key: cord-0775737-lnpu782d
authors: Patel, Ami; Walters, Jewell N.; Reuschel, Emma L.; Schultheis, Katherine; Parzych, Elizabeth; Gary, Ebony N.; Maricic, Igor; Purwar, Mansi; Eblimit, Zeena; Walker, Susanne N.; Guimet, Diana; Bhojnagarwala, Pratik; Adeniji, Opeyemi S.; Doan, Arthur; Xu, Ziyang; Elwood, Dustin; Reeder, Sophia M.; Pessaint, Laurent; Kim, Kevin Y.; Cook, Anthony; Chokkalingam, Neethu; Finneyfrock, Brad; Tello-Ruiz, Edgar; Dodson, Alan; Choi, Jihae; Generotti, Alison; Harrison, John; Tursi, Nicholas J.; Andrade, Viviane M.; Dia, Yaya; Zaidi, Faraz I.; Andersen, Hanne; Abdel-Mohsen, Mohamed; Lewis, Mark G.; Muthumani, Kar; Kim, J Joseph; Kulp, Daniel W.; Humeau, Laurent M.; Ramos, Stephanie J.; Smith, Trevor R.F.; Weiner, David B.; Broderick, Kate E.
title: Intradermal-delivered DNA vaccine induces durable immunity mediating a reduction in viral load in a rhesus macaque SARS-CoV-2 challenge model
date: 2021-09-28
journal: Cell Rep Med
DOI: 10.1016/j.xcrm.2021.100420
sha: eb51c14b2fb9b17ea4aea47b091eed65f9350217
doc_id: 775737
cord_uid: lnpu782d

Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, has had a dramatic global impact on public health, social, and economic infrastructures. Here, we assess the immunogenicity and anamnestic protective efficacy in rhesus macaques of an intradermal (ID)-delivered SARS-CoV-2 spike DNA vaccine, INO-4800, currently being evaluated in clinical trials. Vaccination with INO-4800 induced T cell responses and induced spike antigen and RBD binding antibodies with ADCP and ADCD activity. Sera from the animals neutralized both the D614 and G614 SARS-CoV-2 pseudotype viruses. Several months after vaccination, animals were challenged with SARS-CoV-2 resulting in rapid recall of anti-SARS-CoV-2 spike protein T cell and neutralizing antibody responses. These responses were associated with lower viral loads in the lung. These studies support the immune impact of INO-4800 for inducing both humoral and cellular arms of the adaptive immune system which are likely important for providing durable protection against COVID-19 disease.

COVID-19 was declared a global pandemic on March 11, 2020 by the World Health 45 Organization. There are >160 million confirmed cases worldwide with the total number of 46 deaths estimated to be >3,466,670 (May 24, 2021, gisaid.org) . COVID-19 presents as a 47 respiratory illness, with mild-to-moderate symptoms in many cases (~80%) 1,2 . These symptoms 48 include headache, cough, fever, fatigue, difficulty breathing, and possible loss of taste and smell. 49 The factors involved with progression to severe COVID-19 disease in 20% of cases are unclear; Recent studies support an important role for both cellular and humoral immunity in 81 protection and recovery from COVID-19 disease [26] [27] [28] . T cell-mediated immunity has been shown 82 to be important for other beta-coronaviruses, providing both direct protection and help in the 83 generation of effective humoral responses 29, 30 . Several SARS-CoV-2 studies have now reported 84 that up to 40% of persons can exhibit mild disease which may be associated with T cell 85 immunity 31 or humoral responses 8 , and have indicated the levels of SARS-CoV-2 antibodies 86 wane rapidly in convalescent subjects 32 . This echoes older studies which found that neutralizing 87 antibody levels and memory B cell responses have not been sustained in SARS survivors, while 88 responsive T cell populations have proved more durable [33] [34] [35] . Furthermore, a SARS-CoV-2 89 J o u r n a l P r e -p r o o f challenge study in non-human primates (NHPs) indicated that CD8 T-cells had a major 90 contribution to the reduction in viral loads for animals with low anti-SARS-CoV-2 IgG levels 36 . 91 We have described the immunogenicity and efficacy of a SARS-CoV-2 DNA vaccine 92 (INO-4800), encoding a synthetic spike immunogen in small animal models 37 . This vaccine 93 candidate induced neutralizing and ACE2-blocking antibodies, as well as T cell responses in 94 mice and guinea pigs. Here, we assess vaccine-induced T and B cell responses during the acute 95 expansion phase in rhesus macaques, as well as the impact infection in an NHP viral installation 96 challenge model. Additionally, we studied the effect of the vaccines on the emergence of the 97 dominant SARS-CoV2 variant G614, which now comprises more than 80% of circulating global 98 viral strains and is reportedly associated with increased infectivity and spread 38 . We report 99 vaccine-induced humoral and cellular immunity, including neutralizing antibody responses 100 against both the SARS-CoV-2 D614 virus as well as the G614 strain. Upon SARS-CoV-2 101 challenge more than 3 months following final-dose, vaccinated macaques exhibited a rapid recall NHPs are a valuable model in the development of COVID-19 vaccines and therapeutics as they 111 can be infected with wild-type SARS-CoV-2, and present with early infection that mimics 112 aspects of human disease 18, 39 . Rhesus macaques (n=5) received two immunizations of (1 mg), at weeks 0 and 4 ( Figure 1A ). Naïve control animals (n=5) did not receive vaccine.

