WO2009147196A1 - Hiv-rev-multimerization inhibiting nanobodies - Google Patents

Hiv-rev-multimerization inhibiting nanobodies Download PDF

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WO2009147196A1
WO2009147196A1 PCT/EP2009/056852 EP2009056852W WO2009147196A1 WO 2009147196 A1 WO2009147196 A1 WO 2009147196A1 EP 2009056852 W EP2009056852 W EP 2009056852W WO 2009147196 A1 WO2009147196 A1 WO 2009147196A1
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rev
hiv
multimerization
antibody
protein
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PCT/EP2009/056852
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French (fr)
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Jan Steyaert
Els Pardon
Dirk Daelemans
Thomas Vercruysse
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Vib Vzw
Vrije Universiteit Brussel
Katholieke Universiteit Leuven, K.U.Leuven R&D
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Publication of WO2009147196A1 publication Critical patent/WO2009147196A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1072—Regulatory proteins, e.g. tat, rev, vpt
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to an anti-HIV-Rev antibody. More specifically, the invention relates to a nanobody that is able to inhibit the multimerization of the Rev protein, and by this, it inhibits the nuclear export of single-spliced and unspliced HIV-1 mRNA, and can be used as an anti-HIV agent. The invention relates further to the use of the antibody in the screening of compounds that can inhibit HIV-Rev multimerization.
  • HIV Human Immunodeficiency Virus
  • the prototype member of the lentivirus subfamily of retroviruses Barre-Sinoussi et ai, 1983, GaIIo et ai, 1983
  • AIDS Acquired lmmuno Deficiency Syndrome
  • the replication cycle of HIV can be divided into several essential steps. Infectious virions initially bind to the cellular CD4 receptor and CXCR4 or CCR5 coreceptor on the surface of susceptible cells via its envelope glycoproteins gp120. Fusion of the viral and cellular lipid membranes ensues, and the viral core (nucleocapsid) enters the cytoplasm (Clapham and McKnight, 2002).
  • the single-stranded RNA genome of the virus is then reverse transcribed into a double-stranded linear DNA molecule by the viral enzyme reverse transcriptase; this conversion of RNA to DNA gives retroviruses their name (Ren and Stammers, 2005).
  • retroviruses how to express multiple genes from a single proviral unit (Malim et al., 1989).
  • retroviral transcription is mediated by the human RNA polymerase Il and a combination of cellular basal and promoter (enhancer) specific factors, HIV-1 additionally encodes its own transcriptional activator (Tat).
  • Tat transcriptional activator
  • complex retroviruses utilize alternative splicing of their major full-length transcript to produce the diversity of viral mRNAs required for viral protein expression. The splicing of viral mRNA is mediated by cellular pre mRNA splicing factors.
  • More than 30 different viral mRNA species can be present in HIV-1 infected cells, and at least 4 different 5'-splice sites and 8 different 3'-splice sites are utilized during their biogenesis (Felber et al., 1989; Purcell and Martin, 1993).
  • various multiply spliced mRNAs ⁇ 2-kb
  • ⁇ 4-kb multiply spliced mRNAs
  • Env, Vif, Vpr, or Vpu proteins full-length unspliced RNA ( ⁇ 9-kb) species that encode Gag and Gag-Pol polyproteins.
  • the late unspliced full-length transcripts have three distinct functions: (a) they constitute the genomes of the retroviruses, (b) they serve as templates for translation (Gag and Gag-Pol), and (c) they function as precursor RNAs (pre-mRNAs) for the production of diverse subgenomic mRNAs. Because the first two of these functions depend upon localization to the cytoplasm, retroviruses use dedicated mechanisms for exporting these unspliced RNAs out of the nucleus. Importantly, the translocation of RNAs that harbor functional introns (which, by definition, these RNAs do, as they function as pre-mRNAs) to the cytoplasm is highly unusual.
  • the virally encoded protein Rev directly interacts with a c/s-acting target, an RNA segment in the Env gene known as the Rev responsive element (RRE) (figure 1A), which is present in all incompletely spliced and unspliced viral mRNAs.
  • RRE Rev responsive element
  • the completely spliced viral mRNAs exit the nucleus using pathways that are utilized by fully processed cellular mRNAs.
  • Rev is a 116-residue HIV-encoded protein that is expressed in infected cells. After synthesis in the cytoplasm, Rev is rapidly transported to the nucleus (Cullen, 2003; Arnold et al., 2006) through an interaction of its arginine-rich nuclear localization signal (NLS) with the nuclear import factor importin.
  • NLS nuclear localization signal
  • the same arginine-rich motif comprised of residues 35- 50 (figure 1 B) is responsible for binding to the Rev response element (RRE), located on all incompletely and unspliced HIV mRNAs.
  • the nuclear export of Rev-RRE complexes involves the cellular transport factor CRM1.
  • Human CRM1 localizes to the nuclear pore complex and the nucleoplasm and interacts with nuclear pore proteins (Fornerod et al., 1997).
  • the driving force for the CRM1- mediated nucleocytoplasmic transport is the RanGTP gradient over the nuclear membrane. This gradient involves high concentrations of RanGTP in the nucleus and high levels of RanGDP in the cytosol. RanGTP promotes the association of cargo with nuclear export receptors while RanGDP encourages the dissociation of the export complex.
  • CRM1 binds cargo proteins and RanGTP to form an export complex.
  • Rev can bind to its RRE-high- affinity binding site as a monomer (Cole et al., 1993; Cook et al., 1991 ; MaNm and Cullen, 1991 ; Tiley et al., 1992) and that this binding results in some localized melting of the RNA structure (Daly et al., 1990) as well as stabilization of the protein helix (Tan et al., 1993).
  • Rev molecules then bind and multimerize via a combination of cooperative protein- protein and protein-RNA interactions (Daly et al., 1993) such that eight or more Rev molecules are bound to a single RRE (Daly et al., 1989; Mann et al., 1994).
