Methods and kits for detecting the latent HIV reservoir expressing cell-surface ENV in samples

Abstract

A significant challenge in HIV cure research is the assessment of HIV reservoir protein expression and the quantification of the HIV reservoir size. New strategies are needed to detect and eliminate these reservoirs or achieve durable viral control without lifelong cART. The present application discloses method and kits for detecting latently HIV-infected cells expressing surface Env in a biological sample using a molecule that specifically binds within the Phe43 cavity of gp120 and capable of "opening up" the Env trimer, in combination with a cocktail of three binding agents that target different regions of the HIV-1 Envelope glycoprotein. The first binding agent targets the constant region 1 and 2 (C1-C2) portion of the cluster A region of gp120, the second binding agent targets the bridging sheet of the Co-Receptor Binding Site (CoRBS) of gp120, and the third binding agent targets the cluster I region of gp41.

Description

[0001] METHODS AND KITS FOR DETECTING THE LATENT HIV RESERVOIR EXPRESSING CELL-SURFACE ENV IN SAMPLES CROSS REFERENCE TO RELATED APPLICATIONS

[0002] The present application claims the benefit of U.S. provisional patent application serial No. 63 / 745,017, filed on January 14, 2025, which is incorporated herein by reference in its entirety.

[0003] SEQUENCE LISTING

[0004] A sequence listing is submitted herewith as an XML file named G17004-00023_Seq listing.xml, created on January 13, 2026, and having a size of ~ 36,007 bytes. The content of the aforementioned file is hereby incorporated by reference in its entirety.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with Government support under NIH Grants Nos. R01 AI174908, R01AI176531, UM1AI164562 and R01 AI186809. The Government has certain rights in this invention.

[0007] TECHNICAL FIELD

[0008] The present invention generally relates to the field of virology, and more specifically to the detection of cells expressing the HIV-1 envelope (Env) glycoprotein, such as cells latently infected by the human immunodeficiency virus type 1 (HIV-1).

[0009] BACKGROUND ART

[0010] Combination antiretroviral therapy (cART) effectively controls HIV-1 replication and extends the life-expectancy of people living with HIV-1 (PLWH). However, cART neither eradicates the virus nor prevents long-term complications [1,2], HIV-1 persists in long-lived reservoirs, and the virus rebounds within days to weeks after treatment interruption [3-6], A significant challenge in HIV cure research is the assessment of HIV reservoir protein expression and the quantification of the HIV reservoir size. These measurements are difficult due to the reservoir's small size, which consists of approximately one replication-competent provirus per million cells. Although PCR assays can be used to measure HIV DNA in people living with HIV (PLWH), the majority of integrated HIV proviruses are either hyper-mutated or have large deletions. Consequently, only a very small portion of the detected HIV DNA is replication-competent and accurately reflects the true reservoir.

[0011] Therefore, new strategies are urgently needed to detect and eliminate these reservoirs or achieve durable viral control without lifelong cART.The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

[0012] SUMMARY OF THE INVENTION

[0013] In various aspects and embodiments, the present disclosure provides the following items 1 to 44:

[0014] 1. A method for detecting latently HIV-infected cells in a biological sample comprising CD4+ cells, the method comprising:

[0015] contacting the sample with:

[0016] (a) a molecule that specifically binds within the Phe43 cavity of gp120;

[0017] (b) a first gp120 binding molecule that binds to an epitope within the constant region 1 and 2 (C1-C2) portion of the cluster A region of gp120;

[0018] (c) a second gp120 binding molecule that binds to an epitope within the bridging sheet of the Co-Receptor Binding Site (CoRBS) of gp120; and

[0019] (d) a gp41 binding molecule that binds to an epitope located in the cluster I region of gp41; and

[0020] detecting a signal indicative of latently HIV-infected cells, wherein the signal is generated by the interaction of the first anti-gp120 antibody, the second anti-gp120 antibody, and / or the anti-gp41 antibody with the latently HIV-infected cells.

[0021] 2. The method of item 1, wherein the epitope within the C1-C2 portion of the cluster A region of gp120 is formed by residues 51-54, 56, 58-61, 103, 106-107, 110, 114, 217, and 219-221 of gp120.

[0022] 3. The method of item 1 or 2, wherein the first gp120 binding molecule is an antibody or an antigen-binding fragment thereof.

[0023] 4. The method of item 3, wherein the first gp120 binding molecule is antibody clone A32 or an antigen-binding fragment thereof, or an antibody that competes with antibody clone A32 or an antigen-binding fragment thereof.

[0024] 5. The method of item 4, wherein the first gp120 binding molecule is an antibody or an antigen-binding fragment thereof comprising the complementarity determining regions (CDRs) of antibody clone A32, L9-i 1 or N5-i5.

[0025] 6. The method of item 5, wherein the first gp120 binding molecule is antibody clone A32, L9-i1 or N5-i5, preferably clone A32.

[0026] 7. The method of any one of items 1 to 6, wherein the second gp120 binding molecule binds to the epitope formed by residues 119-122, 200, 202-205, 326-327, 369, 419-423 and 432-437 of gp120.

[0027] 8. The method of any one of items 1 to 7, wherein the second gp120 binding molecule is an antibody or an antigen-binding fragment thereof.9. The method of item 8, wherein the second gp120 binding molecule is antibody clone 17b or an antigen-binding fragment thereof, or an antibody that competes with antibody clone 17b or an antigen-binding fragment thereof.

[0028] 10. The method of item 9, wherein the second sgp120-binding molecule is an antibody or an antigen-binding fragment thereof comprising the complementarity determining regions (CDRs) of antibody clone 17b, X5, L9-i3, N5-i1, N5-i3, N5-i4, N5-i8, N10-i1.1 , N10-i5.3, N12-i1 , N12-i2, N12-i4, N12-i5, N12-i7, N12-i8, N12-i10, N12-i17, N12-i18, 48d, 412d, or N12-i9.

[0029] 11. The method of item 10, wherein the second sgp120-binding molecule is antibody clone 17b, X5, L9-i3, N5-i1, N5-i3, N5-i4, N5-i8, N10-i1.1 , N10-i5.3, N12-i1, N12-i2, N12-i4, N12-i5, N12-i7, N12-i8, N12-i10, N12-i17, N12-i18, 48d, 412d, or N12-i9, preferably antibody clone 17b orX5, more preferably antibody clone 17b.

[0030] 12. The method of any one of items 1 to 11 , wherein the gp41 binding molecule binds to an epitope located in a region defined by amino acids 579 to 623 of gp41.

[0031] 13. The method of item 12, wherein the gp41 binding molecule binds to an epitope located in a region defined by amino acids 579 to 613 of gp41.

[0032] 14. The method of item 12, wherein the gp41 binding molecule binds to an epitope located in a region defined by amino acids 579 to 604 of gp41.

[0033] 15. The method of any one of items 1 to 14, wherein the gp41 binding molecule is an antibody or an antigen-binding fragment thereof.

[0034] 16. The method of item 15, wherein the gp41 binding molecule is antibody clone 7B2, F240, M785-U1, N10-U1, 246D, 240D, 181D, or 98-43, an antigen-binding fragment thereof, or an antibody that competes with antibody clone 7B2, F240, M785-U1, N10-U1, 246D, 240D, 181D, or 98-43 or an antigen-binding fragment thereof.

[0035] 17. The method of item 15 or 16, wherein the gp41 binding molecule is an antibody or an antigen-binding fragment thereof comprising the complementarity determining regions (CDRs) of antibody clone antibody clone 7B2, F240, M785-U1, N10-U1, 246D, 240D, 181D, or 98-43. 18. The method of item 17, wherein the gp41 binding molecule is antibody clone 246D or an antigen-binding fragment thereof.

[0036] 19. The method of any one of items 1 to 18, wherein the molecule that specifically binds within the Phe43 cavity of gp120 is a polypeptide comprising domains 1 and 2 of human CD4 receptor or a CD4 mimetic compound (CD4mc).

[0037] 20. The method of item 19, wherein the CD4mc is NBD-556, NBD-557, DMJ-l-228, JP-lll-48, M48U1, BNM-lll-170, CJFIII-288.

[0038] 21. The method of item 20, wherein the CD4mc is CJFIII-288.

[0039] 22. The method of any one of items 1 to 21, wherein the first anti-gp120 antibody, the second anti-gp120 antibody, and / or the anti-gp41 antibody are conjugated to an affinity tag or a detectable moiety.23. The method of item 22, wherein the affinity tag is a biotin or biotin analog.

[0040] 24. The method of item 23, wherein the method further comprises contacting the sample with a streptavidin or streptavidin analog conjugated to a detectable moiety.

[0041] 25. The method of item 24, wherein the detectable moiety is a fluorophore.

[0042] 26. The method of any one of items 22 to 25, wherein the first anti-gp120 antibody and the second anti-gp120 antibody are conjugated to a first detectable moiety, and the anti-gp41 antibody is conjugated to a second detectable moiety.

[0043] 27. The method of any one of items 1 to 26, wherein the biological sample is blood, plasma, or tissue, preferably a peripheral blood mononuclear cell (PBMC) sample.

[0044] 28. The method of any one of items 1 to 27, further comprising enriching the sample in T lymphocytes prior to said contacting.

[0045] 29. The method of item 28, wherein the T lymphocytes are CD4+ T lymphocytes.

