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• W3047029054 abstract "Simian immunodeficiency virus (SIV) infection of Indian rhesus macaques (RMs) is one of the best-characterized animal models for human immunodeficiency virus (HIV) infection. Monoclonal antibodies (mAbs) have shown promise for prevention and treatment of HIV infection. However, it has been difficult to isolate mAbs that potently neutralize the highly pathogenic SIVmac239 strain. This has been largely due to the low frequency of circulating B cells encoding neutralizing Abs. Here we describe a novel technique to isolate mAbs directly from bone marrow-derived, Ab-secreting plasma cells. We employed an automated micromanipulator to isolate single SIVmac239 SOSIP.664-specific plasma cells from the bone marrow of a SIVmac239-infected RM with serum neutralization titers against SIVmac239. After picking plasma cells, we obtained 44 paired Ab sequences. Ten of these mAbs were SIV specific. Although none of these mAbs neutralized SIVmac239, three mAbs completely neutralized the related SIVmac316 strain. The majority of these mAbs bound to primary rhesus CD4+ T cells infected with SIVmac239 and induced Ab-dependent cellular cytotoxicity. This method is a first step in successful isolation of antigen-specific bone marrow-derived plasma cells from RMs. Simian immunodeficiency virus (SIV) infection of Indian rhesus macaques (RMs) is one of the best-characterized animal models for human immunodeficiency virus (HIV) infection. Monoclonal antibodies (mAbs) have shown promise for prevention and treatment of HIV infection. However, it has been difficult to isolate mAbs that potently neutralize the highly pathogenic SIVmac239 strain. This has been largely due to the low frequency of circulating B cells encoding neutralizing Abs. Here we describe a novel technique to isolate mAbs directly from bone marrow-derived, Ab-secreting plasma cells. We employed an automated micromanipulator to isolate single SIVmac239 SOSIP.664-specific plasma cells from the bone marrow of a SIVmac239-infected RM with serum neutralization titers against SIVmac239. After picking plasma cells, we obtained 44 paired Ab sequences. Ten of these mAbs were SIV specific. Although none of these mAbs neutralized SIVmac239, three mAbs completely neutralized the related SIVmac316 strain. The majority of these mAbs bound to primary rhesus CD4+ T cells infected with SIVmac239 and induced Ab-dependent cellular cytotoxicity. This method is a first step in successful isolation of antigen-specific bone marrow-derived plasma cells from RMs. Monoclonal antibodies (mAbs) are becoming the leading class of newly introduced drugs to the biopharmaceutical market. Over 90 therapeutic mAbs have been approved for a variety of targets and diseases in recent years.1Kaplon H. Muralidharan M. Schneider Z. Reichert J.M. Antibodies to watch in 2020.MAbs. 2020; 12: 1703531Crossref PubMed Scopus (301) Google Scholar Although the field of infectious disease has so far been underrepresented, mAbs are a promising approach for treatment and prophylaxis. In the context of HIV infection, for which there is no vaccine, many potent human immunodeficiency virus (HIV)-neutralizing mAbs (nmAbs) have been isolated, and some are currently being tested in human clinical trials. The simian immunodeficiency virus (SIV)-infected Indian rhesus macaque (RM) model is an excellent method of testing nmAb efficacy in vivo. Indeed, a number of human nmAbs have been tested in RMs infected with chimeric SIV/HIV strains. The simian/human immunodeficiency virus (SHIV) model has been criticized because the pathogenesis of this chimeric