Immunologic insights into the critical epitopes of HIV-1 and structure-based characterization of cross-reactive antibodies

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The virus exploits the error-prone nature of reverse transcriptase and the high mutation rate as key survival strategies. However, the rate of emergence of new variations occurs at a relatively slow pace. In this context, while monospecific antibody responses against invading pathogens are well characterized, the functional relevance of multispecific or cross-reactive antibodies in limiting viral escape remains poorly understood. Interestingly, the immune system often produces cross-reactive antibodies, with an anticipated role in neutralizing point mutations in HIV surface proteins by cross-reacting with mutants and tolerating them. In light of this paradox, we investigated immune evasion in the context of observed antibody cross-reactivity by screening single-chain variable fragment (scFv) antibodies against several crucial HIV-1 gp41 epitopes using a phage display library. Selected cross-reactive scFvs were biochemically characterized for binding affinity and their ability to recognize envelope protein and its mutants expressed on cell surface. Here, high-affinity cross-reactive scFvs showed physiologically relevant affinities with peptide epitopes, their analogs, and the native HIV-1 gp41 protein. We determined the crystal structure of a high-affinity, cross-reactive scFv DE94, and gained insights into the molecular interactions of scFv antibodies with peptide epitopes and their natural mutants using molecular docking studies. This analysis of cross-reactive antibodies could contribute to the development of therapeutics against immune-evading pathogens and pave the way for innovative strategies to combat viral infections, including emerging global threats. HIV-1 membrane-proximal external region (MPER) Kennedy epitope cross-reactive antibodies human scFv phage display library Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The immune system produces antibodies to defend the body against invading pathogens by binding to and neutralizing them. Traditionally, antigen-antibody interactions are perceived as highly specific [ 1 ]. However, accumulating structural data have revealed that host-pathogen interactions are much more complex and exist in dynamic equilibrium. While the immune system is under pressure to eradicate pathogens, selection pressure exists on pathogens to evade detection and establish infection [ 20 ]. Fast-mutating viruses, such as human immunodeficiency virus (HIV-1) and influenza, exploit this balance by incorporating genetic variations in their surface-exposed envelope proteins, thereby evading the immune response. As a result, the immune system can no longer detect mutated antigenic surfaces, disturbing the equilibrium between the host and the pathogen. The immune system must employ some strategy to defend against these viruses and maintain this equilibrium. Several studies have shown that in the case of infection, the regular B-cell repertoire consists of two types of antibody populations: monospecific and multispecific [ 15 ]. The role of monospecific antibodies is well known, as antibodies are specifically generated against invading pathogens. However, the significance and function of multispecific antibodies remain poorly understood and require further investigation. Multispecificity in the affinity-matured antibodies has been previously reported [ 22 ], suggesting a potential role for these antibodies in combating the effect of antigenic variations in fast-mutating viruses. These viruses present a physiologically relevant model for understanding the role of multispecificity in the immune response. A previous study on the influenza hemagglutinin (HA) protein and its mutants investigated the mechanisms of antibody cross-reactivity [ 40 ], suggesting that multispecific antibodies may help prevent immune evasion despite some mutations successfully driving antigenic variation. To gain a deeper understanding of the functions of multispecific antibodies, we extended our investigation to examine multispecific antibodies that target HIV-1. The HIV envelope proteins play a central role in viral replication and are key determinants of the virus’s antigenic and pathogenic properties [ 2 , 26 , 27 ]. The functional envelope is a trimer of two heterodimeric surface glycoproteins, gp120 and gp41. gp120 is essential for the attachment of the virus, whereas gp41 mediates the fusion of the viral and host membranes. The host’s key defense mechanism to combat HIV infection is antibody-mediated neutralization, and many broadly neutralizing antibodies have been identified and studied thus far [ 23 , 24 ]. The gp41 region of the envelope is more conserved than the gp120 region [ 13 , 25 , 32 ]. Within the gp41 region, one of the elusive vaccine targets is the membrane-proximal external region (MPER), which is located outside the viral membrane [ 16 ]. In addition, the Kennedy epitope is another immunogenic site within the cytoplasmic tail (CTT) of gp41 located inside the viral membrane, making it less accessible. However, numerous studies have demonstrated that antibodies directed against the epitope found in the CTT region can neutralize the virion [ 4 , 12 ], indicating that specific epitopes become accessible during viral conformational changes. These antibodies can block the cell-to-cell transmission of HIV by blocking fusion [ 11 , 19 , 35 ]. The contrast between HIV immune evasion strategies and recent studies on antibody promiscuity is fascinating. HIV escapes immune surveillance by incorporating mutations in its envelope proteins. It has been reported that even a point mutation is enough to evade the immune response [ 5 , 28 ]. However, multispecific antibodies are also increased during viral infections [ 29 ], revealing an interesting dichotomy. In light of this, our study aims to address the mechanisms underlying HIV-1 immune evasion and antibody promiscuity. We focused on two critical epitopes- MPER and Kennedy epitopes within gp41 to understand the role of multispecific antibodies in different hotspots of HIV. Using human-based antibody phage display libraries, we screened antibodies against these epitopes and successfully isolated a diverse panel of cross-reactive antibodies against both epitopes. The selected antibodies showed diverse binding affinities, ranging from micromolar to subnanomolar. Intriguingly, antibodies screened against peptide epitopes were able to recognize the epitope within the full-length protein. Crystallographic and in silico analyses have provided insights into the molecular mechanisms of antibody interactions with epitope peptides and their variants. Our findings emphasize the physiological relevance of antibody promiscuity in the natural immune response and its potential implications for therapeutic and vaccine strategies targeting HIV. Methods Peptide selection, synthesis, and conjugation to BSA The neutralizing epitopes were selected from the HIV-1 gp41 region. After two critical epitopes from the MPER and CTT regions were selected, escape variants of both epitopes were chosen from the HIV sequence database (http://www.hiv.lanl.gov/). We analyzed approximately 12000 variants of these epitopes present in the database, resulting in the selection of six variants against each epitope. All the peptides were synthesized with N-terminal cysteine/lysine for BSA conjugation via an automated peptide synthesizer (433A; Applied Biosystems, Foster City, CA, USA). The crude peptides were purified via reverse-phase semipreparative high-performance liquid chromatography (HPLC) (Waters, USA) via a C18 column. The peptide was conjugated with bovine serum albumin (BSA) via either the lysine or cysteine residue of the peptide. Conjugation via lysine residues was performed following the glutaraldehyde cross-linking method, whereas cysteine-based conjugation was performed via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). Biopanning Tomlinson I and Tomlinson J, antibody phage display libraries, KM13 helper phage and Escherichia coli (E. coli) TG1-Tr and HB2151 were procured from the Centre for Protein Engineering, MRC Laboratories, Cambridge, United Kingdom, while HuScL4 libraries were procured from Creative Biolabs, New York, USA. For screening of libraries, MaxiSorp or PolySorp immunotubes (in every alternate round) were immobilized with 4 ml of 100 μg/mL BSA-peptide and BSA in 1x PBS and kept at 4°C overnight. Thereafter, the tubes were washed three times with 1x PBS, and blocking was performed with 2% skim milk in 1x PBS for 2 hours at room temperature (RT). To remove BSA-specific phages, approximately 10 12 phages of amplified Tomlinson I, J, and HuScL4 libraries or amplified phages (from successive rounds of selection) were preincubated with BSA-coated immunotubes for 1 hour, and then the remaining phages were incubated with BSA-peptide for 1 h on a rotating platform and 1 hour of static incubation. The unbound phages were removed, and the samples were washed with 1x PBS with 0.05% Tween 20 (10 times for selection round 1 and increased 10 times after every subsequent round) to remove the weakly bound phages. Affinity elution was performed with 0.45 mL of 1 mg/ml peptide for 30 min to elute the bound phages [40]. The eluted phages were then treated with 50 μl of 10 mg/mL bovine trypsin (Type XIII from Bovine Pancreas, Sigma-Aldrich, St. Louis, MO) for 10 min. The eluted phages were further amplified and used as input phages for the next round. The biopanning was repeated 4-5 times so that the high-affinity phages bound to the peptide epitope. Polyclonal and monoclonal phage ELISA The phages obtained after 4 or 5 rounds of selection were checked for their binding to the peptide epitope. A 96-well plate was coated with 100 μg/mL peptide-BSA conjugate and BSA. The following day, the plates were washed three times with 1x PBS and blocked with 2% skim milk in 1x PBS for 2 h. The polyclonal phages/monoclonal phages were added to the antigen-coated plates and incubated for one hour at RT [21]. The phages were removed, and the plates were washed thoroughly with 0.1% Tween 20 in PBS (PBST). Anti-mouse M13-HRP was added at a 1:5000 dilution for 1 h at room temperature. The plate was washed 4 times with PBST and subsequently developed with ortho-phenylenediamine (OPD) and H 2 O 2 as substrates, and the absorbance was read at 490 nm. Cloning, expression and purification of scFvs The selected scFvs from the Tomlinson libraries were subsequently cloned and inserted into the commercially available pET22b vector (Addgene- Catalog number 69744-3), whereas the scFvs from the HuScL4 library were subsequently cloned and inserted into pET22 b-DS55 (Addgene ID-187175). In the pET22b vector , the SalI and HindIII sites were followed by the NotI, XhoI, and 6X-His tags. We modified this vector such that the Hind-III site is followed by SalI, NotI, XhoI, and 6X-His tags to express scFvs from the HuScL4 library in frame. The amber stop codon present in the scFvs was mutated to glutamine by site-directed mutagenesis. To overexpress scFvs, clones were transformed into the BL21 E. coli strain. The culture was inoculated and incubated at 37 °C for 5 h, followed by induction with isopropyl b-D-1-thiogalactopyranoside (IPTG) and incubation at 18 °C for 16-18 h. The resulting pellet was resuspended in periplasmic extraction buffer I (100 mM Tris_HCl pH=8, 20% sucrose, 1 mM EDTA) and incubated on ice for 45 min. After centrifugation, the pellet was dissolved in periplasmic buffer II (5 mM MgCl 2 in deionized water) and incubated on ice for 30 min. After centrifugation, the supernatants from both processes were mixed and used for immobilized metal affinity chromatography (IMAC). For this, mixed supernatant was loaded on Ni-NTA column (GE Healthcare) and eluted with different concentrations of imidazole with 50mM Tris pH=8 and 300mM NaCl. Further size exclusion chromatography (SEC) was performed via a HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare) by fast-performance liquid chromatography (AKTA pure) at 4°C in 50mM Tris pH=8 and 300mM NaCl buffer. Titration ELISA A 96-well ELISA plate was coated with 10 µg/ml BSA-peptide and BSA in 1x PBS overnight at 4°C. BSA was used as a control. After washing with 1x PBS, the plates were blocked with 2% skim milk in 1x PBS for two hours at room temperature. Purified antibodies were titrated from 50 µg/ml to 16 dilutions and incubated for 1 hour at 37°C. After three washes with PBST, the plates were incubated for 1 hour at 37°C with a 1:5000 dilution of an anti-His HRP-conjugated antibody (Santa Cruz Biotechnology, Cat# sc-8036). The plates were again washed three times with PBST solution. Color development was achieved via the use of the substrate o-phenylenediamine (OPD; HiMedia) in the presence of hydrogen peroxide (H₂O₂; Sigma-Aldrich). The optical density (OD) was measured at 490 nm via a SpectraMax plate reader (Molecular Devices). The EC50 values were calculated with GraphPad Prism version 6.01. Biolayer interferometry The binding of scFvs with peptides as well as proteins was performed via biolayer interferometry (BLI) with an Octet RED96e instrument (ForteBio, Molecular Dimensions). Streptavidin (SA) biosensors were used to immobilize biotinylated BSA-peptide/gp41, and BSA was used as a control until it reached a nanometer shift of 0.8-1.2 nm. The scFv was prepared in 1x PBS via twofold dilution for affinity analysis. The regeneration and neutralization buffers used during the experiment were 10 mM glycine, pH = 2.5, and 1x PBS, respectively. The association and dissociation steps were recorded for 120 s and 200 s, respectively. The data were analyzed via Forte Bio Data analysis software 10.0.0.1. The global data specifying the fit to a 1:1 binding model were used to determine the k on , k off and K D . Flow cytometr ic binding assay and site-directed mutagenesis The scFv phage clones displaying premature termination codon amber (G68, G73, G77, and G80) were mutated to glutamine by site-directed mutagenesis for expression in the BL21 strain. To analyze the binding of scFvs to the full-length gp160 protein, flow cytometry experiments were performed using HEK293T cells. HIV full-length gp160-expressing clones, named CNE8 and SF162, were procured through the NIH AIDS reagent program. Epitope analogs were generated via site-directed mutagenesis via a QuickXL-II SDM kit (Agilent Technologies- Catalog #200521) according to the manufacturer’s protocol. Human embryonic kidney 293T cells (HEK293T; procured from ATCC) were seeded in a 60 mm dish in DMEM containing 10% fetal bovine serum (FBS), 1% penicillin, and streptomycin antibiotics. The next day, cells at a density of 50-60% were transfected with