  • the complete biologically active Rev-response element (RRE) RNA is 351 nucleotides (nt) in length and RRE binds multiple Rev molecules in a cooperative way (Mann et al, 1994).
  • RRE Rev-response element
  • Rev assembly of Rev on the RRE is the mechanism which defines a threshold concentration for the Rev response.
  • RRE thus acts as "molecular rheostat" designed to detect Rev levels during the early stages of the HIV growth cycle.
  • the full molecular structure of Rev is currently unknown, largely because in solution Rev is strongly prone to aggregation at concentrations above 1 ⁇ M, both in the presence and in the absence of RNA (Bogerd et al., 1995).
  • Rev assemblies have a highly ordered, filamentous morphology that has been characterized previously by electron microscopy (Watts et al., 1998). Rev multimerization is essential for its functionality and hence for viral replication (Malim and Cullen, 1991 ) and could therefore be an attractive target for anti-HIV therapy.
  • RevMI O mutant efficiently inhibits the wild- type Rev function and HIV replication (Bevec et al., 1992). Michienzi et al. (2006) demonstrated that a nucleolar localizing RRE inhibits the HIV replication by acting as a decoy for Rev, proving the validity of the target.
  • a first aspect of the invention is an antibody, capable of inhibiting the multimerization of HIV Rev.
  • said HIV Rev is HIV-1 Rev.
  • Said antibody can be any antibody, including but not limited to normal antibodies, single chain antibodies, diabodies and camelid antibodies.
  • Still another aspect of the invention is the use of an antibody according to the invention, or a functional fragment according to the invention for the structure determination of HIV Rev by means of X-ray crystallography or NMR.
  • Another aspect of the invention is the use of an antibody according to the invention, or a functional fragment according to the invention to screen compounds that inhibit HIV Rev multimerization. Indeed, the antibody binds to an epitope comprising a domain that is essential for the multimerization of Rev.
  • compounds can be screened that are quite active in inhibiting the mulimerization.
  • Methods to study protein-protein interaction, and its disruption are known to the person skilled in the art and include, but are not limited to yeast two hybrid, MAPPIT (Tavernier et al., 2002) and reverse MAPPIT (Lemmens et al., 2006).
  • Another aspect of the invention is a compound, isolated with the use of the antibody according to the invention.
  • Fig 1 Domain structures of (A) the Rev responsive element and (B) Rev from HIV-1. NLS: Nuclear Localization Signal, NES: Nuclear Export Signal (Pollard and MaNm, 1998).
  • Fig. 2 (A) Principle of our FRET based Rev-multimerization-assay and (B) typical emission spectrum of samples excited at 430 nm. When FRET occurs (ECFP-Rev + EYFP-Rev) a typical emission peak at 527 nm is detected. As negative control the following pairs were used: free-ECFP + free-EYFP, free-ECFP + EYFP-Rev, ECFP-Rev + free-EYFP.
  • Fig. 3 Amino acid sequences of HIV-Rev specific nanobodies.
  • Nb_190 interacts with Rev in cells. HeLa cells were transfected separately with Rev-
  • Nb_190 fails to inhibit the viral production of a Rev- independent molecular clone.
  • 293T cells were co-transfected with HIV-1 N L4 3 R ⁇ V(-) RRE (-)C TE (+) (0.5 ⁇ g) and different amounts Nb_190, Nb_163, or Dronpa expression plasmids.
  • Expression of HIV-1 NL43R ev(-) RRE (-)c ⁇ E (+) was analyzed by measuring the virus-associated core antigen (p24) in the supernatants of the transfected cells.
  • Nb_190 specifically inhibits the expression of partially and unspliced viral mRNA species. Northern blot analysis of viral mRNA forms in cells transfected with wild-type or mutant HIV-1 NL4.3 molecular clones (see figure 3c).
  • _ 4 3 molecular clone has been previously described (Fisher et al., 1986). HIV NL4 3- Nb 190 and HIV N
  • Transfected HeLa cells were imaged with a laser scanning SP5 confocal microscope (Leica Microsystems, Germany) equipped with an AF 6000 microscope and an AOBS, using a HCX PL APO ⁇ 63 (NA 1.2) water immersion objective magnification.
  • GFP was monitored with the Ar laser using the 488-nm line for excitation, and emission was detected between 492 and 558 nm.
  • mKusabira orange fluorescent protein (mKO) was imaged using the 561-nm line for excitation, and emission was detected between 570 and 670 nm.
  • pTat/Rev represents partial sequence (exons 4 and 7) of HIV-1 multiple spliced RNA encoding Tat and Rev proteins. Results are expressed as number of copies unspliced HIV mRNA per copy of multiple spliced RNA.
  • FRET is the non-radiative transfer of photon energy from an excited fluorophore (the donor, e.g. ECFP) to an acceptor fluorophore (e.g. EYFP) when both are located within close proximity (1-10 nm).
  • the donor e.g. ECFP
  • EYFP acceptor fluorophore
  • a llama was immunized with purified recombinant HIV-Rev protein.
  • the immunization and the isolation of nanobodies have been performed following standard procedures according to Ghahroudi et al. (1997).
  • Llama immunizations and phage displayed libraries of their nanobodies yielded a first series of HIV-Rev specific nanobodies ( Figure 3).
  • All nanobody genes were cloned in a pHENO ⁇ vector for expression with a His-6 tail.
  • the recombinant proteins were produced in Escherichia coli, and purified according to described protocols (Conrath et al., 2001 ). So far, we have been working with the recombinant Rev protein, expressed from pET29b. This protein is tagged at its N-terminus with the S-peptide (Kim and Raines, 1993) and His-tagged at its C-terminus.
  • Nb_188, Nb_227a, Nb_228, Nb_229, Nb_191 , Nb_230, Nb_235, Nb_233, Nb_189, Nb_234, Nb_243, Nb_244 and Nb_190 were tested, together with one irrelevant nanobody (Nb_163). From these, two nanobodies (Nb_190 and Nb_243) reduced the FRET signal significantly (see figure 4A). Nb_190 has a dose-dependent inhibitory effect on the Rev multimerization as shown in figure 4B, while it did not interfere with the FRET signal from an ECFP-EYFP fusion construct.
