Targeting SARS-CoV-2 receptor-binding domain to cells expressing CD40 improves protection to infection in convalescent macaques

Achieving sufficient worldwide vaccination coverage against SARS-CoV-2 will require additional approaches to currently approved viral vector and mRNA vaccines. Subunit vaccines may have distinct advantages when immunizing vulnerable individuals, children and pregnant women. Here, we present a new generation of subunit vaccines targeting viral antigens to CD40-expressing antigen-presenting cells. We demonstrate that targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to CD40 (αCD40.RBD) induces significant levels of specific T and B cells, with long-term memory phenotypes, in a humanized mouse model. Additionally, we demonstrate that a single dose of the αCD40.RBD vaccine, injected without adjuvant, is sufficient to boost a rapid increase in neutralizing antibodies in convalescent non-human primates (NHPs) exposed six months previously to SARS-CoV-2. Vaccine-elicited antibodies cross-neutralize different SARS-CoV-2 variants, including D614G, B1.1.7 and to a lesser extent B1.351. Such vaccination significantly improves protection against a new high-dose virulent challenge versus that in non-vaccinated convalescent animals.


Supplementary Figure 4. Antigen-specific T-cell responses in NHPs. (a-b) Impact of vaccination (a) and
SARS-CoV-2 exposure (b) on the number of IFNγ-secreting cells (n = 6). These cells were analyzed by ELISPOT after ex vivo stimulation with SARS-CoV-2 RBD or N overlapping peptide pools and plotted as spot-forming cells (SFC) per 1.0x10 6 PBMCs. the timepoints in each experimental group were compared using the Wilcoxon signed rank test (* two-tailed p=0.0313). (a) BL: Baseline approximately 1 week before immunization; "Post imm.": Two weeks post immunization. (b) BL: Baseline the day of challenge; "Post expo." Day 9 post SARS-CoV-2 challenge. (c-d) Frequency of IFNγ + (left), IL-2 + (middle) or TFNα + (right) antigen-specific CD4 + T cells (CD154 + ) and CD8 + T cells (CD137 + ) in the total CD4 + T cell (top) or CD8 + T cell (bottom) population, respectively, for each naive macaque (grey symbols, n = 6), non-immunized convalescent macaque (blue symbols, n =6), and αCD40.RBD-vaccinated convalescent macaque (green symbols, n = 6). PBMCs were stimulated overnight with medium (open symbols), SARS-CoV-2 RBD (c) or N (d) overlapping peptide pools (filled symbols). BL: Baseline approximately 1 week before immunization. Bars indicate the mean values for each stimulation. (e-h) Frequency of antigen-specific Th1 CD4 + T cells (CD154 + and IFNγ +/-IL2 +/-TNFα) and antigen-specific CD8 + T cells (CD137 + IFNγ + ) in the total CD4 + T cell (e and g) or CD8 + T cell (f and h) population respectively. PBMC were stimulated overnight with SARS-CoV-2 RBD or N overlapping peptide pools. Time points in each experimental group were compared using the Wilcoxon signed rank test. (e-f) BL: Baseline approximately 1 week before immunization; "Post imm.": Two weeks post immunization. (g-h) BL: Baseline approximately 2 weeks before challenge; "Post expo." Day 9 post SARS-CoV-2 challenge. Groups were compared using the non-parametric Mann-Whitney test (*two-tailed p=0.0152, **twotailed p=0.0022).  plain line  indicates individual values, and the bold dotted lines represent the mean for each experimental group. (a, c, d) The means with the SD are shown. (e) Percentage of macaques with viral gRNA above the limit of detection over time in nasopharyngeal swabs. Experimental groups were compared using the log Rank test. Figure 6. Complete blood counts of SARS-CoV-2 exposed naive and convalescent macaques. Absolute numbers of white blood cells (WBC) (a), lymphocytes (b), monocytes (c), neutrophils (d), basophils (e), eosinophils (f), and platelets (g) in naive (grey), convalescent (blue), and αCD40.RBD-vaccinated convalescent macaques (green) after SARS-CoV-2 exposure. (h) Frequency of cell subsets within WBC at days 0 and 2 post exposure according to NHP group. Each bar indicates the mean of 6 NHPs. and CCL2 (right) at day 2 post-exposure in plasma of naive (n=6, grey), convalescent (n=6, blue), and αCD40.RBD-vaccinated convalescent macaques (n=6, green). Each plot represents one macaque and bars represent median value for each group. Groups were compared using the non-parametric Mann-Whitney test (* Two-tailed p < 0.05).

