key: cord-290788-6y0vjhux
authors: Wang, Qihui; Yan, Jinghua
title: Isolation of Monoclonal Antibodies from Zika Virus-Infected Patient Samples
date: 2020-05-05
journal: Zika Virus
DOI: 10.1007/978-1-0716-0581-3_20
sha: 
doc_id: 290788
cord_uid: 6y0vjhux

The combination of sorting antigen-specific memory B cells with determining immunoglobulin (Ig) genes at the single-cell level enables the isolation of monoclonal antibodies (mAbs) in individuals. This method requires a small amount of blood (usually 10 mL) and is rapid (less than 2 weeks to isolate antigen-specific mAbs). Due to the application of antigens as the bait to capture the specific memory B cells, the majority of isolated mAbs are true binders to the antigen, which increases the isolation efficiency. Here, applying this approach, we describe the characterization of mAbs against Zika virus from a convalescent patient sample. From 10 mL whole blood, we sorted 33 Zika envelope (E) protein-interacting single memory B cells. The Ig genes from 15 cells were determined, and 13 mAbs were found that bind to Zika E protein with varied binding affinities.

Zika virus has caused global concern due to the accumulating evidence suggesting that infection is associated with microcephaly and neurological complications, such as Guillain-Barré syndrome [1] [2] [3] [4] . However, there are currently no approved antivirals against Zika infection [5] . Administration of polyclonal or purified neutralizing monoclonal antibodies (mAbs) to pregnant mice helps to clear the virus and alleviates the neurological disorders in their fetuses [6, 7] , providing proof of concept that neutralizing mAbs can be used to treat Zika virus infections. Currently, multiple strategies have been reported to generate human neutralizing mAbs against Zika infection, including sequencing antigen-specific memory B cells [8, 9] or generating Epstein-Barr virus-immortalized memory B cells from Zika patient samples [10] , and identifying functional mAbs from phage display naïve antibody libraries [11] . In addition, murine mAbs against Zika virus have also been reported [12] .

Here, we introduce a method to apply Zika envelope (E) glycoproteins, which play pivotal roles in virus entry and contain important neutralizing epitopes, to sort single memory B cells from a convalescent Zika patient. Subsequently, the immunoglobulin (Ig) genes encoded by the sorted cells were determined at the single-cell level. The approach to determine the Ig genes from single cells was first reported by Tiller et al. [13] . Later, Scheid et al. modified this approach to sequence single gp140-binding memory B cells, which were isolated from human immunodeficiency virus (HIV) patients [14] . Then the mAbs against human papillomavirus (HPV) or respiratory syncytial virus (RSV) were isolated from immunized or naturally infected human donors, respectively [15, 16] . During the epidemic of Zika virus, we and other groups also applied this strategy to isolate mAbs targeting Zika virus from Zika patients [8, 9] .

From 10 mL whole blood, we were able to pair 15 mAbs from 33 collected single cells. As detected by surface plasmon resonance (SPR), 13 of the mAbs were true binders to Zika E protein [8] .

Although the protocol depicted in this chapter focuses on the isolation of Zika mAbs from a Zika convalescent patient, it could also be expanded to other viruses, and we recently reported the isolation of human neutralizing mAbs against Rift Valley fever virus (RVFV) from a convalescent RVF patient, applying the same strategy [17] . 1. Primers for reverse transcription (RT) are listed in Table 2. 2. Primers for the first round PCR (PCRa) to amplify V H are listed in Table 3. 3. Primers for the PCRa to amplify V κ are listed in Table 4. 4. Primers for the PCRa to amplify V λ are listed in Table 5 .

