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. Author manuscript; available in PMC: 2025 May 29.
Published in final edited form as: Nat Med. 2025 Jan 3;31(2):427–432. doi: 10.1038/s41591-024-03357-0

Sustained virologic suppression of multidrug-resistant HIV in an individual treated with anti-CD4 domain 1 antibody and lenacapavir

M Ali Rai 1, Jana Blazkova 1, Lela Kardava 1, Jesse S Justement 1, Victoria Shi 1, Maegan R Manning 1, Aniqa Shahid 2,3, Winnie Dong 3, Brooke D Kennedy 1, Adeline B Sewack 1, Jeanette Higgins 4, Clarisa M Buckner 1, Kathleen Gittens 5, Raymond E West 6, Aaron S Devanathan 6, Ralph Mangusan 7, Kathryn Lurain 7, Ramya Ramaswami 7, Robert Yarchoan 7, Michael C Sneller 1, Alice K Pau 8, Zabrina L Brumme 2,3, Susan Moir 1, Tae-Wook Chun 1,✉
PMCID: PMC12120854  NIHMSID: NIHMS2080014  PMID: 39753965

Abstract

The clinical management of people with multidrug-resistant (MDR) human immunodeficiency virus (HIV) remains challenging despite continued development of antiretroviral agents. A 58-year-old male individual with MDR HIV and Kaposi sarcoma (KS) was treated with a new antiretroviral regimen consisting of anti-CD4 domain 1 antibody UB-421 and capsid inhibitor lenacapavir. The individual experienced delayed but sustained suppression of plasma viremia and a substantial increase in the CD4+ T cell count. A longitudinal examination of plasma HIV and infectious isolates showed no evidence of viral evolution or the emergence of UB-421- or lenacapavir-resistant viruses. The individual received three cycles of liposomal doxorubicin and five doses of anti-programmed cell death protein 1 (PD-1) monoclonal antibody pembrolizumab that resulted in improvement in KS with flattening of lesions. Our data demonstrate that combination therapy with UB-421 could provide sustained virologic suppression in people harboring MDR HIV with limited therapeutic alternatives.

Modern antiretroviral therapy (ART) is highly effective in suppressing viral replication and has led to transformative improvements in the health of people with human immunodeficiency virus (HIV; PWH)1. Although ART is not curative and requires life-long adherence, the vast majority of PWH receiving antiretroviral drugs achieve sustained virologic suppression, provided that they do not harbor multidrug-resistant (MDR) HIV2–4. PWH with MDR HIV, including those with multiple treatment failures and substantial resistance to existing antiretroviral drugs, present compelling challenges in the clinical management of HIV infection5. The development of MDR HIV can be attributed to numerous factors, such as suboptimal antiretroviral drug regimens, difficulties in adherence to medication, treatment-associated side effects/toxicities or infection with MDR HIV. Although the prevalence of MDR HIV infection remains relatively low in high-resource settings6,7, cases of pretreatment and acquired HIV resistance to antiretroviral drugs, including the integrase inhibitor dolutegravir, are rising in low-resource settings8. In addition, PWH with MDR HIV are at greater risk for disease progression, hospitalization, opportunistic infections and death9. Consequently, there are ongoing efforts to develop therapeutic regimens that contain new drugs with unique mechanisms of action for the treatment of MDR HIV infection. Since the introduction of new drugs, such as the gp120 attachment inhibitor fostemsavir10, CD4 post-attachment inhibitor ibalizumab11 and capsid inhibitor lenacapavir12, the emergence of resistant HIV to these agents has been noted in PWH treated with current salvage regimens13. Therefore, there is a need to continue developing new therapeutic agents, especially those with a high threshold against viral mutation and strong suppression of HIV replication in vivo.

UB-421 is an Fc-aglycosylated, non-T cell-depleting, humanized IgG1 that binds to the domain 1 of the CD4 molecule on human lymphocytes14. In a recent phase 2 study, monotherapy with UB-421 was shown to potently suppress plasma viremia in PWH undergoing treatment interruption of ART, without evidence for the emergence of UB-421-resistant HIV14. In addition, a recent study of 93 infectious viral isolates derived from 11 PWH harboring MDR HIV found no resistance to UB-421 in vitro15. Here we report longitudinal findings in an individual with MDR HIV and Kaposi sarcoma (KS) who received a new therapeutic regimen consisting of weekly infusions of UB-421 and biannual injections of lenacapavir.

