INFECTION

FIELD OF INVENTION The present invention relates to the use of a BLIMP-1 modulator for treating HIV infection.

BACKGROUND OF INVENTION

Despite effective antiretroviral treatment (ART), human immunodeficiency virus (HIV) infection persists and rebounds upon treatment interruption, presumably due to latently infected resting CD4 ⁺ T cells.

Reaching an HIV cure is now recognized as a valid objective but requires, in the context of optimized antiretroviral therapies unable to eradicate the virus, to determine the mechanistic factors responsible for the persistence of HIV reservoirs. The HIV reservoir is established during primary infection. Administration of ART in very early acute infection seems to result in decreasing the size of the viral reservoir. Although early treatment can substantially reduce the size of the total reservoir, a stable population of latently infected CD4 ⁺ T cells transits into the long-lived latent reservoir, and is unaffected by early combination ART. Most HIV pro-viral DNA is detected in CD4 ⁺ T lymphocytes in lymphoid tissue. In blood, most HIV DNA is found in central memory and transitional memory CD4 ⁺ T cells, which maintain the reservoir because of their intrinsic capacity to persist through homeostatic proliferation and renewal. Other cellular reservoirs that might exist include naive CD4 ⁺ T cells, monocytes and macrophages, astrocytes, and microglial cells. During long term effective ART, low level plasma HIV RNA is detected. Chronic production of HIV from a stable reservoir of long-lived infected cells (the latent reservoir) is probably the main source of this persistent HIV. Several therapeutic strategies are considered in HIV cure related research in patients already treated by optimal antiretroviral therapy:

One approach is to disrupt the control of HIV latency in quiescent HIV-infected cells. To do so a strategy is to exploit the ability of histone deacetylase (HDAC) inhibitors to reactivate HIV expression in latently infected cells in the presence of HAART. Chromatin remodeling enzymes like histone deacetylases (HDACs) play a critical role in HIV latency. HDACs are recruited to the highly conserved initiator region of the HIV promoter by several distinct complexes, by means of factors that are both ubiquitous in cell types infected by HIV and also participate in basal and activated viral gene expression. The existence of multiple mechanisms that recruit repressive HDAC compressive HDAC complexes to the proviral promoter raises the possibility that HDAC inhibitors might lead to the activation of HIV in latently infected cells. Such substances are currently being tested in clinical trials (Rasmussen et al, 2013 Jan 31;9(5)). HDAC inhibitors theorically induce a viral pathogenic effect which might enhance the elimination of the latent cells by the immune system but these mechanisms remain unclear and unspecific. However the clinical trial of an HDAC inhibitor, named Vorinostat, in patients on antiretroviral therapy failed at demonstrating such an effect (Archin et al. 2012 Nature Jul 25;487(7408):482-5).

Another strategy based on a DNA methylation inhibitor of histones, such as 5- azacitidine, was also shown to increase mutation frequency within the HIV genome which disrupted its replication (Dapp et al. 2009 J Virol. Nov; 83(22): 11950-8). The problem with these molecules is their action mechanisms within the cells. They induce mutations which enhance highly toxic side effects when administered to patients (Subramanian S et al. 2010 Pharmaceuticals 3, 2751-2767). Another approach is to further decrease the number of infected cells by increasing the number of latently-infected cells HIV latency and/or the control of HIV latency. A strategy is to use agents that block T cell activation and activation of HIV transcription, such as by using immunosuppressive agents (Katlama et al. Lancet 2013). Therefore there is a need to unravel new targets involved in mechanisms related to HIV latency in order to regulate persistence of HIV latency, and to give the patient immune system a better chance to control the virus infection. It also is necessary to design new therapeutic strategies aiming at eliminating latent HIV infection or at restricting the latent pool down to a size bearable by the host immune system, thus allowing reaching a status of functional cure.

For the last few years, a protein named BLIMP-1 was found to be overexpressed in immune cells following an infection. PR domain zinc finger protein 1 also known as B- lymphocyte-induced maturation factor 1 (BLIMP-1) is a 98kDa transcription factor which was originally identified as being induced during the differentiation of a B-cell lymphoma cell line (Turner et al, Cell 77:297, 1994). BLIMP-1 is a protein that in humans is encoded by the PRDMl gene (Genbank accession number: NP_001189.2 and NP_878911.1 for isoforms 1 and 2 respectively). It has been proposed that BLIMP-1 has a pre-eminent role in regulating B-cell terminal differentiation. In T cells, BLIMP-1 regulates the terminal differentiation of CD8 ⁺ T cells (Rutishauser R et al. 2009 Immunity Aug 21;31(2):296-308; Rallies A et al. 2009 Immunity Aug 21;31(2):283-95) and BLIMP-1 is also known to be highly expressed in CD8 ⁺ T cells generated after acute infection (Shin H et al. 2009 Immunity Aug 21; 31(2): 309-320). BLIMP-1 acts as a repressor of beta-interferon (β-IFN) gene expression. The protein binds specifically to the PRDI (positive regulatory domain I element) of the β-IFN gene promoter.

BLIMP-1 has been only poorly investigated in the context of the HIV infection and only thus far to define new molecular pathways for explaining immune exhaustion and T cell dysfunction. Seddiki et al. (Eur J Immunol. 2013 Feb;43(2):510-20) found that BLIMP- 1 expression, as other inhibitory molecules, was highly activated in CD4 ⁺ CD45RO ⁺ T cells (memory T cells) from HIV-infected viremic subjects (patients with progressive chronic HIV infection as set in Seddiki' s paper) compared to Long-Term NonProgressors subjects and healthy subjects, therefore identifying a novel miR- 9/BLIMP-1/IL-2 axis that is dysregulated in progressive HIV infection.

In the present invention, the inventors compared the transcription profile of CD4 ⁺ T cells subsets constituting the ΗΓ reservoir of uninfected subjects with the CD4 ⁺ T cells subsets constituting the HIV reservoir of HIV-infected viremic subjects and Elite Long- Term NonProgressors subjects. They surprisingly found that an overexpression of BLIMP- 1 was observed in a specific CD4 ⁺ T cells subset, the CD4 ⁺ T _CM cells, from Elite Long-Term NonProgressors subjects compared to HrV-infected viremic subjects. In addition, they also found that said BLIMP- 1 overexpression was correlated to HIV pro virus repression in said subset of CD4 ⁺ T cells.

The present invention therefore aims at providing a treatment for HIV infection based on modulation of BLIMP- 1.

SUMMARY

One object of the invention is a modulator of BLIMP- 1 for use in treating HIV infection.

In one embodiment of the invention, said modulator is an inhibitor or an activator of BLIMP- 1 gene expression, protein expression and/or activity. In another embodiment of the invention, said modulator is an inhibitor selected from the group consisting of antisense RNA or DNA molecules, small inhibitory RNAs (siRNAs), short hairpin RNA, micro RNA, DNAzymes, modified or synthetic DNA or RNA degradation-resistant polynucleoside amides, peptide nucleic acids (PNAs), locked nucleic acids (LNAs) and other nucleobase-containing polymers, small molecules, antibodies, aptamers and ribozymes.In another embodiment of the invention, said modulator is a short harpin RNA.

In another embodiment of the invention, said modulator is an activator selected from the group consisting of small molecules or proteins.

Another object of the invention is a method for screening a modulator of BLIMP- 1 for use in treating HIV infection that comprises:

a. providing CD4 ⁺ T cells expressing BLIMP- 1 or a cell line infected by latent HIV infection;

b. incubating said cells with a candidate compound; c. determining whether said candidate compound inhibits BLIMP- 1 or activates BLIMP- 1; and

d. selecting the candidate compound that inhibits BLIMP- 1 or activates BLIMP- 1.

In one embodiment of the invention, said method of the modulation of BLIMP- 1 is determined by measuring whether said candidate compound inhibits or activates IL-2 release.

In one embodiment of the invention, a pharmaceutical composition comprises the said modulator of BLIMP- 1 and a pharmaceutically acceptable excipient for use in treating HIV infection and/or in curing HIV.

In another embodiment of the invention, said pharmaceutical composition further comprises one or more antiretrovirals and/or immunomodulators and/or anti-infectives agents.

In one embodiment of the invention, said BLIMP- 1 modulator or pharmaceutical composition for use as mentioned above is administered in combination with antiretrovirals and/or immunomodulators and/or anti-infectives agents.

In another embodiment of the invention, said modulator of BLIMP- 1 or a pharmaceutical composition for use as mentioned above is administered prior to, concurrent to, or subsequent to the administration of antiretrovirals and/or immunomodulators and/or anti-infectives agents.

Another object of the invention is a BLIMP- 1 biomarker for use for determining whether a subject is in need of a treatment against HIV infection and/or for monitoring a treatment against HIV infection in a subject in need thereof.

