8+ T-cell in the blood and tissues of macaques pre-infected with HTLV-II. infection caused a generalized increase in activation and T-cell proliferation. The magnitude of this increase peaked in the acute phase but remained elevated into the chronic phase. HTLV-II pre-infection did not appear to exacerbate this proliferation; rather, a nonsignificant trend of lower levels of proliferation was found in the co-infected animals. HTLV- II infection has been demonstrated to affect the activation of signal transducers and activators of transcription (STAT) genes in HTLV- II/HIV-l co-infected patients (Bovolenta, C, E. Pilotti, M. Mauri, B. Panzeri, M. Sassi, P. Dall'Aglio, U. Bertazzoni, G. PoIi, and C. Casoli. 2002. J Immunol 169:4443-4449) and the IFNγ gene promoter can be activated by the HTLV-II trans-activator Tax protein (Brown, D. A., F. B. Nelson, E. L. Reinherz, and D. J. Diamond. 1991. Eur. J. Immunol. 21:1879-1885). However, unlike HTLV-I (Migone, T. S., J. X. Lin, A. Cereseto, J. C. Mulloy, J. J. O'Shea, G. Franchini, and W. J. Leonard. 1995. Science 269:79-81), HTLV-II does not induce constitutive STAT-5 activation (Mulloy, J. C, T.-S. Migone, T. M. Ross, N. Ton, P. L. Green, W. J. Leonard, and G. Franchini. 1998. J. Virol. 72:4408-4412).

[00196] The finding reported here that HTLV-II infects and persist in lymphoid and mucosal tissues, suggest that an attenuated HTLV-II may be able to target antigens to the site of SIV_maC25i/HIV-l replication. The induction of effective and persistent T-cell response at the portal of entry of SIV_maC25i/HIV-l could limit infection by the founder virus and be sufficient to inhibit virus dissemination to distal sites. A similar concept has been explored using the RhCMV in Rhesus macaques (Hansen, S. G., C. Vieville, N. Whizin, L. Coyne- Johnson, D. C. Siess, D. D. Drummond, A. W. Legasse, M. K. Axthelm, K. Oswald, C. M. Trubey, M. Piatak, Jr., J. D. Lifson, J. A. Nelson, M. A. Jarvis, and L. J. Picker. 2009. Nat. Med. 15:293-299)( Franchini, G. 2009. Nat. Med.). Live SIV_mac251 -based attenuated vaccines have demonstrated their efficacy at preventing infection in macaques, but their safety for use in humans has been a concern. Hopefully, HTLV-II based vaccine for HIV-I will provide an opportunity to confirm whether persistent antigen expression is a requirement for an effective vaccine for HIV-I. HTLV-II vectored vaccines, even if attenuated, would also pose regulatory issues. However, should their efficacy be proven, strategies to increase their safety, could also be developed.

Table 3 HTLV-II genomic sequence from NC_001488

LOCUS NCJ)01488 8952 bp ss-RNA linear DEFINITION Human T-lymphotropic virus 2, complete genome. ACCESSION NCJ)01488 VERSION NC_001488.1 GL9626726

COMPLETENESS: full length.

FEATURES Loc ation/Qualifier s source 1..8952

/organism="Human T-Lymphotropic virus 2"

/proviral

/mol_type="genomic RNA"

/db_xref="taxon: 11909"

LTR 1..763

/note="5' LTR" gene 6..119

/locus_tag="HTLV2gp 1"

/db_xref="GeneID: 1491947"

CDS 6..119

/locus_tag="HTLV2gp 1 "

/note=" Predicted by GeneMark" /codon_start=l

/product="tax protein"

/protein _id="NP_597787.1 "

/db_xref="GI: 19263402"

/db xref="GeneID: 1491947" /translation="MATSLPSQPPRASHRPKRSDRLTQTIPSKGSDVSPFF" SEQ ID NO: old_sequence 95

/locus tag="HTLV2gpl"

/citation=[4] gene 316..8751

/locus_tag="HTLV2gsl"

/db_xref="GeneID: 1491941 " misc_RNA 316..8751

/locus_tag="HTLV2gsl "

/product=" virion RNA"

/db_xref="GeneID: 1491941" prim_transcript 316..8751

/locus_tag="HTLV2gsl"

/note="env, tax, rex subgenomic mRNAs [2]" repeat_region 316..562

/note=" terminal repeat 5 'copy" intron 450..5043

/locus_tag="HTLV2gsl"

/note="env, tax, rex subgenomic mRNAs intron 1 [2]" misc_binding 766..783

/locus_tag="HTLV2gsl "

/bound_moiety="Pro-tRNA primer" gene 807..2108

/locus_tag="HTLV2gp2"

/db_xref="GeneID: 1491944" CDS 807..2108

/locus_tag="HTLV2gp2"

/codon_start=l

/product="gag polyprotein"

/protein_id="NP_041002.1 "

/db_xref:"GI:9626727"

/db_xref="GeneID: 1491944" /translation="MGQIHGLSPTPIPKAPRGLSTHHWLNFLQ AA YRLQPRPSDFDFQ