Humoral and cellular immune responses were monitored for 15 weeks (~4 months) following 115 prime immunization for antigen-specific T cell responses. All animals seroconverted following a 116 single INO-4800 immunization, with serum IgG titers detected against the full-length S1+S2 117 extracellular domain (ECD), S1, S2, and RBD regions of the SARS-CoV-2 S protein ( Figure 1B   118 and C). Cross-reactive antibodies were also detected against SARS-CoV S1 protein and RBD, 119 but not MERS-CoV ( Figure S1A ). SARS-CoV-2 spike antigen-reactive IgG against the S1 + S2 120 ECD and RBD were detected in bronchoalveolar lavage (BAL) washes at Week 8, 4-weeks 121 following the 2nd immunization dose ( Figure S1B ).

SARS-CoV-2 pseudovirus-neutralizing antibodies were measured in the serum of 123 animals between study weeks 0 and 12 ( Figure 1D ). During the course of the COVID-19 124 pandemic, a D614G SARS-CoV-2 spike variant has emerged that has potentially greater 125 infectivity, and now accounts for >80% of new isolates. We evaluated neutralization against this 126 new variant using a modified pseudovirus developed to express the G614 spike protein ( Figure   127 1E). Similar neutralization ID50 titers were observed against both D614 and G614 spikes, 

To investigate specific receptor-blocking neutralizing antibody activity, we assessed the 136 capacity to occlude ACE2 binding of SARS-CoV-2 spike antigen by vaccine-induced sera. We antibodies that can block the SARS-CoV-2 spike protein from interacting with ACE2 ( Figure   144   1G ). An independent flow cytometry-based assay was also performed to further study the Spike-145 ACE2 interaction. ACE2-expressing 293T cells were co-incubated with Spike with or without 146 the presence of sera. Spike binding to ACE2 was detected by flow cytometry. In this assay, 147 100% of macaques generated vaccine-induced antibodies that inhibited the Spike-ACE2 148 interaction, with a range of 53-96% inhibition ( Figure 1H ). It should be noted this assay was 149 conducted with a 1:27 dilution of sera but subsequent dilutions of the sera yielded similar results 150 with ~50% inhibition.

Antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent complement 152 deposition (ADCD) assays were performed using sera collected at baseline, week 6, and week 8 153 post-immunization to assess the ability of sera from immunized NHPs to engage effector 154 functions. ADCP activity against the SARS-CoV-2 S1 ( Figure S2A ) and RBD ( Figure S2B ) 155 proteins was significantly increased above baseline for immunized NHPs at week 8 (p=0.024 and 156 J o u r n a l P r e -p r o o f p=0.01, respectively). There was also an increased trend of ADCD activity against SARS-CoV-157 2 S1 protein for immunized NHPs at week 6 (p=0.056) ( Figure S2C ). No differences were 158 observed for ADCD activity against the RBD ( Figure S2D ). 159 Total serum antibody and pseudovirus neutralization titers from immunized NHPs were 160 compared with titers detected from 9 human SARS-CoV-2 convalescent sera donors (3, 161 asymptomatic, 3 mild, and 3 moderate infections, sera collected between 30-80 days following 162 positive COVID-19 PCR diagnosis). Overall, ELISA endpoint binding titers against RBD and 163 pseudoneutralization titers at the peak immune response in NHPs (6 weeks post-immunization) 164 were similar to those observed in convalescent donors ( Figure S3A and B). The current study was not designed to address the theoretical concern for vaccine 266 enhanced disease. However, we did not observe an increase in viral replication in the vaccinated 267 animals compared to controls, and no clinical signals such as respiratory distress, changes in 268 heart rates or oxygen saturation levels, temperature spikes, nor significant weight loss. at the time this study was conducted, the G614 strain, was the most prevalent variant of concern 317 and the USA-WA1/2020 isolate was widely used by multiple groups for viral challenge 21,22,38 . 318 We have since performed pseudoneutralization assays with recent VOC, including alpha, beta, 3. Xu, Z., Shi, L., Wang, Y., Zhang, J., Huang, L., Zhang, C., Liu, S., Zhao, P., Liu, H., Zhu, L., et al. 375 (2020) . Pathological findings of COVID-19 associated with acute respiratory distress syndrome. 37. Smith, T.R.F., Patel, A., Ramos, S., Elwood, D., Zhu, X., Yan, J., Gary, E.N., Walker, S.N., Schultheis, 487 K., Purwar, M., et al. (2020) . Immunogenicity of a DNA vaccine candidate for COVID-19. Nat 488

Commun 11, 2601 . 10.1038 Giorgi, E.E., Bhattacharya, T., Foley, B., et al. (2020) . Tracking Changes in SARS-CoV-2 Spike: 491

Evidence that D614G Increases Infectivity of the COVID-19 Virus. Cell 182, 812-827 e819. Sheahan, T., Baric, R., and Subbarao, K. (2008) . Animal models and vaccines for SARS-CoV 505

infection. Virus Res 133, 20-32. 10.1016 /j.virusres.2007 .03.025. 506 43. De Rosa, S.C., Edupuganti, S., Huang, Y., Han, X., Elizaga, M., Swann, E., Polakowski, L., Kalams, 507 S.A., Keefer, M.C., Maenza, J., et al. (2020 . Robust antibody and cellular responses induced by 508 DNA-only vaccination for HIV. JCI Insight 5. 10.1172/jci.insight.137079. 509 44. Patel, A., Reuschel, E.L., Xu, Z., Zaidi, F.I., Kim, K.Y., Scott, D.P., Mendoza, J., Ramos, S., Stoltz, R., 510 Feldmann, F., et al. (2021) . 

SARS vaccines: where are we?

A synthetic consensus anti-spike protein DNA 416 vaccine induces protective immunity against Middle East respiratory syndrome coronavirus in 417 nonhuman primates

A 419 recombinant VSV-vectored MERS-CoV vaccine induces neutralizing antibody and T cell 420 responses in rhesus monkeys after single dose immunization

A single dose of ChAdOx1 424 MERS provides protective immunity in rhesus macaques

Development of an inactivated vaccine candidate for SARS-CoV-2

ChAdOx1 nCoV-19 431 vaccine prevents SARS-CoV-2 pneumonia in rhesus macaques

Single-shot Ad26 vaccine protects against SARS-435

CoV-2 in rhesus macaques

Evaluation of the mRNA-1273 Vaccine against 438 SARS-CoV-2 in Nonhuman Primates

BNT162b vaccines protect rhesus macaques from SARS-CoV-2. 441

NVX-CoV2373 vaccine protects 444 cynomolgus macaque upper and lower airways against SARS-CoV-2 challenge