PEI 25K reagent (Polysciences- Catalog #23966) in OptiMEM media. PEI was mixed with plasmid DNA at a 3:1 ratio and kept at room temperature for 30 min. DNA and PEI complexes were added slowly to the culture dish and incubated for 4-6 h at 37°C in a 5% CO2 incubator. The media was changed to complete media and incubated for 36 hours. The cells were washed to make a single-cell suspension and blocked with 2% FBS in 1x PBS (FACS buffer) for 1 hour at 4°C. The cells were fixed with BioLegend Fixation Buffer (Catalog #420801) and permeabilized with Intracellular Staining Perm Wash Buffer (BioLegend- Catalog #421002) to access the GERDRDR epitope inside the viral membrane. After that, the cells were incubated with scFvs for 1 h at 4°C and washed three times with FACS buffer. His-Tag (D3I1O) XP ® Rabbit mAb (Alexa Fluor ® 647 Conjugate, CST Cat# 14931) was added as a secondary antibody at a dilution of 1:250 for 20 min at RT. The cells were washed three times with FACS buffer and resuspended in 430 μl of 1x PBS, and samples were acquired with a BD LSRFortessa using Diva software (BD Bioscience). X-ray crystallography and structure determination Crystallization of scFv DE94 was performed via the hanging drop vapor diffusion method via a Mosquito LCP nanodispenser (TTP Labtech). Crystals were grown in conditions containing 0.5 M SPG buffer (pH=4), 25% PEG1500, and 10% glycerol. The data were collected at the ID30B beamline, European Synchrotron Radiation Facility (ESRF), with 15% glycerol as a cryoprotectant. Following data collection, HKL3000 software was used to process the data. PHASER from the CCP4 program suite was used to determine the structure by molecular replacement using PDB: 7YUEas a starting model. Refinement was carried out in PHENIX, and Coot was used for visualization. All the figures were prepared via PyMOL and Coot. The PDB validation server checked the final quality of the structure. Molecular docking and MD simulations The scFv-peptide complex was generated by using the ligand docking tool in the Schrödinger Suite [18]. The crystal structure of the scFv obtained was used as a reference. In the ligand docking tool, site specific docking was done while keeping the peptide flexible. The complex was subjected to a molecular dynamics simulations run via the Desmond 3.1 MD package (Schrödinger Inc.) [36]. Mutants of the epitope were generated in PyMOL, and energy minimization of all the complexes was performed to check for steric hindrance in the complexes. Then, Prime MM-GBSA was run. The relative binding affinity of the epitopes was calculated via the Prime MMGBSA module of Schrödinger. The ligand interaction diagram was prepared in PyMOL for interaction studies. Results Selection and design of epitopes The HIV-1 envelope protein comprises two heterodimers, both of which are considered targets for generating monoclonal antibodies. gp41 is particularly important for vaccine development because it plays a crucial role in membrane fusion. During the transition from the prefusion to the postfusion state, gp41 undergoes conformational changes, transiently exposing previously buried epitopes [10,39,41]. Structurally, gp41 consists of an N-terminal region fusion peptide (FP), followed by N-terminal and C-terminal heptad repeats (NHR and CHR), a membrane proximal external region (MPER), a transmembrane region (TM) and a cytoplasmic tail (CTT) [14] (Figure 1A). To understand the effect of antigenic diversity on the immune response, we selected two key neutralizing epitopes within gp41. By analyzing the envelope protein in the HIV database genome browser, we selected a 7-mer neutralizing epitope, 738 GERDRDR 744 , in CTT and a 6-mer neutralizing epitope, 662 ELDKWA 667 , in MPER, both of which are recognized for their neutralization potential [4,12] (Figure 1A). The GERDRDR sequence is a part of the Kennedy epitope ( 724 PRGPDRPEGIEEEG GERDRDR S 745 ), which has been implicated in virus neutralization, incorporation of the envelope in the virion, and viral infectivity. Although no broad neutralizing antibodies have been identified against this epitope, antibodies targeting this region can block cell-to-cell transmission by blocking fusion [11,19,35]. In contrast, ELDKWA is the core epitope of a well-known broadly neutralizing antibody, 2F5, against HIV [8]. Among the several broadly neutralizing antibodies against the MPER region, such as 10E8, LN01, 2F5, and 4E10, the 2F5 antibody binds to the linear N-terminal MPER epitope, which has more natural mutants than other antibodies do [31,32]. The other MPER-directed antibodies recognize helical epitopes located in the C-terminus of the MPER region near the TM region [9]. To understand the extent of degeneracy in antibodies raised against the epitopes GERDRDR and ELDKWA, we selected variants from the HIV sequence database via the web-based AnalyzeAlign tool (www.hiv.lanl.gov) focusing on representative sequences from HIV-1 Groups B, C, and A1, which cover globally prevalent clades. Based on parameters such as percentage abundance, the impact of mutations, and sequence coverage, we selected six variants for each epitope. To comprehensively represent sequence variability, we included single, double, and triple mutants, and each mutation was selected such that it changed the degree of amino acid hypdropathy (Table 1). To identify epitope-specific antibodies, we focused on the use of a human phage display library to screen selected epitopes (Figure 1B). Table 1. Selection of neutralizing epitopes and their natural escape variants from the NIH HIV database. S. No. Mutations Sequence % Abundance Group B Group C Group A1 1 Native epitope GERDRDR 37.17 0.29 0.91 2 Single mutant R ERDRDR 0.02 - - 3 GGRDRDR 0.98 - 1.82 4 GEQDRDR 5.21 41.64 16.82 5 Double mutant GEQDRGR 1.91 4.51 11.82 6 Triple mutant GEQGRGR 0.21 0.44 10 7 GGRDSGR 0.1 - - 1 Native epitope ELDKWA 57.52 - 6.98 2 Single mutant A LDKWA 11.52 0.3 75.24 3 ELDKWD 2.62 0.32 - 4 EWDKWA 0.49 - - 5 Double mutant K LDEWA 1.36 - - 6 A LDKWQ - 0.32 0.47 7 Triple mutant A LGKWD 0.06 28.73 - Biopanning and Characterization of GERDRDR -Specific Antibodies Antibodies can target various regions of the HIV envelope protein involved in virus infection and entry. The GERDRDR epitope is involved in membrane fusion during viral entry. Four rounds of biopanning were performed to isolate epitope-specific antibodies via the human-based semisynthetic Tomlinson I and J scFv phage display libraries. Progressive enrichment of epitope-specific phages was observed in rounds 3 and 4 (Table S1), as confirmed by polyclonal phage ELISA (Figure S1). A total of 400 monoclones from the fourth round of biopanning were tested for binding to the native epitope, and varying degrees of binding reactivity were observed (Figure 2A). Based on peptide/BSA OD 490 values, 100 top binders were selected and further analyzed via ELISA (Figure S2A). To assess the extent of cross-reactivity, monoclonal phages with high binding to the native epitope were tested against cross-clade variants. These phages were examined for their ability to cross-react with single mutants (RERDRDR, GGRDRDR, and GEQDRDR), double mutants (GEQDRGR), and triple mutants (GEQGRGR and GGRDSGR) (Figure 2B). Compared with the native epitope, which was defined as a percentage O.D. value, most of the mutants presented no significant change in the mean OD values, except for GEQDRDR and GEQDRGR, which presented reduced binding. (Figure S2B). This reduced binding suggests that specific amino acid substitutions at the 3rd and 6th positions of the epitope can significantly impact the interaction with the antibodies. Sequencing of 15 cross-reactive high-binding clones resulted in the identification of five unique scFvs (Figure S2C & S3). These scFvs were subsequently cloned and inserted into the pET22b vector, expressed, and purified to high homogeneity (Figure S4A and B). To assess the binding of these selected scFvs to both the native and variant epitopes in soluble form, ELISA was performed in a series of dilutions ranging from 50 µg mL -1 to 1.52 ng mL -1 . All five scFvs bound to the full panel of variants (Figure 2C), although with different binding ranges. The EC 50 values of scFvs G8, G68, G73, G77, and G80 were determined to be 11.72 µM, 5.51 µM, 2.07 µM, 1.26 µM, and 16.60 µM, respectively. Among all the scFvs, G77 presented the highest affinity, followed by G73. Further analysis via biolayer interferometry (BLI) confirmed the multireactive potential and binding kinetics of scFvs to HIV epitope mutants (Table 2). A heatmap of the equilibrium dissociation constant (K D ) revealed that all five scFvs bind in the micromolar range (Figure 2D), reinforcing their potential as broadly reactive antibodies capable of recognizing epitope variants associated with HIV immune evasion. Table 2. BLI derived K D values (M) of scFvs to native epitope, its variants, and the gp41 protein. GERDRDR RERDRDR GGRDRDR GEQDRDR GEQDRGR GEQGRGR GGRDSGR gp41 G8 16.74 x 10 -6 5.74 x 10 -6 8.16 x 10 -6 25.77 x 10 -6 7.69 x 10 -6 9.19 x 10 -6 7.19 x 10 -6 6.86 x 10 -6 G68 5.97 x 10 -6 4.32 x 10 -6 3.37 x 10 -6 8.26 x 10 -6 9.99 x 10 -6 4.08 x 10 -6 2.92 x 10 -6 2.85 x 10 -6 G73 3.44 x 10 -7 2.20 x 10 -6 2.20 x 10 -6 3.91 x 10 -6 4.21 x 10 -6 3.68 x 10 -6 3.66 x 10 -6 5.21 x 10 -6 G77 8.98 x 10 -6 1.54 x 10 -6 2.28 x 10 -6 2.51 x 10 -6 3.42 x 10 -6 1.22 x 10 -6 1.67 x 10 -6 2.83 x 10 -6 G80 9.40 x 10 -6 1.53 x 10 -6 2.18 x 10 -6 2.86 x 10 -6 2.68 x 10 -6 2.56 x 10 -6 3.73 x 10 -6 3.50 x 10 -6 Binding analysis of GERDRDR-specific antibodies to gp41 and gp160 The affinity data clearly revealed that scFvs could bind the native peptide epitope and its variants in a physiologically relevant range. To assess the binding in a more native context, we next evaluated the binding of scFvs with recombinantly expressed full-length gp41 containing the native epitope sequence. Biotinylated gp41 protein was immobilized on a streptavidin (SA) sensor, and different scFvs were used as the analyte at concentrations ranging from 10 µM to 70 nM. Interestingly, all the scFvs bound to the gp41 protein in the micromolar range (Table 2). The binding affinity curves with association and dissociation profiles are shown in Figure 3A. To further investigate whether the scFvs could recognize the native conformation of gp160 in a membrane-anchored state, we tested the binding of the scFvs to the full-length gp160 protein expressed on the mammalian cell surface. The expression of the gp160 protein on the cell surface corresponds approximately to a model of the virus particle in which gp160 is embedded in the viral membrane. Site-directed mutagenesis was performed to generate variants of the full-length gp160 protein via the HIV SF162 plasmid. The RERDRDR and GGRDSGR variants were not successfully generated by site-directed mutagenesis. Flow cytometry analysis revealed that all the scFvs were able to recognize the native gp160 protein and four of its variants, as evidenced by the shift in the fluorescence peak (Figure 3B). The shift in the whole peak with respect to the control peak indicates strong binding to the gp160 protein. The greater the shift in the peak is, the stronger the binding. The disparity in the peak shift with different scFvs indicated different binding strengths. Overall, these results indicated that the scFvs could recognize both recombinant and membrane-bound forms, highlighting their multiple reactive potentials. Biopanning and Characterization of ELDKWA-Specific Antibodies The membrane fusion-associated ELDKWA epitope, which is exposed on the exterior of the viral membrane, was also subjected to screening against the Tomlinson I, Tomlinson J, and HuScL4 libraries as previously described (Figure 1B). Phages specific to this epitope were enriched for 4-5 rounds of biopanning (Table S2), with increased binding observed after each round by polyclonal ELISA (Figure S5). From the fourth and fifth rounds, 1150 monoclonal phages were tested for binding to the native epitope, and 150 high-affinity binders were selected based on the peptide/BSA OD 490 values (Figure 4A, S6A). Of these, 80 top binders were selected for further analysis. To determine the breadth of antigen recognition, the binding of the 80 selected phages to the six selected variants was tested. Remarkably, most phage monoclones recognized the majority of the variants, demonstrating strong cross-reactivity (Figure 4B). Compared with the native epitope, which was defined as a percentage O.D. value of 100%, the triple mutant ALGKWD presented the lowest mean value of 13%, and the single mutant EWDKWA exhibited a mean value of 92%, which was comparable to that of the natural epitope. The single mutants ALDKWA and ELDKWD, showed a mean OD 490 value of 59%, whereas the corresponding values of the double mutants KLDEWA and ALDKWQ were 59% and 54%, respectively (Figure S6B). These results indicate that the phages screened against the native peptide epitope were highly cross-reactive with the escape variants. Sequencing of 20 high-affinity binders yielded four distinct clones from the HuScL4 library and four from the Tomlinson libraries (Figure S6C & S7). These scFvs were subsequently cloned and inserted into the pET22b vector, expressed, and purified (Figure S8A, B). The binding affinities were assessed by ELISA across various dilutions, ranging from 50.00 µg mL -1 to 1.52 ng mL -1 . Interestingly, the eight selected scFvs bound to the native epitope with varying affinities. The EC 50 values for scFvs DE13, DE45, DE62, DE72, DE34, DE37, DE83, and DE94 ranged from 4.27 µM to 0.25 µM, indicating a spectrum of binding affinities ranging from micromolar to submicromolar. Notably, scFvs DE94 and DE34 exhibited the highest binding affinity among all the scFvs (Figure 4C). While scFvs DE13, DE34, DE37, DE83, and DE94 recognized all the tested variants, scFv DE62 failed to bind KLDEWA, and scFv DE72 did not recognize ALGKWD (Figure 4C), indicating differential binding specificities among the clones. To complement the affinity calculations by ELISA, BLI was used to quantify the binding kinetics. BLI analysis revealed the diversity in binding affinities among the scFvs (Figure 4D, Table 3). Some scFvs retained binding across all the selected variants, whereas others lost binding to double or triple mutants, indicating sensitivity to sequence variations. Table 3. Comparative analysis of the K D values (M) of all the scFvs against the native epitope and its variants. scFv ELDKWA ALDKWA ELDKWD EWDKWA KLDEWA ALDKWQ ALGKWD gp41 DE13 2.17 x 10 -7 3.13 x 10 -7 2.35 x 10 -7 1.95 x 10 -7 3.98 x 10 -7 2.66 x 10 -7 2.99 x 10 -7 5.08 x 10 -6 DE45 1.38 x 10 -7 1.85 x 10 -7 1.92 x 10 -7 1.59 x 10 -7 0.58 x 10 -7 3.52 x 10 -7 2.31 x 10 -7 6.46 x 10 -6 DE62 2.49 x 10 -7 3.20 x 10 -7 1.80 x 10 -7 1.98 x 10 -7 No binding 2.42 x 10 -7 