5. Primers for the second round PCR (PCRb) to amplify V H are listed in Table 6. 6. Primers for the PCRb to amplify V κ are listed in Table 7. 7. Primers for the PCRb to amplify V λ are listed in Table 8. 8. Other primers used in this study are listed in Table 9 . Table 4 Primers for the PCRa to amplify V κ Forward primer

Cκ-ext GAG GCA GTT CCA GAT TTC AA Table 5 Primers for the PCRa to amplify V λ Forward primer 11. LB agar plates containing ampicillin: dissolve 5 g of tryptone, 10.0 g of yeast extract, 10.0 g of NaCl, and 15.0 g of agar in 1 L of ddH 2 O. The agar will not dissolve until autoclaved. Autoclave for 15 min and then allow it to cool until the bottle can be held with bare hands. Add 1 mL of ampicillin (100 mg/ mL). Invert to mix thoroughly. Carefully pour out into sterile 8. Trypsin-EDTA (0.05%) and phenol red. Table 9 Other primers used in this study 14. Transfer buffer: Dissolve 6.04 g of Tris base and 28.8 g of glycine together in 1.6 L of ddH 2 O. Then add 200 mL of methanol and mix. At last, add ddH 2 O to a final volume of 2 L.

Characterization for the mAbs 1. Biacore T100 system.

3. Biacore T100 evaluation software, version 1.0.

1. Collect 10 mL of whole blood, using a K 2 EDTA Tube, from a convalescent Zika patient or a healthy donor with informed consent (see Note 5).

2. Dilute the anticoagulated blood with 10 mL PBS (one volume of the original blood).

3. Invert the density gradient medium bottle several times to ensure thorough mixing.

4. Add 13 mL of density gradient medium to a 50 mL centrifuge tube with a conical bottom.

5. Carefully layer the diluted blood sample onto the density gradient medium solution (see Note 6).

6. Centrifuge at 500 Â g for 25 min at room temperature (see Notes 7 and 8).

7. Draw off the upper layer containing plasma and platelets using a sterile pipette, leaving the PBMC layer undisturbed at the interface. Aliquot 1 mL of the upper layer per vial, which contains the plasma, and store them at À80 C ( Fig. 1 ).

8. Transfer the layer of PBMCs to a sterile 50 mL centrifuge tube using a sterile pipette (see Note 9).

9. Add PBS until the total volume reaches 15 mL (1.5 volumes of the original blood). Resuspend the cells by gently drawing them in and out of a pipette.

10. Centrifuge at 500 Â g for 15 min at room temperature, and discard the supernatant.

11. Add 15 mL PBS to resuspend the cells. Mix thoroughly and transfer 50 μL of cell suspension in a 1.5 mL tube for cell counting.

12. Load 10 μL of cell suspension into a cell counting chamber.

13. Insert the chamber into an automated cell counter and run the counting.

14. Repeat step 10 in this section. 15 . Quickly resuspend the cell pellet by adding freezing medium to a cell density of 1 Â 10 7 /mL (see Note 10).

16. Aliquot 1 mL of the cell-medium mixture per vial, and place in a freezing container.

17. Freeze the cells overnight at À80 C. 18 . Transfer vials to a liquid N 2 tank for storage (see Note 11).

Cytometry (0.5 Day)

1. Samples used for staining are designed as follows:

(a) Zika patient PBMCs stained with Zika E protein, as well as a panel of memory B-cell markers (see Note 12). (c) Healthy donor PBMCs stained with MERS-RBD protein (positive control for anti-His antibody).

2. Transfer a vial containing the frozen PBMCs of either a convalescent Zika patient or a healthy donor from the liquid N 2 tank, and place them into a 37 C water bath (see Note 13).

3. Gently swirl the vial in the 37 C water bath, and quickly thaw the cells (<1 min) until there is just a small bit of ice left in the vial.

4. Transfer the vials into a hood. Before opening, wipe the outside of the vials with 70% ethanol.

5. Transfer the thawing cells into a centrifuge tube (15 mL) containing 10 mL of pre-warmed culture medium.

6. Centrifuge the cell suspension at 500 Â g for 5 min at room temperature.

7. Decant the supernatant without disturbing the cell pellet.

8. Gently resuspend the cells in FACS buffer, 400 μL per 10 7 cells. 

11. Keep the mixture on ice for 1 h.

12. Add 10 mL of FACS buffer to the cells and pellet the cells by centrifuging at 500 Â g for 5 min at 4 C. Decant the supernatant without disturbing the cell pellet (see Note 14) .

