Rapid Identification of Antigen-Specific Monoclonal Antibodies from HIV-1 Elite Controllers Using the Beacon Single-Cell Platform

Elite controllers (ECs) are rare human immunodeficiency virus (HIV) infected individuals who manage viral replication without antiretroviral therapy. While their cellular immunity is well-characterized, humoral responses remain limited. Using single-cell functional screening, this study aimed to isolate and characterize HIV-1 envelope (Env) specific monoclonal antibodies (mAbs) from an EC. The Beacon® platform helped in enrichment, activation, and screening of cluster of differentiation 27+ (CD27⁺) memory B cells. Out of ~12,000 single B cells, we identified 40 Env-specific IgG-secreting cells. Although 18 mAbs were recovered, six showed high-affinity binding to multiple Env trimers (average EC50 < 100 ng/mL). Notably, one mAb (2#) neutralized 15 of 17 pseudoviruses with a geometric mean half maximal inhibitory concentration (IC50) of 46.53 μg/mL. Sequence analysis revealed a predominant usage of IGHV1-69 and varied somatic hypermutation levels. However, the high binding affinity could not consistently predict neutralization potency. This study demonstrated the utility of integrated single-cell screening for rapid antibody discovery from rare donors. It also highlighted the need for functional assessment in HIV-1 antibody development and identifying an mAb candidate with moderate breadth and potency for potential therapeutic or vaccine applications. Rapid Identification of Antigen-Specific Monoclonal Antibodies from HIV-1 Elite Controllers Using the Beacon Single-Cell Platform Xiangyu Zhang1, Xinyuan Zheng1, Rishen Liang1, Lina Huang1, Yu Shi2, Zhi Zhang2, Chengrui Yin1, Chengchao Ding1, Jiabing Wu3, Jianjun Wu2,3*, Yong Gao1*. 1The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China 2School of Public Health, Anhui Medical University, Hefei, Anhui, China 3Department of HIV and AIDS Control and Prevention, Anhui Provincial Center for Disease Control and Prevention, Hefei, Anhui, China *Corresponding author. Email: Jianjun Wu, [email protected]; Yong Gao, [email protected] Running head: Beacon-based mAb discovery from Chinese HIV-1 elite controllers Data availability：Data are available from the corresponding author upon reasonable request. Conflict of interest: The authors declare that there are no conflicts of interest. Funding: This work was supported by the Anhui Provincial Natural Science Foundation (No. 2208085MH260) and institutional research funds from the First Affiliated Hospital of the University of Science and Technology of China. Ethics approval statement: This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the First Affiliated Hospital of the University of Science and Technology of China (Approval No. 2025KY079) patient consent statement: Written informed consent was obtained from all study participants prior to enrollment Abstract Elite controllers (ECs) are rare human immunodeficiency virus (HIV) infected individuals who manage viral replication without antiretroviral therapy. While their cellular immunity is well-characterized, humoral responses remain limited. Using single-cell functional screening, this study aimed to isolate and characterize HIV-1 envelope (Env) specific monoclonal antibodies (mAbs) from an EC. The Beacon® platform helped in enrichment, activation, and screening of cluster of differentiation 27+ (CD27⁺) memory B cells. Out of ~12,000 single B cells, we identified 40 Env-specific IgG-secreting cells. Although 18 mAbs were recovered, six showed high-affinity binding to multiple Env trimers (average EC50 < 100 ng/mL). Notably, one mAb (2#) neutralized 15 of 17 pseudoviruses with a geometric mean half maximal inhibitory concentration (IC50) of 46.53 μg/mL. Sequence analysis revealed a predominant usage of IGHV1-69 and varied somatic hypermutation levels. However, the high binding affinity could not consistently predict neutralization potency. This study demonstrated the utility of integrated single-cell screening for rapid antibody discovery from rare donors. It also highlighted the need for functional assessment in HIV-1 antibody development and identifying an mAb candidate with moderate breadth and potency for potential therapeutic or vaccine applications. Keywords: Beacon platform; Broadly neutralizing antibody; Elite controller; HIV-1; Humoral immunity; Monoclonal antibody; Single-cell screening 1. Introduction Human immunodeficiency virus (HIV) infection remains a major global health challenge despite antiretroviral therapy’s (ART) remarkable achievements. As of 2023, an estimated 39.9 million HIV-1-positive individuals are present worldwide, and over one million new infections occur annually.1,2 Although ART effectively suppresses viremia and prolongs the lives of HIV-infected individuals, it is not a cure. Lifelong treatment can induce cumulative drug toxicities. Subsequently, the emergence of drug-resistant viral strains and a substantial economic burden underscore the urgent need for a preventive HIV-1 vaccine or a functional cure.3,4 However, developing an effective HIV-1 vaccine has been challenging, primarily due to the unprecedented genetic diversity of the virus’s envelope (Env) glycoprotein and numerous complex immune evasion mechanisms that allow HIV-1 to evade the host’s humoral and cellular immune responses.5-10 A potential approach to HIV-1 prevention and therapy is the elicitation or administration of broadly neutralizing antibodies (bNAbs). The primary objective of HIV vaccine research is to induce bNAbs—rare antibodies that can neutralize several HIV-1 strains from different clades. These bNAbs typically develop in approximately 10–20% of infected individuals, often after several years of infection. They also exhibit extraordinary genetic and structural features, such as enhanced somatic hypermutation (SHM) levels and abnormally long complementarity-determining region 3 (CDR3) in their heavy chains.3,11-14 Demonstrating