key: cord-1048292-0s3vwnl0
authors: Brocato, Rebecca L.; Kwilas, Steven A.; Kim, Robert K.; Zeng, Xiankun; Principe, Lucia M.; Smith, Jeffrey M.; Hooper, Jay W.
title: Protective efficacy of a SARS-CoV-2 DNA Vaccine in wild-type and immunosuppressed Syrian hamsters
date: 2020-11-10
journal: bioRxiv
DOI: 10.1101/2020.11.10.376905
sha: eae57ad8cf04cadc5a7d177dadb536e44447d44a
doc_id: 1048292
cord_uid: 0s3vwnl0

A worldwide effort to counter the COVID-19 pandemic has resulted in hundreds of candidate vaccines moving through various stages of research and development, including several vaccines in phase 1, 2 and 3 clinical trials. A relatively small number of these vaccines have been evaluated in SARS-CoV-2 disease models, and fewer in a severe disease model. Here, a SARS-CoV-2 DNA targeting the spike protein and delivered by jet injection, nCoV-S(JET), elicited neutralizing antibodies in hamsters and was protective in both wild-type and transiently immunosuppressed hamster models. This study highlights the DNA vaccine, nCoV-S(JET), we developed has a great potential to move to next stage of preclinical studies, and it also demonstrates that the transiently-immunosuppressed Syrian hamsters, which recapitulate severe and prolonged COVID-19 disease, can be used for preclinical evaluation of the protective efficacy of spike-based COVID-19 vaccine.

The COVID-19 pandemic has necessitated the rapid development of candidate vaccines and treatments targeting the SARS-CoV-2. Infection with SARS-CoV-2 results in either asymptomatic infection or disease ranging from mild to severe respiratory symptoms 1 . Many factors contribute to the spread of this virus, including a large number of asymptomatic cases 2 and transmission prior to the onset of symptoms 3 . An effective vaccine would be an invaluable medical countermeasure to protect individuals, prevent transmission, and contribute to containing and ultimately ending this pandemic.

According to the World Health Organization, as of 30 September 2020, there were 41 SARS-CoV-2 vaccines in clinical trials (Phases I, II and III) and 151 vaccines in preclinical development 4 . Of these vaccines in preclinical development several have been tested for immunogenicity in mice and nonhuman primates. Few have been tested in disease models such as the Syrian hamster model. The Syrian hamster has become a leading animal model for SARS-CoV-2 medical countermeasure testing because it does not require a modified virus, or animal, and there are several similarities to human COVID-19 disease including rapid breathing, lethargy, ruffled fur and moderate (<10%) weight loss 5 6 . Histopathology includes areas of lung consolidation, followed by pneumocyte hyperplasia as the virus is cleared. At least three candidate vaccines have been tested for efficacy in the Syrian hamster model [7] [8] [9] .

We have developed a Syrian hamster model of severe COVID-19 disease by using cyclophosphamide (CyP) to transiently immunosuppress the hamsters 10 . In this model, lymphopenia is induced by CyP treatment starting 3 days before exposure to virus. After a relatively low dose of virus (1,000 PFU), the immunosuppressed hamsters develop a protracted disease with >15% weight loss over several days and other indicators of severe disease including high levels of virus in the lungs. Herein, we describe the testing of a jet-injected SARS-CoV-2 DNA vaccine in both wild-type and transiently-immunosuppressed hamsters. Hantavirus DNA vaccines administered at a dosage of 0.2 mg are highly immunogenic in hamsters when administered using jet injection 11 . Therefore, as an initial proof-of-concept, we opted to use the 0.2 mg dose.

A SARS-CoV-2 spike-based DNA vaccine, nCoV-S(JET), was constructed by cloning a humancodon-optimized gene encoding the full-length spike protein into a plasmid vector as described in Methods. The plasmid backbone used for this vaccine, pWRG, has been used for hantavirus DNA vaccines that are currently in phase 1 and 2 clinical trials 12 . Expression of the spike protein from the nCoV-S(JET) was confirmed to express in cell culture (Suppl. Fig. S1 ). In the first vaccine efficacy experiment, groups of 8 hamsters were vaccinated on week 0 and 3 with either 0.2 mg nCoV-S(JET), or 0.2 mg of a MERS-CoV DNA vaccine, or PBS using jet injection ( Fig. 1A) . Sera were collected after 1 vaccination (Wk 3) or 2 vaccinations (Wk 5) and evaluated in a SARS-CoV-2 plaque reduction neutralization test (PRNT) and pseudovirion neutralization assay (PsVNA). SARS-CoV-2 neutralizing antibodies were detected in all of the animals by both assays after the boost (p=0.0156 (PRNT50), p=0.0078 (PsVNA50), Wilcoxon matched-pairs signed rank test, Fig. 1B ; PRNT80 and PsVNA80 titers shown in Suppl. Fig.   2A,B) . Results from the PRNT and PsVNA were acceptably similar (Suppl. Fig. 3) . The MERS DNA vaccine did not elicit SARS-CoV-2 cross-neutralizing antibodies as measured by PRNT or PsVNA, but all of animals vaccinate with that vaccine developed MERS virus neutralizing antibodies as measured by PsVNA (Suppl. Fig. 4 ).

