A class II MHC-targeted vaccine elicits immunity against SARS-CoV-2 and its variants

Significance Vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. Currently available vaccines require cold storage and sophisticated manufacturing capacity, complicating their distribution, especially in less developed countries. We report a protein-based SARS-CoV-2 vaccine that directly and specifically targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (SpikeRBD) fused to a nanobody that recognizes class II major histocompatibility complex antigens (VHHMHCII). Our vaccine elicits robust humoral (high-titer binding and neutralizing antibodies) and cellular immunity against SARS-CoV-2 and its variants in both young and aged mice. VHHMHCII-SpikeRBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy, desirable attributes for logistical reasons.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (Spike RBD ) fused to an alpaca-derived nanobody that recognizes class II major histocompatibility complex antigens (VHH MHCII ). This vaccine elicits robust humoral and cellular immunity against SARS-CoV-2 and its variants. Both young and aged mice immunized with two doses of VHH MHCII -Spike RBD elicit high-titer binding and neutralizing antibodies. Immunization also induces strong cellular immunity, including a robust CD8 T cell response. VHH MHCII -Spike RBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy. nanobody j COVID-19 j vaccine S evere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has caused a global pandemic, infecting over 230 million people, and leading to millions of deaths (1). Rapid distribution of effective vaccines on a global scale is the most effective means of mitigating the political, social, and economic destabilization caused by the SARS-CoV-2 pandemic.
The SARS-CoV-2 spike (S) protein is a trimeric transmembrane protein that binds to the cell surface receptor angiotensin-converting enzyme 2 (ACE2) via its receptorbinding domain (RBD) and mediates fusion with host membranes (2). SARS-CoV-2 S is the primary target for neutralizing antibodies and elicits both CD4 and CD8 T cell responses during infection (3)(4)(5)(6)(7). Most vaccines in current use or in development target S, or fragments of S, as the primary antigen (8). Because several variants of concern have emerged, many of which contain mutations in S that partially resist neutralization by vaccine-elicited and COVID-19-elicited antibodies, vaccines that offer protection against new variants are necessary (9)(10)(11).
Leading vaccine candidates use an array of diverse vaccine platforms. These include inactivated virions, DNA-based vaccines, recombinant subunit preparations, lipid-encapsulated mRNA formulations, as well as live-attenuated, replicationincompetent viral vectored, and replication-competent viral vectored vaccines (8). None of them directly and specifically target antigen-presenting cells (APCs). We hypothesized that targeted delivery of antigen to professional class II MHC + APCs would improve access to the processing and presentation pathways that generate CD4 and CD8 T cell responses, in addition to provoking a robust antibody response. Our earlier efforts to generate an anti-HPV16 CD8 T cell response relied on fusions of an anti-CD11b nanobody to the immunodominant epitope of the HPV16 E7 protein as a vaccine. Its success in eradicating even established tumors inspired us to pursue a similar effort to deliver the RBD of the SARS-CoV2 S protein as a fusion with a nanobody that targets APCs (12). Most vaccines in current use require specialized storage conditions Significance Vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. Currently available vaccines require cold storage and sophisticated manufacturing capacity, complicating their distribution, especially in less developed countries. We report a protein-based SARS-CoV-2 vaccine that directly and specifically targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (Spike RBD ) fused to a nanobody that recognizes class II major histocompatibility complex antigens (VHH MHCII ). Our vaccine elicits robust humoral (high-titer binding and neutralizing antibodies) and cellular immunity against SARS-CoV-2 and its variants in both young and aged mice. VHH MHCII -Spike RBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy, desirable attributes for logistical reasons. (13)(14)(15). The development of vaccines with enhanced stability to allow storage at ambient temperature and rapidly adjustable to emerging variants of the virus therefore remains a priority. Moreover, vaccines that can be produced rapidly in a scalable manufacturing process would improve access.