protective efficacy in experimental models, a passive infusion of bNAbs hinders infection in non-human primates. However, in human clinical trials, bNAbs have shown potential to block or control HIV-1 infections. Hence, these findings establish bNAbs as a crucial immunological protection blueprint and provide critical protection correlates that guide vaccine design.15-18 A very small subset (<1%) of the HIV-1-positive population is designated as elite controllers (ECs), who can naturally suppress viral replication to undetectable levels without undergoing ART.19,20 Thus, ECs might become a unique human model for discovering a “functional cure.” Their robust control of HIV-1 is significantly attributed to exceptionally effective HIV-specific CD8+ T cell responses, favorable host genetic factors like certain human leukocyte antigen (HLA) B alleles, and distinctive innate immune attributes.20-26 Although the major research on ECs has focused on cellular immunity, their humoral immune responses are equally intriguing.27-29 Having low plasma viremia and limited viral diversity, some ECs can generate bNAbs, making them a reservoir of novel antibodies that develop under the specific immune pressures of natural viral control.30-35 However, isolating high-quality, functional antibodies like bNAbs from rare EC donors remains challenging.31,36 The B cells encoding these antibodies are scarce, and traditional antibody discovery methods, such as hybridoma generation or conventional antigen-specific B cell sorting, are labor-intensive and low-throughput. Nowadays, antibody discovery has been revolutionized by several high-throughput single-cell technologies that connect B cell receptor sequences to functional readouts.37,38 One of them is the Beacon® optofluidic platform, which enables parallel screenings of a multitude of individual B cells at one time. Within a single day, this microfluidic platform performs multiple sequential assays on each cell’s secreted antibodies, assessing properties such as antigen specificity, cross-reactivity, and binding affinity in situ. Hence, it has been successfully utilized to discover antibodies for various infections.39-41 Despite these advantages, its application in the context of HIV-1 cases—particularly for isolating bNAbs from naturally infected individuals such as ECs—is unexplored. ECs represent a unique model for studying the humoral immune responses against HIV-1. Since these rare individuals suppress HIV-1 replication without ART, they maintain undetectable viral loads for prolonged periods.20-23,42 Previous studies suggest that ECs harbor distinct immunological features, compared to HIV-infected individuals, including bNAb-enriched unique B cell receptor repertoires.27-29,43,44 Although EC plasma possesses moderate or low neutralizing antibody titers, its memory B-cell pools might harbor rare, potent bNAbs capable of targeting conserved Env epitopes.30,31,35,36,45-48 Thus, using advanced technologies, such as the Beacon® platform, to explore the antibody repertoire of ECs offers promising opportunities for identifying therapeutic bNAbs and provides critical insights for vaccine development. Nevertheless, very few studies have effectively exploited EC-derived B cells due to their scarcity and the limitations of conventional isolation methods. Hence, addressing this gap is essential for understanding protective antibody responses and devising HIV vaccine development strategies. Thus, we hypothesized that the unique immune milieu of an EC might help in obtaining new functional antibodies against HIV-1 Env. Using a Beacon® single-cell optofluidic platform, we conducted a rapid, high-throughput workflow combining immunomagnetic memory B cell enrichment to evaluate this theory. We aimed to isolate and comprehensively characterize HIV-1 Env-specific monoclonal antibodies (mAbs) from an EC donor. By leveraging the Beacon® platform’s unique capabilities, we aimed to discover novel Env-specific mAbs as well as to evaluate their genetic features, antigen-binding affinities, and neutralization activities. Moreover, we also sought to gain significant insights into the antibody responses of ECs and future strategies for HIV-1 vaccine design, as well as antibody-based therapy development. 2. Materials and methods 2.1 Study participants and ethical approval We collected peripheral blood from two HIV-1 ECs, who were ART-naive and displayed undetectable or scanty viral loads. Furthermore, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation and cryopreserved. Ethical approval for this study was obtained from the First Affiliated Hospital of the University of Science and Technology of China. 2.2 Cell lines, recombinant proteins, and plasmids We used serum-free 293 expression medium to cultivate HEK293F cells in suspension for producing mAb and Env trimer. HEK293T cells were maintained in dulbecco’s modified eagle’s medium (DMEM) and used for producing pseudoviruses. Cultured in DMEM, TZM-bl cells were used for neutralization assays. Additionally, SOSIP.664 Env trimers were codon-optimized and cloned into pTT5 vectors with C-terminal His-tags. Co-transfected with a furin plasmid into HEK293F cells, trimers were purified by nickel affinity chromatography. The pSG3ΔEnv backbone and an Env expression plasmid panel helped to generate HIV-1 pseudoviruses. We obtained all plasmids from collaborators or in-house resources. 