key: cord-0820607-d6zm5p1b
authors: Seo, Yong Bok; Suh, You Suk; Ryu, Ji In; Jang, Hwanhee; Oh, Hanseul; Koo, Bon-Sang; Seo, Sang-Hwan; Hong, Jung Joo; Song, Manki; Kim, Sung-Joo; Sung, Young Chul
title: Soluble Spike DNA vaccine provides long-term protective immunity against SAR-CoV-2 in mice and nonhuman primates
date: 2020-10-10
journal: bioRxiv
DOI: 10.1101/2020.10.09.334136
sha: b223d54d2174cdce38f2743b68ed2f287795272f
doc_id: 820607
cord_uid: d6zm5p1b

The unprecedented and rapid spread of SARS-CoV-2 has motivated the need for a rapidly producible and scalable vaccine. Here, we developed a synthetic soluble SARS-CoV-2 spike (S) DNA-based vaccine candidate, GX-19. In mice, immunization with GX-19 elicited not only S-specific systemic and pulmonary antibody responses but also Th1-biased T cell responses in a dose-dependent manner. GX-19 vaccinated nonhuman primate seroconverted rapidly and exhibited detectable neutralizing antibody response as well as multifunctional CD4+ and CD8+ T cell responses. Notably, when the immunized nonhuman primates were challenged at 10 weeks after the last vaccination with GX-19, they did not develop fever and reduced viral loads in contrast to non-vaccinated primates as a control. These findings indicate that GX-19 vaccination provides durable protective immune response and also support further development of GX-19 as a vaccine candidate for SARS-CoV-2 in human clinical trials.

and non-human primate (NHP) models. Notably, the vaccine drives potent cellular and humoral response in NHPs, including neutralizing antibodies that provide potent protective efficacy against SARS-CoV-2 infection.

The data demonstrate that the immunogenicity of this DNA vaccine supports the clinical development to advance the development of this DNA vaccine in response to the current global health crisis.

The representative sequence for the SARS-CoV-2 S protein was generated after analysis of the S protein genomic sequences, which were retrieved from the NCBI SARS-CoV-2 resources. The full-length or entire ectodomain of S gene was codon optimized for increased antigen expression in mammalian cells and Nterminal tissue plasminogen activator (tPA) signal sequence was added, respectively. The insert was then subcloned into the pGX27 vector (21) and the resulting plasmid was designated as pGX27-S and pGX27-S Δ TM (Fig. 1A) .

To assess the immunogenicity of two candidate DNA vaccines, we immunized six-week old female BALB/c mice intramuscularly (IM) twice at 2-week interval. As indicated Fig. 1B , immunized with the both of DNA vaccine candidate induced a robust S protein-specific antibody response compared to the control.

Interestingly, there were higher antibody titers in the pGX27-S Δ TM group than in the pGX27-S group at both post-prime and post-boost. These results were not consistent with the previous report that demonstrated higher binding antibody in full-length S DNA vaccinated macaques compared to S Δ TM DNA vaccinated macaques (8).

pGX27-S

To investigate whether GX-19 was capable of inducing immune responses in a NHP model, three macaques were vaccinated with electroporation (EP)-enhanced delivery three times about 3-week intervals with GX-19, as described in Methods. Blood was collected at week-0, -5.5, and -8 to monitor vaccine-induced immune responses. The binding ELISA results showed that all three macaques immunized with GX-19 seroconverted after a single immunization with anti-S IgG titers tending to increase by boosting vaccination (Fig.   4A ). In addition, sera collected at week-0, -5.5 and -8 were further analyzed for neutralization of wild-type SARS-CoV-2 (Korea CDC) by the 50% plaque reduction neutralization test (PRNT 50 ). Three macaques immunized with GX-19 displayed elevated neutralization titers with mean PRNT 50 titers of 1 : 285 (at week 5.5) and 1 : 996 (at week 8), respectively (Fig. 4B ).