13. Resuspend the cells with 100 μL of FACS buffer per 10 7 cells (see Note 15).

14. Prepare staining master mix in a 1.5 mL microcentrifuge tube (see Table 1 ) and store them at 4 C in the dark before use. 15 . Add 100 μL of the staining master mix to cells that prepared in step 13 in this section. Incubate them on ice for 30 min. 16 . Add 10 mL of FACS buffer to the mixture of cells and antibodies in the last step and pellet the cells by centrifugation at 500 Â g for 5 min at 4 C. Decant the supernatant without disturbing the pellet.

17. Resuspend the cells with 0.5 mL FACS buffer. Place the tube on ice and avoid light before cell sorting.

Controls (1 h) 1. Label a FACS tube for each of the six fluorochromes that will be used in the cell sorting. Add 0.5 mL of FACS buffer into each tube.

2. Mix compensation beads by vigorously inverting at least ten times.

3. Add 50 μL of compensation beads into each tube. 6. Load the Zika PBMCs sample stained with Zika E protein and memory B-cell markers. Gate the target cells as in Fig. 2D. 7. Adjust the flow rate so that the event rate is approximately 8000 events/s. 1. Mix and briefly centrifuge each component in the RT-PCR synthesis system for the first-strand cDNA before use. Table 10 in a 0.5 mL tube. Table 10 ) into each well containing the collected single cells prepared in step 9 in Subheading 3.4.

4. Incubate the plate at 65 C for 5 min and then place it on ice for at least 1 min.

5. Prepare the following cDNA Synthesis Mix in a 1.5 mL tube, adding each component in Table 11 in the indicated order. Table 11 ) to each well, mix gently, and collect by centrifugation at 15,000 Â g at 4 C for 15 s.

7. The RT reaction is performed at 55 C for 60 min and terminated at 85 C for 5 min. Chill on ice and then store at À20 C until use.

8. Prepare primers for the PCRa (see Tables 3-5 ). The amount for 100 reactions is listed in Table 12. 9. Perform the PCRa to amplify the Ig genes. Prepare the reaction system as indicated in Table 13 (see Note 21) . The cycling conditions for the PCRa are shown in Table 14 .

Store the PCRa products at À20 C until further use.

10. Prepare primers for the PCRb (see Tables 6-8 ). The amount listed in Table 15 is for 100 reactions.

11. Perform the PCRb to amplify the Ig genes. Prepare reaction system as indicated in Table 16 (see Notes 21 and 22). The cycling conditions for the PCRb are indicated in Table 17. 12. Load the samples onto an agarose gel (1.2%), and separate the DNAs by electrophoresis. 13 . A typical band size for V H , V κ , and V λ is approximately 400 bp, as displayed in Fig. 3 . 1. Perform the PCRc. Prepare reaction system as indicated in Table 18 . 3. Load the samples onto an agarose gel (1.2%), and separate them by electrophoresis. 4. Cut out the~400 bp bands and extract the DNA segments using a Gel Extraction Kit according to the manufacturer's instructions. Table 19 ) is incubated at 98 C for 2 min, followed by 30 cycles of 98 C for 10 s, 55 C for 30 s, and 72 C for 60 s, with a final incubation at 72 C for 5 min. LF2 new (10 μM)

10Â PCR buffer Table 19 Reaction system for amplification of the constant regions 

10Â PCR buffer 5 5 5 High-Fidelity DNA Polymerase 7. Cut out the~1000 bp bands for C H and~400 bp for C κ and C λ . Extract the DNA segments using a Gel Extraction Kit according to the manufacturer's instructions.

8. Perform overlapping PCR to generate the expression cassette (see Table 20 ). The PCR reaction is incubated at 98 C for 2 min, followed by 30 cycles of 98 C for 10 s, 55 C for 30 s, and 72 C for 2 min, with a final incubation at 72 C for 5 min.

9. Digest the PCR products from the last step with EcoRI and XhoI for both the heavy and lambda chains. Digest the kappa chain with KpnI and XhoI (see Table 21 ). Mix gently and spin down for a few seconds. Incubate at 37 C overnight. 10 . Purify the digested PCR segment using a Universal DNA Purification Kit according to the manufacturer's instruction. 11 . Ligate the expression cassette to the pCAGGS vector (see Table 22 ). For both heavy and lambda chains, the vector was 

10Â PCR buffer 5 5 5 High-Fidelity DNA Polymerase pCAGGS-Z23H encodes the heavy chain of the 23rd sorted cell).