[0046] 30. The method of any one of items 1 to 29, further comprising staining the cells with one or more markers selected from CD3, CD4, and CD8.

[0047] 31. The method of any one of items 1 to 30, further comprising detecting intracellular HIV p24 protein.

[0048] 32. The method of item 31, wherein detecting intracellular HIV p24 protein comprises permeabilizing the cells in the biological sample and contacting the permeabilized cells with one or more antibodies specific for the HIV p24 protein.

[0049] 33. The method of any one of items 1 to 32, wherein the biological sample is from an HIV-1 -infected subject treated with combination antiretroviral therapy (cART).

[0050] 34. The method of any one of items 1 to 33, wherein the method further comprises contacting the sample with an agent that induces HIV reactivation from the latently HIV-infected cells (a latency-reversing agent (LRA)).

[0051] 35. The method of item 34, wherein the LRA comprises phorbol 12-myristate 13-acetate (PMA) and ionomycin, lipid nanoparticles containing HIV Trans- Activator of Transcription (Tat) mRNA (LNP-Tat), a histone deacetylase (HDAC) inhibitor, a histone methyltransferase (HMT) inhibitor, a Bromodomain and Extra-Terminal motif (BET) protein inhibitor, and / or a Protein kinase C (PKC) agonist.

[0052] 36. The method of any one of items 1 to 35, further comprising quantifying the frequency of cells bound by the first anti-gp120 antibody, the second anti-gp120 antibody, and / or the anti-gp41 antibody relative to total CD4+ T cells in the sample.

[0053] 37. The method of any one of items 1 to 36, further comprising sorting cells that are positive for binding by the first anti-gp120 antibody, the second anti-gp120 antibody, and / or the anti-gp41 antibody, and optionally analyzing the sorted cells.

[0054] 38. The method of any one of items 1 to 37, wherein the method is used to monitor changes in the number or frequency of latently HIV-infected cells in a subject over time.39. The method of any one of items 1 to 38, wherein the method is used as a companion diagnostic to evaluate the effect of a latency-reversing or reservoir-reducing therapy by comparing the frequency of cells bound by the combination of antibodies before and after administration of the therapy.

[0055] 40. A kit comprising the molecule that specifically binds to the Phe43 cavity of gp120, the first gp120 binding molecule, the second gp120 binding molecule and the gp41 binding as defined in any one of items 1 to 39.

[0056] 41. The kit of item 40, further comprising an agent that induces HIV reactivation from the latently infected cells (a latency-reversing agent (LRA)), an anti-CD4 antibody, and / or one or more antibodies specific for the HIV p24 protein.

[0057] 42. The kit of item 41, wherein the LRA comprises phorbol 12-myristate 13-acetate (PMA) and ionomycin, lipid nanoparticles containing HIV Trans-Activator of Transcription (Tat) mRNA (LNP-Tat), a histone deacetylase (HDAC) inhibitor, a histone methyltransferase (HMT) inhibitor, a Bromodomain and Extra-Terminal motif (BET) protein inhibitor, and / or a Protein kinase C (PKC) agonist.

[0058] 43. The kit of any one of items 40 to 42, further comprising a streptavidin or streptavidin analog conjugated to a detectable moiety.

[0059] 44. The kit of any one of items 40 to 43, further comprising instructions for detecting latently HIV-infected cells in a biological sample according to the method defined in any one of items 1 to 38.

[0060] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

[0061] BRIEF DESCRIPTION OF DRAWINGS

[0062] In the appended drawings:

[0063] FIG. 1 shows the detection of Env+p24+cells among CD4+ T cells purified from ART-treated PLWH using Env-flow. CD4+ T cells purified from HIV-1 -negative donors or antiretroviral therapy (ART)-treated persons living with HIV (PLWH), were stimulated with phorbol 12-myristate- 13-acetate (PMA) and ionomycin. Surface expression of Env was assessed using Cocktail #9 (comprising biotinylated A32, 17b, and 246D, in addition to the CD4mc CJF-lll-288) and streptavidin-PE, while intracellular p24 detection was performed using anti-p24 KC57 and anti-p24 28B7-APC antibodies. (A) p24 detection with two donors from each group. (B) Env detection among p24-negative (HIV-1 -negative donors) and p24+(PLWH on ART) cells.

[0064] FIG.2 shows the detection of Env+p24+cells among CD4+ T cells purified from untreated viremic PLWH using Env-flow. CD4+ T cells purified from HIV-1 -negative donors or untreated viremic PLWH were left unstimulated. Surface expression of Env was assessed using Cocktail#9 and streptavidin-PE, while intracellular p24 detection was performed using anti-p24 KC57 and anti-p24 28B7-APC antibodies. (A) Example of p24 detection. (B) Example of Env detection among p24-negative (HIV-1-negative donors) and p24+(PLWH on ART) cells.

[0065] FIG.3 shows the frequency of Env+p24+cells detected among CD4+ T cells purified from 7 ART-treated PLWH and 6 untreated PLWH using Env-flow. CD4+ T cells purified from antiretroviral therapy (ART)-treated persons living with HIV (PLWH) were stimulated with PMA / lonomycin, while CD4+ T cells purified from untreated viremic PLWH were left unstimulated. Surface expression of Env was assessed using cocktail #9 and streptavidin-PE, while intracellular p24 detection was performed using anti-p24 KC57 and anti-p24 28B7-APC antibodies. Shown are the frequency of p24+Env+ cells per 106cells detected in 7 ART-treated PLWH (with and without stimulation) and in 6 untreated viremic PLWH.

[0066] FIG. 4 shows the detection of Env in ART-treated PLWH using the Env-Flow assay. CD4+ T cells purified from ART-treated PLWH were stimulated with PM A and ionomycin or lipid nanoparticle containing Tat mRNA (LNP-Tat). CD4+ T cells purified from an HIV-1-negative donor, either unspiked or spiked with HIV- 1 -infected CD4+ T cells, served as negative and positive controls, respectively. Detection of Env-positive cells was performed using the Env-Flow assay with the anti-Env monoclonal antibody A32 (conjugated to Alexa Fluor 488 (AF488)), 17b (conjugated to AF488), and 246D (conjugated to Alexa Fluor 647 (AF647)). The graph shows the number of Env-positive cells detected per 106CD4+ T cells for each sample.

[0067] FIG.5 shows the detection of Env in untreated viremic PLWH using the Env-Flow assay. CD4+ T cells were purified from an untreated viremic PLWH and either left unstimulated or stimulated with PMA / lonomycin. CD4+ T cells from an HIV-1-negative donor, either unspiked or spiked with HIV-1-infected CD4+ T cells, served as negative and positive controls, respectively. Env-positive (Env+) cells were detected using the Env-Flow assay with anti-Env monoclonal antibodies A32-AF488, 17b-AF488, and 246D-AF647. The graph shows the number of Env+ cells detected per 106CD4+ T cells for each sample.

[0068] FIGs.6A-D show the enrichment of HIV-1 -infected cells among sorted Env+ cells. CD4+ T cells were purified from an untreated viremic PLWH and either left unstimulated or stimulated with PMA / lonomycin. CD4+ T cells from an HIV-1 -negative donor were used as a negative control.

[0069] FIG. 6A: Env-positive (Env+) cells from unstimulated CD4+ T cells of the viremic PLWH were sorted based on Env / CD4 co-expression using anti-CD4-FITC monoclonal antibodies and cocktail #9-PE. FIG. 6B: Alternatively, Env+ cells were sorted from PMA / lonomycin-stimulated CD4+ T cells based on binding of A32-AF488, 17b-AF488, and 246D-AF647. FIG. 6C: The relative frequency of HIV-1-infected cells was determined by quantifying HIV-1 DNA relative to a cellular gene (CD3) using a multiplex PCR. The level of enrichment for HIV-infected cells in sorted Env+ cells for each gating strategy is indicated above frequencies. The chronically infected ACH2 cellline was used as a positive control for the PCR. FIG. 6D: The transcriptional profile of sorted CD4l0WEnv+and CD4+Env_cells as determined by single-cell RNA sequencing.

[0070] FIGs. 7A-B show the detection of p24+and Env+p24+cells among CD4+ T cells purified from ART-treated PLWH by the Env-flow assay using different Latency-Reversing Agent (LRA) combinations. CD4+ T cells purified from five antiretroviral therapy (ART)-treated persons living with HIV (PLWH), were stimulated with PMA / lonomycin, LNP-Tat alone, or LNP-Tat in combination with Panobinostat, Romidepsin, Ingenol or JQ-1. Surface expression of Env was assessed using cocktail #9 and streptavidin-PE, while intracellular p24 detection was performed using anti-p24 KC57 and anti-p2428B7-APC antibodies. FIG. 7A: Frequencies of detected p24+(left bars) and Env+p24+(right bars) per 106CD4+ T cells. FIG.7B: Percentage of Env-expressing cells among reactivated p24+

[0071] FIGs. 8A-D show the linearity (FIGs. 8A-B) and sensitivity FIGs. 8C-D) of the Env-flow assay. HIV-infected CD4 T cells were spiked into uninfected CD4 T cells at defined proportion. Surface expression of Env was assessed using cocktail #9 and streptavidin-PE. Infected cells were identified either by HIV-1-mediated CD4 downregulation or by intracellular detection of HIV-1 p24 using the KC57 and 28B7 antibodies. Predicted (dashed line) and experimentally detected (full lines) percentage of CD4l0WEnv+cells (FIG.8A) or p24+Env+cells (FIG. 8B) are shown. CD4+ T cells purified from HIV-1 -negative donors and from untreated or antiretroviral therapy (ART)-treated persons living with HIV (PLWH), were left unstimulated or stimulated with PMA / lonomycin. Surface expression of Env and intracellular p24 detection was assessed as described above. Frequencies of detected CD4l0WEnv+cells (FIG. 8C) or p24+Env+cells (FIG. 8D) per 106CD4 T cells are shown. The dashed line represents the limit of detection.