To determine the impact of the GX-19 on cellular immune response, ELISPOT analysis was used to measure T cell responses in blood of the vaccinated macaques. Three macaques developed T cell responses after single immunization. In addition, all animals exhibited such an elevated responses after boost vaccination indicating vaccine-induced cellular immune responses became progressively stronger in macaques during GX-19 vaccination (Fig. 4C ). To gain further insight into the responses of GX-19-induced T cell responses, we also measured multiple cytokines by ICS as described in Methods. GX-19 vaccination exhibited meaningful induction S-specific CD4 + T cells or CD8 + T cells producing IFN-γ, TNF-α and, to a lesser extent, IL-2 ( 

Induction of long-term immunological memory for T cell and B cell responses is important for effective vaccine development. Unlike other vaccine studies in which NHPs confirmed protective efficacy against SARS-CoV-2 infection within 4 weeks after the last vaccination (8, 9, 26, 27) , we evaluated the protective efficacy approximately 10 weeks after the last vaccination. To assess the protective efficacy of GX-19, macaques were challenged by multiple routes with a total dose of 2.6 x 10 7 50% tissue-culture infectious doses (TCID 50 ), on 10 weeks after the last vaccination. This challenge route and dose were based on a model development study in which we challenged macaques that had no previous exposure to the virus (28) . vaccinated macaques showed no increase in body temperature after viral infection, and showed rapid recovery in lymphocyte reduction compared to unvaccinated macaques (Supplementary Fig. 3A and 3B ). High levels of viral load were observed in the unvaccinated macaques ( Fig. 5A and 5B) with a median peak of 7.54 (range 6.66 -8.01) log 10 viral copies/ml in nasal swab and a median peak of 6.18 (range 6.01 -6.81) log 10 viral copies/ml in throat swab ( Fig. 5C and 5D ). Although there is no statistical significance due to insufficient number of animals, lower levels of viral load were observed in GX-19 vaccinated macaques, including 1.58 and 1.57 log 10 reductions of median peak viral load in nasal swab and throat swab, respectively (Fig 5A -D) . Since the peak viral load does not reflect the presence of total virus over time, the virus load then calculated based on the area under curve (AUC). GX-19 vaccinated macaques had a viral AUC of 6.02 ± 0.23 log 10 in nasal swab and 4.99 ± 0.45 log 10 in throat swab, respectively, which were 1.46 and 1.45 log 10 decreases of the viral AUC compared to unvaccinated macaques respectively ( Fig. 5E and 5F ). To confirm for infectious viruses, TCID 50 assay was performed for nasopharyngeal swab and oropharyngeal swab samples on Vero cells. The infectious viral load showed the similar pattern as viral RNA load. Although there is no statistical significance due to insufficient number of animal, lower levels of infectious viral load were observed in GX-19 vaccinated macaques (Supplementary Fig. 3C and 3D ).

At 4 days post virus inoculation, all animals were euthanized, and tissues were collected. Consistent with previous reports (28) , SARS-CoV-2 infection caused moderate-to-severe inflammation, as evidenced by small airways and the adjacent alveolar interstitia in non-vaccinated macaques. In vaccinated macaques, the viral challenge caused mild histopathologic changes compared to those in control macaques (Fig. 5G ).

In this study, we demonstrated that GX-19 (pGX27-S Δ TM ) exhibited higher S-specific antibody response than pGX27-S. In addition, GX-19 can elicit SARS-CoV-2 S-specific Th1-biased T cell response in mice and NHPs. Vaccination of GX-19 can confer effective protection against SARS-CoV-2 challenge at 10 weeks following the last vaccination.

In a recent study, the low immunogenicity and protective efficacy of S Δ TM was reported in the evaluation of the protective efficacy of DNA vaccine expressing various forms of SARS-CoV-2 S protein (8). In contrast to full-length S DNA here, we showed that GX-19 not only induces excellent antibody responses in mice and NHPs, but also effective protection against SARS-CoV-2 virus challenge in NHPs. The different results can be explained by the difference in the cellular localization of antigen and the difference in the strength of the induced immune responses by vaccination. In a study on the effects of cellular and humoral immune responses according to cellular localization of antigens after DNA immunization, it was found that the DNA encoding secreted OVA produces a much higher immune response than the cytoplasmic or membrane-bound form (29) . In the previous study (8), the vaccines were administered without EP method which significantly enhanced the in vivo delivery efficacy of DNA vaccine by 100 -1,000 folds (30) , and as a result, they induced a weak antibody responses or T cell responses. On the other hand, electroporation-enhanced GX-19 induced robust antibody and T cell responses. In addition, the strength of the immune response increases depending on the strength of the expression vector (31) , and pGX27 vector has about three-time higher expression strength than commercial vector (unpublished data). Here, we believe that the introduction of a high-expression vector (21) into GX-19 along with an effective EP delivery system resulted in the efficient protective effect against SARS-CoV-2 infection through the induction of a strong immune responses. However, further studies will be needed to compare pGX27-S and pGX27-S Δ TM in NHPs under our conditions to confirm this finding.