Here we report the development of a recombinant protein vaccine that consists of the SARS-CoV-2 Spike RBD (Spike RBD ) fused to an alpaca-derived nanobody that targets class II major histocompatibility (MHC II) complex antigens (VHH MHCII -Spike RBD ). This vaccine delivers the antigen directly to class II MHC + APCs. Immunization of both young and aged mice with two doses of VHH MHCII -Spike RBD resulted in robust binding and neutralizing antibody responses against SARS-CoV-2 and emerging variants. Immunization also induced prominent CD8 T cell responses against conserved Spike RBDderived epitopes. VHH MHCII -Spike RBD can be produced in high yield in mammalian cells and tolerates both storage at room temperature for at least 7 d and lyophilization without loss of efficacy.

Results
VHH MHCII -Spike RBD Elicits High-Titer Anti-Spike RBD and Neutralizing Antibodies in Mice. We have characterized a single-domain antibody fragment that binds class II MHC antigens (VHH MHCII ) with nanomolar affinity. Immunization of mice with an influenza A virus (IAV) HA2 antigen, conjugated to VHH MHCII , protected mice against a lethal IAV challenge (16). To apply this vaccine platform to SARS-CoV-2, we generated a recombinant protein that consists of a fusion between VHH MHCII and the SARS-CoV-2 RBD (Fig. 1A). VHH MHCII -Spike RBD was expressed in Expi293 cells and purified by means of its C-terminal His 6 -tag followed by size-exclusion chromatography. The identity of the product was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) (Fig. 1B). A 200-mL culture of Expi293 cell supernatant yielded ∼20 mg of recombinant protein.
To examine the immunogenicity of VHH MHCII -Spike RBD , we used a two-dose immunization regimen. Preimmune serum was collected at À17 d and C57BL/6J mice (H-2 b haplotype) were primed (day À14) intraperitoneally (i.p.) with adjuvanted (poly dIdC and anti-CD40 monoclonal antibody) VHH MHCII -Spike RBD (20 μg), with an equimolar amount of adjuvanted Spike RBD (13.5 μg), or with adjuvant alone. Mice were boosted with each corresponding preparation 14 d postprime (day 0) ( Fig. 1C and SI Appendix, Fig. S1A). Serum was collected from all animals 14 d postboost (day 14). IgG titers were determined by ELISA against recombinant SARS-CoV-2 Spike RBD (Wuhan Hu-1 strain) ( Fig. 1D and SI Appendix, Fig. S1B). Immunization with two doses of VHH MHCII -Spike RBD elicited high levels of anti-Spike RBD antibodies in all animals, reaching mean endpoint titers in excess of 1/23,600,000, an approximate 34,000-fold increase over mice immunized with two doses of Spike RBD . Analysis of immunoglobulin subclasses showed evidence of class switching in mice immunized with VHH MHCII -Spike RBD , judged by the levels of IgA, IgG1, and IgG2b detected at day 14 ( Fig. 1E and SI Appendix, Fig. S1C). Mean endpoint titers were higher in mice immunized with VHH MHCII -Spike RBD than in mice immunized with Spike RBD, with mean endpoint titers reaching >1/7,300 (IgM; fourfold increase over Spike RBD ), 1/8,900 (IgA; 66-fold increase over Spike RBD ), 1/3,160,000 (IgG1; 6,870-fold increase over Spike RBD ), and ∼1/90,000 (IgG2b; 87-fold increase over Spike RBD ). Immunization with VHH MHCII -Spike RBD induced high levels of IgA, which suggests improved mucosal protection in immunized animals. Immunization of mice with two doses of VHH MHCII -Spike RBD thus induces a robust humoral immune response, significantly higher than that induced by the Spike RBD alone.
Since VHH MHCII -Spike RBD binds class II MHC antigens encoded by the I-A locus on target APCs, we immunized a different inbred mouse strain (BALB/cJ; H-2 d haplotype) to confirm efficacy across different MHC haplotypes. Immunization with two doses of VHH MHCII -Spike RBD led to comparable levels of total IgG in C57BL/6J (H-2 b ) and BALB/cJ (H-2 d ) mice (SI Appendix, Fig. S2 A and B). Immunoglobulin class switching was evident in both mouse strains, and mean Spike RBD -specific IgM, IgA, IgG1, and IgG2b titers were comparable (SI Appendix, Fig. S2C).