QLRRFLKLALKTPIWLNPIDYSLLASLIPKGYPGRVVEIINILVKNQVSPSAPAAPVP

TPICPTTTPPPPPPPSPEAHVPPPYVEPTTTQCFPILHPPGAPSAHRPWQMKDLQAIK

QEVSSSALGSPQFMQTLRLAVQQFDPTAKDLQDLLQYLCSSLVVSLHHQQLNTLITEA

ETRGMTGYNPMAGPLRMQANNPAQQGLRREYQNLWLAAFSTLPGNTRDPSWAAILQGL

EEPYCAFVERLNV ALDNGLPEGTPKEPILRSLAYSNANKECQKILQ ARGHTNSPLGEM

LRTCQAWTPKDKTKVLVVQPRRPPPTQPCFRCGKVGHWSRDCTQPRPPPGPCPLCQDP

SHWKRDCPQLKPPQEEGEPLLLDLPSTSGTTEEKNSLRGEI" SEQ ID NO: mat_peptide 807..1214

/locus_tag="HTLV2gp2" /product="pl9-gag protein [2]" /protein_id="NP_954566.1"

/db_xref="GI:40018529" mat_peptide 1215..1856

/locus_tag="HTLV2gp2"

/product="p24-gag protein [2]"

/protein_id="NP_954567.1 "

/db_xref="GI:40018530" mat_peptide 1857..2105

/locus_tag="HTLV2gp2"

/product="pl5-gag protein [2]"

/protein_id="NP_954568.1 "

/db_xref="GI:40018531" gene 2239..5187

/locus_tag="HTLV2gp3"

/db_xref="GeneID: 1491943" CDS <2239..5187

/locus_tag="HTLV2gp3"

/note="NH2-terminus uncertain"

/codon_start=l

/product="pol polyprotein"

/protein_id="NP_041003.1 "

/db_xref="GI:9626728"

/db_xref="GeneID: 1491943"

/translation="HRSRPYGYTPDTRARAGKAPRHPDPRRQWANQHPVQTPPNPPTH

IL ALPKVPR YPFLLPLRHPQQMDHHWKGRPTTMPGASIPPRRPQPPPIAANSHSKHHR

PRTPSPTSPSGPISFKPERLQALNDLVSKALEAGHIEPYSGPGNNPVFPVKKPNGKWR

FIHDLRATNAITTTLTSPSPGPPDLTSLPTALPHLQTIDLTDAFFQIPLPKQYQPYFA

FTIPQPCNYGPGTRYAWTVLPQGFKNSPTLFEQQLAAVLNPMRKMFPTSTIVQYMDDI

LLASPTNEELQQLSQLTLQALTTHGLPISQEKTQQTPGQIRFLGQVISPNHITYESTP

TIPIKSQWTLTELQVILGEIQWVSKGTPILRKHLQSLYSALHGYRDPRACITLTPQQL

HALHAIQQALQHNCRGRLNPALPLLGLISLSTSGTTSVIFQPKQNWPLAWLHTPHPPT

SLCPWGHLLACTILTLDKYTLQHYGQLCQSFHHNMSKQALCDFLRNSPHPSVGILIHH

MGRFHNLGSQPSGPWKTLLHLPTLLQEPRLLRPIFTLSPVVLDTAPCLFSDGSPQKAA

YVLWDQTILQQDITPLPSHETHSAQKGELLALICGLRAAKPWPSLNIFLDSKYLIKYL

HSLAIGAFLGTSAHQTLQAALPPLLQGKTIYLHHVRSHTNLPDPISTFNEYTDSLILA

PLVPLTPQGLHGLTHCNQRALVSFGATPREAKSLVQTCHTCQTINSQHHMPRGYIRRG

LLPNHIWQGDVTHYKYKK YKYCLHVWVDTFSGA VSVSCKKKETSCETISAVLQ AISLL

GKPLHINTDNGP AFLSQEFQEFCTSYRIKHSTHIP YNPTSSGL VERTNGVIKNLLNKY LLDCPNLPLDNAIHKALWTLNQLNVMNPSGKTRWQIHHSPPLPPIPEASTPPKPPPKW FYYKLPGLTNQRWKGPLQSLQEAAGAALLSIDGSPRWIPWRFLKKAACPRPDASELAE

HAATDHQHHG" SEQ ID NO: gene 5121..7663

/locus_tag="HTLV2gp4"

/db_xref="GeneID: 1491945" CDS join(5121..5183,7214..7663)

/locus_tag="HTLV2gp4"

/codon_start=l

/product="rex 26 kD protein"

/protein_id="NP_041004.1 "

/db_xref="GI:9626729"

/db_xref="GeneID: 1491945"

/translation="MPKTRRQRTRRARRNRPPTPWPISQDLDRASYMDTPSTCLAIVY RPIGVPSQVVYVPPAYIDMPSWPPVQSTNSPGTPSMDALSALLSNTLSLASPPSPPRE PQGPSRSLPLPPLLSPPRFHLPSFNQCESTPPTEMDAWNQPSGISSPPSPSPNLASVP

KTSTPPGEKP" SEQ ID NO: gene 5180..8205

/locus_tag="HTLV2gp5"

/db_xref="GeneID: 1491946" CDS join(5180..5183,7214..8205)

/locus_tag="HTLV2gp5"

/codon_start=l

/product="tax protein"

/protein_id="NP_041005.1 "

/db_xref="GI:9626731"

/db_xref="GeneID: 1491946"

/translation="MAHFPGFGQSLLYGYPVYVFGDCVQADWCPVSGGLCSTRLHRHA LLATCPEHQLTWDPIDGRVVSSPLQYLIPRLPSFPTQRTSRTLKVLTPPTTPVSPKVP PAFFQSMRKHTP YRNGCLEPTLGDQLPSLAFPEPGLRPQNIYTTWGKTVVCLYLYQLS PPMTWPLIPHVIFCHPRQLGAFLTKVPLKRLEELLYKMFLHTGTVIVLPEDDLPTTMF QPVRAPCIQTAWCTGLLPYHSILTTPGLIWTFNDGSPMISGPYPKAGQPSLVVQSSLL IFEKFETKAFHPSYLLSHQLIQYSSFHNLHLLFDEYTNIPVSILFNKEEADDNGD" SEQ ID NO: gene 5180..6640

