Broadly neutralizing antibodies to SARS-related viruses can be readily induced in rhesus macaques
Summary (5 min read)
Introduction
- 3IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA 92037, USA 4Consortium for HIV/AIDS Vaccine Development , The Scripps Research Institute, La Jolla, CA 92037, USA.
- 13Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037,.
MAIN
- The rapid development of multiple vaccines to counter the COVID-19 pandemic caused by SARSCoV-2 has been a triumph for science and medicine.
- ID50 neutralization titers showed a similar pattern where both immunization groups developed specific neutralizing antibody responses by week 4, which were enhanced by week 12, post-boost (figs. S1 and S2).
- The elicitation of potent cross-neutralizing responses in rhesus macaques by S-protein immunization is in stark contrast to human SARS-CoV-2 natural infection, which typically results in autologous nAb responses where cross-neutralizing activity against SARS-CoV-1 is rare (Fig. 1E, fig.
- For the human nAbs, the germline-encoded CDRH1 and CDRH2 both contribute substantially to RBD recognition (18, 22), the macaque nAbs predominantly rely on CDRH2 and less on CDRH1 (Fig 6, D, H and I, fig.
Discussion
- Natural infection and current vaccines for SARS-CoV-2 in humans typically induce very limited cross-neutralizing responses.
- One recent study (49) investigated use of different adjuvants with stabilized recombinant SARS-CoV-2 S proteins presented on nanoparticles in macaques and noted nAb responses of varying potency and neutralization breadth.
- One adjuvant produced only a 4.5-fold reduction in neutralizing titer against the B.1.351 variant, whereas another produced a 16-fold reduction.
- Whereas the immunodominant human nAb RBSA/Class 1 response to S-protein largely utilizes the VH3-53 germline gene segment, the immunodominant macaque response leads to a strong VH3-73 response that involves a different epitope and different approach angle to interact with the RBD.
- Differences between human and rhesus macaque antibody repertoires can potentially have important consequences for vaccine model studies and this observation goes beyond SARS-CoV-2.
REFERENCES AND NOTES
- T. F. Rogers et al., Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model.
- Priming a broadly neutralizing antibody response to HIV-1 using a germline-targeting immunogen.
- A. J. Murphy et al., Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice.
FIGURES AND LEGENDS
- Serum and peripheral blood mononuclear cells were collected every two weeks for immune response analysis.
- EC50 binding responses for pre-bleed (PB), postprime (wk4 and wk10) and post-boost (wk14) sample time points are shown.
- The authors structures show germline Y58 would clash, whereas E/H is able to interact with the RBD.
- (J) The Fab/RBD complex structures of the two antibodies are superimposed on the RBD.
Human samples
- Convalescent COVID-19 and SARS-CoV-2 spike vaccinated human sera Sera from convalescent COVID-19 donors (75) and from spike-mRNA vaccinated humans were provided through the “Collection of Biospecimens from Persons Under Investigation for 2019- Novel Coronavirus Infection to Understand Viral Shedding and Immune Response Study” UCSD 33 IRB# 200236.
- Protocol was approved by the UCSD Human Research Protection Program.
- Convalescent serum samples were collected based on COVID-19 diagnosis regardless of gender, race, ethnicity, disease severity, or other medical conditions.
- All human donors were assessed for medical decision-making capacity using a standardized, approved assessment, and voluntarily gave informed consent prior to being enrolled in the study.
Ethics Statement
- The mice and rhesus macaque animal studies were approved and carried out in accordance with protocols provided to the Institutional Animal Care and Use Committee respectively at The Scripps Research Institute (TSRI; La Jolla, CA) under approval number 19-0020 and at Alphagenesis Institutional Animal Care and Use Committee under approval number AUP 19-10.
- The animals at both facilities were kept, immunized, and bled in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and in adherence to the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996).
Immunization and Sampling
- 8 outbred, healthy Indian-origin rhesus macaques (Macaca mulatta) were housed at Alphagenesis, Yemassee, SC.
- Animals were primed (wk0) and boosted (wk10) by subcutaneously (SubQ) injecting 100µg of SARS-CoV-2 S-protein immunogen along with 375µg of a saponin adjuvant (SMNP) (Silva M. et. al., submitted) per animal per immunization.
- All immunizations were administered SubQ divided between the left and right mid-thighs.
- Blood was collected at various time points into CPT tubes for PBMC and plasma isolation.
- Animals were immunized twice subcutaneously by infection immunogen-adjuvant vaccine formulation at prime (wk0) and boost (wk3) and the serum samples were collected at various time points for the analysis of antibody responses.
