INO-4800 DNA vaccine induces neutralizing antibodies and T cell activity against global SARS-CoV-2 variants

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.

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.
npj Vaccines (2021) 6:121 ; https://doi.org/10.1038/s41541-021-00384-7 MAIN TEXT SARS-CoV-2, the causative agent of the COVID-19 pandemic, continues to cause unprecedented levels of mortality and socioeconomic burden. Concerningly, virus surveillance shows the global spread of novel SARS-CoV-2 variants, which are more infectious and display increased transmissibility and pathology [1][2][3] . Some of these variants of concern (VOC) contain mutations in the Spike protein receptor-binding domain (RBD), the region which interacts with the host ACE2 receptor, and to which many SARS-CoV-2 neutralizing antibodies target. The B.1.1.7 lineage, the first emerging VOC, contains the key N501Y and D614G mutations and the 69-70 deletion in the RBD and/or S1 regions and exhibits increased transmissibility, but does not appear to significantly evade neutralizing antibody responses generated by current vaccines approved for use 4,5 . The B.1.351 and P.1 lineages have additional mutations, including E484K in the RBD region 6-8 , as well as unique changes in position K417. Notably, sera isolated from convalescent individuals and vaccinees exposed to the original Wuhan Spike protein sequence have shown significantly lower levels of neutralizing activity against the B.1.351 and P.1 variants [8][9][10][11][12] .
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 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 ( Supplementary 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 0.5, 1, or 2 mg of INO-4800 (Supplementary Table 1). Neutralizing activity was detected against Wuhan and the emerging variants in all serum samples tested (Fig. 1b). The mean ID 50 titers for the Wuhan, B.  (Fig. 2). This is consistent with published results showing that, compared with neutralizing antibody responses, cellular immunity is relatively unimpaired by current VOCs 25 . Here, we show that T cell responses are consistently maintained between Wuhan 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 VOC. Recent studies have shown a dramatic reduction in serum neutralization levels against B.1.351, a 2 to 4-fold reduction against P.1 4,7,18 , and a 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  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. The institutional review board of each clinical site approved the trial. All trial participants provided written informed consent. 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  . 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 Immuno-Spot software. Values are shown as the background-subtracted average of measured triplicates. The ELISpot assay qualification determined that 12 spot forming units were 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 Figs. 1 and 2.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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