[0072] FIG. 9A depicts the amino acid sequences of the heavy and light chain of the anti-gp120 antibody clone A32. The CDR1, CDR2 and CDR3 of the heavy and light chains are underlined (Chothia numbering).

[0073] FIG. 9B depicts the amino acid sequences of the heavy and light chains of anti-gp120 antibody clone 2.2C.2. The CDR1 , CDR2 and CDR3 of the heavy and light chains are underlined (Chothia numbering).

[0074] FIG. 9C depicts the amino acid sequences of the heavy and light chains of anti-gp120 antibody clone n5-i5. The CDR1, CDR2 and CDR3 of the heavy and light chains are underlined (Chothia numbering).

[0075] FIG. 9D depicts the amino acid sequences of the heavy and light chain of the anti-gp120 antibody clone 17b. The CDR1, CDR2 and CDR3 of the heavy and light chains are underlined (Chothia numbering).

[0076] FIG. 9E depicts the amino acid sequences of the heavy and light chains of anti-gp120 antibody clone 412d. The CDR1, CDR2 and CDR3 of the heavy and light chains are underlined (Chothia numbering).FIG. 9F depicts the amino acid sequences of the heavy and light chains of anti-gp120 antibody clone 412d. The CDR1, CDR2 and CDR3 of the heavy and light chains are underlined (Chothia numbering).

[0077] FIG. 9G depicts the amino acid sequences of the heavy and light chains of anti-gp120 antibody clone E51. The CDR1, CDR2 and CDR3 of the heavy and light chains are underlined (Chothia numbering).

[0078] FIG. 9H depicts the amino acid sequences of the heavy and light chains of anti-gp120 antibody clone n12-i2. The CDR1, CDR2 and CDR3 of the heavy and light chains are underlined (Chothia numbering).

[0079] FIG. 9I depicts the amino acid sequences of the heavy and light chains of anti-gp120 antibody clone X5. The CDR1, CDR2 and CDR3 of the heavy and light chains are underlined (Chothia numbering).

[0080] FIG. 9J depicts the amino acid sequences of the heavy and light chains of anti-gp41 antibody clone 246D. The CDR1, CDR2 and CDR3 of the heavy and light chains are underlined (Chothia numbering).

[0081] FIG. 9K depicts the nucleotide (SEQ ID NO:21) and amino acid (SEQ ID NO:22) sequences of the heavy chain of anti-gp41 antibody clone F240. The first 21 amino acids correspond to the Ig-kappa signal peptide, and residues 260-267 correspond to the thrombin cleavage tag, and the sequence also comprises a C-terminal 10-histidine purification tag.

[0082] FIG. 9L depicts the nucleotide (SEQ ID NO:23) and amino acid (SEQ ID NO:24) sequences of the light chain of anti-gp41 antibody clone F240. The first 21 amino acids correspond to the Ig-kappa signal peptide.

[0083] FIG. 9M depicts the amino acid sequences of the heavy (SEQ ID NO:25) and light (SEQ ID NO:26) chains of anti-gp120 antibody clone 48d. The CDR1, CDR2 and CDR3 of the heavy and light chains are underlined (Chothia numbering).

[0084] FIG. 9N depicts the amino acid sequences of the heavy (SEQ ID NO:27) and light (SEQ ID NO:28) chains of anti-gp120 antibody clone 412d. The CDR1, CDR2 and CDR3 of the heavy and light chains are underlined (Chothia numbering).

[0085] FIG. 90 depicts the amino acid sequences of the heavy (SEQ ID NO:29) and light (SEQ ID NQ:30) chains of anti-gp120 antibody clone N60-i3. The CDR1, CDR2 and CDR3 of the heavy and light chains are underlined (Chothia numbering).

[0086] DISCLOSURE OF INVENTION

[0087] In the studies described herein, the present inventors have shown that it is possible to detect Env-expressing cells in PLWH under ART after reactivation with latency-reversing agents (LRAs), using a CD4 mimetic compound (CD4mc) and an antibody cocktail targeting three different regions of Env (gp120 CoRBS, gp120 cluster A, and gp41 cluster I). Since Envexpression is required for viral replication, Env-expressing cells likely constitute the functional reservoir responsible for viral rebound after treatment interruption (i.e., the replication competent reservoir). Importantly, the detection of these cells using the assay described herein does not require the permeabilization of the cells.

[0088] Accordingly, in a first aspect, the present disclosure provides method for detecting latently HIV-infected cells in a biological sample comprising CD4+ cells (e.g., a sample from a PLWH treated with combination antiretroviral therapy (cART)), the method comprising: contacting the sample with:

[0089] (a) a molecule that specifically binds within the Phe43 cavity of gp120,

[0090] (b) a first gp120 binding molecule that binds to an epitope within the constant region 1 and 2 (C1-C2) portion of the cluster A region of gp120;

[0091] (c) a second gp120 binding molecule that binds to an epitope within the bridging sheet of the Co-Receptor Binding Site (CoRBS) of gp120; and

[0092] (d) a gp41 binding molecule that binds to an epitope located in the cluster I region of gp41 ; and

[0093] detecting a signal indicative of latently HIV-infected cells, wherein the signal is generated by the interaction of the first anti-gp120 antibody, the second anti-gp120 antibody, and / or the anti-gp41 antibody with the latently HIV-infected cells.

[0094] In another aspect, the present disclosure provides method for detecting cells expressing the HIV-1 envelope (Env) glycoprotein in a biological sample from a subject (e.g., an HIV-1-infected subject treated with combination antiretroviral therapy (cART)), the method comprising: contacting the sample with:

[0095] (a) a molecule that specifically binds within the Phe43 cavity of gp120;

[0096] (b) a first gp 120-binding molecule that binds to an epitope within the constant region 1 and 2 (C1-C2) portion of the cluster A region of gp120;

[0097] (c) a second gp120-binding molecule that binds to an epitope within the bridging sheet of the Co-Receptor Binding Site (CoRBS) of gp120; and

[0098] (d) a gp41 -binding moleculethat binds to an epitope located in the cluster I region of gp41; and

[0099] detecting a signal indicative of cells expressing the Env glycoprotein, wherein the signal is generated by the interaction of the first anti-gp120 antibody, the second anti-gp120 antibody, and / or the anti-gp41 antibody with the cells expressing the Env glycoprotein.

[0100] In another aspect, the present disclosure provides method for detecting latently HIV-infected cells or cells expressing the HIV-1 envelope (Env) glycoprotein in a biological sample from a subject (e.g., an HIV-1 -infected subject treated with combination antiretroviral therapy (cART)), the method comprising:

[0101] (i) providing a biological sample comprising or suspected of comprising latently HIV-infected cells;(ii) contacting the sample with:

[0102] (a) a molecule that specifically binds within the Phe43 cavity of gp120;

[0103] (b) a first gp 120-binding molecule that binds to an epitope within the constant region 1 and 2 (C1-C2) portion of the cluster A region of gp120;

[0104] (c) a second gp120-binding molecule that binds to an epitope within the bridging sheet of the Co-Receptor Binding Site (CoRBS) of gp120; and

[0105] (d) a gp41 -binding molecule that binds to an epitope located in the cluster I region of gp41; under conditions that allow binding of the binding molecules to Env expressed at the cell surface; and

[0106] (iii) detecting cells to which the first gp 120-binding molecule, second gp 120-binding molecule and gp41 -binding molecule have bound,

[0107] wherein the detection of cells bound by the first gp120-binding molecule, second gp120-binding molecule and gp41-binding molecule is indicative of latently HIV-infected cells.

[0108] The term “latently HIV-infected cell” as used herein means a cell harboring integrated HIV proviral DNA that does not produce detectable levels of infectious virions under basal conditions but has the potential to produce virus upon appropriate stimulation.

[0109] The skilled person would understand that the method described herein is performed on latently HIV-infected cells that have been reactivated. In an embodiment, the method further comprises contacting the sample with an agent that induces HIV reactivation from the latently HIV-infected cells, e.g., a latency-reversing agent.

[0110] In another aspect, the present disclosure provides a kit comprising: (a) a molecule that specifically binds within the Phe43 cavity of gp120; (b) a first gp120 binding molecule that binds to an epitope within the constant region 1 and 2 (C1-C2) portion of the cluster A region of gp120; (c) a second gp120 binding molecule that binds to an epitope within the bridging sheet of the CoReceptor Binding Site (CoRBS) of gp120; (d) a gp41 binding molecule that binds to an epitope located in the cluster I region of gp41; and optionally (e) an agent that induces HIV reactivation from the latently HIV-infected cells. In an embodiment, the kit is for detecting latently HIV-infected cells in a biological sample, or detecting cells expressing the Env glycoprotein in a biological sample from a subject.

