(en)The compositions and methods described herein relate to lentiviral vectors that can be used to generate virions/viruses that exhibit enhanced infectivity with respect to monocyte-derived macrophages (MDM) and dendritic cells (MDDC). Such compositions and methods further relate to production of virions/viruses that can be used as components of vaccines that effectively stimulate innate immune responses. In a particular embodiment, compositions and methods described herein relate to production of virions/viruses that can be used as components of human immunodeficiency-1 (HIV-1) vaccines administered to stimulate innate immune responses to HIV-1.

1.ApplicationNumber: US-201313743877-A
1.PublishNumber: US-2013183334-A1
2.Date Publish: 20130718
3.Inventor: LANDAU NATHANIEL R.
SUNSERI NICOLE
NORTON THOMAS D.

4.Inventor Harmonized: LANDAU NATHANIEL R(US)
SUNSERI NICOLE(US)
NORTON THOMAS D(US)

5.Country: US
6.Claims:
(en)The compositions and methods described herein relate to lentiviral vectors that can be used to generate virions/viruses that exhibit enhanced infectivity with respect to monocyte-derived macrophages (MDM) and dendritic cells (MDDC). Such compositions and methods further relate to production of virions/viruses that can be used as components of vaccines that effectively stimulate innate immune responses. In a particular embodiment, compositions and methods described herein relate to production of virions/viruses that can be used as components of human immunodeficiency-1 (HIV-1) vaccines administered to stimulate innate immune responses to HIV-1.

7.Description:
(en)CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 USC §119(e) from U.S. Provisional Application Ser. No. 61/587,392, filed Jan. 17, 2012, which application is herein specifically incorporated by reference in its entirety.


GOVERNMENTAL SUPPORT
The research leading to the present invention was supported, at least in part, by National Institutes of Health (NIH) grant 5R01 A1067059 and NIH training grant 5T32 A1007180. Accordingly, the Government has certain rights in the invention.


FIELD OF INVENTION
The compositions and methods described herein relate to lentiviral vectors that can be used to generate virions that exhibit enhanced infectivity with respect to monocyte-derived macrophages (MDM) and dendritic cells (MDDC). More particularly, compositions and methods described herein relate to production of virions that can be used as components of vaccines that effectively stimulate innate immune responses. In a particular embodiment, compositions and methods described herein relate to production of virions that can be used as components of human immunodeficiency-1 (HIV-1) vaccines that effectively stimulate innate immune responses.
BACKGROUND OF INVENTION
Unlike simple retroviruses, lentiviruses encode a set of accessory proteins (Nef, Vif, Vpr, Vpu, and Vpx), each of which play a specific role in virus replication and pathogenesis, much of which are directed at evasion of the host adaptive or innate immune response (28, 33, 35, 36, 41, 46). Specifically, Nef downregulates cell surface CD4 molecules and disrupts antigen presentation, and Vif and Vpu serve to counteract the host restriction factors, APOBEC3G and BST-2/tetherin, respectively. Although the roles of Vpx and Vpr are less well understood, they are thought to aid in evasion of a yet uncharacterized restriction to virus replication in macrophages and dendritic cells (3, 7, 10, 18, 20, 22, 24, 27, 51). Sharing significant sequence and structural homology, the two proteins appear to have arisen from the duplication of a common ancestral precursor gene (44). Both are produced late in the viral life cycle and localize to the nucleus (5, 12, 31). A Vpr gene is present in the genome of all known lentiviruses, while Vpx is restricted to HIV-2 and the SIV of sooty mangabey (SIVsm) and macaque (SIVmac). Interestingly, SIV of the African green monkey encodes only Vpr, but this protein has some characteristics of Vpx, sharing the same virion packaging determinant and effect on virus replication in macrophages (1, 9).
A distinguishing feature of Vpr and Vpx is that they are packaged into virions at significant levels (2, 23, 50). Packaging of Vpr and Vpx occurs as the virion assembles and is mediated by amino acid motifs in the carboxy-terminal Gag protein p6, such that deletion of p6 prevents incorporation of both Vpr and Vpx (31, 38, 47). The packaging of Vpr and Vpx is also virus-specific (25). HIV-1 will not package SIVmac Vpr or Vpx, nor will SIVmac package HIV-1 Vpr. By analyzing viruses with mutated or truncated p6, various groups have identified amino acid motifs in p6 that mediate Vpr and Vpx incorporation. The motif 41 LXXLF 44 (SEQ ID NO: 58) near the p6 carboxy-terminus was reported by Kondo et al. as the major determinant required for the packaging of Vpr (30). Subsequently, Zhu et al. (53) reported that mutation of that motif did not prevent Vpr packaging. Instead, in a virus deleted for amino acids 35-52, the 15 FRFG 18 motif near the amino-terminus of p6 was required. This discrepancy has not yet been resolved. In SIVmac, the Vpx packaging motif has also been mapped to the N terminal region of p6, specifically to a conserved 17 DXAXXLL 23 (SEQ ID NO: 60) motif (1). Packaging of SIVagm Vpr was also dependent on this motif, drawing a resemblance with Vpx.
Vpr and Vpx are important for virus replication and pathogenicity in vivo, as demonstrated in the rhesus macaque model, where Vpx-deleted SIVmac 239 is attenuated and Vpx/Vpr-deleted virus is further impaired (17). In vitro, neither Vpr nor Vpx is required for SIVmac replication in activated CD4 T cells (3, 18, 19). However, in monocyte-derived macrophages (MDM) and dendritic cells (MDDC), both proteins enhance SIVmac infection, although Vpr has a more modest effect. The presence of Vpx and Vpr in the virion suggests that they play a role early in virus replication. Vpr and Vpx were initially proposed to aid in nuclear import of the preintegration complex in infection of nondividing cells, a mechanism consistent with their karyophilic properties and their presence in the virion (5, 13). However, this view was challenged by the finding that HIV-1 deleted for Vpr maintained its ability to infect nondividing cells (48). Recently, a role for Vpx and Vpr in counteracting a restriction factor was suggested by the finding that both associated with an E3 ubiquitin ligase composed of damaged DNA binding protein 1 (DDB1), DDB1 cullen associated factor 1 (DCAF1), and Cullin 4A (Cul4A) (6, 21, 26, 39, 40, 42). By analogy with Vif, this finding suggested that Vpx and Vpr might act as substrate receptors that induce the ubiquitination of a host restriction factor (41, 43, 49). Evidence that Vpx plays a role in counteracting an MDM-specific host restriction was provided by somatic cell fusion experiments in which heterokaryons formed between MDM and COS were found to be nonpermissive for Vpx-deleted SIVmac (40). Furthermore, domain mapping of Vpx localized an activation domain at the amino-terminus that might serve as a binding site for the putative host restriction factor (21).
In studies investigating the mechanism by which Vpx promotes the infection of MDDC, Goujon et al. found that Vpx could facilitate infection when introduced into the target cell in trans (18, 20). For this, MDDC were exposed to virus-like particles (VLP) that contained Vpx and then were infected with Vpx-deleted SIVmac. The VLP dramatically enhanced the infectability of the cells. Additionally, Gramberg et al. found that VLP generated with a codon-optimized Vpx expression vector could boost the infection of MDM as much as 100-fold, while Vpr had little effect (21). In these cells, Vpx was found to relieve a block to infection at early reverse transcription or at uncoating. Interestingly, Vpx also dramatically enhanced HIV-1 infection of MDM and MDDC, even though the virus does not itself encode this accessory protein. Recently, Manel et al. used this property to achieve high levels of HIV-1 infected MDDC (32). The infected MDDC strongly induced innate immune defenses, resulting in the production of type-I IFN and upregulation of CD86. Surprisingly, the trigger that induced this response was not the in-coming virus but, rather, the newly produced Gag protein.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.
SUMMARY OF THE INVENTION
To date, Vpx has been used to enhance HIV-1 infection of MDM and MDDC by pretreating cells with VLP. In this study, the present inventors sought to investigate the role of Vpx by generating an HIV-1 vector that would package the protein. Such a vector would obviate the need for Vpx-containing VLP. Virions/viruses generated using the resulting vector would have an enhanced ability to infect MDDC and MDM and be expected to induce an innate immune response in the cells. The present inventors constructed a vector having the desired properties by introducing a fragment of SIVmac 239 p6 into HIV-1 p6. In a further embodiment, the vector comprising the introduced fragment of SIVmac 239 p6 into HIV-1 p6 was further modified by placing vpx in the Nef position. More particularly, vpx was inserted into the first codon of Nef This arrangement prevents Nef expression, because protein synthesis is terminated by the vpx stop codon. Virions generated using the resulting vectors efficiently infected MDM and MDDC and induced high levels of type I IFN. In a MDDC/T cell transfer assay, the engineered virions so produced replicated much more efficiently than those generated using wild-type vector. These findings suggest that an important role of Vpx is to enhance the ability of virus to be transmitted from MDM and MDDC to CD4 T cells. Furthermore, such virions are useful for the production of an HIV vaccine that stimulates innate immune responses and for the generation of lentiviral virions that more efficiently infect these cell types.
In accordance with the present findings, a chimeric vector comprising HIV-1 nucleic acid sequences and SIVmac 239 nucleic acid sequences is presented herein, wherein the SIVmac 239 nucleic acid sequences encode an SIVmac 239 amino acid sequence consisting of a minimal Vpx packaging motif that confers Vpx packaging activity to the chimeric vector. In an embodiment thereof, the chimeric vector does not comprise any SIVmac nucleic acid sequences except for or in addition to the minimal Vpx packaging motif.
In a further embodiment, the chimeric vector further comprises vpx inserted in nef of the HIV-1 nucleic acid sequences. In a particular embodiment thereof, the vpx is SIVmac 239 vpx. In a more particular embodiment, the vpx is a codon-optimized SIVmac 239 vpx. In a still further embodiment, the chimeric vector that further comprises vpx, does not comprise any SIVmac 239 nucleic acid sequences except for or in addition to the minimal Vpx packaging motif and the vpx.
In a further aspect, the minimal Vpx packaging motif consists of at least 10 contiguous amino acids of SIVmac 239 comprising of 17 DPAVDLLKNY 26 (SEQ ID NO: 1), wherein the 5′ terminus of the minimal Vpx packaging motif is the aspartic acid (D) at amino acid position 17 of the SIVmac 239 amino acid sequence. More particularly, the minimal Vpx packaging motif consists of







(SEQ ID NO: 1)


17 DPAVDLLKNY 26 ,





(SEQ ID NO: 2)


17 DPAVDLLKNYM 27 ,





(SEQ ID NO: 3)


17 DPAVDLLKNYMG 28 ,





(SEQ ID NO: 4)


17 DPAVDLLKNYMQL 29 ,





(SEQ ID NO: 5)


17 DPAVDLLKNYMQLG 30 ,





(SEQ ID NO: 6)


17 DPAVDLLKNYMQLGK 31 ,





(SEQ ID NO: 7)


17 DPAVDLLKNYMQLGKQ 32 ,





(SEQ ID NO: 8)


17 DPAVDLLKNYMQLGKQQ 33 ,





(SEQ ID NO: 9)


17 DPAVDLLKNYMQLGKQQRE 34 ,





(SEQ ID NO: 10)


17 DPAVDLLKNYMQLGKQQREK 35 ,





(SEQ ID NO: 11)


17 DPAVDLLKNYMQLGKQQREKQ 36 ,





(SEQ ID NO: 12)


17 DPAVDLLKNYMQLGKQQREKQ 37 ,





(SEQ ID NO: 13)


17 DPAVDLLKNYMQLGKQQREKQR 38 ,





(SEQ ID NO: 14)


17 DPAVDLLKNYMQLGKQQREKQRE 39 ,





(SEQ ID NO: 15)


17 DPAVDLLKNYMQLGKQQREKQRES 40 ,





(SEQ ID NO: 16)


17 DPAVDLLKNYMQLGKQQREKQRESR 41 ,





(SEQ ID NO: 17)


17 DPAVDLLKNYMQLGKQQREKQRESRE 42 ,





(SEQ ID NO: 18)


17 DPAVDLLKNYMQLGKQQREKQRESREK 43 ,





(SEQ ID NO: 19)


17 DPAVDLLKNYMQLGKQQREKQRESREKP 44 ,





(SEQ ID NO: 20)


17 DPAVDLLKNYMQLGKQQREKQRESREKPY 45 ,





(SEQ ID NO: 21)


17 DPAVDLLKNYMQLGKQQREKQRESREKPYK 46 ,





(SEQ ID NO: 22)


17 DPAVDLLKNYMQLGKQQREKQRESREKPYKE 47 ,


or





(SEQ ID NO: 23)