2.10 x 10 -7 1.55 x 10 -5 DE72 1.39 x 10 -7 1.62 x 10 -7 0.96 x 10 -7 0.76 x 10 -7 1.39 x 10 -7 0.82 x 10 -7 No binding 3.39 x 10 -6 DE34 11.90 x 10 -8 9.70 x 10 -8 7.43 x 10 -8 5.76 x 10 -8 6.84 x 10 -8 8.71 x 10 -8 1.09 x 10 -7 2.76 x 10 -6 DE37 1.95 x 10 -6 2.27 x 10 -6 2.85 x 10 -7 4.40 x 10 -7 7.20 x 10 -7 8.35 x 10 -7 1.36 x 10 -6 2.67 x 10 -6 DE83 5.51 x 10 -6 4.49 x 10 -6 3.44 x 10 -6 2.81 x 10 -6 3.41 x 10 -6 2.95 x 10 -6 3.99 x 10 -6 3.64 x 10 -6 DE94 3.44 x 10 -7 4.78 x 10 -7 3.68 x 10 -7 2.48 x 10 -7 5.25 x 10 -7 2.00 x 10 -7 3.73 x 10 -7 0.94 x 10 -6 Binding analysis of ELDKWA-specific antibodies to gp41 and gp160 As described previously, it was necessary to check whether the scFvs screened against the peptide epitope could also recognize the corresponding full-length HIV protein. Binding kinetics with the gp41 protein confirmed that all the scFvs were capable of binding to gp41, with K D values in the micromolar range (Figure 5A, Table 3). The highest binding affinity observed was 0.94 µM for scFv DE94. To explore the multireactive potential of scFvs, a gp160 mammalian expression clone (CNE8) was expressed on HEK293T cells, and surface expression was analyzed via flow cytometry. Variants of native epitopes were introduced into the gp160 mammalian plasmid via site-directed mutagenesis. Intriguingly, five out of eight scFvs showed shifts in fluorescence intensity, indicating binding to gp160 and its variants on the cell surface (Figure 5B), and a shift was observed across all the variants tested. These results suggested that scFvs can bind the whole virus. While all the scFvs bound the soluble recombinant gp41 protein, some exhibited reduced or no binding to the membrane-anchored form, likely due to limited epitope accessibility on the native-like cell surface. The binding of scFvs with all the variants revealed the multireactive potential of these scFvs against the HIV-1 envelope protein. Crystallization and structure determination of scFv DE94 To gain insight into the molecular interaction of scFv with peptide antigens, we attempted to crystallize all anti-GERDRDR antibodies and five anti-ELDKWA antibodies, both in the apo form and with the native peptide, via the hanging drop vapor diffusion method. Crystals of scFv DE94 (anti-ELDKWA antibody) were successfully obtained in the apo form, which diffracted at 2.53 Å resolution and belonged to the monoclinic space group P2 1 , with cell dimensions of a = 43.80 Å, b = 183.92 Å, c = 65.73 Å, and β = 93.60°. DE94 has relatively high binding among all five anti-ELDKWA antibodies. The complete dataset has an Rmerge of 15.0%. Initial phasing of the scFv was performed by molecular replacement via a previously published independent scFv structure (PDB ID: 7YUE). The refined structure contains four molecules in an asymmetric unit of the scFv (Figure 6A). The final R work and R free values were 19.9% and 24.3%, respectively, with 97.59% amino acid residues in the allowed region of the Ramachandran plot. All the molecules of the scFv of an asymmetric unit were superimposed on each other, and no significant structural deviations were observed, as evident from the root mean square deviations (RMSD) values, which were in the range of 0.29-0.41 Å (Figure 6B). The electron density of chain D in DE94 was very weak. The data collection statistics and refinement statistics are shown in Table S3. The scFv DE94 comprises variable light and variable heavy chains connected via a Gly-Ser linker. The six complementary determining regions (CDRs) forming the antigen-binding site are highlighted in Figure 6C. The atomic coordinates and structure factors are available in the RCSB Protein Data Bank under accession code 9V5N. Mapping key residues in the scFv DE94-ELDKWA complex via molecular docking To understand the molecular determinants involved in the recognition of the ELDKWA epitope by the scFv DE94, we conducted extensive crystallographic trials of the scFv-epitope (ELDKWA) complex. As these efforts did not yield a resolved structure, we employed computational tools to investigate the molecular interactions between the scFv and the peptide antigen. Rigid docking was performed at the CDRs of the scFv via the ligand docking tool in the Schrödinger module. The binding strength of scFvs toward peptides was computed via the molecular mechanics generalized Born surface area (MM/GBSA) approach. The binding energy of scFv DE94-ELDKWA was -63.62 kcal/mol. The scFv-peptide complex formed with the lowest Gibbs free energy is shown in Figure 7A. To gain further insight into the binding mechanism and overall stability of the scFv-peptide complex, a long-term (500 ns) all-atom molecular dynamics simulation was performed. After the trajectories were processed, the RMSDs were examined. The system was stable throughout the run, as shown in Figure S8A. The data suggested that scFv had a stable interaction with ELDKWA, which was supported by the finding that the system's RMSD values remained constant (Figure S9A). The backbone Root Mean Square Fluctuation (RMSF) of the scFv was also examined throughout the 500 ns run, and the Gly-Ser linker in the crystal structure connecting the heavy and light chains demonstrated high fluctuations (Figure S9B). To envisage the change in binding affinity with different variants, we mutated the native peptide within the scFv-ELDKWA complex and performed energy minimization for each mutant. The resulting models were used to calculate binding free energies via the MM-GBSA module in Schrödinger. The binding energies of all the epitope variants are shown in Figure S9E. The representative structural overlays of the native and mutant complexes, highlighting the modeled conformations within the binding site, are shown in Figure S9C-D. To elucidate the interactions between the scFv-peptide complexes, hydrogen bonds and hydrophobic interactions were analyzed. Hydrogen bonding was observed between the peptide and the residues located in the CDRH1, CDRH2, CDRH3, CDRL1, and CDRL2 regions of the scFv. In the scFv-ELDKWA complex, E1 of the peptide is involved in H-bond interactions with R61 of CDRH2 and R103 of CDRH3; D3 is involved in H-bond interactions with A35 of CDRH1 and Q55 of CDRH2, with a binding energy of -63.62 kcal/mol. E1, D3, W5, and A7 also exhibited hydrophobic interactions with CDR residues, as shown in Figure 7B. All these interactions made the scFv-epitope complex stable. In the case of the scFv-ALDKWA complex, the binding energy was reduced to -48.73 kcal/mol, which was due to the loss of alanine interactions with the scFv. Similarly, in the case of ALDKWQ (double mutant), the binding energy was -47.26 kcal/mol, as the mutation had no effect at the 6 th position. However, in the case of ALGKWD (triple mutant), the energy was further reduced to -39.95 kcal/mol due to the loss of interaction of glycine at the 3 rd position. In the case of ELDKWD, with a binding energy of -62.64 kcal/mol, there was no change in interaction due to the mutation at the 6 th position. In the double mutant KLDEWA, the binding energy is equivalent to that of the native epitope, as the mutation from E to K at the first position is balanced by the mutation from K to E at the 4 th position. EWDKWA has a more negative ΔG (-67.92 kcal/mol), indicating a more stable complex, which can be verified by the additional interaction of W4 with the CDRs of the scFv (Figure 7C-H). Although there was a difference in the binding energy of the native epitope with its variants, notably, the ΔG values for all the epitopes were in the range of antigen-antibody interactions. Discussion Monoclonal antibodies are recognized as key mediators of protective immunity against HIV-1 [ 3 , 33 ]. There is a need to develop broadly cross-reactive therapeutic antibodies. While cross-reactive antibodies are known to exist in the immune system, immune evasion by HIV-1 is also well-documented. This study aimed to explore antibody cross-reactivity and immune escape mechanisms by comparing antibodies against two critical epitopes of the HIV-1 envelope protein gp41, GERDRDR and ELDKWA, and to draw insights from these findings in the broader context of other fast-mutating viruses. Compared with those against ELDKWA, antibodies targeting the epitope GERDRDR exhibited greater cross-reactivity, reflecting different hotspot behaviors. Together, both epitopes demonstrated structural plasticity at antigen-combining sites. The broader reactivity observed against GERDRDR-specific epitopes can likely be attributed to multiple charged amino acids across the epitope sequence, enabling the binding of scFvs to all the variants. In contrast, antibodies against ELDKWA showed less cross-reactivity, and only five of the eight binders recognized the native epitope and its variants. A plausible explanation for the lack of binding by the remaining three scFvs could be steric hindrance from the membrane-anchored protein. Notably, during HIV-1 infection, polyreactivity is observed in approximately 70–75% of anti-gp160 antibodies, and this property is even more pronounced among anti-gp41 antibodies, of which 85–90% exhibit polyreactivity [ 7 , 9 , 29 ]. This high proportion of polyreactivity highlights the importance of multispecific antibodies in shaping the immune response. Moreover, our findings support the hypothesis that B-cell clones producing multispecific antibodies undergo positive selection during affinity maturation [ 30 ]. This finding is in line with previous studies indicating that the primary factor contributing to antibody multispecificity in response to HIV-1 Env proteins is the inherent conformational flexibility of the antigen-binding site [ 6 , 42 ]. This structural plasticity is not unique to HIV but is also observed in other rapidly mutating viruses. For example, multispecific antibodies against a key antigenic target, the hemagglutinin (HA) protein, have been identified in the influenza virus [ 40 ]. The HA protein undergoes significant antigenic drift, leading to the emergence of new strains. The presence of multispecific antibodies in the humoral immune response is necessary for preventing immune evasion during influenza infections despite mutations that are beneficial for viral escape and lead to antigenic diversification [ 40 ]. This phenomenon is similar to what we observed in the case of HIV-1, where antibodies with broad reactivity can accommodate diverse variants owing to the structural flexibility of the epitopes. Similarly, in SARS-CoV-2, multiple specific antibodies against the spike protein have been identified. Antibodies produced following vaccination against SARS-CoV-2 have the ability to neutralize a range of emerging strains, which may be attributed to their multispecific nature [ 22 , 38 ]. The broad reactivity of such antibodies is crucial for effective immunity, especially as new variants continue to emerge. Previous studies have reported that neutralizing antibodies targeting the MPER of HIV-1 gp41 are cross-reactive [ 2 , 26 ]. Specifically, the broadly neutralizing antibody 2F5, which targets the ELDKWA epitope, relies on DKW residues for interaction [ 8 ], and substitution from DKW to DSW often makes the virus resistant to antibody neutralization [ 17 ]. In the present study, the scFvs demonstrated remarkable recognition of mutations in the DKW region, except for scFv DE62, which failed to recognize the mutant peptide KLDEWA, and scFv DE72, which was unable to recognize the triple mutant peptide ALGKWD, as confirmed through ELISA and BLI assays. Nevertheless, when the variants were exposed on the surface of mammalian cells, cross-reactivity was observed with all the variants. This observation could reflect the conformational constraints imposed by the membrane-anchored protein. These findings correlate with previous findings that the immune system can produce antibodies with degenerate potential, enabling recognition of cross-clade variants [ 6 , 42 ]. To gain deeper insight into these molecular interactions, we performed crystallographic studies on scFv DE94. These studies, combined with in silico analysis of scFv DE94 binding to ELDKWA and its mutants, revealed that the predicted binding energy ranged from − 39.95 to -67.92 kcal mol -1 . Certain mutations were associated with less favorable docking scores, suggesting in potential decrease in binding energy. The structural plasticity observed in both gp41 epitopes highlights the adaptability of multispecific antibodies, which is crucial for combating the high mutation rate of HIV and ensuring an effective immune response. Collectively, the biochemical, cell-based, and structural analyses support our hypothesis that multispecific antibodies can accommodate antigenic diversity, although certain mutations may impact binding efficacy. Our observations are similar to findings in other rapidly mutating viruses, such as influenza virus and SARS-CoV-2, where multispecific antibodies are crucial in neutralizing diverse strains despite antigenic drift [ 22 , 38 ]. HIV has a high mutation rate, with approximately 10 − 3 substitutions per nucleotide per replication cycle, contributing to its genetic diversity [ 34 , 37 ]. However, despite this, its evolution is relatively slow at the population level [ 6 – 8 ]. This could be due to a number of factors, such as less selection pressure over time, a weak immune response, nonbeneficial mutations affecting viral fitness, reversal of patient-specific adaptive changes following transmission and a retrieval mechanism in which the initially infected virus is preferentially transmitted [ 41 , 42 ]. The presence of multispecific antibodies may help constrain the evolution of new strains by providing broad protection against diverse strains. Our findings suggest that not all mutations lead to antigenic drift; only crucial mutations significantly alter viral antigens, rendering existing antibodies less effective. This study highlights the importance of antibody cross-reactivity and structural plasticity in HIV-1 research. The high prevalence of cross-reactive antibodies and their multispecific nature emphasize the need for vaccines and therapeutics that can accommodate viral diversity. Insights gained from crystallographic studies and antibody behavior, particularly regarding the impact of mutations, are valuable for developing novel strategies to increase protection against mutant strains of viruses and improve disease control efforts. Declarations Acknowledgments We thank Mr. Romain Talon for providing support with data collection at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. We would like to thank Mr. Ravinder Kumar for assisting with the cell culture. Funding This work was supported by Department of Biotechnology, Ministry of Science and Technology; India. Ethics declarations Ethics approval and consent to participate Not applicable. Competing interests The authors have no relevant financial or non-financial interests to disclose . Author contributions D.M.S. supervised the project; D.J. and D.M.S. conceived and designed the experiments; D.J. G.K. and K.J.K. synthesized the peptides. D.J. and S.V. carried out the library screening. D.J. carried out the scFv purification, ELISA, BLI, FACS, crystallization, structure determination and MD simulation; D.J. and Z.K.M. carried out the structure refinement. D.J. wrote the original draft; D.M.S. and D.J. reviewed and edited the draft and analyzed the data. All the authors reviewed and approved the final version of the manuscript. Data availability The atomic coordinates and structure factors have been deposited in the Protein Data Bank http://www.wwpdb.org (PDB ID code: 9V5N). References Amit A.G., Mariuzza R.A., Phillips S.E. and Poljak R.J. Three-dimensional structure of an antigen-antibody complex at 2.8 A resolution. Science 233(4765):747–753, 1986. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9072434","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":617978667,"identity":"1c16d6e7-61d9-453b-977f-c64d3cc50034","order_by":0,"name":"Deepika Jaiswal","email":"","orcid":"","institution":"International Centre For Genetic Engineering and Biotechnology New Delhi","correspondingAuthor":false,"prefix":"","firstName":"Deepika","middleName":"","lastName":"Jaiswal","suffix":""},{"id":617978668,"identity":"167441bb-6c6a-4d1f-a394-138d88eec35c","order_by":1,"name":"Sheenam Verma","email":"","orcid":"","institution":"International Centre For Genetic Engineering and Biotechnology New Delhi","correspondingAuthor":false,"prefix":"","firstName":"Sheenam","middleName":"","lastName":"Verma","suffix":""},{"id":617978669,"identity":"a52836c4-54c5-471d-ae7c-da831626bd45","order_by":2,"name":"Zaid K Madni","email":"","orcid":"","institution":"International Centre For Genetic Engineering and Biotechnology New Delhi","correspondingAuthor":false,"prefix":"","firstName":"Zaid","middleName":"K","lastName":"Madni","suffix":""},{"id":617978670,"identity":"d4e3e93f-c7f9-4105-a5cc-966403d506a0","order_by":3,"name":"Gagandeep Kaur","email":"","orcid":"","institution":"National Institute of Immunology","correspondingAuthor":false,"prefix":"","firstName":"Gagandeep","middleName":"","lastName":"Kaur","suffix":""},{"id":617978671,"identity":"cc6fd952-9697-460b-972f-f58c0fbf7c09","order_by":4,"name":"Kanwal J Kaur","email":"","orcid":"","institution":"National Institute of Immunology","correspondingAuthor":false,"prefix":"","firstName":"Kanwal","middleName":"J","lastName":"Kaur","suffix":""},{"id":617978672,"identity":"424f7fb3-ce24-461c-8804-f42fc876f5c8","order_by":5,"name":"Dinakar M. Salunke","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-9003-7373","institution":"ICGEB New Delhi: International Centre For Genetic Engineering and Biotechnology New Delhi","correspondingAuthor":true,"prefix":"","firstName":"Dinakar","middleName":"M.","lastName":"Salunke","suffix":""}],"badges":[],"createdAt":"2026-03-09 11:46:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9072434/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9072434/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106727272,"identity":"803d19ed-9b0e-4db0-93bd-43ef38c1dd42","added_by":"auto","created_at":"2026-04-12 18:38:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":295378,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEpitope selection and antibody biopanning led to epitope-specific phage clones. A. \u003c/strong\u003eSchematic illustration of the gp41 domains consisting of a fusion peptide (FP), N-heptad repeat (NHR), C-heptad repeat (CHR), membrane-proximal external region (MPER), transmembrane domain (TM), and cytoplasmic tail (CTT). Broadly neutralizing antibodies and their corresponding epitopes are depicted on the gp120 and gp41 proteins of the HIV envelope.\u003cstrong\u003e B.\u003c/strong\u003e Schematic of phage display antibody library screening, consisting of 4 main steps: (a) Affinity screening involves immobilization of the BSA-conjugated antigen on immunotubes and the addition of antibody libraries; (b) high-affinity antibodies bind to the antigens while the weak binders are washed off via PBST; (c) bound phages are eluted for the recovery of high-affinity binders, followed by (d) amplificationof the eluted epitope-specific phages. Amplified phages from each round are used as input phages for the next round of biopanning.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9072434/v1/918893a82b40ecc880b798e9.png"},{"id":106727119,"identity":"6c55b57b-b182-4e3c-a847-c98462c6c36d","added_by":"auto","created_at":"2026-04-12 18:38:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":288338,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBinding analysis of monoclonal phages against GERDRDR and their cross-reactivity. A.\u003c/strong\u003e Identification and isolation of GERDRDR-specific monoclonal phages via ELISA. \u003cstrong\u003eB. \u003c/strong\u003eCross-reactivity profile of the selected phage clones against all the mutants. \u003cstrong\u003eC. \u003c/strong\u003eEach scFvs' binding capacity to GERDRDR and its variants was tested via indirect ELISA, which revealed a dose-response curve. Binding graphs are combined per antibody. \u003cstrong\u003eD. \u003c/strong\u003eHeatmap displaying the binding of scFvs to the natural escape variants of the GERDRDR epitope. The -log10 K\u003csub\u003eD\u003c/sub\u003e value range for each epitope is indicated from 0-7, and BSA was used as a negative control.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9072434/v1/ff77483bfecf053bcae89451.png"},{"id":106634100,"identity":"cdfcfeb0-1627-4926-8bf5-a351d8eb593a","added_by":"auto","created_at":"2026-04-10 16:28:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":249712,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBinding analysis of scFvs to the gp41 and gp160 proteins. A. \u003c/strong\u003eSensogram showing the binding of the gp41 protein (ligand) immobilized on the SA sensor to the scFvs (analyte) at different concentrations. The sensogram shows the association and dissociation profiles of all the scFvs. \u003cstrong\u003eB. \u003c/strong\u003eAll the scFvs were analyzed for their binding with the gp160 protein expressed on HEK 293T cells by flow cytometry. Unstained cells were used as a control.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9072434/v1/f8f18a45e1da3e2c5cdf9d0d.png"},{"id":106726876,"identity":"143d924e-8faf-4d56-9213-bedbee0e7ace","added_by":"auto","created_at":"2026-04-12 18:37:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":230004,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBinding analysis of selected monoclonal phages exhibiting cross-reactivity A.\u003c/strong\u003e Identification and isolation of epitope-specific monoclonal phages via ELISA. \u003cstrong\u003eB. \u003c/strong\u003eCross-reactive profile of the selected phage clones against all the mutants. \u003cstrong\u003eC.\u003c/strong\u003e Each scFv binding capacity to ELDKWA and its variants was tested via indirect ELISA. Binding graphs are combined per antibody. \u003cstrong\u003eD.\u003c/strong\u003e Heatmap representing the binding affinities of scFvs to the ELDKWA epitope and its natural escape variants. BSA was used as a negative control. The binding strength is represented by -log\u003csub\u003e10\u003c/sub\u003e K\u003csub\u003eD\u003c/sub\u003e values in the range of 0-7.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9072434/v1/d9c3048f7954449c1e206c07.png"},{"id":106634103,"identity":"e8614e2d-6185-40bf-8091-85fe42add894","added_by":"auto","created_at":"2026-04-10 16:28:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":243055,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBinding analysis of the scFvs against the gp41 and gp160 proteins. A. \u003c/strong\u003eBLI kinetics curve showing the association and dissociation profilesof scFvs against the gp41 protein at different concentrations. \u003cstrong\u003eB. \u003c/strong\u003escFvs were analyzed for their binding with the gp160 protein expressed on HEK293T cells by flow cytometry. Unstained cells were used as a control.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9072434/v1/2e2ade0dd907806e1e6d973b.png"},{"id":106727121,"identity":"d08516cd-4e85-4067-8a83-ae0269a0cece","added_by":"auto","created_at":"2026-04-12 18:38:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":647159,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCrystal structure of scFv DE94. A. \u003c/strong\u003eStereoscopic view of scFv DE94 containing four monomers per asymmetric unit. The complementarity determining regions (CDRs) of each monomer are highlighted. \u003cstrong\u003eB.\u003c/strong\u003eStereoscopic view of the superimposition of Cα atoms of all monomeric units. \u003cstrong\u003eC. \u003c/strong\u003eStructure of a monomeric unit of scFv DE94. The heavy and light chains of the scFvs are colored green and cyan, respectively. CDRs on heavy and light chains are shown in brown and magenta, respectively. All the figures are generated via PyMOL\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9072434/v1/392b85fd3da15bc35562b2e3.png"},{"id":106726918,"identity":"a3b6a53a-3f21-4c54-8712-341fdf8b3ce7","added_by":"auto","created_at":"2026-04-12 18:37:41","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":646974,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModel of the scFv DE94-ELDKWA complex and stereo view of interactions with ELDKWA and its variants A.\u003c/strong\u003e Overall view of the docked complex of scFv-ELDKWA,where scFv is shown in surface representation and ELDKWA in stick form. \u003cstrong\u003eB-H\u003c/strong\u003e. Stereoscopic view of\u003cstrong\u003e the \u003c/strong\u003ehydrogen bond interactions and hydrophobic interactions of scFv DE94 with ELDKWA, ALDKWA, ELDKWD, EWDKWA, KLDEWA, ALDKWQ, and ALGKWD is shown along with its Gibbs free energy. Ligand interaction diagrams were prepared in PyMOL with a maximum distance of hydrophobic interactions at 4.0 Å and hydrogen bonds at 3.5 Å. Heavy and light chains are represented in green and cyan, respectively, and peptides are shown in magenta.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9072434/v1/ad208d742659aa4bb114b12a.png"},{"id":106728045,"identity":"b972c868-3915-40d6-b401-ec2fd19b87ad","added_by":"auto","created_at":"2026-04-12 18:41:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3697598,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9072434/v1/b95ecfd9-c7df-4624-9d84-2e89fbf23db0.pdf"},{"id":106726921,"identity":"11876cd8-4b24-406c-bf72-55cf2425ed87","added_by":"auto","created_at":"2026-04-12 18:37:41","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2453952,"visible":true,"origin":"","legend":"","description":"","filename":"Jaiswaletalsupplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-9072434/v1/4eaee8d7a8a5239fe47df97d.docx"}],"financialInterests":"","formattedTitle":"Immunologic insights into the critical epitopes of HIV-1 and structure-based characterization of cross-reactive antibodies","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe immune system produces antibodies to defend the body against invading pathogens by binding to and neutralizing them. Traditionally, antigen-antibody interactions are perceived as highly specific [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, accumulating structural data have revealed that host-pathogen interactions are much more complex and exist in dynamic equilibrium. While the immune system is under pressure to eradicate pathogens, selection pressure exists on pathogens to evade detection and establish infection [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Fast-mutating viruses, such as human immunodeficiency virus (HIV-1) and influenza, exploit this balance by incorporating genetic variations in their surface-exposed envelope proteins, thereby evading the immune response. As a result, the immune system can no longer detect mutated antigenic surfaces, disturbing the equilibrium between the host and the pathogen. The immune system must employ some strategy to defend against these viruses and maintain this equilibrium. Several studies have shown that in the case of infection, the regular B-cell repertoire consists of two types of antibody populations: monospecific and multispecific [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The role of monospecific antibodies is well known, as antibodies are specifically generated against invading pathogens. However, the significance and function of multispecific antibodies remain poorly understood and require further investigation.\u003c/p\u003e \u003cp\u003eMultispecificity in the affinity-matured antibodies has been previously reported [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], suggesting a potential role for these antibodies in combating the effect of antigenic variations in fast-mutating viruses. These viruses present a physiologically relevant model for understanding the role of multispecificity in the immune response. A previous study on the influenza hemagglutinin (HA) protein and its mutants investigated the mechanisms of antibody cross-reactivity [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], suggesting that multispecific antibodies may help prevent immune evasion despite some mutations successfully driving antigenic variation. To gain a deeper understanding of the functions of multispecific antibodies, we extended our investigation to examine multispecific antibodies that target HIV-1.