virus may not mimic that of HIV or SIV. To address the shortcomings of the SHIV-infected RM model, several groups have attempted to isolate mAbs against SIV isolates and pathogenic clones that might more closely resemble HIV infection. To date, it has proven difficult to isolate tier 3 SIV-specific nmAbs, and only one group has reported consistent isolation of different SIV-specific nmAbs.2Gorman J. Mason R.D. Nettey L. Cavett N. Chuang G.Y. Peng D. Tsybovsky Y. Verardi R. Nguyen R. Ambrozak D. et al.Isolation and Structure of an Antibody that Fully Neutralizes Isolate SIVmac239 Reveals Functional Similarity of SIV and HIV Glycan Shields.Immunity. 2019; 51: 724-734.e4Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar Many different technologies have facilitated isolation of antigen-specific mAbs, including single-cell sorting with fluorescent bait, B cell immortalization, and B cell culture.3Walker L.M. Burton D.R. Passive immunotherapy of viral infections: ‘super-antibodies’ enter the fray.Nat. Rev. Immunol. 2018; 18: 297-308Crossref PubMed Scopus (174) Google Scholar However, these methods require probes, high-throughput screening of recombinant mAb libraries, or maintenance of thousands of sorted immortalized B cells or antibody (Ab)-secreting cells in culture. In fact, Gorman et al.2Gorman J. Mason R.D. Nettey L. Cavett N. Chuang G.Y. Peng D. Tsybovsky Y. Verardi R. Nguyen R. Ambrozak D. et al.Isolation and Structure of an Antibody that Fully Neutralizes Isolate SIVmac239 Reveals Functional Similarity of SIV and HIV Glycan Shields.Immunity. 2019; 51: 724-734.e4Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar isolated the first SIVmac239 nmAbs after screening ~5,700 cultured CD20+ immunoglobulin D (IgD)− IgM− total B cells and 118 mAbs from sorted gp140 FT+ CD20+ IgG+ IgM− memory B cells. The generation of B cells encoding high-affinity Abs is usually associated with extensive selection and proliferation in the germinal center. Although terminally differentiated plasma cells in the bone marrow are responsible for sustaining the majority of circulating Ig,4Slifka M.K. Ahmed R. Long-term humoral immunity against viruses: revisiting the issue of plasma cell longevity.Trends Microbiol. 1996; 4: 394-400Abstract Full Text PDF PubMed Scopus (83) Google Scholar, 5Slifka M.K. Antia R. Whitmire J.K. Ahmed R. Humoral immunity due to long-lived plasma cells.Immunity. 1998; 8: 363-372Abstract Full Text Full Text PDF PubMed Scopus (953) Google Scholar, 6McHeyzer-Williams M.G. Ahmed R. B cell memory and the long-lived plasma cell.Curr. Opin. Immunol. 1999; 11: 172-179Crossref PubMed Scopus (204) Google Scholar, 7Manz R.A. Hauser A.E. Hiepe F. Radbruch A. Maintenance of serum antibody levels.Annu. Rev. Immunol. 2005; 23: 367-386Crossref PubMed Scopus (435) Google Scholar memory B cells have been the most exploited source to isolate SIV-specific mAbs because they can be easily sorted based on their surface Ig specificity. Thus, isolation of Abs from plasma cells represents another potential source of mAbs. However, there are several limitations associated with working with these cells. Although the plasma cell phenotype from RMs has been defined previously as CD138+ CD31+ B cells,8Martinez-Murillo P. Pramanik L. Sundling C. Hultenby K. Wretenberg P. Spångberg M. Karlsson Hedestam G.B. CD138 and CD31 Double-Positive Cells Comprise the Functional Antibody-Secreting Plasma Cell Compartment in Primate Bone Marrow.Front. Immunol. 