In this study, we observed that GX-19 induced concurrent antibody, CD4 + T, and CD8 + T cell response in both mice and NHPs models. Indeed, successful DNA vaccination effectively induces complete complementation of the immune responses, including humoral and cellular responses (CD8 + and Th1 cellular responses) similar to those achieved by live attenuated viruses (14, 15, 32, 33) . This can be explained by the nature of DNA vaccine, presumably because both class-I antigen-processing pathways (i.e., intracellular processing of viral proteins into peptides and subsequent loading onto MHC class-I molecules) and class-II antigen-processing pathways (i.e., specifically engineered in the S signal sequence that cause an increased export of viral surface antigens) are possible. Among T cell responses, the balanced Th1/Th2 responses are important because vaccine-associated enhanced respiratory disease (VAERD) is associated with Th2-biased immune response. Indeed, immunopathologic complications characterized by Th2-biased immune responses have been reported in animal model of the SARS-CoV or MERS-CoV challenge (22, (34) (35) (36) (37) (38) , and similar phenomena have been reported in clinics vaccinated with whole-inactivated virus vaccines against RSV and measles virus (39, 40) . In addition, the importance of T cell responses has been highlighted by recent study of asymptomatic and mild SARS-CoV-2 convalescent (41) . These results collectively suggest that vaccines capable of generating balanced antibody responses and T cell responses may be important in providing protection against SARS-CoV-2 diseases. Here, we show that GX-19 induces Th1-biased responses, suggesting DNA vaccination can avoid Th2-biased immune response associated with VARED. In fact, there were subtle pathologic changes in the SARS-CoV-2 infected GX-19 vaccine group. These results were also demonstrated in the other respiratory infection DNA vaccine such as SARS-CoV and MERS-CoV (42), . This suggests that DNA vaccine platform can be a good alternative in vaccine development for emerging infections where the balanced T cell response as well as antibody response is important.

It is desirable that the SARS-CoV-2 vaccine can prevent infection or disease and induce long-term immunity. Virus-specific T cell responses play an important role in antiviral and disease control. Immunemodulatory cytokines (e.g., IFN-γ, TNF-α, and IL-2) released from virus-specific CD4 + T and CD8 + T cells play a key role in several antiviral responses and act in synergy with type I IFNs to inhibit viral replication (43, 44) .

Patients with impaired IFN-γ activity were reported to have 5-fold increased susceptibility to SARS (45) .

Clinical cases of asymptomatic virus infection indicate that virus-specific T cells can control disease even in the absence of neutralizing antibodies (41, 46) . Here, we showed that GX-19 induce potent antigen-specific CD4 + and CD8 + T cell activation and robust release of immune-modulatory cytokines in mice and NHPs, indicating that GX-19 can effectively control the disease of SARS-CoV-2. Next, clinical cases in which antibody responses rapidly decrease and disappear after SARS-CoV-2 infection indicate the importance of vaccines that can induce long-term immunological memory (47) (48) (49) . GX-19 induced potent CD4 + and CD8 + T cells in both animal models and it may confer long-lasting immunity against coronaviruses as indicated in SARS survivors, where CD8 + T cell immunity persisted up to 11 years (43, 50) . Although the results of the long-term immune response were not covered here, we observed an effective protection against viral infection about 10 weeks after the last vaccination, indicating that the GX-19-induced memory immune response is also effective against long-term infection. In summary, these results explain the promising immunogenicity of the GX-19 and, in particular, Pathogens (accession number 43326), via combined routes (intratracheal, oral, conjunctival, intranasal, and intravenous route) as previously described (28) . After viral challenge, macaques were anesthetized with a ketamine sodium (10 mg/kg) and tiletamine/zolazepam (5 mg/kg) at 0, 1, 2, 3, and 4 days post-infection (dpi) and conducted the following procedure: checking body temperature, weight, and respiration rate and collecting nasopharyngeal, oropharyngeal swab samples in universal transport medium. Swab samples were centrifuged at 1600 x g for 10 min and filtered with 0.2 μ m pore size syringe filters for further virus quantification. Antigen binding ELISA. Serum and BAL fluid was collected at each time point was evaluated for binding titers. Ninety-six well immunosorbent plates (NUNC) were coated with 1 μ g/mL recombinant SARS-CoV-2 1 S1+S2 ECD protein (Sino Biological 40589-V08B1), S1 protein (Sino Biological 40591-V08H) in PBS overnight at 4°C. Plates were washed 3 times with 0.05% PBST (Tween 20 in PBS) and blocked with 5% skim milk in 0.05% PBST (SM) for 2-3 hours at room temperature. Sera or BAL fluid were serially diluted in 5% SM, added to the wells and incubated for 2 hours at 37°C. Following incubation, plates were washed 5 times with 0.05% PBST and then incubated with horseradish peroxidase ( 