\u003c/p\u003e \u003cp\u003eThe HIV envelope proteins play a central role in viral replication and are key determinants of the virus\u0026rsquo;s antigenic and pathogenic properties [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The functional envelope is a trimer of two heterodimeric surface glycoproteins, gp120 and gp41. gp120 is essential for the attachment of the virus, whereas gp41 mediates the fusion of the viral and host membranes. The host\u0026rsquo;s key defense mechanism to combat HIV infection is antibody-mediated neutralization, and many broadly neutralizing antibodies have been identified and studied thus far [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The gp41 region of the envelope is more conserved than the gp120 region [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Within the gp41 region, one of the elusive vaccine targets is the membrane-proximal external region (MPER), which is located outside the viral membrane [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In addition, the Kennedy epitope is another immunogenic site within the cytoplasmic tail (CTT) of gp41 located inside the viral membrane, making it less accessible. However, numerous studies have demonstrated that antibodies directed against the epitope found in the CTT region can neutralize the virion [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], indicating that specific epitopes become accessible during viral conformational changes. These antibodies can block the cell-to-cell transmission of HIV by blocking fusion [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe contrast between HIV immune evasion strategies and recent studies on antibody promiscuity is fascinating. HIV escapes immune surveillance by incorporating mutations in its envelope proteins. It has been reported that even a point mutation is enough to evade the immune response [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, multispecific antibodies are also increased during viral infections [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], revealing an interesting dichotomy. In light of this, our study aims to address the mechanisms underlying HIV-1 immune evasion and antibody promiscuity. We focused on two critical epitopes- MPER and Kennedy epitopes within gp41 to understand the role of multispecific antibodies in different hotspots of HIV. Using human-based antibody phage display libraries, we screened antibodies against these epitopes and successfully isolated a diverse panel of cross-reactive antibodies against both epitopes. The selected antibodies showed diverse binding affinities, ranging from micromolar to subnanomolar. Intriguingly, antibodies screened against peptide epitopes were able to recognize the epitope within the full-length protein. Crystallographic and in silico analyses have provided insights into the molecular mechanisms of antibody interactions with epitope peptides and their variants. Our findings emphasize the physiological relevance of antibody promiscuity in the natural immune response and its potential implications for therapeutic and vaccine strategies targeting HIV.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch4\u003e\u003cstrong\u003ePeptide\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eselection, synthesis, and conjugation to BSA\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eThe neutralizing epitopes were selected from the HIV-1 gp41 region. After two critical epitopes from the MPER and CTT regions were selected, escape variants of both epitopes were chosen from the HIV sequence database (http://www.hiv.lanl.gov/). We analyzed approximately 12000 variants of these epitopes present in the database, resulting in the selection of six variants against each epitope. All the peptides were synthesized with N-terminal cysteine/lysine for BSA conjugation via an automated peptide synthesizer (433A; Applied Biosystems, Foster City, CA, USA). The crude peptides were purified via reverse-phase semipreparative high-performance liquid chromatography (HPLC) (Waters, USA) via a C18 column. The peptide was conjugated with bovine serum albumin (BSA) via either the lysine or cysteine residue of the peptide. Conjugation via lysine residues was performed following the glutaraldehyde cross-linking method, whereas cysteine-based conjugation was performed via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP).\u003c/p\u003e\n\u003ch5 id=\"_Toc122270620\"\u003e\u003cstrong\u003eBiopanning\u003c/strong\u003e\u003c/h5\u003e\n\u003cp\u003eTomlinson I and Tomlinson J, antibody phage display libraries, KM13 helper phage and \u003cem\u003eEscherichia coli (E. coli)\u003c/em\u003e TG1-Tr and HB2151 were procured from the Centre for Protein Engineering, MRC Laboratories, Cambridge, United Kingdom, while HuScL4 libraries were procured from Creative Biolabs, New York, USA. For screening of libraries, MaxiSorp or PolySorp immunotubes (in every alternate round) were immobilized with 4 ml of 100 \u0026mu;g/mL BSA-peptide and BSA in 1x PBS and kept at 4\u0026deg;C overnight. Thereafter, the tubes were washed three times with 1x PBS, and blocking was performed with 2% skim milk in 1x PBS for 2 hours at room temperature (RT). To remove BSA-specific phages, approximately 10\u003csup\u003e12\u003c/sup\u003e phages of amplified Tomlinson I, J, and HuScL4 libraries or amplified phages (from successive rounds of selection) were preincubated with BSA-coated immunotubes for 1 hour, and then the remaining phages were incubated with BSA-peptide for 1 h on a rotating platform and 1 hour of static incubation. The unbound phages were removed, and the samples were washed with 1x PBS with 0.05% Tween 20 (10 times for selection round 1 and increased 10 times after every subsequent round) to remove the weakly bound phages. Affinity elution was performed with 0.45 mL of 1 mg/ml peptide for 30 min to elute the bound phages [40]. The eluted phages were then treated with 50 \u0026mu;l of 10 mg/mL bovine trypsin (Type XIII from Bovine Pancreas, Sigma-Aldrich, St. Louis, MO) for 10 min. The eluted phages were further amplified and used as input phages for the next round. The biopanning was repeated 4-5 times so that the high-affinity phages bound to the peptide epitope.\u003c/p\u003e\n\u003ch5\u003e\u003cstrong\u003ePolyclonal and monoclonal phage ELISA\u003c/strong\u003e\u003c/h5\u003e\n\u003cp\u003eThe phages obtained after 4 or 5 rounds of selection were checked for their binding to the peptide epitope. A 96-well plate was coated with 100 \u0026mu;g/mL peptide-BSA conjugate and BSA. The following day, the plates were washed three times with 1x PBS and blocked with 2% skim milk in 1x PBS for 2 h. The polyclonal phages/monoclonal phages were added to the antigen-coated plates and incubated for one hour at RT [21]. The phages were removed, and the plates were washed thoroughly with 0.1% Tween 20 in PBS (PBST).\u0026nbsp;Anti-mouse M13-HRP was added at a 1:5000 dilution for 1 h at room temperature. The plate was washed 4 times with PBST and subsequently developed with ortho-phenylenediamine (OPD) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as substrates, and the absorbance was read at 490 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCloning,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eexpression\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and purification of scFvs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003c/strong\u003eThe selected scFvs from the Tomlinson libraries were subsequently cloned and inserted into the commercially available pET22b vector (Addgene- Catalog number 69744-3), whereas the scFvs from the HuScL4 library were subsequently cloned and inserted into pET22 b-DS55 (Addgene ID-187175). In the pET22b vector , the SalI and HindIII sites were followed by the NotI, XhoI, and 6X-His tags. We modified this vector such that the Hind-III site is followed by SalI, NotI, XhoI, and 6X-His tags to express scFvs from the HuScL4 library in frame. The amber stop codon present in the scFvs was mutated to glutamine by site-directed mutagenesis. To overexpress scFvs, clones were transformed into the BL21 \u003cem\u003eE. coli\u003c/em\u003e strain. The culture was inoculated and incubated at 37 \u0026deg;C for 5 h, followed by induction with isopropyl b-D-1-thiogalactopyranoside (IPTG) and incubation at 18 \u0026deg;C for 16-18 h. The resulting pellet was resuspended in periplasmic extraction buffer I (100 mM Tris_HCl pH=8, 20% sucrose, 1 mM EDTA) and incubated on ice for 45 min. After centrifugation, the pellet was dissolved in periplasmic buffer II (5 mM MgCl\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ein deionized water) and incubated on ice for 30 min. After centrifugation, the supernatants from both processes were mixed and used for immobilized metal affinity chromatography (IMAC). For this, mixed supernatant was loaded on Ni-NTA column (GE Healthcare) and eluted with different concentrations of imidazole with 50mM Tris pH=8 and 300mM NaCl. Further size exclusion chromatography (SEC) was performed via a HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare) by fast-performance liquid chromatography (AKTA pure) at 4\u0026deg;C in 50mM Tris pH=8 and 300mM NaCl buffer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTitration ELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 96-well ELISA plate was coated with 10 \u0026micro;g/ml BSA-peptide and BSA in 1x PBS overnight at 4\u0026deg;C. BSA was used as a control. After washing with 1x PBS, the plates were blocked with 2% skim milk in 1x PBS for two hours at room temperature. Purified antibodies were titrated from 50 \u0026micro;g/ml to 16 dilutions and incubated for 1 hour at 37\u0026deg;C. After three washes with PBST, the plates were incubated for 1 hour at 37\u0026deg;C with a 1:5000 dilution of an anti-His HRP-conjugated antibody (Santa Cruz Biotechnology, Cat# sc-8036). The plates were again washed three times with PBST solution. Color development was achieved via the use of the substrate o-phenylenediamine (OPD; HiMedia) in the presence of hydrogen peroxide (H₂O₂; Sigma-Aldrich). The optical density (OD) was measured at 490 nm via a SpectraMax plate reader (Molecular Devices). The EC50 values were calculated with GraphPad Prism version 6.01.\u003c/p\u003e\n\u003ch4 id=\"_Toc122270633\"\u003e\u003cstrong\u003eBiolayer\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;interferometry\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eThe binding of scFvs with peptides as well as proteins was performed via biolayer interferometry (BLI) with an Octet RED96e instrument (ForteBio, Molecular Dimensions). Streptavidin (SA) biosensors were used to immobilize biotinylated BSA-peptide/gp41, and BSA was used as a control until it reached a nanometer shift of 0.8-1.2 nm. The scFv was prepared in 1x PBS via twofold dilution for affinity analysis. The regeneration and neutralization buffers used during the experiment were 10 mM glycine, pH = 2.5, and 1x PBS, respectively. The association and dissociation steps\u0026nbsp;were recorded for\u0026nbsp;120 s\u0026nbsp;and\u0026nbsp;200 s, respectively. The data were analyzed\u0026nbsp;via\u0026nbsp;Forte Bio Data analysis software 10.0.0.1. The global data specifying the fit\u0026nbsp;to a 1:1 binding model\u0026nbsp;were\u0026nbsp;used to determine the k\u003csub\u003eon\u003c/sub\u003e, k\u003csub\u003eoff\u003c/sub\u003e and K\u003csub\u003eD\u003c/sub\u003e.\u003c/p\u003e\n\u003ch4 id=\"_Toc122270634\"\u003e\u003cstrong\u003eFlow cytometr\u003c/strong\u003e\u003cstrong\u003e\u003cspan id=\"_Toc122270625\"\u003eic binding assay and site-directed mutagenesis\u003c/span\u003e\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eThe scFv phage clones displaying premature termination codon amber (G68, G73, G77, and G80) were mutated to glutamine by site-directed mutagenesis for expression in the BL21 strain. To analyze the binding of scFvs to the full-length gp160 protein, flow cytometry experiments were performed using HEK293T cells. HIV full-length gp160-expressing clones, named CNE8 and SF162, were procured through the NIH AIDS reagent program. Epitope analogs were generated via site-directed mutagenesis\u0026nbsp;via a\u0026nbsp;QuickXL-II SDM kit (Agilent Technologies- Catalog #200521)\u0026nbsp;according to\u0026nbsp;the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003cp\u003eHuman embryonic kidney 293T cells (HEK293T; procured from ATCC) were seeded in a 60 mm dish in DMEM containing 10% fetal bovine serum (FBS), 1% penicillin, and streptomycin antibiotics. The next day, cells at a density of 50-60% were transfected with PEI 25K reagent (Polysciences- Catalog #23966) in OptiMEM media. PEI was mixed with plasmid DNA at a 3:1 ratio and kept at room temperature for 30 min. DNA and PEI complexes were added slowly to the culture dish and incubated for 4-6 h at 37\u0026deg;C in a 5% CO2 incubator. The media was changed to complete media and incubated for 36 hours. The cells were washed to make a single-cell suspension and blocked with 2% FBS in 1x PBS (FACS buffer) for 1 hour at 4\u0026deg;C.\u0026nbsp;The cells\u0026nbsp;were fixed\u0026nbsp;with\u0026nbsp;BioLegend Fixation\u0026nbsp;Buffer\u0026nbsp;(Catalog #420801) and permeabilized\u0026nbsp;with\u0026nbsp;Intracellular Staining Perm Wash Buffer (BioLegend- Catalog #421002) to access the GERDRDR epitope inside the viral membrane. After that, the\u0026nbsp;cells were incubated with scFvs for 1 h at 4\u0026deg;C and washed\u0026nbsp;three times\u0026nbsp;with FACS buffer. His-Tag (D3I1O) XP\u003csup\u003e\u0026reg;\u003c/sup\u003e Rabbit mAb (Alexa Fluor\u003csup\u003e\u0026reg;\u003c/sup\u003e 647 Conjugate, CST Cat# 14931) was added as a secondary antibody at a dilution of 1:250 for 20 min at RT. The cells were washed three times with FACS buffer and resuspended in 430 \u0026mu;l of 1x PBS, and samples were acquired with a BD LSRFortessa using Diva software (BD Bioscience).\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eX-ray crystallography and structure determination\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eCrystallization of scFv DE94 was performed via the hanging drop vapor diffusion method via a Mosquito LCP nanodispenser (TTP Labtech). Crystals were grown in conditions containing\u0026nbsp;0.5 M SPG buffer\u0026nbsp;(pH=4), 25% PEG1500,\u0026nbsp;and\u0026nbsp;10% glycerol. The data were collected at\u0026nbsp;the\u0026nbsp;ID30B beamline,\u0026nbsp;European Synchrotron Radiation Facility (ESRF),\u0026nbsp;with\u0026nbsp;15%\u0026nbsp;glycerol as\u0026nbsp;a\u0026nbsp;cryoprotectant. Following data collection, HKL3000 software was used to process the data. PHASER from the CCP4 program suite was used to determine the structure by molecular replacement using PDB: 7YUEas a starting model. Refinement was carried out in PHENIX, and Coot was used for visualization. All the figures were prepared via\u0026nbsp;PyMOL and Coot. The PDB validation server checked the final quality of the structure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular docking and MD simulations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe scFv-peptide complex was generated by using the ligand docking tool in the Schr\u0026ouml;dinger Suite [18]. The crystal structure of the scFv obtained was used as a reference. In the ligand docking tool, site specific docking was done while keeping the peptide flexible. The complex was subjected to a molecular dynamics simulations run via\u0026nbsp;the Desmond 3.1 MD package (Schr\u0026ouml;dinger Inc.)\u0026nbsp;[36].\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMutants of the epitope were generated in PyMOL, and energy minimization of all the complexes was performed to check for steric hindrance in the complexes. Then, Prime MM-GBSA was run. The relative binding affinity of the epitopes was calculated via the Prime MMGBSA module of Schr\u0026ouml;dinger. The ligand interaction diagram was prepared in PyMOL for interaction studies.