Three weeks after the boost all of the hamsters were exposed to 100,000 PFU SARS-CoV-2 by the intranasal route (Day 0). Daily weight change data demonstrated that animals vaccinated with nCoV-S(JET) lost significantly less weight than the animals vaccinated with PBS on Day 4 (p=0.0044, Kruskal-Wallis test, Fig. 1C ). In contrast, animals vaccinated with the MERS-CoV DNA vaccine were not protected from weight loss. No significant changes in viral RNA load from pharyngeal swabs between nCoV-S(JET)-vaccinated and PBS animals were observed at any timepoint (Fig. 1D) Neutralizing antibodies were detected in all of the animals by both assays after the boost (Fig.   2B ). In contrast, hamsters vaccinated with PBS had undetectable neutralizing antibodies in both assays. Previously we demonstrated that transient immunosuppression using CyP results in a severe disease model in Syrian hamsters 10 . Here, hamsters were treated with CyP on Day -3, 1, 5, and 9 relative to challenge. On Day 0 prior to challenge, hamsters were bled for hematology to confirm lymphopenia (Fig. 2C) . Hamsters were then exposed to 1,000 PFU SARS RNA labeling in areas of inflammation and respiratory epithelial cells by ISH (Fig. 2H,I) .

Noteworthy, these lungs were collected on Day 13 whereas those collected in the experiment with wild-type hamsters were collected on Day 5. Together, these data indicate the nCoV-S(JET) vaccine had a protective effect in a SARS-CoV-2 infection model with severe and prolonged disease, in which animals that were transiently immunosuppressed before exposure to virus.

Unprotected animals lost >15% of their weight and still harbored infectious virus in their lungs almost two weeks after exposure.

The COVID-19 pandemic has spurred an unprecedented global effort to develop a vaccine to prevent this disease. Nearly every conceivable vaccine platform has been brought to bear on the problem including both RNA-and DNA-based vaccines. Nucleic acid vaccines can be produced rapidly once a target immunogen sequence is known and can be modified rapidly if changes in the sequence become necessary; however, delivering the nucleic acid to cells for immunogen expression remains a technical challenge. For RNA vaccines, efficient vaccine delivery requires formulation with lipid nanoparticles (LNPs) or other modalities to protect the RNA and get it across cell membranes. The safety and efficacy of LNP-formulated RNA is currently being assesses in multiple COVID vaccine trials (Clinicaltrials.gov). DNA delivered by needle and syringe can be immunogenic without LNP formulation, even in nonhuman primates 13 ; however, the use of other techniques such as electroporation or jet injection can increase immunogenicity while reducing dosing requirements. At least one COVID-19 DNA vaccine delivered by electroporation (Inovio) has advanced into the clinic (Clinicaltrials.gov).

To our knowledge, there are no reports of a COVID-19 DNA delivered by jet injection advancing into the clinical-or even progressed to animal efficacy testing. This is surprising because of the logistical and regulatory advantages of disposable syringe jet injection over electroporation. There are several contract manufacturing organizations around the world capable of rapidly producing GMP plasmid for use in humans. Thus, the drug substance could be produced rapidly and the safety profile for DNA vaccines has been established over decades. The drug product delivery system, disposable syringe jet injection, such as PharmaJet's Stratis, is U.S. FDA 510(k)-cleared and has CE Mark and WHO PQA certification. Disadvantages of the DNA vaccine is that at least one booster vaccination, and possibly two in humans, would likely be needed and the dosage would be milligrams rather than micrograms, as is the case for LNPformulated mRNA vaccines.