2016; 7: 242Crossref PubMed Scopus (15) Google Scholar there is selective loss of these critical phenotype-defining markers following freezing, limiting our ability to use previously frozen bone marrow samples. In addition, antigen-specific plasma cells cannot be easily isolated using traditional flow cytometry because they do not have membrane-bound Ig. To date, only two groups have successfully isolated antigen-specific bone marrow-derived plasma cells from mice, rabbits, and rats.9Clargo A.M. Hudson A.R. Ndlovu W. Wootton R.J. Cremin L.A. O’Dowd V.L. Nowosad C.R. Starkie D.O. Shaw S.P. Compson J.E. et al.The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method.MAbs. 2014; 6: 143-159Crossref PubMed Scopus (39) Google Scholar,10Reddy S.T. Ge X. Miklos A.E. Hughes R.A. Kang S.H. Hoi K.H. Chrysostomou C. Hunicke-Smith S.P. Iverson B.L. Tucker P.W. et al.Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells.Nat. Biotechnol. 2010; 28: 965-969Crossref PubMed Scopus (225) Google Scholar Based on the methodology described by Clargo et al.,9Clargo A.M. Hudson A.R. Ndlovu W. Wootton R.J. Cremin L.A. O’Dowd V.L. Nowosad C.R. Starkie D.O. Shaw S.P. Compson J.E. et al.The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method.MAbs. 2014; 6: 143-159Crossref PubMed Scopus (39) Google Scholar we established a fluorescence-based platform to isolate SIVmac239 SOSIP.664-specific plasma cells from r10051, a SIVmac239-infected Indian RM with high neutralizing titers against this pathogenic clone. Here we used an automated micromanipulator linked to a fluorescence microscope to identify antigen-specific plasma cells without performing direct functional assays on cultured cells. Typically, HIV nmAbs have high levels of somatic hypermutations (SHMs), insertions or deletions (indels), and often long variable heavy (VH)-chain CDR3s. Plasma cells from a RM making neutralizing Abs should have accumulated such characteristics necessary for SIV neutralization because of repeated antigen exposure and multiple selection cycles. Using the ALS CellCelector, we picked plasma cells and subsequently isolated RNA encoding the VH and variable light (VL) chains of 44 complete Ab pairs. Ten of these 44 mAbs were SIV specific and targeted different areas of the viral Envelope (Env) glycoprotein. Although none of these mAbs neutralized SIVmac239, three potently neutralized 100% of the related tier 1 SIVmac316 strain. In addition, these mAbs bound to primary rhesus CD4+ T cells infected with SIVmac239, and seven induced Ab-dependent cellular cytotoxicity (ADCC), which could have implications for their in vivo utility. We previously reported the serum neutralization titers of our cohort of 34 Indian RMs at the Wisconsin National Primate Research Center.11Pedreño-Lopez N. Dang C.M. Rosen B.C. Ricciardi M.J. Bailey V.K. Gutman M.J. Gonzalez-Nieto L. Pauthner M.G. Le K. Song G. et al.Induction of Transient Virus Replication Facilitates Antigen-Independent Isolation of SIV-Specific Monoclonal Antibodies.Mol. Ther. Methods Clin. Dev. 2020; 16: 225-237Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar There we identified a conventional progressor, r10051, that developed SIVmac239-neutralizing titers of 1:2,157 80 weeks post-infection. After 2 additional years on antiretroviral therapy, its serum neutralization titers remained elevated (1:1,610). Additionally, we detected SIVmac239-specific Ab-secreting cells in the bone marrow by Ig enzyme-linked immunospot ELISpot (Figure 1A). Although approximately 7% of the total bone marrow cells were Ab-secreting cells, 20% of these Ab-secreting cells were reactive against the SIVmac239 gp140 FT protein.12Mason R.D. Welles H.C. Adams C. Chakrabarti B.K. Gorman J. Zhou T. Nguyen R. O’Dell S. Lusvarghi S. Bewley C.A. et al.Targeted Isolation of Antibodies Directed against Major Sites of SIV Env Vulnerability.PLoS Pathog. 