In one embodiment of the invention, said inhibitor of BLIMP- 1 is for use in a subject that has a latent HIV infection and overexpresses BLIMP- 1 in at least one of the CD4 ⁺ T cells subsets, preferably in the CD4 ⁺ T _CM cells. In another embodiment of the invention, latent HIV infection is determined by measuring the HIV provirus expression in at least one of the CD4 ⁺ T cells or memory subsets, preferably CD4 ⁺ T _CM cells, isolated from a blood sample obtained from the subject, and wherein a lower level of HIV provirus in the CD4 ⁺ T _CM cells compared to the level of HIV provirus in CD4 ⁺ T _CM cells from HIV-infected viremic subjects, is indicative of a positive outcome of the treatment.

In another embodiment of the invention, said activator of BLIMP- 1 is for use in an HIV-infected antiretroviral-treated subject and wherein BLIMP- 1 is repressed in at least one of the CD4 ⁺ T cells subsets, preferably in the CD4 ⁺ T _CM cells.

Another object of the invention is a method for determining whether a subject is to be treated with the BLIMP- 1 modulator that comprises:

a. isolating CD4 ⁺ T cells or memory subsets from a blood sample obtained from the subject;

b. measuring BLIMP- 1 and optionally HIV provirus expression in CD4 ⁺ T cells subsets, or in at least one of the memory CD4 ⁺ T cells subsets, preferably CD4 ⁺ T _CM cells, wherein the level of BLIMP- 1 in CD4 ⁺ T cells, preferably T _CM cells, determines the treatment of the subject with a BLIMP- 1 modulator. Another object of the invention is a method of treatment of HIV infection comprising the administration in a subject in need thereof of a therapeutically effective amount of a modulator of BLIMP- 1.

DEFINITIONS

In the present invention, the following terms have the following meanings: - "Subjects": refers to a mammal, preferably a human.

"Human immunodeficiency virus infection (HIV)" refers to a disease of the human immune system caused by an infection by the human immunodeficiency virus (HIV). HIV can be divided into two major types: HIV type 1 (HIV-1) and HIV type 2 (HIV-2). Although HIV-1 and HIV-2 cause the infection, HIV-1 group M viruses predominate and are responsible for the pandemic infection.

"Long-Term Nonprogressors (LTNPs) subjects" refers to a subset of HIV- infected subjects having prolonged elevation in CD4 ⁺ T cell counts for many years in the absence of ART.

"Elite controllers (ECs) subjects" refers to a subset of HIV-infected subjects having the ability to spontaneously control plasma viral load without ART.

"Elite Long-Term Nonprogressors (E-LTNPs) subjects" refers to rare subjects who are infected with HIV, but control the infection without ART. Many of these patients have been HIV positive for 30 years without progressing to the point of needing to take medication in order not to develop acquired immunodeficiency syndrome (AIDS). They display long-term virtually undetectable viremia, stable CD4 ⁺ T cell counts and extremely low levels of HIV reservoir, in the absence of ART.

"HIV-infected viremic subjects (VH)": refers to a subject infected by the human immunodeficiency virus (HIV) in whom HIV replication is detectable as assessed with plasma viremia (HIV RNA) above the threshold of detection; a retrovirus that causes (AIDS) by infecting helper T cells of the immune system. During the initial infection, a person may experience a brief period of influenza-like illness. This is typically followed by a prolonged period without symptoms. As the illness progresses, it interferes more and more with the immune system, making the person much more likely to get infections, including opportunistic infections and tumors that do not usually affect people who have working immune systems.

"Uninfected subjects (UI)": refers to a subject non-infected by the human immunodeficiency virus.

"Resting CD4 ⁺ T cells subsets": refers in the present invention to subtypes of resting CD4 ⁺ T cells which lack cell surface expression of classical activation markers as: CD25 ^" , CD69 ^" , HLA-DR ^" and that represent 92% of total CD4 ⁺ T cells. The resting CD4 ⁺ T cells comprise the resting CD4 ⁺ T naive cells (T _N : CD45RA ⁺ CCR7 ⁺ CD27 ⁺ ) and the cells from the memory compartment that includes central memory cells (T _CM : CD45RA ^" CCR7 ⁺ CD27 ⁺ ), transitional memory cells (T™: CD45RA ^" CCR7 ^" CD27 ⁺ ), and effector memory cells (T _EM : CD45RA ^" CCR7 ^" CD27 ^" ).

"Viremia": refers to a medical condition where viruses enter the bloodstream and hence have access to the rest of the body. Primary viremia refers to the initial spread of virus in the blood from the first site of infection. Secondary viremia occurs when primary viremia has resulted in infection of additional tissues via bloodstream, in which the virus has replicated and once more entered the circulation.

"A modulator" refers to a natural or synthetic compound that has a biological effect to activate or inhibit the expression of a gene and/or a protein, or that has a biological effect to activate or inhibit the biological activity of a protein. Consequently, "a BLIMP-1 modulator" refers to a natural or synthetic compound that has a biological effect to activate or inhibit the expression of BLIMP-1 gene and/or BLIMP-1 protein, or that has a biological effect to activate or inhibit the biological activity of BLIMP-1.

"An inhibitor": refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce or down-regulate the expression of a gene and/or a protein or that has a biological effect to inhibit or significantly reduce the biological activity of a protein. Consequently, "a BLIMP-1 inhibitor" refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce or down-regulate the expression of the gene encoding for BLIMP-1 and/or the expression of the BLIMP-1 protein and/or the biological activity of BLIMP-1.

"An antagonist" refers to a natural or synthetic compound which binds to the protein and blocks the biological activation of the protein, and thereby the action of the said protein. Consequently, "a BLIMP-1 antagonist" includes any chemical entity that, upon administration to a patient, results in inhibition or down-regulation of a biological activity associated with activation of BLIMP-1 in the patient, including any of the downstream biological effects otherwise resulting from the binding to BLIMP- 1 of its natural ligand. Such BLIMP-1 antagonists include any agent that can block BLIMP-1 activation or any of the downstream biological effects of BLIMP-1 activation. "An activator": refers to a natural or synthetic compound which binds to the protein and stimulates the expression of a gene and/or a protein or that has a biological effect to stimulate the biological activity of a protein. Consequently, "a BLIMP-1 activator" refers to a natural or synthetic compound that has a biological effect to stimulate the expression of the gene encoding for BLIMP-1 and/or the expression of the BLIMP-1 protein and/or the biological activity of BLIMP-1. The activator usually mimics the action of a natural activator that binds to the transcription factor.

"An agonist"; refers to a natural or synthetic compound which binds to the protein and stimulates the biological activation of the protein, and thereby the action of the said protein. Consequently, "a BLIMP-1 agonist" includes any chemical entity that, upon administration to a patient, result in stimulation of a biological activity associated with repression of BLIMP-1 in the patient, including any of the downstream biological effects otherwise resulting from the binding to BLIMP-1 of its natural ligand. Such BLIMP-1 agonists include any agent that can stimulate BLIMP-1 expression or any of the downstream biological effects of BLIMP-1 repression. In one embodiment, the binding of the agonist to the transcription factor on said transcription factor activates the transcription factor.

"Therapeutically effective amount" refers the total amount of each active component of the composition and method that is sufficient to show a meaningful patient benefit, i.e., inhibiting, ameliorating, or healing of acute conditions characterized by inhibition of the HIV infection. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

"Pharmaceutically" or "pharmaceutically acceptable": refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi- solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. "Treat, treating, treatment": "Treating" or "treatment" or "alleviation" refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully "treated" for an infection if, after receiving the treatment according to the present invention, the subject or mammal shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of pathogenic cells; reduction in the percent of total cells that are pathogenic; and/or relief to some extent, one or more of the symptoms associated with the specific disease or condition; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

"HIV cure" refers to the eradication of HIV virus in a subject. The treatment enhancing a disappearance or almost disappearance of all HIV-DNA copies, from the peripheral blood and tissues.

"Functional cure of HIV or remission" refers to the decrease of the HIV reservoirs down to levels so low that the virus production will be subsequently maintained at very low levels and for prolonged periods of time after treatment interruption.

"Biomarker" refers to a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. A biomarker may be used to diagnose a specific infection and/or disease as early as possible (diagnostic biomarker), to predict the risk of developing an infection and/or a disease (risk biomarker), to predict the evolution of an infection and/or a disease (prognostic biomarker), to predict the response and the toxicity to a given treatment (companion biomarker). Particularly, a companion biomarker can serve to improve the risk/benefit profile of the treatment of a previously diagnosed condition through a classification of patients. DETAILED DESCRIPTION

The present invention relates to a modulator of BLIMP- 1 for use in treating HIV infection. Preferably, said modulator of BLIMP- 1 is used in combination with antiretroviral for use in treating HIV infection and/or curing HIV. In one embodiment, the present invention relates to an inhibitor of BLIMP- 1 for use in treating HIV infection.