[0111] Molecules that specifically bind within the Phe43 cavity of gp120 (mimicking CD4's Phe43 interaction with Env) and that are capable to 'opening up' the Envtrimer and thereby enabling the binding of CD4-induced non-neutralizing antibodies (nnAbs) are well known in the art and include “small CD4 mimetic” or “CD4mc” such as NBD-556, NBD-557, DMJ-l-228, JP-lll-48, MCG-IV-210 , TFH-070-A6, TFH-l-116-D1 , ZXC-l-090, ZXC-l-092, DL-l-101, and DL-l-102, BNM-lll-170, CF-I I I-049-S and CJF-111-288. CD4mc are disclosed, for example, in PCT publications Nos. WO 2013 / 090696, WO 2020 / 028482, and WO 2023 / 235756, as well as in references [10, 16 and 17], Molecules that specifically bind to the Phe43 cavity of gp120 also includes peptides andpolypeptides, such as peptides or polypeptides derived from the human CD4 receptor, such as the miniCD4 peptide M48U1. In an embodiment, the molecule that specifically binds to a CD4-binding domain of gp120 is a soluble human CD4 peptide or polypeptide (sCD4). In a further embodiment, the soluble human CD4 peptide or polypeptide comprises a sequence having at least 70%, 75%, 80%, 85%, 90% or 95% with the sequence of the D1 and D2 domains of human CD4, e.g., residues 26 to 208). In a further embodiment, the soluble human CD4 peptide or polypeptide comprises residues 26 to 208 of human CD4 (Richard etal., mBio, Vol. 12(5): e1405-21). In an embodiment, the molecule that specifically binds within the CD4-binding domain (i.e., the Phe43 cavity) of gp120 is a CD4mc, for example CJF-111-288.

[0112] As used herein, the term “gp120-binding molecule” or “gp41 -binding molecule” refers to any molecule capable of binding to the specified domain or epitope of gp120 or gp41. The term “binding molecule” encompasses antibodies, antibody fragments and non-antibody binding agents, for example antibody mimetics such as those described in Yu et al. (2017) Annu Rev Anal Chem 10(1):293-320. Thus, the gp120- or gp41-binding molecules defined herein may be ligands of gp120 or gp41 (natural or synthetic), antibodies, antibody fragments, antibody mimetics, adnectins, affibodies, affilins, affimers, affitins, alphabodies, anticalins, aptamers, armadillo repeat protein-based scaffolds, atrimers, avimers, DARPins, fynomers, knottins, Kunitz domain peptides, monobodies, and nanofitins.

[0113] In embodiments, the first and / or second gp120-binding molecules are antibodies or antigen-binding fragments thereof. In further embodiments, the first and second gp120-binding molecules are antibodies or antigen-binding fragments thereof. In an embodiment, the gp41-binding molecule is an antibody or an antigen-binding fragment thereof. In a further embodiment, the gp41 -binding molecule is an antibody or an antigen-binding fragment thereof.

[0114] The terms “antibody” and “antibodies” refer to naturally occurring forms including monoclonal or polyclonal antibodies, or recombinant antibodies such as chimeric antibodies or humanized antibodies. The term “antibody fragment” includes, for example, F(ab), F(ab’)2, Fv, single chain antibodies or diabodies. In an embodiment, the antibody is a full-length human antibody.

[0115] In an embodiment, the first gp120-binding molecule is antibody clone A32, or a gp120-binding molecule (e.g., an antibody or an antigen-binding fragment thereof) that competes with antibody clone A32 for binding to gp120 (j.e., a competing antibody or an antigen-binding fragment thereof).

[0116] The term “competing antibody” refers to an antibody that binds to the same epitope (or an overlapping epitope) as another antibody and, as a result, competes with that antibody for binding. Thus, an antibody or an antigen-binding fragment thereof that competes with antibody clone A32 (a competing antibody of clone A32) is an antibody or antibody fragment that blocks or reduces binding of antibody clone A32 to gp120 in a standardized competition assay (e.g., ELISA,surface plasmon resonance). In an embodiment, the competing antibody reduces the binding of the reference antibody (e.g., A32 or any other antibody described herein) by at least 50%, and preferably by at least 60, 70, 80 or 90% as measured by a standardized competition assay (e.g., ELISA, surface plasmon resonance).

[0117] Examples of gp120-binding molecules that compete with antibody clone A32 include antibody clones L9-i1, N5-i5, N60-i3, 2.2c (Guan, Yongjun et al. “Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding.” Proceedings of the National Academy of Sciences of the United States of America, vol.

[0118] 110, 1 (2013): E69-78. doi:10.1073 / pnas.1217609110; Tolbert et al., “Structural Basis for Epitopes in the gp120 Cluster A Region that Invokes Potent Effector Cell Activity”, Viruses. 2019 Jan; 11(1): 69). In an embodiment, the first gp120-binding molecule specifically binds to an epitope formed by residues within the following regions: 51-54, 56, 58-61, 68-80, 103, 106-107, 110, 113-114, 217, and 219-221 of gp120.

[0119] In an embodiment, the first gp120-binding molecule is an antibody or an antigen-binding fragment thereof comprising the complementarity determining regions (CDRs) of antibody clone A32, N60-i3, L9-i1, 2.2c, 2.2c.2 or N5-i5, preferably A32. In an embodiment, the first gp120-binding molecule is an antibody or an antigen-binding fragment thereof comprising the variable regions of antibody clone A32, L9-i1, 2.2c, 2.2.C.2 or N5-i5, preferably A32. In a further embodiment, the first gp120-binding molecule is antibody clone A32, L9-i1, 2.2c, 2.2.C.2 or N5-i5, preferably A32. The amino acid sequences of the heavy and light chains of antibody clone A32 are depicted in FIG.9A, the amino acid sequences of the heavy and light chains of antibody clone 2.2c.2 are depicted in FIG.9B, the amino acid sequences ofthe heavy and light chains of antibody clone N5-i5 are depicted in FIG. 9C, and the amino acid sequences ofthe heavy and light chains of antibody clone N60-i3 are depicted in FIG. 90.

[0120] In an embodiment, the second gp120-binding molecule is antibody clone 17b ora gp120-binding molecule (e.g., an antibody or an antigen-binding fragment thereof) that competes with antibody clone 17b for binding to gp120. Examples of gp 120-binding molecules that compete with antibody clone 17b include antibody clones X5, 412d, 48d, E51, L9-i3, N5-i1, N5-i3, N5-i4, N5-i8, N10-i1.1, N10-i5.3, N12-i1, N12-i2, N12-i4, N12-i5, N12-i7, N12-i8, N12-i10, N12-i17, N12-i18, and N12-i9 (Guan, Yongjun et al. “Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding.” Proceedings ofthe National Academy of Sciences of the United States of America, vol. 110,1 (2013): E69-78. doi:10.1073 / pnas.1217609110). In an embodiment, the second gp120-binding molecule specifically binds to an epitope formed by residues within the following regions: 119-122, 200, 202-205, 326-327, 369, 419-423 and 432-437 of gp120.

[0121] In an embodiment, the second gp120-binding molecule is an antibody or an antigenbinding fragment thereof comprising the complementarity determining regions (CDRs) of antibodyclone 17b, X5, 412d, 48d, E51, L9-I3, N5-I1, N5-I3, N5-I4, N5-I8, N10-I1.1, N10-I5.3, N12-I1, N12-I2, N12-I4, N12-I5, N12-I7, N12-I8, N12-I10, N12-I17, N12-I18, or N12-I9, preferably 17b. In an embodiment, the second gp120-binding molecule is an antibody or an antigen-binding fragment thereof comprising the variable regions of antibody clone 17b, X5, 412d, 48d, E51, L9-i3, N5-i1, N5-i3, N5-i4, N5-i8, N10-i1.1 , N10-i5.3, N12-i1, N12-i2, N12-i4, N12-i5, N12-i7, N12-i8, N12-i10, N12-i17, N12-i18, or N12-i9, preferably 17b. In a further embodiment, the second gp120-binding molecule is antibody clone 17b, X5, 412d, 48d, E51, L9-i3, N5-i1, N5-i3, N5-i4, N5-i8, N10-i1.1 , N10-I5.3, N12-i1, N12-i2, N12-i4, N12-i5, N12-i7, N12-i8, N12-i10, N12-I17, N12-I18, or N12-i9, preferably 17b. The amino acid sequences of the heavy and light chains of antibody clone 17b are depicted in FIG.9D, the amino acid sequences of the heavy and light chains of antibody clone 412d are depicted in FIG. 9E, the amino acid sequences of the heavy and light chains of antibody clone 48d are depicted in FIG. 9F, the amino acid sequences of the heavy and light chains of antibody clone E51 are depicted in FIG. 9G, the amino acid sequences of the heavy and light chains of antibody clone N12-i2 are depicted in FIG. 9H, the amino acid sequences of the heavy and light chains of antibody clone X5 are depicted in FIG. 9I, the amino acid sequences of the heavy and light chains of antibody clone 48d are depicted in FIG. 9M, and the amino acid sequences of the heavy and light chains of antibody clone 412d are depicted in FIG. 9N.

[0122] Gp41 -binding molecules that binds to an epitope located in the cluster I region of gp41 are known in the art and include antibody clones 246D, F240, 7B2, 181D, 98-43, M785-U1 and N10-U1. Some of these antibodies are described in reference

[0011] ,

[0123] In an embodiment, the gp41 -binding molecule is antibody clone 246D, F240, 7B2, 181D, 98-43, M785-U1 or N10-U1, or a gp120-binding molecule (e.g., an antibody or an antigen-binding fragment thereof) that competes with antibody clone 246D, F240, 7B2, 181D, 98-43, M785-U1 or N10-U1, for binding to gp120.