2016; 12: e1005537Crossref PubMed Scopus (38) Google Scholar To identify bone marrow-derived SIVmac239-specific Ab-secreting cells from r10051, we selected the recently available SIVmac239 SOSIP.664 trimer as our screening tool because of its native-like structure.13von Bredow B. Andrabi R. Grunst M. Grandea 3rd, A.G. Le K. Song G. Berndsen Z.T. Porter K. Pallesen J. Ward A.B. et al.Differences in the Binding Affinity of an HIV-1 V2 Apex-Specific Antibody for the SIVsmm/mac Envelope Glycoprotein Uncouple Antibody-Dependent Cellular Cytotoxicity from Neutralization.MBio. 2019; 10: e01255-19Crossref PubMed Scopus (12) Google Scholar Unlike other SIV subunits (i.e., gp140), SOSIP.664 contains quaternary epitopes that facilitate proper formation of the Env spike apex, mimicking the native Env protein. This soluble protein is usually purified using an affinity column with PGT145, an Ab that recognizes a quaternary epitope at the trimer apex and allows exclusion of non-trimeric Env protein. We verified by ELISA that our SIVmac239 SOSIP.664 had the correct trimeric conformation (Figure S1). Thus, to isolate SIVmac239 SOSIP.664-specific plasma cells using our newly developed fluorescence method, we first coated 24-well plates (with imprinted 50 × 50 μm nanowells) with streptavidin overnight and then added biotinylated SIVmac239 SOSIP.664 the following day (Figure 1B). Bone marrow aspirates were obtained from both femora and both humeri of r10051 at different time points during SIV infection and while this animal was on antiretroviral therapy (ART). To prevent false positives with our fluorescence-based method, we pre-incubated total bone marrow cells with different mouse anti-Fcγ receptor (FcγR) I–III Abs to block the FcγRs present on the surface of certain types of cells, including macrophages and dendritic cells. These cells were then resuspended in medium containing recombinant a proliferation-inducing ligand (APRIL), interleukin-6 (IL-6), and mouse anti-monkey IgG fluorescein isothiocyanate (FITC) and added to the SIVmac239 SOSIP.664 plates.8Martinez-Murillo P. Pramanik L. Sundling C. Hultenby K. Wretenberg P. Spångberg M. Karlsson Hedestam G.B. CD138 and CD31 Double-Positive Cells Comprise the Functional Antibody-Secreting Plasma Cell Compartment in Primate Bone Marrow.Front. Immunol. 2016; 7: 242Crossref PubMed Scopus (15) Google Scholar,14Cassese G. Arce S. Hauser A.E. Lehnert K. Moewes B. Mostarac M. Muehlinghaus G. Szyska M. Radbruch A. Manz R.A. Plasma cell survival is mediated by synergistic effects of cytokines and adhesion-dependent signals.J. Immunol. 2003; 171: 1684-1690Crossref PubMed Scopus (366) Google Scholar,15Kawano M.M. Mihara K. Huang N. Tsujimoto T. Kuramoto A. Differentiation of early plasma cells on bone marrow stromal cells requires interleukin-6 for escaping from apoptosis.Blood. 1995; 85: 487-494Crossref PubMed Google Scholar After a 12-h incubation period, we used the fluorescence microscope of the ALS CellCelector to visualize fluorescent halos around cells that had secreted SOSIP.664-specific Abs (Figure 1C). We then used the micromanipulator to pick single cells with 30-μm oil-filled glass capillaries and transferred them into 96-well plates containing lysis buffer for subsequent PCR amplification. This technique allowed us to functionally screen 10 million bone marrow cells in less than a day. Of the picked bone marrow-derived cells, we amplified 46 unique VH chains and 56 VL chains, which resulted in a total of 44 complete Ab pairs. Ten of these wells were positive for multiple kappa and lambda chains, indicating