A 58-year-old male individual with MDR HIV receiving a failing ART regimen was referred to the HIV Outpatient Clinic at the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), for the evaluation of alternative treatment options. He had an extensive antiretroviral drug treatment history dating back to 1989, when he was first diagnosed with HIV infection and generalized lymphadenopathy during a routine physical examination. He was started on zidovudine monotherapy. Over the next three decades, he cycled through multiple ART regimens (Extended Data Table 1). When he was referred to our clinic, his ART regimen comprised fostemsavir, ibalizumab, darunavir/ritonavir and tenofovir/emtricitabine. Of note, phenotypic resistance testing confirmed the presence of fostemsavir-resistant virus in his plasma. His HIV plasma viremia was 295,656 copies per ml and CD4+ T cell count was 23 cells per mm3. In addition, he had developed lesions that were diagnosed as KS by biopsy and lymphedema in his left ankle. The genotypic analysis of his plasma HIV revealed resistance to nucleoside reverse transcriptase inhibitors including resistance to emtricitabine and tenofovir, non-nucleoside reverse transcriptase inhibitors, protease inhibitors including darunavir/ritonavir, and integrase inhibitors including dolutegravir (Extended Data Table 2). Further testing of multiple autologous, replication-competent HIV isolates derived from the patient revealed resistance to ibalizumab, but not UB-421 (Extended Data Fig. 1a,b). Of note, the baseline (before initiation of UB-421 and lenacapavir) viral isolates were also highly resistant to all but one of the HIV-specific broadly neutralizing antibodies (bNAbs) being considered for clinical development (Extended Data Fig. 1a). Given the ineffectiveness of his ART regimen in suppressing his HIV plasma viremia as well as the low CD4+ T cell count and the presence of KS, we sought expanded access to UB-421 from the Food and Drug Administration (FDA). Upon receiving approval from the FDA and the Institutional Review Board of the NIH, he provided written informed consent and discontinued ibalizumab and fostemsavir, initiating weekly infusions of UB-421 (5 mg kg−1) and injections of lenacapavir every 6 months. He remained on tenofovir/emtricitabine along with prophylactic trimethoprim/sulfamethoxazole and azithromycin. As shown in Fig. 1a, his HIV plasma viremia declined substantially within the first two weeks after receiving UB-421 and lenacapavir, then continued to decrease at a slower rate over time. Although his CD4+ T cell count gradually increased, the KS lesions around his ankle spread and increased in number, possibly facilitated by either the prolonged period of a severely compromised immune system before UB-421 and lenacapavir treatment or by immune reconstitution inflammatory syndrome (Fig. 1b). The patient was started on liposomal doxorubicin (Fig. 1a,b). However, he only received three cycles (administered at weeks 12, 15 and 19) of liposomal doxorubicin, which were limited by worsening fatigue, neutropenia and prolonged diarrhea. The gastrointestinal pathogen panel revealed the presence of norovirus, which persisted for several months. After discontinuing liposomal doxorubicin, his CD4+ T cell counts gradually increased, while his HIV plasma viremia continued to decline, reaching <20 copies per ml for the first time at week 29 (Fig. 1a). However, his KS lesions worsened, and he was administered the PD-1 monoclonal antibody (mAb) pembrolizumab as a lymphocyte-sparing treatment16. He received five cycles of pembrolizumab, 200 mg, administered intravenously every 3 weeks without immune-related adverse effects, which resulted in improvement in KS with flattening of several of his lesions (Fig. 1b).

Fig. 1 |. Clinical parameters, pharmacodynamics and efficacy findings in the patient with KS and MDR HIV following initiation of UB-421 and lenacapavir.

Fig. 1 |

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a, The patient’s longitudinal HIV plasma viremia and CD4+ T cell count. The green triangles and purple diamonds indicate the administration of UB-421 and lenacapavir, respectively. The red dotted horizontal line indicates the limit of detection of the assay (20 copies of HIV RNA per ml). The white triangles indicate undetectable plasma viremia. The timing of liposomal doxorubicin and pembrolizumab administration is shown (teal/black diamonds and orange/brown down-pointing triangles, respectively). b, KS lesions on the ankle of the patient over time. c, The percent receptor occupancy of UB-421 on CD4+ T cells (left) and the in vitro sensitivity to UB-421 of autologous, replication-competent HIV isolates derived from the patient at baseline and week 12 (right). Measurements of percent receptor occupancy were performed once at each time point. Measurements of IC80 values were performed in duplicate for each viral isolate. d, The plasma pharmacokinetics of lenacapavir over time (left) and the in vitro sensitivity to lenacapavir of autologous, replication-competent HIV isolates derived from the patient at baseline and week 12 (right). Measurements of lenacapavir concentration in plasma were performed once at each time point. Measurements of IC80 values were performed in duplicate for each viral isolate. P values were determined using the two-tailed Mann–Whitney test.

The initiation of UB-421 and lenacapavir led to a rapid reduction in the patient’s HIV plasma viremia, followed by a protracted second-phase decay lasting more than 30 weeks (Fig. 1a). The slower-than-expected second-phase decay of plasma viremia prompted us to longitudinally examine the pharmacodynamics of and in vitro autologous virus susceptibility to UB-421 and lenacapavir. Domain 1 of CD4 on his T cells remained fully occupied by UB-421 throughout the study period, except for the two time points when UB-421 administration was delayed due to the patient’s scheduling conflict (Fig. 1c). The sensitivity (80% inhibitory concentration, IC80) to UB-421 of his infectious HIV isolates remained unchanged at week 12 compared to baseline (Fig. 1c). Plasma lenacapavir concentrations remained above the in vitro protein-adjusted 95% effective concentration (EC95; 3.87 ng ml−1)17 throughout the 48 weeks (Fig. 1d), consistent with previous studies18. The IC80 of lenacapavir against the infectious HIV isolates remained unchanged (Fig. 1d). These data suggest that despite the persistence of residual plasma viremia for a prolonged period, the viruses in the patient remained sensitive to both UB-421 and lenacapavir.