/locus_tag="HTLV2gp6"

/db_xref="GeneID: 1491942" CDS 5180..6640

/locus_tag="HTLV2gp6"

/note=" (putative; first expressed exon); putative"

/number=2

/codon_start=l

/product="env propeptide"

/protein id="NP_041006.1"

/db_xref="GI:9626730"

/db_xref="GeneID: 1491942"

/translation="MGNVFFLLLFSLTHFPLAQQSRCTLTIGISSYHSSPCSPTQPVC

TWNLDLNSLTTDQRLHPPCPNLITYSGFHKTYSLYLFPHWIKKPNRQGLGYYSPSYND

PCSLQCPYLGCQAWTSAYTGPVSSPSWKFHSDVNFTQEVSQVSLRLHFSKCGSSMTLL VDAPGYDPLWFITSEPTQPPPTSPPLVHDSDLEHVLTPSTSWTTKILKFIQLTLQSTN YSCMVCVDRSSLSSWHVLYTPNISIPQQTSSRTILFPSLALP APPSQPFPWTHCYQPR LQAITTDNCNNSIILPPFSLAPVPPPATRRRRAVPIAVWLVSALAAGTGIAGGVTGSL SLASSKSLLLEVDKDISHLTQAIVKNHQNILRVAQYAAQNRRGLDLLFWEQGGLCKAI QEQCCFLNISNTHVSVLQERPPLEKR VITGWGLNWDLGLSQW ARE ALQTGITIL ALLL

LVILFGPCILRQIQALPQRLQNRHNQYSLINPETML" SEQ ID NO: mat-peptide 6104..6637

/locus_tag=" HTLV2gp6 " /product="p21E envelope protein" /protein_id="NP_954569.1 " /db_xref="GI:40018532" intron 5184..7213

/locus_tag="HTLV2gp4" /note="tax, rex subgenomic RNA intron 2" variation 6229

/locus_tag=" HTLV2gp6 " /note="c in [3]; g in [2]" /replace="g"

LTR 8190..8952 /note="3' LTR" polyA_signal 8458..8463

/locus_tag="HTLV2gsl " /note="mRNA polyadenylation signal" repeat_region 8505..8751 /note=" terminal repeat 3' copy"

ORIGIN

1 tgacaatggc gactagcctc ccaagccagc cacccagggc gagtcatcga cccaaaaggt 61 cagaccgtct cacacaaaca atcccaagta aaggctctga cgtctccccc tttttttagg

121 aactgaaacc acggccctga cgtccctccc ccctaggaac aggaacagct ctccagaaaa

181 aaatagacct cacccttacc cacttcccct agcgctgaaa aacaaggctc tgacgattac

241 cccctgccca taaaatttgc ctagtcaaaa taaaagatgc cgagtctata aaagcgcaag

301 gacagttcag gaggtggctc gctccctcac cgaccctctg gtcacggaga ctcaccttgg

361 ggatccatcc tctccaagcg gcctcggttg agacgccttc cgtgggaccg tctcccggcc

421 tcggcacctc ctgaactgct cctcccaagg taagtctcct ctcaggtcga gctcggctgc

481 cccttaggta gtcgctcccc gagggtcttt agagacaccc gggtttccgc ctgcgctcgg

541 ctagactctg ccttaaactt cacttccgcg ttcttgtctc gttctttcct cttcgccgtc

601 actgaaaacg aaacctcaac gccgccctct tggcaggcgt cccggggcca acatacgccg

661 tggagcgcag caagggctag ggcttcctga acctctccgg gagaggtcta ttgctatagg

721 caggcccgcc ctaggagcat tgtcttcccg gggaagacaa acaattgggg gctcgtccgg

781 gatttgaatt cctccattct cacattatgg gacaaatcca cgggctttcc ccaactccaa

841 tacccaaagc ccccaggggg ctatcaaccc accactggct taactttctc caggctgctt

901 accgcttgca gcctaggccc tccgatttcg acttccagca gctacgacgc tttctaaaac

961 tagcccttaa aacgcccatt tggctaaatc ctattgacta ctcgctttta gctagcctta 1021 tccccaaggg atatccagga agggtggtag agattataaa tatccttgtc aaaaatcaag 1081 tctcccctag cgcccccgcc gccccagttc cgacacctat ctgccctact actactcctc 1141 cgccacctcc ccccccttcc ccggaggccc atgttccccc cccttacgtg gaacccacca 1201 ccacgcaatg cttccctatc ttacatcccc caggagcccc ctcagctcat aggccctggc 1261 agatgaaaga cttacaggcc atcaagcagg aggtcagctc ctctgctctt ggcagccccc 1321 agttcatgca gaccctccgg ctggcggtac aacagtttga ccccaccgcc aaggacttac 1381 aagatctcct ccagtaccta tgctcctccc tcgtagtttc cttacaccat cagcagctta 1441 acacactaat taccgaggct gagacccgcg ggatgacagg ctacaacccc atggcagggc 1501 ccctaagaat gcaggctaat aaccccgccc agcaaggtct tagacgggag taccagaatc 1561 tttggctggc tgctttctcc accctgccag gcaatacccg tgacccctct tgggcagcta 1621 tcctacaggg gctggaggaa ccctattgcg cgttcgtaga gcgccttaac gtggcccttg 1681 acaacggcct ccccgagggt acccccaaag agcccatctt acgttcccta gcgtactcaa