2.3 Expression and purification of Env trimers and monoclonal antibodies Using polyethyleneimine (PEI), codon-optimized SOSIP.664 Env trimer plasmids were transfected into HEK293F cells at a 1:1 ratio with Furin. Moreover, cells were cultured in serum-free medium at 37 °C with 5% CO₂ while being shaken at 120 rpm. Supernatants were harvested five days after transfection, clarified, and purified by nickel affinity chromatography with stepwise imidazole elution. After a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) analysis, fractions were pooled, buffer-exchanged into PBS, and stored at 4 °C. Under identical solutions, paired heavy and light chain expression plasmids were co-transfected into HEK293F cells to produce mAbs. Culture supernatants were collected after 5–7 days and purified by Protein A affinity chromatography using an AKTA system. Following neutralization, the eluted antibodies were buffer-exchanged into PBS, quantified by BCA assay, and subsequently stored at 4 °C or –80 °C. 2.4 Antigen preparation and biotinylation BG505 and CAP45.2.00 SOSIP.664 trimers were biotinylated using NHS-PEG4-biotin (Thermo Fisher) and desalted using Zeba™ spin columns to remove excess reagent. A BCA assay was used to measure protein concentrations. At 4 °C overnight, biotinylated Env trimers were coupled to streptavidin-coated polystyrene beads (6–8 µm, Spherotech) and washed with PBS + 0.1% BSA. Flow cytometry was used to validate the bead coating efficiency by using HIV+ plasma and PE-conjugated anti-human IgG. After acquiring data on a BD FACSAria III, data were analyzed using FlowJo software. 2.5 Enrichment and in vitro activation of memory B cells After thawing, PBMCs were rinsed with RPMI 1640, underwent DNase I treatment, and sifted through a 37 µm filter. Negative selection helped in enriching the CD27⁺ memory B cells by the EasySep™ Human Memory B Cell Isolation Kit (STEMCELL Technologies) by negative selection. Enriched cells were resuspended in serum-free medium (Opto® Memory B Discovery Sample Prep Kit, Bruker/Berkeley Lights) and seeded at 5 × 10³ cells/well in 96-well round-bottom plates. After 5 days of culture at 37 °C and 5% CO₂, cells were harvested, pooled, and assessed for viability by trypan blue staining. An approximately 3-fold expansion in cell number was typically observed. 2.6 Single-cell screening using the Beacon® platform Light-based dielectrophoresis was used to transfer activated memory B cells into OptoSelect™ 11k chips (Berkeley Lights) and position them into NanoPen chambers. Although cells were maintained in survival medium, they were screened in situ for IgG secretion and antigen binding. Anti-human IgG-coated beads and fluorescent secondary antibodies helped to detect IgG secretion. Initially, BG505- and CAP45-coated beads were introduced, followed by PE-labeled anti-human IgG Fc antibodies. The localized fluorescence around NanoPens helped in visualizing antigen-binding events. Positive cells were identified using Beacon Analysis Suite and individually exported into 96-well polymerase chain reaction (PCR) plates that contained lysis buffer. Furthermore, the plates were either frozen at –80 °C or immediately used for synthesizing cDNA. 2.7 Single-cell antibody recovery, cloning, and expression A template-switch-based 5′RACE protocol was used to reverse-transcribe the RNA from exported single cells. Moreover, V_H and V_L regions were amplified by semi-nested PCR using constant region-specific primers. After being gel-verified, the PCR products were cloned into human IgG1 and κ/λ light chain expression vectors by Gibson assembly. Subsequently, recombinant plasmids were sequence-verified and co-transfected into 293-F suspension cells. Protein A affinity chromatography was used to produce and purify the antibodies in suspension, after which they were buffer-exchanged and quantified by BCA assay. 2.8 ELISA binding assays The binding activity of mAbs to HIV-1 Env trimers was assessed by ELISA. We evaluated four in-house SOSIP.664 gp140 trimers: BG505 (clade A), SF162 and SF33 (clade B), and CAP45.2.00 (clade C). At 4 °C overnight, high-binding 96-well plates (Nunc MaxiSorp) were coated with 2 µg/mL of each trimer in PBS (100 µL/well). After a PBST (PBS + 0.05% Tween-20) wash, plates were blocked with 5% non-fat milk (Beyotime, Cat# P0216) in PBST for an hour at room temperature. Five-fold serial dilutions of mAbs (starting at 10 µg/mL) were added to wells and incubated for one hour. HRP-conjugated goat anti-human IgG Fc antibody (1:5000) was used to detect bound IgG, and TMB substrate was added for 5 min. The reaction was halted with TMB stop solution, and absorbance at 450 nm (with a reference of 620 nm) was measured. Furthermore, binding curves and EC₅₀ values were calculated by using four-parameter nonlinear regression (GraphPad Prism). Consequently, antibodies with EC₅₀<500 ng/mL were considered high-affinity binders. We used the bNAb N6 as a positive control. 2.9 Neutralization assays The neutralizing activity of mAbs was evaluated using 17 HIV-1 Env-pseudotyped viruses, encompassing Tier 1, Tier 2, and Tier 3 strains from clades A, B, C, CRF01_AE, CRF02_AG, and 07_BC. Furthermore, pseudoviruses were cultivated by co-transfecting HEK293T cells with Env expression plasmids and the pSG3ΔEnv backbone. This was followed by supernatant harvest, filtration, and storage at –80° C. For the neutralization assay, serial 3- or 5-fold dilutions (starting at 50–100 µg/mL) of mAbs were incubated with pseudoviruses (~50,000–150,000 RLU) at 37 °C in 96-well plates for 45–90 min. We added 10,000 TZM-bl cells per well in complete medium containing 12.5 µg/mL polybrene. At 37 °C, plates were incubated with 5% CO₂ for 48–72 h. Luciferase activity was measured by the Britelite Plus Reporter Gene Assay System (Revvity, Cat# 6066769). Post-cell lysis, 100 µL of PBS and substrate each were added per well, incubated at room temperature for 2 min, and transferred to black 96-well plates for a luminometer reading. We also calculated the percent neutralization relative to virus-only controls. IC₅₀ values were determined by nonlinear regression (GraphPad Prism), and antibodies were considered neutralizing if the IC₅₀ level was < 50 µg/mL. Neutralization breadth was defined as the number of viruses neutralized out of 17, and geometric mean IC₅₀ was calculated among susceptible strains. 