Isolation of antibody V(D)J sequences from single cell sorted rhesus macaque B cells

Sundling, Christopher; Phad, Ganesh E.; Douagi, Iyadh; Navis, Marjon; Hedestam, Gunilla B. Karlsson

doi:10.1016/j.jim.2012.09.003

Cited by 71 publications

(148 citation statements)

References 45 publications

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“…Out of nine positive clones that bound to the three GPs by ELISAs, two clones showed an OD of Ͼ1.5 to GP⌬muc and approximately 0.5 to 0.7 to GP⌬TM to at least two species. Heavy and light chain variable Ig genes were amplified by seminested PCR with specific macaque primers (32). After sequencing and reamplifying with cloning primers, the VH and VL PCR fragments were cloned into expression vectors (35) that contain constant region sequences of human Ig␥1 heavy and Ig light chains.…”

Section: Resultsmentioning

confidence: 99%

“…The methods for generating the yeast display single-chain variable fragment (scFv) library, selection of ebolavirus GPspecific scFv, and production of IgG1 chimeric macaque-human monoclonal antibodies (M/HMAbs) were as described previously (31). Primers used in primary PCRs to amplify macaque gamma heavy chain and kappa and lambda light chains were described previously (32). Briefly, the yeast library was grown in SG-CAA growth medium (0.51% Na 2 HPO 4 ·7H 2 O, 0.43% Na 2 HPO 4 ·H 2 O, 0.25% Casamino Acids, 1% galactose) for 48 h at 18°C.…”

Section: Methodsmentioning

confidence: 99%

“…The IgH, Ig, and Ig V genes were amplified separately by nested PCR starting from 1 l of cDNA directly following RT and the nested PCR on 1 l of the first-round PCR product as described previously (32) with the following modifications. All PCRs were performed in a total volume of 20 l containing nuclease-free water, 4 l of 5ϫ buffer, 0.4 l of 10 mM dNTP mix, 0.8 l of 40 M mixture of forward and reverse primers (32), and 0.4 l of PHusion polymerase. The PCR instrument was programmed for 5-min incubation at 94°C, followed by 40 cycles, with one cycle consisting of 30 s at 94°C, 30 s at 55°C (for the first round of PCR) or 60°C (for the nested round of PCR), and 60 s at 70°C, with a final elongation step at 70°C for 7 min before cooling to 4°C.…”

Section: Methodsmentioning

confidence: 99%

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Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein

Keck

Enterlein

Howell

et al. 2016

J Virol

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Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models. IMPORTANCEFiloviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus. Since the nature of future outbreaks cannot be predicted, there is an urgent need for therapeutics with broad protective efficacy against multiple filoviruses. Here we describe a set of monoclonal antibodies cross-reactive with multiple filovirus species. These antibodies target novel conserved epitopes within the envelope glycoprotein and exhibit protective efficacy in mice. We further present novel concepts for combination of cross-reactive antibodies against multiple epitopes that show enhanced efficacy compared to monotherapy and provide complete protection in mice. These findings set the stage for further evaluation of these antibodies in nonhuman primates and development of effective pan-filovirus immunotherapeutics for use in future outbreaks. F iloviruses consisting of Marburg virus (MARV), Ravn virus (RAVV), and five species of ebolavirus, Ebola virus (EBOV), Sudan virus (SUDV), Bundibugyo virus (BDBV), Reston virus (RESTV), and Taï Forest virus (TAFV), are causative agents of severe hemorrhagic fever in humans and nonhuman primates (NHPs) (1, ...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein

Keck

Enterlein

Howell

et al. 2016

J Virol

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“…The sorted cells were lysed with the lysis buffer, followed by single-cell reverse transcription and PCRs to amplify Ig sequences as described previously (15,27,52). After sequencing verification of correct V(D)J sequences, the Ig variable domains of selected B cell clones were cloned into Ab IgG heavy-and light-chain expression vectors, respectively, as described previously (15).…”

Section: Methodsmentioning

confidence: 99%

HIV-1 Cross-Reactive Primary Virus Neutralizing Antibody Response Elicited by Immunization in Nonhuman Primates

Wang

O’Dell

Turner

et al. 2017

J Virol

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Elicitation of broadly neutralizing antibody (bNAb) responses is a major goal for the development of an HIV-1 vaccine. Current HIV-1 envelope glycoprotein (Env) vaccine candidates elicit predominantly tier 1 and/or autologous tier 2 virus neutralizing antibody (NAb) responses, as well as weak and/or sporadic cross-reactive tier 2 virus NAb responses with unknown specificity. To delineate the specificity of vaccine-elicited cross-reactive tier 2 virus NAb responses, we performed single memory B cell sorting from the peripheral blood of a rhesus macaque immunized with YU2gp140-F trimers in adjuvant, using JR-FL SOSIP.664, a native Env trimer mimetic, as a sorting probe to isolate monoclonal Abs (MAbs). We found striking genetic and functional convergence of the SOSIP-sorted Ig repertoire, with predominant VH4 or VH5 gene family usage and Env V3 specificity. Of these vaccine-elicited V3-specific MAbs, nearly 20% (6/33) displayed cross-reactive tier 2 virus neutralization, which recapitulated the serum neutralization capacity. Substantial similarities in binding specificity, neutralization breadth and potency, and sequence/structural homology were observed between selected macaque cross-reactive V3 NAbs elicited by vaccination and prototypic V3 NAbs derived from natural infections in humans, highlighting the convergence of this subset of primate V3-specific B cell repertories. Our study demonstrated that cross-reactive primary virus neutralizing B cell lineages could be elicited by vaccination as detected using a standardized panel of tier 2 viruses. Whether these lineages could be expanded to acquire increased breadth and potency of neutralization merits further investigation.IMPORTANCE Elicitation of antibody responses capable of neutralizing diverse HIV-1 primary virus isolates (designated broadly neutralizing antibodies [bNAbs]) remains a high priority for the vaccine field. bNAb responses were so far observed only in response to natural infection within a subset of individuals. To achieve this goal, an improved understanding of vaccine-elicited responses, including at the monoclonal Ab level, is essential. Here, we isolated and characterized a panel of vaccine-elicited cross-reactive neutralizing MAbs targeting the Env V3 loop that moderately neutralized several primary viruses and recapitulated the serum neutralizing antibody response. Striking similarities between the cross-reactive V3 NAbs elicited by vaccination in macaques and natural infections in humans illustrate commonalities between

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“…[5][6][7][8] Antibody secreting cells originating from activated memory B cells possess unique properties including their ability to produce large amounts of IG in response to ongoing infection or immunization, and are enriched in specificity for the antigens of interest. Therefore, antigen-specific IG can be isolated from single antibody secreting cells efficiently without antigen pre-screening.…”

Section: Introductionmentioning

confidence: 99%

Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells

Meng

Xiong

et al. 2015

mAbs

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Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n D 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs.

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Isolation of antibody V(D)J sequences from single cell sorted rhesus macaque B cells

Cited by 71 publications

References 45 publications

Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein

Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein

HIV-1 Cross-Reactive Primary Virus Neutralizing Antibody Response Elicited by Immunization in Nonhuman Primates

Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells

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