In one embodiment, the present invention relates to the use of an inhibitor of BLIMP- 1 gene expression.

In another embodiment, the present invention relates to the use of an inhibitor of BLIMP- 1 protein expression.

In another embodiment, the present invention relates to the use of an inhibitor that blocks any biological activity and/or downstream biological effects of BLIMP- 1.

The inhibitors of BLIMP- 1 gene expression or BLIMP- 1 protein expression which may be used according to the invention advantageously provide significant inhibition of BLIMP- 1 gene expression and/or protein expression and/or BLIMP- 1 activity, by comparison with BLIMP- 1 endogenous expression and/or activity.

A significant inhibition of BLIMP- 1 corresponds to a decrease of at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and 100% in BLIMP- 1 gene expression and/or BLIMP- 1 protein expression and/or BLIMP- 1 activity and/or downstream molecules of BLIMP- 1.

In one embodiment, inhibitors of BLIMP- 1 for use in the present invention may be based on anti-sense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of BLIMP- 1 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of BLIMP- 1, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding BLIMP- 1 can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

In another embodiment, small inhibitory RNAs (siRNAs) can also function as inhibitors of BLIMP- 1 for use in the present invention. BLIMP- 1 gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that BLIMP- 1 gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

In another embodiment, short hairpin RNA (shRNA) can also function as inhibitors of BLIMP- 1 for use in the present invention. A small hairpin RNA or short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. The promoter choice is essential to achieve robust shRNA expression. At first, polymerase III promoters such as U6 and HI were used; however, these promoters lack spatial and temporal control. As such, there has been a shift to using polymerase II promoters to regulate expression of shRNA. shRNA is an advantageous mediator of RNAi in that it has a relatively low rate of degradation and turnover.

In another embodiment, microRNA (miRNA) can also function as inhibitors of BLIMP- 1 for use in the present invention. A microRNA (miRNA) is a sequence of RNA that can be used to silence target gene expression via RNA interference (RNAi). Expression of miRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. Micro RNAs (miRNAs) constitute non coding RNAs of 21 to 25 nucleotides, which controls genes expression at post-transcriptional level. miRNAs are synthesized from ARN polymerase II or ARN polymerase III in a premiRna of 125 nucleotides. Pre-miRNA are cleaved in the nucleus by the enzyme Drosha, giving rise to a precursor called imperfect duplex hairpin RNA (or miRNA-based hairpin RNA). These imperfect duplex hairpin RNAs are exported from the nucleus to the cytoplasm by exportin-5 protein, where it is cleaved by the enzyme DICER, giving rise to mature miRNAs. miRNAs combine with RISC complex which allows total or partial annealing with the homologous single-stranded target mRNA. Partial annealing with the mRNA leads to the repression of protein translation, whereas total annealing leads to cleavage of the singlestranded mRNA.

In another embodiment, ribozymes can also function as inhibitors of BLIMP- 1 gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalysing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyse endonucleolytic cleavage of BLIMP- 1 mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful as inhibitors of BLIMP- 1 gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing BLIMP- 1. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art. In another embodiment, inhibitor of BLIMP- 1 also include DNAzymes, modified or synthetic DNA or RNA (including but not limited to nucleic acids comprising synthetic and naturally- occurring base analogs, or other sugars, and thiols). Other exemplary oligonucleotides include degradation-resistant polynucleoside amides, peptide nucleic acids (PNAs), locked nucleic acids (LNAs) and other nucleobase-containing polymers. A common PNA has a backbone consisting of repeating N-(2-aminoethyl) glycine unit, linked by amide bonds. Unlike DNA or RNA, PNA often does not contain any sugar or phosphate groups.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high- efficiency transduction of genes in vivo. Standard protocols for producing replication- deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell line with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991).

Preferred viruses for certain applications are the adenoviruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hematopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno- associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion. Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.

In a particular embodiment, the inhibitor, preferably a nucleic acid, is formulated in a nanoparticle. siRNA especially may be delivered by means of nanoparticles. Generally speaking, nanoparticle-based delivery systems are delivery reagents that compact siRNA into particles in the optimal size range of hundreds of nanometers that are on the order of 100,000,000 Daltons in mass. The predominant packaging strategy is to utilize the anionic charge of the siRNA backbone as a scaffold for electrostatic interaction with the delivery reagent. Cationic lipids, cationic polymers, and cationic peptides, which can advantageously be combined with cholesterol, are used to engage the negatively charged phosphodiester backbone and organize large numbers of siRNA molecules into nanoparticle structures prior to cellular treatment in vitro or systemic administration in vivo (Whitehead et al., 2009. See also e.g. WO 2010/080724; US 2006/0240554 and US 2008/0020058). Beyond cationic motifs required for siRNA nanoparticle formation, additional motifs are applied to the delivery reagent. A large variety of lipids, cell targeting ligands, antibodies, and cell penetrating peptides, to list a few, can be covalently tethered to the cationic packaging motifs so that the resulting nanoparticles that are formed will have cellular delivery properties (Whitehead et al., 2009).

Another aspect of the present invention is the use of a BLIMP- 1 antagonist to inhibit BLIMP- 1.

In one embodiment, the affinity of an antagonist for BLIMP- 1 may be quantified by measuring the activity of BLIMP- 1 in the presence of a range of concentrations of said antagonist in order to establish a dose-response curve. From that dose response curve, an IC ₅₀ value may be deduced which represents the concentration of antagonist necessary to inhibit 50% of the response to an agonist in defined concentration. The IC ₅₀ value may be readily determined by the one skilled in the art by fitting the dose- response plots with a dose-response equation as described by De Lean et al. (1979). IC ₅₀ values can be converted into affinity constant (Ki) using the assumptions of Cheng and Prusoff (1973). In one embodiment, the BLIMP- 1 antagonists may be low molecular weight antagonists, e. g. a small molecule.

In another embodiment the BLIMP- 1 antagonist may consist in an antibody (the term including antibody fragment) that can block BLIMP- 1 activation.

In particular, the BLIMP- 1 antagonist may consist in an antibody or fragments thereof directed against the BLIMP- 1 or a ligand of BLIMP- 1, in such a way that said antibody impairs the binding of a BLIMP- 1 ligand to said transcription factor.

Antibodies directed against BLIMP- 1 can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against BLIMP- 1 or ligands of BLIMP- 1 can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see, e.g., U.S. Pat. No. 4,946,778) can be adapted to produce anti-BLIMP-1, or anti-BLIMP-1 ligands single chain antibodies. BLIMP- 1 antagonists useful in practicing the present invention also include anti- BLIMP-1, or anti-BLIMP-1 ligands antibody fragments including but not limited to F(ab') ₂ fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab') ₂ fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to BLIMP- 1. Divalent (or bivalent) single-chain variable fragments (di-scFvs, bi-scFvs) can be engineered by linking two scFvs. Another possibility is the creation of scFvs with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Diabodies have been shown to have dissociation constants up to 40-fold lower than corresponding scFvs, meaning that they have a much higher affinity to their target.

Humanized anti-BLIMP-1 or anti-BLIMP-1 antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397).

Then after raising antibodies directed against the BLIMP- 1 transcription factor as above described, the skilled man in the art can easily select those blocking BLIMP- 1 activation.

In another embodiment the BLIMP- 1 antagonist is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

Then after raising aptamers directed against BLIMP- 1 as above described, the skilled man in the art can easily select those blocking BLIMP- 1 activation.

In another embodiment, the present invention relates to an activator of BLIMP- 1 for use in treating HIV infection.

In one embodiment, the present invention relates to the use of an activator of BLIMP- 1 gene expression. In another embodiment, the present invention relates to the use of an activator of BLIMP- 1 protein expression.

In another embodiment, the present invention relates to the use of an activator that stimulates any biological activity and/or downstream biological effects of BLIMP- 1. The activators of BLIMP- 1 gene expression or BLIMP- 1 protein expression which may be used according to the invention advantageously provide significant activation of BLIMP- 1 gene expression and/or protein expression and/or BLIMP- 1 activity, by comparison with BLIMP- 1 endogenous expression and/or activity.

A significant activation of BLIMP- 1 corresponds to an increase of at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and 100% in BLIMP- 1 gene expression and/or BLIMP- 1 protein expression and/or BLIMP- 1 activity and/or downstream molecules of BLIMP- 1.

Another aspect of the present invention is the use of a BLIMP- 1 agonist to activate BLIMP- 1. In one embodiment, the BLIMP- 1 agonists may be low molecular weight agonists, e. g. a small molecule.