1741 acgccaacaa agaatgccaa aaaatcttac aagcccgcgg acacactaac agcccccttg

1801 gggagatgct ccggacatgt caggcgtgga cacccaagga caaaaccaag gtccttgtgg 1861 tccaaccacg gaggcccccc cccacacagc cctgctttcg ttgtggcaag gtaggacact 1921 ggagtcggga ctgtacccag ccacgccccc ctcctggccc ctgcccccta tgccaagatc

1981 cttctcactg gaaaagggac tgcccacaac tcaaaccccc tcaggaggaa ggggaacccc

2041 tcctgttgga tctcccttcc acctcaggca ctactgagga aaaaaactcc ttaagggggg

2101 agatctaatc tccccccatc ccgatcaaga catctcgata ctcccactca tccccctgcg

2161 gcagcaacag caaccaattc taggggtccg gatctccgtt atgggacaaa cacctcagcc

2221 tacccaagcg ctacttgaca caggagccga ccttacggtt ataccccaga cactcgtgcc

2281 cgggccggta aagctccacg acaccctgat cctaggcgcc agtgggcaaa ccaacaccca

2341 gttcaaactc ctccaaaccc ccctacacat attcttgccc ttccgaaggt cccccgttat

2401 cctttcctcc tgcctcttag acacccacaa caaatggacc atcattggaa gggacgccct

2461 acaacaatgc caggggcttc tatacctccc agacgacccc agcccccacc aattgctgcc

2521 aatagccact ccaaacacca taggcctcga acaccttccc ccacctcccc aagtggacca

2581 atttccttta aacctgagcg cctccaggcc ttaaatgacc tggtctccaa ggccctggag

2641 gctggtcaca ttgaaccata ctcaggacca ggcaataacc ccgtcttccc cgttaaaaaa

2701 ccaaatggta aatggaggtt cattcatgac ctaagagcca ccaatgccat tactaccacc

2761 ctcacctctc cttccccagg gccccccgat ctcactagcc taccgacagc cttaccccac

2821 ctacagacca tagatcttac tgacgccttt ttccaaatcc ccctccccaa gcagtaccag

2881 ccatacttcg ccttcaccat tccccagcca tgtaactatg gccccgggac cagatatgca

2941 tggactgtcc ttccacaggg gtttaaaaac agccccaccc tcttcgaaca acaattagca

3001 gccgtcctca accccatgag gaaaatgttt cccacatcga ccattgtcca atacatggat

3061 gacatacttt tagccagccc caccaatgag gaattacaac aactctccca gctaaccctc

3121 caggcactga ccacgcatgg ccttccaatt tcccaggaaa aaacacaaca aaccccaggc

3181 caaatacgct tcttaggaca ggtcatctcc cctaatcaca ttacatatga gagtacccct

3241 actattccca taaaatccca atggacactc actgaattac aagttatcct aggagagatc

3301 cagtgggtct ctaaaggaac acccatcctt cgcaaacacc tacaatccct atattctgcc

3361 cttcacgggt accgggaccc aagagcttgt atcaccctca ccccacaaca actccatgcg

3421 ttacatgcca ttcaacaagc tctacaacat aactgccgtg gccgcctcaa ccccgcccta

3481 cctctccttg gcctcatctc gttaagtaca tctggtacaa catctgtcat ctttcaaccc

3541 aagcaaaatt ggcccctggc ttggctccac accccccacc ctccgaccag tttatgtcct

3601 tggggtcacc tactggcctg caccatctta actctagaca aatataccct acaacattat

3661 ggccagctct gccaatcttt ccaccacaac atgtcaaagc aagccctttg cgacttcctg

3721 aggaactccc ctcatccaag tgtcggcatc ctcattcacc acatgggtcg attccataac

3781 cttggcagcc aaccgtctgg tccgtggaag actctcttac acctcccaac ccttctccag

3841 gaaccacgac tcctcaggcc aattttcacc ctctcccccg tcgtgcttga cacggccccc

3901 tgcctttttt ccgatggctc ccctcaaaag gcagcgtacg ttctctggga ccagactatc

3961 cttcaacagg acatcactcc cctgccctct cacgaaacac attccgcaca aaagggggag

4021 ctccttgcac ttatctgtgg actacgtgct gccaagccat ggccttccct taacatcttt

4081 ttagactcta aatatttaat caaataccta cattccctcg ccattggggc cttcctcggc

4141 acttccgccc atcaaaccct ccaggcggcc ttgccacccc tactgcaggg caagaccatc

4201 tacctccacc atgtccgcag ccacaccaac ctccccgacc caatttccac cttcaatgaa

4261 tacacagact cccttatctt agctcccctt gttcccctga cgccccaagg cctccacggc

4321 ctcacccatt gcaatcaaag ggctctagtc tcttttggcg ccacaccaag ggaagccaag

4381 tcccttgtac agacttgcca tacctgtcaa accatcaact cacaacatca tatgcctcga

4441 gggtacattc gccggggcct cttgcccaac cacatatggc aaggtgatgt aacccattat

4501 aagtacaaaa aatacaaata ctgcctccac gtctgggtag acaccttctc cggtgcggtt

4561 tccgtctcct gtaaaaagaa agaaaccagc tgtgagacta tcagcgccgt tcttcaggcc

4621 atttccctcc tagggaaacc actccacatt aacacagata atgggccagc cttcctatca

4681 caagaattcc aggagttttg tacctcctat cgcatcaagc attctaccca tataccatac

4741 aaccccacca gctcaggcct ggtcgagaga accaatggtg taatcaaaaa cttactaaat

4801 aaatatctac tagactgtcc taaccttccc ctagacaatg ccattcacaa agccctttgg

4861 actctcaatc agctaaatgt catgaacccc agtggtaaaa cccgatggca aatccaccac

4921 agtcctccac taccacccat tcctgaagcc tctacccctc ccaaaccacc tcccaaatgg

4981 ttctattata aactccccgg ccttaccaat cagcggtgga aaggtccatt gcaatccctc

5041 caggaagcgg ccggggcagc cttgctctcc atagacggct ccccccggtg gatcccgtgg

5101 cgattcctga aaaaagctgc atgcccaaga ccagacgcca gcgaactcgc cgagcacgcc

5161 gcaacagacc accaacacca tgggtaatgt tttcttccta cttttattca gtctcacaca

5221 ttttccacta gcccagcaga gccgatgcac actcacgatt ggtatctcct cctaccactc

5281 cagcccctgt agcccaaccc aacccgtctg cacgtggaac ctcgacctta attccctaac

5341 aacggaccaa cgactacacc ccccctgccc taacctaatt acttactctg gcttccataa

5401 gacttattcc ttatacttat tcccacattg gataaaaaag ccaaacagac agggcctagg

5461 gtactactcg ccttcctaca atgacccttg ctcgctacaa tgcccctact tgggctgcca

5521 agcatggaca tccgcataca cgggccccgt ctccagtcca tcctggaagt ttcattcaga 5581 tgtaaatttc acccaggaag tcagccaagt gtcccttcga ctacacttct ctaagtgcgg