Plasmid construction
- To generate the expression plasmids for soluble S ectodomain proteins from SARS-CoV-1 (residues 1-1190; GenBank: AAP13567) and SARS-CoV-2 (residues 1-1208; GenBank: MN908947), spike genes were synthesized by GeneArt (Life Technologies).
- The ectodomains of SARS-CoV-1 and SARS-CoV-2 were constructed by PCR amplification and Gibson assembly (NEB, E2621L) cloning into the vector phCMV3 (Genlantis, USA).
- To stabilize soluble S proteins in the trimeric prefusion state, the authors made the following changes: double proline substitutions (2P) in the S2 subunit, replacement of the furin cleavage site by “GSAS” in SARS-CoV-2 (residues 682–685), and SARS-CoV-1 (residues 664–667), and incorporation of a C-terminal T4 fibritin trimerization motif (76, 77).
- To aid purification and biotinylation, the HRV-3C protease cleavage site, 6x HisTag, and AviTag spaced by GS-linkers were added to the C-terminus.
- The original secretion signal or the Tissue Plasminogen Activator (TPA) leader sequence were cloned in frame with the gene fragments.
Transient transfection
- To express antibodies, the corresponding HC and LC plasmids were transiently transfected into the Expi293 cell (Life Technologies) at 3 x 106 cells/mL with FectoPRO PolyPlus transfection reagent (Polyplus Cat # 116-040).
- To boost the protein yield, the authors fed cells with 300 mM sodium valproic acid solution and glucose solution 1 day post transfection.
- To purify monoclonal antibodies, the supernatants were harvested 4 days post transfection.
- After resting at room temperature for 30 mins, the mixture was poured into 1L HEK293F cells.
Protein purification
- After incubation, the Protein A Sepharose was loaded into Econo-Pac columns (BioRad #7321010).
- Three bed volumes of wash buffer (25 mM Imidazole, pH 7.4) 36 were used to wash away the nonspecific binding proteins before the elution step.
- The purified proteins were buffer exchanged into PBS using Amicon tubes.
- Protein and beads were passed through a 0.22 µm spin filter.
- Endosafe nexgen-MCS (Charles River) and Endosafe LAL cartridges (Charles River #PTS201F) were used to measure endotoxin levels.
Cell lines
- To generate HeLa-hACE2 cells, the human ACE2 lentivirus was transduced into HeLa cells.
- The stable cell line post transduction was selected and scaled up for use in the neutralization assay.
- The authors used 293FreeStyle expression medium (Life Technologies) to culture the cell line in the shaker at 150 rpm, 37°C with 8% CO2.
- The authors used Expi293 Expression Medium (Life Technologies) in the same condition as the HEK293F cell line.
ELISA
- 96-well half-area plates (Corning cat. #3690, Thermo Fisher Scientific) were coated overnight at 4°C with 2 μg/mL of mouse anti-His-tag antibody (Invitrogen cat.
- DEN3 was used as a negative control for all 3 spike proteins.
- After washes, a secondary antibody conjugated with alkaline phosphatase (AffiniPure goat anti-human IgG Fc fragment specific, Jackson ImmunoResearch Laboratories cat. #109-055-008) diluted 1:1000 in 1% BSA/PBST, was added to each well.
- The absorbance was measured after 10 and 20 minutes and was recorded at an optical density of 405 nm (OD405) using a VersaMax microplate reader (Molecular Devices), where data were collected using SoftMax version 5.4 (Molecular Devices).
Pseudovirus production
- To generate the pseudoviruses, the MLV-gag/pol and MLV-CMV-Luciferase plasmids were cotransfected with full-length or variant SARS-CoV-1 or SARS-CoV-2 plasmid into HEK293T cells by using Lipofectamine 2000 (Thermo Fisher Scientific, 11668019).
- To evaluate the neutralization efficiency, the supernatants were removed from the well 48 h post infection.
- All of the sera and mAbs were tested in duplicate for each experiment.
- 39 For neutralization of anti-RBD antibody depleted macaque sera, the immune sera were depleted by two rounds of sequential incubation with SARS-CoV-2 RBD-conjugated with magnetic beads.
Bio-layer interferometry (BLI)
- Binding of NHP mAbs to SARS-CoV-1, SARS-CoV-2 spike proteins and truncated proteins was analyzed on an Octet RED384 system using a Protein A biosensor (18-5010, Sartorius) to capture the mAbs. 10 μg/mL of each NHP mAbs were loaded on the hydrated biosensor for 60s.
- Biosensors were subsequently moved into blank buffer (1x PBS + 0.1% Tween 20) for 60s to remove unbound protein and provide a baseline.
- The biosensors were moved to immerse in the protein solutions with 200 nM spike/truncated proteins for 120s to acquire an association signal.
- To monitor disassociation, the biosensors were moved into blank buffer for 240s.