17 DPAVDLLKNYMQLGKQQREKQRESREKPYKEV 48 .












































































In a still further aspect, the minimal Vpx packaging motif consists of 17 DPAVDLLKNY 26 (SEQ ID NO: 1).
Chimeric vectors described herein may comprise SIVmac 239 nucleic acid sequences encoding the minimal Vpx packaging motif, wherein SIVmac 239 nucleic acid sequences comprise at least one codon optimized nucleic acid sequence.
In a particular embodiment, the SIVmac 239 nucleic acid sequences encoding the minimal Vpx packaging motif are inserted into the HIV-1 nucleic acid sequences encoding p6 of HIV-1 Gag polyprotein.
In a more particular embodiment, the SIVmac 239 nucleic acid sequences encoding the minimal Vpx packaging motif are inserted into the HIV-1 nucleic acid sequences encoding p6 of HIV-1 Gag polyprotein to generate a hybrid HIV-1/SIVmac 239 nucleic acid sequence that encodes a hybrid HIV-1/SIVmac 239 p6 wherein amino acids 1-14 of HIV-1 p6 are linked directly to the minimal Vpx packaging motif consisting of at least 10 contiguous amino acids of SIVmac 239 p6 comprising of 17 DPAVDLLKNY 26 (SEQ ID NO: 1), wherein the 5′ terminus of the minimal Vpx packaging motif is the aspartic acid (D) at amino acid position 17 of the SIVmac 239 p6 amino acid sequence. See, for example, FIG. 1 .
In another aspect, the chimeric vector comprises HIV-1 nucleic acid sequences that encode Gag and Pol.
In yet another aspect, the chimeric vector comprising HIV-1 nucleic acid sequences, wherein the HIV-1 nucleic acid sequences comprise HIV-1 p6 or pNL.Ba.L.
In a further aspect, a method of making a plurality of virions/viruses having enhanced infectivity for monocyte-derived macrophages (MDM) and dendritic cells (MDDC) is presented, the method comprising transfecting a population of cells with a lentiviral vector comprising 5′ and 3′ long terminal repeats (LTRs) and a nucleic acid sequence encoding at least one immunogen of a peptide or protein, a vector encoding vesicular stomatitis virus (VSV) envelope glycoprotein, a vector encoding Vpx, and a chimeric vector comprising HIV-1 nucleic acid sequences and SIVmac 239 nucleic acid sequences (as described herein), wherein the SIVmac 239 nucleic acid sequences encode an SIVmac 239 amino acid sequence consisting of a minimal Vpx packaging motif that confers Vpx packaging activity to the chimeric vector, to generate a transfected population of cells, wherein the transfected population of cells produces the plurality of virions having enhanced infectivity for MDM and dendritic cells MDDC.
In an embodiment thereof, the vector comprising 5′ and 3′ LTRs further comprises a nucleic acid sequence encoding at least one dendritic cell activator protein and/or at least one cytokine. An exemplary dendritic cell activator protein useful for such purposes is CD40 ligand (CD40L). Cytokines and cell surface proteins that would activate DC or could be expressed in DCs to activate T cells include: IL-2 (GenBank No. S82692), IL-12 (GenBank No. NM — 000882), TNF-alpha (GenBank No. XO2910.1), and CD28 (GenBank No. NM — 006139.3).
In a particular aspect, the above method may further comprise the step of administering the plurality of virions having enhanced infectivity for MDM and dendritic cells MDDC or a composition thereof to a subject in a therapeutically effective amount sufficient to enhance innate immune responses to the peptide or protein in the subject. In a particular embodiment thereof, the plurality of virions/viruses comprises at least one immunogen of an HIV-1 encoded peptide or protein, and the plurality of virions/viruses or the composition thereof is administered to a subject in a therapeutically effective amount sufficient to enhance innate immune responses to HIV-1 in the subject. In a particular embodiment thereof, the subject is infected with HIV-1 or suspected to be infected with HIV-1. In a more particular embodiment, the subject is a mammal. In an even more particular embodiment, the mammal is a primate or a human.
Also encompassed herein is a plurality of virions/viruses having enhanced infectivity for MDM and MDDC, which are suitable for use in humans. As described herein, the plurality of virions/viruses having enhanced infectivity for MDM and MDDC is produced by a method comprising transfecting a population of cells with a lentiviral vector comprising 5′ and 3′ long terminal repeats (LTRs) and a nucleic acid sequence encoding at least one immunogen of a peptide or protein, a vector encoding vesicular stomatitis virus (VSV) envelope glycoprotein, a vector encoding Vpx, and a chimeric vector comprising HIV-1 nucleic acid sequences and SIVmac 239 nucleic acid sequences (as described herein), wherein the SIVmac 239 nucleic acid sequences encode an SIVmac 239 amino acid sequence consisting of a minimal Vpx packaging motif that confers Vpx packaging activity to the chimeric vector, to generate a transfected population of cells, wherein the transfected population of cells produces the plurality of virions having enhanced infectivity for MDM and dendritic cells MDDC. Compositions comprising the plurality of virions/viruses having enhanced infectivity for MDM and MDDC and pharmaceutically acceptable carriers are also envisioned. The plurality of virions/viruses made in accordance with the present method is suitable for use in humans at least in part because it is produced using the chimeric vector comprising HIV-1 nucleic acid sequences and SIVmac 239 nucleic acid sequences described herein.
In a further aspect, a method of enhancing innate immune responses to a peptide or protein in a subject is presented, the method comprising: administering the plurality of virions/viruses having enhanced infectivity for MDM and MDDC or a composition thereof to the subject, wherein the plurality of virions/viruses comprises the at least one immunogen of the peptide or protein, and wherein the plurality of virions/viruses or a composition thereof is administered to the subject in a therapeutically effective amount sufficient to enhance innate immune responses to the peptide or protein in the subject. In a particular embodiment, the subject is a mammal. In an even more particular embodiment, the mammal is a primate or a human.
In a still further aspect, a method of enhancing innate immune responses to human immunodeficiency virus 1 (HIV-1) in a subject is presented, the method comprising: administering the plurality of virions/viruses having enhanced infectivity for MDM and MDDC or a composition thereof to the subject, wherein the plurality of virions/viruses comprises the at least one immunogen of the peptide or protein and the peptide or protein is an HIV-1 encoded peptide or protein, and wherein the plurality of virions/viruses or the composition thereof is administered to the subject in a therapeutically effective amount sufficient to enhance innate immune responses to HIV-1 in the subject. In a particular embodiment thereof, the subject is infected with HIV-1 or suspected to be infected with HIV-1. In a more particular embodiment, the subject is a mammal. In an even more particular embodiment, the mammal is a primate or a human.
Other objects and advantages will become apparent to those skilled in the art from a review of the following description which proceeds with reference to the following illustrative drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-D . Identification of the minimal Vpx packaging motif in SIVmac p6 by transfer into HIV-1 p6. A. Alignment of NL4-3 and SIVmac 239 p6 amino acid sequence. The two proposed alpha helices (α1 and α2) are shaded and the PTAPP late domain and TSG101 binding site are in bold underline. Binding sites for ALIX and Vpx are in bold underline. Below are diagrams of the HIV-1/SIVmac p6 chimeras with white for HIV-1 and black for SIV sequence. The SIV sequence is inserted at position 14 of HIV-1 p6, except in chimera 17-23(a), where the Vpx binding motif is displaced to position 21 to not alter the 14 FRFG 18 Vpr packaging motif. B. Immunoblot analysis shows packaging of SIVmac 239 Vpx in chimeras containing the Vpx packaging motif 293T cells were cotransfected with pNL4-3 containing a wild-type or chimeric p6 and pcVpx.myc or empty vector. Two days later, cell lysate and virions were pelleted by ultracentrifugation and analyzed on an immunoblot. The immunoblot was probed with antibody to myc-tagged Vpx, HIV-1 p24 CA, or tubulin. A pcVpx-myc alone transfection was included to rule-out nonspecific release of Vpx. C. HIV-1 p6 chimeras that map amino acids required for Vpx packaging. Virions were prepared by transfection and analyzed on an immunoblot as in (B). D. Effects of p6 mutations on packaging of HIV-1 Vpr. 293T were cotransfected with wild-type or chimeric p6 pNL4-3 and pcVpr.myc or empty vector. Cell lysate and virions were analyzed on an immunoblot as in (B).
FIGS. 2A-C . Relative contribution of the two proposed Vpr packaging motifs of HIV-1. A. Sequence of the two reported Vpr packaging motifs of HIV-1 p6. The two motifs are underlined in bold. B. Immunoblot analysis of Vpr packaged by motif 1 mutant virions. Virions were generated by transfection of 293T cells with pNL4-3 containing a wild-type or mutant p6 and a pcVpr.myc expression vector or empty vector. The virions were pelleted from the culture supernatants by ultracentrifugation and analyzed on an immunoblot probed with antibody to myc-tagged Vpr, HIV-1 CA p24, or tubulin. C. Immunoblot analysis of Vpr packaged by motif 2. Mutant virions prepared as in (B). M1A is F15, R16, F17, and G18 in motif 1 mutated to alanine. M2Aa is L35, L38, L41, and L44 in motif 2 mutated to alanine. M2Ab is M2Aa with the addition of S40, S43, and F45 mutated to alanine. M1 and 2a is the combination of M1A and M2Aa. M1 and 2b is the combination of M1A and M2Ab.
FIGS. 3A-C . Chimeric HIV-1 containing the Vpx packaging motif has an enhanced ability to infect MDDC and MDM. A. Expression and packaging of Vpx provided in trans. Luciferase reporter viruses were generated by cotransfection of 293T cells with wild-type or the 17-26 chimeric proviral reporter virus plasmid (18 μs) and increasing amounts of pcVpx.myc (0.3 μg, 0.6 μg, 3.0 μg, 6.0 μs) with the total mass of DNA held constant by the addition of pcDNA plasmid. The resulting virions were analyzed on an immunoblot probed with antibody to myc-tagged Vpx, HIV-1 CA p24, or tubulin. B. The effect of Vpx on MDDC and MDM infection. MDDC (upper panel) and MDM (lower panel) were infected with VSV-G pseudotyped luciferase reporter viruses normalized for infectivity on 293T cells. After 4 days, the cultures were harvested and the luciferase activity was determined. The data are displayed as the fold-enhancement of the virus containing
Vpx divided by virus lacking Vpx. Error bars indicate the standard deviation of triplicate measurements. Results from three MDDC and two MDM donors are shown. C. P24 analysis of MDDC and MDM infection by HIV-1 containing Vpx. MDDC (upper panels) and MDM (lower panels) were infected with luciferase reporter virus. Three days later, the cells were collected and intracellular p24-FITC was determined by flow cytometry.
FIGS. 4A-B . Comparison of SIVmac Vpx, SIVagm Vpr, and HIV-2 rod Vpx. Luciferase reporter viruses were generated by cotransfection of 293T cells with wild-type or p6 chimeric HIV-1 and expression vectors for SIVmac 239 Vpx, HIV-2 rod Vpx, or SIVagm Vpr. A. Expression and packaging of codon-optimized SIVmac 239 Vpx, HIV-2 rod Vpx, and SIVagm Vpr. Cell lysates and pelleted virions were analyzed on an immunoblot probed with antibody to myc-tagged Vpx , myc-tagged Vpr, HIV-1 p24 CA, or tubulin. B. Effect of HIV-1 packaging SIVmac 239 Vpx, HIV-2 rod Vpx, or SIVagm Vpr on MDDC and MDM infection. MDDC and MDM were infected with the indicated virus, normalized by luciferase activity on 293T, and, 4 days postinfection, luciferase activity was determined. Error bars indicate the standard deviation of triplicates. Three MDDC and MDM donors are shown.
FIGS. 5A-C . CCR5-using chimeric virus with Vpx in cis replicates more efficiently in MDDC and MDM. Wild-type and chimeric p6 NL.Ba.L viruses that express Vpx in cis were generated by transfection of 293T cells. A. Immunoblot of SIVmac 239 Vpx, HIV-2 rod Vpx and SIVagm Vpr expressed in cis in the cell lysates of cells treated for 5 h with 20 μM MG132. The cell lysates were analyzed on an immunoblot probed for myc-tagged Vpx, myc-tagged Vpr, or tubulin. B. The effect of p6 chimeric virus with vpx in cis on MDM infection. MDM were infected with wild-type or p6 chimeric vpx in cis virus. After 24 and 48 h, total DNA was prepared and the reverse transcripts were quantified by qRT-PCR using primers specific for the late products and 2-LTR circles. Early products were not quantified as these were found to be present in the virions prior to infection as a result of endogenous reverse transcription. AZT (25 μM) was added to the sample infected with the p6 chimeric virus encoding Vpx and served to control for plasmid contamination and intra-virion reverse transcripts. C. MDDC infection by p6 chimeric virus with vpx or vpr in cis. MDDC were infected with virus normalized for p24. After 3 days, the cells were collected, stained for intracellular p24 with anti-p24-FITC, and analyzed by flow cytometry. AZT (25 μM) was used to control for contamination with input virus. Nelfinavir (3 μM) was used to limit replication to a single cycle. Results from two donors are shown.
FIG. 6 . Infection of MDDC with Vpx-containing virus stimulates an innate immune response. MDDC from two healthy donors were infected with the p6 chimeric virus complemented in trans with Vpx. AZT (25 μM) or Raltegravir (10 μM) was added to the indicated samples. The cultures were harvested 24 and 48 h postinfection and IFN-β mRNA was quantified by qRT-PCR. The data are presented relative to G6PDH mRNA amplified in parallel.
FIGS. 7A-C . Exposure of MDDC to Vpx-containing virus allows for efficient transfer of virus to T cells. A. MDDC were infected with NL.Ba.1 wild-type or p6 chimeric virus containing vpx in cis and with or without additional Vpx complementation in trans. After 6 h, free virus was removed, and after 48 h, CD3/CD28-activated autologous CD4 T cells were added. Supernatant p24 was measured over 10 days. Results are representative of MDDC and CD4 T cells from two donors. B. MDDC were infected as in (A). After 6 h, free virus was removed. After 48 h, media or CD3/CD28-activated autologous T cells were added. In parallel, T cells alone were infected as in (A) and after 6 h, free virus was removed. Supernatant p24 was measured over 14 days. The results shown are representative of MDDC and CD4+ T cells from two donors. C. MDDC and T cells were infected as in (A) and cells were collected at day 3 post coculture. The cells were incubated with anti-CD11c-APC and anti-p24-FITC and analyzed by flow cytometry. The cell populations were gated on CD11c and then evaluated for intracellular p24 using FACS. Result shown is representative of three donors tested.
FIG. 8 . Nucleotide sequences of codon-optimized SIVmac239 Vpx, HIV-2rod Vpx, and SIVagm Vpr open reading frames. Total length is indicated following the final base.
FIG. 9 . P6 chimeric mutants package reduced amounts of HIV-1 Vpr. 293T were cotransfected with wild-type or chimeric p6 pNL4-3 and pcVpr.myc or empty vector. Cell lysate and virions were analyzed on an immunoblot probed with antibody against myc-tagged Vpr, HIV-1 p24 CA, or tubulin.
FIGS. 10A-C . Alteration of gag-pol to contain SIVp6 17-26 allows for packaging of Vpx and enhanced infection of MDDC. pGK-NGFR-IRES GFP lentivirus was generated by cotransfecting 293T with the proviral plasmid pGK-NGFR-IRES-GFP, the wild-type or p6 chimeric gag/pol packaging plasmid (pMDLg/pRRE or pMDLg-SIVp6 — 17-26, respectively), pcRSV-REV, pcVSV-G, and pcDNA6 or pcVpx.mychis. A. Immunoblot analysis of Vpx packaging by lentivirus with wild-type or p6 chimeric gag. Cell lysates and pelleted virions were analyzed on an immunoblot probed with antibody to myc-tagged Vpx, HIV-2 p24 CA, or tubulin. B. Effect of Vpx packaging on MDDC infection by pGK-NGFR-IRES-GFP. MDDC (1.25×10 5 ) were infected with pGK-NGFR-IRES-GFP at 5 or 25 ng p24, and MDDC (3.0×10 5 ) were infected with 120 ng p24. Three days after infection, cells were collected and examined for GFP expression by FACS. C. Effect of Vpx packing on 293T infection by pGK-NGFR-IRES-GFP. 293T (2.5×10 5 ) were infected with pGK-NGFR-IRES-GFP at 120 ng p24. Three days after infection, cells were collected and examined for GFP expression by FACS.
FIG. 11 shows nucleic and amino acid sequences of NL4.3 Sequence—Full Length with Chimeric p6, 17-26
FIG. 12 shows nucleic and amino acid sequences of NL4.3 Sequence—Full Length, Wild Type
FIG. 13 shows SIVmac239 Vpx wild-type and codon-optimized sequences.
FIG. 14 . The structure of LV vectors used to transduce DCs. The vectors express a CMV promoter-driven CD40L, GFP or CD40-P2A-Flu fusion protein (CD40L-Flu) and a PGK-driven puromycin resistance gene or ER-retention signal linked to Flu matrix amino acids 58-66, GILGFVFTL (Chang et al. Methods in Molecular Biology 614:161-171, 2010), (Flu). The vectors were produced as viruses that contained or lacked Vpx by transfection of 293T cells with the LV vector plasmid, the Gag/Pol packaging vector, HIV-1 Rev, and SIVmac Vpx expression vector pcVpx and pseudotyped with VSV-G.
FIG. 15 . Packaged Vpx allows for efficient DC transduction. DCs were transduced with LV vector indicated below each panel. Transduction efficiency was determined by analysis of GFP and CD40L expression. (Vpx+ constructs on top).
FIGS. 16A-B . Transduction of DCs with Vpx-containing vectors expressing CD40L induces DC maturation. DCs were transduced with the LV vectors that contained or lacked Vpx. After 48 and 72 h, the number of cells that expressed CD83 and CD86 were determined by flow cytometry. A. CD83 expression at 72 h post-transduction (Vpx+ constructs on top). CD83 expression for each infected DC population (blue) is contrasted against uninfected DCs (red). B. CD83 expression on the DC of four donors
FIGS. 17A-B . Transduction of DCs with Vpx-containing LV vectors expressing CD40L induces a high level of IL-12. Cytokine bead array analysis on culture supernatants shows the IL-12 levels secreted by DCs from four different donors at 72 h post transduction with different LV constructs as compared to IL-12 levels secreted from uninfected DCs.
FIG. 18 . DCs transduced with peptide and CD40L expressing lentiviral vectors activate a CTL response. Transduced DCs were cultured with CTL clone. After 24 h, the supernatant IFN-γ was quantified by CBA. As a control, the cells were pulsed with synthetic flu peptide, T cells alone, T cell clones co-cultured with uninfected DCs, and T cell clones co-cultured with uninfected DCs and then pulsed with synthetic flu peptide.
FIG. 19 . DCs transduced with Vpx-containing LV vectors encoding CD40L and a peptide epitope from influenza enhance antigen-specific CD8+ memory T cell responses. To assess for a Flu-specific memory response, flu-specific tetramer staining was performed on autologous CD8+CD3+ T cells two weeks after being co-cultured with DCs transduced by different LV vector constructs (Vpx+ constructs on top). Analysis on T cells co-cultured with uninfected DC and uninfected DC pulsed with synthetic flu peptide were performed as controls.
FIG. 20 . DCs transduced with CD40L expressing lentiviral vectors packaged with Vpx induce reactivation of latent HIV from ACH-2 cells. DCs were transduced with indicated LV vector and then co-cultured with ACH-2 cells. Infectious virus in the supernatant was quantified by TZM-bl assay to assess reactivation of HIV-1 proviruses.