DNA vaccine protection against SARS-CoV-2 in 448 rhesus macaques

A neutralizing human antibody binds to the N-terminal domain of the Spike protein of 451 SARS-CoV-2

Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and 454 Treatment

Broad and strong memory CD4(+) and CD8(+) T cells induced by SARS-CoV-2 457 in UK convalescent individuals following COVID-19

Further information and requests for resources and reagents should be directed to and will be 602 fulfilled by the Lead Contact

Requests for resources and reagents should be directed to and will be fulfilled by the Lead 606

This includes plasmids, cell lines, and pseudoviruses. All reagents 607 will be made available on request following completion of a Material Transfer Agreement

610 Data: All data for this study are available without restriction from the Lead Contact upon 611 request

Code: This study did not generate code

General Statement: Any additional information required to reanalyze the data reported in this 614 study is available from the lead contact upon request

Experimental Model and Subject Details: 617 Cell lines: ACE2-CHO and ACE2-293T cells were maintained in Dulbecco's Modified Eagles

GIBCO) at 37°C, 5% CO 2 . All cell lines were tested to be mycoplasma

Ten Chinese rhesus macaques 625 (ranging from 4.55kg-5.55kg) were randomly assigned to be immunized (3 males and 2 females) 626 or naïve (2 males and 3 females). The sex and age in years old on day 0 for each animal were

At week 17, all animals were 632 challenged with 1.2x10 8 VP (1.1x10 4 PFU) SARS-CoV-2 isolate

Virus was administered as 1 ml by the intranasal route 636 (0.5 ml in each nostril) and 1 ml by the intratracheal route. Nasal swabs were collected during 637 the challenge period using Copan flocked swabs and placed into 1 ml PBS. Blood was collected 638 at indicated pre-challenge and post-challenge time points to analyse blood chemistry and to 639 isolate peripheral blood mononuclear cells (PBMC), and serum. Bronchoalveolar lavage was 640 collected at Week 8 and on Days 1, 2, 4, 7 post challenge to assay lung viral loads. BAL from , INO-4800: The highly optimized DNA sequence encoding SARS-CoV-2 IgE-645 spike was created using Inovio's proprietary in silico Gene Optimization Algorithm to enhance 646 expression and immunogenicity 37

BamHI and XhoI, and cloned into the expression vector pGX0001 under the control of the 648 human cytomegalovirus immediate-early promoter and a bovine growth hormone 649 polyadenylation signal

The tubes were centrifuged to 653 separate plasma and lymphocytes, according to the manufacturer's protocol. Samples were 654 transported by same-day shipment on cold-packs from Bioqual to The Wistar Institute for PBMC 655 isolation. PBMCs were washed and residual red blood cells were removed using ammonium-656 chloride-potassium (ACK) lysis buffer

Coulter) and resuspended in RPMI 1640 (Corning), supplemented with 10% fetal bovine serum 658 (Atlas), and 1% penicillin/streptomycin (Gibco). Fresh cells were then plated for IFNγ ELISpot 659 Assays and flow cytometry

Following PBMC 665 isolation, 200 000 cells were added to each well in the presence of 1) peptide pools (15-mers 666 with 9-mer overlaps) corresponding to the SARS-CoV-1, SARS-CoV-2, or MERS-CoV spike 667 proteins (5ug/mL/well final concentration), 2) R10 with DMSO (negative control), or 3) anti-668 CD3 positive control (Mabtech, 1:1000 dilution). All samples were plated in triplicate. Plates 669 were incubated overnight at 37°C, 5% CO 2 . After 18-20 hours, the plates were washed in PBS 670 and spots were developed according to the manufacturer's protocol. Spots were imaged using a 671 CTL Immunospot plate reader and antigen-specific responses were

Ninety-six well immunosorbent plates (NUNC) were coated with 1ug/mL 677 recombinant SARS-CoV-2 S1+S2 ECD protein (Sino Biological 40589-V08B1), S1 protein 678 (Sino Biological 40591-V08H), S2 protein (Sino Biological 40590-V08B), or receptor-binding 679 domain (RBD) protein (Sino Biological 40595-V05H

