key: cord-0964641-a451243b
authors: Andrade, Viviane M.; Christensen-Quick, Aaron; Agnes, Joseph; Tur, Jared; Reed, Charles; Kalia, Richa; Marrero, Idania; Elwood, Dustin; Schultheis, Katherine; Purwar, Mansi; Reuschel, Emma; McMullan, Trevor; Pezzoli, Patrick; Kraynyak, Kim; Sylvester, Albert; Mammen, Mammen P.; Tebas, Pablo; Kim, J. Joseph; Weiner, David B.; Smith, Trevor R.F.; Ramos, Stephanie J.; Humeau, Laurent M.; Boyer, Jean D.; Broderick, Kate E.
title: INO-4800 DNA Vaccine Induces Neutralizing Antibodies and T cell Activity Against Global SARS-CoV-2 Variants
date: 2021-04-14
journal: bioRxiv
DOI: 10.1101/2021.04.14.439719
sha: 70885faea27a22323bfe0b751ca0e0516c182518
doc_id: 964641
cord_uid: a451243b

Global surveillance has identified emerging SARS-CoV-2 variants of concern (VOC) associated with broadened host specificity, pathogenicity, and immune evasion to vaccine induced immunity. Here we compared humoral and cellular responses against SARS-CoV-2 VOC in subjects immunized with the DNA vaccine, INO-4800. INO-4800 vaccination induced neutralizing antibodies against all variants tested, with reduced levels detected against B.1.351. IFNγ T cell responses were fully maintained against all variants tested.

INO-4800 is a SARS-CoV-2 Spike DNA-based vaccine that is delivered intradermally followed by electroporation (EP) using CELLECTRA ® 2000 and is currently undergoing clinical development. In a Phase 1 clinical trial, INO-4800 vaccination induced a balanced immune response characterized by both functional antibody and T cell responses in vaccinated subjects [13] . Both humoral and cellular immune responses have been shown to be important components of protection against betacoronaviruses [14] [15] [16] . In the present study, we have assessed the humoral and T cell responses against SARS-CoV-2 B.1.1.7, B.1.351 and P.1 variants elicited after INO-4800 vaccination ( Figure S1A ).

In INO-4800 vaccinated subjects, serum IgG antibody binding titers to SARS-CoV-2 full-length Spike proteins were evaluated by ELISA using proteins specific for B.1.1.7, B.1.351, and P.1 variants (Fig. 1A , and S1A). IgG binding titers were not negatively impacted between WT and B.1.1.7 or B.1.351 variants. An average 1.9-fold reduction was observed for the P.1 variant in subjects tested at week 8 after receiving two doses of INO-4800 (Fig. 1A) .

We performed a SARS-CoV-2 pseudovirus neutralization assay using sera collected from thirteen subjects two weeks after administration of a third dose of either 0.5 mg, 1 mg, or 2 mg of INO-4800 (Table S1 ). Neutralizing activity was detected against WT and the emerging variants in all serum samples tested (Fig. 1B) . The mean ID50 titers for the WT, B.1.1.7, B.1.351 and P.1. were 643, 295, 105, and 664, respectively (Table S1 ). Compared to WT, there was a 2.1 and 6.9-fold reduction for B.1.1.7 and B.1.351, respectively, while there was no difference between WT and the P.1 variant. These results are consistent with other recent studies, which have demonstrated a significant reduction in neutralizing activity in vaccinated individuals towards the B.1.351 (≥6fold reduction), while the B.1.1.7 lineage has demonstrated a reduced activity of 2-fold or less [4, [7] [8] [9] 17] . Strikingly, while the P.1 strain presents with similar RBD mutations as B.1.351, we did not observe any reduction in neutralizing activity compared to the WT strain in INO-4800 vaccinated individuals [18, 19] . The P.1 lineage has similar changes in the RBD to the B.1.351 lineage as they contain the N501Y mutation found in the B.1.1.7 lineage and identical E484K mutations. However, they have similar but different mutations at position K417, with a change to T for the P.1 and to N for the B.1.351 lineage. While both are changes to polar uncharged side chains, the B1.351 N mutation carries an additional amide group. It is possible that this position and this change allows for the impact of neutralizing responses observed.