DETAILED DESCRIPTION OF THE INVENTION
The lentiviral Vpx accessory protein is thought to facilitate the infection of macrophages and dendritic cells by counteracting a yet unidentified host restriction. Although HIV-1 does not encode Vpx, it can be provided to monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) through virus-like particles to dramatically enhance their susceptibility to HIV-1. Vpx and the related accessory protein Vpr are packaged into virions through a virus-specific interaction with the p6 carboxy-terminal domain of Gag. Here, the present inventors determined the minimal Vpx packaging motif of SIVmac 239 p6 and introduced this ten amino acid sequence into the p6 region of an infectious HIV-1 molecular clone. The chimeric virus efficiently packaged Vpx provided in trans and was substantially more infectious on MDDC and MDM. The present inventors further engineered the virus to express Vpx in cis by introducing the coding sequence in place of nef. The resulting virus produced less Vpx but was significantly enhanced in its infectivity on MDDC and MDM. Infection of the cells with Vpx-containing HIV-1 induced a potent type I interferon response. In a co-culture system, Vpx-containing HIV-1 was enhanced in its ability to be transmitted from MDDC to T cells. These findings suggest that Vpx could facilitate dendritic cell to T cell virus transmission in vivo. See, for example, Examples 1-2 herein below. The chimeric viruses described herein are envisioned as useful for the design of dendritic cell vaccines that induce an innate immune response in MDDC. Additionally, this approach can be useful for designing and generating engineered lentiviral vectors to produce virions that can be used to transduce these relatively resistant cells.
Further to the above, dendritic cells (DCs) are central to the induction of innate and adaptive immune responses yet are difficult to manipulate for therapeutic purposes due to their resistance to the introduction of transfection or transduction. Lentiviral vectors offer the ability to express genes stably in cells but are restricted in DCs by SAMHD1, a host factor that blocks retroviruses at reverse transcription. Examples 1 and 2 illustrate that the present inventors have developed lentiviral vectors and have used same to produce lentiviral vector virions that escape the restriction in DCs by virtue of packaging the SIV accessory protein Vpx. Such lentiviral vector virions comprise the lentiviral vector used to produce the virions. As a proof of principal, the present inventors have constructed lentiviral vectors that express an HLA A2-restricted influenza (Flu) peptide and immunostimulatory CD40L. As described in Example 3, the Flu peptide was expressed as a fusion protein with CD40L separated by the P2A self-cleaving peptide. Because CD40L is a type II transmembrane protein, this configuration places the carboxy-terminal Flu peptide in the endoplasmic reticulum (ER) where it can be shunted into the antigen presentation pathway during biosynthesis. The Vpx-containing vectors infected DCs 60-fold better than control Vpx-negative viruses. Vpx-containing vectors induced the DCs to mature as judged by CD83 and CD86 upregulation and induced Th1-skewing inflammatory cytokines. The transduced DCs, moreover, stimulated a Flu-peptide-specific CD8+ CTL clone and antigen-specific CD8+ cells in PBMC from healthy human donors, as assessed by IFNγ and tetramer staining. This system, therefore, provides the basis for the development of an effective DC-targeted anti-HIV-1 vaccine.
In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, “Molecular Cloning: A Laboratory Manual” (1989); “Current Protocols in Molecular Biology” Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A Laboratory Handbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocols in Immunology” Volumes I-III [Coligan, J. E., ed. (1994)]; “Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic Acid Hybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “Transcription And Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “Animal Cell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning” (1984).
Therefore, if appearing herein, the following terms shall have the definitions set out below.
A. Terminology
The term “specific binding member” describes a member of a pair of molecules which have binding specificity for one another. The members of a specific binding pair may be naturally derived or wholly or partially synthetically produced. One member of the pair of molecules has an area on its surface, or a cavity, which specifically binds to and is therefore complementary to a particular spatial and polar organisation of the other member of the pair of molecules. Thus the members of the pair have the property of binding specifically to each other. Examples of types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. This application is concerned with antigen-antibody type reactions.
The term “antibody” describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. CDR grafted antibodies are also contemplated by this term. An “antibody” is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Pat. Nos. 4,816,397 and 4,816,567. The term “antibody(ies)” includes a wild type immunoglobulin (Ig) molecule, generally comprising four full length polypeptide chains, two heavy (H) chains and two light (L) chains, or an equivalent Ig homologue thereof (e.g., a camelid nanobody, which comprises only a heavy chain); including full length functional mutants, variants, or derivatives thereof, which retain the essential epitope binding features of an Ig molecule, and including dual specific, bispecific, multispecific, and dual variable domain antibodies; Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Also included within the meaning of the term “antibody” is any “antibody fragment”.
An “antibody fragment” means a molecule comprising at least one polypeptide chain that is not full length, including (i) a Fab fragment, which is a monovalent fragment consisting of the variable light (VL), variable heavy (VH), constant light (CL) and constant heavy 1 (CH1) domains; (ii) a F(ab′)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a heavy chain portion of an Fab (Fd) fragment, which consists of the VH and CH1 domains; (iv) a variable fragment (Fv) fragment, which consists of the VL and VH domains of a single arm of an antibody, (v) a domain antibody (dAb) fragment, which comprises a single variable domain (Ward, E. S. et al., Nature 341, 544-546 (1989)); (vi) a camelid antibody; (vii) an isolated complementarity determining region (CDR); (viii) a Single Chain Fv Fragment wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (ix) a diabody, which is a bivalent, bispecific antibody in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementarity domains of another chain and creating two antigen binding sites (WO94/13804; P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, (1993)); and (x) a linear antibody, which comprises a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementarity light chain polypeptides, form a pair of antigen binding regions; (xi) multivalent antibody fragments (scFv dimers, trimers and/or tetramers (Power and Hudson, J Immunol. Methods 242: 193-204 9 (2000)); and (xii) other non-full length portions of heavy and/or light chains, or mutants, variants, or derivatives thereof, alone or in any combination.
As antibodies can be modified in a number of ways, the term “antibody” should be construed as covering any specific binding member or substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023 and U.S. Pat. Nos. 4,816,397 and 4,816,567.
An “antibody combining site” is that structural portion of an antibody molecule comprised of light chain or heavy and light chain variable and hypervariable regions that specifically binds antigen.
The phrase “antibody molecule” in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.
Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab′, F(ab′) 2 and F(v), which portions are preferred for use in the therapeutic methods described herein.
Antibodies may also be bispecific, wherein one binding domain of the antibody is a specific binding member of the invention, and the other binding domain has a different specificity, e.g. to recruit an effector function or the like. Bispecific antibodies of the present invention include wherein one binding domain of the antibody is a specific binding member of the present invention, including a fragment thereof, and the other binding domain is a distinct antibody or fragment thereof, including that of a distinct anti-cancer or anti-tumor specific antibody. The other binding domain may be an antibody that recognizes or targets a particular cell type, as in a neural or glial cell-specific antibody. In the bispecific antibodies of the present invention the one binding domain of the antibody of the invention may be combined with other binding domains or molecules which recognize particular cell receptors and/or modulate cells in a particular fashion, as for instance an immune modulator (e.g., interleukin(s)), a growth modulator or cytokine (e.g. tumor necrosis factor (TNF), and particularly, the TNF bispecific modality demonstrated in U.S. Ser. No. 60/355,838 filed Feb. 13, 2002 incorporated herein in its entirety) or a toxin (e.g., ricin) or anti-mitotic or apoptotic agent or factor.
The phrase “monoclonal antibody” in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may also contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.
The term “antigen binding domain” describes the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may bind to a particular part of the antigen only, which part is termed an epitope. An antigen binding domain may be provided by one or more antibody variable domains. Preferably, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
The term “specific” may be used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s). The term is also applicable where e.g. an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.
The term “adjuvant” refers to a compound or mixture that enhances the immune response, particularly to an antigen. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response (Hood et al., Immunology, Second Ed, 1984, Benjamin/Cummings: Menlo Park, Calif., p. 384). Often, a primary challenge with an antigen alone, in the absence of an adjuvant, will fail to elicit a humoral or cellular immune response. Previously known and utilized adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvant such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum . Mineral salt adjuvants include but are not limited to: aluminum hydroxide, aluminum phosphate, calcium phosphate, zinc hydroxide and calcium hydroxide. Preferably, the adjuvant composition further comprises a lipid of fat emulsion comprising about 10% (by weight) vegetable oil and about 1-2% (by weight) phospholipids. Preferably, the adjuvant composition further optionally comprises an emulsion form having oily particles dispersed in a continuous aqueous phase, having an emulsion forming polyol in an amount of from about 0.2% (by weight) to about 49% (by weight), optionally a metabolizable oil in an emulsion-forming amount of up to 15% (by weight), and optionally a glycol ether-based surfactant in an emulsion-stabilizing amount of up to about 5% (by weight).
As used herein, the term “immunomodulator” refers to an agent which is able to modulate an immune response. An example of such modulation is an enhancement of cell activation or of antibody production.
The term “effective amount” of an immunomodulator refers to an amount of an immunomodulator sufficient to enhance a vaccine-induced immune response, be it cell-mediated, humoral or antibody-mediated. An effective amount of an immunomodulator, if injected, can be in the range of about 0.1-1,000 μg, preferably 1-900 μg, more preferably 5-500 μg, for a human subject, or in the range of about 0.01-10.0 μg/Kg body weight of the subject animal. This amount may vary to some degree depending on the mode of administration, but will be in the same general range. If more than one immunomodulator is used, each one may be present in these amounts or the total amount may fall within this range. An effective amount of an antigen may be an amount capable of eliciting a demonstrable immune response in the absence of an immunomodulator. For many antigens, this is in the range of about 5-100 μg for a human subject. The appropriate amount of antigen to be used is dependent on the specific antigen and is well known in the art.
The exact effective amount necessary will vary from subject to subject, depending on the species, age and general condition of the subject, the severity of the condition being treated, the mode of administration, etc. Thus, it is not possible to specify an exact effective amount. However, the appropriate effective amount may be determined by one of ordinary skill in the art using only routine experimentation or prior knowledge in the vaccine art.
An “immunological response” to a composition or vaccine comprised of an antigen is the development in the host of a cellular- and/or antibody-mediated immune response to the composition or vaccine of interest. Usually, such a response consists of the subject producing antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells directed specifically to an antigen or antigens included in the composition or vaccine of interest.
The term “immunogen” refers to a substance that provokes an immune response.
The term “comprise” is generally used in the sense of include, that is to say permitting the presence of one or more features or components.
The term “consisting essentially of” refers to a product, particularly a peptide sequence, of a defined number of residues which is not covalently attached to a larger product. In the case of the peptide of the invention referred to above, those of skill in the art will appreciate that minor modifications to the N- or C-terminal of the peptide may however be contemplated, such as the chemical modification of the terminal to add a protecting group or the like, e.g. the amidation of the C-terminus.
The term “isolated” refers to the state in which specific binding members of the invention, or nucleic acid encoding such binding members will be, in accordance with the present invention. Members and nucleic acid will be free or substantially free of material with which they are naturally associated such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practised in vitro or in vivo. Members and nucleic acid may be formulated with diluents or adjuvants and still for practical purposes be isolated—for example the members will normally be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy.
As used herein, “pg” means picogram, “ng” means nanogram, “ug” or “μg” mean microgram, “mg” means milligram, “ul” or “μl” mean microliter, “ml” means milliliter, “l” means liter.
The amino acid residues described herein are preferred to be in the “L” isomeric form. However, residues in the “D” isomeric form can be substituted for any L-amino acid residue, as long as the desired fuctional property of immunoglobulin-binding is retained by the polypeptide. NH 2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969), abbreviations for amino acid residues are shown in the following Table of Correspondence:







TABLE OF CORRESPONDENCE






SYMBOL








1-Letter
3-Letter
AMINO ACID





Y
Tyr
tyrosine


G
Gly
glycine


F
Phe
phenylalanine


M
Met
methionine


A
Ala
alanine


S
Ser
serine


I
Ile
isoleucine


L
Leu
leucine


T
Thr
threonine


V
Val
valine


P
Pro
proline


K
Lys
lysine


H
His
histidine


Q
Gln
glutamine


E
Glu
glutamic acid


W
Trp
tryptophan


R
Arg
arginine


D
Asp
aspartic acid


N
Asn
asparagine


C
Cys
cysteine










































It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues. The above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.
A “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
A “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
A “DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
An “origin of replication” refers to those DNA sequences that participate in DNA synthesis.
A DNA “coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.
Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the −10 and −35 consensus sequences.
An “expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
A “signal sequence” can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.
The term “oligonucleotide,” as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.
The term “primer” as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
The primers herein are selected to be “substantially” complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5′ end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.
As used herein, the terms “restriction endonucleases” and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
A cell has been “transformed” by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
Two DNA sequences are “substantially homologous” when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
It should be appreciated that also within the scope of the present invention are DNA sequences encoding protein or peptide sequences as provided herein, or comprising sequences which are degenerate thereto. DNA sequences having the nucleic acid sequence encoding the peptides of the invention are contemplated, including degenerate sequences thereof encoding the same, or a conserved or substantially similar, amino acid sequence. By “degenerate to” is meant that a different three-letter codon is used to specify a particular amino acid. It is well known in the art that the following codons can be used interchangeably to code for each specific amino acid:








Phenylalanine (Phe or F)
UUU or UUC

Leucine (Leu or L)
UUA or UUG or CUU or CUC or CUA or


CUG

Isoleucine (Ile or I)
AUU or AUC or AUA

Methionine (Met or M)
AUG

Valine (Val or V)
GUU or GUC of GUA or GUG

Serine (Ser or S)
UCU or UCC or UCA or UCG or AGU or


AGC

Proline (Pro or P)
CCU or CCC or CCA or CCG

Threonine (Thr or T)
ACU or ACC or ACA or ACG

Alanine (Ala or A)
GCU or GCG or GCA or GCG

Tyrosine (Tyr or Y)
UAU or UAC

Histidine (His or H)
CAU or CAC

Glutamine (Gln or Q)
CAA or CAG

Asparagine (Asn or N)
AAU or AAC

Lysine (Lys or K)
AAA or AAG

Aspartic Acid (Asp or D)
GAU or GAC

Glutamic Acid (Glu or E)
GAA or GAG

Cysteine (Cys or C)
UGU or UGC

Arginine (Arg or R)
CGU or CGC or CGA or CGG or AGA or


AGG

Glycine (Gly or G)
GGU or GGC or GGA or GGG

Tryptophan (Trp or W)
UGG

Termination codon
UAA (ochre) or UAG (amber) or UGA (opal)





































It should be understood that the codons specified above are for RNA sequences. The corresponding codons for DNA have a T substituted for U.
Mutations can be made in the sequences encoding the protein or peptide sequences of the invention, such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible. A substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. A non-conservative change is more likely to alter the structure, activity or function of the resulting protein. The present invention should be considered to include sequences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein. Further, variants and subtypes of HIV are known and recognized and any such variants or subtype corresponding protein or peptide sequences of the invention (e.g., Vpx) are encompassed and contemplated herein.
Codon-optimized forms of vpx, for example, are used to advantage in the chimeric viruses and lentiviral vectors described herein.
The following is one example of various groupings of amino acids:
Amino acids with nonpolar R groups
Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine
Amino acids with uncharged polar R groups
Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine
Amino acids with charged polar R groups (negatively charged at Ph 6.0)

Aspartic acid, Glutamic acid

Basic amino acids (positively charged at pH 6.0)

Lysine, Arginine, Histidine (at pH 6.0)
Another grouping may be those amino acids with phenyl groups:
Phenylalanine, Tryptophan, Tyrosine
Another grouping may be according to molecular weight (i.e., size of R groups):













Glycine
75
Alanine
89


Serine
105
Proline
115


Valine
117
Threonine
119


Cysteine
121
Leucine
131


Isoleucine
131
Asparagine
132


Aspartic acid
133
Glutamine
146


Lysine
146
Glutamic acid
147


Methionine
149
Histidine (at pH 6.0)
155


Phenylalanine
165
Arginine
174


Tyrosine
181
Tryptophan
204
























Particularly preferred substitutions are:
Lys for Arg and vice versa such that a positive charge may be maintained; Glu for Asp and vice versa such that a negative charge may be maintained; Ser for Thr such that a free -OH can be maintained; and Gln for Asn such that a free NH 2 can be maintained.