[0124] In an embodiment, the gp41-binding molecule is an antibody or an antigen-binding fragment thereof comprising the complementarity determining regions (CDRs) of antibody clone 246D, F240, 7B2, 181D, 98-43, M785-U1 or N10-U1, preferably 246D. In an embodiment, the gp41 -binding molecule is an antibody or an antigen-binding fragment thereof comprising the variable regions of antibody clone 246D, F240, 7B2, 181D, 98-43, M785-U1 or N10-U1, preferably 246D. In a further embodiment, the gp41-binding molecule is antibody clone 246D, F240, 7B2, 181D, 98-43, M785-U1 orN10-U1, preferably 246D. The amino acid sequences of the heavy and light chains of antibody clone 246D are depicted in FIG. 9J, the amino acid sequences of the heavy and light chains of antibody clone 412d are depicted in FIGs. 9E, and the amino acid sequences of the heavy and light chains of antibody clone F240 are depicted in FIGs. 9K-L, Relevant information concerning the antibody clones described herein may be found, e.g., in the HIV database (Los Alamos National Laboratory, HIV Molecular Immunology 2021, Editors: Elizabeth-Sharon David-Fung, Bette T. M. Korber, Christian Brander, Dan Barouch, Robde Boer, Barton F. Haynes, Richard Koup, John P. Moore, Bruce D. Walker, and David I. Watkins. Publisher: Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, New Mexico. LA- UR-21-32446, https: / / www.hiv.lanl.gov / content / index), the RCSB Protein Data Bank (PDB) (H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne. (2000). The Protein Data Bank. Nucleic Acids Research, 28: 235-242, https: / / www.rcsb.org / ), and the references cited herein. Based on the sequences of the heavy and light chains of the antibodies disclosed herein (see, e.g., FIGs. 9A-9O), the skilled person would be able to easily identify the amino acid sequences corresponding to the CDRs and FRs of these antibodies based on common general knowledge in the field of antibodies, for example using commonly available tools such as the abYsis tool (Swindells et al., J Mol Biol. 2017 Feb 3;429(3):356-364. doi: 10.1016 / j.jmb.2016.08.019. Epub 2016 Aug 22 http: / / www.abysis.org / abysis / ), the AbRSA tool (Li et al., Protein Sci. 2019 Aug; 28(8): 1524-1531, or the GenSmart™ Variable Domain Sequence Analyzer from Genscript.

[0125] In an embodiment, the molecule that specifically binds within the Phe43 cavity of gp120 is CJF-lll-288, the first gp120-binding molecule is an antibody or an antigen-binding fragment thereof comprising the CDRs or variable regions of antibody clone A32, the second gp120-binding molecule is an antibody or an antigen-binding fragment thereof comprising the CDRs or variable regions of antibody clone 17b, and the gp41 -binding molecule is an antibody or an antigen-binding fragment thereof comprising the CDRs or variable regions of antibody clone 246D.

[0126] The first gp120-binding molecule, the second gp120-binding molecule, and / or the gp41-binding molecule may be directly or indirectly labelled for detection or separation.

[0127] In an embodiment, the first gp120-binding molecule, the second gp120-binding molecule, and / or the gp41-binding molecule is / are conjugated to a detectable label or reporter protein. In another embodiment, the first gp120-binding molecule, the second gp120-binding molecule, and / or the gp41-binding molecule is / are not directly conjugated to a detectable label or reporter protein, and the detection agent further comprises a secondary agent that specifically binds to the first gp120-binding molecule, the second gp 120-binding molecule, and / or the gp41-binding molecule, such as an anti-human antibody conjugated to a detectable label or reporter protein. The first gp120-binding molecule, the second gp120-binding molecule, and / or the gp41-binding molecule may also be modified with an affinity tag. Affinity tag systems are well known in the art and include, for example, NTA- “His-Tag” systems, biotin - avidin / streptavidin systems, glutathione S-transferase (GST) - glutathione systems, Maltose Binding Protein (MBP) - amylose systems, as well as antigen - antibody systems. In an embodiment, the first gp120-binding molecule, the second gp120-binding molecule, and / or the gp41-binding molecule is / are conjugated to biotin, and the method further comprises contacting the sample with avidin / streptavidin conjugated to a detectable label or reporter protein.The term “detectable label or reporter protein” as used herein refers to a moiety emitting a signal (e.g., light) that may be detected using an appropriate detection system. Any suitable detectable label may be used in the method described herein. Detectable labels and reporter proteins include, for example, enzyme or enzyme substrates, reactive groups, chromophores such as dyes or colored particles, luminescent moieties including bioluminescent, phosphorescent or chemiluminescent moieties, and fluorescent moieties. In an embodiment, the detectable label is a fluorescent moiety. Fluorophores that are commonly used include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL), 5-(2'-aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS), Alexa™ dyes (Molecular Probes), fluor dye, Bodipy dye™ (Life technologies), Cy dye™(Life technologies), dansyl, umbelliferone (7- hydroxycoumarin), fluorescent microsphere, luminescent nanocrystal, Marina blue™ (Life technologies), Cascade blue™ (Life technologies), Cascade yellow™ (Life technologies), Pacific blue™ (Life technologies), Oregon green™ (Life technologies), Tetramethylrhodamine, Rhodamine, Texas red™(Life technologies), as well as fluorescent proteins such as green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and the like. Enzymes used for labeling proteins for immunoassays are known in the art, and include for example peroxidases (e.g., horseradish peroxidase, HRP) and alkaline phosphatase (AP). In an embodiment, the detectable label or reporter protein is an enzyme, preferably a peroxidase such as HRP.

[0128] The signal emitted by the detectable label or reporter protein (a signal that is proportional to the amount of detectable label or reporter protein present in the assay mixture) may be measured by any method known in the art. For example, if the detectable label or reporter protein is an enzyme, a substrate for the enzyme, preferably a chromogenic or fluorogenic substrate, is added to the assay mixture, and the signal is measured by detecting the level of a detectable product generated by catalysis of the substrate by the enzyme. For a chromogenic or fluorogenic substrate, the signal is measured by assessing the color or fluorescence intensity in the assay mixture using a suitable device, such as a spectrometer. Examples of chromogenic HRP substrates include 3,3',5,5'-tetramethylbenzidine (TMB), 3,3'-Diaminobenzidine (DAB) and 2,2' -azino-di-[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS). In an embodiment, the detectable label is a fluorescent molecule and the signal is detected by flow cytometry.

[0129] In an embodiment, the first gp120-binding molecule, the second gp120-binding molecule, and / or the gp41 -binding molecule may be conjugated to magnetic particles (for magnetic cell separation).

[0130] In an embodiment, the first gp120-binding molecule, the second gp120-binding molecule, and / or the gp41 -binding molecule may be labelled with distinct detectable labels (e.g.,fluorophores), which may be useful to perform multi-parametric flow cytometry. In some embodiment, two of the binding molecules (e.g., the first and second gp120-binding molecules) may be labelled with the same detectable label (e.g., fluorophore), and the other binding molecule (e.g., the gp41 -binding molecule) may be labelled with another detectable label (e.g., fluorophore).

[0131] In embodiments, the first gp120-binding molecule, the second gp120-binding molecule, and / or the gp41-binding molecule attached to a solid support. The coating of binding molecules (e.g., antibodies or antigen-binding fragments thereof) on a solid support may be performed by contacting the solid support with a solution (e.g., a buffer) comprising a suitable amount of the binding molecules. In an embodiment, the method further comprises at least one washing step after the coating step to remove the uncoated gp120-binding and / or gp41 -binding molecule. The gp120-binding and / or gp41 -binding molecule may be attached or coated on the solid support using a suitable tag, through its fragment crystallizable (Fc) domain (for antibodies), or through molecular interactions such as hydrophobic or hydrophilic intermolecular interactions. The above-mentioned solid support may be any solid support which permits the binding (e.g. , immobilization) of the gp120-binding and / or gp41-binding molecule and which may be used for the desired application. It includes for example beads / resins, etc.

[0132] Agent that induces HIV reactivation from the latently HIV-infected cells, often referred to as Latency-Reversing Agents (LRA), are well known in the art, and include, for example, PMA / ionomycin, anti-CD3 / CD28 antibodies, Tat vaccine (such as lipid nanoparticle containing HIV Trans-Activator of Transcription (Tat) mRNA (LNP-Tat), Tat Oyi vaccine, Tat-R5M4 protein), HDAC inhibitors (such as Trichostatin A, trapoxin, suberoylanilide hydroxamic acid (SAHA), romidepsin, vorinostat, entinostat, valproic acid, fimepinostat, chidamideand and panobinostat), Histone methyltransferase inhibitors (such as Chaetocin and BIX-01294), BET inhibitors such as JQ1, l-BET, I-BET151, MMQO, RVX-208, and PFI-1, DNA methylation inhibitors such as 5-aza-cytidine, 5-aza-deoxycytidine, and Zebularine, Protein kinase C (PKC)ZNF-KB agonists (such as Bryostatin, Prostratin, ingenols and derivatives thereof such as ingenol-3,20-dibenzoate, ingenol 3-hexanoate, and ingenol-3-angelate, EK-16A and GSK445A), Second mitochondria-derived activator of caspases mimetics (SMACm) (such as AZD5582 and Ciapavir), STAT5 sumoylation inhibitors (such as 1-hydroxybenzotriazol, 1-hydroxy-7-amino benzotriazole, and 3-hydroxy-1,2,3-benzotriazin-4(3H)-one and analogs), cytokines, cytokine analogs or cytokine receptor agonists (such as IL-15 and N-803), toll-like receptor (TLR) agonists (such as Pam3CSK4 (TLR2), lefitolimod (TLR-9) and vesatolimod (TLR-7)), as well as immune checkpoint inhibitors (such as PD1 / PD-L1, CTLA-4, LAG-3 and TIGIT inhibitors) (see references [12-15]).