  • Nb_190 is able to compete with the ECFP- Rev:EYFP-Rev complex formation since it is equally active whether it is added before or after ECFP-Rev:EYFP-Rev complex formation (figure 4B).
  • the irrelevant Nb_163 had no effect on the Rev multimerization.
  • Nb_190 behaves as an intrabody and binds wild-type Rev in living cells.
  • our multimerization inhibitor (Nb_190) and the multimerization impaired single V16D and triple Y23D-S25D-N26L. Rev mutations provoke very similar effects on the cellular distribution of Rev. Indeed, the Nb_190-
  • Nb190 interferes with the HIV-1 expression
  • Nb_190 was cloned in a mammalian expression vector (pcDNA3.1 ) and co-transfected with pNL4.3 (an HIV-1 molecular clone) in HEK 293T cells.
  • Expression of NL 43 was analyzed by measuring the virus-associated core antigen (p24) in the supernatants of transfected cells using ELISA.
  • p24 is a measure for the amount of produced virus particles present in the supernatant.
  • Figure 7A shows the effect of Nb_163 and Nb_190 on the expression of NL 43 .
  • Nb_190 reduces virus expression by more than 70% compared to the Nb_163 control.
  • Nb_190 was unable to reduce the expression of this Rev- independent virus, further establishing the anti-viral effect of this anti-multimerization nanobody closely correlates with inhibition of the Rev function (Fig. 8a).
  • DaIyJJ. Doten,R.C, Rennert,P., Auer,M., Jaksche,H., Donner,A., Fisk,G., and RuscheJ.R. (1993). Biochemical characterization of binding of multiple HIV-1 Rev monomeric proteins to the Rev responsive element. Biochemistry 32, 10497-10505. DaIyJJ., RuscheJ.R., MaioneJ.E., and Frankel,A.D. (1990). Circular dichroism studies of the HIV-1 Rev protein and its specific RNA binding site. Biochemistry 29, 9791-9795.
  • CRM1 is an export receptor for leucine-rich nuclear export signals.

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Abstract

The present invention relates to an anti-HIV-Rev antibody. More specifically, the invention relates to a nanobody that is able to inhibit the multimerization of the Revprotein, and by this, it inhibits the nuclear export of single-spliced and unspliced HIV-1 m RNA, and can be used as an anti-HIV agent. The invention relates further to the use of the antibody in the screening of compounds that can inhibit HIV-Rev multimerization.

Description

HIV-REV-MULTIMERIZATION INHIBITING NANOBODIES
The present invention relates to an anti-HIV-Rev antibody. More specifically, the invention relates to a nanobody that is able to inhibit the multimerization of the Rev protein, and by this, it inhibits the nuclear export of single-spliced and unspliced HIV-1 mRNA, and can be used as an anti-HIV agent. The invention relates further to the use of the antibody in the screening of compounds that can inhibit HIV-Rev multimerization.
HIV (Human Immunodeficiency Virus), the prototype member of the lentivirus subfamily of retroviruses (Barre-Sinoussi et ai, 1983, GaIIo et ai, 1983), is the causative agent of AIDS (Acquired lmmuno Deficiency Syndrome). The replication cycle of HIV can be divided into several essential steps. Infectious virions initially bind to the cellular CD4 receptor and CXCR4 or CCR5 coreceptor on the surface of susceptible cells via its envelope glycoproteins gp120. Fusion of the viral and cellular lipid membranes ensues, and the viral core (nucleocapsid) enters the cytoplasm (Clapham and McKnight, 2002). The single-stranded RNA genome of the virus is then reverse transcribed into a double-stranded linear DNA molecule by the viral enzyme reverse transcriptase; this conversion of RNA to DNA gives retroviruses their name (Ren and Stammers, 2005).
At a point thereafter, the DNA enters the nucleus as a nucleic acid-protein complex (the preintegration complex) and is incorporated into the host cell's genome by the action of a second viral enzyme, integrase (I N) (Al-Mawsawi and Neamati, 2007). The covalently integrated form of viral DNA, which is defined as the provirus, serves as the template for viral transcription. Retroviral RNAs are synthesized, processed, and then transported to the cytoplasm, where they are translated to produce the viral proteins. The proteins that form the viral core, encoded by the gag and pol genes, initially assemble into immature polyprotein complexes together with two copies of full-length viral RNA. As these structures bud through the plasma membrane, they become encapsulated by a layer of membrane that also harbors the viral Env glycoproteins. Coincident with budding, the viral protease (PR) cleaves the GAG and POL polyproteins into their final forms and produces mature virions. All FDA-approved HIV/AIDS drugs interfere with one of four essential steps of the viral replication cycle (Mocroft et ai, 1998; Pomerantz and Horn, 2003; De Clercq 2007). The fusion inhibitor (enfuvirtide) and the CCR5-coreceptor inhibitor (maraviroc) interfere with the entry of the virus. Reverse transcriptase inhibitors (nucleosides: zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir; non-nucleosides: nevirapine, delavirdine) interfere with the conversion of the viral RNA into double stranded proviral DNA. The integrase inhibitor raltegravir interferes with the HIV integrase activity. The protease inhibitors (saquinavire, ritonavir, indinavir, nelfinavir) prevent the processing of the GAG and POL polyproteins. Examination of the proviral organization immediately reveals a critical gene expression problem that must be surmounted by retroviruses: how to express multiple genes from a single proviral unit (Malim et al., 1989). Although retroviral transcription is mediated by the human RNA polymerase Il and a combination of cellular basal and promoter (enhancer) specific factors, HIV-1 additionally encodes its own transcriptional activator (Tat). Importantly, complex retroviruses utilize alternative splicing of their major full-length transcript to produce the diversity of viral mRNAs required for viral protein expression. The splicing of viral mRNA is mediated by cellular pre mRNA splicing factors. More than 30 different viral mRNA species can be present in HIV-1 infected cells, and at least 4 different 5'-splice sites and 8 different 3'-splice sites are utilized during their biogenesis (Felber et al., 1989; Purcell and Martin, 1993). In the early phase of mRNA expression, various multiply spliced mRNAs (±2-kb) are produced that encode the regulatory proteins Tat, Rev, or Nef. In a later phase, the virus produces singly (partially) spliced mRNAs (±4-kb) encoding Env, Vif, Vpr, or Vpu proteins and full-length unspliced RNA (±9-kb) species that encode Gag and Gag-Pol polyproteins.