In one embodiment of the invention, modulators of BLIMP- 1 can be further identified by screening methods. The screening methods of the invention can be carried out according to known methods. The screening method may measure the binding of a candidate compound to BLIMP- 1, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound. Alternatively, a screening method may involve measuring or, qualitatively or quantitatively, detecting the competition of binding of a candidate compound to BLIMP-1 with a labelled competitor (e.g., antagonist or agonist). Further, screening methods may test whether the candidate compound results in a signal generated by an antagonist or an agonist of the transcription factor, using detection systems appropriate to cells bearing the transcription factor. Antagonists and/or agonists can be assayed in the presence of a known agonist or antagonist such as a scfv antibody against BLIMP- 1 and an effect on activation by the agonist or the antagonist by the presence of the candidate compound is observed. Competitive binding using known agonists and/or antagonists is also suitable.

Furthermore, screening methods may test whether the candidate compound results in a signal generated by an antagonist of BLIMP- 1 or an agonist of BLIMP- 1, using appropriate detection systems.

In general, such screening methods involve providing appropriate cells which express BLIMP- 1. In particular, a nucleic acid encoding BLIMP- 1 may be employed to transfect cells to thereby express the encoded protein of the invention. Such a transfection may be accomplished by methods well known in the art.

In a particular embodiment, the transfected cells are selected from the group consisting of CD4 ⁺ T cells, preferably memory CD4 ⁺ T cells, and most preferably, CD4 ⁺ T _CM cells.

The screening method of the invention may be employed for determining an inhibitor or an activator by contacting such cells with compounds to be screened and determining whether such compound inhibits or activates BLIMP- 1 activity.

The determination of the inhibition of BLIMP- 1 can be assessed by determining the activity of downstream molecules such as for example: IL-2, IFN-γ, c-myc, IL-10. In particular, one skilled in the art can determine whether the candidate compound is acting on the inhibition or activation of IL-2.

According to an embodiment of the invention, the candidate compound may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo or natural compounds. The candidate compound may be selected from the group of (a) proteins or peptides, (b) nucleic acids and (c) organic or chemical compounds (natural or not). Illustratively, libraries of pre-selected candidate nucleic acids may be obtained by performing the SELEX method as described in documents US 5,475,096 and US 5,270,163. Further illustratively, the candidate compound may be selected from a group of antibodies directed against BLIMP- 1.

In a particular embodiment, the screening method of the invention comprises the step consisting of:

a) providing CD4 ⁺ T cells expressing BLIMP- 1 or a cell line infected by latent HIV infection;

b) incubating said cells with a candidate compound;

c) determining whether said candidate compound inhibits BLIMP- 1 or

activates BLIMP- 1; and

d) selecting the candidate compound that inhibits or activates BLIMP- 1.

In a particular embodiment, the screening method of the invention comprises the step consisting of:

a) providing CD4 ⁺ T cells expressing BLIMP- 1 or a cell line infected by latent HIV infection;

b) incubating said cells with a candidate compound;

c) determining whether said candidate compound inhibits or activates IL-2 release; and

d) selecting the candidate compound that inhibits or activates IL-2 release. The present invention relates to a pharmaceutical composition comprising a BLIMP- 1 modulator.

In one embodiment, said pharmaceutical composition comprises a BLIMP- 1 inhibitor.

In one embodiment, the pharmaceutical composition comprises the inhibitor of BLIMP- 1 gene expression as described hereabove. In one embodiment, the pharmaceutical composition comprises the inhibitor of BLIMP- 1 protein expression as described hereabove.

In another embodiment, the pharmaceutical composition comprises the BLIMP- 1 antagonist as described hereabove. In another embodiment, said pharmaceutical composition comprises a BLIMP- 1 activator.

In one embodiment, the pharmaceutical composition comprises the activator of BLIMP- 1 gene expression as described hereabove. In one embodiment, the pharmaceutical composition comprises the activator of BLIMP- 1 protein expression as described hereabove.

In another embodiment, the pharmaceutical composition comprises the BLIMP- 1 agonist as described hereabove.

In another embodiment, the pharmaceutical composition comprises a modulator of BLIMP- 1 activity. In another embodiment, the pharmaceutical composition comprises a modulator of the activity of the biological effects induced by BLIMP- 1 as described hereabove.

In one embodiment, said modulator is an inhibitor of BLIMP- 1 activity. In another embodiment, said modulator is an inhibitor of the activity of the biological effects induced by BLIMP- 1 as described hereabove. In another embodiment, said modulator is an activator of BLIMP- 1 activity. In another embodiment, said modulator is an activator of the activity of the biological effects induced by BLIMP- 1 as described hereabove.

In one embodiment of the invention, the pharmaceutical composition may further comprise pharmaceutically acceptable excipients. Suitable pharmaceutically acceptable excipients include but are not limited to: solvents and dispersion media containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils, such as, for example, peanut oil and sesame oil; isotonic agents, such as, for example, sugars or sodium chloride; coating agents, such as, for example, lecithin; agents delaying absorption, such as, for example, aluminum monostearate and gelatin; preservatives, such as, for example, benzethonium chloride, chlorobutanol, thimerosal and the like; buffers, such as, for example, boric acid, sodium and potassium bicarbonate, sodium and potassium borates, sodium and potassium carbonate, sodium acetate, sodium biphosphate and the like; tonicity agents, such as, for example, dextran 40, dextran 70, dextrose, glycerin, potassium chloride, sodium chloride; antioxidants and stabilizers, such as, for example, sodium bisulfite, sodium metabisulfite, sodium thiosulfite, thiourea and the like; nonionic wetting or clarifying agents, such as, for example, polysorbate 80, polysorbate 20, poloxamer 282 and tyloxapol; viscosity modifying agents, such as, for example dextran 40, dextran 70, gelatin, glycerin, hydroxyethylcellulose, hydroxmethylpropylcellulose, lanolin, methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulose; and the like. In one embodiment of the invention, the pharmaceutical composition may further comprise one or more other active agent for treating HIV infection.

In one embodiment of the invention, the BLIMP- 1 modulator (the BLIMP- 1 inhibitor or the BLIMP- 1 activator) may be co-formulated with one or more other active agent for treating HIV infection. In another embodiment, the pharmaceutical composition of this invention may be administered alone or in combination with effective amounts of one or more other active agent for treating HIV infection, surgical operation, hormonal therapy, a drug and/or biological response controllers.

Examples of antiretrovirals against HIV infection, immunomodulators, anti-infectives, or vaccines, include, but are not limited to, those in the following tables:

Drug name Manufacturer Indication

097 Hoechst Bayer HIV infection, AIDS, ARC (non- nucleoside reverse transcriptase (RT) inhibitor)

Amprenavir Glaxo Wellcome HIV infection, AIDS, ARC (protease

141 W94 inhibitor)

GW 141

Abacavir (1592 U89) Glaxo Wellcome HIV infection, AIDS, ARC (RT inhibitor)

GW 1592

Acemannan Carrington Labs ARC

(Irving, TX)

Acyclovir Burroughs Wellcome HIV infection, AIDS, ARC

AD-439 Tanox Biosystems HIV infection, AIDS, ARC AD-519 Tanox Biosystems HIV infection, AIDS, ARC

Adefovir dipivoxil Gilead Sciences HIV infection

AL-721 Ethigen (Los Angeles, HIV positives, AIDS

CA)

Alpha interferon Glaxo Wellcome Kaposi's sarcoma, HIV in combination w/Retrovir

Ansamycin Adria Laboratories ARC

LM 427 Dublin (OH) Antibody which Advanced Biotherapy

Erbamont (Stamford, CT) AIDS, ARC Neutralizes pH Concepts

Labile alpha aberrant (Rockville, MD)

Antibody which neutralizes Advanced Biotherapy AIDS, ARC

pH Labile alpha aberrant Concepts

interferon

AR177 Aronex Pharm HIV infection, AIDS, ARC

Beta-fluoro-ddA Nat 1 Cancer Institute AIDS-associated diseases

BMS-234475 Bristol-Myers Squibb/ HIV infection

CGP-61755 Novartis AIDS, ARC (protease inhibitor)

CI-1012 Warner-Lambert HIV-1 infection

Cidofovir Gilead Science CMV retinitis, herpes, papillomavirus

Curdlan sulfate AJI Pharma USA HIV infection

Cytomegalovirus Immune Medlmmune CMV retinitis

globin

Cytovene Syntex Sight threatening

Ganciclovir

CMV peripheral CMV retinitis

Darunavir Tibotec-J & J HIV infection, AIDS, ARC (protease inhibitor)

Delaviridine Pharmacia-Upj ohn HIV infection, AIDS, ARC (RT inhibitor)