Exemplary and preferred conservative amino acid substitutions include any of: glutamine (Q) for glutamic acid (E) and vice versa; leucine (L) for valine (V) and vice versa; serine (S) for threonine (T) and vice versa; isoleucine (I) for valine (V) and vice versa; lysine (K) for glutamine (Q) and vice versa; isoleucine (I) for methionine (M) and vice versa; serine (S) for asparagine (N) and vice versa; leucine (L) for methionine (M) and vice versa; lysine (L) for glutamic acid (E) and vice versa; alanine (A) for serine (S) and vice versa; tyrosine (Y) for phenylalanine (F) and vice versa; glutamic acid (E) for aspartic acid (D) and vice versa; leucine (L) for isoleucine (I) and vice versa; lysine (K) for arginine (R) and vice versa.
Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced a potential site for disulfide bridges with another Cys. A His may be introduced as a particularly “catalytic” site (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces β-turns in the protein's structure.
Two amino acid sequences are “substantially homologous” when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90 or 95%) are identical, or represent conservative substitutions.
A “heterologous” region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
A DNA sequence is “operatively linked” to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence. The term “operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.
The term “standard hybridization conditions” refers to salt and temperature conditions substantially equivalent to 5×SSC and 65° C. for both hybridization and wash. However, one skilled in the art will appreciate that such “standard hybridization conditions” are dependent on particular conditions including the concentration of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatch, percent formamide, and the like. Also important in the determination of “standard hybridization conditions” is whether the two sequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20° C. below the predicted or determined T m with washes of higher stringency, if desired.
The term ‘agent’ means any molecule, including polypeptides, antibodies, polynucleotides, chemical compounds and small molecules. In particular the term agent includes compounds such as test compounds or drug candidate compounds.
The term ‘agonist’ refers to a ligand that stimulates the receptor the ligand binds to in the broadest sense or stimulates a response that would be elicited on binding of a natural binder to a binding site.
The term ‘assay’ means any process used to measure a specific property of a compound or agent. A ‘screening assay’ means a process used to characterize or select compounds based upon their activity from a collection of compounds.
The term ‘preventing’ or ‘prevention’ refers to a reduction in risk of acquiring or developing a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop) in a subject that may be exposed to a disease-causing agent, or predisposed to the disease in advance of disease onset.
The term ‘prophylaxis’ is related to and encompassed in the term ‘prevention’, and refers to a measure or procedure the purpose of which is to prevent, rather than to treat or cure a disease. Non-limiting examples of prophylactic measures may include the administration of vaccines; the administration of low molecular weight heparin to hospital patients at risk for thrombosis due, for example, to immobilization; and the administration of an anti-malarial agent such as chloroquine, in advance of a visit to a geographical region where malaria is endemic or the risk of contracting malaria is high.
‘Therapeutically effective amount’ means that amount of a drug, compound, antimicrobial, antibody, or pharmaceutical agent that will elicit the biological or medical response of a subject that is being sought by a medical doctor or other clinician. As an example, with regard to immune response, the term “effective amount” is intended to include an effective amount of a compound or agent that will bring about a biologically meaningful increase in the amount of or extent of immune response, activation indicator and/or a biologically meaningful increase in the amount or extent of dendritic cell, T cell and/or B cell effects. The phrase “therapeutically effective amount” is used herein to mean an amount sufficient to promote, and preferably increase by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant change in the immune response or immune cell indicator or response, or in a patient's response to an antigen, vaccine, or other immune agent, or in a patient's clearance of an infectious agent, or other feature of pathology such as for example, elevated activated T or B cells, activated DC cell count, fever or white cell count.
The term ‘treating’ or ‘treatment’ of any disease or infection refers, in one embodiment, to ameliorating the disease or infection (i.e., arresting the disease or growth of the infectious agent or bacteria or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof). In another embodiment ‘treating’ or ‘treatment’ refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, ‘treating’ or ‘treatment’ refers to modulating the disease or infection, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In a further embodiment, ‘treating’ or ‘treatment’ relates to slowing the progression of a disease or reducing an infection.
The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
As used herein, the term “replication defective HIV vector” refers to a vector that encodes HIV peptides/proteins, but does not encode live virus.
HIV-1 comprises several major genes that encode structural proteins that are common to all lentiviruses and several nonstructural genes. The gag (group specific antigen) gene encodes the Gag polyprotein, which is processed during maturation to matrix protein (MA, p17), capsid protein (CA, p24), spacer peptide 1 (SP1, p2), nucleocapsid protein (NC, p7), spacer peptide 2 (SP2, p 1) and p6. These proteins provide for the physical infrastructure of the virus. The pol gene encodes viral enzymes for reverse transcriptase (RT), integrase (IN), and HIV protease (PR).
B. Detailed Disclosure
Lentiviral vectors are currently used to stably express proteins and small RNAs in a variety of cell types, including primary cells. However, myeloid cells, such as macrophages and dendritic cells, are relatively resistant to viral transduction by current lentiviral systems. Macrophages and dendritic cells are initiators of adaptive immune responses that confer immunity during vaccination and also mediate innate immune responses to pathogens. A lentiviral vector that efficiently transduces this cell type would facilitate improved expression of therapeutic proteins and RNAs in macrophages and dendritic cells. Methods using such lentiviral vectors would, moreover, be useful in the design of vaccines against HIV and/or other pathogens to induce a stronger immune response to the immunogen.
Vpx is a lentiviral accessory protein encoded by a subset of lentiviruses, including SIVmac of the rhesus macaque, but is absent from HIV-1. Consequently, the Vpx protein is packaged in SIV virions, but not in those of HIV-1 or lentiviral vectors based on HIV-1. In SIVmac, Vpx greatly enhances the ability of the virus to infect MDM and MDDC.
The invention described herein allows Vpx to be delivered and packaged into HIV-1 and HIV-1 derived lentiviral vector virions. To allow for Vpx packaging, the present inventors altered the p6 region of HIV-1 to encode a ten amino acid sequence in p6 of SIVmac 239 . As defined herein, the ten amino acid sequence ( 17 DPAVDLLKNY 26 ) is the minimal sequence required for Vpx packaging. To generate the modified virions, 293 cells were transfected with lentiviral vector DNA, VSV-G expression plasmid and p6-chimeric HIV-1 Gag/pol expression vector. After 3 days, virus-containing culture supernatant is collected and frozen for later use. Details pertaining to making these constructs and virions and their composition are presented in, for example, the Drawings and Examples.
The constructs, vectors, and virions described herein can be used to improve the efficiency of transduction of myeloid cells by lentiviral vectors. The virions contain high copy numbers of functional Vpx, increasing their infectivity 100-fold in MDM and MDDC.
The present constructs, vectors, and virions differ in many respects when compared to those generated by other investigators. The introduction of the minimal Vpx packaging motif into HIV-1 based lentiviral vectors generates a new genus of lentiviral vector having unique structural features (e.g., nucleic acid sequences) that confer new and surprising functional properties, predominant among these properties is the enhanced ability of HIV-1 virions generated therefrom to infect MDM and MDDC. More particularly, the presence of the minimal Vpx packaging motif in the new genus of HIV-1 based lentiviral vectors makes it possible to package Vpx supplied in trans into virus like particles generated therefrom that can promote HIV-1 infection of MDM and MDDC. In a further embodiment, the present inventors have modified the new genus of HIV-1 based lentiviral vectors comprising the minimal Vpx packaging motif to include SIV vpx. Accordingly, in a second generation of HIV-1 based lentiviral vectors, Vpx is supplied in cis, thus eliminating the need for additional constructs/vectors/VLPs encoding Vpx. Methods described herein thus produce lentiviral vector particles which comprise packaged Vpx protein.
The methods described herein can be used to generate lentiviral vectors that express a protein or RNA of interest that have an improved ability to infect MDM and MDDC. This is useful for basic research into signal transduction pathways in these specific cells types. The approach may also be used advantageously in the clinic for gene therapy to achieve stable expression of protein or RNA in vivo for the treatment of genetic deficiencies.
The method may also find application for vaccination approaches that rely on transduction of MDM and MDDC. The chimeric vectors, constructs, and VLPs described herein will enhance the delivery and subsequent expression of antigens in such cells and thus, lead to more effective vaccine responses. Indeed, current efforts to generate dendritic cell-based cancer vaccines would be significantly enhanced by incorporating the chimeric vectors and constructs described herein.
Both HIV-1 p6 and pNL.Ba.L are identical to the HIV-1 reference strain NL4-3, as present in Genbank and presented herein. The essential genes of HIV-1 p6 and pNL.Ba.L with respect to the present chimeric vectors and constructs include the following:
GAG Polyprotein Precursor: 168-1670 Nucleotide Sequence (SEQ ID NO: 24) atg ggtgcgagag cgtcggtatt aagcggggga gaattagata aatgggaaaa aattcggtta aggccagggg gaaagaaaca atataaacta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctttta gagacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca atagcagtcc tctattgtgt gcatcaaagg atagatgtaa aagacaccaa ggaagcctta gataagatag aggaagagca aaacaaaagt aagaaaaagg cacagcaagc agcagctgac acaggaaaca acagccaggt cagccaaaat taccctatag tgcagaacct ccaggggcaa atggtacatc aggccatatc acctagaact ttaaatgcat gggtaaaagt agtagaagag aaggctttca gcccagaagt aatacccatg ttttcagcat tatcagaagg agccacccca caagatttaa ataccatgct aaacacagtg gggggacatc aagcagccat gcaaatgtta aaagagacca tcaatgagga agctgcagaa tgggatagat tgcatccagt gcatgcaggg cctattgcac caggccagat gagagaacca aggggaagtg acatagcagg aactactagt acccttcagg aacaaatagg atggatgaca cataatccac ctatcccagt aggagaaatc tataaaagat ggataatcct gggattaaat aaaatagtaa gaatgtatag ccctaccagc attctggaca taagacaagg accaaaggaa ccctttagag actatgtaga ccgattctat aaaactctaa gagccgagca agcttcacaa gaggtaaaaa attggatgac agaaaccttg ttggtccaaa atgcgaaccc agattgtaag actattttaa aagcactggg accaggagcg acactagaag aaatgatgac agcatgtcag ggagtggggg gacccggcca taaagcaaga gttttggctg aagcaatgag ccaagtaaca aatccagcta ccataatgat acagaaaggc aattttagga accaaagaaa gactgttaag tgtttcaatt gtggcaaaga agggcacata gccaaaaatt gcagggcccc taggaaaaag ggctgttgga aatgtggaaa ggaaggacac caaatgaaag attgtactga gagacaggct aattttttag ggaagatctg gccttcccac aagggaaggc cagggaattt tcttcagagc agaccagagc caacagcccc accagaagag agcttcaggt ttggggaaga gacaacaact ccctctcaga ggcaggagcc gatagacaag gaactgtatc ctttagcttc cctcagatca ctctttggca gcgacccctc gtcacaataa agataggggg Translation (SEQ ID NO: 25) MGARASVLSGGELDKWEKIRLRPGGKKQYKLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTIAVLYCVH QRIDVKDTKEALDKIEEEQNKSKKKAQQAAADTGNNSQVSQNYPIVQNLQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEG ATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWII LGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGP GHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRP GNFLQSRPEPTAPPEESFRFGEETTTPSQRQEPIDKELYPLASLRSLFGSDPSSQ POL Polyprotein Precursor: 1463-4474 Nucleotide Sequence (SEQ ID NO: 26) ttttttag ggaagatctg gccttcccac aagggaaggc cagggaattt tcttcagagc agaccagagc caacagcccc accagaagag agcttcaggt ttggggaaga gacaacaact ccctctcaga ggcaggagcc gatagacaag gaactgtatc ctttagcttc cctcagatca ctctttggca gcgacccctc gtcacaataa agataggggg gcaattaaag gaagctctat tagatacagg agcagatgat acagtattag aagaaatgaa tttgccagga agatggaaac caaaaatgat agggggaatt ggaggtttta tcaaagtaag acagtatgat cagatactca tagaaatctg cggacataaa gctataggta cagtattagt aggacctaca cctgtcaaca taattggaag aaatctgttg actcagattg gctgcacttt aaattttccc attagtccta ttgagactgt accagtaaaa ttaaagccag gaatggatgg cccaaaagtt aaacaatggc cattgacaga agaaaaaata aaagcattag tagaaatttg tacagaaatg gaaaaggaag gaaaaatttc aaaaattggg cctgaaaatc catacaatac tccagtattt gccataaaga aaaaagacag tactaaatgg agaaaattag tagatttcag agaacttaat aagagaactc aagatttctg ggaagttcaa ttaggaatac cacatcctgc agggttaaaa cagaaaaaat cagtaacagt actggatgtg ggcgatgcat atttttcagt tcccttagat aaagacttca ggaagtatac tgcatttacc atacctagta taaacaatga gacaccaggg attagatatc agtacaatgt gcttccacag ggatggaaag gatcaccagc aatattccag tgtagcatga caaaaatctt agagcctttt agaaaacaaa atccagacgt agtcatctat caatacatgg atgatttgta tgtaggatct gacttagaaa tagggcagca tagaacaaaa atagaggaac tgagacaaca tctgttgagg tggggattta ccacaccaga caaaaaacat cagaaagaac ctccattcct ttggatgggt tatgaactcc atcctgataa atggacagta cagcctatag tgctgccaga aaaggacagc tggactgtca atgacataca gaaattagtg ggaaaattga attgggcaag tcagatttat gcagggatta aagtaaggca attatgtaaa cttcttaggg gaaccaaagc actaacagaa gtagtaccac taacagaaga agcagagcta gaactggcag aaaacaggga gattctaaaa gaaccggtac atggagtgta ttatgaccca tcaaaagact taatagcaga aatacagaag caggggcaag gccaatggac atatcaaatt tatcaagagc catttagaaa tctgaaaaca ggaaagtatg caagaatgaa gggtgcccac actaatgatg tgaaacaatt aacagaggca gtacaaaaaa tagccacaga aagcatagta atatggggaa agactcctaa atttaaatta cccatacaaa aggaaacatg ggaagcatgg tggacagagt attggcaagc cacctggatt cctgagtggg agtttgtcaa tacccctccc ttagtgaagt tatggtacca gttagagaaa gaacccataa taggagcaga aactttctat gtagatgggg cagccaatag ggaaactaaa ttaggaaaag caggatatgt aactgacaga ggaagacaaa aagttgtccc cctaacggac acaacaaatc agaagactga gttacaagca attcatctag ctttgcagga ttcgggatta gaagtaaaca tagtgacaga ctcacaatat gcattgggaa tcattcaagc acaaccagat aagagtgaat cagagttagt cagtcaaata atagagcagt taataaaaaa ggaaaaagtc tacctggcat gggtaccagc acacaaagga attggaggaa atgaacaagt agataaattg gtcagtgctg gaatcaggaa agtactattt ttagatggaa tagataaggc ccaagaagaa catgagaaat atcacagtaa ttggagagca atggctagtg attttaacct accacctgta gtagcaaaag aaatagtagc cagctgtgat aaatgtcagc taaaagggga agccatgcat ggacaagtag actgtagccc aggaatatgg cagctagatt gtacacattt agaaggaaaa gttatcttgg tggcagttca tgtagccagt ggatatatag aagcagaagt aattccagca gagacagggc aagaaacagc atacttcctc ttaaaattag caggaagatg gccagtaaaa acagtacata cagacaatgg cagcaatttc accagtacta cagttaaggc cgcctgttgg tgggcgggaa tcaagcagga atttggcatt ccctacaatc cccaaagtca aggagtaata gaatctatga ataaagaatt aaagaaaatt ataggacagg taagagatca ggctgaacat cttaagacag cagtacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga gatccagttt ggaaaggacc agcaaagctc ctctggaaag gtgaaggggc agtagtaata caagataata gtgacataaa agtagtgcca agaagaaaag caaagatcat cagggattat ggaaaacaga tggcaggtga tgattgtgtg gcaagtagac aggatgagga ttaa Translation (SEQ ID NO: 27) FFREDLAFPQGKAREFSSEQTRANSPTRRELQVWGRDNNSLSEAGADRQGTVSFSFPQITLWQRPLVTIKIGGQLKEALLDTGADDTVL EEMNLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNFPISPIETVPVKLKPGMDGPKVKQ WPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKQKKSVTVLDVG DAYFSVPLDKDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQCSMTKILEPFRKQNPDVVIYQYMDDLYVGSDLEIGQHRTK IEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKINGKLNWASQIYAGIKVRQLCKLLRGTKALT EVVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFRNLKTGKYARMKGAHTNDVKQLTEAVQKIATES IVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIIGAETFYVDGAANRETKLGKAGYVTDRGRQKVVP LTDTTNQKTELQAIHLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSAGIR KVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDKCQLKGEAMHGQVDCSPGIWQLDCTHLEGKVILVAVHVASGYIEA EVIPAETGQETAYFLLKLAGRWPVKTVHTDNGSNFTSTTVKAACWWAGIKQEFGIPYNPQSQGVIESMNKELKKIIGQVRDQAEHLKTA VQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQKQITKIQNFRVYYRDSRDPVWKGPAKLLWKGEGAVVIQDNSDIKVVPRRK AKIIRDYGKQMAGDDCVASRQDED VIF Protein: 4419-4997 Nucleotide Sequence (SEQ ID NO: 28) at ggaaaacaga tggcaggtga tgattgtgtg gcaagtagac aggatgagga ttaacacatg gaaaagatta gtaaaacacc atatgtatat ttcaaggaaa gctaaggact ggttttatag acatcactat gaaagtacta atccaaaaat aagttcagaa gtacacatcc cactagggga tgctaaatta gtaataacaa catattgggg tctgcataca ggagaaagag actggcattt gggtcaggga gtctccatag aatggaggaa aaagagatat agcacacaag tagaccctga cctagcagac caactaattc atctgcacta ttttgattgt ttttcagaat ctgctataag aaataccata ttaggacgta tagttagtcc taggtgtgaa tatcaagcag gacataacaa ggtaggatct ctacagtact tggcactagc agcattaata aaaccaaaac agataaagcc acctttgcct agtgttagga aactgacaga ggacagatgg aacaagcccc agaagaccaa gggccacaga gggagccata caacgaatgg acactag Translation (SEQ ID NO: 29) MENRWQVMIVWQVDRMRINTWKRLVKHHMYISRKAKDWFYRHHYESTNPKISSEVHIPLGDAKLVITTYWGLHTGERDWHLGQGVSIEW RKKRYSTQVDPDLADQLIHLHYFDCFSESAIRNTILGRIVSPRCEYQAGHNKVGSLQYLALAALIKPKQIKPPLPSVRKLTEDRWNKPQ KTKGHRGSHTTNGH VPR Protein: 4937-5227 Nucleotide Sequence (SEQ ID NO: 30) atgg aacaagcccc agaagaccaa gggccacaga gggagccata caacgaatgg acactagagc ttttagagga acttaagagt gaagctgtta gacattttcc taggatatgg ctccataact taggacgaca tatctatgaa acttacgggg atacttgggc aggagtggaa gccataataa gaattctgca acaactgccg tttatccatt tcagaattgg gtgtcgacat agcagaatag gcgttactcg acagaggaga gcaagaaatg gagccagtag atcctag Translation (SEQ ID NO: 31) MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFMTKALGISYGRKKRRQRRRAHQNSQTHQASLSKQPTSQSRGDPTGPKE TAT Protein: 5208-7792 (translate 5208-5422, 7747-7792) Nucleotide Sequence (SEQ ID NO: 32) atg gagccagtag atcctagact agagccctgg aagcatccag gaagtcagcc taaaactgct tgtaccaatt gctattgtaa aaagtgttgc tttcattgcc aagtttgttt catgacaaaa gccttaggca tctcctatgg caggaagaag cggagacagc gacgaagagc tcatcagaac agtcagactc atcaagcttc tctatcaaag cagtaagtag tacatgtaat gcaacctata atagtagcaa tagtagcatt agtagtagca ataataatag caatagttgt gtggtccata gtaatcatag aatataggaa aatattaaga caaagaaaaa tagacaggtt aattgataga ctaatagaaa gagcagaaga cagtggcaat gagagtgaag gagaagtatc agcacttgtg gagatggggg tggaaatggg gcaccatgct ccttgggata ttgatgatct gtagtgctac agaaaaattg tgggtcacag tctattatgg ggtacctgtg tggaaggaag caaccaccac tctattttgt gcatcagatg ctaaagcata tgatacagag gtacataatg tttgggccac acatgcctgt gtacccacag accccaaccc acaagaagta gtattggtaa atgtgacaga aaattttaac atgtggaaag atgacatggt agaacagatg catgaggata taatcagttt atgggatcaa agcctaaagc catgtgtaaa attaacccca ctctgtgtta gtttaaagtg cactgatttg aagaatgata ctaataccaa tagtagtagc gggagaatga taatggagaa aggagagata aaaaactgct ctttcaatat cagcacaagc ataagagata aggtgcagaa agaatatgca ttcttttata aacttgatat agtaccaata gataatacca gctataggtt gataagttgt aacacctcag tcattacaca ggcctgtcca aaggtatcct ttgagccaat ccccatacat tattgtgccc cggctggttt tgcgattcta aaatgtaata ataagacgtt caatggaaca ggaccatgta caaatgtcag cacagtacaa tgtacacatg gaatcaggcc agtagtatca actcaactgc tgttaaatgg cagtctagca gaagaagatg tagtaattag atctgccaat ttcacagaca atgctaaaac cataatagta cagctgaaca catctgtaga aattaattgt acaagaccca acaacaatac aagaaaaagt atccgtatcc agaggggacc agggagagca tttgttacaa taggaaaaat aggaaatatg agacaagcac attgtaacat tagtagagca aaatggaatg ccactttaaa acagatagct agcaaattaa gagaacaatt tggaaataat aaaacaataa tctttaagca atcctcagga ggggacccag aaattgtaac gcacagtttt aattgtggag gggaattttt ctactgtaat tcaacacaac tgtttaatag tacttggttt aatagtactt ggagtactga agggtcaaat aacactgaag gaagtgacac aatcacactc ccatgcagaa taaaacaatt tataaacatg tggcaggaag taggaaaagc aatgtatgcc cctcccatca gtggacaaat tagatgttca tcaaatatta ctgggctgct attaacaaga gatggtggta ataacaacaa tgggtccgag atcttcagac ctggaggagg cgatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct gatatagtgc agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caaacagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg aacagatttg gaataacatg acctggatgg agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc acctcccaat cccgagggga cccgacaggc ccgaaggaat ag Translation (SEQ ID NO: 33) MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFMTKALGISYGRKKRRQRRRAHQNSQTHQASLSKQPTSQSRGDPTGPKE REV Protein: 5347-8021 (translate 5347-5422, 7747-8021) Nucleotide Sequence (SEQ ID NO: 34) atgg caggaagaag cggagacagc gacgaagagc tcatcagaac agtcagactc atcaagcttc tctatcaaag cagtaagtag tacatgtaat gcaacctata atagtagcaa tagtagcatt agtagtagca ataataatag caatagttgt gtggtccata gtaatcatag aatataggaa aatattaaga caaagaaaaa tagacaggtt aattgataga ctaatagaaa gagcagaaga cagtggcaat gagagtgaag gagaagtatc agcacttgtg gagatggggg tggaaatggg gcaccatgct ccttgggata ttgatgatct gtagtgctac agaaaaattg tgggtcacag tctattatgg ggtacctgtg tggaaggaag caaccaccac tctattttgt gcatcagatg ctaaagcata tgatacagag gtacataatg tttgggccac acatgcctgt gtacccacag accccaaccc acaagaagta gtattggtaa atgtgacaga aaattttaac atgtggaaag atgacatggt agaacagatg catgaggata taatcagttt atgggatcaa agcctaaagc catgtgtaaa attaacccca ctctgtgtta gtttaaagtg cactgatttg aagaatgata ctaataccaa tagtagtagc gggagaatga taatggagaa aggagagata aaaaactgct ctttcaatat cagcacaagc ataagagata aggtgcagaa agaatatgca ttcttttata aacttgatat agtaccaata gataatacca gctataggtt gataagttgt aacacctcag tcattacaca ggcctgtcca aaggtatcct ttgagccaat ccccatacat tattgtgccc cggctggttt tgcgattcta aaatgtaata ataagacgtt caatggaaca ggaccatgta caaatgtcag cacagtacaa tgtacacatg gaatcaggcc agtagtatca actcaactgc tgttaaatgg cagtctagca gaagaagatg tagtaattag atctgccaat ttcacagaca atgctaaaac cataatagta cagctgaaca catctgtaga aattaattgt acaagaccca acaacaatac aagaaaaagt atccgtatcc agaggggacc agggagagca tttgttacaa taggaaaaat aggaaatatg agacaagcac attgtaacat tagtagagca aaatggaatg ccactttaaa acagatagct agcaaattaa gagaacaatt tggaaataat aaaacaataa tctttaagca atcctcagga ggggacccag aaattgtaac gcacagtttt aattgtggag gggaattttt ctactgtaat tcaacacaac tgtttaatag tacttggttt aatagtactt ggagtactga agggtcaaat aacactgaag gaagtgacac aatcacactc ccatgcagaa taaaacaatt tataaacatg tggcaggaag taggaaaagc aatgtatgcc cctcccatca gtggacaaat tagatgttca tcaaatatta ctgggctgct attaacaaga gatggtggta ataacaacaa tgggtccgag atcttcagac ctggaggagg cgatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct gatatagtgc agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caaacagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg aacagatttg gaataacatg acctggatgg agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc acctcccaat cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga gaggcagaga cagatccatt cgattagtga acggatcctt agcacttatc tgggacgatc tgcggagcct gtgcctcttc agctaccacc gcttgagaga cttactcttg attgtaacga ggattgtgga acttctggga cgcagggggt gggaagccct caaatattgg tggaatctcc tacagtattg gagtcaggaa ctaaagaata g Translation (SEQ ID NO: 35) MAGRSGDSDEELIRTVRLIKLLYQSNPPPNPEGTRQARRNRRRRWRERQRQIHSISERILSTYLGRSAEPVPLQLPPLERLTLDCNEDC GTSGTQGVGSPQILVESPTVLESGTKE VPU Protein: 5439-5684 Nucleotide Sequence (SEQ ID NO: 36) at gcaacctata atagtagcaa tagtagcatt agtagtagca ataataatag caatagttgt gtggtccata gtaatcatag aatataggaa aatattaaga caaagaaaaa tagacaggtt aattgataga ctaatagaaa gagcagaaga cagtggcaat gagagtgaag gagaagtatc agcacttgtg gagatggggg tggaaatggg gcaccatgct ccttgggata ttgatgatct gtag Translation (SEQ ID NO: 37) MQPIIVAIVALVVAIIIAIVVWSIVIIEYRKILRQRKIDRLIDRLIERAEDSGNESEGEVSALVEMGVEMGHHAPWDIDDL ENV Polyprotein Precursor: 5599-8163 Nucleotide Sequence (SEQ ID NO: 38) at gagagtgaag gagaagtatc agcacttgtg gagatggggg tggaaatggg gcaccatgct ccttgggata ttgatgatct gtagtgctac agaaaaattg tgggtcacag tctattatgg ggtacctgtg tggaaggaag caaccaccac tctattttgt gcatcagatg ctaaagcata tgatacagag gtacataatg tttgggccac acatgcctgt gtacccacag accccaaccc acaagaagta gtattggtaa atgtgacaga aaattttaac atgtggaaag atgacatggt agaacagatg catgaggata taatcagttt atgggatcaa agcctaaagc catgtgtaaa attaacccca ctctgtgtta gtttaaagtg cactgatttg aagaatgata ctaataccaa tagtagtagc gggagaatga taatggagaa aggagagata aaaaactgct ctttcaatat cagcacaagc ataagagata aggtgcagaa agaatatgca ttcttttata aacttgatat agtaccaata gataatacca gctataggtt gataagttgt aacacctcag tcattacaca ggcctgtcca aaggtatcct ttgagccaat ccccatacat tattgtgccc cggctggttt tgcgattcta aaatgtaata ataagacgtt caatggaaca ggaccatgta caaatgtcag cacagtacaa tgtacacatg gaatcaggcc agtagtatca actcaactgc tgttaaatgg cagtctagca gaagaagatg tagtaattag atctgccaat ttcacagaca atgctaaaac cataatagta cagctgaaca catctgtaga aattaattgt acaagaccca acaacaatac aagaaaaagt atccgtatcc agaggggacc agggagagca tttgttacaa taggaaaaat aggaaatatg agacaagcac attgtaacat tagtagagca aaatggaatg ccactttaaa acagatagct agcaaattaa gagaacaatt tggaaataat aaaacaataa tctttaagca atcctcagga ggggacccag aaattgtaac gcacagtttt aattgtggag gggaattttt ctactgtaat tcaacacaac tgtttaatag tacttggttt aatagtactt ggagtactga agggtcaaat aacactgaag gaagtgacac aatcacactc ccatgcagaa taaaacaatt tataaacatg tggcaggaag taggaaaagc aatgtatgcc cctcccatca gtggacaaat tagatgttca tcaaatatta ctgggctgct attaacaaga gatggtggta ataacaacaa tgggtccgag atcttcagac ctggaggagg cgatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct gatatagtgc agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caaacagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg aacagatttg gaataacatg acctggatgg agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc acctcccaat cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga gaggcagaga cagatccatt cgattagtga acggatcctt agcacttatc tgggacgatc tgcggagcct gtgcctcttc agctaccacc gcttgagaga cttactcttg attgtaacga ggattgtgga acttctggga cgcagggggt gggaagccct caaatattgg tggaatctcc tacagtattg gagtcaggaa ctaaagaata gtgctgttaa cttgctcaat gccacagcca tagcagtagc tgaggggaca gatagggtta tagaagtatt acaagcagct tatagagcta ttcgccacat acctagaaga ataagacagg gcttggaaag gattttgcta taa Translation (SEQ ID NO: 39) MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLVNV TENFNMWKDDMVEQMHEDIISLWDQSLKPCVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNISTSIRDKVQKEYAFFYK LDIVPIDNTSYRLISCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEED VVIRSANFTDNAKTIIVQLNTSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNATLKQIASKLREQFGNNET IIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQEVGKAMYAPPISGQIRCSS NITGLLLTRDGGNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAVGIGALFLGFLGAAGSTMGAASMT LTVQARQLLSDIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNN MTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSIVNRVRQGYSP LSFQTHLPIPRGPDRPEGIEEEGGERGRDRSIRLVNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYW SQELKNSAVNLLNATAIAVAEGTDRVIEVLQAAYRAIRHIPRRIRQGLERILL NEF Protein: 8165-8785 Nucleotide Sequence (SEQ ID NO: 40) atgggt ggcaagtggt caaaaagtag tgtgattgga tggcctgctg taagggaaag aatgagacga gctgagccag cagcagatgg ggtgggagca gtatctcgag acctagaaaa acatggagca atcacaagta gcaatacagc agctaacaat gctgcttgtg cctggctaga agcacaagag gaggaagagg tgggttttcc agtcacacct caggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaaagaa gacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct gattggcaga actacacacc agggccaggg gtcagatatc cactgacctt tggatggtgc tacaagctag taccagttga gccagataag gtagaagagg ccaataaagg agagaacacc agcttgttac accctgtgag cctgcatgga atggatgacc ctgagagaga agtgttagag tggaggtttg acagccgcct agcatttcat cacgtggccc gagagctgca tccggagtac ttcaagaact gctga Translation (SEQ ID NO: 41) MGGKWSKSSVIGWPAVRERMRRAEPAADGVGAVSRDLEKHGAITSSNTAANNAACAWLEAQEEEEVGFPVTPQVPLRPMTYKAAVDLSH FLKEKGGLEGLIHSQRRQDILDLWIYHTQGYFPDWQNYTPGPGVRYPLTFGWCYKLVPVEPDKVEEANKGENTSLLHPVSLHGMDDPER EVLEWRFDSRLAFHHVARELHPEYFKNC HIV-1 p6*: sequence of this virus is presented in the Drawings attached hereto. pNL.Ba.L. The DNA sequence of the Ba.L Env sequence is shown below (SEQ ID NO: 42). This sequence was used to replace the Env sequence of NL4.3 in constructs with the Ba.L envelope. Unannotated (sense strand only; this sequence replaces the NL4.3 envelope sequence in the constructs with the Ba.L envelope): CCAACATAGCAGAATAGGTATTATTCAACAGAGGAGAGCAAGAAATGGAGCCAGTAGATCCTAAACTAGAGCCCTGGAAGCATCCAGGA AGTCAGCCTAAGACTGCTTGTACCACTTGCTATTGTAAAAAGTGTTGCTTTCATTGCCAAGTTTGCTTCATAACAAAAGGCTTAGGCAT CTCCTATGGCAGGAGGAAGCGGAGACAGCGACGAAGAGCTCCTCAAGACAGTCAGACTCATCAAGTTTCTCTATCAAAGCAGTAAGTAG TACATGTAATGCAAGCTTTACAAATATCAGCAATAGTAGGATTAGTAGTAGCAGCAATAATAGCAATAGTTGTGTGGACCATAGTATTC ATAGAATATAGGAAAATATTAAGGCAAAGAAAAATAGACAGGTTAATTGATAGAATAACAGAAAGAGCAGAAGACAGTGGCAATGAGAG TGATGGAGATCAGGAGGAATTATCAGCACTGGTGGAGATGGGGCATCATGCTCCTTGGGATGTTAATGATCTGTAATGCTGAAGAAAAA TTGTGGGTCACAGTCTATTATGGGGTACCTGTGTGGAAAGAAGCAACCACCACTCTATTTTGTGCATCAGATGCTAAAGCATATGATAC AGAGGTACATAATGTTTGGGCCACACATGCCTGTGTACCCACAGACCCCGACCCACAAGAAGTAAAATTGGAAAATGTGACAGAAAATT TTAACATGTGGAAAAATAAAATGGTAGAACAGATGCATGAGGATATAATCAGTTTATGGGATCAAAGCCTAAAGCCATGTGTAAAATTA ACTCCACTCTGTGTTACTTTAAATTGCACTGTAAGCGGGGGAATGATGGGGGGAGGAGAAATGAAAAATTGCTCTTTCAATATCACCAC AAACATAAGAGGTAAGGTGCAGAAAGAATATGCACTTTTTTATGAACTTGATATAGTACCAATAGATAATAAAAATGATAGCTATAGGT TGATAAGTTGTAACACCTCAGTCATTACACAGGCCTGTCCAAAGGTATCCTTTGAGCCAATTCCCATACATTATTGTGCCCCGGCTGGT TTTGCGATTCTAAAGTGTAAAGATAATAAGTTCAATGGAAAAGGACCATGTACAAATGTCAGCACAGTACAATGTACACATGGAATTAG GCCAGTAGTATCAACTCAACTGCTGTTAAATGGCAGTCTAGCAGAAGAAGAGGTAGTAATTAGATCCGAAAATTTCACGAACAATGCTA AAACCATAATAGTACAGCTGAATGAATCTGTAGTAATTAATTGTACAAGACCCAACAACAATACAAGAAAAAGTATAAATATAGGACCA GGCAGAGCATTTTATACAACAGGAGAAATAATAGGAGATATAAGACAAGCACATTGTAACCTTAGTAGAGCAAAATGGAATGACACTTT AAATAAGATAGTTATAAAATTAAGAGAACAATTTGGGAATAAAACAATAGTCTTTAAGCACTCCTCAGGAGGAGACCCAGAAATTGTGA CGCACAGTTTTAATTGTGGAGGGGAATTTTTCTACTGTAATTCAACACAACTGTTTAATAGTACTTGGAATGTTACTGAAGAGTCAAAT AACACTGTAGAAAATAACACAATCACACTCCCATGCAGAATAAAACAAATTATAAACATGTGGCAGGAAGTAGGAAGAGCAATGTATGC CCCTCCCATCAGAGGACAAATTAGATGTTCATCAAATATTACAGGGCTGCTATTAACAAGAGATGGTGGTCCTGAGGACAACAAGACCG AGGTCTTCAGACCTGGAGGAGGAGATATGAGGGATAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGA GTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTGTGTTCCTTGGGTTCTTGGGAGC AGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACTATTATTGTCTGGTATAGTGCAACAGCAGAACAATC TGCTGAGAGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAGTCCTGGCTGTGGAA AGATACCTAAGGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATCTGCACCACTGCTGTGCCTTGGAATGCTAGTTG GAGTAATAAATCTCTGAATAAGATTTGGGATAACATGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCATAATATACA GCTTAATTGAAGAATCGCAGAACCAACAAGAAAAGAATGAACAAGAATTATTAGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTT GACATAACAAAATGGCTGTGGTATATAAAAATATTCATAATGATAGTAGGAGGCTTGATAGGTTTAAGAATAGTTTTTTCTGTACTTTC TATAGTGAATAGAGTTAGGCAGGGATACTCACCATTATCGTTTCAGACCCACCTCCCAGCCTCGAGGGGACCCGACAGGCCCGGAGGAA TCGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCGGTTCATTAGTGAACG CD40 Ligand: Accession: CAA48077.1 GI: 38412 CD40 Ligand Nucleic Acid Sequence (786 base pairs; SEQ ID NO: 43): ATG ATCGAAACATACAACCAAACTTCTCCCCGATCTGCGGCCACTGGACTGCCCATCAGCATGAAAATTTTTATGTATTTACTTACTGT TTTTCTTATCACCCAGATGATTGGGTCAGCACTTTTTGCTGTGTATCTTCATAGAAGGTTGGACAAGATAGAAGATGAAAGGAATCTTC ATGAAGATTTTGTATTCATGAAAACGATACAGAGATGCAACACAGGAGAAAGATCCTTATCCTTACTGAACTGTGAGGAGATTAAAAGC CAGTTTGAAGGCTTTGTGAAGGATATAATGTTAAACAAAGAGGAGACGAAGAAAGAAAACAGCTTTGAAATGCAAAAAGGTGATCAGAA TCCTCAAATTGCGGCACATGTCATAAGTGAGGCCAGCAGTAAAACAACATCTGTGTTACAGTGGGCTGAAAAAGGATACTACACCATGA GCAACAACTTGGTAACCCTGGAAAATGGGAAACAGCTGACCGTTAAAAGACAAGGACTCTATTATATCTATGCCCAAGTCACCTTCTGT TCCAATCGGGAAGCTTCGAGTCAAGCTCCATTTATAGCCAGCCTCTGCCTAAAGTCCCCCGGTAGATTCGAGAGAATCTTACTCAGAGC TGCAAATACCCACAGTTCCGCCAAACCTTGCGGGCAACAATCCATTCACTTGGGAGGAGTATTTGAATTGCAACCAGGTGCTTCGGTGT TTGTCAATGTGACTGATCCAAGCCAAGTGAGCCATGGCACTGGCTTCACGTCCTTTGGCTTACTCAAACTC TGA Translation of CD40 Ligand (261 amino acids, SEQ ID NO: 44): MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKS QFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFC SNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL HIV Peptides: Peptides Protein of origin a Sequence A9M Pol (188-196) ALVEICTEM (SEQ ID NO: 45) 19V Pol (464-472) YLKEPVHGV (SEQ ID NO: 46) K9L Env gp120 (120-128) YLTPLCVSL (SEQ ID NO: 47) T9V Gag p24 (19-27) YLNAWVKVV (SEQ ID NO: 48) V9L Pol (334-342) YIYQYMDDL (SEQ ID NO: 49) P10L Nef (134-143) YLTFGWCFKL (SEQ ID NO: 50) V11V Pol (263-273) VLDVGDAYFSV (SEQ ID NO: 51) P9L Pol (576-584) YLVKLWYQL (SEQ ID NO: 52) S9L Gag p17 (77-85) SLYNTVATL (SEQ ID NO: 53) E9V Gag p24 (212-221) YMMTACQGV (SEQ ID NO: 54) L10V Pol (79-88) LLDTGADDTV (SEQ ID NO: 55) L9V Pol (956-964) LLWKGEGAV (SEQ ID NO: 56) K9L (T) Env gp120 (120-128) YLTPLCVTL (SEQ ID NO: 57)
See Iglesias et al. Mol Ther 15, 1203-1210, doi:10.1038/sj.mt.6300135 (2007), the entire content of which is incorporated herein by reference, with respect to the HIV-1 peptides indicated above.

The invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention and should in no way be construed, however, as limiting the broad scope of the invention.
EXAMPLE 1
Methods
Cells and cell culture. 293T cells were cultured in Dulbecco's modified Eagle's Medium (DMEM)-10% fetal bovine serum (FBS). MDM, MDDC, and T cells were cultured in RPMI 1640-5% Human AB. Peripheral blood mononuclear cells (PBMC) were purified from normal human donor blood by Ficoll density gradient. Monocytes were purified from healthy donor PBMC by adherence to plastic or by positive selection on anti-CD14-coated magnetic beads (Miltenyi Biotec Inc.). Bead purified monocytes were typically >98% CD14 + . The monocytes were differentiated to MDM by culturing for 4-6 days in medium containing 50 ng/ml GM-CSF (Invitrogen Inc.). MDDC were generated by culturing the monocytes for 5-6 days in medium containing 50 ng/ml GM-CSF and 100 ng/ml IL-4 (R&D Systems). Autologous T cells were obtained from the CD 14-negative fraction by positive selection on anti-CD4-conjugated magnetic beads (Miltenyi Biotec Inc.). The T cells were then activated using anti-CD3/CD28 beads (Invitrogen) and cultured in medium containing 10 ng/ml IL-2 (R&D Systems).
Plasmids. Codon-optimized, epitope-tagged SIVmac 239 and HIV-2 rod Vpx and SIVagm Vpr expression vectors were generated by overlapping PCR and cloned into pcDNA6 (Invitrogen Inc.) at the EcoR-I and Xho-I sites ( FIG. 8 ). HIV-1 p6 chimeras and point mutants were generated in the pNL4-3-based luciferase reporter virus, pNL.Luc3. An Apa-I to EcoR-I subclone containing the p6 region was subcloned into pBluescript-KS+ (Fermentas Life Sciences) that had been modified to remove the multi-cloning site Pst-I site. Mutations were introduced by overlapping PCR and the mutated fragment was cloned back into the subcloned fragment in pBS-KS+ at the Apa-I and Pst-I sites. The mutant fragment was then cloned into NL4-3 at the Apa-I and EcoR-I sites. Vpr and vpx were cloned into the nef position of NL.Luc3 by removing the luciferase gene with Not-I and Xho-I and replacing it with codon optimized vpr and vpx amplicons that had been amplified with primers that introduced Not-I and Sal-I sites.
Codon optimization of vpx was undertaken where non-optimal codons were present in the original vpx sequence. Non-optimal codons were altered to be the most commonly used mammalian codon which encodes the indicated amino acid. An alignment of the wildtype and codon-optimized Vpx sequences is shown in FIG. 8 .
Virus preparation and infections. HIV-1 and SIVmac 239 luciferase reporter viruses were generated as described previously (10, 34). Briefly, to produce trans complemented reporter viruses, 293T cells were cotransfected using lipofectamine 2000 with pNL-luc3-E − R − , pcVSV-G, and pcVpr, pcVpx, or pcDNA at a mass ratio of 2:1:1. Supernatants were harvested 48 h posttransfection, passed through 0.45 μm pore-size filters, aliquoted, and frozen at −80° C. Luciferase reporter virus infectivity was normalized for luciferase activity on 293T cells by infecting 2.0×10 4 cells with 50 μl of virus. Three days later, luciferase activity was measured using SteadyLite HTS reagents (PerkinElmer), typically yielding 1.0-3.0×10 6 counts per second (cps) per 50 μl. P24 content of virus-containing supernatants was quantified by ELISA using commercially available capture and sandwich antibodies (Aalto Bioreagents, LTD)
MDM (1.0×10 5 ) and MDDC (1.25×10 5 ) were seeded in a 96-well plate and then infected with reporter virus corresponding to 3.0×10 5 cps. MDM were spin-infected with reporter virus for 2 h at 500×g. The data are presented as the average cps of triplicate infections with error bars indicating the standard deviation. For intracellular p24 detection, MDM (3.0×10 5 ) or MDDC (3.0×10 5 ) were infected with 20 ng p24. For infections in which Vpx was produced in cis, MDM (5.0×10 5 ) were infected with 200 ng p24 and MDDC (1.25×10 5 ) were infected with 50 ng of p24. To control for input virus, one sample was treated with 25 μM AZT (AIDS Research and Reference Reagent Program, NIH) prior to infection. To limit replication to one round, 3 μM Nelfinavir (AIDS Research and Reference Reagent Program, NIH) was added to the samples 6 hours post infection. The cells were fixed, permeabilized, and stained as described below. For MDDC:T cell trans infection assays, either 2.5×10 5 or 1.0×10 5 MDDC were seeded in a 96 well culture plate. The next day, the cells were treated with 50 or 25 ng p24, respectively, and, after 6 h, washed three times with medium. After 48 h, autologous CD3/CD28-activated T cells (1.0×10 6 or 4.0×10 5 ) were added to wells designated for co-culture, resulting in a final ratio of MDDC to T cell of 1:4. To test T cell infection in the absence of MDDC, CD3/CD28-activated T cells (4.0×10 5 ) were infected with 25 ng virus, and, after 6 h, media was changed. Supernatant was collected either at 3, 5, 7, and 10 days or 3, 5, 7, 9, and 14 days post coculture, and p24 was then quantified. To detect intracellular p24, cells were collected at 3 days post coculture, fixed, permeabilized, and stained as described below.
Quantitative Real-time PCR (qRT-PCR). To quantify HIV-1 reverse transcripts in newly infected cells, 3×10 5 MDM were infected with 5 ng p24 of virus treated for 1 h with 50 U/ml of Benzonase (Invitrogen, Inc.). To the sample infected with the p6 chimeric virus encoding Vpx, 25 μM AZT was added prior to infection to control for residual plasmid DNA. After 24 h and 48 h, DNA was isolated (Qiagen). Late reverse transcripts and 2-LTR circles were quantitated by qRT-PCR using 250 ng DNA template as described previously (8, 21). Amplicons were detected with SYBR Green (Applied Biosystems) using an ABI Prism 7300 (Applied Biosystems). Absolute copy numbers were determined by normalization to standard curves generated from serially diluted proviral plasmid and two-LTR plasmid.
Induction of interferon-β 1 (IFN-β 1 ) in response to HIV-1 in MDDC was measured by qRT-PCR. MDDC were infected with 50 ng p24. To two samples, 25 μM AZT or 10 μM of Raltegravir (Merck) was added prior to infection. After 24 h and 48 h, RNA was isolated using Triazol (Invitrogen Inc.), and cDNA was generated using Transcriptor reverse transcriptase (Roche) primed with oligo-(dT). IFN-β 1 and glucose-6-phosphate dehydrogenase (G6PDH) were then amplified with primers previously described by Di Domizio et al. (11) Relative threshold Cycle (Ct) value for IFN-β 1 was normalized to G6PDH.
Immunoblot analysis. Virions were harvested from culture supernatants two days posttransfection, filtered, pelleted through 20% sucrose at 100,000×g for 20 min. at 4° C., and then lysed in buffer containing 0.5% NP40. Cell and virus proteins were analyzed on immunoblots as previously described (21). The filters were probed with anti-myc MAb 9E10 (Covance), anti-p24 MAb #183-H12-SC (AIDS Research and Reference Reagent Program, NIH), and anti-α-tubulin (Sigma). The filters were then hybridized with biotinylated goat anti-mouse immunoglobulin and Streptavidin DyeLight 680 or 800 conjugate (Pierce) and imaged on an Odyssey Infrared Imaging System (LiCOR) at 700 or 800 nm, respectively.
Intracellular p24 Staining. The cells were removed from culture dishes in phosphate-buffered saline (PBS) containing 5.0 mM EDTA, fixed and permeabilized with BD Cytofix/Cytoperm (BD-Pharmingen), and washed with BD Perm/Wash (BD-Pharmingen) according to the manufacturer's instructions. The cells were then stained with a 1:200 dilution of either PE- or FITC-conjugated anti-p24 MAb KC57 (Beckman Coulter) and analyzed by flow cytometry using Flojo software. The cells were gated for forward and side scatter and analyzed for PE or FITC fluorescence with mock infected cells as a negative control.
For analysis of the cells in the MDDC:T cell trans infection assays, the cells in the mixed cultures were removed from the culture dishes and stained with a 1:25 dilution of APC-conjugated CD11c (BD Pharmingen). They were then fixed and permeabilized and stained with for p24. T cells and MDDC were gated on CD11c and intracellular p24 was detected by FITC fluorescence. Mock-infected cells were used as a negative control.
Results
Mapping of the Vpx packaging determinant in Gag. Packaging of Vpx into virions is mediated by its interaction with the C-terminal domain of the Gag polyprotein precursor, p6 (1, 47). The interaction is virus-specific, such that Vpx is packaged by SIVmac but not by HIV-1(25). P6 is structured as two alpha helices with the PTAPP late domain motif that binds TSG101 adjacent to helix 1 and the ALIX binding motif overlapping helix 2 (14, 52) ( FIG. 1A ). Accola et al. mapped the Vpx packaging site in p6 to the amino acid sequence 17 DPAVDLL 23 , just C-terminal to the PTAPP late domain motif (1). As a first step in engineering an HIV-1 that would package SIVmac Vpx, we determined the minimal SIVmac p6 amino acid sequence that needed to be introduced into HIV-1 to allow Vpx packaging. To do this, we generated chimeric HIV-1 genomes 17-23(a), 17-23(b), 17-28, 17-38, and 17-48, where the indicated region of SIVmac p6 was transferred to HIV-1 p6 ( FIG. 1A ). In chimera 17-23(a), the SIV sequence was displaced by six codons to preserve amino acids 14-18 that contain the 14 FRFG 18 motif previously reported to play a role in Vpr packaging (53). We determined the ability of the chimeric viruses to package Vpx by cotransfecting 293T cells with the chimeric proviral DNA and two different amounts of the myc-tagged SIVmac Vpx expression vector, pcVpx.myc. After two days, the culture supernatant was harvested and the virions were pelleted by ultracentrifugation and analyzed on an immunoblot ( FIG. 1B ). The results showed that chimeras 17-23(a) and 17-23(b) failed to package Vpx, but the addition of five more amino acids from SIVmac p6 in chimera 17-28 allowed for packaging of Vpx. Extension of the chimeric region in chimeras 17-38 and 17-48 did not further increase Vpx packaging at either of the two amounts of cotransfected pcVpx.myc. The failure of chimera 17-23 to package Vpx was unexpected as this chimera contained the previously described 17 DPAVDLL 23 motif, suggesting that the packaging motif extends further C-terminal. To more precisely define the Vpx packaging motif, we generated chimeras 17-24, 17-25, 17-26, 17-27, and 17-28, where single amino acids from positions 24 to 28 were added ( FIG. 1C ). Immunoblot analysis of virions derived from these chimeric genomes showed that addition of SIVmac p6 amino acids 24 and 25 did not allow for Vpx packaging. When the chimeric region was extended to position 26, packaging was restored. We conclude that the minimal Vpx packaging motif required to allow efficient packaging of SIVmac Vpx into HIV-1 virions is 17 DPAVDLLKNY 26 (SEQ ID NO: 1).
Mapping the Vpr packaging determinant in p6. Since p6 also contains the packaging determinant for Vpr, it was possible that alteration of this region would affect Vpr packaging. Previous reports have mapped the Vpr packaging motif in p6 to two separate locations. Kondo et al. mapped the determinant in HIV-1 and SIVmac to a 41 LXXLF 45 (SEQ ID NO: 58) motif near the C-terminus of p6 that overlaps with the ALIX binding motif (here termed “motif 2”; FIG. 2A ) (30). Subsequently, Zhu et al. reported that p6 deleted for motif 2 retained the ability to package Vpr (53). Instead, they found that the 15 FRFG 18 (SEQ ID NO: 59) motif (motif 1) near the N-terminus of p6 was required for Vpr packaging. In that study, motif 1 was tested only in the context of a truncated p6 that lacked motif 2. To evaluate the roles of the two motifs in Vpr packaging, we generated p6 point mutants in both motifs and tested the resulting virions for Vpr packaging. For motif 1, mutants F15A and F17A decreased packaging by about 80%, while mutants R16A and G18A were similar to wild-type ( FIG. 2B ). Mutation of the entire 15 FRFG 18 (SEQ ID NO: 59) motif to alanine (M1A) did not further reduce the amount of Vpr packaged below that of the point mutants. In the case of motif 2, single mutations to alanine had little effect ( FIG. 2C ). L38 mutated virions appeared to contain a reduced amount of Vpr; however, production of the virions was also reduced, suggesting that the Vpr content per virion had been unaltered. Mutation of amino acids S43 and S45 did not decrease the amount of Vpr packaged. In fact, both appeared to package more Vpr than wild-type virus. These two mutant viruses generated virions that were defective for Gag processing, probably because those amino acids are close to the proteolytic processing site and interfere with recognition by the viral protease. Unprocessed virions are highly stable and as a result may be more effective at packaging Vpr. Although single amino acid mutations in motif 2 had little effect, mutation of the four conserved leucines together (M2Aa) blocked Vpr packaging, confirming the importance of motif 2 in Vpr packaging. Combination of the motif 1 and motif 2 mutations (M1 and 2a and M1 and 2b) also prevented Vpr packaging. We conclude that both motifs play a role in Vpr packaging.
Having identified both motifs as important, we next measured Vpr packaging by the p6 chimeras. To do this, we produced the chimeric virions in cells cotransfected with a Vpr expression vector and then analyzed the resulting virions on an immunoblot. We found that chimera 17-23a, in which the 15 FRFG 18 (SEQ ID NO: 59) motif is intact, maintained its ability to package Vpx, while the other chimeras, in which the 15 FRFG 18 (SEQ ID NO: 59) motif has been altered, packaged about one third as much Vpr ( FIGS. 2D and 9 ). For reasons that are not clear, chimera 17-28 reproducibly packaged Vpr somewhat better (70% of wild-type) despite lacking the 15 FRFG 18 motif, perhaps due to a conformational effect on motif 2.
HIV-1 p6 chimera containing SIVmac Vpx efficiently infects MDDC and MDM. The introduction of Vpx into MDM and MDDC using VLP increases the infectivity of HIV-1 (18, 21). These findings predict that HIV-1 virions packaging Vpx should have an increased ability to infect MDM and MDDC. To determine whether this is the case, we used the 17-26 p6 chimeric virus that contains the minimum SIVmac 239 Vpx packaging sequence. Chimeric p6 and wild-type luciferase reporter viruses were produced as VSV-G pseudotypes in 293T cells cotransfected with increasing amounts of pcVpx.myc. The viruses were normalized for infectivity on 293T cells and were then used to infect MDDC and MDM from three donors. Immunoblot analysis of the virions showed that wild-type virions contained only small amounts of Vpx over the Vpx expression vector titration curve, while the p6 chimeric virions packaged Vpx proportional to the amount of transfected Vpx expression vector ( FIG. 3A ). On MDDC, the wild-type virus that lacked Vpx was poorly infectious. Complementation with the highest amount of Vpx significantly enhanced its infectivity, suggesting that the small amount of Vpx packaged was sufficient to provide an effect on the target cell ( FIG. 3B ). The p6 chimeric virus complemented with increasing amounts of Vpx became even more infectious. The smallest amount of Vpx, which was barely detectable in the virion by immunoblot analysis, enhanced the infectivity of the virus an average of 100-fold. Increasing amounts of Vpx further enhanced the infectivity to 600-800-fold, depending on the donor. Similar results were obtained using cell preparations from 7 additional donors. Vpx was also active in the infection of MDM, although the fold-enhancement was not as dramatic (about 3-fold lower). The titration curve on the MDM suggests that they are more sensitive to a low level of packaged Vpx, with infectivity reaching nearly half-maximal with the lowest amount of cotransfected Vpx expression vector.
The luciferase reporter virus does not distinguish between effects on the number of cells infected and the provirus transcriptional activity per infected cell. To determine whether packaged Vpx increases the number of infected cells, we quantified intracellular p24 in the infected MDM and MDDC by flow cytometry ( FIG. 3C ). We found that the wild-type and p6 chimeric viruses lacking Vpx were poorly infectious on MDDC, as was the wild-type virus complemented with Vpx. In contrast, complementation of the p6 chimeric virus with Vpx increased the number of infected cells 98-fold. On MDM, complementation of the chimeric virus increased its infectivity by 84-fold. On these cells, the Vpx-complemented wild-type virus was also considerably enhanced, resulting in nearly 40% as many infected cells as the complemented p6 chimeric virus. This result suggests that MDM are less restrictive to virus lacking Vpx and are therefore sensitive to low levels of packaged Vpx. The mean fluorescence intensity of the p24-positive cells was not affected, indicating that Vpx did not affect transcription of the provirus or translation of the viral proteins.
Comparison of the activities of lentiviral Vpx. Besides SIVmac, HIV-2 encodes a Vpx. In addition, SIVagm encodes a protein that has been termed Vpr but is more similar to Vpx, sharing its ability to enhance infection of MDM and inability to induce G 2 arrest (9, 45). To determine whether these proteins enhance HIV-1 infection, we complemented wild-type or p6 chimeric viruses with expression vectors for SIVmac 239 Vpx, HIV-2 rod Vpx, or SIVagm Vpr. Immunoblot analysis showed that each of the accessory proteins could be packaged into the p6 chimeric virus but not into the wild-type virus ( FIG. 4A ). SIVagm Vpr and HIV-2 rod Vpx were packaged in smaller amounts due to their relatively low level expression in the cell. On MDDC, the three accessory proteins had no effect on the infectivity of the wild-type virus. SIVmac 239 Vpx and HIV-2 rod Vpx both enhanced the infectivity of the p6 chimeric virus ( FIG. 4B , top panel). HIV-2 rod Vpx was only about 8% as active as SIVmac 239 Vpx, but this may be the result of its relatively low expression level. SIVmac 239 Vpx and HIV2 rod Vpx also enhanced the infectivity of the p6 chimeric virus on MDM ( FIG. 4B , bottom panel). As in the earlier experiments, the MDM were overall less restrictive than MDDC. SIVagm Vpr had no detectable effect on infection of MDDC and the less restrictive MDM. The lack of enhancement may indicate a species-restriction to the AGM protein. Intracellular p24 staining of infected MDDC further supported these findings (not shown).
A p6 chimeric virus that encodes Vpx. In the experiments described above, the Vpx containing virions were produced by trans complementation in cells cotransfected with a replication-defective reporter virus and a Vpx expression vector. Expression of Vpx in cis would allow for Vpx expression through multiple rounds of virus replication and would obviate the need to complement by cotransfection. To generate a Vpx-containing virus that expressed Vpx in cis, we placed a codon-optimized vpx open reading frame in place of nef, a position that has been found to allow for expression of an inserted reporter gene without affecting virus replication (10, 29, 34). We considered placing the vpx in the vpr position; however, this would disrupt the overlapping reading frames and splice signals in this region of the viral genome. The cis virus is based on pNL.Ba.L, an NL4-3 that contains the CCR5-specific envelope glycoprotein of Ba.L. The provirus was further modified by introduction of the Vpx packaging residues 17-26 of SIVmac 239 p6 and a codon-optimized SIVmac 239 vpx, HIV-2 rod vpx, or SIVagm vpr in nef. Immunoblot analysis of virions produced by 293T cells transfected with each proviral DNA showed that they expressed low levels of the accessory proteins ( FIG. 5A ). These could be detected in cell lysates treated with MG132 to stabilize the relatively short-lived proteins. Due to the low levels of Vpx production, we measured the infectivity of the cis viruses in newly infected cells by the sensitive method of qRT-PCR quantification. For this, we infected MDM with cis virus encoding SIVmac 239 Vpx and, after 24 and 48 h, measured the early reverse transcription products and 2-LTR circles. The analysis showed that at both time points the p6 chimeric virus encoding Vpx generated about 4-fold more late cDNA molecules than virus with wild-type p6 or virus that lacked Vpx. After 48 h, the p6 chimeric virus with vpx in cis generated six-fold more 2-LTR circles ( FIG. 5B ). To determine the relative numbers of infected cells, we infected MDDC with the cis viruses and analyzed them for intracellular p24 by flow cytometry. The analysis showed that the p6 chimeric virus encoding SIVmac 239 Vpx infected 8-fold more MDDC compared to viruses lacking Vpx ( FIG. 5C ). The HIV2 rod and SIVagm viruses were not significantly enhanced compared to the controls. Addition of the protease inhibitor, Nelfinavir, to the SIVmac 239 Vpx-encoding p6 chimeric virus decreased the number of infected cells by an average of 58%, suggesting that the virus had replicated beyond the first cycle. We conclude that the cis virus containing SIVmac 239 Vpx replicated in the cells with enhanced efficiency despite having small amounts of Vpx.