18. The generation of vectors for mAb expression is displayed in schematic diagram in Fig. 4 . Fig. 4 Strategy to clone and express Zika E-specific human mAbs. The Ig genes from the sorted cells were determined and cloned into the expression vectors by a reported approach with some modifications [13, 18] . The Ig transcripts in the collected Zika E-interacting memory B cells were first reverse-transcribed into cDNA using the Ig gene-specific primer mix at the single-cell level. Then the variable regions for heavy, kappa, and lambda chains were amplified by nested RT-PCR. The first round PCRs were performed with the forward primer mix specific for the leader region and reverse primers specific for the constant regions of heavy, kappa, and lambda chain, respectively. The second round PCRs were performed with the forward primer mix specific for framework segment FR1 and respective nested reverse primers specific for the heavy, kappa, and lambda constant regions. Then the PCR products were separated by electrophoresis and sent for sequencing. In terms of the correct segments, another round of PCR (PCRc) was performed with the forward primer containing the signal peptide of mouse Ig κ and reverse primer paired with the framework segment FR4. Then the resultant PCR segments were overlapped with the respective constant region to get the full expression cassettes for each chain, which were then ligated to the linearized pCAGGS vector. All expression plasmids were sequenced and aligned with those in PCRb 10. Load the samples onto a precast SDS-PAGE gel (4-10%), and separate the proteins by electrophoresis.

11. Transfer the proteins onto a nitrocellulose membrane.

12. Perform western blotting to assess the expression of each mAb using anti-hIgG/HPR (diluted by 1:1000). As indicated in Fig. 5 

1. EtBr stains DNA by intercalating between the bases of DNA. It will also intercalate into human DNA, so wear gloves to prevent contact with it. A separate space is also recommended for performing all experiments using EtBr-containing materials. Do not pull the comb out too soon, as it causes the wells to collapse. It will take 15-20 min to gel. If the gel cannot be used in 1 h, it is recommended to transfer the gel without comb into a tank containing 1Â TAE buffer.

3. The primers used for the amplification of Ig genes are the same as reported (see Tables 2-8 ) [18] . However, after sequencing, cloning of the expression vectors was designed based on the pCAGGS vector.

4. All primers are stored in small aliquots to avoid repeated freezing and thawing.

5. The blood sample of the convalescent Zika patient was collected 20 days post onset of fever, headache, and dizziness. It is reported that after immunization, antigen-specific B cells with a memory phenotype could be detected in the blood within 1 week [19] . In the presence of antigens, memory B cells undergo affinity maturation, and their B-cell receptors have increased affinities for the antigen. Studies on memory B cells after smallpox vaccination in humans indicated that antigenspecific memory B cells initially declined postimmunization (1 year) but then reached a plateau~tenfold lower than peak and were stably maintained for >50 years after vaccination [20] . Thus, considering the time that needed for affinity maturation, we recommend collecting the blood 1 month-1 year post onset of symptoms to isolate specific mAbs.

6. When layering the sample, do not mix the density gradient medium solution and the diluted blood sample. Keep them in separate layers.

7. The break should be turned off at this step. For other steps using centrifuging, set the break on.

8. If the blood has been collected for >2 h, extend the centrifuging time to 30 min.

9. Usually, 3-4 mL of solutions containing PBMCs will be pipetted out from 10 mL of the blood. 10 . For example, if the cell density is determined to be 1 Â 10 6 / mL at step 13 in Subheading 3.1, the total cell number is 1Â 10 6 /mL Â 15 mL ¼ 1.5 Â 10 7 . Thus, we need to add 1.5 mL freezing medium to resuspend the cells.

11. Freshly prepared PBMCs exert higher efficiencies for Ig gene amplification than frozen cells. Thus, freshly prepared PBMCs are recommended for the isolation of mAbs. In case the following cell sorting cannot be performed immediately, the methods using the freezing cells are provided in Subheading 3.2.