2.10 Antibody sequence analysis We analyzed variable region sequences of the mAbs to determine immunogenetic features, including germline gene usage, CDR3 characteristics, and SHM levels. Initially, paired heavy (V_H) and light (V_L) chain nucleotide sequences obtained from single-cell PCR were trimmed to include the complete variable regions (FR1-FR4), based on IMGT definitions. The NCBI IgBLAST tool was used to conduct germline gene assignment (https://www.ncbi.nlm.nih.gov/igblast), thereby aligning each sequence against the IMGT reference database. Subsequently, the best-matched V, D, and J gene segments were identified for both chains. As per IMGT numbering. CDR3 was defined from the conserved second cysteine in FR3 to the conserved phenylalanine/tryptophan in the J region. Moreover, CDR3 amino acid lengths were calculated individually for heavy and light chains, respectively. SHM rates were calculated by counting nucleotide mismatches between each V_H or V_L sequence and its closest inferred germline gene, while mutation frequency was expressed as a percentage of the total V-region length. Summary statistics included V gene family usage, CDR3 lengths, and SHM percentages of all antibodies. These data helped to assess the maturation levels and functional diversity of Env-specific antibodies derived from the EC donor. 3. Results 3.1 Single-cell screening of Env-specific memory B cells using the Beacon® platform Although ECs are capable of controlling HIV-1 replication without ART, the precise mechanisms underlying B cell-mediated humoral immunity in these individuals remain ambiguous. Moreover, the contribution of memory B cells in producing antigen-specific antibodies warrants exploration. B cell receptor (BCR) repertoire sequencing has now emerged as a crucial strategy to investigate HIV-specific antibody responses. However, one of the major challenges in isolating mAbs from human B cells lies in the limited ex vivo survival of B cells and the absence of a robust long-term culture system. This hampers sustained antibody production in vitro. To circumvent these limitations, current high-throughput screening strategies rely on enriching the memory B cells, which, post-activation, can secrete antibodies suitable for downstream selection and characterization. In this study, CD27⁺ memory B cells were rapidly enriched from PBMCs of ECs using magnetic-activated cell sorting (MACS). Using a commercial human memory B cell activation kit, the enriched cells were activated and cultured for five days, during which their cell viability and fold expansion were assessed. To enable high-throughput and antigen-specific screening, we employed the Beacon® platform to isolate and analyze memory B cells that secreted IgG antibodies specific to the HIV-1 envelope trimer proteins, BG505 and CAP45.2.00. Peripheral blood from two elite controllers (EC_5 and EC_6) was collected and screened for baseline humoral responses against HIV-1 Env. Plasma binding activity to Env trimers and neutralization potency against a 17-virus panel were assessed (Supplementary Figure S1a, S1b), confirming detectable Env-specific antibodies in both donors. Figure 1 displays the representative schematic of the Beacon®-based single-cell antibody discovery workflow. This includes donor PBMC isolation, CD27⁺ memory B cell enrichment and activation, as well as the identification and recovery of antigen-specific antibody-secreting cells. Using this workflow, we successfully isolated 40 Env-specific memory B cells from two ECs (EC_5 and EC_6). Subsequent recovery and molecular cloning of the antibody genes from these single cells helped to identify 18 unique HIV-1-specific mAbs. Initially, flow cytometry was used to validate biotinylated BG505 and CAP45.2.00 SOSIP.664 Env trimers for effective labeling. Streptavidin-coated beads conjugated with these Env trimers that were bound with HIV-positive plasma antibodies, and confirmed antigen functionality (Figure 2a) . CD27⁺ memory B cells were enriched from two HIV EC donors and stimulated in vitro for five days, thereby expanding from approximately 7 × 10⁴ to 2.12 × 10⁵ cells (~3.0-fold increase) with activated B cells were loaded on a Beacon® single-cell chip. Approximately 33% of NanoPen chambers (~3,800) contained IgG-secreting cells. Using fluorescently labeled Env-coated beads, we detected 40 cells that secreted Env-specific IgG (38 BG505-reactive, 35 CAP45-reactive, with overlap). This yielded an overall antigen-specific hit rate of approximately 0.30–0.33%. A background binding assay using isotype control antibodies confirmed specificity due to an absence of significant fluorescence. All 40 Env-specific cells were subsequently exported for downstream analysis. Table 1 summarizes the single-cell screening metrics, including their loading efficiency, IgG+ frequency, and antigen-specific hit rates (Table 1). Additionally, paired heavy and light chain genes were successfully amplified and sequenced from 18 cells. The resulting monoclonal antibodies (designated mAb 1–18) were expressed in HEK293F cells and subsequently purified. SDS-PAGE and Western blotting analyses confirmed their accurate molecular sizes and Env-specific reactivity and plasmid verification (Supplementary Figure S2a and 2b). Figure 2b depicts the representative NanoPen images. 