5641 ctcctccatg accctcctag tagatgcccc tggatatgat cctttatggt tcatcacctc

5701 agaacccact cagcctccac caacttctcc cccattggtc catgactccg accttgaaca

5761 tgtcctaacc ccctccacgt cctggacgac caaaatactc aaatttatcc agctgacctt

5821 acagagcacc aattactcct gcatggtttg cgtggataga tccagcctct catcctggca

5881 tgtactctac acccccaaca tctccattcc ccaacaaacc tcctcccgaa ccatcctctt

5941 tccttccctt gccctgcccg ctcctccatc ccaacccttc ccttggaccc attgctacca

6001 acctcgccta caggcgataa caacagataa ctgcaacaac tccattatcc tccccccttt

6061 ttccctcgct cccgtacctc ctccggcgac aagacgccgc cgtgccgttc caatagcagt

6121 gtggcttgtc tccgccctag cggccggaac aggtatcgct ggtggagtaa caggctccct

6181 atctctggct tccagtaaaa gccttctcct cgaggttgac aaagacatct cccaccttac

6241 ccaggccata gtcaaaaatc atcaaaacat cctccgggtt gcacagtatg cagcccaaaa

6301 tagacgagga ttagacctcc tattctggga acaagggggt ttgtgcaagg ccatacagga

6361 gcaatgttgc ttcctcaaca tcagtaacac tcatgtatcc gtcctccagg aacggccccc

6421 tcttgaaaaa cgtgtcatca ccggctgggg actaaactgg gatcttggac tgtcccaatg

6481 ggcacgagaa gccctccaga caggcataac cattctcgct ctactcctcc tcgtcatatt

6541 gtttggcccc tgtatcctcc gccaaatcca ggcccttcca cagcggttac aaaaccgaca

6601 taaccagtat tcccttatca acccagaaac catgctataa tagacctgct agcttctgca

6661 gcaaatcccc taggttcgtc cccctaccat tgacccatcc acagtcctct ataccagatg

6721 agtcgccccc gatgtccagc cctaactcga ttctgaataa ttgcctcaaa tagttcctct

6781 aacccccgct cacattcctc ccataggacc ttcttttccc cttcaggaaa tccacataac

6841 cctgaagcaa gtcacaaaac ccatcaaaac ccaggagtcc tatacactcc aactgctgat

6901 gcctttcttc cctctcccgg cgcttttgat ccttttcccg caggcgctcc tttctgcgcc

6961 gctcccgctc ctcacgctcc tgcagaagtt ttaagatctc ccgctgctcc tccgccaaca

7021 gtctccgacg agagtctcgc acctgctcgc tgaccgatcc cgaccccaga gggcgacctt

7081 ttgctgtcct tctcggttcc tctccagggg gaggcacacc agatgtcaga ctcgcctctc

7141 cctggtctcc taacggcaat ctcctaaaat agtctaaaaa atcacacata attacaatcc

7201 tgtctcctct cagcccattt cccaggattt ggacagagcc tcctatatgg ataccccgtc

7261 tacgtgtttg gcgattgtgt acaggccgat tggtgtcccg tctcaggtgg tctatgttcc

7321 acccgcctac atcgacatgc cctcctggcc acctgtccag agcaccaact cacctgggac

7381 cccatcgatg gacgcgttgt cagctctcct ctccaatacc ttatccctcg cctcccctcc

7441 ttccccaccc agagaacctc aaggaccctc aaggtcctta cccctcccac cactcctgtc

7501 tcccccaagg ttccacctgc cttctttcaa tcaatgcgaa agcacacccc ctaccgaaat

7561 ggatgcctgg aaccaaccct cggggatcag ctcccctccc tcgccttccc cgaacctggc

7621 ctccgtcccc aaaacatcta caccacctgg ggaaaaaccg tagtatgcct atacctatac

7681 cagctttccc cacccatgac atggccactt ataccccatg tcatattctg ccaccccaga

7741 caattaggag ccttcctcac caaggtgcct ctaaaacgat tagaagaact tctatacaaa

7801 atgttcctac acacagggac agtcatagtc ctcccggagg acgacctacc caccacaatg

7861 ttccaacccg tgagggctcc ctgtatccag actgcctggt gtacaggact tctcccctat

7921 cactccatct taacaacccc aggtctaata tggaccttca atgacggctc accaatgatt

7981 tccggccctt accccaaagc agggcagcca tctttagtag ttcagtcctc cctattaatc

8041 ttcgaaaaat tcgaaaccaa agccttccat ccctcctatc tactctctca tcagcttata

8101 caatactcct ccttccataa ccttcacctt ctattcgatg aatacaccaa catccctgtc

8161 tctattttat ttaataaaga agaggcggat gacaatggcg actagcctcc cgagccagcc

8221 acccagggcg agtcatcgac ccaaaaggtc agaccgtctc acacaaacaa tcccaagtaa

8281 aggctctgac gtctccccct ttttttagga actgaaacca cggccctgac gtccctcccc

8341 cctaggaaca ggaacagctc tccagaaaaa aatagacctc acccttaccc acttccccta

8401 gcgctgaaaa acaaggctct gacgattacc ccctgcccat aaaatttgcc tagtcaaaat

8461 aaaagatgcc gagtctataa aagcgcaagg acagttcagg aggtggctcg ctccctcacc

8521 gaccctctgg tcacggagac tcaccttggg gatccatcct ctccaagcgg cctcggttga

8581 gacgccttcc gtgggaccgt ctcccggcct cggcacctcc tgaactgctc ctcccaaggt

8641 aagtctcctc tcaggtcgag ctcggctgcc ccttaggtag tcgctccccg agggtcttta

8701 gagacacccg ggtttccgcc tgcgctcggc tagactctgc cttaaacttc acttccgcgt