that more than one B cell had been picked using the ALS CellCelector. In these instances, we selected both light chains and verified their binding specificities in functional assays. We next analyzed V-gene use, VH CDR3 length, percent divergence from the germline, and presence of indels of the 44 VH/VL Ab pairs isolated from the bone marrow of r10051. IGHV4-2∗01 was used by over 40% of the total amplified mAbs, which was also true for the 10 SIVmac239 SOSIP.664-specific mAbs (Figure 2A). Of note, we identified three sets of two Ig V-region genes that were clonally related but had unique VH CDR3s. We then analyzed the amino acid length distributions of the plasma cell-derived VH CDR3 sequences. VH CDR3 length ranged from 9–23 amino acids, indicating considerable VH CDR3 length diversity (Figure 2B). Although the average SHM of IgGs isolated from healthy donors is 5% nucleotide divergence from the germline,16Francica J.R. Sheng Z. Zhang Z. Nishimura Y. Shingai M. Ramesh A. Keele B.F. Schmidt S.D. Flynn B.J. Darko S. et al.NISC Comparative Sequencing ProgramAnalysis of immunoglobulin transcripts and hypermutation following SHIV(AD8) infection and protein-plus-adjuvant immunization.Nat. Commun. 2015; 6: 6565Crossref PubMed Scopus (64) Google Scholar the total mAbs isolated in this study diverged from their putative germline predecessors by 14.0% ± 4.7%. Interestingly, the four most mutated mAbs were all SIV specific and diverged from their germline sequences by up to 24.7% (Figure 2C). Previous studies have suggested that development of neutralization breadth is usually associated with extensive SHM in the antigen-binding site of the V-gene in conjunction with in-frame indels.17Kepler T.B. Liao H.X. Alam S.M. Bhaskarabhatla R. Zhang R. Yandava C. Stewart S. Anasti K. Kelsoe G. Parks R. et al.Immunoglobulin gene insertions and deletions in the affinity maturation of HIV-1 broadly reactive neutralizing antibodies.Cell Host Microbe. 2014; 16: 304-313Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar Kepler et al.17Kepler T.B. Liao H.X. Alam S.M. Bhaskarabhatla R. Zhang R. Yandava C. Stewart S. Anasti K. Kelsoe G. Parks R. et al.Immunoglobulin gene insertions and deletions in the affinity maturation of HIV-1 broadly reactive neutralizing antibodies.Cell Host Microbe. 2014; 16: 304-313Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar determined that HIV-infected patients have higher numbers of indels compared with non-HIV-infected individuals, and 25% of the HIV broadly nmAb genes contain indels, a 7-fold higher rate compared with 13,000 V-genes extracted from NCBI GenBank.17Kepler T.B. Liao H.X. Alam S.M. Bhaskarabhatla R. Zhang R. Yandava C. Stewart S. Anasti K. Kelsoe G. Parks R. et al.Immunoglobulin gene insertions and deletions in the affinity maturation of HIV-1 broadly reactive neutralizing antibodies.Cell Host Microbe. 2014; 16: 304-313Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar In our study, 37% of the total plasma cell-derived mAbs and 40% of the SIV-specific mAbs contained indels. To evaluate the Abs expressed by the plasma cells of r10051, we first determined their epitope specificity by ELISA with different Env subunits and mutants (Table 1), as we described previously.11Pedreño-Lopez N. Dang C.M. Rosen B.C. Ricciardi M.J. Bailey V.K. Gutman M.J. Gonzalez-Nieto L. Pauthner M.G. Le K. Song G. et al.Induction of Transient Virus Replication Facilitates Antigen-Independent Isolation of SIV-Specific Monoclonal Antibodies.Mol. Ther. Methods Clin. Dev. 2020; 16: 225-237Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar Of the 44 mAbs, 10 were SIVmac239 Env specific (22.7% of