PBS-T) and blocked 683 with 5% skim milk in PBS-T (5% SM) for 90 minutes at 37°C. Sera or BAL from INO-4800 684 vaccinated and control macaques were serially diluted in 5% SM, added to the washed ELISA 685 plates, and then incubated for 1 hour at 37°C. Following incubation, plates were washed 4 times 686 with PBS-T and an anti-monkey IgG conjugated to horseradish peroxidase (Southern Biotech 687 4700-5) 1 hour at 37°C. Plates were washed 4 times with PBS-T and one-step TMB solution

ACE2 Competition ELISA Non-human primates: 96-well half area plates (Corning) were coated 693 at room temperature for 3 hours with 1 µg/mL PolyRab anti-His antibody

983B), followed by overnight blocking with blocking buffer containing 1x PBS, 5% SM, 1% 695 FBS, and 0.2% Tween-20. The plates were then incubated with 10 µg/mL of His6x-tagged

Sino Biological, 40589-V08B1) at room temperature for 1-2 hours

NHP sera (Day 0 or Week 6) was serially diluted 3-fold with 1XPBS containing 1% FBS and 698

The pre-699 mixture was then added to the plate and incubated at room temperature for 1-2 hours. The plates 700 were further incubated at room temperature for 1 hour with goat anti-mouse IgG H+L HRP 701 (A90-116P, Bethyl Laboratories) at 1:20,000 dilution followed by addition of one-step TMB 702 ultra substrate (ThermoFisher) and then quenched with 1M H 2

Single cell cloning 708 was done on these cells to generate cell lines with equivalent expression of ACE2-GFP

This mixture was then transferred to 712 150,000 293T-ACE2-GFP cells and incubated on ice for 90 min. Following this, the cells were 713 washed 2x with PBS followed by staining for surelight® APC conjugated anti-his antibody 714 (Abcam, ab72579) for 30 min on ice. As a positive control, spike protein was pre-incubated with 715 recombinant human ACE2 before transferring to 293T-ACE2-GFP cells. Data was acquired 716 using a BD LSRII

GeneJammer (Agilent) using IgE-SARS-CoV-2 spike plasmid (Genscript) 720 and pNL4-3.Luc.R-E-plasmid (NIH AIDS reagent) at a 1:1 ratio. Forty-eight hours post 721 transfection, supernatant was collected, enriched with FBS to 12% final volume, steri-filtered 722 (Millipore Sigma), and aliquoted for storage at -80°C. SARS-Cov-2 pseudovirus neutralization 723 assay was set up using D10 media

SARS-Cov-2 pseudovirus were titered to yield 726 greater than 20 times the cells only control relative luminescence units (RLU) after 72h of 727 infection. 10,000 CHO-ACE2 cells/well were plated in 96-well plates in 100ul D10 media and 728 rested overnight at 37˚C and 5% CO2 for 24 hours. The following day, sera from INO-4800 729 vaccinated and control groups were heat inactivated and serially diluted. Sera were incubated 730 with a fixed amount of SARS-Cov-2 pseudovirus for 90 minutes at RT. The sera+virus mix was 731 then added to the plated CHO-ACE2 cells and allowed to incubate in a standard incubator (37% 732 humidity, 5% CO2) for 72h. Cells were then lysed using Britelite plus luminescence reporter 733 gene assay system

Neutralization titers (ID50) were calculated using GraphPad Prism 8 and 735 control wells after subtraction of background RLU in cell control wells

on sera samples collected at pre-challenge (Weeks 0, 6, 740 15) and post-challenge. Vero 76 cells (ATCC No. CRL-1586) were plated at 175,000 cells/well 741 in DMEM + 10% FBS + gentamicin, overnight at 37°C, 5% CO 2 . Serially diluted serum 742 samples were mixed with SARS-CoV-2 virus (isolate USA-WA1/2020) for a final 30 pfu/well 743 virus per well and final starting 1:20 serum dilution