HIV-1 containing Vpx induces Type I IFN in MDDC. Using pretreatment with Vpx-containing VLP, Manel et al. recently showed that HIV-1 infection of MDDC activates an innate immune response, resulting in the induction of CD86 and type I IFN (32). Induction of the response appeared to be triggered by newly synthesized viral Gag protein that had activated an as yet unidentified sensor. These findings predict that Vpx-containing HIV-1 will induce an innate immune response in MDDC. To determine whether this is the case, we infected MDDC with p6 chimeric virus containing or lacking Vpx and, after 24 and 48 h, quantified IFN-Iβ mRNA. The results showed a robust Vpx-dependent induction of IFN-β 48 h postinfection ( FIG. 6 ). The IFN-β induction was blocked by AZT and Raltegravir, inhibitors that block reverse transcription and integration, respectively. This result suggests that the response was triggered post-integration and not induced by the in-coming virion. Furthermore, the lack of induction at 24 h is also consistent with a late event in virus replication, supporting the findings of Manel et al. (32). These results suggest that the p6 chimeric virus induced an innate immune response in the MDDC.
Vpx facilitates the transfer of HIV-1 from MDDC to T cells. In vitro, MDDC can be shown to transmit HIV-1 to CD4 T cells by trans-infection, a process in which the virus binds to cell surface lectin-like proteins on MDDC, such as DC-SIGN, and is then directly transferred to the T cell (16). In trans-infection, the MDDC does not become infected but binds the virus at the plasma membrane. Since Vpx enhances infection of MDDC, we hypothesized that, in addition to transmission through trans-infection, HIV-1 virions containing Vpx could be efficiently transmitted from MDDC to CD4 T cells through a direct infection mechanism analogous to cell-cell transmission by T cells (37). To test whether Vpx would allow for enhanced transmission of HIV-1 from MDDC to CD4 T cells, we infected MDDC with the NL.Ba.L p6 chimeric virus containing Vpx (Vpx+) or lacking Vpx (Vpx−) in cis. The free virus was removed and two days later, autologous activated CD4 T cells were added. We harvested the culture supernatant over 10 days for p24 quantification. The results showed that the Vpx-containing chimeric virus replicated with more rapid kinetics, producing greater than 8-fold more p24 at earlier timepoints than virus lacking Vpx ( FIG. 7A ).
The increased p24 production in this experiment could have been due simply to production from the MDDC. Alternatively, it could have been due to an effect of Vpx on HIV-1 replication in the activated CD4 T cells. To better determine the relative contribution of the two cell-types to virus replication, we established three cultures: MDDC alone, activated CD4 T cells alone, and a coculture of activated CD4 T cells and MDDC. All three cultures were infected with the NL.Ba.L chimeric p6 virus either containing Vpx (Vpx+) or lacking Vpx (Vpx−) in cis. In the case of the coculture, the MDDC were infected and, after 6 h, the virus was removed. Two days later, activated CD4 T cells were then added. We collected supernatants over the next 14 days for p24 quantification. In the MDDC alone culture, the Vpx− virus produced undetectable p24, while the Vpx+ virus produced moderate p24 after 10 days ( FIG. 7B ). In the coculture, the Vpx− virus replicated to a similar moderate level, while the Vpx+ virus replicated faster and to a higher p24 amount. This result could be explained either by more efficient transfer of the Vpx+ virus from MDDC to CD4 T cell or, alternatively, by better replication of the Vpx+ virus in the T cells independent of the MDDC. In the CD4 T cell alone culture, the Vpx+ and Vpx− viruses replicated with similar kinetics. This result suggests that Vpx does not enhance virus replication directly in
CD4 T cells, consistent with earlier findings. Thus, in the cocultures the increased replication was due to transfer of the virus from infected MDDC to activated CD4 T cell.
To determine the number of infected CD4 T cells and MDDC in the cocultures, we stained the cells for intracellular p24 and gated on the CD11c+ (MDDC) and CD11c− (T cell) populations. The analysis showed that both the CD11c+ and CD11c− cells were infected more efficiently by the Vpx+ as compared to the Vpx− virus ( FIG. 7C ). As Vpx does not have a significant effect on HIV-1 replication in T cells, the result further suggests a role for Vpx in transmission of virus from MDDC to CD4 T cell.
Discussion
We report here on the development of an HIV-1 with a p6 that has been modified to allow packaging of Vpx. With the addition of a ten amino acid Vpx packaging motif from SIVmac 239 , the engineered virus efficiently packaged SIVmac 239 Vpx, HIV-2 rod Vpx, or SIVagm Vpr expressed in trans in the producer cell. The virus containing SIVmac 239 Vpx was dramatically enhanced in its ability to infect MDDC and MDM, induced a strong innate immune response, and replicated more efficiently in MDDC:CD4 T cell co-cultures than wild-type virus. Replication-competent, R5-tropic p6 chimeric virus that expressed Vpx in cis produced a relatively small amount of the accessory protein, but, in the case of SIVmac 239 Vpx, this was sufficient to provide a significant replicative advantage in MDDC. MDDC were more restrictive than MDM, an effect that was apparent in infections with viruses that had packaged only a limited amount of Vpx. In addition, there was variability in the stringency of the restriction in the cells of different donors. Vpx had no detectable effect on replication of the virus in activated CD4 T cells. As Vpx has been proposed to counteract a yet unidentified host restriction factor, our results imply that MDDC express more of the hypothetical restriction factor than MDM and that donors differ in how much of the factor their cells express.
Because Vpx packaging is virus-specific, engineering the virus required the introduction of the amino acid motif of SIVmac 239 that mediates Vpx packaging into HIV-1 p6 (25). To construct the virus, we first showed that the SIVmac 239 p6 sequence 17 DPAVDLLKNY 26 (SEQ ID NO: 1) was the minimal motif needed to confer Vpx packaging on HIV-1. This sequence includes the 17 DxAxxLL 23 (SEQ ID NO: 60) identified as the Vpx packaging motif by Accola et al., with the addition of three carboxy-terminal amino acids (1). Introduction of the motif adjacent to the PTAPP (SEQ ID NO: 61) late domain resulted in a chimeric virus that efficiently packaged SIVmac 239 Vpx, HIV-2 rod Vpx, and SIVagm Vpr. When SIVmac 239 Vpx was provided in trans, the resulting virus was 10-100-fold more infectious on MDM and MDDC. Due to low level expression, HIV-2 rod Vpx only slightly enhanced MDM and MDDC infection. SIVagm Vpr, which shares properties with Vpx, did not enhance the infection in human MDM or MDDC, suggesting that its function may be species-specific. When expressed in cis, only SIVmac 239 Vpx enhanced the infection of the MDM and MDDC.
The packaging of Vpr is mediated by a separate amino acid motif in p6. Two groups have reported on p6 residues required for Vpr packaging but with different results. Kondo et al. showed that p6 could be transferred to MuLV to allow virion packaging of Vpr and that the critical region required for packaging was the leucine motif, termed here motif 2 (30). In contrast, Zhu et al. found that virus containing a truncation of p6 at amino acid 35 that deletes motif 2 maintained Vpr packaging (53). In the truncated virus, the motif required for Vpr packaging mapped to 15 FRFG 18 , here termed motif 1. In this study, we show that in the context of full-length Gag both motifs play a role in Vpr packaging, although our results differ somewhat from both groups. Kondo et al. found that single amino acid mutations in motif 2 at positions 41, 44, or 45 prevented Vpr packaging. In contrast, we found that the individual mutations had no effect, but mutation of all four hydrophobic residues prevented Vpr packaging. This difference is probably because Kondo et al. tested the p6 mutants in the context of MuLV Gag, whereas we analyzed them in the context of HIV-1 Gag. Our results differed from those of Zhu et al. in that single amino acid mutations in the motif 1 15 FRFG 18 (SEQ ID NO: 59) sequence partially reduced but did not prevent Vpr packaging. The difference is probably because they analyzed the mutations in the context of truncated Gag, whereas we tested them in full-length Gag. A likely explanation for the role of the two motifs in Vpr packaging is that one serves as a binding site and the other affects p6 conformation. It is difficult to distinguish between these roles because both motifs lie in predicted alpha helices (14). However, it is likely that the hydrophobic alpha helical leucines of motif 2 face inward in the protein and thus are more likely to play a conformational role than serve as a binding site. Based on the importance of both motifs, the chimeric 17-26 p6 virus was able to maintain a low level of Vpr packaging despite the alteration of the overlapping 15 FRFG 18 (SEQ ID NO: 59) motif.
Despite the use of a codon optimized open reading frame, expression of Vpx in cis resulted in a low level of intracellular expression and suboptimal packaging. The reduced level of Vpx packaging was probably caused by two factors. First, Vpx is rapidly degraded in the cell through a proteasomal pathway (data not shown). When expressed in trans by a high copy expression vector, the protein overwhelms the capacity of the proteasomal pathway, thereby increasing the half-life of Vpx. Second, expression in the nef position may not be optimal due to the temporal regulation of this position. Nef is expressed early from a fully-spliced mRNA (15). In contrast, virion assembly and Vpx packaging occurs later when there is a bias towards the Rev-induced production of unspliced mRNAs that encode the structural proteins. Due to its short half-life, Vpx produced early is likely to be degraded before it can be packaged.
In vivo, MDDC activate T cells by costimulation of the TCR and CD28 through a cell contact-dependent mechanism (4). This cell:cell interaction could provide an effective means by which the virus spreads throughout the body. Our findings with the replication of the chimeric virus in MDDC:T cell cocultures supports such a mechanism. In the previously described trans-infection mechanism, virus binds to the MDDC surface through DC-SIGN and other type C lectins. The virus does not infect the cell but is transferred to the CD4 T cell through an infectious synapse. We propose that Vpx can provide a second mechanism of transmission in which virus produced in the infected MDDC is transmitted to a CD4 T cell. It is unlikely that Vpx would act through trans-infection as it is thought to act in the target cell and not in the virion attached to the cell surface. Whether the transmission is through a virological synapse or cell-free virus could not be distinguished in the coculture system. It is remarkable that HIV-1 is susceptible to the hypothetical host restriction factor yet lacks the gene for Vpx to counteract it (19, 21). This role does not appear to have been subsumed by Vpr, which in culture does not have the potency of Vpx in increasing MDDC infection. It is conceivable that MDDC to T cell spread of virus is more important for a virus such as SIVmac than for HIV-1, where the infection may be driven more by T cell to T cell spread, especially once the virus has developed the ability to use CXCR4.
The infected MDDC were induced to produce IFNβ by infection with the Vpx-complemented HIV-1, a property that might interfere with transmission of the virus to T cells. Manel et al. suggested that IFNβ production by infected MDDC may inhibit virus spread to T cells (32). In our experiments this effect did not appear to interfere with replication of the Vpx-containing virus in T cells, perhaps due to our use of lower virus doses in the infections. It is possible that there is a balance between the enhancing and inhibitory activities of infected MDDC and it is difficult to know which condition exists in vivo.
The chimeric virus developed here will provide a tool for studying the role of Vpx in infection and may also provide a tool for vaccine and lentiviral vector design. Because of the role of MDDC in presenting antigens to T cells, the chimeric p6 virus could be a vehicle to enhance immune responses to lentiviral vector encoded immunogens. Additionally, lentiviral vectors that are engineered to package Vpx could provide a means to increase the transduction of MDDC and MDM.
EXAMPLE 2
Generation of an Exemplary Gag/pol Packaging Vector that Produces HIV-1 Based Lentiviral Vectors with Enhanced Dendritic Cell and Macrophage Tropism
Methods:
Cells and cell culture. 293T cells were cultured in Dulbecco's modified Eagle's Medium (DMEM)-10% fetal bovine serum (FBS). MDDC were cultured in RPMI 1640-5% Human AB Serum. Peripheral blood mononuclear cells (PBMC) were purified from normal human donor blood by Ficoll density gradient. Monocytes were purified from healthy donor PBMC by adherence to plastic. Monocyte-derived dendritic cells (MDDC) were generated by culturing the monocytes for 5-6 days in medium containing 50 ng/ml GM-CSF and 100 ng/ml IL-4 (R&D Systems).
Plasmids. Codon-optimized, epitope-tagged SIVmac 239 Vpx expression vector was generated by overlapping PCR and cloned into pcDNA6 (Invitrogen Inc.) at the EcoR-I and Xho-I sites. pMDLg-SIVp6 — 17-26/pRRE was generated by cloning in the chimeric p6 region of pNL-luc3-E − R/SIVp6 — 17-26 into the corresponding gag region in pMDLg/pRRE using the Age-I and Mfe-I sites.
Virus preparation and infections. To produce lentivirus using the lentiviral vector pGK-NGFR-IRES-GFP, 293T cells were cotransfected using lipofectamine 2000 with pGK-NGFR-IRES-GFP (14 μg), pcVSV-G (3.5 μg), pRSV-Rev (2.5 μg), pMDLg/pRRE or pMDLg-SIVp6 — 17-26/pRRE (5 μg), and pcVpx.mychis or pcDNA6 (4.7 μg). Supernatants were harvested 48 h post-transfection, passed through 0.45 μm pore-size filters, aliquoted, and frozen at −80° C. P24 content of virus-containing supernatants was quantified by ELISA using commercially available capture and sandwich antibodies (Aalto Bioreagents, LTD).
MDDC (1.25×10 5 ) were seeded in a 96-well plate and then infected with lentivirus at either 5 or 25 ng p24. MDDC (3×10 5 ) and 293T (2.5×10 5 ) were seeded in a 24-well and 12-well plate, respectively, and then were infected with lentivirus at 120 ng p24. Three days post-infection, the cells were fixed and analyzed by flow cytometry using Flojo software (BD Biosciences). The cells were gated for forward and side scatter and analyzed for FITC fluorescence with mock infected cells as a negative control.
Background: Lentiviral vectors have found great utility for the expression of proteins in cell culture. They are also starting to be used in human gene therapy trials. Although the vectors are generally produced as VSV-G pseudotypes and thus can infect all cell-types, they have poor infectivity with respect to macrophages and dendritic cells. Such cells are important in the immune system due to their ability to present peptide antigens to T cells. They are also important because of they contribute to activation of innate immune pathways. In one approach, dendritic cell based vaccines are being developed as cancer vaccines. The present inventors have discovered and developed reagents and devised a method using same that can be used to generate lentiviral vectors that exhibit a greatly enhanced ability to infect macrophages and dendritic cells relative to lentiviral vectors generated via other means, including those generated as VSV-G pseudotypes. Such vectors are useful for vaccine development and/or for expression of exogenous proteins in gene therapy.
The lentiviral accessory protein Vpx has been shown to facilitate HIV-1 infection of myeloid cells, such as macrophages and dendritic cells, although this virus does not encode it. In order to take advantage of this property, Vpx is added separately or independently from HIV-1 by pre-treating the target cells with SIVmac viral-like particles (VLP). VLPs lack a viral genome but contain the proteins normally delivered to the target cell, such as Vpx. To the best of the inventors' knowledge, Vpx has never been added to HIV-1 via any other means. The system described herein circumvents the use of VLPs. By placing a ten amino acid SIVmac Vpx packaging sequence into the p6 of HIV-1, the present inventors constructed an HIV-1 that is able to directly package Vpx. The resulting virions are also dramatically enhanced in their ability to infect MDDC, in some donors up to 100-fold (based on intracellular p24). By not having to add Vpx separately from the actual virus, the present system prevents the chance of artifacts or immune responses arising due to the presence of large amounts of VLPs. This may become especially important in vaccine design.
Results:
Lentivirus constructed with p6 chimeric gag-pol packages Vpx and infects MDDC. As shown in Example 1 above, the present inventors showed that when placed into HIV-1 p6 the residues 17 DPAVDLLKNY 26 (SEQ ID NO: 1) of SIVmac 239 p6 are sufficient to allow for Vpx packaging. Furthermore, the resulting virus was significantly enhanced in its ability to infect MDDC by an average of 100-fold. Current HIV-1 gag-pol expression plasmids used in generating lentivirus are unable to allow for efficient packaging of Vpx. These results predict that the addition of these SIVmac 239 p6 residues to a gag-pol plasmid used with third generation lentiviral vector systems would boost the ability of the resulting virus to transduce dendritic cells, a cell type that is relatively resistant to infection.
To test this hypothesis, the present inventors constructed a p6 chimeric gag-pol plasmid by modifying the pMDLg/pRRE plasmid to contain the SIVmac 239 p6 residues 17 DPAVDLLKNY 26 (SEQ ID NO: 1) in gag. The pGK-NGFR-IRES-GFP lentivirus was generated by cotransfecting 293T with this proviral plasmid, the wild-type or chimeric packaging plasmid (pMDLg/pRRE or pMDLg-SIVp6 — 17-26/pRRE), pcRSV-Rev, and pcDNA6 or pcVpx.mychis. After two days, the supernatant was harvested. To examine whether the chimeric gag allowed for packaging of Vpx, the virions were pelleted through ultracentrifugation and subsequently analyzed on an immunoblot ( FIG. 10A ). Lentivirus made with wild-type gag failed to efficiently package Vpx. In contrast, lentivirus produced in the presence of a p6 chimeric gag incorporated significant amounts of Vpx.
To test infection, the present inventors normalized the virus for CA p24 and infected monocyte-derived dendritic cells (MDDC) and 293T. The number of cells infected after three days was measured by examining GFP expression by FACS. The present inventors found that viruses that lacked Vpx infected few MDDC ( FIG. 10B ). In contrast, virus that packaged Vpx infected significantly more cells at all amounts, with the increase substantially greater for the p6 chimeric lentivirus. The viruses all infected 293T to a similar extent ( FIG. 10C ). These findings demonstrate that the Vpx and/or the alteration of Gag were not deleterious to the virus and that the Vpx effect is cell-type specific.
Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety.
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Additional references are cited in Example 3 below, each of which is incorporated herein by reference in its entirety.