the total isolated mAbs; Table S1; GenBank: MT_678464–MT_678483). Four of these mAbs targeted the gp41 fusion protein, and four additional mAbs bound the variable (V) loops. Interestingly, we identified one mAb that only bound SOSIP.664 trimer but not gp140, and another mAb that bound gp140 but did not bind gp41 or the V loops.Table 1Functional Characteristics of the Bone Marrow-Derived SIV-Specific Abs Isolated from r10051Env SpecificityBinding to Infected CD4+ T Cells at 100 μg/mLSIVmac239 IC50 (μg/mL)SIVmac316 IC50 (μg/mL)SIVmac316 VmaxP1A05gp140N/B>50>50N/DP1B05V1V2V3>40%>500.019100%P1D06V1V2V3>40%>50>50N/DP1F04gp41<20%>50>50N/DP1H06V1V2V3>40%>500.006100%P2C02SOSIP.664<20%>50>50N/DP2F06V4>40%>500.002100%P2H02gp41<20%>50>50NDP2H04gp41NB>50>50NDP2H06gp41<20%>50>50ND5L7 (Pos Ctl)V4>40%>500.003100%IC50, inhibitory mAb concentration at which 50% virus neutralization was attained; Vmax, maximum percentage neutralization; N/B, non-binding; N/D, not determined; Pos Ctl, Positive control. Open table in a new tab IC50, inhibitory mAb concentration at which 50% virus neutralization was attained; Vmax, maximum percentage neutralization; N/B, non-binding; N/D, not determined; Pos Ctl, Positive control. We then tested the neutralization activity of the isolated mAbs against SIVmac239 and SIVmac316.18Daniel M.D. Letvin N.L. King N.W. Kannagi M. Sehgal P.K. Hunt R.D. Kanki P.J. Essex M. Desrosiers R.C. Isolation of T-cell tropic HTLV-III-like retrovirus from macaques.Science. 1985; 228: 1201-1204Crossref PubMed Scopus (655) Google Scholar,19Mori K. Ringler D.J. Kodama T. Desrosiers R.C. Complex determinants of macrophage tropism in env of simian immunodeficiency virus.J. Virol. 1992; 66: 2067-2075Crossref PubMed Google Scholar SIVmac316 is a tier 1 strain sensitive to neutralization that was isolated from alveolar macrophages of a SIVmac239-infected RM.20Desrosiers R.C. Hansen-Moosa A. Mori K. Bouvier D.P. King N.W. Daniel M.D. Ringler D.J. Macrophage-tropic variants of SIV are associated with specific AIDS-related lesions but are not essential for the development of AIDS.Am. J. Pathol. 1991; 139: 29-35PubMed Google Scholar Although none of the mAbs neutralized SIVmac239, two V1V2V3-specific mAbs and one V4-specific mAb exhibited 100% neutralization of SIVmac316, with 50% inhibitory concentration (IC50) values ranging from 2–19 ng/mL. Plasma cells reside in the bone marrow and produce large quantities of circulating Abs.21Nutt S.L. Hodgkin P.D. Tarlinton D.M. Corcoran L.M. The generation of antibody-secreting plasma cells.Nat. Rev. Immunol. 2015; 15: 160-171Crossref PubMed Scopus (749) Google Scholar We therefore examined the potential effector role of these non-neutralizing mAbs in vivo. First, we analyzed the capacity of these mAbs to bind to primary rhesus CD4+ T cells infected with SIVmac239 (Figure 3). All but two of the plasma-cell-derived mAbs isolated in this study, P1A05 and P2H04, bound to infected CD4+ T cells, albeit with varying affinities. In fact, our V4-specific mAb, P2F06, bound CD4+ T cells similarly as 5L7 IgG1, a non-neutralizing V4-specific mAb that protected a single macaque from six successive SIVmac239 intravenous challenges when present at serum concentrations greater than 200 μg/mL.22Fuchs S.P. Martinez-Navio J.M. Piatak Jr., M. Lifson J.D. Gao G. Desrosiers R.C. AAV-Delivered Antibody Mediates Significant Protective Effects against SIVmac239 Challenge in the Absence of Neutralizing Activity.PLoS Pathog. 