Recent reports have supported an important role for T cell immunity in protecting against COVID-19 in the absence of antibodies to SARS-CoV-2 [20, 21] . Through optimized DNA construct design, combined with intradermal enhanced delivery, Inovio's DNA platform technology has been demonstrated to drive balanced humoral and cellular immune responses to a wide range of infectious disease and tumor antigen targets [22] [23] [24] . We therefore compared cellular immune responses to WT and SARS-CoV- (Fig. 2 ). This is consistent with published results showing that, compared with neutralizing antibody responses, cellular immunity is relatively unimpaired by the current variants of concern [25] . Here, we show that T cell responses are consistently maintained between WT and all SARS-CoV-2 variants tested, including B.1.351 and P.1. Cells stimulated with peptides against these variants generated IFNγ responses as well as cytokines associated with CD8+ cytotoxic T cell responses (data not shown).

There is a growing concern regarding the protective efficacy of vaccines recently approved for emergency use and those vaccines currently in development against the variants of concern. Recent studies have shown a reduction in serum neutralization levels against B.1.351 and P.1 [4, 7, 18] , with minimal impact against B.1.1.7 [17] . In addition, vaccine trials have shown a considerable reduction in protective efficacy against B.1.351 [11, 12, 26] . Interestingly, a small reduction in protective efficacy is observed in Latin American countries including Brazil, suggesting that P.1 may have emerged around the time of clinical trials were in effect [26] . Here we report the neutralizing antibody and T cell activity measured in INO-4800 vaccinated subjects against emerging SARS-CoV-2 variants first detected in the United Kingdom, South Africa, and Brazil. The neutralization levels against B.1.351 and B.1.1.7 for the INO-4800 SARS-CoV-2 Spike DNA vaccine are consistent with previous reports of subjects receiving vaccines encoding for the ancestral Spike protein [7, 27] . Surprisingly, despite recent reports showing a reduction in neutralizing activity against the P.1 variant [8, 18] , INO-4800 generates robust neutralizing antibodies at levels comparable to the WT strain. INO-4800 induces cross-reactive T cell responses against B.1.1.7, B.1.351, and P.1 variants that are comparable to the WT strain. Taken together, these data demonstrate maintenance of one or both cellular and humoral arms of the immune response against emerging SARS-CoV-2 variants for the INO-4800 vaccine, which will likely be critical factors to impact the ongoing COVID-19 pandemic. 

Clinical Trial Subject Samples: Serum and PBMC samples were acquired from participants of the phase I INO-4800 clinical trial (NCT04336410) described previously [13] . The trial has since been expanded to include participants of 51-64 and 64+ years of age as separate groups in addition to the original 18-50 age group. A 0.5 mg dose group was also added. Sera from 20 subjects out of the 120 total study participants were selected for analysis on variant Spike protein binding ELISAs and variant pseudovirus neutralization assays. The samples analyzed by pseudovirus neutralization assay were collected from subjects two weeks after a third dose of INO-4800, and the samples used for other ELISA and ELISpot were collected after two doses.

Antigen Binding ELISA: Binding ELISAs were performed as described previously [13] , except different variants of SARS-CoV-2 S1+S2 proteins were used for plate coating. The S1+S2 wildtype Spike protein (Acro Biosystems #SPN-C52H8) contained amino acids 16-1213 of the full Spike protein (Accession #QHD43416.1) with R683A and R685A mutations to eliminate the furin cleavage site. The B.1.1.7, B.1.351, and P.1 S1+S2 variant proteins (Acro Biosystems #SPN-C52Hc,#SPN-C52H6, and #SPN-C52Hg, respectively) additionally contained the following proline substitutions for trimeric protein stabilization: F817P, A892P, A899P, A942P, K986P, and V987P. The B.1.1.7 protein contained the following variant-specific amino acid substitutions: HV69-70del, Y144del, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H; the B.1.351 protein contained the following substitutions: L18F, D80A, D215G, R246I, K417N, E484K, N501Y, D614G, A701V; and the P.1 protein contained the following: L18F,T20N,P26S, D138Y,R190S,K417T,E484K,N501Y,D614G,H655Y,T1027I,V1176F. Assay plates were coated using 100 µL of 2 µg/mL of protein.

