key: cord-0859091-5h8xk617
authors: Sieling, P.; King, T.; Wong, R.; Nguyen, A.; Wnuk, K.; Gabitzsch, E. R.; Rice, A.; Adisetiyo, H.; Hermreck, M.; Verma, M.; Zakin, L.; Shin, A.; Morimoto, B.; Higashide, W.; Dinkins, K.; Balint, J.; Peykov, V.; Taft, J.; Patel, R.; Buta, S.; Martin-Fernandez, M.; Bogunovic, D.; Spilman, P.; Sender, L.; Reddy, S.; Robinson, P.; Rabizadeh, S.; Niazi, K.; Soon-Shiong, P.
title: Single Prime hAd5 Spike (S) + Nucleocapsid (N) Dual Antigen Vaccination of Healthy Volunteers Induces a Ten-Fold Increase in Mean S- and N- T-Cell Responses Equivalent to T-Cell Responses from Patients Previously Infected with SARS-CoV-2
date: 2021-04-07
journal: nan
DOI: 10.1101/2021.04.05.21254940
sha: 748bfe58723709c110b837dba8f30385c9a216a7
doc_id: 859091
cord_uid: 5h8xk617

In response to the need for a safe, efficacious vaccine that provides broad immune protection against SARS-CoV-2 infection, we have developed a dual-antigen COVID-19 vaccine. The vaccine delivers both the viral spike (S) protein modified to increase cell-surface expression (S-Fusion) and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to enhance MHC class I and II presentation and T-cell responses. The vaccine antigens are delivered using a human adenovirus serotype 5 (hAd5) platform with E1, E2b, and E3 regions deleted that has been shown in previous cancer vaccine studies to be effective in the presence of pre-existing hAd5 immunity. Here, we demonstrate the hAd5 S-Fusion + N-ETSD (hAd5 S + N) vaccine antigens when expressed by dendritic cells (DCs) of previously SARS-CoV-2-infected patients elicit Th1 dominant activation of autologous patient T cells, indicating the vaccine antigens have the potential for generating immune responses in patients previously infected or vaccinated. We further demonstrate that participants in our open-label Phase 1b study of the dual-antigen hAd5 S + N vaccine generate Th1 dominant S- and N- specific T cells after a single prime subcutaneous injection and that the magnitude of these responses were comparable to those seen for T cells from previously infected patients. We further present our in silico prediction of T-cell epitope HLA binding for both the first-wave SARS-CoV-2 A strain and the K417N, E484K, and N501Y S as well as the T201I N variants that suggests T-cell responses to the hAd5 S + N vaccine will retain efficacy against these variants. These findings that the dual-antigen hAd5 S + N vaccine elicits SARS-CoV-2-relevant T-cell responses and that such cell-mediated protection is likely to be sustained against emerging variants supports the testing of this vaccine as a universal booster that would enhance and broaden existing immune protection conferred by currently approved S-based vaccines.

To address the need for an efficacious COVID-19 vaccine that elicits broad immunity against 51 SARS-CoV-2, is suitable for global distribution and use, and has a high likelihood of maintaining 52 efficacy against emerging SARS-CoV-2 variants, we have developed a dual-antigen COVID-19 53 'T cell' vaccine. This dual-antigen vaccine expresses both the virus spike (S) and nucleocapsid (N) 54 proteins using a next-generation human adenovirus serotype 5 (Ad5) platform. The S antigen is 55 full-length S including SD1 receptor binding domain, S1 and S2 domains modified to enhance 56 surface expression (S-Fusion); 1 and full-length N protein modified with an Enhanced T cell 57

Stimulation Domain (ETSD) to direct N to the endo-/lysosomal compartment for increased MHC 58 class I and II expression. The hAd5 platform used has deletions in the E1, E2b, and E3 gene 59 regions, thereby minimizing host anti-vector immune responses 2,3 and enabling efficient antigen 60 cargo expression and cognate T cell activation even in the presence of existing anti-adenovirus 61 immunity, as demonstrated previously in clinical studies targeting tumor-associated antigens in 62 cancer patients. 4,5 63