After incubation, 746 0.5% methylcellulose media (Sigma #M0512-100G) was added to each well and plates were 747 incubated at 37 o C, 5% CO 2 for 3 days. Each well was then washed and fixed with methanol, then 748 stained with 0.2% crystal violet (Sigma #HT901-8FOZ) in 20% MeOH for 30 min at room 749 temperature

Antibody-dependent cellular phagocytosis (ADCP): Biotinylated SARS-CoV-2 (COVID-19) S1

Acro Biosystems) were combined with fluorescent NeutrAvidin beads

Beads were washed with 1 ml buffer and spun at 14,000 x g 756 for 2 min at room temperature. Antigen-coated beads were resuspended in a final volume of 1 ml 757 in PBS-BSA. 10 μl beads were added into each well of a round-bottom 96-well culture plate after 758 which 5 µl of heat-inactivated monkey plasma were added and incubated for 2 h at 37 o C. A 200 759 µl suspension of THP-1 cells at 2.5 x 10 5 cells/ml were to each well, for a total of 5 x 10 4 THP-1 760 cells per well. After mixing, the cell-bead mixtures were incubated overnight at 37°C. The 761 following day, 100 μl of supernatant from each well were removed and 100 μl of BD Cytofix 762 were added to each well. Cells were analyzed by flow cytometry and data collected were 763 analyzed in FlowJo software. The percentage of fluorescent

COVID-19) S1 and RBD proteins (Acro Biosystems) for 768 30 min at 37 o C. Excess, unbound antigens were removed by washing cells once with complete 769 medium. Heat-inactivated monkey plasma (10 µl) were added to the antigen-pulsed cells and 770 incubated for another 30 min at 37 o C. Freshly resuspended lyophilized guinea pig complement 771 (Cedarlane) diluted 1:20 with veronal buffer 0

BioProducts) were added to the cells for 2 h at 37°C. Following a wash with 1X PBS, cells were 773 assessed for complement deposition by staining with goat anti-guinea pig C3-FITC (MP 774 biomedicals). After fixing, cells were analyzed by flow cytometry and ADCD are reported as 775 MFI of FITC+ cells

Viral RNA assay: RT-PCR assays were utilized to monitor viral loads, essentially as previously 778 described 56 . Briefly, RNA was extracted using a QIAcube HT (Qiagen,Germany) and the Cador 779 pathogen HT kit from bronchoalveolar lavage (BAL) supernatant and nasal swabs

Applied Biosystems) according to 782 manufacturer's specifications. Viral loads were calculated of viral RNA copies per mL or per 783 swab and the assay sensitivity was 50 copies. The target for amplification was the SARS-CoV2

N (nucleocapsid) gene. The primers and probes for the targets were

To generate a standard curve, 791 the SARS-CoV-2 E gene sgmRNA was cloned into a pcDNA3.1 expression plasmid; this insert 792 was transcribed using an AmpliCap-Max T7 High Yield Message Maker Kit (Cellscript) to 793 obtain RNA for standards. Prior to RT-PCR, samples collected from challenged animals or 794 standards were reverse-transcribed using Superscript III VILO (Invitrogen) according to the 795 manufacturer's instructions. A Taqman custom gene expression assay (ThermoFisher Scientific) 796 was designed using the sequences targeting the E gene sgmRNA 41 . Reactions were carried out 797 on a QuantStudio 6 and 7 Flex Real-Time PCR System (Applied Biosystems) according to the 798 manufacturer's specifications. Standard curves were used to calculate sgmRNA in copies per ml 799 or per swab

All bar 803 graphs, scatter plots, and line graphs display individual animals or the mean value, and error bars 804 represent the standard deviation. A two-tailed Mann-Whitney test was performed for viral load 805 comparison between vaccinated and unvaccinated animals. Samples and animal groups with a p 806 value < 0.05 were considered statistically significant

demonstrate that immunization with a DNA vaccine encoding the SARS-CoV-2 spike antigen, INO-4800, induces durable immune responses in rhesus macaques and is associated with reduced viral loads after challenge