8761 tcttgtctcg ttctttcctc ttcgccgtca ctgaaaacga aacctcaacg ccgccctctt

8821 ggcaggcgtc ccggggccaa catacgccgt ggagcgcagc aagggctagg gcttcctgaa

8881 cctctccggg agaggtctat tgctataggc aggcccgccc taggagcatt gtcttcccgg

8941 ggaagacaaa ca SEQ ID NO:

Table 6

Description Sequence and SEQ ID NO

Plasmid tcgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggt sequence agcgcaacgaccgcaaaaaggtatccgaggcggggggactgctcgtagtgtttttagctgcgagttcagtctcca including

ORI, ggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccga

AmpR, and ccgctttgggctgtcctgatatttctatggtccgcaaagggggaccttcgagggagcacgcgagaggacaaggct bacterial promoter ccctgcaaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccacgctgta sequences gggacgtttctatggtccgcaaagggggaccttcgagggagcacgcgagaggacaaggctgggacggtgcgacat ggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgct ccatagagtcaagccacatccagcaagcgaggttcgacccgacacacgtgcttggggggcaagtcgggctggcga gcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactg cgcggaataggccattgatagcagaactcaggttgggccattctgtgctgaatagcggtgaccgtcgtcggtgac gtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctaca cattgtcctaatcgtctcgctccatacatccgccacgatgtctcaagaacttcaccaccggattgatgccgatgt ctagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgat gatcttcttgtcataaaccatagacgcgagacgacttcggtcaatggaagcctttttctcaaccatcgagaacta ccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggat ggccgtttgtttggtggcgaccatcgccaccaaaaaaacaaacgttcgtcgtctaatgcgcgtctttttttccta ctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttgg gagttcttctaggaaactagaaaagatgccccagactgcgagtcaccttgcttttgagtgcaattccctaaaacc taatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagta attactctaatagtttttcctagaagtggatctaggaaaatttaatttttacttcaaaatttagttagatttcat tatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttc atatactcatttgaaccagactgtcaatggttacgaattagtcactccgtggatagagtcgctagacagataaag gttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtg caagtaggtatcaacggactgaggggcagcacatctattgatgctatgccctcccgaatggtagaccggggtcac ctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccg gacgttactatggcgctctgggtgcgagtggccgaggtctaaatagtcgttatttggtcggtcggccttcccggc agcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagta tcgcgtcttcaccaggacgttgaaataggcggaggtaggtcagataattaacaacggcccttcgatctcattcat gttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggta caagcggtcaattatcaaacgcgttgcaacaacggtaacgatgtccgtagcaccacagtgcgagcagcaaaccat tggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggtta accgaagtaagtcgaggccaagggttgctagttccgctcaatgtactagggggtacaacacgttttttcgccaat gctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgc cgaggaagccaggaggctagcaacagtcttcattcaaccggcgtcacaatagtgagtaccaataccgtcgtgacg ataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgag tattaagagaatgacagtacggtaggcattctacgaaaagacactgaccactcatgagttggttcagtaagactc aatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactt ttatcacatacgccgctggctcaacgagaacgggccgcagttatgccctattatggcgcggtgtatcgtcttgaa taaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagtt attttcacgagtagtaaccttttgcaagaagccccgcttttgagagttcctagaatggcgacaactctaggtcaa cgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaa gctacattgggtgagcacgtgggttgactagaagtcgtagaaaatgaaagtggtcgcaaagacccactcgttttt caggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttc gtccttccgttttacggcgttttttcccttattcccgctgtgcctttacaacttatgagtatgagaaggaaaaag aatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaac ttataataacttcgtaaatagtcccaataacagagtactcgcctatgtataaacttacataaatctttttatttg aaataggggttccgcgcacatttccccgaaaagtgccacctgacgacgacggatcgggagatcaacttgtttatt tttatccccaaggcgcgtgtaaaggggcttttcacggtggactgctgctgcctagccctctagttgaacaaataa gcagcttataatggttacaaggcgcc SEQ ID NO: cgtcgaatattaccaatgttccgcgg SEQ ID NO: ( in rever se )