For mouse samples, the Mouse IFN-γ ELISPOT set (BD 551083) was used as directed by the manufacturer. ELISPOT plates were coated with purified anti-mouse IFN-γ capture antibody and incubated overnight at 4°C. Plates were washed and blocked for 2 hours with RPMI + 10% FBS (R10 media) Five hundred thousand splenocytes were added to each well and stimulated for 24 hours at 37°C in 5% CO 2 with R10 media (negative control), concanavalin A (positive control), or specific peptide antigens ( 

for 3 days at 37℃, for virus isolation to calculate the values of TCID50/mL using the Reed and Muench method.

The viral RNA genome was extracted from the supernatant using QIAamp Viral RNA Mini Kit (Qiagen) and stored at -80℃ in the ABL-3 facility until use. RT-qPCR was performed with a primer set targeting partial regions of the ORF1b gene in the SARS-CoV-2 virus using the QIAGEN OneStep RT-PCR kit (Qiagen) as previously reported (51) . For all RT-qPCR analyses, SARS-CoV-2 RNA standard and negative samples were run in parallel for determination of virus copy number.

Histological evaluation. Six lobes of the lung samples (three lobes in the right and left lung, namely upper, middle and lower lobe) of infected macaques were fixed in 4% paraformaldehyde for a minimum of 7 days, embedded in paraffin, and 4-to 5-μm sections were stained with hematoxylin and eosin.

Statistical analysis. Analysis of virologic and immunologic data was performed using GraphPad Prism 5 (GraphPad Software). Comparison of data between groups was performed using two-sided Mann-Whitney tests. Schematic diagram of COVID-19 DNA vaccine expressing soluble SARS-CoV-2 S protein (S Δ TM ) or full-length SARS-CoV-2 S protein (S) (A). BALB/c mice (n=4-10/group) were immunized at week 0 and 2 with pGX27-S Δ TM , pGX27-S or pGX27 (empty control vector) as described in the methods. Sera were collected 2 weeks post-prime (blue) and 2 weeks post-boost (red) and evaluated for SARS-CoV-2 S-specific IgG antibodies (B). 

of SARS-CoV-2 S-specific IFN-γ secreting cells in PBMCs was determined by IFN-γ ELISPOT assay after stimulation with peptide pools spanning the SARS-CoV-2 S protein. Shown are spot-forming cells (SFC) per 10 6 PBMCS in triplicate wells (C). The frequency of S-specific CD4 + or CD8 + T cells producing IFN-γ, TNF-α, or IL-2 was determined by intracellular cytokine staining assays stimulated with SARS-CoV-2 S peptide pools.

Shown are the frequency of S-specific CD4 + or CD8 + T cells after subtraction of background (DMSO vehicle) (D). Mouse splenocytes were stimulated with specific peptide pools and then analyzed with multicolor flow cytometry to simultaneously detect SARS-CoV-2 S-specific expression of IFN-γ, TNF-α, and IL-2. Gating strategy to identify CD4 + or CD8 + T cells (A). The representative plots show the frequencies of IFN-γ, TNF-α, IL-2 producing CD4 + or CD8 + T cells, respectively (B).

Cryopreserved PBMCs of GX-19 vaccinated macaques were stimulated with specific peptide pools and then analyzed with multicolor flow cytometry to simultaneously detect SARS-CoV-2 S-specific expression of IFN-γ, TNF-α, and IL-2. Gating strategy to identify CD4 + or CD8 + T cells (A). The representative plots show the frequencies of IFN-γ, TNF-α, IL-2 producing CD4 + or CD8 + T cells, respectively (B). 

After viral challenge, macaques were anesthetized for checking body temperature (A), and blood lymphocyte count (B) at 0, 1, 2, 3, and 4 days post-infection (dpi). TCID 50 /mL was measured in nasopharyngeal (C), and oropharyngeal (D) at multiple time-points following challenge.

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The pathogen resources (NCCP43326) for this study were provided by the National Culture Collection for Pathogens.