The emergence of SARS-CoV-2 variants that have the potential to evade immune responses 64 generated in response to currently available vaccines has spurred renewed interest in the potential 65

The hAd5 dual antigen vaccine construct includes sequences designed to target N to MHC 127 class II antigen loading compartments. To confirm this occurs, we evaluated the localization of N-128 ETSD in human monocyte-derived dendritic cells (MoDCs) compared to its untargeted 129 cytoplasmic counterpart (N). MoDCs from healthy subjects were infected with hAd5 N-ETSD or 130 hAd5 N and localization was determined by immunocytochemistry. N-ETSD showed localization 131 to discrete vesicles, some coincident with CD71, a marker of recycling endosomes ( Studies have shown that lysosomes fuse with autophagosomes to enhance peptide processing 135 and MHC class II presentation. 37,38 Thus we examined whether N-ETSD localized in 136 autophagosomes in MoDCs by co-labeling with the autophagosome marker LC3a/b 39 to identify 137 another potential site of localization relevant to MHC class II antigen presentation. 40 We found 138 that N-ETSD also displayed some co-localization with the autophagosome marker ( The Immune Recall Study with Previously SARS-CoV-2 Infected Patients 156

For the 'recall' studies described below, plasma samples were collected from four individuals 159 convalescing from SARS-CoV-2 infection as confirmed by antibody assays and patient history. 160

The presence of anti-Spike IgG, and neutralizing antibodies by both a surrogate SARS-CoV-1 161 neutralization 44 and live virus assays, were confirmed in all patient samples (Suppl. Fig. S1 ). 162 Samples from four virus-naïve individuals were used as controls. 163 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In additional studies to validate immune responses to SARS-CoV-2 antigens, the binding of 164 previously SARS-CoV-2 infected patient and virus-naïve control individual plasma to human 165 embryonic kidney (HEK) 293T cells transfected with either hAd5 S-Fusion alone or hAd5 S-166 Fusion + N-ETSD was assessed (Suppl. Fig. S2 ). This binding reflects the presence of antibodies 167 in plasma that recognize antigens expressed by the hAd5 vectored vaccines. Quantification of 168 histograms showed little or no binding of virus-naïve plasma antibodies to cells expressing 169 constructs, with the highest previously SARS-CoV-2 infected patient plasma binding to cells 170 expressing the dual antigen S-Fusion + N-ETSD construct (Suppl. Fig. S2R ). 171

To confirm the T cells from previously SARS-CoV-2-infected patients were reactive to 172 SARS-CoV-2 peptides, isolated T cells from the patients were incubated with S1 (containing the 173 S receptor binding domain), S2 and N peptides pools. T cells from all four patients showed 174 reactivity (Suppl. Fig. S3 ). Selected CD4+ and CD8+ T cells from 2 patients were activated by S1, 175 S2, and N peptides, revealing CD8+ T cells were more reactive to N (Suppl. Fig. S3D and E). 176

To evaluate the immune significance of endo/lysosome-localized N-ETSD versus cytoplasmic 179 N, MoDCs were infected with hAd5 constructs (Null, N-ETSD or N) then incubated with 180 autologous CD3+ and CD4+-or CD8+-selected T cells ( Fig. 2A) . CD3+ T cells from previously 181 infected SARS-CoV-2 patients showed significantly greater IFN-g secretion in response to N-182 ETSD than both Null and cytoplasmic N in the two patients where N-ETSD and N were compared 183 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 7, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 7, 2021. ; https://doi.org/10.1101/2021.04.05.21254940 doi: medRxiv preprint

To compare CD3+, CD4+, CD8+ memory T-cell responses to the components of the hAd5 S-201 Fusion + N-ETSD vaccine, MoDCs were infected with hAd5 S-Fusion, hAd5 N-ETSD, or the dual 202 antigen vaccine (Fig. 3A) . 203

For unselected CD3+ T cells, IFN-g responses were similar to S-Fusion + N-ETSD and N-204 ETSD with responses to S-Fusion being relatively low ( (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 7, 2021. ; https://doi.org/10.1101/2021.04.05.21254940 doi: medRxiv preprint