3.2 Sequence features of Env-specific monoclonal antibodies To further characterize the detected antibodies’ immunogenetic features, we performed sequence alignment and annotation using the NCBI IgBLAST tool. These analyses included variable (V), diversity (D), and joining (J) gene assignments, CDR3 length calculation, and estimation of SHM frequencies for both heavy and light chain variable regions. These parameters helped to evaluate the origin of the antibody’s germline, affinity maturation status, and repertoire diversity. These factors are critical for identifying antibodies with high affinity and potential neutralizing activity. Table 2 summarizes the key immunogenetic features of representative mAbs, including V gene usage, CDR3 lengths, and SHM frequencies for both heavy and light chains. All details for all 18 antibodies are provided in Supplementary Table S1 . IgBLAST analysis (Table 2) revealed that the 18 mAbs used varied immunoglobulin germline genes. Among the successfully annotated heavy chains (n = 15), 40% were derived from the IGHV1-69 family, while the remaining antibodies were extracted from IGHV2-5 (13.3%), IGHV3-23 (13.3%), IGHV3-47 (13.3%), IGHV3-7 (6.7%), IGHV3-71 (6.7%), and IGHV3-9 (6.7%) families, respectively. Notably, IGHV1-69 is frequently used in bNAbs against HIV, especially those targeting the membrane-proximal external region (MPER) of gp41. This is a highly conserved domain on the viral envelope that is a critical vulnerable site. IGHV1-69 germline-based antibodies possess comprehensive neutralization capacity and effectiveness against diverse HIV-1 strains, making them crucial for HIV vaccine design and subsequent therapeutic development. Although the CDR3 lengths of the isolated antibodies ranged from 7 to 24 amino acids, light chain CDR3 lengths spanned from 9 to 12 amino acids. The SHM frequencies in heavy and light chains varied from approximately 2.4% to 22.1% and 2.4% to 12.3%, indicating a range of affinity maturation, respectively (Table 2) . Collectively, these findings reflect a polyclonal and antigen-driven B cell response with IGHV1-69-derived clones’ significant enrichment, which supports the notion that ECs possess a memory B cell repertoire that can generate highly somatically mutated and potentially functional anti-HIV antibodies. 3.3 Env binding levels of isolated antibodies We evaluated the binding capability of all 18 mAbs to four SOSIP.664 Env trimers like BG505 clades A, SF162 and SF33 clades B, as well as CAP45.2.00 clades C. ELISA binding curves demonstrated an extensive range of reactivities (Supplementary Figure S3) . Subsequently, the EC 50 values were calculated (Table 3) . Although most of the antibodies exhibited low binding activity (EC 50 > 1000 ng/mL), six mAbs (2#, 4#, 8#, 9#, 13#, and 16#) displayed a high affinity, with an average EC 50 of < 100 ng/mL in all four trimers. Notably, mAb 16# showed the strongest overall binding affinity (average EC 50 = 18.45 ng/mL), surpassing even the bNAb N6 (average EC 50 ≈ 24.11 ng/mL). Additional analysis revealed that most of the high-affinity antibodies originated from the IGHV1-69 gene family, suggesting a potential role for IGHV1-69 in generating high-affinity Env-specific antibodies. Sequence analysis indicated that mAb 16# exhibited lower SHM frequencies (heavy chain strong binding affinity. This suggests that high antigen-binding capability might not be correlated directly with high SHM levels, and that other structural features, such as specific V-gene usage or CDR3 characteristics, might contribute to its high affinity. Specific mAbs demonstrated clade-specific binding activities. For example, mAb 4# bound strongly to SF162 and SF33 (EC 50 = 2.71 and 3.14 ng/mL, respectively), while mAb 5# preferentially bound to BG505 and CAP45.2.00 (EC 50 < 9 ng/mL). Moreover, sequence analysis revealed distinct characteristics between mAb 4# and mAb 5#. Specifically, mAb 4# had a CDR3 length of 16 amino acids and a high heavy chain SHM frequency of 20.6%, while mAb 5# possessed a slightly longer CDR3 of 21 amino acids with a lower SHM level of 14.5%. These differences might have contributed to their distinct Env trimer binding preferences, thereby reflecting recognition of different epitopes or conformational regions. Their divergent subtype-specific binding patterns also suggest that they might target structurally or immunogenically distinct determinants on the HIV Env. Interestingly, several antibodies with moderate average EC 50 values exhibited robust binding activity against specific individual Env trimers, suggesting recognition of distinct or conformationally restricted epitopes. This characteristic was especially evident in mAb 1#, which selectively bound to CAP45.2.00 (EC 50 = 204.4 ng/mL) but showed no significant binding to the other three Env trimers, thereby indicating subtype-restricted specificity. Contrary to our initial hypothesis, repertoire sequencing showed that mAb 1# was derived from the IGHV3-2304/IGKV1-1602 germline pair, and had a long CDR3 region (22 amino acids). This indicates that the limited Env recognition of mAb 1# is not due to a shorter CDR3 length or unusual germline usage, but might be because of unique paratope-epitope interactions or an enhanced conformational specificity toward CAP45.2.00 (Table 3) . In summary, all 18 antibodies exhibited significant heterogeneity in their antigen-binding capabilities, reflecting the diversity within the Env-specific memory B cell repertoire of the EC donor. Additionally, combined analyses of sequence features and binding properties clarified how specific V-gene usage, CDR3 length, and SHM levels can collectively influence antibody specificity and affinity maturation level. These findings provide significant insights into the diversity and unique features of humoral immune responses in ECs. 3.4 Neutralization activity across a 17-virus panel Seven high-binding antibodies (2#, 4#, 5#, 8#, 9#, 13#, and 16#) were evaluated against a 17-virus panel, such as Tier 1, 2, and 3 HIV-1 pseudoviruses from clades A, B, C, CRF01_AE, CRF02_AG, and 07_BC, respectively. Consequently, only mAb 2# showed broad and consistent neutralization, neutralized 15 out of 17 pseudoviruses with IC 50 < 100 µg/mL (Supplementary Figure S4), and displayed an average NT_IC 50 of 46.53 µg/mL (Table 4) . Moreover, mAb 2# displayed maximum potency against the autologous BG505 strain (IC 50 = 12.23 µg/mL) as well as several Tier 1 and 2 viruses (e.g., Q23, SF162, BJOX2000, CE0217), with IC 50 values under 25 µg/mL, respectively. Conversely, mAbs 4# and 9# neutralized 6 of 17 viruses, while 5#, 8#, and 13# neutralized only three viruses. Exhibiting minimal activity, mAb 16# neutralized only one virus (BAL.01, IC 50 ≈ 85 µg/mL). None of the isolated mAbs matched the potency or breadth of our positive control