HTLV-II tgacaatggcgactagcctcccaagccagccacccagggcgagtcatcgacccaaaaggtcagaccgtctcacac 5' LTR actgttaccgctgatcggagggttcggtcggtgggtcccgctcagtagctgggttttccagtctggcagagtgtg aaacaatcccaagtaaaggctctgacgtctccccctttttttaggaactgaaaccacggccctgacgtccctccc tttgttagggttcatttccgagactgcagagggggaaaaaaatccttgactttggtgccgggactgcagggaggg ccctaggaacaggaacagctctccagaaaaaaatagacctcacccttcccacttcccctagcgctgaaaaacaag gggatccttgtccttgtcgagaggtctttttttatctggagtgggaagggtgaaggggatcgcgactttttgttc gctctgacgattaccccctgcccataaaatttgcctagtcaaaataaaagatgccgagtctataaaagcgcaagg cgagactgctaatgggggacgggtattttaaacggatcagttttattttctacggctcagatattttcgcgttcc acagttcaggaggtggctcgctccctcaccgaccctctggtcacggagactcaccttggggatcaatcctctcca tgtcaagtcctccaccgagcgagggagtggctgggagaccagtgcctctgagtggaacccctagttaggagaggt agcggcctcggttgagacgccttccgtgggaccgtctcccggcctcggcacctcctgaactgctcctcccaaggt tcgccggagccaactctgcggaaggcaccctggcagagggccggagccgtggaggacttgacgaggagggttcca aagtctcctctcaggtcgagctcggctgccccttaggtagtcgctccccgagggtctttagagacacccgggttt ttcagaggagagtccagctcgagccgacggggaatccatcagcgaggggctcccagaaatctctgtgggcccaaa ccgcctgcgctcggctagactctgccttaaacttcacttccgcgttcttgtctcgttctttcctcttcgccgtca ggcggacgcgagccgatctgagacggaatttgaagtgaaggcgcaagaacagagcaagaaaggagaagcggcagt ctgaaaacgaaacctcaacgccgccctcttggcaggcgtcccggggccaacatacgccgtggagcgcagcaaggg gacttttgctttggagttgcggcgggagaaccgtccgcagggccccggttgtatgcggcacctcgcgtcgttccc ctagggcttcctgaacctctccgggagaggtctattgctataggcaggcccgccctaggagcattgtcttcccgg gatcccgaaggacttggagaggccctctccagataacgatatccgtccgggcgggatcctcgtaacagaagggcc ggaagacaaac SEQ ID NO: ccttctgtttg SEQ ID NO: (in reverse)

HTLV-II gattgggggctcgtccgggatttaaattcctccattctcacattatgggacaaatccacgggctttccccaactc gag ψ ctaacccccgagcaggccctaaatttaaggaggtaagagtgtaataccctgtttaggtgcccgaaaggggttgag signal and PPT caatacccaaagcccccagggggctatcaacccaccactggcttaactttctccaggctgcttaccgcttgcagc gttatgggtttcgggggtcccccgatagttgggtggtgaccgaattgaaagaggtccgacgaatggcgaacgtcg ctaggccctccgatttcgacttccagcagctacgacgctttctaaaactagcccttaaaacgcccatttggctaa gatccgggaggctaaagctgaaggtcgtcgatgctgcgaaagattttgatcgggaattttgcgggtaaaccgatt

HTLV-II tgacaatggcgactagcctcccgagccagccacccagggcgagtcatcgacccaaaaggtcagaccgtctcacac 3'LTR actgttaccgctgatcggagggctcggtcggtgggtcccgctcagtagctgggttttccagtctggcagagtgtg aaacaatcccaagtaaaggctctgacgtctccccctttttttaggaactgaaaccacggccctgacgtccctccc tttgttagggttcatttccgagactgcagagggggaaaaaaatccttgactttggtgccgggactgcagggaggg ccctaggaacaggaacagctctccagaaaaaaatagacctcacccttacccacttcccctagcgctgaaaaacaa gggatccttgtccttgtcgagaggtctttttttatctggagtgggaatgggtgaaggggatcgcgactttttgtt ggctctgacgattaccccctgcccataaaatttgcctagtcaaaataaaagatgccgagtctataaaagcgcaag ccgagactgctaatgggggacgggtattttaaacggatcagttttattttctacggctcagatattttcgcgttc gacagttcaggaggtggctcgctccctcaccgaccctctggtcacggagactcaccttgggaatccatcctctcc ctgtcaagtcctccaccgagcgagggagtggctgggagaccagtgcctctgagtggaacccttaggtaggagagg aagcggcctcggttgagacgccttccgtgggaccgtctcccggcctcggcacctcctgaactgctcctcccaagg ttcgccggagccaactctgcggaaggcaccctggcagagggccggagccgtggaggacttgacgaggagggttcc taagtctcctctcaggtcgagctcggctgccccttaggtagtcgctccccgagggtctttagagacacccgggtt attcagaggagagtccagctcgagccgacggggaatccatcagcgaggggctcccagaaatctctgtgggcccaa tccgcctgcgctcggctagactctgccttaaacttcacttccgcgttcttgtctcgttctttcctcttcgccgtc aggcggacgcgagccgatctgagacggaatttgaagtgaaggcgcaagaacagagcaagaaaggagaagcggcag actgaaaacgaaacctcaacgccgccctcttggcaggcgtcccggggccaacatacgccgtggagcgcagcaagg tgacttttgctttggagttgcggcgggagaaccgtccgcagggccccggttgtatgcggcacctcgcgtcgttcc gctagggcttcctgaacctctccgggagaggtctattgctataggcaggcccgccctaggagcattgtcttcccg cgatcccgaaggacttggagaggccctctccagataacgatatccgtccgggcgggatcctcgtaacagaagggc gggaagacaaacaagatcta SEQ ID NO: cccttctgtttgttctagat SEQ ID NO: (in reverse)