HIV-induced CD4+ T cell lymphopenia is associated with wide-ranging immune cell dysregulation, including among B cell and T cell compartments19. Before the initiation of UB-421 and lenacapavir, over 95% of the B cells in the peripheral blood mononuclear cells (PBMCs) of the patient consisted of clusters with immature or transitional and naïve phenotypes (Fig. 2a and Extended Data Fig. 2a), as previously reported for advanced HIV disease19. During the treatment period, there was a modest decrease in immature or transitional B cells and an increase in early memory B cells, punctuated by large transient expansions of a cluster of ectonucleotidase CD73-expressing naïve and immature or transitional B cells during the two periods of KS exacerbation (Fig. 2a). During the treatment period, the overall composition of T cell subsets remained relatively stable (Fig. 2a and Extended Data Fig. 2b). Notably, a population of terminally differentiated (TTD) senescent CD8+ T cells (cluster 1) persisted throughout the treatment period; however, the overall percentage of these cells decreased over time. In addition, there was an expansion of central memory (TCM) and transitional memory (TTM) CD4+ T cell populations with a TH2 and T regulatory phenotype (cluster 13).

Fig. 2 |. Immunologic and virologic analyses of the peripheral blood and plasma of the patient following the initiation of UB-421 and lenacapavir.

Fig. 2 |

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a, High-dimensional immunophenotyping of B and T cells over time. High-dimensional immunophenotyping was conducted by spectral flow cytometry to identify B and T cell populations in PBMCs. b, The longitudinal frequency of CD4+ T cells carrying intact and defective HIV proviral DNA (top) and cell-associated HIV RNA (bottom). c, Maximum-likelihood phylogenies inferred from HIV gag and HIV env sequences derived from longitudinal plasma HIV RNA, with baseline and post-therapy sequences denoted by unique symbols. The phylogenetic trees are outgroup-rooted using the subtype B sequence KU678055, which is the genetically closest publicly available full-genome HIV sequence to the patient. Scale bars are shown in estimated substitutions per nucleotide site. The clade branching near the root of the env tree that contains one week 25 and one week 32 sequence, which is mentioned in the population genetic structure analysis, is highlighted in blue. The bottom graphs show the root-to-tip distances of HIV gag and HIV env sequences sampled post-therapy, analyzed using simple linear regression.

Next, we longitudinally examined the kinetics of HIV reservoirs in the patient’s PBMCs. The frequency of cells carrying intact, 5′- and 3′-defective HIV proviral DNA decreased by 0.64, 0.59 and 0.25 logs, respectively, between baseline and week 68, but infected cells persisted throughout the treatment period (Fig. 2b). The level of cell-associated HIV RNA decreased rapidly by week 8 but remained stable thereafter (Fig. 2b).

To address the genetic makeup and potential source of residual plasma viremia, we conducted phylogenetic analyses of plasma HIV RNA gag and env sequences sampled at baseline and longitudinally following the initiation of UB-421 and lenacapavir. The diversity of baseline gag and env was very high and, following treatment with UB-421 and lenacapavir, viral sequences interspersed through the phylogenetic trees and remained highly diverse (Fig. 2c and Extended Data Fig. 3). Notably, post-therapy sequences did not increase in divergence from the root over time (Fig. 2c, bottom). In fact, post-therapy gag and env sequences were comparable to baseline sequences in terms of divergence (Extended Data Fig. 4). Analysis of pre- and post-treatment gag sequences also revealed no evidence of temporal changes in population genetic structure imposed by the treatment (KST 0.01, P = 0.2), and post-treatment HIV gag sequences showed no evidence of emerging resistance mutations to lenacapavir (Supplementary Table 1, https://hivdb.stanford.edu/hivdb-capsid/by-sequences/). There was also no significant evidence that the treatment induced any temporal changes in population genetic structure within the env region (KST 0.02, P = 0.07), after we excluded two post-treatment env sequences (highlighted in blue) from weeks 25 and 32 that clustered in a distinct clade near the root of the tree, suggesting that they originated from older viral reservoirs (Fig. 2c, blue highlighting in env tree). Collectively, these observations suggest that persistent residual plasma viremia during the treatment period was attributable to a continuous release of virions from diverse and short-lived cellular reservoirs, not ongoing HIV evolution during therapy.

In this study, we present a case of a patient harboring near pan-resistant HIV with a history of extensive treatment, failing multiple antiretroviral drug regimens and a severely compromised immune system that contributed to the development of KS. This case reports the successful treatment of MDR HIV with UB-421 as a part of a salvage drug regimen. For 70 consecutive weeks, infusions of UB-421 were well-tolerated without any marked side effects and resulted in sustained virologic suppression and a slow but steady increase of CD4+ T cells. There was no negative interaction between UB-421 and the KS-directed therapies.