bNAb N6 (reported NT_IC 50 ≈ 0.038 µg/mL, (Table 4) . Additional neutralization curve analysis (Supplementary Figure S4) showed that mAb 2# effectively inhibited multiple viral subtypes, including clade A, B, and CRF01_AE viruses, with IC50 values below 100 µg/mL. These results indicate that mAb 2# exhibited broader neutralizing activity compared to other isolated antibodies. Despite their enhanced binding activities in ELISA, mAbs 4#, 5#, and 13# demonstrated limited neutralization breadth. For instance, mAb 5#, which showed a strong binding to BG505 and CAP45.2.00 (EC 50 < 9 ng/mL), could not effectively neutralize the corresponding pseudoviruses. This indicates that the bound epitope may be conformationally masked or not at all involved in viral entry. Similarly, mAb 9# showed a low potency against Tier 2/3 viruses despite being effective against Tier 1 strains like SF162 and CH119. This pattern suggests a limited epitope accessibility or insufficient affinity under native Env structural constraints. These observations are consistent with a key feature of humoral immunity in ECs, i.e., neutralization is not solely dependent on binding affinity but is also influenced by epitope exposure, antibody orientation, and somatic maturation. Notably, mAb 2# displayed a moderate SHM rate (VH: 17.2%) and a CDR3 length of 16 amino acids. These parameters might have contributed to its functional balance between breadth and potency. Conversely, mAb 16#, despite exhibiting the strongest ELISA binding (average EC 50 = 18.45 ng/mL), showed minimal neutralizing activity, thereby underscoring the decoupling between binding and neutralization. Repertoire analysis revealed that mAb 16# displayed a low SHM level and a moderate CDR3 length of 20 amino acids, potentially limiting its structural adaptability or paratope diversity required for broad neutralization. Thus, these results suggest that only mAb 2# possessed moderate breadth and potency, although multiple mAbs displayed strong Env binding. This disparity highlights that high binding affinity cannot ensure neutralization functionality and reflects the complex, selective nature of humoral responses in ECs. Overall, this study demonstrates that the antibody repertoire in ECs might contain a few broadly reactive clones among a comprehensive background of non-neutralizing or strain-specific antibodies. This reinforces the need for high-throughput, function-based screening approaches like Beacon to analyze functional immunity. 4. Discussion We employed the Beacon® single B-cell screening platform to isolate 18 HIV-1 Env-specific mAbs from two EC donors. Although most of the antibodies exhibited measurable binding to SOSIP.664 trimers from diverse HIV-1 clades, only one clone, 2#, demonstrated a moderate neutralization breadth, by achieving IC50<100 µg/mL against 15 of 17 pseudoviruses. These findings illustrate the utility of high-throughput, function-first screening and the inherent difficulty in identifying bNAbs from natural infections. Notably, a high frequency of IGHV1-69 germline gene usage was found in 40% of isolated clones. This gene family is frequently associated with MPER-targeting bNAbs. However, in our study, most of these IGHV1-69-derived antibodies showed a weak or absent neutralization despite high binding affinities. This highlights a dissociation between germline origin and neutralizing function. This finding also aligns with our sequence analysis, which showed that even antibodies with long CDRH3 regions or intermediate SHM levels—typical of bNAbs—cannot guarantee functional activity. Various structural factors, such as glycan shielding, epitope orientation, and conformational masking, might contribute to this finding. Although several antibodies exhibited sub-nanomolar EC50 values across Env trimers, they failed to neutralize matching pseudoviruses. This underscores the limitations of SOSIP-based binding assays in predicting functional potential, as they might expose non-neutralizing or decoy epitopes that are not accessible on native virions. This was evident for clone 5#, which bound to BG505 and CAP45.2.00 with a high affinity but lacked corresponding neutralization activity. Similar discrepancies have been reported in antibodies that target variable loops or surface-exposed non-functional Env regions. Although the Beacon® platform offers significant advantages, like rapid screening, real-time functional assessment, and single-cell resolution, our findings also highlight its limitations. Unlike phage display or bulk sequencing coupled with antigen baiting, Beacon® relies heavily on soluble, recombinant antigens presented in artificial microenvironments. Despite having a stabilized structure, SOSIP trimers might not truly mimic the glycan shield and native virions’ conformational states. Consequently, antibodies binding to non-neutralizing conformers might be preferentially recovered. Additionally, the platform’s reliance on IgG secretion within a short in vitro culture period might produce a bias against low-frequency, resting memory B cells that produce potent but infrequent bNAbs. These constraints might have contributed to the limited neutralization breadth observed in our panel. However, the Beacon® platform still enables rapid identification and recovery of diverse, antigen-specific mAbs from a rare donor source without any labor-intensive sorting, cloning, or expansion steps, as seen in older technologies. Our study had a few limitations. First, it involved only two donors, thereby limiting antibody diversity. Second, although our pseudovirus panel was subtype-diverse, it might not have captured global HIV-1 antigenic breadth. Third, we conducted no structural assays to precisely map epitope targets. Lastly, functional analyses were restricted to neutralization. Fc-dependent functions, such as ADCC or phagocytosis, which contribute to in vivo efficacy, were not assessed. Additional studies should integrate immune repertoire sequencing, epitope mapping, and Fc-effector profiling to refine antibody characterization. Enhancing antigen design—for example, by displaying membrane-anchored or native-like Env trimers on nanofluidic platforms—might also improve recovery of functional bNAbs. Although we could not identify any potent bNAbs, our data demonstrate the feasibility of associating memory B-cell activation with real-time functional screening to recover rare Env-specific antibodies from challenging donors. 