For the ELISpot assay results shown here, T cells were isolated from patients in cohort 2 of 231 the Phase 1b study of the ImmunityBio hAd5 COVID-19 'T cell' vaccine (NCT04591717). These 232 participants were vaccinated subcutaneously with 1x10 11 hAd5 S-Fusion + N-ETSD viral particles 233

The studies presented here are experimental and all assays and analyses were performed in-235 house at ImmunityBio, Inc. Formal analyses of the primary and secondary end points are ongoing 236 pending completion of the trial. 237

T-cell responses to S, N, and SARS-CoV-2 membrane (M) peptides were determined by 240

ELISpot for clinical study participants pre-vaccination (Day 1) and at two time points following 241 the prime injection: Days 14-16 and 21-23. 242

By Day 14-16, T-cell secretion of IFN-g in response to N peptides increased more than 10-243 fold compared to pre-vaccination (Day 1); these responses were sustained on Day 21-23 and were 244 significant (Fig. 4A ). The changes in T-cell responses to N for individual participants from pre-to 245 post-vaccination are shown in Fig. 4B . 246

T-cell secretion of IFN-g in response to S peptides also increased post-prime only vaccination, 247 increasing more than 3-fold by Day 14-16 and more than 10-fold by Day 21-23, when differences 248 were significant (Fig. 4C) . T-cell responses to S for individual participants are shown in Fig. 4D . 249

When responses to S, N and SARS-CoV-2 membrane (M) peptides are compared, the greatest 250 responses are seen to N peptides ( Fig. 4F and G). As expected, there were very low/no responses 251 to M peptide, not delivered in the vaccine. Importantly, IL-4 secretion in response to peptide 252 stimulation was very low, indicating all responses to vaccine antigens were highly Th1 dominant. 253 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

As shown in Figure 5 , T-cell responses to both N and S for previously SARS-CoV-2 infected 267 patients (Pt) and hAd5 S-Fusion + N-ETSD vaccinated participants (IB Vac) on Day 21-23 were 268 comparable. For responses to N, both Pt and IB Vac were ~40-fold greater than Unex; and for 269 responses to S, both Pt and IB Vac were >15-fold higher than Unex. As expected, T cells from 270 previously SARS-CoV-2 infected patients, but not vaccinated participants, reacted to M due to its 271 presence in the live virus. The M antigen is not present in the vaccine. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The key questions asked here were: how does mutation change HLA availability and which 309 mutations favor immune evasion? Not all mutations lead to evasion; while a mutation may lead to 310 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 7, 2021.

an amino acid substitution that has potential functional consequences, that same mutation may still 311 generate an epitope that retains HLA binding affinity and does not contribute to escape. The unique 312 binding HLAs for each S variants when compared to the A strain are shown in Figure 1 . For class 313 I, there are only 2 K417N (B.1.351) HLAs and for the A strain there are 6 (Fig. 6A) ; for E484K 314 there are 8 and for the A strain 7 (Fig. 6B) ; and for N501Y there are 8 and only 1 for the A strain 315 (Fig. 6C ). There are no unique class II HLAs for these same comparisons (Fig. 6D-F) . 316

In the B.1.351 strain, there is only one N mutation -T205I. Similarly to the S variants, there 317 are few unique binding HLAs. For class I, the T201I mutation generates 2 and the A strain 3 unique 318 binding HLAs (Fig. 6G) . For class II, the T205I generates 4 and the A strain just 1 unique binding 319 HLA(s) (Fig. 6H) . 320 321 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The emergence and rapid spread of SARS-CoV-2 variants is raising concern that first 330 generation monovalent wild-type S-targeting vaccines may eventually lose effectiveness at 331 protecting against COVID-19. In addition to updating S sequences to include variants in current Here we show that our strategy of including N-ETSD, confirmed to be directed to the endo-347 /lysosomal compartment with S optimized for cell-surface display 1,47 results in recognition of the 348

All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 7, 2021. We also thank Phil Yang of ImmunityBio for his ongoing coordination of project updates for the 366 studies described herein. 367