Dextran Sulfate Ueno Fine Chem. AIDS, ARC, HIV asymptomatic

Ind. Ltd. (Osaka, positive

Japan)

ddC Hoffman-La Roche HIV infection, AIDS

Dideoxycytidine ARC

ddl Bristol-Myers Squibb HIV infection, AIDS

Dideoxyinosine ARC; combination with AZT/d4T

DMP-450 AVID HIV infection, AIDS, ARC (protease

(Camden, NJ) inhibitor)

Efavirenz Bristol Myers Squibb HIV infection,

(DMP 266, Sustiva®) AIDS, ARC

(-)6-Chloro-4-(S)- (non- (non-nucleoside reverse transcriptase nucleoside RT inhibitor)

cyclopropylethynyl- inhibitor) 4(S)-trifluoro- methyl- 1 ,4-dihydro- 2H- 3,1-benzoxazin- 2-one,

STOCRINE

EL10 Elan Corp, PLC HIV infection

(Gainesville, GA)

Etravirine Tibotec/J & J HIV infection, AIDS, ARC (non- nucleoside reverse transcriptase inhibitor) Famciclovir Smith Kline herpes zoster, herpes simplex

GS 840 Gilead HIV infection, AIDS, ARC (reverse transcriptase inhibitor)

HBY097 Hoechst Marion Roussel HIV infection, AIDS, ARC (non- nucleoside reverse transcriptase inhibitor)

Hypericin VIMRx Pharm. HIV infection, AIDS, ARC

Recombinant Human Triton Biosciences AIDS, Kaposi's

Interferon Beta

(Almeda, CA) sarcoma, ARC

Interferon alfa-n3 Interferon Sciences ARC, AIDS

Indinavir Merck HIV infection, AIDS, ARC, asymptomatic

HIV positive, also in combination with AZT/ddl/ddC

ISIS 2922 ISIS Pharmaceuticals CMV retinitis

KNI-272 Nat'l Cancer Institute HIV-assoc. diseases

Lamivudine, 3TC Glaxo Wellcome HIV infection, AIDS, ARC (reverse transcriptase inhibitor); also with AZT

Lobucavir Bristol-Myers Squibb CMV infection

Nelfinavir Agouron Pharmaceuticals HIV infection, AIDS, ARC (protease inhibitor)

Nevirapine Boeheringer HIV infection, AIDS, ARC (RT inhibitor)

Ingleheim

Novapren Novaferon Labs, Inc. HIV inhibitor

(Akron, OH)

Peptide T Peninsula Labs AIDS

Octapeptide Sequence (Belmont, CA)

Trisodium Astra Pharm. Products, CMV retinitis, HIV infection, other CMV Phosphonoformate Inc. infections

PNU- 140690 Pharmacia Upjohn HIV infection, AIDS, ARC (protease inhibitor)

Probucol Vyrex HIV infection, AIDS

RBC-CD4 Sheffield Med. Tech HIV infection, AIDS, ARC

(Houston, TX)

Ritonavir Abbott HIV infection, AIDS, ARC (protease inhibitor)

Saquinavir Hoffmann-LaRoche HIV infection, AIDS, ARC (protease inhibitor)

Stavudine; d4T Bristol-Myers Squibb HIV infection, AIDS, ARC

Didehydrodeoxy-

Thymidine

Tipranavir Boehringer Ingelheim HIV infection, AIDS, ARC (protease inhibitor)

Valaciclovir Glaxo Wellcome Genital HSV & CMV infections

Virazole Viratek/ICN (Costa Mesa, asymptomatic HIV positive, LAS, ARC Ribavirin CA)

VX-478 Vertex HIV infection, AIDS, ARC

Zalcitabine Hoffmann-LaRoche HIV infection, AIDS, ARC, with AZT

Zidovudine; AZT Glaxo Wellcome HIV infection, AIDS, ARC, Kaposi's sarcoma, in combination w/ other therapies Tenofovir disoproxil, Gilead HIV infection, AIDS, (reverse fumarate salt (Viread ®) transcriptase inhibitor)

Emtriva® (Emtricitabine) Gilead HIV infection, AIDS, (reverse (FTC) transcriptase inhibitor)

Combivir® GSK HIV infection, AIDS, (reverse transcriptase inhibitor)

Abacavir succinate (or GSK HIV infection, AIDS, (reverse Ziagen ®) transcriptase inhibitor)

Reyataz® Bristol-Myers Squibb HIV infection, AIDs, protease inhibitor (or atazanavir)

Fuzeon® Roche/Trimeris HIV infection, AIDs, viral Fusion

(Enfuvirtide or T-20) inhibitor

Lexiva® (or GSK/Vertex HIV infection, AIDs, viral protease Fosamprenavir calcium) inhibitor

Selzentry Pfizer HIV infection, AIDS (CCR5 antagonist in

Maraviroc (UK 427857) development)

Trizivir® GSK HIV infection, AIDS (three drug combination)

Sch-417690 (vicriviroc) Schering-Plough HIV infection AIDs, (CCR5 antagonist, in development)

TAK-652 Takeda HIV infection AIDs, (CCR5 antagonist, in development)

GSK 873140 GSK/ONO HIV infection, AIDs, (CCR5 antagonist, (ONO-4128) in development)

Integrase inhibitor MK- Merck HIV infection, AIDS

0518

Raltegravir Truvada® Gilead Combination of Tenofovir disoproxil fumarate salt (Viread®) and Emtriva® (Emtricitabine)

Integrase Inhibitor Gilead/Japan Tobacco HIV Infection, AIDs

GS917/JTK-303

Elvitegravir in development

Triple drug combination Gilead/Bristol-Myers Combination of Tenofovir Atripla ®

Squibb disoproxil fumarate salt (Viread®),

Emtriva® (Emtricitabine), and Sustiva ® (Efavirenz)

Festinavir® Oncolys BioPharma HIV infection AIDs in development

CMX-157 Chimerix HIV infection, AIDs

Lipid conjugate of

nucleotide tenofovir

GSK1349572 GSK HIV infection, AIDs

Integrase inhibitor

Voronistat (HDAC Merck HIV latent infection

inhibitor)

NCH-51 (histone Merck HIV latent infection

deacetylase inhibitor

Dacogen (DNA Janssen HIV latent infection

methyltransferase inhibitor)

Table 1: antiretrovirals Drug name Manufacturer Indication

AS-101 Wyeth-Ayerst AIDS

Bropirimine Pharmacia Upjohn Advanced AIDS

Acemannan Carrington Labs ARC

(Irving, TX)

CL246J38 Wyeth Lederle Labs AIDS, Kaposi's sarcoma

FP-21399 Fuki ImmunoPharm Blocks HIV fusion with CD4+ cells

Gamma interferon Genentech ARC in combination w/TNF

Granulocyte Genetics Institute Sandoz, AIDS

Macrophage Colony

Stimulating Factor

Granulocyte Hoechst-Roussel AIDS

Macrophage Colony Immunex

Stimulating Factor

Granulocyte Schering-Plough AIDS, combination Stimulating Factor

Macrophage Colony w/AZT

Stimulating Factor

HIV Core Particle Rorer Seropositive HIV Immunostimulant

IL2 receptor antibody Novartis AIDS, in combination w/AZT

Basiliximab

IL2 receptor antibody Hoffmann-La Roche AIDS, in combination w/AZT

Daclizumab

Immune Globulin Cutter Biological AIDS, in combination w/AZT

Intravenous (human) Pediatric(Berkeley, CA)

IMREG-1 Imreg (New Orleans, LA) AIDS, Kaposi's sarcoma, ARC, PGL

IMREG-2 Imreg (New Orleans, LA) AIDS, Kaposi's sarcoma, ARC, PGL

Imuthiol Diethyl Merieux Institute AIDS, ARC

Dithio Carbamate Schering Plough Kaposi's sarcoma w/AZT, AIDS

Alpha-2 Interferon

Methionine- Enkephalin TNI Pharmaceutical AIDS, ARC

(Chicago, IL)

MTP-PE Muramyl- Ciba-Geigy Corp. Kaposi's sarcoma

Tripeptide

Granulocyte Colony Amgen AIDS, in combination w/AZT

Stimulating Factor

PD1 antibody Bristol-Myers Squibb, AIDS, in combination w/AZT

BMS-936558, MDX- Ono Pharmaceuticals

1106

PDL1 antibody Bristol-Myers Squibb, AIDS, in combination w/AZT

MDX-1105 Medarex

BMS-936559

PDL1 antibody Genentech AIDS, in combination w/AZT

MPDL3280A

Remune Immune Response Corp. Immunotherapeutic

rCD4 Recombinant Genentech AIDS, ARC

Soluble Human CD4

rCD4-IgG hybrids AIDS, ARC

Recombinant Biogen AIDS, ARC

Soluble Human CD4 Interferon alfa 2a Hoffmann-La Roche Kaposi's sarcoma, AIDS, ARC, in combination w/AZT

SK&F106528 Smith Kline HIV infection

Soluble T4

Thymopentin Immunobiology Research HIV infection

Institute (Annandale, NJ)

Tumor necrosis factor Genentech ARC in combination w/gamma interferon (TNF)

Table 2: Immunomodulators

Table 3: Anti-infectives

In the scope of combinations of the pharmaceutical composition of this invention with HIV antiretrovirals, immunomodulators, anti-infectives, HIV entry inhibitors or vaccines, these one are not limited to the lists in the above Tables but include, in principle, any combination with any pharmaceutical composition useful for the treatment of HIV infection.