The late unspliced full-length transcripts have three distinct functions: (a) they constitute the genomes of the retroviruses, (b) they serve as templates for translation (Gag and Gag-Pol), and (c) they function as precursor RNAs (pre-mRNAs) for the production of diverse subgenomic mRNAs. Because the first two of these functions depend upon localization to the cytoplasm, retroviruses use dedicated mechanisms for exporting these unspliced RNAs out of the nucleus. Importantly, the translocation of RNAs that harbor functional introns (which, by definition, these RNAs do, as they function as pre-mRNAs) to the cytoplasm is highly unusual. Under normal circumstances, intron-containing pre-RNAs are retained in the nucleus by the interaction of splicing factors (sometimes referred to as commitment factors) until they are either spliced to completion or degraded (Chang and Sharp, 1989; Legrain and Rosbash, 1989). The same export problem also confronts the late incompletely spliced transcripts of HIV-1 ; all these RNAs contain at least one functional intron but must enter the cytoplasm unspliced to act as templates for the synthesis of structural proteins. Complex retroviruses exploit dedicated posttranscriptional mechanisms by which their intron- containing mRNAs circumvent nuclear sequestration and are exported to the cytoplasm (Kohler and Hurt, 2007). In the case of HIV, the virally encoded protein Rev, directly interacts with a c/s-acting target, an RNA segment in the Env gene known as the Rev responsive element (RRE) (figure 1A), which is present in all incompletely spliced and unspliced viral mRNAs. In contrast to the full-length transcripts and the partially spliced viral mRNAs, the completely spliced viral mRNAs exit the nucleus using pathways that are utilized by fully processed cellular mRNAs.
Rev is a 116-residue HIV-encoded protein that is expressed in infected cells. After synthesis in the cytoplasm, Rev is rapidly transported to the nucleus (Cullen, 2003; Arnold et al., 2006) through an interaction of its arginine-rich nuclear localization signal (NLS) with the nuclear import factor importin. The same arginine-rich motif comprised of residues 35- 50 (figure 1 B) is responsible for binding to the Rev response element (RRE), located on all incompletely and unspliced HIV mRNAs. After transport to the nucleus, the formation of multimeric complexes between Rev and its RRE-containing target RNA is thought to mask the NLS and expose a nuclear export signal (NES) of Rev, contained in residues 73-83, that mediates the interaction of Rev with the cellular nucleocytoplasmic transport factor CRM1/exportin 1 and other cellular cofactors (Bogerd et al., 1998). Dissociation of RNA from Rev in the cytoplasm is believed to expose the NLS, leading to binding to importin-β and transport into the nucleus resulting in successive rounds of nucleocytoplasmic transport (Bevec ef a/., 1996).
As mentioned earlier, the nuclear export of Rev-RRE complexes involves the cellular transport factor CRM1. Human CRM1 localizes to the nuclear pore complex and the nucleoplasm and interacts with nuclear pore proteins (Fornerod et al., 1997). The driving force for the CRM1- mediated nucleocytoplasmic transport is the RanGTP gradient over the nuclear membrane. This gradient involves high concentrations of RanGTP in the nucleus and high levels of RanGDP in the cytosol. RanGTP promotes the association of cargo with nuclear export receptors while RanGDP encourages the dissociation of the export complex. Within the nucleus CRM1 binds cargo proteins and RanGTP to form an export complex. This trimeric complex is transported to the cytoplasm, where the hydrolysis of RanGTP to RanGDP results in the dissociation of the complex releasing cargo into the cytoplasm. In this pathway Rev is a chaperone between the HIV mRNA and the cellular transport factor CRM1. Through in vitro RRE-binding assays, it has been shown that Rev can bind to its RRE-high- affinity binding site as a monomer (Cole et al., 1993; Cook et al., 1991 ; MaNm and Cullen, 1991 ; Tiley et al., 1992) and that this binding results in some localized melting of the RNA structure (Daly et al., 1990) as well as stabilization of the protein helix (Tan et al., 1993). Additional Rev molecules then bind and multimerize via a combination of cooperative protein- protein and protein-RNA interactions (Daly et al., 1993) such that eight or more Rev molecules are bound to a single RRE (Daly et al., 1989; Mann et al., 1994). The complete biologically active Rev-response element (RRE) RNA is 351 nucleotides (nt) in length and RRE binds multiple Rev molecules in a cooperative way (Mann et al, 1994). At low Rev concentrations, binding is confined to the high affinity Stem Nb in the RRE (Figure 1A). As Rev levels rise in the infected cells, cooperative binding reactions lead to the coating of Stem Na and Stem I. It seems likely that assembly of Rev on the RRE is the mechanism which defines a threshold concentration for the Rev response. RRE thus acts as "molecular rheostat" designed to detect Rev levels during the early stages of the HIV growth cycle. The full molecular structure of Rev is currently unknown, largely because in solution Rev is strongly prone to aggregation at concentrations above 1 μM, both in the presence and in the absence of RNA (Bogerd et al., 1995). Rev assemblies have a highly ordered, filamentous morphology that has been characterized previously by electron microscopy (Watts et al., 1998). Rev multimerization is essential for its functionality and hence for viral replication (Malim and Cullen, 1991 ) and could therefore be an attractive target for anti-HIV therapy.