Production of all hAd5 [E1-, E2b-, E3-] (Fig. 7A) constructs and virus particles were carried 371 out as previously described. 49 In brief, high titer adenoviral stocks were generated by serial 372 propagation in the E1-and E2b-expressing E.C7 packaging cell line, followed by CsCl2 373 purification, and dialysis into storage buffer (2.5% glycerol, 20 mM Tris pH 8, 25 mM NaCl) by 374 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 7, 2021. The

Blood was collected with informed consent via venipuncture from volunteer patients (Pt) who 395 had either not been exposed (UNEX) to SARS-CoV-2 as confirmed by ELISA and multiple 396 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 7, 2021. ; https://doi.org/10.1101/2021.04.05.21254940 doi: medRxiv preprint negative SARS-CoV-2 tests or who had recovered from COVID-19 as indicated by recent medical 397 history and a positive SARS-CoV-2 antibody test. The presence of anti-S IgG and of neutralizing 398 antibodies in plasma from previously SARS-CoV-2 infected patients was confirmed by ELISA, a 399 surrogate neutralization assay, 44 and a live virus assay as shown in Supplementary Fig. S1 . A third 400 source of whole blood was apheresis of healthy subjects from a commercial source (HemaCare). 401

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by density 402 gradient centrifugation and plasma was collected after density gradient centrifugation. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 7, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 7, 2021. ; https://doi.org/10.1101/2021.04.05.21254940 doi: medRxiv preprint secondary antibody. The number of spots per well (1 x 10 5 cells), was counted using an ELISpot 446 plate reader. IL-4 was measured by ELISpot using a kit (MabTech) with wells precoated with anti-447 IL-4 antibody and following the manufacturer's instructions. Remaining steps for IL-4 detection 448 were identical to those for IFN-g, but with alkaline phosphatase detection rather than peroxidase. Study participants comprised healthy adult volunteers between the ages of 18 and 55. Subjects 466 were randomized to cohorts described above. All the participants provided informed, written 467 consent before enrollment. 468 469 470 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

T-cell responses in PBMCs were measured at baseline (Day 1) and after prime vaccination 480 alone on Day 14-16 and 21-23 by ELISpot for secretion of interferon g (IFNg) and interleukin 4 481 (IFN-4). T helper cell type 1 dominance was determined based on the IFNg/IL-4 ratio. 482

ELISpot assays were used to enumerate S-and N-specific IFN-γ-secreting T cells in fresh 484

PBMCs isolated from the blood of subjects vaccinated with hAd5-S-Fusion+N-ETSD. Whole 485 blood was collected by venipuncture pre-and post-vaccination. PBMCs were isolated from whole 486 blood by standard density gradient centrifugation and frozen in liquid nitrogen until use. On the 487 day of assay, PBMCs were thawed and re-suspended in RPMI 10% human AB serum, then 488 incubated with 2 µg/ml of SARS-CoV-2 S or N peptide pools (JPT Peptide Technologies catalogue 489 # PM-WCPV-S and PM-WCPV-NCAP-1, respectively). Internal positive controls were incubated 490 with 2 µg/ml of CEFT peptide pool consisting of epitopes derived from human Cytomegalovirus, 491

Epstein-Barr virus, Influenza A, and Clostridium tetani (JPT Peptide Technologies catalogue # 492 PM-CEFT-4). Positive assay controls were incubated with a CD3/CD28/CD2 agonist (StemCell 493

Technologies catalogue # 10970). Negative controls were incubated with media alone or 2 µg/ml 494 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 7, 2021. ; https://doi.org/10.1101/2021.04.05.21254940 doi: medRxiv preprint of SARS-CoV-2 membrane peptide pool (JPT Peptide Technologies catalogue # PM-WCPV-495 VME). The number of spot-forming cells (SFCs) per well (2.5-4 x 10 5 PBMCs) were enumerated 496 using an ELISpot plate reader (Cellular Technologies Limited model # S6UNIV-01-7115). All 497 IFN-γ ELISpot reagents were obtained from a commercial kit and used according to manufacturer 498 protocol (Cellular Technologies Limited catalogue # Hu IFN-g/IL-4). 499 500