12. Memory B cells express CD19 and CD27, but not CD38. In this study, we focus on the IgG + memory B cells.

13. If fresh PBMCs are used here, please skip to step 8. If using frozen cells, please follow step 2.

14. Before turning the tube right side up, it is recommended to aspirate the liquid left around the tube orifice.

15. It is difficult to decant the entire buffer. Usually,~50 μL of buffer will be left. Thus, we add 50 μL FACS buffer for 10 7 cells or 150 μL for 2 Â 10 7 cells.

16. There are three antibodies conjugated with PE-Cy ® 5. They are anti-human CD3/PE-Cy ® 5, anti-human CD16/PE-Cy ® 5, and anti-human CD235a/PE-Cy ® 5. Any of the three are suitable for preparation of the compensation beads for PE-Cy ® 5.

17. The steps in this section are specific to, but not limited to, a BD FACSAria III cytometer.

18. Here, we use the markers for T cells, NK cells, and platelets for negative selection, to exclude their disturbance.

19. Here, we include FSC-A and FSC-H to exclude cell aggregates.

We are not sure about the proportion of memory B cells that bind to Zika E protein. Thus, it is difficult to set the threshold to distinguish antigen-specific memory B cells from those targeting other antigens in the sample of Zika patient PBMCs. In previous work, we studied the interaction between the receptor-binding domain (RBD) of Middle East respiratory syndrome coronavirus (MERS-CoV) and its receptor CD26, which is widely expressed on lymphocytes, including T cells.

Here, we used the MERS-RBD, which is also tagged at its C-terminus with 6Â His like the Zika E protein, to stain PBMCs from a healthy donor. Then, anti-His/PE was applied to bind to the His tag. Through comparison between the results in Fig. 2B and C, we could determine the threshold to gate the Zika E-specific memory B cells, as indicated in Fig. 2D . 21 . The PCR reactions are performed in 96-well plates.

22. The combined primers are used to amplify Ig genes in the PCRb. However, different primers in a single tube might disturb each other and reduce their annealing efficiency. Thus, in terms of the single cells that yield typical bands for the kappa or lambda chain but not for heavy chain in gel electroporation, we usually repeat PCRb using the separate primers. In addition, MgCl 2 exerts effects on the activities of DNA polymerase (for sing-cell PCR). Varied concentrations of MgCl 2 , ranging from 1.5 to 4.5 mM, can also be applied to amplify the variable region of the heavy chain from single cell, whose light chains' variable regions have been sequenced.

23. From PMBCs isolated from 10 mL whole blood of a convalescent patient, we finally sequenced 15 paired mAbs, which have been published previously [8] .

Qihui Wang and Jinghua Yan 24. Here, we introduce the traditional method to double digest the PCR segments with two restriction enzymes and clone them into the same sites in the linearized pCAGGS vector using T4 ligase. However, other methods (e.g., In-Fusion reaction [21] ) can be used.

25. Here we displayed the results for Z6, Z20, and Z23.

26. The steps in this section are specific to, but not limited to, a Biacore T100 system.

27. In addition to SPR, enzyme-linked immunosorbent assay is another typical method to assess the interaction between mAbs and antigens. However, for Zika E-specific mAbs, we found that some of them displayed relatively low response to the antigens coated on the plate (data not shown here). It is possible that Zika E proteins undergo some conformational changes when adsorbing to the plate, which results in the decreased binding to certain mAbs. In terms of SPR experiments, the mAbs were captured on the chip through interactions with protein A, which binds to the Fc region. Thus, the CDRs of mAbs orient to the buffer flowing over the chip surface. In addition, the Zika E proteins in the buffer of HBS-EP are label-free and more prone to be in its native conformation than those coated on the plate. Thus, we chose SPR assay to detect the interaction between mAbs and antigens. 29. Due to the different binding kinetics between an antigen and its mAb, we set varied dissociation time. For example, mAb Z6 dissociates with Zika E with very low rate; thus we set 600 s for their dissociation. However, for both Z20 and Z23, they are ready to dissociate with the antigen, and 60 s was used for their dissociation (Fig. S2 G, J, and L in the paper [8] ).

30. Due to the different binding kinetics between mAbs and Zika E protein, the data could be fit by either a steady state affinity model or 1:1 (Langmuir) binding model.

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