Production: SARS-CoV-2 pseudovirus stocks encoding for the WT, B.1.1.7, B.1.351 or P.1 Spike protein were produced using HEK 293T cells transfected with Lipofectamine 3000 (ThermoFisher) using IgE-SARS-CoV-2 S plasmid variants (Genscript) cotransfected with pNL4-3.Luc.R-E-plasmid (NIH AIDS reagent) at a 1:8 ratio. 72h post transfection, supernatants were collected, steri-filtered (Millipore Sigma), and aliquoted for storage at -80°C.

CHO cells stably expressing ACE2 (ACE2-CHOs) were used as target cells plated at 10,000 cells/well. SARS-CoV-2 pseudovirus were tittered to yield greater than 30 times the cells only control relative luminescence units (RLU) after 72h of infection. Sera from 13 INO-4800 vaccinated subjects were heat inactivated and serially diluted two-fold starting at 1:16 dilution. Sera were incubated with SARS-CoV-2 pseudovirus for 90 min at room temperature. After incubation, sera-pseudovirus mixture was added to ACE2-CHOs and allowed to incubate in a standard incubator (37% humidity, 5% CO2) for 72h. After 72h, cells were lysed using Bright-Glo™ Luciferase Assay (Promega) and RLU was measured using an automated luminometer. Neutralization titers (ID50) were calculated using GraphPad Prism 8 and defined as the reciprocal serum dilution at which RLU were reduced by 50% compared to RLU in virus control wells after subtraction of background RLU in cell control wells. . Cells were incubated overnight with peptide pools at a concentration of 1 μg per ml per peptide in a precoated ELISpot plate, (MabTech, Human IFNγ ELISpot Plus). Cells were then washed off, and the plates were developed via a biotinylated anti-IFN-γ detection antibody followed by a streptavidin-enzyme conjugate resulting in visible spots. After plates were developed, spots were scanned and quantified using the CTL S6 Micro Analyzer (CTL) with ImmunoCapture and ImmunoSpot software. Values are shown as the backgroundsubtracted average of measured triplicates. The ELISpot assay qualification determined that 12 spot forming units was the lower limit of detection. Thus, anything above this cutoff signal is an antigen specific cellular response.

Statistical Methods: GraphPad Prism 8.1.2 (GraphPad Software, San Diego, USA) was used for graphical and statistical analysis of data sets. P values of <0.05 were considered statistically significant. A nonparametric two-tailed student t-test Wilcoxon signed-rank test was used to assess statistical significance in Figures 1 and 2 .

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

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We acknowledge the members of the Inovio Pharmaceuticals R&D department for significant technical assistance. This work is funded by Coalition for Epidemic Preparedness Innovations (CEPI). 

T.R.F.S., J.J.K., D.E., S.J.R., A.CQ., I.M., J.A., C.R., J.T., T.M., K.S., P.P., T.H., V.M.A., J.D.B., L.M.H., and K.E.B. are employees of Inovio Pharmaceuticals and as such receive salary and benefits, including ownership of stock and stock options, from the company. D.B.W. has received grant funding, participates in industry collaborations, has received speaking honoraria, and has received fees for consulting, including serving on scientific review committees and board services. Remuneration received by D.B.W. includes direct payments or stock or stock options, and in the interest of disclosure he notes potential conflicts associated with this work with Inovio and possibly others. In addition, he has a patent DNA vaccine delivery pending to Inovio. All other authors report there are no competing interests.

Correspondence and requests for materials should be addressed to Kate E. Broderick.