EXAMPLE 3
Vpx-Containing Lentiviral Vector Targets Dendritic Cells to Induce Antigen-specific CD8+ T Cell Responses
Methods:
Plasmid construction. To construct the pLenti.CD40L.Puro LV vector, a DNA fragment containing the CD40L coding sequence and 5′ and 3′ BamHI and Sal-I ends was generated by RT-PCR from human lymphocyte RNA using specific primers. The fragment was cleaved with BamHI and SalI and ligated to similarly cleaved pLenti CMV GFP Puro (658-5 Addgene™), replacing the GFP sequence. To construct pLenti.GFP.Flu, an amplicon was generated that encoded the influenza virus matrix protein epitope GILGFVFTL 20 (SEQ ID NO: 62) fused to the ER retention signal MRYMILGLLALAAVCSAA. 22 (SEQ ID NO: 63) The DNA fragment was cloned into the Pst-I and Xho-I sites of pLenti CMV GFP Puro, replacing the puro r sequence. To construct pLenti.CD40LP2AF1u.Puro, an amplicon was constructed in which the CD40L coding sequence was fused to the self-cleaving picornavirus 2A (P2A) sequence GSGATNFSLLKQAGDVEENPGP 21 (SEQ ID NO: 64) and influenza peptide using specific primers and overlapping PCR. The amplicon was cloned into pLenti.GFP.Puro at the BamHI and Sal-I sites, replacing GFP.
Cell culture. 293T cells were cultured in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. Monocyte-derived dendritic cells (MDDC) were cultured in RPMI supplemented with 1 mM HEPES, gentamicin, and 5% heat inactivated pooled human serum (PHS). T cells were cultured in Iscove's Modified Dulbecco's Medium supplemented with 1.0 mM HEPES, 2 mM L-glutamine, penicillin/streptomycin, MEM nonessential amino acids, and 5% heat-inactivated PHS. Peripheral blood mononuclear cells (PBMCs) and cord blood of anonymous healthy donors were obtained from the New York Blood Center. Buffy coats were prepared from the cells by Ficoll density gradient centrifugation and the cells were typed for MHC Class I HLA-A2 by flow cytometry with an anti-A2 monoclonal antibody (mAb). The monocytes were purified by plastic adherence and then cultured for 4 days in medium supplemented with 100 U/mL granulocyte-macrophage colony stimulating factor (GM-CSF; Invitrogen Inc.) and 300 U/mL interleukin-4 (IL-4; R&D systems) to generate MDDC. The cells were fed every other day with medium containing fresh cytokines. To isolate autologous CD8+ T cells, the non-adherent fraction was sorted with anti-CD8-conjugated magnetic beads (Miltenyi Biotec). The cells were frozen in PHS/10% dimethyl sulfoxide prior to use.
Virus preparation and infections. To prepare LV vector stocks, 293T cells were cotransfected using calcium phosphate coprecipitation with LV plasmid, the P6-modified HIV-1 Gag/Pol expression vector pMDL-X, pcRev, pcVSV-G and pcVpx or pcDNA at a mass ratio of 28:10:7:5:2. After 48 h, virus-containing supernatant was harvested, passed through a 0.45-μm filter, concentrated 10-fold through a 100K MWCO centrifugal filter (Millipore) and frozen at −80° C. in aliquots. GFP and CD40L expressing viruses were titered on 293 cells by flow cytometry to determine the number of GFP+ or CD40L+ cells per ml of virus.
Lentiviral vector-induced DC maturation and activation. DCs (2×10 5 ) were plated in a 96 well dish and then infected with titered LV vector stock at a multiplicity of infection (MOI) of 2. The number of GFP+ and CD40L+ cells was determined at 48 and 72 h post-infection by flow cytometry. The differentiation status of DCs was determined 48 and 72 h post-infection by CD83 and CD86 staining and measurement of supernatant IL-12p70, TNF-α, IL-6, and IL-1β using the Human Inflammatory Cytokine Cytometric Bead Array (BD Pharmingen).
Naive and memory responses to LV-transduced DC. DCs (2×10 5 ) were plated in a 96 well plate and infected with LV vectors at an MOI of 2. After 48 h, 5.0×10 4 transduced DCs were co-cultured with 5×10 4 Flu MP (58-66) A0201-restricted CD8+ T cell clones in a 96 well plate at an effector to target ratio of 1:1. As a control for maximal stimulation, 1 μg/mL Flu peptide (58-66) was added. After 24 h, supernatant IFN-γ was measured by Cytokine Cytometric Bead Array (BD Pharmingen).
To determine the ability of transduced DC to present peptide antigens and to activate memory CD8+ T cells, transduced DC (2.5×10 4 ) were co-cultured with thawed autologous CD8+ T cells (2×10 5 ) in a 96 well dish in medium supplemented with 25 U/ml interleukin 2 (IL-2) and 5 ng/ml interleukin 7 (IL-7) and replenished every 2 to 3 days. After 14 days, half of the cells were harvested for Flu T cell receptor (TCR) quantification by staining with allophycocyanin (APC)-conjugated Flu-specific tetramer and analysis by flow cytometry. The remaining autologous cells were stimulated with 5.0 μg/ml Flu peptide. Brefeldin A (10 μg/ml) was added one hour later and after another five hours, the cells were stained with antibodies against CD3 and CD8 and then fixed in 1% paraformaldehyde. The cells were permeabilized with PBS containing 0.1% bovine serum albumin and 0.1% saponin and the intracellular IFN-γ and TNF-α was stained and analyzed by flow cytometry. Naive T cell responses were measured as for the memory response with the exception that cord blood T cells were used instead of adult CD8 T cells.
Measuring ACH-2 cells provirus reactivation response to LV-transduced DC. DC were transduced with LV vector and after 24 h were cultured with 2.0×10 5 ACH-2 cells 23 in a 96 well plate. After 48 h the supernatant was harvested and applied to 2.0×10 4 TZM-bl cells in a 96 well plate in serial 3-fold dilutions with 2.0 μg/mL polybrene (Millipore). After 48 h, the cells were fixed in PBS containing 2% gluaraldehyde (Sigma) and 2% formaldehyde (Sigma). The cells were then stained with X-gal for 2 h at 37° C., fixed in 100% methanol for 5 min, and dried. Infectious centers were counted using an Elispot reader.
Background:
Dendritic Cells (DCs) are professional antigen presenting cells that activate antigen-specific CD4+ and CD8+ T cells to initiate an immune response. Immature DCs efficiently take-up and process antigens on class I and class II major histocompatibility complexes (WIC), and upon maturation, induce the expression of cytokines and cell surface proteins that stimulate antigen-specific CD4+ and CD8+ T cells. DC-based vaccine strategies currently under development involve isolation of DCs from the individual, ex vivo pulsing with antigenic peptide, and subsequent reinfusion to stimulate T cell response; 1-5 a strategy that results in short-term antigen presentation. Alternatively, DCs can be transduced with lentiviral (LV) vectors to express peptide antigens. LV vectors are advantageous because they infect non-dividing cells and result in transgene expression over the lifetime of the cell and in daughter cells. 6-8 The vectors have the disadvantage that they are blocked in DCs by a post-entry restriction mediated by endogenous SAMHD1. SAMHD1 is a host nucleotide phosphohydrolyase that dephosphorylates intracellular dNTPs, thereby diminishing the pool of nucleotide precursors used by the virus in the synthesis of its genome. 9 In HIV-2 and some SIVs, SAMHD1 is counteracted by the viral Vpx accessory protein that targets SAMHD1 for degradation and counteracts the restriction. 10,11 HIV-1 does not encode such a protein, yet engineering HIV-1 to package Vpx protein results in virus with significantly improved ability to infect DC. The present inventors recently reported on viral vectors that allow for the production of Vpx-containing HIV-1 based LV vectors and showed that they efficiently infected primary human DC in culture. 12
DC maturation results following antigen and cytokine exposure and is essential for optimizing T cell responses. CD40 ligand (CD40L), also known as CD 154, is a transmembrane protein expressed on activated T-helper cells that promotes activation, maturation, and enhanced survival of DCs upon engagement with the CD40 receptor. 13-15 It has also been shown to help induce memory T cells and activate humoral immunity by promoting the proliferation of B cells, their differentiation to antibody-secreting plasma cells and memory B cells, and immunoglobulin class-switching. 14,16
CD40-CD40L interactions play a significant role in the production of several Th1-skewing and pro-inflammatory cytokines, such as IL-12, TNF-α and IL-6 that may lead to enhanced cell mediated immunity. 13,14,17 Endogenous expression of CD40L by LV vector transduction of DCs induces autonomous maturation as evidenced by enhanced expression of immunologically relevant markers (CD83, CD80) and secretion of IL-12. 17 With respect to HIV, immunization with chimeric CD40L/SHIV virus-like particles was found to induce DC activation and enhance humoral and cellular responses to the SIV Gag and HIV Env proteins in mouse models, lending credence to the possibility that incorporating CD40L may be helpful in developing effective HIV vaccines. 18 Moreover, the use CD40L appears to be safe in humans. 19
Long-term highly active antiretroviral therapy (HAART) reduces viral loads in many patients to low levels and may even completely suppress virus replication yet does not result in eradication of the virus. As a result, patients must remain on life-long therapy. The ability of the virus to integrate into the host chromosomal DNA allows it to remain in quiescent T cells. Strategies to reactivate latently infected cells in patients are under development and have met with some success, yet are not thought to result in purging of the reservoir, as virus production in itself is not sufficient to induce cellular apoptosis and the immune response under long-term HAART is not adequate to lyse infected cells. If such a strategy is to work it will require both reactivation of the proviruses in latently infected cells coupled with a mechanism by which the cells are targeted. To devise such an approach, the present inventors have taken advantage of a LV vector system described herein that allows for highly efficient transduction of myeloid cells. The vectors also facilitate stable expression of antigenic peptides and immunostimulatory proteins. The former feature stimulates antigen-specific CD4+ and CD8+ effector cells and the latter enhances the immune response and induces viral production from latently infected cells.
Accordingly, the present inventors tested the ability of Vpx-containing LV vectors to induce antigen-specific CD8+ T cell responses. As shown herein, these vectors have a markedly improved ability to transduce DCs and cells transduced thereby are very effective at activating antigen-specific CD8+ T cells. The vectors expressed the HLA-A2.1 restricted influenza peptide 20 fused to CD40L via a self-cleaving picornavirus 2A peptide. 21 As CD40L is a type II transmembrane protein, fusion of the peptide to CD40L proved an effective method of antigen presentation since CD40L is targeted to the ER lumen with its C-terminal domain, thereby enabling the peptide to be cleaved directly into the ER lumen for efficient processing. Furthermore, by capitalizing on the immunostimulatory effect of CD40L, the ability to augment the antigen-specific Th1 response and induce quiescent HIV proviruses from latently infected cells is demonstrated, thereby establishing a DC-targeted LV vector system that serves as the basis for developing an HIV immunotherapeutic or antigen-specific vaccine.
Results:
Vpx-containing LV vectors that encode a CD40L-Flu peptide epitope. Lentiviral vectors in which the virions contain packaged SIV Vpx escape SAMHD1-mediated restriction and thus infect cells such as DC with significantly improved efficiency. The present inventors used this principle to generate lentiviral vectors encoding a CMV promoter-driven nominal peptide antigen and an immunostimulatory gene. As nomimal antgen, an influenza matrix protein peptide that forms an immunodominant epitope to which the CD8 T cells of most HLA-A2.1 donors respond was used. In the vector, pLenti.CD40L.Flu, the peptide is expressed as a carboxy-terminal fusion to CD40L with an intervening picornavirus-2A (P2A) self-cleaving peptide ( FIG. 14 ). Because CD40L is a type II transmembrane protein, it is synthesized with its carboxy-terminus facing the ER lumen such that cleavage of the peptide during biosynthesis releases it into the ER lumen where it has direct access to the antigen presentation pathway. The CD40L, a T cell expressed protein that binds to CD40L on DC, serves to induce maturation and augment antigen presentation pathways of the transduced DC. As controls, vectors were constructed that encode (i) CMV promoter-driven CD40L, (ii) GFP, and (iii) CMV-driven GFP with PGK promoter-driven Flu peptide. Virus stocks in which the virions contained or lacked packaged Vpx were produced by cotransfecting 293 cells with vector plasmid and pMDL-X, an HIV-1 Gag/Pol expression vector in which the Vpx packaging motif has been placed in P6, and with or without Vpx expression vector. The viruses were normalized for infectious titer on 293 cells.
To test the efficiency with which the vectors transduced DC, HLA-A2+ donor DC were infected at an MOI=1 equivalent as measured by infectivity on 293 cells. After 48 and 72 h the number of GFP+ and CD40L+ DCs was quantified by flow cytometry. Viruses that lacked Vpx, failed to transduce the DC with detectable efficiency. In contrast, viruses that contained Vpx transduced 36% to 65% of the cells ( FIG. 15 ). In some cases the GFP vectors appeared to be more infectious than the CD40L-expressing vectors; however this was likely the result of less sensitive detection of CD40L as compared to GFP. The results demonstrated that the packaged Vpx had allowed for much more efficient transduction of the DC.
CD40L-peptide fusion protein induces DC maturation. As DC mature they secrete stimulatory cytokines and upregulate CD83 and CD86. To test whether transduction of the DC with the CD40L-peptide peptide expressing lentiviral vectors would cause the DC to mature, DCs were transduced with the panel of LV and 48 and 72 h later their CD83 and CD86 expression levels were quantified. The results showed that viruses produced without Vpx induced a moderate amount of maturation, with between 36% to 50% of the cells becoming CD83+ and only a small effect of CD40L. In contrast, for Vpx-containing viruses that expressed CD40L or CD40L-Flu, induced nearly all of the DCs to mature, resulting in 84% and 88% CD83+ cells, respectively ( FIG. 16 ). Vpx-containing vectors that lacked CD40L (GFP or GFP.Flu vectors) induced no maturation over base-line. The cause of the maturation by Vpx-lacking virions is not clear but is most likely the result of an innate immune response to the virions themseleves and not the result of direct infection, as these viruses are largely uninfectious with respect to DCs. The results demonstrated that the Vpx-containing lentviral vectors that expressed CD40L potently stimulated the DC to mature.
To determine whether the vectors induced Th1/Th2 skewing cytokines in the DC, the DC of four donors were transduced with the panel of LV containing or lacking Vpx and after 48 h and 72 h IL-12 and TNF-α were measured in the culture supernaants. Vectors that lacked Vpx induced little IL-12 over base-line, uninfected cells. In contrast, when the viruses contained Vpx, the vectors that expressed CD40L and CD40L-Flu induced IL-12 production ( FIG. 17 ). IL-12 reached levels up to 7,000 times greater than controls. The results show that the Vpx-containing vectors that expressed CD40L efficiently induced the maturation of the DC and induced these cells to produce cytokines that enhance the Th1 response.
DC transduced with Vpx-containing LV vectors present antigens to CTL. The ability of the Vpx-containing vectors to induce DC to present antigen and stimulate antigen-specific T cells was then assessed. To this end, DCs were transduced with various vectors described herein and after 48 h, co-cultured with an A2-restricted CTL clone specific for the influenza peptide epitope. After 24 h, IFN-γ in the culture medium was quantified as a measure of the T cell response. The present inventors found that the Vpx-containing vectors that encoded the Flu peptide were more potent than the control vectors and that CD40L enhanced the response of the CTL clone. The vectors encoding the peptide epitope induced a 10,000-fold increase in IFN-γ production and the vector that expressed the peptide epitope and CD40L induced a 70,000 fold increase as compared to the controls ( FIG. 18 ). The response was comparable and in some cases higher than that induced by pulsing the DC with synthetic peptide. To see the response at the single cell level, the present inventors also analyzed the IFN-γ response by intracellular staining. The results showed that the Vpx-containing vectors encoding Flu peptide activated a similar number of the CTL clone regardless of whether or not they encoded CD40L, however, the addition of CD40L increased the intensity of the IFN-γ response, suggesting that CD40L amplified the amount of IFN-γ activation of the responding cells and not the number of cells that respond. To determine the potency of the transduced DCs, the effect of reducing the DC:CTL ratio was evaluated. The titration showed that as the DC:CTL ratio was decreased from 1:1 to 1:40, the Vpx-containing vectors that expressed CD40L and peptide epitope induced much greater levels of IFN-γ compared to controls.
Transduced DCs elicit antigen-specific memory and naive CD8 responses. To assess whether the transduced DCs would expand a CD8 memory response from primary donor T cells, the DC were transduced with various vectors described herein and after 48 h, autologous CD8 T cells were added. The cells were allowed to expand for two weeks after which the peptide-specific T cells were quantified by tetramer staining ( FIG. 19 ). Results showed that DC transduced with Vpx-containing LV vectors encoding Flu peptide induced a significant antigen-specific memory response compared to LV vectors that lacked Vpx. Additional ICS analysis performed revealed that IFN-γ and TNF-α secretion by the autologous cytotoxic T cells following reticulation with synthetic influenza peptide mirrored the tetramer results.
Transduced DCs activate quiescent HIV-1 provirus expression from latently infected cells. To determine if the transduced DCs could reactivate latent HIV, transduced DCs were cocultured with ACH-2 cells, a cell line that harbors a quiescent provirus. 23 After 48 h, supernatants were assessed for HIV reactivation using TZM-bl cell infection as a function of Tat-induced luciferase reporter gene expression after a single round of HIV infection. TZM-bl cells contain the reporter gene for E. coli β-galactosidase under the control of an HIV-1 LTR. Expression of β-galactosidase is induced by viral Tat protein, and when X-gal is added to the cells it is cleaved by the enzyme to form a blue product. The number of blue spots (infected cells) observed is directly proportional to the number of infectious virions present in the analyte and can be counted using an Elispot reader. Results showed that DC transduced with the Vpx-containing LV vector encoding CD40L induced 5-fold more infectious virions than vectors lacking CD40L or Vpx. These levels were on par or greater than those induced by control PMA suggesting the Vpx-containing LV vectors encoding CD40L may stimulate HIV reactivation from the latent viral reservoir.
Discussion
DCs have been targeted for vaccine development due to their role as antigen-presenting cells and their capacity to stimulate adaptive immunity by inducing durable antigen-specific memory T cell responses. LV vectors have been explored as a method of gene delivery given their ability to transduce non-dividing cells and maintain long-term transgene expression via integration into the host genome. While genome integration poses theoretical safety concerns, particularly given the cases of lymphoid leukemia and myelodysplasia seen in trials using murine leukemia virus (MLV)-based γ-retroviral vectors, 24-26 LV vectors based on HIV have shown low oncogenic potential in animal models. 27 Moreover, several trials utilizing LV vectors are being pursued that will hopefully help to further delineate this potential risk.
With respect to DCs, however, LV vector infectivity is limited due to a post-entry restriction to infection mediated by SAMHD1. SAMHD1 hydrolyzes intracellular deoxynucleotide triphosphates (dNTPs) thereby lowering the concentration of dNTPs below that required for synthesis of viral DNA by reverse transcriptase and thus limiting infection. SIV Vpx bypasses this restriction by degrading SAMHD1 to allow for productive infection. Given that, the present inventors used the LV vector system described herein that packages SIV Vpx to improve DC transduction and enhance antigen presentation. Results presented herein clearly show that DC transduction efficiency is markedly improved with LV vectors that package Vpx. Indeed, the absolute degree of DC infectivity derived from Vpx-containing LV vectors varies from donor to donor, possibly owing to varying levels of donor DC SAMHD1 expression, however, LV vectors lacking Vpx consistently show negligible infectivity via flow cytometry analysis.
By modifying the Vpx-packaged LV vectors to encode the immunostimulatory protein, CD40L, the present inventors demonstrated that these vectors can be used to enhance DC maturation, as evident by upregulation of DC surface markers CD83 and CD86. This is significant, since DC maturation is directly related to effective antigen presentation. However, maturation alone does not dictate the type of immune response induced by DCs. Mature DCs are capable of inducing various T cell responses, including T H 1, T H 2, and T H 17 responses, and it is the cytokines produced by DCs that influence direction in which T cell differentiation is skewed. Examination of the supernatants of transduced DCs revealed that the Vpx-containing LV vectors encoding CD40L described herein stimulated increased secretion of pro-T H 1 cytokines, namely IL-12 and TNF-α, thereby favoring priming of an adaptive immune response.
In turn, to assess antigen presentation efficiency, the WIC class I conserved major epitope of influenza was encoded into our LV vectors and then co-cultured DCs transduced with such vectors with an influenza-specific cytotoxic T cell clone and measured T cell IFN-γ production. Results presented herein showed that not only is Vpx packaging necessary for efficient antigen presentation, but that the combination of CD40L and Vpx markedly increased the intensity of IFN-γ production, reaching levels over 70,000 times greater (pg/mL) than control vectors. These findings confirm the validity of the present inventors' hypothesis that enhancing DC maturation and T H 1 skewing cytokine production can lead to amplification of antigen-specific cytotoxic T cell immune responses. Further intracellular staining analysis for IFN-γ on the T Cell clone co-cultured with transduced DC supported this finding as demonstrated by the observation that while Vpx-containing vectors encoding influenza stimulated similar proportions of the cytotoxic T cell clones, vectors that encoded CD40L significantly intensified IFN-γ secretion. The present inventors also showed that the amount of synthetic influenza peptide needed to stimulate detectable clone IFN-γ release was significantly less when the clone was cocultured with DC transduced with Vpx-packaged LV vectors encoding CD40L than when transduced with vectors lacking CD40L. One pitfall of using an antigen-specific clone, however, is that the cells are already terminally differentiated, such that TCR upregulation cannot be assessed. In turn, the present inventors also co-cultured transduced DC with autologous CD8+ T cells to evaluate induction of an antigen-specific memory response and showed that not only do Vpx-containing LV vectors effectively expand a CD8 memory response, but that this too is bolstered by the addition of CD40L.
Results presented herein affirm the LV vector system described herein can be modified to encode specific antigens and thus, provide the basis for either a DC-targeted antigen specific vaccine or immunotherapeutic. With respect to HIV-1, infected persons can readily achieve virologic control with highly active antiretroviral therapy (HAART), however, a cure remains unattainable due to the persistence of a latently infected viral reservoir that reemerges upon cessation of therapy. 28 One theory to eradicate this reservoir is to provide a trigger that can awaken HIV proviruses from latently infected cells to allow for subsequent targeting of the infected cells as a result of viral cytopathic effects (CPEs) or host immune responses. However, Siliciano et al. (2012, Immunity 36(3):491-501) showed that while the histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), induced virus reactivation from resting CD4+ T cells, reactivation did not result in death of infected cells and that the CTLs from patients on HAART failed to kill latently infected CD4+ T cells, thereby suggesting that reactivation of latent HIV-1 alone will not purge the viral latent reservoir unless there is stimulation of HIV-1 specific CTL responses prior to reactivation. 29 Given results presented herein showing that DC transduced with Vpx-packaged LV vectors encoding CD40L reactivate HIV-1 proviruses from ACH-2 cells 5-fold greater than control vectors, the present inventors envision that by encoding HIV-1 specific antigens into LV vectors described herein, such vectors will be able to supply both the “kick and kill” mechanisms necessary eradicate the latent reservoir.
In summary, the present inventors have established a DC targeted LV vector virion that matures and activates DC to facilitate an efficient antigen-specific CTL response and shows potential to be the basis of an HIV immunotherapeutic and/or vaccine that can be used to induce quiescent HIV proviruses from latently infected cells and target them for elimination.
References Relating to Example 3


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This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all aspects illustrate and not restrictive, the scope of the invention being indicated by the appended claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.