We next measured total and subclass immunoglobulin levels at days 7, 14, and 21 in mice immunized with one or two doses of adjuvanted VHH MHCII -Spike RBD or control (SI Appendix, Fig. S3A). Mice immunized with two doses of VHH MHCII -Spike RBD quickly achieved peak IgG titers on day 7, with levels persisting until at least day 21 (SI Appendix, Fig. S3A), while animals immunized with a single dose produced lower levels of total IgG and other subclass immunoglobulins (SI Appendix, Fig. S3B).
Because SARS-CoV-2 variants with mutations in S that enable partial immune escape have emerged, we next examined whether serum from mice immunized with VHH MHCII -Spike RBD recognized recombinant Spike RBD with the K417T, E484K, and N501Y mutations. These mutations are found individually in many S variants and in combination in the P.1 (Gamma or Brazil) variant and partially in the B.1.351 (Beta or South Africa) variant which has K417N instead of K417T (17). Immunization with two doses of adjuvanted wild-type Spike RBD induced antibodies (day 14) with a low capacity to bind the triple-mutant Spike RBD , while immunization with two doses of adjuvanted VHH MHCII -Spike RBD elicited antibodies still capable of recognizing the mutant Spike RBD to high titers (mean endpoint titer of 1/178,000) ( Fig. 1F and SI Appendix, Fig.  S1D). Two doses of VHH MHCII -Spike RBD were required to maintain high titers of IgG against the mutant Spike RBD (SI Appendix, Fig. S3C).
A Single Dose of VHH MHCII -Spike RBD Elicits Strong Cellular Immunity.
Because cellular immunity, particularly that exerted by T cells, is important for protection against and memory of SARS-CoV-2 infection, we next examined the T cell response in animals immunized with a single dose of adjuvanted Spike RBD or VHH MHCII -Spike RBD ( Fig. 2A) (5). Splenocytes harvested 7 d postimmunization were stimulated with a library of Spike RBD 15-mer peptides with 11-residue overlap and evaluated by an ELISpot assay ( Fig. 2B and Table 1). While mouse class I MHC products prefer peptides in the eight to nine residue range, we chose to use synthetic 15-mers, as they are known to contain shorter peptides that serve as class I MHC ligands or can be processed to yield proper class I MHC ligands (22,23). The use of longer peptides would enable identification of both class I and class II restricted epitopes. We observed robust production of IFNγ in splenocytes from mice immunized with VHH MHCII -Spike RBD and, to a lesser extent, from mice immunized with Spike RBD (Fig. 2C). IFNγ production was highest when splenocytes were stimulated with peptides 42, 47, 48, 49, and 50, indicative of at least two unique stimulatory regions. Peptide 42 contains residue E484, which is frequently mutated to K or Q in SARS-CoV-2 variants, and S494, which is sometimes mutated to P in the B.1.1.7 lineage. Peptides 47 to 50, which correspond to spike residues 503 to 529, are conserved among all Centers for Disease Control-designated variants (17). Indeed, coculture of splenocytes from VHH MHCII -Spike RBD -immunized mice with Spike RBD peptides 42 and 47 to 50 resulted in secretion not only of high levels of IFNγ but also high levels of the proinflammatory cytokines IL-6 and TNFα. These levels are higher than those induced by immunization with Spike RBD and indicate proliferation of memory CD4 and CD8 T cells (Fig. 2D) (24,25). We did not observe high levels of IL-2 secretion. Cellular immunity elicited by immunization with VHH MHCII -Spike RBD is strong and may persist upon infection with circulating variants.
To distinguish between CD4 and CD8 T cells as the dominant cytokine-producing cell type, we performed intracellular cytokine staining. A significant proportion of the inflammatory cytokines are derived from the CD8 T cell compartment (Fig. 2 E and F and SI Appendix, Fig. S5). Recombinant VHH MHCIIantigen fusion proteins are therefore subject to crosspresentation and can induce an efficacious CD8 T cell response against Spike RBD . The presence of a strong humoral immune response that includes class switching likewise implies the contribution by CD4 T cells ( Fig. 2F and SI Appendix, Fig. S5).