Without willing to be bound to a theory, the inventors suggest that inhibiting BLIMP- 1 will lead to an induction of HIV provirus expression, therefore allowing HIV antiretrovirals, immunomodulators and/or anti-infectives to be more efficient.

Without willing to be bound to a theory, the inventors suggest that activating BLIMP- 1 will lead to a repression of HIV provirus expression, therefore allowing treatment of HIV infection and/or HIV cure. In one embodiment, preferred combinations with the composition according to the invention include but is not limited to BLIMP- 1 inhibitor and antiretrovirals, BLIMP- 1 inhibitor and HDAC I inhibitors and antiretrovirals, BLIMP- 1 inhibitor and demethylating agent and antiretrovirals.

In one embodiment, preferred combinations with the composition according to the invention include but is not limited to BLIMP- 1 activator and antiretrovirals, BLIMP- 1 activator and HDAC I inhibitors and antiretrovirals, BLIMP- 1 activator and demethylating agent and antiretrovirals.

In one embodiment, the form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

In another embodiment, the pharmaceutical compositions of the present invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

According to one embodiment of the invention, the administration dose of the pharmaceutical composition is determined by the skilled artisan and personally adapted to each subject.

In one embodiment, the pharmaceutical composition of the present invention and other active agents may be administered separately or in conjunction. In another embodiment, the administration of one element may be prior to, concurrent to, or subsequent to the administration of other agent(s).

In another embodiment, the pharmaceutical composition of the present invention may be administered to a subject in periods of pre-exposure and/or post-exposure with antiretrovirals, immunomodulators and/or anti-infectives agents.

In one embodiment, the composition for use according to the invention is for preventing, ameliorating or healing HIV infection, i.e. reaching an HIV cure as defined by the disappearance or almost disappearance of all HIV-DNA copies, or a functional cure of HIV that is the decrease of the HIV reservoirs down to levels so low that the virus production will be subsequently maintained at very low levels and for prolonged periods of time after treatment interruption.

In one embodiment, the composition for use according to the invention is for preventing, ameliorating or healing the symptoms associated with HIV infection. In one embodiment, the amelioration or heal of a symptom corresponds to a diminution or eradication of HIV pro virus expression in CD4 ⁺ T cells.

In one embodiment, the composition for use according to the invention is for curing HIV infection.

In a preferred embodiment, the amelioration or heal of a symptom corresponds to both a decrease in the number of occurrence of said symptom and in a decrease in the intensity of said symptom.

The skilled artisan knows how to evaluate the efficacy of a treatment by measuring the HIV pro virus expression, the HIV mRNA transcripts in CD 4 ⁺ T cells or the production of HIV on the basis of the HIV viremia. In one embodiment of the invention, the subject is a mammal and preferably a human. In one embodiment of the invention, the subject is a female. In another embodiment of the invention, the subject is a male.

In another embodiment of the invention, the subject is affected, preferably is diagnosed with HIV. In another embodiment of the invention, the subject is an LTNP subject.

In another embodiment of the invention, the subject is an EC subject.

In another embodiment of the invention, the subject is an E-LTNP subject.

In another embodiment, the subject is an HIV-infected viremic subject.

In another embodiment, the subject is a LTNP, EC, E-LTNP, or HIV-infected subject treated with antiretrovirals. In another embodiment, the subject is an HIV-infected subject treated with antiretrovirals, with or without undetectable viremia.

In another embodiment, the subject has a BLIMP- 1 overexpression in its CD4+ T _CM subset cells. In another embodiment, the subject is an E-LTNP subject having a BLIMP- 1 overexpression in its CD4+ T _CM subset cells.

In another embodiment, the subject has a BLIMP- 1 underexpression in its CD4+ T _CM subset cells.

The present invention also relates to a method for treating and/or curing viral infections such as HIV infection in a subject in need thereof, wherein said method comprises administering to the subject a therapeutically effective amount of a modulator (inhibitor or activator) of BLIMP- 1.

The present invention also relates to a method for preventing, ameliorating, or healing HIV infection characterized by inhibition of the HIV infection in a subject in need thereof, wherein said method comprises administering to the subject a therapeutically effective amount of a BLIMP- 1 modulator (inhibitor or activator).

The BLIMP- 1 modulator (inhibitor or activator) used in the methods of the present invention are as defined above. In particular, it can be an inhibitor of BLIMP- 1 gene expression, an inhibitor BLIMP- 1 protein expression or an inhibitor that blocks any biological activity and/or downstream biological effects of BLIMP- 1. It can also be an activator of BLIMP- 1 gene expression, an activator of BLIMP- 1 protein expression or an activator that stimulates any biological activity and/or downstream biological effects of BLIMP- 1. In one embodiment, the method for treating HIV infection comprises the administration alone of the pharmaceutical composition.

In another embodiment, the method for treating HIV infection comprises the administration of the pharmaceutical composition prior to, concurrent to, or subsequent to the administration of other agent(s), such as the antiretrovirals, immunomodulators, anti-infectives as described here above. The present invention also relates to personalized medicine in particular to the use of BLIMP- 1 as a biomarker.

In one embodiment of the invention, BLIMP- 1 is a biomarker or a companion biomarker to determine or predict whether a subject is in need of a treatment against HIV infection.

In one embodiment of the invention, BLIMP- 1 is a biomarker companion to monitor a treatment against HIV infection administered in a subject in need thereof.

Another object of the invention is an in vitro companion diagnostic device comprising BLIMP- 1. Another object of the invention is a method for predicting responsiveness to treatment against HIV infection with a BLIMP- 1 modulator.

Another object of the invention is a method for predicting responsiveness to treatment against HIV infection with a BLIMP- 1 inhibitor, comprising determining in the subject to be treated whether there is a high BLIMP- 1 expression. Another object of the invention is a method for predicting responsiveness to treatment against HIV infection with a BLIMP- 1 activator, comprising determining in the subject to be treated whether there is a low in levels of BLIMP- 1 expression.

The high or low level of BLIMP- 1 may be determined by comparison with the level ranges of BLIMP- 1 associated with HIV control or lack of HIV control to reference/control CD4 ⁺ T cell samples from HIV-infected patients, and to reference/control uninfected CD4 ⁺ T cell samples.

Another object of the invention is a method for use of BLIMP- 1 inhibitor in combination with antiretrovirals for treating HIV infection wherein said inhibitor increases HIV provirus expression. Another object of the invention is a method for use of BLIMP- 1 activator in combination or not with antiretrovirals wherein said activator decreases HIV provirus expression.

The present invention also relates to a personalized treatment of HIV infection in a subject, comprising :

a. isolating CD4 ⁺ T cells or memory subsets from a blood sample obtained from the subject,

b. measuring BLIMP- 1 and optionally HIV provirus expression in at least one of the CD4 ⁺ T cells subsets, preferably CD4 ⁺ T _CM cells, wherein the level of BLIMP- 1 expression in the CD4 ⁺ T cells, preferably T _CM cells, determines the treatment of the subject with a BLIMP- 1 modulator.

Another object of the invention is a method for monitoring the efficacy of the HIV infection treatment with a BLIMP- 1 modulator and antiretrovirals comprising :

a. isolating CD4 ⁺ T cells or memory subsets from a blood sample obtained from the subject,

b. measuring HIV provirus expression in CD4 ⁺ T cells or at least one of the memory CD4 ⁺ T cells subsets, preferably CD4 ⁺ T _CM cells, wherein a lower level of ΗΓ provirus expression in the CD4 ⁺ T _CM cells compared to the level of HIV provirus expression before treatment is indicative of a positive outcome of the treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a histogram showing a distribution of CD4 ⁺ T cell subsets in UI, E-LTNP, VH (% of all studied CD4 ⁺ T cell subsets). Figure 2 is a histogram showing the distribution of the HIV reservoirs in the patients from the study group. It represents the levels of total ΗΓ DNA as measured by PCR in each purified CD4 ⁺ T cell subset from VH and E-LTNP patients. Figure 3 shows a number of differentially expressed genes between T _CM , _TM and T _EM from UI, E-LTNP and VH patients as compared to T from UI.