The reference strain and COVID19 variants used for epitope analysis was assembled from 503 75,000 individual sequences deposited in the GISAID database. Binding analysis was run on the 504 consensus sequence generated using our in-house binding algorithm. 51 were determined by ELISpot. Statistical analysis performed using One-way ANOVA and 792

Dunnett's post-hoc multiple comparison analysis to compare each peptide pool to Veh (shown 793 above the bar) or between peptide pools (above line) where *p<0.05, **p<0.01 and 794 ****p<0.00001. Data graphed as mean and SEM; n = 3. 795 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 7, 2021. ; https://doi.org/10.1101/2021.04.05.21254940 doi: medRxiv preprint

Complete Protection of Nasal and Lung Airways Against SARS-514

CoV-2 Challenge by Antibody Plus Th1 Dominant N-and S-Specific T-Cell Responses 515 to Subcutaneous Prime and Thermally-Stable Oral Boost Bivalent hAd5 Vaccination in 516 an NHP Study. bioRxiv

Next-generation adenoviral vectors: new and improved

New recombinant SAd5 vector overcomes Ad5 immunity 521 allowing for multiple safe, homologous, immunizations

Anti-tumor immunotherapy despite immunity to adenovirus using a 524 novel adenoviral vector Ad5

A Phase I Trial Using a Multitargeted Recombinant Adenovirus 527 5 (CEA/MUC1/Brachyury)-Based Immunotherapy Vaccine Regimen in Patients with 528 Advanced Cancer

What are the roles of antibodies versus a durable, high quality T-cell 530 response in protective immunity against SARS-CoV-2? Vaccine X 6

CD8+ T cell responses in COVID-19 convalescent individuals target 533 conserved epitopes from multiple prominent SARS-CoV-2 circulating variants

cells recognize recent SARS-CoV-2 variants

Negligible impact of SARS-CoV-2 variants on CD4+ and CD8+ T cell 539 reactivity in COVID-19 exposed donors and vaccinees. bioRxiv

Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine

Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine

Interim Results of a Phase 1-2a Trial of Ad26

T cell and antibody responses induced by a single dose of ChAdOx1 548 nCoV-19 (AZD1222) vaccine in a phase 1/2 clinical trial

Phase 1-2 Trial of a SARS-CoV-2 Recombinant Spike Protein 551 Nanoparticle Vaccine

Safety and Immunogenicity of SARS-CoV-2 mRNA-1273 Vaccine 554 in Older Adults

COVID-19 vaccine BNT162b1 elicits human antibody and T(H)1 T cell 556 responses

Vaccines and SARS-CoV-2 variants: the urgent need for a correlate of 558 protection

Efficacy of the ChAdOx1 nCoV-19 Covid-19 Vaccine against the 560 B.1.351 Variant

Preliminary Efficacy of the NVX-CoV2373 Covid-19 Vaccine Against 563 the B.1.351 Variant. medRxiv

Novavax COVID-19 Vaccine Demonstrates 89.3% Efficacy in UK Phase 3

Candidate Met Primary Endpoints in Interim Analysis of its Phase 3 ENSEMBLE Trial

Reduced binding and neutralization of infection-and vaccine-induced 571 antibodies to the B.1.351 (South African) SARS-CoV-2 variant. bioRxiv

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating 574 variants

mRNA-1273 vaccine induces neutralizing antibodies against spike mutants 576 from global SARS-CoV-2 variants. bioRxiv

A combined nucleocapsid vaccine induces vigorous SARS-CD8+ T-cell 578 immune responses

Characterization and application of monoclonal antibodies against N 580 protein of SARS-coronavirus

Long-lived memory T lymphocyte responses against SARS coronavirus 583 nucleocapsid protein in SARS-recovered patients

Nucleocapsid-independent specific 586 viral RNA packaging via viral envelope protein and viral RNA signal

Biochemical characterization of SARS-CoV-2 nucleocapsid protein. 589 Biochemical and biophysical research communications

Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans 592 with COVID-19 Disease and Unexposed Individuals

SARS-CoV-2-specific T cell immunity in cases of COVID-19 and 595 SARS, and uninfected controls

Robust T cell immunity in convalescent individuals with asymptomatic 597 or mild COVID-19

SARS-CoV-2 T cell immunity: Specificity, function, 599 durability, and role in protection

T cell responses in patients with COVID-19

CD4+ T cells are required to sustain 604 CD8+ cytotoxic T-cell responses during chronic viral infection

The multifaceted role of CD4(+) T cells in 607 CD8(+) T cell memory

New insights into autophagosome-lysosome fusion

Autophagosome-Lysosome Fusion

LC3, a mammalian homologue of yeast Apg8p, is localized in 613 autophagosome membranes after processing

Antigen-loading compartments for major 616 histocompatibility complex class II molecules continuously receive input from 617 autophagosomes

Intersection of autophagy with pathways of antigen 619 presentation

Autophagy and Its Role in MHC-Mediated Antigen 621 Presentation

The crosstalk between autophagic and endo-/exosomal pathways in antigen 624 processing for MHC presentation in anticancer T cell immune responses

A SARS-CoV-2 surrogate virus neutralization test based on antibody-627 mediated blockage of ACE2-spike protein-protein interaction

GISAID. SARS-CoV-2 Lineages

Broad and strong memory CD4(+) and CD8(+) T cells induced by SARS-631

CoV-2 in UK convalescent individuals following COVID-19

The Dual-Antigen Ad5 COVID-19 Vaccine Delivered as an Intranasal Plus 634 Subcutaneous Prime Elicits Th1 Dominant T-Cell and Humoral Responses in CD-1 Mice. 635 bioRxiv

Comparative computational analysis of SARS-CoV-2 nucleocapsid 637 protein epitopes in taxonomically related coronaviruses

Production and Characterization of Improved Adenovirus Vectors 640 with the E1, E2b, and E3 Genes Deleted

CD1 molecule 643 expression on human monocytes induced by granulocyte-macrophage colony-stimulating 644 factor

Data, disease and diplomacy: GISAID's innovative 646 contribution to global health

The Immune Epitope Database (IEDB): 2018 update

A SARS-CoV-2 surrogate virus neutralization test based on antibody-650 mediated blockage of ACE2-spike protein-protein interaction

No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. format and 24 hours later, cells were incubated with antibodies or heat inactivated plasma 710 previously serially diluted in 3-fold steps in

This incubation 713 did not include cells to allow for neutralizing activity to occur prior to infection. For detection of 714 neutralization, the virus/sample mixture (120 µL) was transferred to the Vero E6 cells and 715 incubated (48 hours) before fixation with 4% PFA. Each well received virus (60 µl) or an infectious 716 dose of 600 TCID50. Control wells, including six wells on each plate for no virus and virus-only 717 controls, were used. The percent neutralization was calculated as 100-((sample of interest

Transfection of HEK 293T cells and detection of plasma antibody binding 722 723 To assess binding of plasma from a previously infected patient to antigens expressed by hAd5

HEK 293T cells (2.5 x 726 10 5 cells/well in 24 well plates) were grown in DMEM (Gibco) with 10% FBS and 1X PSA (100 727 units/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL Amphotericin B) at 37 o C. Cells were 728 either left untransfected or were transfected with 0.5 µg of S-Fusion or S-Fusion + N-ETSD hAd5 729 plasmid DNA using a JetPrime transfection reagent (Polyplus) according to the manufacturer's 730 instructions. Twenty-four hours later, cells were incubated for 30 min

Plasma IgG was labeled using a goat anti-human IgG-phycoerythrin conjugated and labeled cells 733 were acquired using the Thermo-Fisher Attune NxT flow cytometer and analyzed using FlowJo 734 Software to determine Mean Fluorescence Intensity (MFI) values of both the untransfected and 735 transfected cells. Results were graphed as the difference in