VHH MHCII -Spike RBD Elicits a Strong Humoral Response Regardless of the Route of Administration, Storage Temperature, Formulation, and Age of the Mice. We next compared different routes of administration, including i.p., intramuscular (i.m.), and intranasal (i.n.) routes, to elicit an immune response. Mice were primed with adjuvant only (i.p.) or received adjuvanted VHH MHCII -Spike RBD (i.p., i.m., or i.n.) and boosted 14 d later with a dose of the corresponding vaccine and route of administration ( Fig. 3A and SI Appendix, Fig. S6A). Mean total Spike RBD -specific IgG in the blood, as well as mean IgM, IgG1, and IgG2b levels, were comparable across the three different routes of administration, although i.p. and i.m. administration led to an increase in mean IgA levels ( Fig. 3B and SI Appendix, Fig. S6A). While i.n. administration did not induce significant levels of IgA in the blood, it remains to be determined whether these animals produced significant levels of IgA in the respiratory tract.
Because the SARS-CoV-2 vaccines in use require specific formulations and cold storage conditions, we determined whether recombinant VHH MHCII -Spike RBD protein would retain its efficacy at inducing an antibody response upon storage at ambient temperature or lyophilization. We immunized mice with two doses of adjuvant only, adjuvanted VHH MHCII -Spike RBD stored at À20˚C, 4˚C, or 25˚C for 1 wk, or lyophilized and resuspended VHH MHCII -Spike RBD (Fig. 3A and SI Appendix,  Fig. S6B). Mean total serum IgG, IgA, IgG1, IgG2b, and IgM levels between the different conditions were comparable, indicating that neither temperature of storage nor storage in liquid form is required for the vaccine to retain its efficacy (Fig. 3C).
Because the ability to produce a robust and durable immune response can decrease with age, we also determined whether immunization of aged mice with VHH MHCII -Spike RBD would elicit a strong immune response (26). We immunized 8-to 12wk-old mice with two doses of adjuvant only or adjuvanted VHH MHCII -Spike RBD and 72-wk-old [equivalent to humans aged 56 y to 69 y (27)] with two doses of VHH MHCII -Spike RBD (Fig. 3A and SI Appendix, Fig. S6C). While switching to some immunoglobulin subclasses (IgA and IgG1) decreased in aged mice, consistent with diminished T cell help, mean total IgG levels in the blood were unchanged when compared to 8-to 12-wk-old mice (Fig. 3D). This may be due, in part, to high levels of IgG2b in aged mice and slightly higher levels of IgM (SI Appendix, Fig. S6C).
Humanized VHH hMHCII -Spike RBD Elicits Both Humoral and Cellular Immunity in a Transgenic Mouse Model. Because the anti-MHC class II nanobody used for these experiments recognizes murine antigens independent of haplotype, we also generated a version of the vaccine that could be applied in a clinical setting. We used HLA-DR4-IE-transgenic C57BL/6 IAb null mice, which lack wildtype murine MHC class II products and instead express transgenic hybrid MHC class II molecules composed of the peptide-binding portion of human HLA-DR4 and the membrane-proximal domains of mouse I-E (DR4-IE). We used a previously characterized nanobody (VHH hMHCII ) that recognizes nearly all allelic variants of human class II MHC molecules (HLA-DR specific, with the exception of HLA-DR-03*01) (VHH hMHCII -Spike RBD ) (28). Flow cytometry on splenocytes from HLA-DR4-IE-transgenic C57BL/6 IAb null mice confirmed that VHH hMHCII recognizes these hybrid DR4-IE molecules (Fig. 4A). We then generated a genetic fusion between VHH hMHCII and either the Wuhan Hu-1 or B.1.1.7+E484K SARS-CoV-2 Spike RBD , as an illustration of rapid and straightforward vaccine adjustment in anticipation of emerging mutations in the RBD. Both constructs expressed well in mammalian cells. A 200-mL culture of Expi293 cell supernatant yielded 15 and 12.5 mg of VHH hMHCII -Spike RBD (Wuhan Hu-1) and VHH hMHCII -Spike RBD (B.1.1.7+E484K), respectively (Fig. 4B).