Table 7

Claims

CLAIMSWhat is claimed is:

1. An isolated viral vector comprising at least a portion of the HTLV-II genome encoding the gag, pro, and pol genes and lacking all or a portion of the pX region and a polynucleotide encoding all or a portion of a protein that corresponds to a viral protein from a heterologous virus located within the deletion in the pX region.

2. The viral vector of claim 1, wherein HTLV-II genome encodes gag, pro, pol, env, tax, and rex.

3. The viral vector of claim 1, comprising the polynucleotide sequence of Table 3 with a deletion of nucleotides 6645 to 7153.

4. The viral vector of claim 1, wherein the heterologous virus is SIV or HIV.

5. The viral vector of claim 1, wherein the protein is a consensus sequence from several clades for anyone of the viral proteins selected from the group consisting of Vif, Tat, Gag, Env, Rev, gpl20, gp41, p24, p7, pl7, Tev, and combinations thereof or immunogenic fragments thereof.

6. The viral vector of claim 1, wherein the vector can replicate in a human T cell line or a human dendritic cell.

7. A composition comprising a viral vector of claim 1 and a carrier.

8. The composition of claim 7, further comprising at least two different viral vectors, wherein each of the viral vectors comprise a polynucleotide encoding a different viral protein.

9. The composition of claim 7, further comprising an adjuvant or immunomodulator and wherein the adjuvant or immunomodulator is encoded by a polynucleotide.

10. A method for increasing an immune response to a virus infection in a subject, comprising administering to the subject a composition of claim 7, wherein the subject is infected with a virus that corresponds to the heterologous viral protein encoded by the polynucleotide in the viral vector.

11. The method of claim 10, wherein the virus is HIV or SIV.

12. A method for inhibiting a viral infection in a subject, comprising administering to the subject a composition of claim 10, wherein the subject is infected with a virus that corresponds to the heterologous viral protein encoded by the polynucleotide in the viral vector.

13. The method of claim 12, further comprising screening the subject for the presence of antibodies to HTLV-II antigens and for the presence of HTLV-II proviral DNA.

14. The method of claim 12, wherein the virus is HIV or SIV.

15. An isolated viral vector comprising an HTLV-II 5'LTR and HTLV-II 3'LTR having a HTLV-II gag signal, one or more IRES sequences, one or more SV40 poly A signal sequences and a CTE encompassed therebetween and wherein one or more genes of interest can be insterted between the HTLV-II 5'LTR and HTLV-II 3'LTR.

16. The isolated viral vector of claim 15 further comprising one to five genes of interest and wherein the one to five genes of interest are in appropriate functional relationship to the HTLV-II gag signal, one or more IRES sequences, one or more SV40 poly A signal sequences and the CTE.

17. The isolated viral vector of claim 16 wherein one of the genes of interest encodes all or a portion of a viral protein.

18. The isolated viral vector of claim 16 wherein one of the genes of interest encodes all or a portion of a tumor antigen.

19. The isolated viral vector of claim 16 having two genes of interest, one gene of interest oriented in a forward direction and one gene of interest in a reverse direction.

20. The isolated viral vector of claim 16 having three genes of interest, one gene of interest oriented in a forward direction and two genes of interest in the reverse direction.

21. The isolated viral vector of claim 20 wherein the gene of interest in the forward direction encodes at least a portion of an adjuvant polypeptide.

22. A composition comprising a viral vector of claim 16 and a carrier.

23. A method for increasing an immune response to a virus infection in a subject, comprising administering to the subject a composition of claim 16, wherein the subject is infected with a virus that corresponds to the heterologous viral protein encoded by the one or more genes of interest in the viral vector.

24. The method of claim 23 wherein the virus is HIV, SIV, HCV, HSV, CMV, or EBV.

25. A method for increasing a cytotoxic immune response to a tumor in a subject, comprising administering to the subject a composition of claim 16, wherein the subject has a tumor that corresponds to the tumor antigen encoded by the one or more genes of interest in the viral vector.

26. A kit, comprising:

(a) a viral vector or a composition of claim 16.

27. Use of the viral vector of claim 16 in the preparation of a medicament for inhibiting HIV or SIV infection in a subject.

28. Use of the viral vector claim 16 in the preparation of a medicament for inducing an immune response to a HIV, SIV, HCV, HSV, CMV, or EBV protein in a subject.

29. The use of claim 28, wherein the subject is a non human primate.

0. The use of claims 28, wherein the subject is a human.