The central role of Rev in the replication makes it an interesting target for the development of novel anti-HIV agents. Indeed, the transdominant RevMI O mutant efficiently inhibits the wild- type Rev function and HIV replication (Bevec et al., 1992). Michienzi et al. (2006) demonstrated that a nucleolar localizing RRE inhibits the HIV replication by acting as a decoy for Rev, proving the validity of the target. Duan et al. (1994) describe anti-Rev single chain antibodies that inhibit HIV-1 replication by sequestering Rev in the cytoplasm. Wu et al. (1996) showed that anti-Rev single chain variable fragments directed against the Rev activation domain could inhibit HIV replication. However, antibodies directed to the C-terminus seemed far less efficient and could not achieve the same level of inhibition as for the activation domain. None of these antibodies act on the multimerization of the Rev protein; indeed, although the multimerization has been studied, and models have been developed (Jain and Belsaco, 2001 ), the isolation and screening of such antibodies is not evident. Surprisingly, we obtained nanobodies that are able to inhibit the Rev multimerization. Even more surprisingly, these antibodies efficiently inhibit the HIV replication in cell culture. A first aspect of the invention is an antibody, capable of inhibiting the multimerization of HIV Rev. Preferably, said HIV Rev is HIV-1 Rev. Said antibody can be any antibody, including but not limited to normal antibodies, single chain antibodies, diabodies and camelid antibodies. Preferably, said antibody is a nanobody. A nanobody is known to the person skilled in the art, and consists of one single variable domain derived from a camelid antibody devoid of light chains. Even more preferably, said nanobody is selected from the group comprising SEQ ID N°1 (Seq Nb_190, fig 3) and SEQ I D N° 2 (Seq Nb_243, fig 3), or a functional fragment thereof. A functional fragment, as used here, is one of the CDR loops. Preferably, said antibody consists of SEQ ID N° 1 (Seq Nb_190, fig 3), or SEQ ID N° 2 (Seq Nb_243, fig 3).
Another aspect is a functional fragment, derived of a nanobody according to the invention, capable of inhibiting the multimerization of HIV Rev. Preferably, said HIV Rev is HIV-1 Rev. A functional fragment as used here is one of the CDR loops. Preferably, said functional fragment is CDR3. Another aspect of the invention is the use of an antibody according to the invention, or a functional fragment according to the invention to inhibit HIV-1 replication. Said antibody can be used in a therapeutic treatment, e.g. by the expression of the antibody by gene therapy. Gene therapeutic approaches for treatment of HIV are known to person skilled in the art and are reviewed by Strayer et al. (2002).
Still another aspect of the invention is the use of an antibody according to the invention, or a functional fragment according to the invention for the structure determination of HIV Rev by means of X-ray crystallography or NMR.
Another aspect of the invention is the use of an antibody according to the invention, or a functional fragment according to the invention to screen compounds that inhibit HIV Rev multimerization. Indeed, the antibody binds to an epitope comprising a domain that is essential for the multimerization of Rev. By studying the disruptance of this protein-protein interaction by a compound, compounds can be screened that are quite active in inhibiting the mulimerization. Methods to study protein-protein interaction, and its disruption, are known to the person skilled in the art and include, but are not limited to yeast two hybrid, MAPPIT (Tavernier et al., 2002) and reverse MAPPIT (Lemmens et al., 2006). Another aspect of the invention is a compound, isolated with the use of the antibody according to the invention.
BRIEF DESCRIPTION OF THE FIGURES
Fig 1 : Domain structures of (A) the Rev responsive element and (B) Rev from HIV-1. NLS: Nuclear Localization Signal, NES: Nuclear Export Signal (Pollard and MaNm, 1998). Fig. 2: (A) Principle of our FRET based Rev-multimerization-assay and (B) typical emission spectrum of samples excited at 430 nm. When FRET occurs (ECFP-Rev + EYFP-Rev) a typical emission peak at 527 nm is detected. As negative control the following pairs were used: free-ECFP + free-EYFP, free-ECFP + EYFP-Rev, ECFP-Rev + free-EYFP. Fig. 3: Amino acid sequences of HIV-Rev specific nanobodies. Fig 4: Inhibition of the HIV Rev multimerization using our FRET-based multimerization assay. (A) Nb_190 and Nb_243 are identified as Rev-multimerization inhibitors. (B) Dose dependent inhibition of the Rev multimerization by Nb_190 as measured by FRET. Nb_190 has no effect on the FRET signal coming from an ECFP-EYFP fusion construct. As negative control an irrelevant Nb_163 was used. Fig. 5: Effect of Nb_190 on the Rev-RRE ribonucleoprotein complex formation. Gel mobility shifts of labeled highaffinity stem NB in complex with Rev were performed in the presence of increasing amounts of Nb_190 (lanes 3 to 6; 6, 30, 150 and 750 nM respectively). Bands corresponding to free high-affinity binding stem NB RRE (lane 1 ), IIB/Rev (lane 2) and IIB/Rev/Nb190 complexes (lanes 4 to 6) are indicated. Nb_163 (lane 7) was included as negative control.
Fig. 6: Nb_190 interacts with Rev in cells. HeLa cells were transfected separately with Rev-
GFP, Rev-V16D-GFP, Rev-M4-GFP (Y23D-S25D-N26L), Nb_190-mKO, and Nb_163-mKO expression plasmids or co-transfected with Rev-GFP or Rev-M4-GFP and Nb190-mKO or Nb163-mKO as indicated. Subcellular localization of the proteins was visualized by fluorescence confocal microscopy in both GFP and mKO channels.
Fig 7: Effect of the intracellular expression of Nb_190 on the expression of the NL43 molecular HIV-1 clone. (A) HEK 293T cells were co-transfected with pNL4.3 and pcDNA3.1-Nb_190 plasmid DNA in different relative concentrations. The amount of pcDNA3.1-Nb_190 plasmid DNA was kept constant (1 ) while the amount of pNL4.3 was decreased (1 , 0.5, 0.2 or 0.1 ). One day after transfection virus-associated p24 in the supernatant was monitored using p24 ELISA. The intracellular expression of Nb_163 was used as control. (B) A mutated molecular clone of NL43 expressing the Nb_190 or Nb_163 in place of the nef gene was constructed. After transfection of the respective plasmid DNA into HEK 293T cells, virus-associated p24 in the supernatant was monitored using p24 ELISA.