IMMUNOLOGY AND INFLAMMATION
Pishesha et al.
A class II MHC-targeted vaccine elicits immunity against SARS-CoV-2 and its variants protein-based SARS-CoV-2 vaccine that specifically targets APCs. This preparation is easy to both produce and store. Immunization of mice with two doses of VHH MHCII -Spike RBD elicited high-titer binding and neutralizing antibodies against SARS-CoV-2 and several of its circulating variants, including B.1.1.7, P.1, and B.1.351. Strong immune responses were evoked in both young and aged mice, largely independent of the route of administration of the vaccine. A single dose was sufficient to induce cellular immunity to conserved regions of the RBD, as evident from cytokine production by CD8 T cells. The vaccine maintained its potency regardless of storage conditions, including ambient temperature and lyophilization. Humoral and cellular immune responses were both more consistent and potent in mice immunized with VHH MHCII -Spike RBD compared to immunization with the Spike RBD . A version of this vaccine suitable for clinical translation elicits robust immunity in a humanized mouse model. This approach would therefore complement ongoing active and passive immunization strategies. Most currently used vaccines are difficult to manufacture and/or require specialized storage conditions. Vaccines with enhanced stability that tolerate lyophilization, such as the proteinbased vaccine reported here or a different, nanoparticle-based vaccine, allow stockpiling at ambient temperature (29). This is an important attribute for distribution in countries where access to cold storage and/or effective transportation is a challenge. A unique feature of this vaccine approach is its ability to deliver antigen directly to MHC class II + cells. Humoral and cellular immunity directed against the Spike RBD develops within a week of the first dose, unlike mRNA-based vaccines and nontargeting protein-based vaccines, which usually take longer (30).
In conclusion, we report a purely protein-based vaccine preparation that is unique, in that it directly targets professional APCs. The robust immunity afforded by this vaccine, combined with its ease of manufacture and stability, indicates the potential for a rapidly adjustable vaccine. Vaccination of mice with the VHH MHCII -Spike RBD adduct elicits CD4 and CD8 T cell as well as B cell responses, resulting in the formation of antibodies that neutralize not only recombinant VSV expressing the SARS-CoV-2 spike but also a SARS-CoV2 isolate. Further studies in small animal models and nonhuman primates are needed to establish whether immunity elicited by VHH MHCII -Spike RBD protects against a SARS-CoV-2 challenge and to establish breadth of coverage. We suggest that this approach merits consideration for use in a clinical setting as a complement to ongoing active and passive immunization strategies.

Design, Expression, and Purification of Recombinant VHHs and VHH Fusions.
Sequences encoding the Spike RBD and VHH MHCII -Spike RBD were synthesized (Integrated DNA Technologies) as double-stranded DNA. Inserts were assembled in the pVRC vector (a gift from Stephen Harrison, Harvard Medical School). Constructs were transfected into Expi293F cells (Thermo Fischer Scientific) using Polyethyleneimine "Max" (Polysciences). Cell cultures were maintained in Expi293 Media (Thermo Fischer Scientific) at 37°C for 4 d following transfection. Proteins were harvested by centrifugation at 5,000 × g for 30 min at 4°C, followed by affinity chromatography with HisPur Ni-NTA Resin (Thermo Fischer Scientific) and size exclusion chromatography with a Hi-Load 16/600 S75 column (Cytivia). Protein samples (2 μg each) were prepared by   boiling for 5 min in sample buffer containing 1% (wt/vol) SDS and 1% (vol/vol) BME. Samples were analyzed on 10% or 15% SDS/PAGE. Gels were stained with Instant Blue (Abcam) and destained with ddH 2 O.
Mouse Models. All animals were housed in the animal facility of Boston Children's Hospital (BCH) and were maintained according to protocols approved by the BCH Committee on Animal Care. C57BL/6J (CD45.2+) and BALB/c mice were either purchased from the Jackson Laboratory or bred in house. DR4-IE transgenic mice were purchased from Taconic. Only female mice aged 8 to 12 wk were used in this study unless indicated otherwise.