5. Conclusion We have reported the isolation and characterization of 18 HIV-1 Env-specific mAbs from two ECs using the Beacon® single B-cell screening platform. Although only clone 2# exhibited moderate neutralization breadth, other antibodies demonstrated high-affinity binding to diverse Env trimers. Underscoring the disconnect between binding affinity and neutralization potency, our findings reflect both the advantages and limitations of real-time, function-first single-cell screening. Notably, the reliance on recombinant Env trimers and in vitro IgG secretion might impede the recovery of rare bNAbs. Nevertheless, this study confirms that memory B-cell activation and optofluidic screening can be combined for rapid antibody discovery from limited clinical data. Our work can contribute to a better understanding of humoral responses in ECs and provide a technical foundation for refining future bNAb discovery strategies. This might include improved antigen design and complementary sequencing-based approaches. Acknowledgments We thank all participants, for their contributions to this study. We also appreciate the colleagues at the First Affiliated Hospital of USTC and the Department of HIV and AIDS Control and Prevention, Anhui CDC.

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Structural basis for broad HIV-1 neutralization by the MPER-specific human broadly neutralizing antibody LN01. Cell Host Microbe 2019;26:623-637. https://doi.org/10.1016/j.chom.2019.09.016. Figure 1. Schematic overview of the Beacon®-based single-cell antibody discovery workflow from an HIV-1 elite controller. Peripheral blood mononuclear cells (PBMCs) were isolated from two antiretroviral therapy-naïve elite controllers (ECs) with undetectable viral loads. CD27⁺ memory B cells were enriched via magnetic negative selection and activated in vitro for five days, yielding a Activated cells (~12,000–15,000) were loaded onto a Beacon® OptoSelect™ 11k chip, where single cells were screened in real time for IgG secretion and antigen specificity using fluorescently labeled BG505 and CAP45.2.00 SOSIP.664 Env trimer-coated beads. Antigen-specific IgG⁺ cells were identified and exported individually for reverse transcription, antibody gene amplification, and cloning into IgG1 expression vectors. Recombinant monoclonal antibodies (mAbs) were expressed in HEK293F cells, purified by Protein A affinity chromatography, and subjected to downstream binding and neutralization analyses. Figure 2a. Validation of biotinylated HIV-1 Env trimer coupling to streptavidin-coated beads by flow cytometry. Streptavidin (SA)-coated polystyrene beads were conjugated with biotinylated BG505 SOSIP.664 Env trimers and incubated with HIV-positive plasma at serial dilutions (1:40 to 1:5000), followed by PE-conjugated anti-human IgG detection. Representative dot plots show dose-dependent binding of plasma antibodies to Env-coated beads. An irrelevant-antigen control confirmed assay specificity, with negligible background binding. The percentage of PE-Antigen⁺ beads is indicated in each plot. Figure 2b. Representative NanoPen images from Beacon® single-cell screening for HIV-1 Env-specific IgG secretion. Activated memory B cells from an HIV-1 elite controller were individually captured in NanoPen chambers and assayed for antigen-specific IgG secretion. Streptavidin-coated beads were conjugated with either BG505 (left) or CAP45.2.00 (middle) SOSIP.664 Env trimers and incubated with secreted antibodies, followed by fluorescent anti-human IgG detection. Bright fluorescence halos surrounding beads indicate Env-specific IgG secretion. An anti-IgG bead assay (right) served as a positive control for total IgG secretion. Images were acquired directly on the Beacon® platform under real-time functional screening conditions. Table 1. Single-cell screening metrics and antigen-specific hit rates obtained using the Beacon® platform. Activated CD27⁺ memory B cells from HIV-1 elite controllers were loaded onto an OptoSelect™ chip and subjected to real-time functional screening. The table summarizes (i) chip loading efficiency (total pens, penned cells, single-cell occupancy), (ii) cell export rates for downstream antibody gene recovery, and (iii) the number and percentage of IgG-secreting NanoPens detected with BG505- or CAP45.2.00-coated beads. Anti-IgG beads served as a positive control to measure total IgG secretion frequency. Table 2. Immunogenetic features of HIV-1 Env-specific monoclonal antibodies isolated from an elite controller. Paired heavy (VH) and light (VL) chain variable region gene usage, complementarity-determining region 3 (CDR3) amino acid lengths, and somatic hypermutation (SHM) frequencies are summarized for 18 monoclonal antibodies recovered by single-cell screening on the Beacon® platform. VH and VL germline assignments were determined using IgBLAST. Mutation frequencies were calculated as the percentage of nucleotide differences from the closest germline genes. These features provide insights into germline origins, CDR3 diversity, and affinity maturation levels among Env-specific antibodies from elite controllers. Table 3. Binding activity of isolated HIV-1 Env-specific monoclonal antibodies against four Env trimers. Eighteen monoclonal antibodies (mAbs) isolated from an HIV-1 elite controller were tested for binding activity to Env trimers derived from clades A (BG505), B (SF162 and SF33), and C (CAP45.2.00) using ELISA. Half-maximal effective concentrations (EC50) are reported in ng/mL and represent