2015; 11: e1005090Crossref PubMed Scopus (69) Google Scholar 5L7 IgG1 also lowered peak and set point viremia and increased the number of challenges required to achieve infection at lower concentrations. Not surprisingly, the mAbs that targeted the V loops bound to higher proportions of SIVmac239-infected CD4+ T cells than gp41-specific mAbs. To determine whether mAbs might have a potential effect in vivo, we measured their ADCC activity. We employed a luciferase-based assay that measures natural killer (NK) cell-mediated ADCC activity independent of neutralization or complement activity.23Alpert M.D. Heyer L.N. Williams D.E. Harvey J.D. Greenough T. Allhorn M. Evans D.T. A novel assay for antibody-dependent cell-mediated cytotoxicity against HIV-1- or SIV-infected cells reveals incomplete overlap with antibodies measured by neutralization and binding assays.J. Virol. 2012; 86: 12039-12052Crossref PubMed Scopus (80) Google Scholar As expected, P1A05, P2H04, and P2C02 had no detectable ADCC activity (Figure 4). mAbs P1A05 and P2H04 did not bind to SIVmac239-infected CD4+ T cells, and P2C02, which only binds the SOSIP.664 trimer, bound weakly. Six of the 10 SIV-specific bone marrow-derived mAbs efficiently recruited NK cells and induced ADCC activity similar to 5L7, with 50% ADCC activity (ADCC50) values of close to 0.1 μg/mL. Surprisingly, P2F06, the only V4-specific mAb tested besides 5L7, had a 10-fold lower ADCC50 than 5L7 of 0.03 μg/mL. Here we developed a fluorescence-based platform to isolate SIV-specific mAbs from an Indian RM with high serum neutralization titers against SIVmac239 using the trimeric SIVmac239 SOSIP.664 Env protein. Utilizing this methodology, we picked bone marrow-derived plasma cells and isolated 44 rhesus VH/VL paired sequences. Ten of these 44 mAbs were SIV specific and targeted different parts of the trimeric Env glycoprotein, including the V loops and the gp41 fusion protein. Although none of these mAbs neutralized SIVmac239, three of them exhibited 100% neutralization of the tier 1 SIVmac316 strain. In addition, the majority of these mAbs bound to primary rhesus CD4+ T cells infected with SIVmac239 and induced ADCC activity against SIVmac239-infected cells, suggesting that these mAbs could have a role in vivo. The scientific community has commonly used memory B cells as the main source of cells for mAb isolation. However, plasma cells contribute to the serum neutralization responses detected in conventional functional assays.4Slifka M.K. Ahmed R. Long-term humoral immunity against viruses: revisiting the issue of plasma cell longevity.Trends Microbiol. 1996; 4: 394-400Abstract Full Text PDF PubMed Scopus (83) Google Scholar, 5Slifka M.K. Antia R. Whitmire J.K. Ahmed R. Humoral immunity due to long-lived plasma cells.Immunity. 1998; 8: 363-372Abstract Full Text Full Text PDF PubMed Scopus (953) Google Scholar, 6McHeyzer-Williams M.G. Ahmed R. B cell memory and the long-lived plasma cell.Curr. Opin. Immunol. 1999; 11: 172-179Crossref PubMed Scopus (204) Google Scholar, 7Manz R.A. Hauser A.E. Hiepe F. Radbruch A. Maintenance of serum antibody levels.Annu. Rev. Immunol. 2005; 23: 367-386Crossref PubMed Scopus (435) Google Scholar Unfortunately, these cells cannot be sorted using fluorescent bait because they do not express surface Ig. To date, only two studies have isolated antigen-specific plasma cells from bone marrow of different rodents,9Clargo A.M. Hudson A.R. Ndlovu W. Wootton R.J. Cremin L.A. O’Dowd V.L. Nowosad C.R. Starkie D.O. Shaw S.P. Compson J.E. et al.The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method.MAbs. 2014; 6: 143-159Crossref PubMed Scopus (39) Google Scholar,10Reddy S.T. Ge X. Miklos A.E. Hughes R.A. Kang S.H. Hoi K.H. Chrysostomou C. Hunicke-Smith S.P. Iverson B.L. Tucker P.W. et al.Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells.Nat. Biotechnol. 2010; 28: 965-969Crossref PubMed Scopus (225) Google Scholar and no studies have focused on isolation of antigen-specific long-lived plasma cells from bone marrow of RMs. Clargo et al.9Clargo A.M. Hudson A.R. Ndlovu W. Wootton R.J. Cremin L.A. O’Dowd V.L. Nowosad C.R. Starkie D.O. Shaw S.P. Compson J.E. et al.The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method.MAbs. 2014; 6: 143-159Crossref PubMed Scopus (39) Google Scholar utilized an automated micromanipulator to identify bone marrow-derived mAbs that targeted tumor necrosis factor alpha (TNF-α), a highly conserved molecule. Automated micromanipulators systems, like the ALS CellCelector, have been used primarily to isolate circulating tumor cells.24Neumann M.H. Schneck H. Decker Y. Schömer S. Franken A. Endris V. Pfarr N. Weichert W. Niederacher D. Fehm T. Neubauer H. Isolation and characterization of circulating tumor cells using a novel workflow combining the CellSearch® system and the CellCelector™.Biotechnol. Prog. 2017; 33: 125-132Crossref PubMed Scopus (38) Google Scholar, 25Lampignano R. Yang L. Neumann M.H.D. Franken A. Fehm T. Niederacher D. Neubauer H. A Novel Workflow to Enrich and Isolate Patient-Matched EpCAMhigh and EpCAMlow/negative CTCs Enables the Comparative Characterization of the PIK3CA Status in Metastatic Breast Cancer.Int. J. Mol. Sci. 2017; 18: 1885Crossref PubMed Scopus (36) Google Scholar, 26Nelep C. Eberhardt J. Automated rare single cell picking with the ALS cellcelector™.Cytometry A. 2018; 93: 1267-1270Crossref PubMed Scopus (20) Google Scholar, 27Reinhardt F. Franken A. Meier-Stiegen F. Driemel C. Stoecklein N.H. Fischer J.C. Niederacher D. Ruckhaeberle E. Fehm T. Neubauer H. Diagnostic Leukapheresis Enables Reliable Transcriptomic Profiling of Single Circulating Tumor Cells to Characterize Inter-Cellular Heterogeneity in Terms of Endocrine Resistance.Cancers (Basel). 2019; 11: 903Crossref Scopus (19) Google Scholar However, Ogunniyi et al.28Ogunniyi A.O. Thomas B.A. Politano T.J. Varadarajan N. Landais E. Poignard P. Walker B.D. Kwon D.S. Love J.C. Profiling human antibody responses by integrated single-cell analysis.Vaccine. 2014; 32: 2866-2873Crossref PubMed Scopus (27) Google Scholar employed this methodology in combination with V(D)J gene amplification to evaluate human humoral responses present in blood and mucosal tissue from HIV-infected patients. After characterization of the samples, they verified the phenotype of eight Env-specific mAbs, two of which partially neutralized two HIV-1 tier 1 and 2 strains. There are multiple factors that might affect the likelihood of isolating a SIVmac239-specific nmAb. The most relevant include the levels of neutralizing titers against SIVmac239 in r10051, which might influence our ability to isolate nmAbs. Use of a relevant native-like probe, such as SOSIP.664, would theoretically enhance our ability to isolate nmAbs. The number of screened B cells or cloned Abs is also a factor. In our case, the limited number of mAbs screened in this study was probably the main reason why we did not identify such a nmAb. It is worth noting that, although the described method has some throughput limitations compared with other methods, it might be possible to increase the number of screened cells with a pre-enrichment step. Unfortunately, other high-throughput methods (e.g., flow cytometry) are not suitable for isolating antigen-specific plasma cells. Although Martinez-Murillo et al.8Martinez-Murillo P. Pramanik L. Sundling C. Hultenby K. Wretenberg P. Spångberg M. Karlsson Hedestam G.B. 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