For the studies described below, plasma samples were collected from four individuals 743 convalescing from SARS-CoV-2 infection as confirmed by antibody assays and patient history as 744 described Methods. The presence of anti-Spike IgG, and of neutralizing antibodies by both a 745 surrogate SARS-CoV-1 neutralization assay 53 and live virus assays, were confirmed in all patient 746 samples

No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity

S1 Plasma samples from patients (Pt) previously infected with SARS-CoV-2 contain 750 neutralizing antibodies. (A) The levels of anti-spike IgG as determined by OD at 450 nm are 751 shown. (B) All four Pt plasma samples contained antibodies that blocked RBD-ACE2 binding and 752 (C) contained SARS-CoV-2 neutralizing antibodies

CoV-2-infected patient plasma binds to vaccine-transfected HEK-293T cells 754 755 In additional studies to validate immune responses to SARS-CoV-2 antigens, the binding of 756 previously SARS-CoV-2 infected patient and virus-naïve control individual plasma to human 757 embryonic kidney (HEK) 293T cells transfected with either hAd5 S-Fusion alone or

This binding reflects the presence of antibodies 759 in plasma that recognize antigens expressed by the hAd5 vectored vaccines. Quantification of 760 histograms showed little or no binding of virus-naïve plasma antibodies to cells expressing either 761 construct, and the highest binding of plasma antibodies from a previously SARS-CoV-2 infected 762 patient to cells expressing the dual antigen S-Fusion + N-ETSD construct

S2 Plasma from previously SARS-CoV-2 infected patients shows greater binding to S-Fusion

ETSD surface antigens compared to S-Fusion alone after transfection of HEK 293T cells

HEK 293T cells that were either uninfected (pink) or infected (blue) with S-Fusion alone or 767 bivalent S-Fusion + N-ETSD were exposed to plasma from either unexposed (UnEx) controls or 768 previously infected SARS-CoV-2 patients (Pt). Flow histograms for (A-D) UnEx with S-Fusion

When the DMFI (difference in binding to transfected versus 771 untransfected cells) is graphed, it is apparent that compared to (Q) S-Fusion

CoV-2-infected patient T cells are activated by S and N peptide pools 776 777 The reactivities of T cells from the previously SARS-CoV-2-infected patients was confirmed 778 by incubation with S1 (containing the spike receptor binding domain

Supplementary Materials 667 668Methods 669 670Quantification of anti-SARS-CoV-2 spike antibodies in plasma 671 672The presence of anti-SARS-CoV-2 spike antigen antibodies in previously infected patient 673 plasma used in studies here was validated. IgG against SARS-CoV-2 spike was detected in plasma 674 using ELISA. Briefly, EIA/RIA plates were coated with a solution of purified recombinant SARS-675CoV The GenScript cPass TM (https://www.genscript.com/cpass-sars-cov-2-neutralization-691 antibody-detection-Kit.html) kit for detection of neutralizing antibodies was used according to the 692 manufacturer's instructions. 44 The kit detects circulating neutralizing antibodies against SARS-693CoV-2 that block the interaction between the S RBD with the ACE2 cell surface receptor. It is 694 suitable for all antibody isotypes. 695 696To evaluate the levels of anti-SARS-CoV-2 neutralizing antibodies in plasma, dilutions of 697 plasma were incubated with horseradish peroxidase-conjugated spike RBD (37°C, 30min). The 698 RBD-plasma mixture was added to a microtiter plate coated with ACE2 and incubated at 37°C (15 699 min). Plates were washed and substrate (TMB) added at room temperature (15 min). The reaction 700was stopped and plates read on a plate reader at 450 nm. 701 702Live virus assay of plasma neutralization of infection 703 704The ability of previously SARS-CoV-2 infected patient plasma used in studies here to 705 neutralize SARS-CoV-2 infection in vitro was also validated. All aspects of the assay utilizing 706 virus were performed in a BSL3 containment facility according to the ISMMS Conventional 707Biocontainment Facility SOPs for SARS-CoV-2 cell culture studies. Vero E6 kidney epithelial 708 cells from Cercopithecus aethiops (ATCC CRL-1586) were plated (20,000 cells/well) in a 96-well 709