The clinical management of patients with extensive MDR HIV remains challenging despite the continued development of antiretroviral drugs20,21. New classes of drugs, such as lenacapavir, have recently become available for the treatment of MDR HIV infection12. However, lenacapavir-resistant HIV has already been reported in PWH harboring MDR viruses who were unintentionally receiving a monotherapeutic regimen13,22. There is an urgent need to address the rapid emergence of HIV resistance to antiretroviral agents, especially in heavily treatment-experienced patients. This can best be achieved using drugs and biological agents with potent antiretroviral activity and a high threshold for the appearance of resistant viruses. The prolonged history of failing treatments and extensive HIV resistance profile of our patient strongly suggested that no single effective antiretroviral agent, including lenacapavir, could have provided clinically meaningful virologic suppression or immunologic recovery. While it is not possible to determine the individual contribution of each drug (UB-421 and lenacapavir) to the antiviral activity that ultimately led to the suppression of plasma viremia in this patient, it is highly likely that UB-421 was critical to the effectiveness of the treatment regimen. The unique ability of UB-421 to inhibit viral entry and prevent the selection of resistant HIV14 likely had an important role in the successful suppression of HIV plasma viremia in our patient. Although his residual plasma viremia (>20 copies per ml) persisted for more than 30 weeks following the initiation of UB-421 and lenacapavir, there was no evidence of viral evolution in the plasma or the emergence of resistant viruses against these drugs.

One of the primary reasons we sought expanded access to UB-421 for our patient was to suppress his HIV plasma viremia, enable the improvement of his immune system and aid in the control of his KS23. The KS lesions, which were present at baseline, progressively worsened despite the suppression of HIV plasma viremia and therefore warranted KS-directed treatment with liposomal doxorubicin and later pembrolizumab. Although further studies would be necessary to delineate the interplay between host immunity and KS in the setting of MDR HIV infection, it is likely that HIV-targeted treatment alone was not sufficient to prevent the progression of KS in the absence of any substantial immunologic recovery during the follow-up period. This was evident from the slow recovery and normalization of B cells and T cells in the peripheral blood of our patient and the transient expansions of CD73-expressing B cells but not T cells, which may have been related to KS-associated effects on B cells24. Of note, the treatment with pembrolizumab led to an improvement in the KS in our patient, suggesting that immune exhaustion may have played a role in the persistence of KS during the period of CD4+ T cell recovery16. Despite a steady increase in CD4+ T cell counts over time, it remains to be determined whether a substantial immunologic recovery or normalization of CD4+ T cells is feasible in our patient, given the prolonged severe lymphopenia and high plasma viremia before the initiation of UB-421 and lenacapavir11. Additional therapeutic interventions may be necessary to promote the recovery of CD4+ T cells in PWH harboring MDR HIV.

In summary, we demonstrated that by targeting domain 1 of CD4 with UB-421, sustained virologic control could be achieved in a patient harboring MDR HIV with a history of multiple treatment failures and no other viable clinical options. Further investigation is needed to enhance the in vivo pharmacodynamics of UB-421. However, UB-421 could be considered as a direct-acting or companion drug to treat PWH harboring multidrug- or pan-resistant viruses with virus-associated cancer or a life-threatening condition. In addition, monotherapy with UB-421 could potentially contribute to the treatment of PWH with MDR HIV by substantially lowering the initial plasma viral burden, thus allowing other therapeutic agents that require lower plasma viremia thresholds to be considered.

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Methods

UB-421 product and dose administration

The active ingredient of UB-421 is a CHO-expressed, Fc-aglycosylated, humanized IgG1/κ mAb that was produced under good manufacturing practices and supplied at a concentration of 10 mg ml−1. The patient received intravenous infusions of 5 mg kg−1 weekly.

Determination of CD4+ T cell count and receptor occupancy

The CD4+ T cell count was determined using an anti-CD4 domain 3 antibody (BioLegend, 317416). Mean fluorescence intensity (MFI) of CD4 domain 1 was calculated using Multitest 6-Color TBNK mAb (BD Biosciences, 662967). Cells were stained with antibodies, then lysed with BD FACS Lysing solution (BD Biosciences, 349202) and analyzed immediately on a FACSLyric flow cytometer (BD Biosciences) using BD FACSuite Clinical v2.5.0.24. The percent receptor occupancy was calculated using the following formula: (baseline MFI-post-treatment MFI)/baseline MFI × 100.

Quantification of lenacapavir concentrations in plasma

Lenacapavir concentrations were quantified in the plasma using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) with a previously validated method25. Briefly, a UPLC–MS/MS system consisting of a Thermo Scientific Vanquish UPLC and Thermo Scientific TSQ Altis equipped with a heated ESI (HESI) source was used. The parent → product ion transitions used for quantitation were m/z 968.3 → 869.0 for lenacapavir and m/z 972.8 → 873.8 for (13C, 2H3)-lenacapavir. Chromatographic separation of the samples was accomplished with a Waters CORTECS T3 (2.1 × 50 mm, 1.6 μm) column with a gradient elution using water with 0.15% formic acid (A) and methanol (B) at a flow rate of 0.5 ml min−1. The linear range of the standard curves for lenacapavir was 0.1–500 ng ml−1 (r2 ≥ 0.9960) with an interassay precision and accuracy within 15% using Xcalibur Software 4.2.47 (Thermo Fisher Scientific).