Figure 4 is a histogram showing a heatmap and clustering analysis based on the expression of 38 genes that differentiate T _N , T _CM , T _TM and T _EM , regardless of the patient' s status (UI, E-LTNP or VH) .

Figure 5 represents histograms of the levels of several signatures in the different CD4 ⁺ T cell subsets from E-LTNP compared to uninfected T _N cells.

Figure 6 represents graphs showing a correlation between PRDMl expression intensity in T _CM and total HIV DNA contents in PBMCs of HIV infected aviremic LTNPs. Figure 7 represents graphs showing quantitative RT-PCR analysis of (A) BLIMP- 1 gene expression. The log2 values of the ratios calculated between infected E-LTNP and VH patients and uninfected donors in subpopulations of CD4 ⁺ T cells are shown. In (B), unspliced form of HIV- 1 RNA in subpopulations of E-LTNP and subpopulations of VH patients are shown.

EXAMPLES

The present invention is further illustrated by the following examples. Materials and Methods The study group: The study group included 13 untreated LTNP selected from the previously described ANRS CO 15 cohort (Magierowska Blood 1999) initially recruited on the basis of an asymptomatic HIV infection for at least 8 years and CD4 ⁺ T cell counts above 600/mm ³ in the absence of antiretroviral therapy. The patients were separated into 2 groups according to the HIV DNA contents in total peripheral blood mononuclear cells (PBMC) and to the plasmatic viremia at the study time point. Six patients met the definition of HIV controllers (Lambotte CID 2005) and were classified as "Elite LTNP" (E-LTNP) on the basis of a plasma HIV-RNA viral load below 400/mm (mean, 167 copies/mm ³ , 107-299) and extremely low levels of HIV DNA in total PBMC (below 100 copies/10 ⁶ PBMC, mean, 42 copies/10 ⁶ PBMC, 5-85), as measured by real-time polymerase chain reaction (RT-PCR). The 7 remaining patients were classified as viremic HIV-infected patients (VH); they significantly differed from the E-LTNP group only by plasma HIV-RNA viral loads (mean, 14351 copies/ml, 934-54000, p=0.003) and cell-associated HIV-DNA rates in total PBMC (mean, 358 copies/10 ⁶ PBMC, 117- 802, p=0.03). Seven uninfected blood donors (UI, uninfected individuals) were also included (Table 4).

PI ELTNP M,40 27 38 11 None 299 828 5

P10 ELTNP M,33 44 14 23 None 112 690 68

P9 ELTNP F,69 51 44 11 None 154 1011 25

P12 ELTNP M,37 14 40 9 None 223 790 85

P3 ELTNP F,43 14 27 11 None 107 807 33

Pl l ELTNP M,59 08 35 10 None 107 565 33

Mean

47 13 167 782 42 (ELTNP)

P2 VH M,61 07 57 20 None 934 317 275

VH

P4 M,44 50 57 15 None 1015 1428 152

VH

P6 F,35 05 08 11 None 1075 392 117

VH

P8 F,35 14 18 12 None 10147 623 463

VH

P5 M,31 35 14 14 None 54000 949 461

VH

P13 M,35 51 52 10 None 28000 544 217

VH

P14 M,45 44 49 10 None 5286 590 802

Mean

41 13 14351 692 358 (VH)

P 0.51 0.56 0.003 0.29 0.03 Table 4. Clinical, genetic and immunovirological parameters of the patients in the study group. E-LTNP: Elite Long-Term Nonprogressors, F: female, HAART: highly active antiretroviral therapy, M:male, PBMC=peripheral blood mononuclear cells, VH: viremic HIV-infected patient, yrs=years. p=p- values, nonparametric Mann-Whitney U- test, between VH and E-LTNP. *heterozygous mutation

Microarray experiments

Total RNA was purified from each sorted CD4 ⁺ T cell subset and treated with DNAse (RNAqueous kit, Ambion) according to the manufacturer's protocol. The purity of all RNA samples was checked with an ND-1000 spectrophotometer (NanoDrop Technologies). Total RNA was reverse-transcribed and amplified using SPIA amplification (Ovation PicoSL WTA System V2, NuGEN Technologies) and biotin- labeled (Encore BiotinIL Module Module, NuGEN Technologies). cDNA samples were purified on a filtration column (MinElute PCR Purification Kit, Qiagen). The integrity of all cDNA samples was checked by electrophoresis on a 2100 Bio Analyzer (Agilent Technologies). 750 ng of biotinylated cDNA from each sample were hybridized on niumina® HT-12 V4 Expression BeadChip, stained with streptavidin-Cy3 and washed. Arrays were scanned on iScan System and fluorescence intensities associated to each probe were converted into numerical data using GenomeStudio™ Data Analysis Software.

Microarray data analysis

Fluorescence intensities were background corrected and quantile normalized using negative and positive control probes according to the NormExp Background correction and Normalization method (neqc) provided in the Lumi package of R. Fluorescence intensity data were log2 transformed.

Gene filtering and ponderation, quality controls Samples that were performed in duplicates clustered together on principal component analysis. Differentially expressed genes were calculated between groups using the Ebayes method.

Example 1: Isolation of CD4 ⁺ T cell subpopulations Live resting CD4 ⁺ subsets were sorted using a FACSAria flow cytometer (BD Biosciences) in a L3 facility. Cryopreserved peripheral blood mononuclear cells (PBMC) from study persons were thawed and stained with an amine-reactive fluorescent viability dye (Life Technologies) and with the following monoclonal antibodies: anti-CD3-PB, anti-CD4-Alexa700, anti-CD69-FITC, anti-HLA-DR-FITC (Becton Dickinson), anti-CD25-FITC, anti-CD45RA-ECD (Beckman Coulter), anti- CCR7-PE-Cy7, anti-CD27-APC (Becton Dickinson). The resting (CD25 ^" , CD69 ^" , HLA- DR ^" ) CD4 ⁺ T cells (mean, 92% of total CD4 ⁺ T cells, 83-96) were sorted as resting CD4 ⁺ T naive (T _N : CD45RA ⁺ CCR7 ⁺ CD27 ⁺ ) and memory cells, including central (T _CM : CD45RA ^" CCR7 ⁺ CD27 ⁺ ), transitional (T™: CD45RA ^" CCR7 ^" CD27 ⁺ ), and effector memory (T _EM : CD45RA CCR7 CD27 ) cells.

Example 2: characterization of CD4 ⁺ T cells subsets

First, the distribution of CD4 ⁺ T cell subsets was studied in the 3 conditions (UI, E- LTNP and VH). In HIV-infected patients as compared to UI, the naive compartment was reduced (46% in VH, 30% in E-LTNP and 67% in UI, p=0.02) whereas the memory compartment (T _CM , T _TM and T _EM ) was more represented in proportion of all studied CD4 ⁺ T cells subsets (Figure 1). In particular, T _CM were significantly higher in E-LTNP (39%), in percentage of all studied CD4 ⁺ T cells, than in UI (23%, p=0.01) and VH patients (p=0.01).

As shown in Figure 2, T™ and T _CM were the main contributors to the HIV reservoir in resting CD4 ⁺ T cells in VH and E-LTNP patients. In particular, in E-LTNP, the central memory CD4 ⁺ T cell subset represented 43% of the total HIV DNA in resting CD4 ⁺ T cells, and was therefore the most infected CD4 ⁺ T cell subset. Example 3: Transcriptome analysis in CD4 T ⁺ cell from UI, ELTNP and VH patients

According to Figure 3, there is a very strong similarity, within each subset, between the three groups of patients with no differentially expressed gene (DEG) using a false- discovery rate (FDR-adjusted p-value below 0.05). We then compared the transcriptional profiles of each resting memory CD4 ⁺ T cell subset to a single comparator, i.e. the naive CD4 ⁺ T cells from UI (UI-T _N ) as the single reference parameter. In UI, as expected, a gradient of differences followed the differentiation pathways. Among the two LTNP sub-groups however this observation was true only in Vir-LTNPs with T _EM more different from T than T _TM , T _TM more different from T than T _CM while the T _CM had the closest transcriptome to naive cells in UI and Vir-LTNPs among all subsets of memory cells. In contrast in VL, the T _CM were the most different subset from T with: 255 DEG in TC _M , 101 DEG in T _TM and 182 DEG in T _EM as compared to UTT _N . Altogether the T _EM from Vir-LTNPs had the most distant transcriptome from UI-T _N with 373 DEG differing from VH UI-T _N , while only 182 DEG differed between TEM from E-LTNP and UI-TN, and 106 DEG between UI-T _EM and UI-T _N . These observations suggest that in this setting, HIV reservoirs and plVL were major factors affecting the effector memory CD4 ⁺ T cells differentiation at the transcriptional level. Example 4: Transcriptome analysis in CD4 ⁺ T cell subsets from UI, ELTNP and VH patients

When analyzing in more details the transcriptional signature of each resting CD4 ⁺ T cell subset, as shown in the heat-map analysis (Figure 4). We observed a segregation of most genes according to the differentiation stage, with 9, 5 and 35 genes always overexpressed in T _CM , T _TM and T _EM respectively compared to the UI-T _N . A group of 4 genes associated with (FAM129A, MAF, KLRB 1, ITGB1) was overexpressed in each memory subset (T _CM , T _TM and T _EM ) from the 3 study sub-groups regardless of the patient's condition. Interestingly compared to UI-T _N the CCR4 and CCR6 genes were also over-expressed in T _CM , CCL5 in both T _TM and T _EM , the ANXA2, ANXA2P1 and LINC0052 in both T _CM and T _EM , while a series of 30 other genes were overexpressed in T _EM versus T in all of the 3 conditions. Most of these T _EM 35 genes encoded a protein whose function was related to cell-to-cell adherence, cell cytotoxicity or intracellular signaling.