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSelection and design of epitopes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eHIV-1 envelope protein comprises two heterodimers, both of which are considered targets for generating monoclonal antibodies. gp41 is particularly important for vaccine development because it plays a crucial role in membrane fusion. During the transition from the prefusion to the postfusion state, gp41 undergoes conformational changes, transiently exposing previously buried epitopes [10,39,41]. Structurally, gp41 consists of an N-terminal region fusion peptide (FP), followed by N-terminal and C-terminal heptad repeats (NHR and CHR), a membrane proximal external region (MPER), a transmembrane region (TM) and a cytoplasmic tail (CTT) [14] (Figure 1A). To understand the effect of antigenic diversity on the immune response, we selected two key neutralizing epitopes within gp41. By analyzing the envelope protein in the HIV database genome browser, we selected a 7-mer neutralizing epitope, \u003csup\u003e738\u003c/sup\u003eGERDRDR\u003csup\u003e744\u003c/sup\u003e, in CTT and a 6-mer neutralizing epitope, \u003csup\u003e662\u003c/sup\u003eELDKWA\u003csup\u003e667\u003c/sup\u003e,\u003csup\u003e\u0026nbsp;\u003c/sup\u003ein MPER, both\u0026nbsp;of which are\u0026nbsp;recognized for their neutralization potential\u0026nbsp;[4,12]\u0026nbsp;(Figure 1A). The GERDRDR sequence is a part of the Kennedy epitope (\u003csup\u003e724\u003c/sup\u003ePRGPDRPEGIEEEG\u003cstrong\u003eGERDRDR\u003c/strong\u003eS\u003csup\u003e745\u003c/sup\u003e), which has been implicated in virus neutralization, incorporation of the envelope in the virion, and viral infectivity. Although no broad neutralizing antibodies have been identified against this epitope, antibodies targeting this region can block cell-to-cell transmission by blocking fusion\u0026nbsp;[11,19,35]. In contrast, ELDKWA\u003csup\u003e\u0026nbsp;\u003c/sup\u003eis the core epitope of \u0026nbsp;a well-known broadly neutralizing antibody, 2F5, against HIV [8]. Among the several broadly neutralizing antibodies against the MPER region, such as 10E8, LN01, 2F5, and 4E10, the 2F5 antibody binds to the linear N-terminal MPER epitope, which has more natural mutants than other antibodies do [31,32]. The other MPER-directed antibodies recognize helical epitopes located in the C-terminus of the MPER region near the TM region [9].\u003c/p\u003e\n\u003cp\u003eTo understand the extent of degeneracy in antibodies raised against the epitopes GERDRDR and ELDKWA, we selected variants from the HIV sequence database via the web-based AnalyzeAlign tool (www.hiv.lanl.gov) focusing on representative sequences from HIV-1 Groups B, C, and A1, which cover globally prevalent clades. Based on parameters such as percentage abundance, the impact of mutations, and sequence coverage, we selected six variants for each epitope. To comprehensively represent sequence variability, we included single, double, and triple mutants, and each mutation was selected such that it changed the degree of amino acid hypdropathy (Table 1). To identify epitope-specific antibodies, we focused on the use of a human phage display library to screen selected epitopes (Figure 1B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Selection of neutralizing\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eepitopes\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003etheir\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;natural escape variants from\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ethe\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eNIH HIV database.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eS. No.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eMutations\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eSequence\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Abundance\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGroup B\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGroup C\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGroup A1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNative epitope\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGERDRDR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e37.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eSingle mutant\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003cstrong\u003eERDRDR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGGRDRDR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGEQDRDR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e41.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDouble mutant\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGEQDRGR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eTriple mutant\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGEQGRGR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGGRDSGR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNative epitope\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eELDKWA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e57.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.98\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eSingle mutant\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003eLDKWA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e75.24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eELDKWD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eEWDKWA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eDouble mutant\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eK\u003c/strong\u003e\u003cstrong\u003eLDEWA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003eLDKWQ\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eTriple mutant\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003eLGKWD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e28.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eBiopanning and Characterization of GERDRDR -Specific Antibodies\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntibodies can target various regions of the HIV envelope protein involved in virus infection and entry. The GERDRDR epitope is involved in membrane fusion during viral entry. Four rounds of biopanning were performed to isolate epitope-specific antibodies via the human-based semisynthetic Tomlinson I and J scFv phage display libraries. Progressive enrichment of epitope-specific phages was observed in rounds 3 and 4 (Table S1), as confirmed by polyclonal phage ELISA (Figure S1). A total of 400 monoclones from the fourth round of biopanning were tested for binding to the native epitope, and varying degrees of binding reactivity were observed (Figure 2A). \u0026nbsp;Based on peptide/BSA OD\u003csub\u003e490\u003c/sub\u003e values, 100 top binders were selected and further analyzed via ELISA (Figure S2A).\u003c/p\u003e\n\u003cp\u003eTo assess the extent of cross-reactivity, monoclonal phages with high binding to the native epitope were tested against cross-clade variants. These phages were examined for their ability to cross-react with single mutants (RERDRDR, GGRDRDR, and GEQDRDR), double mutants (GEQDRGR), and triple mutants (GEQGRGR and GGRDSGR) (Figure 2B). Compared with the native epitope, which was defined as a percentage O.D. value, most of the mutants presented no significant change in the mean OD values, except for GEQDRDR and GEQDRGR, which presented reduced binding. (Figure S2B). This reduced binding suggests that specific amino acid substitutions at the 3rd and 6th positions of the epitope can significantly impact the interaction with the antibodies. Sequencing of 15 cross-reactive high-binding clones resulted in the identification of five unique scFvs (Figure S2C \u0026amp; S3). These scFvs were subsequently cloned and inserted into the pET22b vector, expressed, and purified to high homogeneity (Figure S4A and B).\u003c/p\u003e\n\u003cp\u003eTo assess the binding of these selected scFvs to both the native and variant epitopes in soluble form, ELISA was performed in a series of dilutions ranging from 50 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e to 1.52 ng mL\u003csup\u003e-1\u003c/sup\u003e. All five scFvs bound to the full panel of variants (Figure 2C), although with different binding ranges. The EC\u003csub\u003e50\u003c/sub\u003e values of scFvs G8, G68, G73, G77, and G80 were determined to be 11.72 \u0026micro;M, 5.51 \u0026micro;M, 2.07 \u0026micro;M, 1.26 \u0026micro;M, and 16.60 \u0026micro;M, respectively. Among all the scFvs, G77 presented the highest affinity, followed by G73. Further analysis via biolayer interferometry (BLI) confirmed the multireactive potential and binding kinetics of scFvs to HIV epitope mutants (Table 2). A heatmap of the equilibrium dissociation constant (K\u003csub\u003eD\u003c/sub\u003e) revealed that all five scFvs bind in the micromolar range (Figure 2D), reinforcing their potential as broadly reactive antibodies capable of recognizing epitope variants associated with HIV immune evasion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eBLI derived K\u003csub\u003eD\u003c/sub\u003e values (M) of scFvs to native epitope, its variants, and the gp41 protein.\u003c/p\u003e\n\u003ctable style=\"width: 4.6e+2pt\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGERDRDR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eRERDRDR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGGRDRDR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGEQDRDR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGEQDRGR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGEQGRGR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGGRDSGR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003egp41\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eG8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16.74 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.74 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.16 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e25.77 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.69 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9.19 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.19 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.86 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eG68\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.97 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.32 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.37 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.26 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9.99 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.08 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.92 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.85 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eG73\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.44 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.20 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.20 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.91 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.21 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.68 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.66 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.21 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eG77\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.98 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.54 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.28 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.51 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.42 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.22 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.67 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.83 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eG80\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9.40 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.53 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.18 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.86 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.68 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.56 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.73 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.50 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eBinding analysis of GERDRDR-specific antibodies to gp41 and gp160\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe affinity data clearly revealed that scFvs could bind the native peptide epitope and its variants in a physiologically relevant range. To assess the binding in a more native context, we next evaluated the binding of scFvs with recombinantly expressed full-length gp41 containing the native epitope sequence. Biotinylated gp41 protein was immobilized on a streptavidin (SA) sensor, and different scFvs were used as the analyte at concentrations ranging from 10 \u0026micro;M to 70 nM. Interestingly, all the scFvs bound to the gp41 protein in the micromolar range (Table 2). The binding affinity curves with association and dissociation profiles are shown in Figure 3A.\u003c/p\u003e\n\u003cp\u003eTo further investigate whether the scFvs could recognize the native conformation of gp160 in a membrane-anchored state, we tested the binding of the scFvs to the full-length gp160 protein expressed on the mammalian cell surface. The expression of the gp160 protein on the cell surface corresponds approximately to a model of the virus particle in which gp160 is embedded in the viral membrane. Site-directed mutagenesis was performed to generate variants of the full-length gp160 protein via the HIV SF162 plasmid. The RERDRDR and GGRDSGR variants were not successfully generated by site-directed mutagenesis. Flow cytometry analysis revealed that all the scFvs were able to recognize the native gp160 protein and four of its variants, as evidenced by the shift in the fluorescence peak (Figure 3B). The shift in the whole peak with respect to the control peak indicates strong binding to the gp160 protein. The greater the shift in the peak is, the stronger the binding. The disparity in the peak shift with different scFvs indicated different binding strengths. Overall, these results indicated that the scFvs could recognize both recombinant and membrane-bound forms, highlighting their multiple reactive potentials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiopanning and Characterization of ELDKWA-Specific Antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe membrane fusion-associated ELDKWA epitope, which is exposed on the exterior of the viral membrane, was also subjected to screening against the Tomlinson I, Tomlinson J, and HuScL4 libraries as previously described (Figure 1B). Phages specific to this epitope were enriched for 4-5 rounds of biopanning (Table S2), with increased binding observed after each round by polyclonal ELISA (Figure S5). From the fourth and fifth rounds, 1150 monoclonal phages were tested for binding to the native epitope, and 150 high-affinity binders were selected based on the peptide/BSA OD\u003csub\u003e490\u003c/sub\u003e values (Figure 4A, S6A). Of these, 80 top binders were selected for further analysis.