[0133] The method may include one or more washing steps.

[0134] In an embodiment, the above-mentioned biological sample comprising CD4+ cells is a biological fluid, e.g., urine, saliva, lymph, cervicovaginal fluid, cerebrospinal fluid or a blood-derived sample. The term “blood-derived sample” as used herein refers to blood (e.g., fresh blood, stored blood) or to a fraction thereof, such as serum, plasma and the like. It also refers to any sample that may be obtained following one or more purification, enrichment, and / or treatment steps using blood (obtained by venous puncture, for example) as starting material (e.g., buffy coat, isolated peripheral blood mononuclear cells (PBMCs)). In an embodiment, the above-mentioned blood-derived sample is plasma. The biological sample comprising CD4+ cells may also be a tissue sample, for example lymphoid tissue (e.g., lymph node biopsies, tonsil, spleen), a gut-associated lymphoid tissue sample, ora bone marrow sample), or in vitro cell cultures (e.g., infected cell lines or ex vivo infected primary cells).

[0135] In an embodiment, the method further comprises enriching the sample in T cells, such as CD4+ T cells, prior to contacting the sample with the binding molecules. Such enriching step may be performed by depleting one or more non-T cell lineages from the sample prior to contacting the sample with the binding molecules (negative selection). Methods for depleting one or more non-T cell lineages refer to any processes or step(s) aiming at reducing or removing cells other than T lymphocytes in a biological sample. Depletion may be achieved by physical removal using binding agents (or affinity agents) targeting non-T cell lineage cells. In this context, non-limiting examples of target non-T cell lineage populations include B cells (e.g., CD19+, CD20+), plasma cells (CD138+), NK cells (CD56+), innate lymphoid cells, myeloid cells (monocytes / macrophages, dendritic cells, neutrophils, eosinophils, basophils, mast cells), and myeloid-derived suppressor cells (MDSCs; monocytic or polymorphonuclear).

[0136] Such enriching step may be performed using binding molecules specific for one or more T cell markers such as CD3, CD4 and / or CD8 (positive selection).

[0137] Kits for enriching T cells such as CD4 T cells are commercially available from various providers (e.g., EasySep™ Human T Cell Enrichment Kit from STEMCELL Technologies, MagniSort™ Human T cell Enrichment Kit from ThermoFisher Scientific, Akadeum’s Human T Cell Isolation Kit, Human Pan T Cell Isolation Kit from Miltenyi Biotec, etc.).

[0138] In an embodiment, the method further comprises contacting the sample with a labelled anti-CD3 antibody, an anti-CD4 antibody and / or an anti-CD8 antibody, and determining / assessing the binding of the first gp120-binding molecule, the second gp120-binding molecule, and / or the gp41 -binding molecule on CD3+CD4+ cells in the biological sample.

[0139] The presence of latently infected cells in the sample may be further confirmed by various methods such as single-cell PCR for proviral DNA, viral outgrowth from sorted Env-positive cells, and / or detection of cell-associated HIV RNA or p24 after subsequent activation.

[0140] In another embodiment, the method further comprises detecting intracellular HIV p24 protein in the cells. Such detecting may involve permeabilizing the cells in the biological sample (after contacting the cells with the first gp120-binding molecule, the second gp120-binding molecule, and the gp41 -binding molecule (and optionally the labelled anti-CD3 antibody, anti-CD4 antibody and / or anti-CD8 antibody), and contacting the permeabilized cells with one or more binding agents (e.g., antibodies) specific for the HIV p24 protein. Binding agents (e.g., antibodies) specific for the HIV p24 protein are well known in the art and commercially available from various providers. In an embodiment, the one or more antibodies specific for the HIV p24 protein comprise the KC57 clone antibody from Beckman Coulter™ (Cat. No. 6604667). In an embodiment, the one or more antibodies specific for the HIV p24 protein comprise the 28B7 clone antibody from MediMabs (Cat. No. MM-0289). In an embodiment, the one or more antibodies specific for the HIV p24 protein comprise the KC57 and 28B7 clone antibodies.

[0141] The method described herein may be useful for various applications including

[0142] • measurement of latent reservoir in clinical samples, e.g., estimating the frequency of Env-positive latent reservoir cells, tracking changes in reservoir size over time or in response to therapeutic interventions, characterizing the phenotype of reservoir cells (e.g., memory subset, exhaustion markers);

[0143] • isolation and characterization of reservoir cells, e.g., sorting by fluorescence-activated cell sorting (FACS) for downstream analyses, analyzing the isolated cells by single-cell transcriptomics, epigenetic profiling, and / or integrated provirus sequencing, and testing the susceptibility / response to latency-reversing agents and / or immune-based therapies;

[0144] • Companion diagnostics and therapeutic monitoring, e.g., efficacy of latency reversing strategies (“shock and kill”) by monitoring changes in Env positive cell frequencies, efficacy of reservoir-reducing strategies (“block and lock”) by assessing diminution of detectable Env-positive latent cells overtime.

[0145] In an embodiment, the method further comprises isolating or purifying the latently HIV-infected cells (or Env-expressing cells) from the sample. In an embodiment, the method further comprises characterization the latently HIV-infected cells (or Env-expressing cells) identified by the method. Such characterization may include gene expression profiling, protein expression profiling, expression of cell surface markers, response to drugs or drug candidates, etc.

[0146] In an embodiment, the method further comprising quantifying the number or frequency of latently HIV-infected cells (or Env-expressing cells) in the sample. The number or frequency may be reported for example, in absolute number or relative to the number of cells or of a cell subset (e.g., CD4+ T cells) in the sample.

[0147] In an embodiment, the kit according to the present disclosure may comprise, in addition to elements (a)-(d) (and optional element (e)) defined above, one or more additional reagents useful to perform the method described herein such as secondary detection antibodies, anti-CD3, an anti-CD4 and / or anti-CD8 antibodies, anti-p24 antibodies, control antibodies or isotypecontrols, reagents for sample preparation, permeabilization agents, solid supports, streptavidin, buffers, solutions, etc.

[0148] In an embodiment, the kit according to the present disclosure may be divided into separate packages or compartments containing the respective reagent components explained above.

[0149] In addition, such a kit may optionally comprise one or more of the following: (1) instructions for using the reagents for detecting latently HIV-infected cells (or Env-expressing cells) in a biological sample according to the methods described herein; (2) one or more containers; and / or (3) appropriate controls / standards. Such a kit can include reagents for collecting a biological sample from a patient and reagents for processing the biological sample. The kits featured herein can also include an instruction sheet describing how to perform the method for detecting latently HIV-infected cells (or Env-expressing cells) in a biological sample.

[0150] Informational material included in the kits can be descriptive, instructional, marketing or other material that relates to the methods described herein and / or the use of the reagents for the methods described herein. For example, the informational material of the kit can contain contact information, e.g., a physical address, email address, website, or telephone number, where a user of the kit can obtain substantive information about performing the method described herein and interpreting the results.

[0151] MODE(S) FOR CARRYING OUT THE INVENTION

[0152] The present invention is illustrated in further details by the following non-limiting examples.

[0153] Example 1: Detection of Env-expressing cells in PLWH under ART Materials and Methods

[0154] Antibodies. The anti-Env monoclonal antibodies (mAbs) A32, 17b, and 246D were used as primary antibodies to detect Env-expressing cells. These mAbs were biotinylated using the EZ-Link Sulfo-NHS-LC-Biotinylation Kit, following the manufacturer’s instructions (Thermo Fisher Scientific, 21435). Biotinylated antibody binding was detected using PE-conjugated streptavidin (Thermo Fisher Scientific, S866). Alternatively, A32 and 17b were directly conjugated with Alexa Fluor™ 488, and 246D was conjugated with Alexa Fluor™ 647, using the manufacturer’s protocol (Thermo Fisher Scientific) for Env detection. For extracellular staining, the following mAbs were used: anti-CD3 BUV395 (Clone UCHT1, BD, 563546), anti-CD4 BUV496 (Clone OKT4, BD Biosciences, 750980), and anti-CD8 BV786 (Clone RPA-T8, BD Biosciences, 563824). Intracellular p24 expression was detected using a combination of FITC-conjugated mouse anti-p24 (Clone KC57, Beckman Coulter, 6604665) and APC-conjugated human anti-p24 (Clone 28B7, MediMabs, MM-0289-APC). Viable cells were identified using the LIVE / DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific, L34957).Small CD4-mimetics. The small-molecule CD4-mimetic compounds (CD4mc) CJF-III-288 was synthesized as described previously (Fritschi et al., 2023 PMID:36961924). The compound was dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM and then diluted in phosphate-buffered saline (PBS) for cell-surface staining.