Example 5: analysis of the transcriptional signature of T _rM cells in ELTNPs patients

Next, as T _CM from E-LTNP patients were highly represented among all CD4 ⁺ T cell subsets (Figure 1), as they were the main contributors to the HIV reservoir in these patients (Figure 2), and as their transcriptome was very different from that of naive cells from UI (Figure 3) we focused on the genes that were specific to the T _CM of E- LTNP patients. To this purpose, we made the following comparisons:

-T _CM E-LTNP versus T _N UI,

-TC _M E-LTNP versus T _CM UI,

-TC _M VH versus T _N UI,

-TC _M UI versus T _N UI,

-T _CM VH versus T _N VH.

The 151 genes that were differentially expressed in the 2 first comparisons and not differentially expressed in the 3 others were considered "specific" to T _CM from E-LTNP patients. Indeed, if they were characteristic of the HIV infection, they would be differentially expressed in the third comparison, and if they were characteristic of the CD4 ⁺ T cell subset, they would be differentially expressed in the latter two comparisons.

151 DEG were found specific for T _CM E-LTNP as differing from both UI-T _N (FDR adjusted p-value<0.05) and UI-T _CM (unadjusted p-value<0.05) but not in the 3 other comparisons. These 151 DEG defined 3 main signatures for the E-LTNP T _CM (Figure 5) involving an upregulation of (i) TCR and costimulation signaling genes and (ii) PRDM1 (BLIMP-1), but a downregulation of (iii) the IFN- stimulated genes as compared to T _CM from UI and from VH, though to a lesser extent.

The first signature among the 151 "E-LTNP-specific genes in T _CM ', was composed of 5 genes related to TCR and costimulation signaling. Four of them were upregulated in T _CM E-LTNP as compared to UI and VH. Three genes showed a 2 to 3 fold upregulation compared to UI-T _CM and up to 2 fold compared to Vir-LTNPs including the PIK3R1, (phosphoinositide-3-kinase, regulatory subunit 1) which binds to activated phosphorylated protein-Tyr kinases and plays a role in TCR, growth factor receptors and costimulation (CD28, CTLA4) signaling, CD3E, part of the CD3 multimeric complex, SH3KBP1 i.e. the SH3-domain kinase binding protein- 1, involved in TCR signaling. A fourth gene, the Protein tyrosine phosphatase C (PTPRC) required for T- cell activation through the TCR and positive regulator of T-cell coactivation was only 1.5 to 1.8 fold up-regulated compared to both UI and Vir-LTNP T _CM - The fifth gene coding for the phosphodiesterase 4D (PDE4D) induced by TCR engagement in CD4 ⁺ T cells, activates IL-2 synthesis and regulates the cellular levels, localization and duration of action of the cAMP and/or cGMP second messengers, was down -regulated in T _CM from E-LTNP compared to UI and Vir-LTNP.

The second signature was composed of the PRDMl gene, was 3.7 and 3 fold upregulated in T _CM from ELTNP compared to T _CM from VH and from UI T _CM

-7 -5

(unadjusted p=10 ^~ and 6.10 ^" , respectively), as well as in T _TM from ELTNP, though only 2.5 and 2.6 fold upregulated, as compared to T _TM from VH and from UI (unadjusted p=10 ^"3 and 3.10 ^"4 , respectively). Interestingly PRDMl encodes a transcription factor (BLIMP- 1) initially shown to bind the interferon gene regulatory element in the beta-interferon promoter and to negatively regulate the expression of beta-interferon in fibroblasts. In addition PRDMl negatively regulates IL-2, IL-10 and IFN-gamma expression, as well as the T _EM CD4 ⁺ T cell differentiation into effector cells, while it regulates CD8 ⁺ T cell exhaustion during murine chronic viral infection.

The third signature among the 151 DEG specific to "E-LTNP-T _CM '\ was composed of 0 other genes related to interferon-signaling that were all significantly (p<0.05) down- regulated in T _CM from E-LTNP as compared to T _CM from UI (and to T _CM from VH for 3 of them). Among them 9 are interferon-stimulated genes (ISG) including IFIT3, OAS1, OASL, HIF1A, FTSJD2, IFI6, IFIT2, PYHIN1=IFIX and IFI44, whereas the IFNAR genes (encoding the type-I IFN-receptors) were not differentially expressed nor were the genes encoding type-I IFN, JAK1/2 or STATs. In addition the DDX21 which encodes a RNA-helicase sensing double- stranded RNA in dendritic cells and activates the type-I IFN pathway was also significantly down-regulated. This "Antiinflammatory" T cell profile characterized by the downregulation of type-I ISG in E- LTNPs compared to UI could seem paradoxical but similar patterns have been found in the nonpathogenic model of SIV infection, i.e. the SIV-infected African green monkeys compared to SIV-infected Rhesus macaques.

Example 6: BLIMP-1 and HIV levels in CD4 ⁺ T cells subsets

Among the signatures defined above in T _CM from the E-LTNPs compared to the VH we found that the PRDMl expression in T _CM was negatively correlated to the total HIV DNA content in the sorted CD4 ⁺ T _CM subset from both E-LTNP and Vir-LTNPs ( Figure 6). Three PRDMl gene isoforms (3840113, 5260392, 6220288) were negatively correlated to the HIV-DNA copy numbers in the T _CM subsets (r=-0.538, -0.610, -0.492; unadjusted p=0.0158, 0.0070, 0.022, respectively). We thus quantified in parallel by RT-PCR the amounts of PRDM-1 genes and the cell- associated, unspliced form of HIV-1 gag RNA in the different CD4 ⁺ T cell subsets sorted from E-LTNPs and VH PBMCs. RNA samples from each sorted CD4 ⁺ T cell subset used for the microarrays were pooled and then analyzed by quantitative RT-PCR (qRT-PCR) experiments. Single- stranded complementary DNA (cDNA) was synthesized from 100 ng of total RNA using the High Capacity cDNA Reverse Transcription Kit (AB Applied Biosystem), according to the manufacturer's instructions. Quantitative PCR was performed on a 7300 Real-Time PCR System (Applied Biosystems), using Mesa Green qPCR Master Mix Plus (Eurogentec). The cycling conditions were 5 min at 95°C, 40 cycles of 5 sec at 95°C, 1 min at 60°C. Relative quantification by RT-PCR provides accurate comparison between the initial levels of cDNA in each sample, but requires an endogenous control to correct for differences in RNA input. The RPS14 transcript mRNA was chosen as an endogenous control because it had the lowest standard deviation calculated between all the different T cell subset, and therefore it was used as an endogenous control in relative quantification assays to determine the cell-associated HIV RNA and the PRDM1 expression. Results were analyzed using 7300 System Sequence Detection Software vl.4 and RQ Study Application (Applied Biosystems), and are presented as relative quantitation or log2 (mean ddCT infected= mean ddCT non infected), with ddCT being the fold change (relative quantity) of target mRNA, normalized to RPS14 transcript mRNA and relative to a calibrator sample.

In Figure 7, the possibility of the presence of the HIV-1 viral RNA was investigated in the CD4 ⁺ T cell subsets of the VH and E-LTNP patients, by measuring the level of the cell- associated, unspliced form of HIV RNA. Quantitative measurements of viral RNA revealed much higher levels in T _CM from VH as compared to T _CM from E-LTNP (327 copies/ 6 copies, per 100 ng of RNA), and to a lesser extent, in T _TM from VH as compared to T _TM from E-LTNP (45 copies/ 5 copies, per 100 ng of RNA) patients, suggesting that perhaps these subsets of T cells may support replication of HIV in vivo.