In-tandem epitope binning by BLI
- In-tandem epitope binning was carried out using the Octet RED384 in order to distinguish binding epitopes of mAbs from each other or from human mAbs or from hACE2-Fc. 100 nM of His-tagged SARS-CoV2-RBD protein antigens were captured using Anti-HIS (HIS2) biosensors (18-5114, Sartorius).
- The biosensor was loaded with antigen for 5 min and then moved into the mAbs at saturating concentration of 100 µg/mL for 10 min.
Competition BLI
- To inspect the binding epitope of the NHP sera compared with known human SARS-CoV-2 mAbs or hACE2-FC, the authors did in-tandem epitope binning experiments using the Octet RED384 system.
- The biosensor was loaded with antigen for 5 min and then moved into the saturating mAbs at a concentration of 100 µg/mL for 10 min.
- The biosensors were then moved into 1:50 diluted NHP sera for 5 min to measure binding in the presence of saturating antibodies.
- The percent (%) inhibition in binding is calculated with the formula: [Percent (%) binding inhibition = 1- (serum binding response in presence of the competitor antibody / binding response of the corresponding control serum antibodies without the competitor antibody)].
Random biotinylation of proteins
- The spike proteins and truncated proteins were randomly biotinylated using an EZ-Link NHS-PEG Solid-Phase Biotinylation Kit (Thermo Scientific #21440).
- The reagent in each tube were dissolved 41 with 10 μL DMSO into stock solution.
- The spike protein or truncated proteins were concentrated to 7- 9 mg/mL using Amicon tubes in PBS before biotinylation.
- The biotinylated proteins were evaluated by BioLayer Interferometry.
- BirA biotinylation of proteins for B cell sorting.
Isolation of monoclonal antibodies (mAbs)
- The sorting strategy of antigen-specific memory B cells was described in previous papers (78- 80).
- The BirA biotinylated SARS-CoV-2 spike was coupled to streptavidin-AF488 (Thermo Fisher S32354), streptavidin-BV421 (BD Biosciences 563259) separately.
- 15 min later, the stained cells were washed with cold 10 mL FACS buffer and resuspended in 500 μL FACS buffer for each 10 million cells.
- Purified DNA fragments of heavy and light chain variable regions were subsequently cloned into expression vectors encoding human IgG1, and Ig kappa/lambda constant domains, respectively, using Gibson assembly (NEB, E2621L) according to the manufacturer’s instructions.
Fab production
- The truncated heavy chains were co-transfected with the corresponding light chains in 293Expi cells to produce the Fab.
- RBD protein that was secreted into the supernatant was then concentrated using a 10 kDa MW cutoff Centramate cassette (Pall Corporation).
- Crystals were grown for 7 days and then flash cooled in liquid nitrogen.
- Diffraction data were collected at cryogenic temperature (100 K) at the Stanford Synchrotron Radiation Lightsource (SSRL) on the Scripps/Stanford beamline 12-1 with a wavelength of 0.9793 Å, and processed with HKL2000 (86).
Phylogenetic analysis
- Heavy chain sequences of neutralizing and non-neutralizing antibodies collected from animals K398 and K288 were processed using DiversityAnalyzer tool (92).
- (B) Summary table showing peak of the BLI binding responses for each antibody with given monomeric RBD proteins.
- Sequence similarity of macaque IGHV3-73 germline with the closest human IGHV germline genes.
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Q1. What have the authors contributed in "Broadly neutralizing antibodies to sars-related viruses can be readily induced in rhesus macaques" ?
Here, the authors show that immunization of macaques with SARS-CoV-2 spike ( S ) protein generates potent receptor binding domain crossneutralizing antibody ( nAb ) responses to both SARS-CoV-2 and SARS-CoV-1, in contrast to human infection or vaccination where responses are typically SARS-CoV-2-specific. CC-BY-NC-ND 4. 0 International license available under a was not certified by peer review ) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Furthermore, the macaque nAbs are equally effective against SARS-CoV-2 variants of concern. Human antibodies generally target epitopes strongly overlapping the ACE2 receptor binding site ( RBS ), whereas the macaque antibodies recognize a relatively conserved region proximal to the RBS that represents another potential pan-SARS-related virus site rarely targeted by human antibodies. B cell repertoire differences between the two primates appear to significantly influence the vaccine response and suggest care in the use of rhesus macaques in evaluation of vaccines to SARS-related viruses intended for human use. Broadly neutralizing antibodies to an unappreciated site of conservation in the RBD in SARSrelated viruses can be readily induced in rhesus macaques because of distinct properties of the naïve macaque B cell repertoire that suggest prudence in the use of the macaque model in SARS vaccine evaluation and design.