\u003c/p\u003e\n\u003cp\u003eTo determine the breadth of antigen recognition, the binding of the 80 selected phages to the six selected variants was tested. Remarkably, most phage monoclones recognized the majority of the variants, demonstrating strong cross-reactivity (Figure 4B). Compared with the native epitope, which was defined as a percentage O.D. value of 100%, the triple mutant ALGKWD presented the lowest mean value of 13%, and the single mutant EWDKWA exhibited a mean value of 92%, which was comparable to that of the natural epitope. The single mutants ALDKWA and ELDKWD, showed a mean OD\u003csub\u003e490\u003c/sub\u003e value of 59%, whereas the corresponding values of the double mutants KLDEWA and ALDKWQ were 59% and 54%, respectively (Figure S6B). These results indicate that the phages screened against the native peptide epitope were highly cross-reactive with the escape variants. Sequencing of 20 high-affinity binders yielded four distinct clones from the HuScL4 library and four from the Tomlinson libraries (Figure S6C \u0026amp; S7). These scFvs were subsequently cloned and inserted into the pET22b vector, expressed, and purified (Figure S8A, B).\u003c/p\u003e\n\u003cp\u003eThe binding affinities were assessed by ELISA across various dilutions, ranging from 50.00 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e to 1.52 ng mL\u003csup\u003e-1\u003c/sup\u003e. Interestingly, the eight selected scFvs bound to the native epitope with varying affinities. The EC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003evalues for scFvs DE13, DE45, DE62, DE72, DE34, DE37, DE83, and DE94 ranged from 4.27 \u0026micro;M to 0.25 \u0026micro;M, indicating a spectrum of binding affinities ranging from micromolar to submicromolar. Notably, scFvs DE94 and DE34 exhibited the highest binding affinity among all the scFvs (Figure 4C). While scFvs DE13, DE34, DE37, DE83, and DE94 recognized all the tested variants, scFv DE62 failed to bind KLDEWA, and scFv DE72 did not recognize ALGKWD (Figure 4C), indicating differential binding specificities among the clones.\u003c/p\u003e\n\u003cp\u003eTo complement the affinity calculations by ELISA, BLI was used to quantify the binding kinetics. BLI analysis revealed the diversity in binding affinities among the scFvs (Figure 4D, Table 3). Some scFvs retained binding across all the selected variants, whereas others lost binding to double or triple mutants, indicating sensitivity to sequence variations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Comparative analysis of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ethe\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eK\u003csub\u003eD\u003c/sub\u003e values (M) of all the scFvs against\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ethe\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003enative epitope and its variants.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003escFv\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eELDKWA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eALDKWA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eELDKWD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eEWDKWA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eKLDEWA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eALDKWQ\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eALGKWD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003egp41\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDE13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.17 \u003cstrong\u003ex\u0026nbsp;\u003c/strong\u003e10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.13 \u003cstrong\u003ex\u0026nbsp;\u003c/strong\u003e10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.35 \u003cstrong\u003ex\u0026nbsp;\u003c/strong\u003e10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.95 \u003cstrong\u003ex\u0026nbsp;\u003c/strong\u003e10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.98 \u003cstrong\u003ex\u0026nbsp;\u003c/strong\u003e10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.66 \u003cstrong\u003ex\u0026nbsp;\u003c/strong\u003e10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.99 \u003cstrong\u003ex\u0026nbsp;\u003c/strong\u003e10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.08 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDE45\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.38 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.85 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.92 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.59 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.58 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.52 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.31 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.46 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDE62\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.49 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.20 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.80 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.98 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNo binding\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.42 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.10 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.55 x 10\u003csup\u003e-5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDE72\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.39 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.62 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.96 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.76 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.39 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.82 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNo binding\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.39 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDE34\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11.90 x 10\u003csup\u003e-8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9.70 x 10\u003csup\u003e-8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.43 x 10\u003csup\u003e-8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.76 x 10\u003csup\u003e-8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.84 x 10\u003csup\u003e-8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.71 x 10\u003csup\u003e-8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.09 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.76 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDE37\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.95 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.27 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.85 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.40 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.20 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.35 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.36 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.67 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDE83\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.51 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.49 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.44 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.81 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.41 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.95 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.99 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.64 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDE94\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.44 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.78 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.68 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.48 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.25 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.00 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.73 x 10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.94 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eBinding analysis of ELDKWA-specific antibodies to gp41 and gp160\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs described previously, it was necessary to check whether the scFvs screened against the peptide epitope could also recognize the corresponding full-length HIV protein. Binding kinetics with the gp41 protein confirmed that all the scFvs were capable of binding to gp41, with K\u003csub\u003eD\u003c/sub\u003e values in the micromolar range (Figure 5A, Table 3). The highest binding affinity observed was 0.94 \u0026micro;M for scFv DE94.\u003c/p\u003e\n\u003cp\u003eTo explore the multireactive potential of scFvs, a gp160 mammalian expression clone (CNE8) was expressed on HEK293T cells, and surface expression was analyzed via flow cytometry. Variants of native epitopes were introduced into the gp160 mammalian plasmid via site-directed mutagenesis. Intriguingly, five out of eight scFvs showed shifts in fluorescence intensity, indicating binding to gp160 and its variants on the cell surface (Figure 5B), and a shift was observed across all the variants tested. These results suggested that scFvs can bind the whole virus. While all the scFvs bound the soluble recombinant gp41 protein, some exhibited reduced or no binding to the membrane-anchored form, likely due to limited epitope accessibility on the native-like cell surface. The binding of scFvs with all the variants revealed the multireactive potential of these scFvs against the HIV-1 envelope protein.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCrystallization and structure determination of scFv DE94\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo\u0026nbsp;gain insight\u0026nbsp;into the molecular interaction of scFv with peptide\u0026nbsp;antigens, we attempted\u0026nbsp;to crystallize\u0026nbsp;all anti-GERDRDR antibodies and five anti-ELDKWA antibodies, both in\u0026nbsp;the\u0026nbsp;apo form and with the native peptide, via\u0026nbsp;the hanging drop vapor diffusion method.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eCrystals of scFv DE94 (anti-ELDKWA antibody) were successfully obtained in the apo form, which\u0026nbsp;diffracted at 2.53 \u0026Aring; resolution and belonged to the monoclinic space group P2\u003csub\u003e1\u003c/sub\u003e, with cell dimensions of a = 43.80 \u0026Aring;, b = 183.92 \u0026Aring;, c = 65.73 \u0026Aring;, and \u0026beta; = 93.60\u0026deg;. DE94 has relatively high binding among all five anti-ELDKWA antibodies. The complete\u0026nbsp;dataset\u0026nbsp;has\u0026nbsp;an\u0026nbsp;Rmerge of 15.0%. Initial phasing of the scFv was performed by molecular replacement\u0026nbsp;via\u0026nbsp;a previously published independent scFv structure (PDB ID: 7YUE). The refined structure contains four molecules in an asymmetric unit of\u0026nbsp;the\u0026nbsp;scFv (Figure 6A). The final R\u003csub\u003ework\u003c/sub\u003e and R\u003csub\u003efree\u003c/sub\u003e values were 19.9% and 24.3%, respectively, with 97.59% amino acid residues in the allowed region of the Ramachandran plot. All the molecules of the scFv of an asymmetric unit were superimposed on each other, and no significant structural deviations were observed, as evident from the root mean square deviations (RMSD) values, which were in the range of 0.29-0.41 \u0026Aring; (Figure 6B). The electron density of chain D in DE94 was very weak. The data collection statistics and refinement statistics are shown in Table S3. The scFv DE94 comprises variable light and variable heavy chains connected via a Gly-Ser linker. The six complementary determining regions (CDRs) forming the antigen-binding site are highlighted in Figure 6C. The atomic coordinates and structure factors are available in the RCSB Protein Data Bank under accession code 9V5N.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMapping key residues in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ethe\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003escFv DE94-ELDKWA complex via molecular docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo understand the molecular determinants involved in the recognition of\u0026nbsp;the\u0026nbsp;ELDKWA epitope by\u0026nbsp;the\u0026nbsp;scFv DE94, we conducted extensive crystallographic trials of\u0026nbsp;the\u0026nbsp;scFv-epitope (ELDKWA) complex. As these efforts did not yield a resolved structure, we employed computational tools to investigate the molecular interactions between\u0026nbsp;the\u0026nbsp;scFv and the peptide antigen. Rigid docking was performed at\u0026nbsp;the\u0026nbsp;CDRs of\u0026nbsp;the\u0026nbsp;scFv\u0026nbsp;via\u0026nbsp;the ligand docking tool in the Schr\u0026ouml;dinger module.\u0026nbsp;The binding strength of scFvs toward\u0026nbsp;peptides\u0026nbsp;was computed\u0026nbsp;via the\u0026nbsp;molecular mechanics generalized Born surface area (MM/GBSA) approach. The binding energy of scFv DE94-ELDKWA was -63.62 kcal/mol. The\u0026nbsp;scFv-peptide\u0026nbsp;complex formed with the lowest Gibbs free energy is shown in Figure 7A. To gain\u0026nbsp;further insight\u0026nbsp;into the binding mechanism and overall stability of\u0026nbsp;the\u0026nbsp;scFv-peptide complex, a long-term\u0026nbsp;(500 ns) all-atom molecular dynamics simulation was performed. After the trajectories\u0026nbsp;were processed, the\u0026nbsp;RMSDs\u0026nbsp;were examined. The system was stable throughout the run, as\u0026nbsp;shown\u0026nbsp;in Figure S8A. The data suggested that scFv had a stable interaction with ELDKWA, which was supported by the finding that the system\u0026apos;s RMSD values remained constant (Figure S9A). The backbone Root Mean Square Fluctuation (RMSF) of the scFv was also examined throughout the 500 ns run,\u0026nbsp;and the Gly-Ser linker in the crystal structure connecting the heavy and light chains demonstrated high\u0026nbsp;fluctuations\u0026nbsp;(Figure S9B).