B cell and T cell immunophenotyping

PBMCs were isolated from peripheral blood by Ficoll-Hypaque density gradient centrifugation. Immunophenotyping was performed by spectral flow cytometry to identify major B cell and T cell populations among PBMCs (see Supplementary Tables 2 and 3 for lists and sources of antibodies). Freshly isolated 1 × 106 PBMCs were stained with the viability reagent Zombie NIR (BioLegend, 423106) and cocktails of 21 (for B cells) and 28 (for T cells) monoclonal antibodies in a staining buffer (2% FBS/PBS) supplemented with Super Bright Complete Staining Buffer and CellBlox Blocking Buffer (Thermo Fisher Scientific). The stained cells were fixed with BD FACS Lysing solution (BD Biosciences, 349202), acquired on an Aurora spectral cytometer using SpectroFlo Software v3.2.1 (Cytek Biosciences) and analyzed using FlowJo v10.10 (BD Biosciences). Gated CD45+CD3−CD19+ B cells and CD45+CD3+T cells were exported from all time points for downstream analysis.

UMAP embedding and FlowSOM clustering analyses

FlowSOM clustering and Uniform Manifold Approximation and Projection (UMAP) embedding were performed on exported FCS files using the OMIQ platform (omiq.ai). B cell panel markers CD20, CD10, CD38, CD21, CD27, IgM, IgD, IgG, IgA, CD71, CD11c, CXCR3, CD95, CD11a, CD29, CD39, CD45RB, and CD73 and T cell panel markers CD4, CD8, CD45RA, CCR7, CD27, CD28, CD38, HLA-DR, CD226, TIGIT, PD-1, 2B4, CD160, CD161, CCR4, CCR5, CCR6, CXCR3, CXCR4, CD69, CD62L, KLRG1, CD57, CD127, CD25, and CD95 were used to perform UMAP embedding and FlowSOM clustering with the number of clusters set to 20 for B cells and 15 for T cells.

Intact proviral DNA assay

The frequency of CD4+ T cells carrying intact HIV proviruses was determined by modified Intact proviral DNA assay (IPDA)26,27 using the QIAcuity Digital PCR system (Qiagen). Genomic DNA was isolated using QIAamp DNA mini kit (Qiagen) according to the manufacturer’s instructions. Digital PCR was conducted using two sets of primers and probes specific for HIV gag and env and for two segments of housekeeping gene RPP30 in two separate reactions. A total of 910 ng DNA was used for the HIV quantification using the following primers and probes: HIV gag specific, gag forward—5′-GACTAGCGGAGGCTAGAAGGAGAGA-3′ (nt 764–788 in HXB2; GenBank, K03455.1), gag reverse—5′-CTAATTCTCCCCCGCTTAATAYTGACG-3′ (nt 829–803) and gag LNA probe—5′−6FAM-A + T + GGG + TG + CGAGA-IABkFQ-3′ (nt 790–801); HIV env specific, env forward—5′-AGTGGTGCAGAGAGAAAAAAGAGC-3′ (nt 7736–7759), env reverse—5′-GTCTGGCCTGTACCGTCAGC-3′ (nt 7851–7832) and env probes—5′-VIC-CCTTGGGTTCTTGGGA-MGB-3′ (nt 7781–7798 and unlabeled hypermutated probe 5′-CCTTAGGTTCTTAGGAGC-MGB-3′). A total of 9.1 ng DNA was used for the RPP30 quantification using the following primers and probes: RPP30–1 forward—5′-GATTTGGACCTGCGAGCG-3′ (nt 29–46; GenBank: NC_000010.11), RPP30–1 reverse—5′-GCGGCTGTCTCCACAAGT-3′ (nt 90–73) and RPP30–1 probe—5′−6FAM-TTCTGACCTGAAGGCTCTGCGC-IABkFQ-3′ (nt 49–70); RPP30–2 forward—5′-GTGTGAGTCAATCACTAGACAGAA-3′ (nt 7012–7035), RPP30–2 reverse—5′-AAACTGCAACAACATCATAGAGC-3′ (nt 7135–7113) and RPP30–2 probe—5′-HEX-AGAGAGCAACTTCTTCAAGGGCCC-IABkFQ-3′ (nt 7088–7091). Three technical replicates were performed for each sample. Intact HIV DNA copy numbers (gag and env double-positive partitions) were normalized per 1 × 106 CD4+ T cells and adjusted for DNA shearing using DNA shearing index (DSI) based on a ratio of double-positive RPP30 partitions.