Supplementary Figure 8
Supplementary Figure 8. Representative transversal slices of lung CT scans from SARS-CoV-2exposed naive and convalescent macaques. Imaging was performed at baseline and days 2 and 6 postexposure. Images are presented for each macaque according to their experimental group, with a window level of -300 and a window width of 1,600. The blue and red arrows indicate vaccination and viral exposure, respectively. (b) Neutralization ED50 values of the three viral isolates at the day of challenge (4 w.p.im). Each plot represents one macaque (n=6 NHP/group) and bars indicate mean value for each group. Groups were compared using the nonparametric Mann-Whitney test (** Two-tailed p=0.0022).

Supplementary Tables
Supplementary Table I

αCD40.RBD vaccine
Production and quality assurance of the αCD40.RBD vaccine Vectors and sequences for humanized anti-human CD40 12E12 IgG4 and control human IgG4 antibodies have been described previously 1-3 . GenBank sequences HQ738666.1 and KP684037 describe the human IgG4 chimeric forms of the 12E12, anti-human CD40 H and L chains. Methods for expression vectors and protein production and purification, via transient or stable CHO-S (Chinese Hamster Ovary cells; ThermoFisher Scientific) transfection and quality assurance including CD40 binding specificity were as are described. CHOoptimized codons encoding SARS-CoV-2 RBD residues 318-541 of sequence ID: YP_009724390.1 with appended residues encoding a C-tag (EPEA) and a stop codon were inserted between the vector Nhe I and Not I sites positioned distal to the H chain C-terminal codon. Expression plasmids encoding the antibody H chain RBD fusion and the L chain were transiently transfected into Expi-CHO cells (ThermoFisher Scientific) with TransIT-PRO Pro reagent (Mirus Bio) using the manufacturers protocol. The product was purified by protein A affinity capture of the culture medium followed by elution with a gradient of 1M L-Arginine monohydrochloride in H2O, from pH 8.0 and pH 1.8. Product was formulated in phosphate buffered saline (pH 7.4) with 125 mM cyclodextrin (average MW 1420). The LPS value was .037 ng/mg. Using a solid phase assay direct binding assay previously described 3 these was no significant difference in the CD40 binding affinity of anti-CD40 12E12 (EC50 30 pM) versus anti-CD40 12E12-RBD (EC50 35 pM).

DREP-S vaccine
DREP-S vaccine constructs were made by cloning the sequences encoding S of SARS-CoV-2 spike protein into the Semliki Forest Virus (SFV) DREP plasmid vector backbone 3 using BamHI and SpeI restriction sites 4 . The S construct encodes the surface glycoprotein of SARS-CoV-2 (Wuhan-Hu-1) with an 18-aa deletion in the cytoplasmic tail (D18). The synthesis of the construct with the appropriate restriction sites was ordered from Twist bioscience. The spike variant was codon optimized for human expression and the construct's sequence was confirmed by sequencing. Plasmid DNA of the DREP-S vaccine candidate was purified from bacterial cultures using the EndoFree Plasmid Maxi or Giga Kit (QIAGEN) and the concentration and purity was measured on a NanoDrop One (ThermoFisher).

T-cells response in hu-mice
To analyze the SARS-CoV-2 RBD protein-specific T cell using functional recall assay, we used fifteenmer peptides (n = 70) overlapping by 11 amino acids (aa) and covering the vaccine RBD sequence (aa281-571 from Spike) synthesized by JPT Peptide Technologies (Berlin, Germany) and used at a final concentration of 1 µg/mL. We also used HLA class I PE labelled tetramers purchased from ProImmune Ltd (Oxford, UK). We used the following two specificities: SARS-CoV-2 A*0201 KIA (KIADYNYKL), SARS-CoV-2 A*0301 KCY (KCYGVSPTK).
Cryopreserved hu-mice spleen cells from 6 weeks after the priming immunization (one week after final immunization) were thawed and counted. Cells were rested overnight in RPMI 1640 media with L-Glutamax supplemented with Penicillin / Streptomycin and 10% of human serum. Subsequently, cells from HLA-A*0201 and HLA-A*0301 donors were pooled together for the mock group and group 2 plus 3 vaccinated hu-mice, then cultured at 0.6x10 6 cells per condition with 1µg/mL of 15-mers peptides JPT Peptide Technologies (Berlin, Germany). As a negative control no stimulant was added, and as a positive control 1 µL of DynabeadsTM CD3/CD28 (ThermoFischer Scientific) were used. IL-2 (100 IU/mL, R&D System) was added on day 2, half of the volume of each culture well was refreshed with fresh media containing IL-2 (10 U/mL) at day 5 and with fresh media without IL-2 at day 7. On day 8, cells were re-stimulated:

SARS-CoV-2 S protein-specific B cell analysis
Hu-mice PBMC from 3 weeks after the priming immunization and hu-mice PBMC and spleen cells from 6 weeks (one week after the last recall injection) were incubated first with the biotinylated SARS-CoV-2 S protein for 30 min at 4°C. After a washing step, cells were stained for 30 min at 4°C with streptavine- S-Fuse neutralization assay: The assay was performed as described 8 . U2OS-ACE2 GFP1-10 or GFP 11 cells (obtained from U2OS cells, ATCC HTB-96), also termed S-Fuse cells, become GFP + cells when they are productively infected with SARS-CoV-2 9 . Cells were tested negative for mycoplasma. Cells were mixed (at a 1:1 ratio) and plated at 8 × 10 3 cells per well in a μClear 96-well plate (Greiner Bio-One).
The indicated SARS-CoV-2 strains were incubated with sera at the indicated concentrations or dilutions for 15 min at room temperature and added to S-Fuse cells. Sera were heat inactivated 30 min at 56 °C before use. Then, 18 h later, cells were fixed with 2% paraformaldehyde, washed and stained with Hoechst (1:1,000 dilution; Invitrogen). Images were acquired with an Opera Phenix high-content confocal microscope (PerkinElmer). The GFP area, the number of syncytia and nuclei were quantified using the Harmony software (PerkinElmer). The percentage of neutralization was calculated using the number of syncytia as the value with the following formula: 100 × (1 − (value with serum − value in 'noninfected')/(value in 'no serum' − value in 'noninfected')). Neutralizing activity of each sera was expressed as the ED50.

Antigen specific T cell assays using non-human primate cells
To analyze the SARS-CoV-2 protein-specific T cell using functional assay, 15-mer peptides (n = 70) overlapping by 11 amino acids (aa) and covering the vaccine RBD sequence (n=70, aa 281-571 from Spike) and the SARS-CoV-2 Nucleoprotein sequence (n=102, aa 1-419 from N) synthesized by JPT Peptide Technologies (Berlin, Germany) and used at a final concentration of 2 µg/mL. Permeabilized cell samples will be stored at -80 °C before the staining procedure. Antibody staining was performed in a single step following permeabilization. After 30 min of incubation at 4°C, in the dark, cells were washed in BD Perm/Wash buffer then acquired on the ZE5 flow cytometer (Biorad). Analysis was performed on FlowJo v.10 software.

Virus quantification in cynomolgus macaque samples
Upper respiratory (nasopharyngeal and tracheal) and rectal specimens were collected with swabs (Viral Transport Medium, CDC, DSR-052-01). Tracheal swabs were performed by insertion of the swab above the tip of the epiglottis into the upper trachea at approximately 1.5 cm of the epiglottis. All specimens were stored between 2°C and 8°C until analysis by RT-qPCR with a plasmid standard concentration range containing an RdRp gene fragment including the RdRp-IP4 RT-PCR target sequence (Supplementary table II). The limit of detection was estimated to be 2.67 log10 copies of SARS-CoV-2 gRNA per mL and the limit of quantification was estimated to be 3.67 log10 copies per mL. SARS-CoV-2 E gene subgenomic mRNA (sgRNA) levels were assessed by RT-qPCR using primers and probes previously described (Corman et al., 2020; Wölfel et al., 2020) (Supplementary table SII). The protocol describing the procedure for the detection of SARS-CoV-2 is available on the WHO website (https://www.who.int/docs/default-source/coronaviruse/real-time-rt-pcr-assays-for-the-detectionof-sars-cov-2-institut-pasteur-paris.pdf?sfvrsn=3662fcb6_2). The limit of detection was estimated to be 2.87 log10 copies of SARS-CoV-2 sgRNA per mL and the limit of quantification was estimated to be 3.87 log10 copies per mL 10,11 .