[0155] Env-Flow assay. Upon thawing of PBMCs, CD4+ T cells were isolated by negative magnetic selection using the EasySep™ Human CD4 T Cell Enrichment Kit (StemCell Technology, 19052). 1x107CD4+ T cells were resuspended at 2x106cells / mL in RPMI + 20% Fetal Bovine Serum and antiretroviral drugs were added to the culture (200 nM raltegravir, 200 nM lamivudine). Cells were rested in presence of antiretroviral drugs at least 1h before stimulation with 1 pg / mL ionomycin (Sigma, I9657) and 162 nM PMA (Sigma, P8139) for24h orTat LNPs (at 1 pg per 1x106cells) for40h. After stimulation, cells were collected in 5 mL flow cytometry tubes, resuspended in PBS and stained with the Aqua Live / Dead staining kit for 30 min at 4°C. Cells were washed with PBS + 4% human serum (Atlanta Biologicals, 540110) and then incubated with biotinylated A32, 17b and 246 (at 2.5 pg / ml) in PBS + 4% human serum for 30 min at 37°C, in the presence of the CD4mc, CJF-lll-288 (at 50 pM). Cells were washed twice with PBS + 4% human serum and then incubated 20 min at room temperature (RT) with a mix containing a brilliant stain buffer (BD Biosciences), the surface markers for CD4+ T cells detection (anti-CD3 and anti-CD4 mAbs) and CD8 exclusions (anti-CD8 mAbs) and PE-conjugated streptavidin. Cells were washed twice with PBS + 4% human serum and fixed at RT with 4% formaldehyde for 15 min. After one wash with PBS + 4% human serum, cells were permeabilized for 30 min at 4°C using the PermWash™ buffer (BD Biosciences, 554723) + 4% human serum, and stained with anti-p24 KC57 and anti-p24 28B7 antibodies for an additional 45 min at RT. Cells were then washed and resuspended in PBS for subsequent analysis. Alternatively, to detect Env-expressing cells without requiring permeabilization or p24 detection, cells were stained with Alexa-Fluor™ 488-conjugated A32 mAbs, Alexa-Fluor™ 488-conjugated 17b mAbs and Alexa-Fluor™ 647-conjugated 246D mAbs (at 2.5 pg / ml) in PBS + 4% human serum for 30 min at 37°C in the presence of the CD4mc, CJF-lll-288 (at 50pM). After three washes with PBS containing 4% human serum, the cells were fixed with 2% formaldehyde. Following fixation, the cells were washed and resuspended in PBS for subsequent analysis. Samples were acquired using a FORTESSA™ flow cytometer (BD Biosciences), and data analysis was conducted with FlowJo™ v10.5.3 software (Tree Star, Ashland, OR, USA). The frequencies of p24+Env+, Env+, or CD4l0WEnv+cells were determined by gating on viable CD3+CD8“ cells

[0156] Results

[0157] A new CD4mc / Ab cocktail (named Cocktail #9) that is effective at detecting and quantitating HIV-1 -infected cells in vitro was developed. Cocktail #9 comprises the CD4mc CJF-lll-288 as well as three antibodies that target highly conserved regions of Env. The first antibody,A32, targets the cluster A domain of gp120, the second antibody, 17b, targets the CoRBS domain of gp120, and the third antibody, 246D, targets the cluster I domain of gp41. Results demonstrating that Cocktail #9 can detect rare Env-expressing cells upon treatment of primary CD4+ T cells from PLWH with PMA / lonomicyn or Tat mRNA-LNPs were generated. This approach was named “Env-Flow” as it parallels the “HIV-Flow” assay described in Pardons et al.

[0158] [9], While the HIV-Flow assay detects intracellular p24 by combining two different anti-p24 (KC57 from BD and 28B7 from MediMabs), Env-Flow identifies Env-expressing cells among the p24+ cells using Cocktail #9

[0010] (FIGs. 1-3 and 6A). To enhances sensitivity, the antibodies (A32, 17b and 246D) from Cocktail #9 were biotinylated, and antibody binding is detected with PE-conjugated streptavidin. Interestingly, the results provide evidence that not all reactivated p24+ cells from PLWH under ART express Env.

[0159] Alternatively, to detect Env-expressing cells without requiring permeabilization or p24 detection, anti-gp120 mAbs (A32, 17b) and the anti-gp41 Ab (246D) from Cocktail #9 were conjugated to two distinct fluorochromes, Alexa Fluor™ 488 (AF488) and Alexa Fluor™ 647 (AF647), respectively (FIGs.4, 5 and 6B). By using these two different approaches — Cocktail #9 combined with anti-p24 antibodies or anti-gp120 and anti-gp41 mAbs conjugated with two fluorochromes, Env-expressing cells from a PLWH were successfully sorted and HIV-1-infected cells were substantially enriched (FIG. 6C). The detection of Env-expressing cells using the CD4 / Env co-staining uniquely enables the identification and sorting of Env-expressing cells without the need for fixation and permeabilization, representing a major advantage for downstream single-cell RNA sequencing analyses. Accordingly, CD4l0WEnv+cells were isolated using Env-flow and subjected to single-cell RNA sequencing, and their transcriptional profiles were compared to those of autologous sorted uninfected CD4+Env“ cells. Env-expressing cells displayed a distinct transcriptional signature relative to uninfected cells (FIG. 6D). These results highlight the unique advantage of Env-flow for studying the transcriptional profiles of HIV-infected cells at single-cell resolution.

[0160] CD4-targeted delivery of the viral transactivator Tat mRNA using lipid nanoparticles (LNP-Tat) has been shown to potently reactivate latent HIV, making it a strong latency-reversing agent (LRA) candidate for in vivo applications. Its capacity to induce HIV-1 Env expression, either alone or in combination with distinct classes of LRAs, including agents previously tested in clinical trials, was evaluated. CD4+T cells isolated from ART-treated people with HIV (PWH) were stimulated for 24 h with LNP-Tat alone or in combination with the histone deacetylase inhibitors panobinostat (Pano) and romidepsin (RMD), the protein kinase C (PKC) agonist ingenol, or the BET bromodomain inhibitor JQ1. Cells stimulated with PMA and ionomycin served as positive control. Surface Env expression was detected using antibody cocktail #9 followed by streptavidin-PE, while infected cells were identified by intracellular detection of HIV-1 p24 using the KC57 and 28B7 antibodies. As shown in FIG. 7A, CD4-targeted LNP-Tat robustly induced HIV-1 p24expression in CD4+T cells from ART-treated PWH, reaching frequency of p24+cells comparable to those obtained upon PMA / ionomycin stimulation. Co-treatment with panobinostat, romidepsin, or ingenol further increased the frequency of p24+cells. Although LNP-Tat alone induced lower levels of cell-surface Env expression relative to PMA / ionomycin, combination with panobinostat, romidepsin, or ingenol markedly enhanced the frequency of p24+Env+cells. Among these, the LNP-Tat / ingenol combination was particularly potent, resulting in a 34-fold increase in the frequency of p24+Env+cells relative to LNP-Tat alone. This combination also significantly increased the proportion of reactivated p24+cells that expressed Env at the cell surface (FIG.

[0161] 7B). Together, these results demonstrate that Env-flow is a powerful tool for identifying optimal LRA combinations that promote HIV-1 Env expression in cure-oriented strategies, while also defining experimental conditions that enable the detection and characterization of these rare reactivated cells.

[0162] To assess the linearity of the Env-flow assay, human primary CD4+T cells infected in vitro with HIV-1 were spiked into autologous uninfected CD4+T cells at defined ratios. Surface Env expression was detected using antibody cocktail #9 followed by streptavidin-PE. Infected cells were identified either by HIV-1 -mediated CD4 downregulation or by intracellular HIV-1 p24 staining. Both Env / CD4 (FIG. 8A) and Env / p24 co-staining (FIG. 8B) reliably and reproducibly detected Env-expressing infected cells across all dilution points, including the lowest proportion of spiked infected cells. These results demonstrate the linearity and robustness of the Env-flow assay for detecting Env-expressing cells in human samples.

[0163] To evaluate the sensitivity and specificity of Env-flow, the frequency of Env-expressing cells among CD4+T cells isolated from untreated PWH was quantified, and these values were compared to those obtained from H IV- 1 -negative donors using Env / CD4 co-detection (FIG. 8C).

[0164] CD4l0WEnv+cells were readily detected in all untreated PWH, with a median frequency of 28.9 CD4l0WEnv+cells per million CD4+T cells, whereas false-positive events were exceedingly rare in HIV-1 -negative donors (only 1 CD4l0WEnv+event among 6.5 million CD4+T cells analyzed). The frequency of Env-expressing cells in unstimulated CD4+T cells from untreated PWH and in PMA / ionomycin-stimulated CD4+T cells from ART-treated PWH using the Env / p24 co-detection was also measured, and these results were compared to those from HIV-1-negative donors (FIG.

[0165] 8D). No false-positive events were detected among all cells analyzed from HIV-1 -negative donors. In contrast, p24+Env+cells were readily detected in all untreated PWH and in the majority of ART-treated PWH (21 / 34). Based on the observed false-positive rates, the limit of detection for Env-flow was estimated at 1.1 CD4l0WEnv+and 0.6 p24+Env+cells per million CD4+T cells.