Fig. 8: Mechanism of Nb_190 action. (A) Nb_190 fails to inhibit the viral production of a Rev- independent molecular clone. 293T cells were co-transfected with HIV-1 NL43RΘV(-)RRE(-)CTE(+) (0.5 μg) and different amounts Nb_190, Nb_163, or Dronpa expression plasmids. Expression of HIV-1 NL43Rev(-)RRE(-)cτE(+) was analyzed by measuring the virus-associated core antigen (p24) in the supernatants of the transfected cells. p24 production levels are presented relative to control cells expressing HIV-1 NL43Rev(-)RRE(-)cτE(+) only. Results are mean ± s.e.m. (B) Nb_190 specifically inhibits the expression of partially and unspliced viral mRNA species. Northern blot analysis of viral mRNA forms in cells transfected with wild-type or mutant HIV-1 NL4.3 molecular clones (see figure 3c). (C) Levels of late unspliced relative to early multiple spliced viral mRNA forms in transfected cells as measured by real-time RT-qPCR. Results are mean ± s.e.m, with n = 4, P < 0.025.
EXAMPLES
Materials and methods to the examples
Nanobody generation, selection and purification.
Two mg of purified Hiv-Rev was injected in a llama {Lama glama) over a period of 6 weeks. The immunization, library construction and selection have been performed following standard procedures according to Conrath et al. (Conrath et al., 2001 ) with minor modifications: total RNA was extracted from the peripheral blood lymphocytes according to the method of Chomczynski et al. (Chomczynski and Sacchi, 1987) 50μg of total RNA was used to prepare cDNA using SupersScript Il RNaseH (Invitrogen) and a dN6 primer according to the manufactures instruction. Phages were recovered by incubating the HIV-Rev coated wells with 10OmM triethylamine pH10 for 10min, this eluate containing phages was neutralized by adding TrisHCI pH 6,8 and added to freshly grown TG1 cells. These HIV-Rev coated wells were then washed once with TrisHCI pH 6,8 and several times with PBS and freshly grown TG1 cells were added to the wells to recover the non eluted phages. Maxisorb 96-well plates (Nunc) were coated with purified HIV-Rev overnight at 4 0C at 1 μg/ml in sodium bicarbonate buffer pH 8.2. Residual protein binding sites in the wells were blocked for two hours at room temperature with 2% milk in PBS. Detection of antigen-bound nanobodies was performed with a mouse anti-haemaglutinin-decapeptide-tag (clone 16B12, BAbCO) or a mouse anti-histidine-tag (Serotec), as appropriate. Subsequent detection of the mouse anti-tag antibodies was done with an alkaline phosphatase anti-mouse-lgG conjugate (Sigma), respectively. The absorption at 405 nm was measured 15 min after adding the enzyme substrate p-nitrophenyl phosphate.
Finally, all selected nanobody genes were cloned in a pHEN6 vector for expression with a His- tag in E. coli (Conrath et al., 2001 ).
Cell culture, transfections, plasmids, and microscopy
HeLa, human epithelial cells, and human embryonal kidney 293T cells were maintained using standard procedures. For transfection of HeLa, cells were plated onto glass bottom microwell dishes (MatTek corporation, Ashland, MA) at 0.1 x 106 cells/plate and cultured until 50% confluent. Then cells were washed with PBS and transfected with 1 μg of plasmid DNA using SuperFect® transfection reagent (Qiagen, Valencia, California) according to the manufacturers manual and incubated overnight. 293T cells were cultured in microwell dishes until 50% confluency and transfected by the calcium phosphate coprecipitation technique (Graham FL and Van der Eb AJ 1973 Virology). Prokaryotic and eukaryotic expression vectors were constructed using standard molecular cloning techniques.
The HIV-1 N|_43 molecular clone has been previously described (Fisher et al., 1986). HIVNL43- Nb 190 and HIVN|_43-Nb_i63 were generated by subcloning the Nb sequence into the Blp\-Xho\ site of p83-10 ((Gibbs, 1994.).). The resulting plasmids were obtained by replacing the EcoR\-Xho\ fragment from HIV-1 NL43 with the same EcoR\-Xho\ fragment from p83-10 containing the Nb sequences.
Transfected HeLa cells were imaged with a laser scanning SP5 confocal microscope (Leica Microsystems, Germany) equipped with an AF 6000 microscope and an AOBS, using a HCX PL APO χ63 (NA 1.2) water immersion objective magnification. GFP was monitored with the Ar laser using the 488-nm line for excitation, and emission was detected between 492 and 558 nm. mKusabira orange fluorescent protein (mKO) was imaged using the 561-nm line for excitation, and emission was detected between 570 and 670 nm. Protein expression and purification pET29b(+) and pET21 b(+) constructs encoding respectively the Rev protein and fusion proteins ECFP-Rev, EYFP-Rev, ECFP-EYFP and free ECFP were transformed in Escherichia coli BL21 (DE3) and expressed by a 3.5 hour induction with 1 mM IPTG. Cells were lysed using the SLM Amino French Pressure Cell Press (Beun-DeRonde) and the proteins were purified via Ni-NTA affinity chromatography. For immunization of the llama the Rev protein was further purified by anion exchange chromatography (Akta Explorer 1 OS, GE Healthcare).