\u003c/p\u003e\n\u003cp\u003eTo envisage the change in binding affinity with different variants, we mutated the native peptide within the scFv-ELDKWA complex and performed energy minimization for each mutant. The resulting models were used to calculate binding free energies\u0026nbsp;via\u0026nbsp;the MM-GBSA module in Schr\u0026ouml;dinger. The binding energies of all\u0026nbsp;the\u0026nbsp;epitope variants are shown in Figure S9E. The representative structural overlays of\u0026nbsp;the\u0026nbsp;native and mutant complexes, highlighting the modeled conformations within the binding site,\u0026nbsp;are shown in Figure S9C-D. To elucidate the\u0026nbsp;interactions\u0026nbsp;between the scFv-peptide\u0026nbsp;complexes, hydrogen\u0026nbsp;bonds\u0026nbsp;and hydrophobic\u0026nbsp;interactions\u0026nbsp;were analyzed. Hydrogen bonding was observed between the peptide and the residues located in the CDRH1, CDRH2, CDRH3, CDRL1, and CDRL2 regions of the scFv. In the scFv-ELDKWA complex, E1 of the peptide is involved in H-bond\u0026nbsp;interactions\u0026nbsp;with R61 of CDRH2 and R103 of CDRH3; D3 is involved in H-bond\u0026nbsp;interactions\u0026nbsp;with A35 of CDRH1 and Q55 of CDRH2, with\u0026nbsp;a\u0026nbsp;binding energy of -63.62 kcal/mol. E1, D3, W5, and A7 also\u0026nbsp;exhibited\u0026nbsp;hydrophobic\u0026nbsp;interactions\u0026nbsp;with CDR residues,\u0026nbsp;as shown in Figure 7B. All these interactions made the scFv-epitope complex stable. In the case of\u0026nbsp;the\u0026nbsp;scFv-ALDKWA complex,\u0026nbsp;the\u0026nbsp;binding energy\u0026nbsp;was\u0026nbsp;reduced to -48.73 kcal/mol, which\u0026nbsp;was\u0026nbsp;due to the loss of alanine\u0026nbsp;interactions\u0026nbsp;with\u0026nbsp;the\u0026nbsp;scFv. Similarly, in the case of ALDKWQ (double mutant),\u0026nbsp;the\u0026nbsp;binding energy was -47.26 kcal/mol, as the mutation\u0026nbsp;had\u0026nbsp;no effect at the 6\u003csup\u003eth\u003c/sup\u003e position. However, in the case of ALGKWD (triple mutant), the energy was further reduced to -39.95 kcal/mol due to the loss of interaction of glycine at the 3\u003csup\u003erd\u003c/sup\u003e position. In the case of ELDKWD, with a binding energy of -62.64 kcal/mol, there was no change in interaction due to the mutation at the 6\u003csup\u003eth\u003c/sup\u003e position. In the double mutant KLDEWA, the binding energy is equivalent to that of the native epitope, as the mutation from E to K at the first position is balanced by the mutation from K to E at the 4\u003csup\u003eth\u003c/sup\u003e position. EWDKWA has a more negative \u0026Delta;G (-67.92 kcal/mol), indicating a more stable complex, which can be verified by the additional interaction of W4 with the CDRs of the scFv (Figure 7C-H). Although there was a difference in the binding energy of the native epitope with its variants, notably, the \u0026Delta;G values for all the epitopes were in the range of antigen-antibody interactions.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMonoclonal antibodies are recognized as key mediators of protective immunity against HIV-1 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. There is a need to develop broadly cross-reactive therapeutic antibodies. While cross-reactive antibodies are known to exist in the immune system, immune evasion by HIV-1 is also well-documented. This study aimed to explore antibody cross-reactivity and immune escape mechanisms by comparing antibodies against two critical epitopes of the HIV-1 envelope protein gp41, GERDRDR and ELDKWA, and to draw insights from these findings in the broader context of other fast-mutating viruses.\u003c/p\u003e \u003cp\u003eCompared with those against ELDKWA, antibodies targeting the epitope GERDRDR exhibited greater cross-reactivity, reflecting different hotspot behaviors. Together, both epitopes demonstrated structural plasticity at antigen-combining sites. The broader reactivity observed against GERDRDR-specific epitopes can likely be attributed to multiple charged amino acids across the epitope sequence, enabling the binding of scFvs to all the variants. In contrast, antibodies against ELDKWA showed less cross-reactivity, and only five of the eight binders recognized the native epitope and its variants. A plausible explanation for the lack of binding by the remaining three scFvs could be steric hindrance from the membrane-anchored protein.\u003c/p\u003e \u003cp\u003eNotably, during HIV-1 infection, polyreactivity is observed in approximately 70\u0026ndash;75% of anti-gp160 antibodies, and this property is even more pronounced among anti-gp41 antibodies, of which 85\u0026ndash;90% exhibit polyreactivity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This high proportion of polyreactivity highlights the importance of multispecific antibodies in shaping the immune response. Moreover, our findings support the hypothesis that B-cell clones producing multispecific antibodies undergo positive selection during affinity maturation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This finding is in line with previous studies indicating that the primary factor contributing to antibody multispecificity in response to HIV-1 Env proteins is the inherent conformational flexibility of the antigen-binding site [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. This structural plasticity is not unique to HIV but is also observed in other rapidly mutating viruses. For example, multispecific antibodies against a key antigenic target, the hemagglutinin (HA) protein, have been identified in the influenza virus [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The HA protein undergoes significant antigenic drift, leading to the emergence of new strains. The presence of multispecific antibodies in the humoral immune response is necessary for preventing immune evasion during influenza infections despite mutations that are beneficial for viral escape and lead to antigenic diversification [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This phenomenon is similar to what we observed in the case of HIV-1, where antibodies with broad reactivity can accommodate diverse variants owing to the structural flexibility of the epitopes. Similarly, in SARS-CoV-2, multiple specific antibodies against the spike protein have been identified. Antibodies produced following vaccination against SARS-CoV-2 have the ability to neutralize a range of emerging strains, which may be attributed to their multispecific nature [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The broad reactivity of such antibodies is crucial for effective immunity, especially as new variants continue to emerge.\u003c/p\u003e \u003cp\u003ePrevious studies have reported that neutralizing antibodies targeting the MPER of HIV-1 gp41 are cross-reactive [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Specifically, the broadly neutralizing antibody 2F5, which targets the ELDKWA epitope, relies on DKW residues for interaction [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and substitution from DKW to DSW often makes the virus resistant to antibody neutralization [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In the present study, the scFvs demonstrated remarkable recognition of mutations in the DKW region, except for scFv DE62, which failed to recognize the mutant peptide KLDEWA, and scFv DE72, which was unable to recognize the triple mutant peptide ALGKWD, as confirmed through ELISA and BLI assays. Nevertheless, when the variants were exposed on the surface of mammalian cells, cross-reactivity was observed with all the variants. This observation could reflect the conformational constraints imposed by the membrane-anchored protein. These findings correlate with previous findings that the immune system can produce antibodies with degenerate potential, enabling recognition of cross-clade variants [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo gain deeper insight into these molecular interactions, we performed crystallographic studies on scFv DE94. These studies, combined with in silico analysis of scFv DE94 binding to ELDKWA and its mutants, revealed that the predicted binding energy ranged from \u0026minus;\u0026thinsp;39.95 to -67.92 kcal mol\u003csup\u003e-1\u003c/sup\u003e. Certain mutations were associated with less favorable docking scores, suggesting in potential decrease in binding energy. The structural plasticity observed in both gp41 epitopes highlights the adaptability of multispecific antibodies, which is crucial for combating the high mutation rate of HIV and ensuring an effective immune response. Collectively, the biochemical, cell-based, and structural analyses support our hypothesis that multispecific antibodies can accommodate antigenic diversity, although certain mutations may impact binding efficacy.\u003c/p\u003e \u003cp\u003eOur observations are similar to findings in other rapidly mutating viruses, such as influenza virus and SARS-CoV-2, where multispecific antibodies are crucial in neutralizing diverse strains despite antigenic drift [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. HIV has a high mutation rate, with approximately 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e substitutions per nucleotide per replication cycle, contributing to its genetic diversity [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, despite this, its evolution is relatively slow at the population level [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This could be due to a number of factors, such as less selection pressure over time, a weak immune response, nonbeneficial mutations affecting viral fitness, reversal of patient-specific adaptive changes following transmission and a retrieval mechanism in which the initially infected virus is preferentially transmitted [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The presence of multispecific antibodies may help constrain the evolution of new strains by providing broad protection against diverse strains. Our findings suggest that not all mutations lead to antigenic drift; only crucial mutations significantly alter viral antigens, rendering existing antibodies less effective.\u003c/p\u003e \u003cp\u003eThis study highlights the importance of antibody cross-reactivity and structural plasticity in HIV-1 research. The high prevalence of cross-reactive antibodies and their multispecific nature emphasize the need for vaccines and therapeutics that can accommodate viral diversity. Insights gained from crystallographic studies and antibody behavior, particularly regarding the impact of mutations, are valuable for developing novel strategies to increase protection against mutant strains of viruses and improve disease control efforts.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Mr. Romain Talon for providing support with data collection at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. We would like to thank Mr. Ravinder Kumar for assisting with the cell culture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Department of Biotechnology, Ministry of Science and Technology; India.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.M.S. supervised the project; D.J. and D.M.S. conceived and designed the experiments; D.J. G.K. and K.J.K. synthesized the peptides. D.J. and S.V. carried out the library screening. D.J. carried out the scFv purification, ELISA, BLI, FACS, crystallization, structure determination and MD simulation; D.J. and Z.K.M. carried out the structure refinement. D.J. wrote\u0026nbsp;the original draft; D.M.S. and D.J. reviewed and edited the draft and analyzed the data. All the authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe atomic coordinates and structure factors have been deposited in the Protein Data Bank http://www.wwpdb.org (PDB ID code: 9V5N).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAmit A.G., Mariuzza R.A., Phillips S.E. and Poljak R.J. Three-dimensional structure of an antigen-antibody complex at 2.8 A resolution. 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PLoS One 7(2):e31555, 2012.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"HIV-1, membrane-proximal external region (MPER), Kennedy epitope, cross-reactive antibodies, human scFv phage display library","lastPublishedDoi":"10.21203/rs.3.rs-9072434/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9072434/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHIV-1 escape from neutralizing antibodies, even in the presence of strong host immunity, is associated with variations in envelope proteins that drive antigenic diversification. The virus exploits the error-prone nature of reverse transcriptase and the high mutation rate as key survival strategies. However, the rate of emergence of new variations occurs at a relatively slow pace. In this context, while monospecific antibody responses against invading pathogens are well characterized, the functional relevance of multispecific or cross-reactive antibodies in limiting viral escape remains poorly understood. Interestingly, the immune system often produces cross-reactive antibodies, with an anticipated role in neutralizing point mutations in HIV surface proteins by cross-reacting with mutants and tolerating them. In light of this paradox, we investigated immune evasion in the context of observed antibody cross-reactivity by screening single-chain variable fragment (scFv) antibodies against several crucial HIV-1 gp41 epitopes using a phage display library. Selected cross-reactive scFvs were biochemically characterized for binding affinity and their ability to recognize envelope protein and its mutants expressed on cell surface. Here, high-affinity cross-reactive scFvs showed physiologically relevant affinities with peptide epitopes, their analogs, and the native HIV-1 gp41 protein. We determined the crystal structure of a high-affinity, cross-reactive scFv DE94, and gained insights into the molecular interactions of scFv antibodies with peptide epitopes and their natural mutants using molecular docking studies. This analysis of cross-reactive antibodies could contribute to the development of therapeutics against immune-evading pathogens and pave the way for innovative strategies to combat viral infections, including emerging global threats.\u003c/p\u003e","manuscriptTitle":"Immunologic insights into the critical epitopes of HIV-1 and structure-based characterization of cross-reactive antibodies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-10 16:28:44","doi":"10.21203/rs.3.rs-9072434/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor Revision","date":"2026-05-17T07:07:02+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2026-04-07T23:03:19+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-06T03:57:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-11T11:46:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2026-03-10T16:32:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d4a6e43c-1636-42a6-9d8c-0bd81ce7b360","owner":[],"postedDate":"April 10th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Minor Revision","date":"2026-05-17T07:07:02+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-17T11:07:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-10 16:28:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9072434","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9072434","identity":"rs-9072434","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}