[0166] Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word "comprising" is usedas an open-ended term, substantially equivalent to the phrase "including, but not limited to". The singular forms "a", "an" and "the" include corresponding plural references unless the context clearly dictates otherwise.

[0167] REFERENCES

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Claims

WHAT IS CLAIMED IS:

1. A method for detecting latently HIV-infected cells in a biological sample comprising CD4+ cells, comprising:contacting the sample with:(a) a molecule that specifically binds within the Phe43 cavity of gp120;(b) a first gp120 binding molecule that binds to an epitope within the constant region 1 and 2 (C1-C2) portion of the cluster A region of gp120;(c) a second gp120 binding molecule that binds to an epitope within the bridging sheet of the Co-Receptor Binding Site (CoRBS) of gp120; and(d) a gp41 binding molecule that binds to an epitope located in the cluster I region of gp41; anddetecting a signal indicative of latently HIV-infected cells, wherein the signal is generated by the interaction of the first anti-gp120 antibody, the second anti-gp120 antibody, and / or the anti-gp41 antibody with the latently HIV-infected cells.

2. The method of claim 1 , wherein the epitope within the C1-C2 portion of the cluster A region of gp120 is formed by residues 51-54, 56, 58-61, 103, 106-107, 110, 114, 217, and 219-221 of gp120.

3. The method of claim 1 or 2, wherein the first gp120 binding molecule is an antibody or an antigen-binding fragment thereof.

4. The method of claim 3, wherein the first g p 120 binding molecule is antibody clone A32 or an antigen-binding fragment thereof, or an antibody that competes with antibody clone A32 or an antigen-binding fragment thereof.

5. The method of claim 4, wherein the first gp120 binding molecule is an antibody or an antigen-binding fragment thereof comprising the complementarity determining regions (CDRs) of antibody clone A32, L9-i 1 or N5-i5.

6. The method of claim 5, wherein the first gp120 binding molecule is antibody clone A32, L9-i1 or N5-i5, preferably clone A32.

7. The method of any one of claims 1 to 6, wherein the second gp120 binding molecule binds to the epitope formed by residues 119-122, 200, 202-205, 326-327, 369, 419-423 and 432-437 of gp120.

8. The method of any one of claims 1 to 7, wherein the second gp120 binding molecule is an antibody or an antigen-binding fragment thereof.

9. The method of claim 8, wherein the second gp120 binding molecule is antibody clone 17b or an antigen-binding fragment thereof, or an antibody that competes with antibody clone 17b or an antigen-binding fragment thereof.

10. The method of claim 9, wherein the second sgp120-binding molecule is an antibody or an antigen-binding fragment thereof comprising the complementarity determining regions (CDRs) of antibody clone 17b, X5, L9-i3, N5-i1, N5-i3, N5-i4, N5-i8, N10-i1.1 , N10-i5.3, N12-i1 , N12-i2, N12-i4, N12-i5, N12-i7, N12-i8, N12-i10, N12-i17, N12-i18, 48d, 412d, or N12-i9.

11. The method of claim 10, wherein the second sgp120-binding molecule is antibody clone 17b, X5, L9-i3, N5-i1, N5-i3, N5-i4, N5-i8, N10-i1.1 , N10-i5.3, N12-i1, N12-i2, N12-i4, N12-i5, N12-i7, N12-i8, N12-i10, N12-i17, N12-i18, 48d, 412d, or N12-i9, preferably antibody clone 17b orX5, more preferably antibody clone 17b.

12. The method of any one of claims 1 to 11 , wherein the gp41 binding molecule binds to an epitope located in a region defined by amino acids 579 to 623 of gp41.

13. The method of claim 12, wherein the gp41 binding molecule binds to an epitope located in a region defined by amino acids 579 to 613 of gp41.

14. The method of claim 12, wherein the gp41 binding molecule binds to an epitope located in a region defined by amino acids 579 to 604 of gp41.

15. The method of any one of claims 1 to 14, wherein the gp41 binding molecule is an antibody or an antigen-binding fragment thereof.

16. The method of claim 15, wherein the gp41 binding molecule is antibody clone 7B2, F240, M785-U1, N10-U1, 246D, 240D, 181D, or 98-43, an antigen-binding fragment thereof, or an antibody that competes with antibody clone 7B2, F240, M785-U1, N10-U1, 246D, 240D, 181D, or 98-43 or an antigen-binding fragment thereof.

17. The method of claim 15 or 16, wherein the gp41 binding molecule is an antibody or an antigen-binding fragment thereof comprising the complementarity determining regions (CDRs) of antibody clone antibody clone 7B2, F240, M785-U1, N10-U1, 246D, 240D, 181D, or 98-43.

18. The method of claim 17, wherein the gp41 binding molecule is antibody clone 246D or an antigen-binding fragment thereof.

19. The method of any one of claims 1 to 18, wherein the molecule that specifically binds within the Phe43 cavity of gp120 is a polypeptide comprising domains 1 and 2 of human CD4 receptor or a CD4 mimetic compound (CD4mc).

20. The method of claim 19, wherein the CD4mc is NBD-556, NBD-557, DMJ-l-228, JP-lll-48, M48U1, BNM-lll-170, CJFIII-288.

21. The method of claim 20, wherein the CD4mc is CJFIII-288.

22. The method of any one of claims 1 to 21, wherein the first anti-gp120 antibody, the second anti-gp120 antibody, and / or the anti-gp41 antibody are conjugated to an affinity tag or a detectable moiety.

23. The method of claim 22, wherein the affinity tag is a biotin or biotin analog.

24. The method of claim 23, wherein the method further comprises contacting the sample with a streptavidin or streptavidin analog conjugated to a detectable moiety.

25. The method of claim 24, wherein the detectable moiety is a fluorophore.

26. The method of any one of claims 22 to 25, wherein the first anti-gp120 antibody and the second anti-gp120 antibody are conjugated to a first detectable moiety, and the anti-gp41 antibody is conjugated to a second detectable moiety.

27. The method of any one of claims 1 to 26, wherein the biological sample is blood, plasma, or tissue, preferably a peripheral blood mononuclear cell (PBMC) sample.

28. The method of any one of claims 1 to 27, further comprising enriching the sample in T lymphocytes prior to said contacting.

29. The method of claim 28, wherein the T lymphocytes are CD4+ T lymphocytes.

30. The method of any one of claims 1 to 29, further comprising staining the cells with one or more markers selected from CD3, CD4, and CD8.

31. The method of any one of claims 1 to 30, further comprising detecting intracellular HIV p24 protein.

32. The method of claim 31, wherein detecting intracellular HIV p24 protein comprises permeabilizing the cells in the biological sample and contacting the permeabilized cells with one or more antibodies specific for the HIV p24 protein.

33. The method of any one of claims 1 to 32, wherein the biological sample is from an HIV-1-infected subject treated with combination antiretroviral therapy (cART).

34. The method of any one of claims 1 to 33, wherein the method further comprises contacting the sample with an agent that induces HIV reactivation from the latently HIV-infected cells (a latency-reversing agent (LRA)).

35. The method of claim 34, wherein the LRA comprises phorbol 12-myristate 13-acetate (PMA) and ionomycin, lipid nanoparticles containing HIV Trans-Activator of Transcription (Tat) mRNA (LNP-Tat), a histone deacetylase (HDAC) inhibitor, a histone methyltransferase (HMT) inhibitor, a Bromodomain and Extra-Terminal motif (BET) protein inhibitor, and / or a Protein kinase C (PKC) agonist.

36. The method of any one of claims 1 to 35, further comprising quantifying the frequency of cells bound by the first anti-gp120 antibody, the second anti-gp120 antibody, and / or the anti-gp41 antibody relative to total CD4+ T cells in the sample.

37. The method of any one of claims 1 to 36, further comprising sorting cells that are positive for binding by the first anti-gp120 antibody, the second anti-gp120 antibody, and / or the anti-gp41 antibody, and optionally analyzing the sorted cells.

38. The method of any one of claims 1 to 37, wherein the method is used to monitor changes in the number or frequency of latently HIV-infected cells in a subject over time.

39. The method of any one of claims 1 to 38, wherein the method is used as a companion diagnostic to evaluate the effect of a latency-reversing or reservoir-reducing therapy by comparing the frequency of cells bound by the combination of antibodies before and after administration of the therapy.

40. A kit comprising the molecule that specifically binds to the Phe43 cavity of gp120, the first gp120 binding molecule, the second gp120 binding molecule and the gp41 binding as defined in any one of claims 1 to 39.

41. The kit of claim 40, further comprising an agent that induces HIV reactivation from the latently infected cells (a latency-reversing agent (LRA)), an anti-CD4 antibody, and / or one or more antibodies specific for the HIV p24 protein.

42. The kit of claim 41, wherein the LRA comprises phorbol 12-myristate 13-acetate (PMA) and ionomycin, lipid nanoparticles containing HIV Trans-Activator of Transcription (Tat) mRNA (LNP-Tat), a histone deacetylase (HDAC) inhibitor, a histone methyltransferase (HMT) inhibitor,a Bromodomain and Extra-Terminal motif (BET) protein inhibitor, and / or a Protein kinase C (PKC) agonist.

43. The kit of any one of claims 40 to 42, further comprising a streptavidin or streptavidin analog conjugated to a detectable moiety.

44. The kit of any one of claims 40 to 43, further comprising instructions for detecting latently HIV-infected cells in a biological sample according to the method defined in any one of claims 1 to 38.

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