the mean values from two independent experiments. The broadly neutralizing antibody N6 served as a positive control. Lower EC50 values indicate stronger antigen binding affinity. Table 4. Neutralization activity of HIV-1 Env-specific monoclonal antibodies against a 17-virus pseudovirus panel. Seven high-binding monoclonal antibodies (mAbs) were tested for neutralizing activity using a TZM-bl assay against a panel of HIV-1 pseudoviruses representing multiple clades (A, B, C, CRF01_AE, CRF02_AG, and 07_BC) and Tier 1–3 neutralization sensitivities. Half-maximal inhibitory concentrations (IC50) are reported in μg/mL and represent the average of two independent experiments. The broadly neutralizing antibody N6 was included as a positive control. IC50 values below 50 μg/mL indicate strong neutralizing potency. Supplementary Figure 1a. Plasma binding activity of HIV-1 elite controller (EC) against Env SOSIP.664 trimers. Plasma samples from EC donors (EC_1–EC_7) were serially diluted and tested by ELISA for binding to HIV-1 Env trimers representing clades A (BG505), B (SF162, SF33), and C (CAP45.2.00). OD₄₅₀ values were plotted against the logarithm of plasma dilution, and binding curves were fitted using a four-parameter nonlinear regression model (GraphPad Prism). Data represent mean values from two independent experiments. Supplementary Figure 1b. HIV-1 neutralization activity of plasma samples from elite controller donors. Plasma from EC donors (EC_1–EC_7) were serially diluted and tested for neutralizing activity against a 12-virus pseudovirus panel representing multiple HIV-1 clades (A, B, C, CRF01_AE, CRF02_AG) and different neutralization sensitivities (Tier 1 and Tier 2). Percent neutralization was plotted against the logarithm of plasma dilution, and IC50 values were calculated using a four-parameter logistic regression model (GraphPad Prism). Curves illustrate inter-donor variability in neutralization breadth and potency. Supplementary Figure S2a. Purity assessment of recombinant monoclonal antibodies by SDS-PAGE and Western blot (WB). Recombinant antibodies expressed in HEK293F cells were purified from culture supernatants using Protein A affinity chromatography. Antibody samples were analyzed under reducing conditions on 10% SDS-PAGE gels, followed by Coomassie blue staining (upper panel). Western blotting (lower panel) was performed using HRP-conjugated anti-human IgG to confirm heavy (~50 kDa) and light (~25 kDa) chain expression. Representative data are shown for selected Env-specific antibodies. Supplementary Figure S2b. Electrophoretic validation of cloned antibody plasmid DNA. Heavy and light chain expression plasmids recovered from single antigen-specific B cells were analyzed by agarose gel electrophoresis. Supplementary Figure 3. ELISA binding curves of 18 Env-specific monoclonal antibodies isolated from an HIV-1 elite controller. Eighteen monoclonal antibodies (mAbs) obtained by single-cell screening were tested for binding activity against four HIV-1 Env SOSIP.664 trimers representing clades A (BG505), B (SF162, SF33), and C (CAP45.2.00). Serial dilutions of purified antibodies were analyzed by ELISA, and OD₄₅₀ values were plotted against the logarithm of antibody concentration. Binding curves were fitted using a four-parameter logistic regression model (GraphPad Prism). The broadly neutralizing antibody N6 was included as a positive control. Supplementary Figure 4. Neutralization curves of Env-specific monoclonal antibodies against a 17-virus HIV-1 pseudovirus panel. Selected monoclonal antibodies (mAbs) were tested for their ability to neutralize pseudoviruses from diverse HIV-1 clades (A, B, C, CRF01_AE, CRF02_AG, and 07_BC) encompassing Tier 1–3 neutralization phenotypes. Serial dilutions of purified mAbs were incubated with pseudoviruses, and residual infectivity in TZM-bl cells was measured by luminescence readout. Percent neutralization was plotted against the logarithm of antibody concentration, and IC50 values were determined using a four-parameter logistic regression model (GraphPad Prism). The broadly neutralizing antibody N6 was included as a positive control. Supplementary Table S1. HCDR3 amino acid sequences of HIV-1 Env-specific monoclonal antibodies. The table lists the deduced amino acid sequences of the heavy chain complementarity-determining region 3 (HCDR3) from 18 Env-specific monoclonal antibodies recovered from an HIV-1 elite controller using the Beacon® single-cell screening platform. Information includes: Antibody clone identifiers (mAb 1–18), Corresponding HCDR3 amino acid sequences, CDR3 lengths (number of amino acids). These sequences were determined from paired heavy chain variable region genes amplified from single B cells and analyzed using IgBLAST. The data illustrate the junctional diversity of antigen-specific B cell clones and highlight potential structural determinants for HIV-1 Env recognition. Supplementary Material File (figure 1-beacon platform antibody discovery workflow.tif) - Download - 1.43 MB File (figure 2a-streptavidin-coated beads test.tif) - Download - 1.68 MB File (table1. beacon cell sorting result.xlsx) - Download - 10.22 KB File (table2. antibody sequence characteristics.xlsx) - Download - 10.23 KB File (table3. antibody elisa ec50 binding level.xlsx) - Download - 10.69 KB File (table4. antibody neutralization ntic50 results.xlsx) - Download - 10.88 KB Information & Authors Information Version history Copyright This work is licensed under a Non Exclusive No Reuse License.

Authors Metrics & Citations Metrics Article Usage 366views 152downloads Citations Download citation Xiangyu Zhang, Xinyuan Zheng, Rishen Liang, et al. Rapid Identification of Antigen-Specific Monoclonal Antibodies from HIV-1 Elite Controllers Using the Beacon Single-Cell Platform. Authorea. 14 August 2025. DOI: https://doi.org/10.22541/au.175515681.13775821/v1 DOI: https://doi.org/10.22541/au.175515681.13775821/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.