Quantitation of cell-associated HIV RNA

The level of CD4+ T cell-associated HIV RNA was measured by RT–PCR. Total RNA was isolated from PBMCs using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using qScript XLT cDNA Master Mix (Quanta Biosciences), followed by digital PCR (Qiagen) using the following primers: HIV-specific primers, HIV-US-F—5′-TCTCTAGCAGTGGCGCCCGAACA-3′ (nt 626–648), HIV-US-R—5′-TCTCCTTCTAGCCTCCGCTAGTC-3′ (nt 786–764) and HIV-US-probe—5′−6FAM-CAAGCCGAGTCCTGCGTCGAGAG-IABkFQ-3′ (nt 705–683), and housekeeping gene encoding TATA box-binding protein (TBP)-specific primers, TBP-F—5′-CACGAACCACGGCACTGATT-3′ (nt 863–882; GenBank, NM_003194.5), TBP-R—5′-TTTTCTTGCTGCCAGTCTGGAC-3′ (nt 951–930) and TBP-probe—5′-HEX-TGTGCA CAGGAGCCAAGAGTGAAGA/3-IABkFQ-3′ (nt 902–926). Copy numbers of cell-associated HIV RNA were normalized per 1 × 106 copies of TBP.

Single-genome plasma HIV RNA sequencing

Total nucleic acids were extracted from 500 μl to 1 ml plasma aliquots using the NucliSENS EasyMag (BioMerieux) and were DNAse treated. For the post-therapy time points, up to 8 ml plasma was extracted. cDNA was generated using HIV-specific reverse primers, listed below, and was then endpoint-diluted such that the subsequent nested PCR reactions amplifying the full gag and near full-length env genes (~1,300 to 1,500 bases) yielded no more than 30% positive reactions. For gag, first round primers were Gag+2 forward—5′-GATCTCTCGACGCAGGACTCGGCT-3′ (nt 680–703) and PR.R1 reverse—5′-CTGAAATCTACTAATTTYCTCCA-3′ (nt 2782–2760). The second round primers were Gag+3 forward—5′-GCGGCGACTGGTGAGTACGC-3′ (nt 734–753) and ProC– reverse—5′-GAGTATTGTATGGATTTTCAGGCCCAAT-3′ (nt 2724–2697). For env, first round primers were ENVF.1 forward—5′-GAAAGAGCAGAAGACAGTGGCA-3′ (nt 6203–6224) and CO602 reverse—5′-GCCCATAGTGCTTCCTGCTGCTCCCAAGAACC-3′ (nt 7817–7786). The second round primers were MK603 forward—5′-CAGAAAAATTGTGGGTCACAGTCTATTATGGGGTACCT-3′ (nt 6316–6353) and CD4R reverse—5′-TATAATTCACTTCTCCAATTGTCC-3′ (nt 7675–7652).

Negative controls were included in every PCR run and control reactions performed without reverse transcription using more concentrated templates generated no amplicons, confirming that DNAse treatment was successful. Amplicons were sequenced using a 3730xl Automated DNA Sequencer (Applied Biosystems). Chromatograms were analyzed using Sequencher (v5.4.6; GeneCodes). Sequences containing nucleotide mixtures were excluded from the analysis. Sequences were also screened for hypermutation (using Hypermut2.0 (ref. 28)) and suspected within-host recombination (using RDP4 (ref. 29)). No sequences with these properties were identified.

Phylogenetic inference

Within-host gag and env sequences were aligned in a codon-aware manner in MAFFT version 7.471 (ref. 30) and manually edited in AliView version 1.26 (ref. 31). Phylogenies were inferred with IQ-TREE 232 following automated model selection using ModelFinder with an AIC selection criterion33. Here the HIV subtype B sequence KU678055, which represented the most genetically similar full-genome HIV sequence to the study participant in the Los Alamos HIV database (www.hiv.lanl.gov), was used as an outgroup. Branch support values were derived from 1,000 bootstraps. Phylogenies were visualized using the R (v4.1.2) package ggtree, version 3.21 (ref. 34).

Lenacapavir resistance interpretations

Within-host gag sequences were analyzed for the presence of resistance mutations to lenacapavir using the HIV-1 Capsid: Sequence Analysis tool, hosted by the Stanford University HIV Drug Resistance Database (https://hivdb.stanford.edu/hivdb-capsid/by-sequences/).

Tests for temporal genetic population structure pre- and post-therapy

We applied Hudson, Boos and Kaplan’s nonparametric test for population structure (KST)35, implemented in hyPhy36. This test compares the mean pairwise genetic distances between categories (in this case, pretherapy versus all post-therapy sequences) and within the same category. Population structure is supported if within-category distances are smaller than between-category distances, where statistical significance is assessed via a population-structure randomization test. Here we analyzed all distinct sequences per category (that is, identical sequences were collapsed to a single representative per group before analysis). The KST test statistic ranges from 0 (no evidence for population structure) to 1 (fully distinct populations), where P values are derived from 1,000 permutations.

Statistical analysis

Comparisons between two groups were performed using the Mann–Whitney U test. Relationships between continuous variables were assessed using linear regression, conducted in Prism 10.0.2 (GraphPad).