Rev multimerization assay
To assess the effect of Nb_190 on the Rev multimerization by FRET, variable concentrations of Nb_190 were mixed (in a 96-well plate in 100 μl PBS) with 0.1 μM ECFP-Rev and 0.2 μM EYFP-Rev either prior to or after (15 min of incubation) the assembly of Rev multimers. A sample with only ECFP-Rev and EYFP-Rev was used as a positive control for FRET, while the combination of free ECFP plus EYFP-Rev was used as negative control. After 30 min incubation the FRET percentage was determined using a spectrofluorometer (Safire2, Tecan). Hence emission was measured at 480 ± 5 nm (C) and 530 ± 5 nm (F) after excitation with 430 ± 5 nm and at 530 ± 5 nm (Y) after excitation with 490 ± 5 nm. The relative FRET efficiency was then calculated as follows. The total FRET signal (F) was first corrected for the 'bleed through' of the donor ECFP (i.e. 44% of C) and the 'direct excitation' of the acceptor EYFP (i.e. 5% of Y) resulting in the 'real FRET W: W = F - (0.44 x C) - (0.05 x B) The FRET efficiency (E) is then given by:
W + γ.C where γ is the ratio between the quantum yields of acceptor and donor, i.e. 1.53. The relative FRET percentage was obtained by expressing the FRET efficiency (E) relative to the FRET efficiency of both the positive control (100%) and the negative control (0%).
Northern blot analysis mRNA was extracted using the Oligotex Direct mRNA kit (Qiagen). After contaminating DNA was digested using RNase-free DNase I (Invitrogen), the mRNA was denatured by heating to 500C in glyoxal load dye and separated by agarose electrophoresis under denaturing conditions. mRNA was blotted using the Northern Max-Gly system (Ambion) according to manufacturers manual. The RNA probe spanning exon 7 from the SamHI to the BgIW site (nt 8475 through 9056) of the NL43 genome was produced by in vitro transcription from T7 primer
PCR products. Quantitative RT-PCR
Two days after transfection, total mRNA from cells was extracted using the Oligotex Direct mRNA kit (Qiagen) followed by DNA digestion using RNase-free DNase I (Invitrogen). DNase I treated mRNA was used to generate cDNA along with Thermoscript reverse transcriptase (Invitrogen) and oligo(dT)2o. q RT-PCR for unspliced and multiple spliced HIV mRNA was performed according to a protocol described earlier (Pasternak et al., 2008) that we adapted slightly. For unspliced mRNA, 0.2 mM primers GAG1 and SK431 and 0.2 mM FAM-BHQ1 fluorescent probe GAG3 was used. For multiple spliced mRNA 0.2 mM primers mf83 and mf84 and 0.2 mM FAM-BHQ1 fluorescent probe ks2-tq was used. Control reactions omitted reverse transcriptase, and the number of cDNA copies was determined using the HIV-1 NL43 molecular clone or the pTat/Rev DNA as standards. pTat/Rev represents partial sequence (exons 4 and 7) of HIV-1 multiple spliced RNA encoding Tat and Rev proteins. Results are expressed as number of copies unspliced HIV mRNA per copy of multiple spliced RNA.
Mobility Shift assays
High-affinity NB RNA labelled with Alexa633 was purchased from Sigma. Full-length RRE RNA transcripts were prepared by a T7 in vitro transcription, and purified by electrophoresis on a 6% polyacylamide gel. These transcripts where then labelled with Alexa647 by an Ulysis Nucleic Acid Labelling Kit (Molecular Probes). Typically, 5 nM of NB RRE and 100 nM of Rev protein, or 3 nM of full-length RRE, 60 nM of Rev and 300 nM of Nb190 or Nb163 were used. Binding of Rev to RNA was performed in buffer 20 mM Tris-HCI (pH 8.0), 100 mM NaCI, 10 mM DTT with 0.1 mg/ml Bakers Yeast tRNA. The samples were incubated for 20 min at room temperature and then loaded onto a 6% polyacrylamide gel for 1 hour. The gels were visualized using an Ettan DIGE Imager (GE Healthcare).
Example 1: A Rev-multimerization assay based on FRET
We developed a powerful Rev-multimerization-assay (Daelemans et al., 2004) that is based on FRET. FRET is the non-radiative transfer of photon energy from an excited fluorophore (the donor, e.g. ECFP) to an acceptor fluorophore (e.g. EYFP) when both are located within close proximity (1-10 nm). sing FRET one can resolve intimate interactions between two protein partners. In our biochemical Rev-multimerization assay, Rev protein fused to cyan-fluorescent protein (ECFP-Rev) is mixed with Rev that is fused to yellow-fluorescent protein (EYFP-Rev) as shown in figure 2, resulting in a FRET efficiency of about 20-35% while the negative controls (ECFP + EYFP-Rev) resulted in a FRET efficiency of only 1.6% (figure 2B). Therefore this assay is suitable for the detection of Rev multimerization and inhibitors thereof. Example 2: Characterization of HIV-Rev specific nanobodies as multimerization inhibitors
A llama was immunized with purified recombinant HIV-Rev protein. The immunization and the isolation of nanobodies have been performed following standard procedures according to Ghahroudi et al. (1997). Llama immunizations and phage displayed libraries of their nanobodies yielded a first series of HIV-Rev specific nanobodies (Figure 3). All nanobody genes were cloned in a pHENOΘ vector for expression with a His-6 tail. The recombinant proteins were produced in Escherichia coli, and purified according to described protocols (Conrath et al., 2001 ). So far, we have been working with the recombinant Rev protein, expressed from pET29b. This protein is tagged at its N-terminus with the S-peptide (Kim and Raines, 1993) and His-tagged at its C-terminus.
A set of selected nanobodies have been analyzed and two nanobodies were found to interfere with the multimerization of Rev. In a first set of experiments, the serum of the Rev immunized llama was tested and compared with that of a control animal in the Rev-multimerization assay (examplei ). The data clearly show that the serum of the immunized llama reduces the FRET signal significantly. This is not the case for the control serum. In a next step, all purified HIV- Rev specific nanobodies (Nb_188, Nb_227a, Nb_228, Nb_229, Nb_191 , Nb_230, Nb_235, Nb_233, Nb_189, Nb_234, Nb_243, Nb_244 and Nb_190) were tested, together with one irrelevant nanobody (Nb_163). From these, two nanobodies (Nb_190 and Nb_243) reduced the FRET signal significantly (see figure 4A). Nb_190 has a dose-dependent inhibitory effect on the Rev multimerization as shown in figure 4B, while it did not interfere with the FRET signal from an ECFP-EYFP fusion construct. Nb_190 is able to compete with the ECFP- Rev:EYFP-Rev complex formation since it is equally active whether it is added before or after ECFP-Rev:EYFP-Rev complex formation (figure 4B). The irrelevant Nb_163 had no effect on the Rev multimerization.