ELISA. Serum samples were collected on the indicated days and stored in BD Vacutainers; 96-well plates were coated with 2 μg/mL of either recombinant Spike RBD or Spike RBD (K417T, E484K, N501Y) proteins in phosphate-buffered  , and IgG2b levels were measured by ELISA against recombinant Spike RBD . ELISA data were summarized as endpoint titers and presented as means 6 SEM. (E) Total IgG levels were measured from immunized mice by ELISA against recombinant Spike RBD containing the mutations K417T, E484K, and N501Y. ELISA data were summarized as endpoint titers and presented as means 6 SEM. (F) ELISpot assay measuring IFNγ-secreting cells in splenocytes of DR4-IE-transgenic C57BL/6 IAb null mice (n = 3 per condition) immunized with one dose of adjuvant only or adjuvanted VHH hMHCII -Spike RBD . Splenocytes were harvested at day 7 post immunization. (G) Flow cytometry analyses of splenocytes after incubation for 6 h in the presence of pooled peptides (42 and 47 to 50) and monensin. All data are presented as means 6 SEM; *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t test with Holm-Sidak adjustment. saline (PBS) overnight at 4°C and incubated in blocking buffer (0.05% Tween20 + 2% bovine serum albumin in PBS). Plates were incubated with diluted serum samples for 2 h at room temperature. Plates were then washed four times with PBS and incubated with goat anti-mouse IgG-HRP, anti-mouse IgM-HRP, anti-mouse IgG1-HRP, or IgG2b-HRP (SouthernBiotech) at 1:10,000 or with IgA-HRP at 1:2,000 in blocking buffer for 1 h. Plates were developed with 3,3',5,5'-tetramethylbenzidine liquid substrate reagent (Sigma). The reaction was stopped with 1 N HCl, and absorbance was read at 450 nm.
Recombinant VSV Neutralization Assays. Serum samples were heat inactivated at 56°C for 30 min. Neutralization assays were performed similarly to what has been described (19,20). Briefly, threefold serial dilutions of sera, starting with a 1:20 dilution, were performed in 384-well plates and were incubated with 10 6 plaque-forming units (pfu) of VSV-SARS-CoV-2 expressing eGFP and the Spike of Wuhan Hu-1+D614G, B.1.1.7, P.1, or B.1.351 for 1 h at 37°C. Vero E6 cells then were added to the human serum-virus complexes in 384-well plates at 3 × 10 3 cells per well and incubated at 37°C for 16 h. Cells were fixed at room temperature in 4% formaldehyde and then rinsed with PBS. Cells were stained at room temperature with NucRed Live 647 (Invitrogen) for 30 min. Images were acquired using an InCell 6500 confocal imager (Cytiva) to visualize nuclei and infected cells (4× objective, 1 field per well). Images were segmented using InCarta (Cytiva). Infected cells were identified by comparing them to the uninfected threshold in Spotfire (Tibco). Cells were gated based on nuclear parameters. Additional VSV neutralization assays were performed similarly to what has been described (19). Briefly, serial dilutions of serum samples were incubated with ∼10 2 pfu of VSV-SARS-CoV-2 for 1 h at 37°C. Antibody-virus complexes were then added to Vero CCL-81 cells in black 96-well plates for 7.5 h at 37°C. Cells were then fixed in 2% (vol/vol) formaldehyde (Millipore Sigma) containing 10 mg/mL Hoechst 33342 nuclear stain (Invitrogen) for 45 min at room temperature. Following incubation, nuclear stain and fixative were removed and replaced with PBS. Images were acquired with the InCell 2000 Analyzer (GE Healthcare) automated microscope in both the DAPI and fluorescein isothiocyanate (FITC) channels to visualize nuclei and infected cells (i.e., eGFPpositive cells), respectively (4× objective, four nonoverlapping fields per well, covering the entire well). Images were analyzed using the InCell Investigator Developer Toolbox v1.9 (GE Healthcare). Nuclei and GFP-positive cells were identified and segmented from their respective images using the Object Segmentation function, which uses a variation of the top-hat approach for initial segmentation followed by a binarization operation to define the target sets. Target linking was then performed to determine the total number of GFP-positive nuclei per well. Data were processed using Prism software (GraphPad Prism 9.0).