HIV suppression assay

Near-clonal replication-competent HIV isolates were derived from coculturing CD8-depleted PBMCs of the patient with CD8-depleted anti-CD3 stimulated PBMCs from HIV-seronegative donors, as previously described37,38. The concentration of infectious viral isolates was determined by HIV p24 ELISA, and then each isolate was subsequently titrated using TZM-bl cells. Each viral isolate was incubated with 10 μg ml−1 of bNAbs and anti-CD4 antibodies for 90 min followed by incubation with TZM-bl cells for 48 h. Cells were subsequently lysed and substrate (Promega) was added to measure the luciferase activity (Tecan). The level of viral suppression by each bNAb was assessed over media controls.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Extended Data

Extended Data Fig. 1 |. Neutralization and suppression capacity of bNAbs and anti-CD4 antibodies against replication-competent viral isolates derived from the patient with MDR HIV.

Extended Data Fig. 1 |

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a, The y axis indicates percentage suppression over control, as determined by the TZM-bl neutralization assay. Measurements for each viral isolate were performed in duplicate. Black bars represent the median values. b, The IC80 values of ibalizumab against the infectious clones obtained from the patient. Measurements for each viral isolate were performed in duplicate. The P value was determined using the two-tailed Mann–Whitney test.

Extended Data Fig. 2 |. High-dimensional phenotypic profiling of B and T cells following initiation of UB-421 and lenacapavir.

Extended Data Fig. 2 |

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a,b, UMAP and heatmaps of the patient CD19+ B cells (a) and CD3+ T cells (b). The heatmaps show intensities of expression for individual markers in each of the 15 clusters shown. Only the top 15 of 20 clusters are shown for B cells. UMAP plots show intensity of expression for each marker (left) and identification of B and T cell clusters generated by FlowSOM (right).

Extended Data Fig. 3 |. Within-host gag and gp120 phylogenies in rectangular format.

Extended Data Fig. 3 |

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These are the same rooted trees as shown in Fig. 2c. Asterisks denote nodes with bootstrap support values ≥ 70%. Scale in estimated substitutions per nucleotide site.

Extended Data Fig. 4 |. Root-to-tip distances of pre- versus post-therapy sequences.

Extended Data Fig. 4 |

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Horizontal black lines denote medians. Each measurement was performed once at each time point. P values were computed using the two-tailed Mann–Whitney U test.

Extended Data Table 1 |.

Antiretroviral drug treatment history

Timeline Antiretroviral drugs
1989–2004 Regimens included: Zidovudine, Didanosine, Zalcitabine, Stavudine, Indinavir, Saquinavir, Amprenavir, Efavirenz, Nevirapine
06/2004 Abacavir, Lamivudine, Zidovudine, Tenofovir, Lopinavir/ritonavir
03/2005 Abacavir, Lamivudine, Azidothymidine, Tenofovir, Lopinavir/ritonavir, Atazanavir
09/2008 Abacavir, Tenofovir, Emtricitabine, Darunavir/ritonavir, Raltegravir
Early 2012 Tenofovir, Emtricitabine, Lopinavir/ritonavir, Atazanavir
10/2014 Tenofovir, Emtricitabine, Darunavir/ritonavir, Dolutegravir, Maraviroc
10/2015 Tenofovir, Emtricitabine, Darunavir/ritonavir, Maraviroc
08/2016 Tenofovir, Emtricitabine, Darunavir/ritonavir
01/2020 Tenofovir, Emtricitabine, Darunavir/ritonavir, Fostemsavir, Ibalizumab
08/2021 Tenofovir, Emtricitabine, Darunavir/ritonavir, Fostemsavir, Ibalizumab
02/2022 Tenofovir, Emtricitabine, Fostemsavir, Ibalizumab
06/2022 – 03/2023 Tenofovir, Emtricitabine, Darunavir/ritonavir, Fostemsavir, Ibalizumab
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Extended Data Table 2 |.

HIV drug resistance mutationsa in the case patient prior to initiation of UB-421 and lenacapavir