Gel-shift experiments of Rev binding to the high-affinity NB hairpin of RRE reveal that Nb_190 does not interfere with the RNA-protein interaction (Fig. 5). However, addition of increasing amounts of Nb_190 causes a reduction of the mobility of the IIB/Rev complex, confirming the dose-dependent binding of this nanobody to this RNA-protein complex.
Example 3: Effect of NbJl 90 in vivo
We also investigated the interaction of Nb_190 with Rev in living cells using confocal microscopy (Fig. 6). For these experiments we fused Rev to GFP and the nanobodies to the orange-coloured mKO. When transiently expressed in HeIa cells, the steady-state localization of Rev-GFP is nucleolar (Fig. 6 panel A). The Nb_190-mKO distributes evenly over the nucleus and the cytoplasm (Fig. 6, panel B) as does the irrelevant Nb_163-mKO (Fig. 6, Panel
C). In contrast to control experiments with Nb_163-mKO (Fig 6, panel E), Rev-GFP and Nb_190-mKO both redistribute and co-localize to the cytoplasm when co-expressed (Fig. 6, panel D).
Taken together, these co-localization experiments emphasize that Nb_190 behaves as an intrabody and binds wild-type Rev in living cells. Most importantly, our multimerization inhibitor (Nb_190) and the multimerization impaired single V16D and triple Y23D-S25D-N26L. Rev mutations provoke very similar effects on the cellular distribution of Rev. Indeed, the Nb_190-
Rev complex (Fig. 6, panel D) and these V16D and Y23D-S25D-N26L Rev mutants (Fig. 6, panels F and G) similarly redistribute to the cytoplasm. Interestingly, Nb_190-mKO does not interact with the multimerization deficient Y23D-S25D-N26L Rev mutant (Fig. 6, panel H), suggesting that the Y23-N26 segment of Rev is contained in the epitope that is recognized by
Nb 190.
Example 3: Nb190 interferes with the HIV-1 expression Next, the effect of Nb_190 on the expression of HIV-1 was analyzed. Therefore, Nb_190 was cloned in a mammalian expression vector (pcDNA3.1 ) and co-transfected with pNL4.3 (an HIV-1 molecular clone) in HEK 293T cells. Expression of NL43 was analyzed by measuring the virus-associated core antigen (p24) in the supernatants of transfected cells using ELISA. p24 is a measure for the amount of produced virus particles present in the supernatant. Figure 7A shows the effect of Nb_163 and Nb_190 on the expression of NL43. Nb_190 reduces virus expression by more than 70% compared to the Nb_163 control.
To further establish the inhibitory effect of this anti-Rev-multimerization nanobody on viral replication, we constructed a heterologous suicide variant of the molecular clone HIV-1 NL43 by inserting the nanobody coding sequence in the nef open reading frame (Fig. 7B). Virus production from this HIV-1 NL43-Nb_i 90 clone was reduced by 300-fold compared to the expression of wild-type HIV-1 NL43 or a control virus expressing the irrelevant nanobody Nb_163 (Fig. 7B). Finally, we assessed the effect of Nb_190 on the expression of a Rev-independent HIV-1 molecular clone HIV-1 NL43ReV(-)RRE(-)cτE(+) ■ In this engineered infectious HIV-1 clone Rev and RRE are substituted by the constitutive transport element (CTE) from the Mason-Pfizer monkey virus. Upon co-transfection, Nb_190 was unable to reduce the expression of this Rev- independent virus, further establishing the anti-viral effect of this anti-multimerization nanobody closely correlates with inhibition of the Rev function (Fig. 8a). Compelling independent confirmation of the Rev-specific effects of the multimerization inhibitor Nb_190 was obtained by analyzing the effect of Nb_190 on the expression levels of the different viral mRNA species. Wild type viruses, such as HIV-1 Nι_43 produce three predominant size classes of viral mRNAs with sizes of 9.3, 4, and 2 kb. The late unspliced 9.3 kb and single spliced 4 kb species are expressed in a Rev-dependent manner whereas the expression of the early multiply spliced 2 kb mRNA is Rev-independent. In northern blot (Fig 8b) and real-time RT-qPCR (Fig. 8c) experiments on RNA extracted from cells transfected with the suicide HIV-1 NL4.3-Nb_190 molecular clone we find that the ratio of the late unspliced transcripts over the early fully spliced mRNA is greatly reduced compared to cells transfected with the wild-type HIV-1 NL43 θr the HIV-1 NL43-Nb 163 virus.
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Claims

1. An anti HIV-1 Rev antibody, whereby said antibody inhibits HIV Rev multimerization.
2. An antibody according to claim 1 whereby said antibody is a nanobody.
3. A nanobody according to claim 2, whereby said nanobody is selected from the group comprising SEQ ID N°1 and SEQ ID N°2, or a functional fragment thereof.
4. The use of an antibody, according to any of the claims 1-3 to inhibit HIV replication.
5. The use of a functional fragment according to claim 3 to inhibit HIV replication.
6. The use of an antibody, according to any of the claims 1-3, to crystallize HIV-Rev and/or to solve its structure by NMR.
7. The use of a functional fragment according to claim 3, to crystallize HIV-Rev and/or to solve its structure by NMR.
8. The use of an antibody, according to any of the claims 1-3, to screen compounds that inhibit HIV-1 Rev multimerization.
9. The use of a functional fragment according to claim 3, to screen compounds that inhibit
HIV-1 Rev multimerization.
10. A compound, isolated with the use of an antibody according to claim 8 or 9.
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