Drug class Drug Mutation list Scoreb Rangec Interpretation
Nucleoside Abacavir M184V, M41L, D67E, T69ins, A62V 90 5 High-Level Resistance
reverse Zidovudine M184V, T215D, M41L, D67E, T69ins, A62V 105 5 High-Level Resistance
transcriptase Stavudine M184V, T215D, M41L, D67E, T69ins, A62V 105 5 High-Level Resistance
inhibitors Didanosine M184V, T215D, M41L, D67E, T69ins, A62V 105 5 High-Level Resistance
Emtricitabine M184V, T69ins 90 5 High-Level Resistance
Lamivudine M184V, T69ins 90 5 High-Level Resistance
Tenofovir M184V, M41L, D67E, T69ins, A62V 65 5 High-Level Resistance
Non-nucleoside Doravirine Y181C, K101E, G190A, A98G 60 5 High-Level Resistance
reverse Efavirenz Y181C, K101E, G190A, A98G 115 5 High-Level Resistance
transcriptase Etravirine Y181C, K101E, G190A, A98G 90 5 High-Level Resistance
inhibitors Nevirapine Y181C, K101E, G190A, A98G 190 5 High-Level Resistance
Rilpivirine Y181C, K101E, G190A, A98G 135 5 High-Level Resistance
Protease Atazanavir/r K20T, V32I, L33F, M46L, I54L, I84V, L90M 165 5 High-Level Resistance
inhibitors Darunavir/r V32I, L33F, I54L, L89V, I84V 85 5 High-Level Resistance
Fosamprenavir/r K20T, V32I, L33F, M46L, I54L, L89V, I84V, L90M 255 5 High-Level Resistance
Indinavir/r K20T, V32I, L33F, M46L, I54L, L89V, I84V, L90M 190 5 High-Level Resistance
Lopinavir/r V32I, L33F, M46L, I54L, L89V, I84V, L90M 130 5 High-Level Resistance
Nelfinavir K20T, V32I, L33F, K43T, M46L, I54L, L89V, I84V, L90M 270 5 High-Level Resistance
Saquinavir/r K20T, L33F, M46L, I54L, I84V, L90M 160 5 High-Level Resistance
Tipranavir/r V32I, L33F, K43T, M46L, I54L, I84V, L90M 60 5 High-Level Resistance
Integrase Bictegravir G140GS, Q148QH 45 4 Intermediate Resistance
inhibitors Cabotegravir G140GS, Q148QH 60 5 High-Level Resistance
Dolutegravir G140GS, Q148QH 45 4 Intermediate Resistance
Elvitegravir G140GS, Q148QH 90 5 High-Level Resistance
Raltegravir G140GS, Q148QH 90 5 High-Level Resistance
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b

The Score indicates the sum of each mutation penalty score for a drug. Scores less than 10 indicate susceptible; between 10 and 14 indicate potential low-level resistance; between 15 and 29 indicate low-level resistance; and between 30 and 59 indicate intermediate resistance.

c

The range of 1 and 5 indicate susceptible and high-level resistance to a drug, respectively.

Supplementary Material

supp

Acknowledgements

We are grateful to the dedication of the patient who was the participant of this study. We thank United BioPharma for providing UB-421, H. C. Lane, the NIH Clinical Center and NIAID HIV Outpatient Clinic staff for their assistance in the execution of this study, the British Columbia Centre for Excellence in HIV/AIDS laboratory for support and expertise in HIV sequence analysis and the NIH AIDS Reagent Program for providing HIV-specific mononuclear antibodies.

This work was supported by the Division of Intramural Research of the NIAID and the National Cancer Institute (NCI), NIH, and the services of the University of Pittsburgh Small Molecule Biomarker Core, which was partially funded by NIH through S10OD028540. The plasma HIV RNA sequencing was supported by the Martin Delaney ‘REACH’ Collaboratory (NIH grant 1-UM1AI164565–01 to Z.L.B.). A.S. was supported by a Doctoral Award from the Canadian Institutes for Health Research. Z.L.B. was supported by a Scholar Award from Michael Smith Health Research BC. The funders had no role in study design, data collection and analysis, the decision to publish or preparation of the manuscript.

Footnotes

Competing interests

R.Y., R.R. and K.L. report receiving research support from Celgene (now Bristol-Myers Squibb), CTI BioPharma (a Sobi A.B. Company), PDS Biotech, and Janssen Pharmaceuticals, drugs for clinical trials from Merck, EMD-Serano and Eli Lilly, and preclinical material from Lentigen Technology through CRADAs or MTAs with the NCI. R.Y. is a co-inventor of US Patent 10,001,483 entitled ‘Methods for the treatment of Kaposi’s sarcoma or KSHV-induced lymphoma using immunomodulatory compounds and uses of biomarkers’. An immediate family member of R.Y. is a co-inventor on patents or patent applications related to internalization of target receptors, epigenetic analysis and ephrin tyrosine kinase inhibitors. All rights, title, and interest to these patents have been assigned to the U.S. Department of Health and Human Services; the government conveys a portion of the royalties it receives to its employee inventors under the Federal Technology Transfer Act of 1986 (P.L. 99–502). The other authors declare no competing interests.

Additional information

Extended data is available for this paper at https://doi.org/10.1038/s41591-024-03357-0.

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41591-024-03357-0.

Data availability

External requests for data will be evaluated by the corresponding author and requests will be subject to patient confidentiality and NIH policies. Requests for data related to the paper should be made to the corresponding author and will be processed within 8 weeks. The nucleotide sequences reported in this paper are available in GenBank (gag accessions, PP907225–PP907368; env-gp120 accessions, PP907369–PP907528). HIV subtype B sequence KU678055 can be found in the Los Alamos HIV database (www.hiv.lanl.gov).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

supp
NIHMS2080014-supplement-supp.pdf (593.3KB, pdf)

Data Availability Statement

External requests for data will be evaluated by the corresponding author and requests will be subject to patient confidentiality and NIH policies. Requests for data related to the paper should be made to the corresponding author and will be processed within 8 weeks. The nucleotide sequences reported in this paper are available in GenBank (gag accessions, PP907225–PP907368; env-gp120 accessions, PP907369–PP907528). HIV subtype B sequence KU678055 can be found in the Los Alamos HIV database (www.hiv.lanl.gov).

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