There are a limited number of published reports of COVID-19 vaccine efficacy testing in animal models of COVID-19 disease. These include the testing of self-amplify mRNA in the K18-hACE2 mouse model 14 , a VSV-vectored vaccine in the hACE2 transduced mouse model 15 , and at least four virus-vectored (yellow fever, adenovirus, VSV, and inactivated Newcastle disease virus) vaccines in SARS-CoV-2 adapted mouse and/or the Syrian hamster model 7, 9, 16, 17 . In all of the aforementioned efficacy experiments, the vaccines were based on the full-length spike protein and neutralizing antibodies were predictive of protection. Here we used a used a jet injection technique to deliver a SARS2 spike-based DNA vaccine to Syrian hamsters. Jet injection technology is not widely available for small animal use. We used a human intradermal jet injection technology to deliver vaccines intramuscularly to the hamsters. We had previously demonstrated approximately 300-fold increases in neutralizing antibodies when this jet injection technique was used relative to a needle and syringe in hamsters vaccinated with hantavirus DNA vaccines 11 .

The immunogenicity parameter we focused on was neutralizing antibody. We measured neutralizing antibodies against live virus by PRNT and a non-replicating VSV-based PsVNA.

These assays showed significant correlation (p< 0.0001) (Suppl. Fig 3A,B) . The neutralizing antibody levels rose significantly after the booster vaccination reaching a PRNT80 geometric mean titer (GMT) of 207 and PRNT50 GMT of 761 that are comparable or exceeding titers of other DNA vaccines evaluated in nonhuman primates 13 and mice 18 . The PRNT50=761 is similar to the 50% titers elicited in hamsters vaccinated with single-dose, live-virus vectored vaccines: Ad26-vectored vaccine PsVNA50 <1000; VSV-vectored vaccine PRNT50 <1000, and Yellow Fever-vectored vaccine PRNT50 <1000 [7] [8] [9] . Neutralization titer was plotted against viral RNA detected in lung tissue collected at the time of euthanasia. Negative correlation was observed (Suppl. Fig. 5) ; however, this did not reach statistical significance. There was no crossneutralizing antibodies against MERS pseudovirions, and those animals were not protected from disease in the hamster model. PRNT. An equal volume of complete media (EMEM containing 10% heat-inactivated FBS, 1%

Pen/Strep, 0.1% Gentamycin, 0.2% Fungizone, cEMEM) containing SARS-CoV-2 was combined with 2-fold serial dilutions of cEMEM containing antibody and incubated at 37°C in a 5% CO2 incubator for 1 hour (total volume 222µl). 180 µl per well of the combined virus/antibody mixture was then added to 6-well plates containing 3-day old, ATCC Vero 76 monolayers and allowed to adsorb for 1 hour in a 37°C, 5% CO2 incubator. 3mL per well of agarose overlay (0.6% SeaKem ME agarose, EBME with HEPES, 10% heat-inactivated FBS, 100X NEAA, 1% Pen/Strep, 0.1% Gentamycin and 0.2% Fungizone) was then added and allowed to solidify at room temperature. The plates were placed in a 37°C, 5% CO2 incubator for 2 days and then 2mL per well of agarose overlay containing 5% neutral red and 5% heatinactivated FBS is added. After 1 additional day in a 37°C, 5% CO2 incubator, plaques were visualized and counted on a light box. PRNT50 and PRNT80 titers are the reciprocal of the highest dilution that results in an 50% and 80% reduction in the number of plaques relative to the number of plaques visualized in the cEMEM alone (no antibody) wells.

The PsVNA used to detect neutralizing antibodies in sera utilized a non-replicating vesicular stomatitis (VSV)-based luciferase expressing system described previously 20 . For the MERS PsVNA there were no modifications, for SARS-CoV-2 assays there were two modifications: 1) no complement was used to parallel the SARS-CoV-2 PRNT assay, 2) a monoclonal anti-VSV-G (IE9F9) was added at 100ng/ml to eliminate any residual VSV activity in the pseudotype preparation. PsVNA50 and PsVNA80 titers were interpolated from 4-parameter curves, and GMTs were calculated.

Pseudovirions were produced using the pWRG/CoV-S(opt)Δ21 or MERS-CoV plasmid described above. HEK293T cells were seeded in T75 tissue culture flasks to be ∼80% confluent the following day and were transfected with the plasmid of interest using Fugene 6 (Promega).

After ∼18 h the transfection media was removed and the cells were infected with VSVΔG * rLuc at a multiplicity of infection of ∼0.07 for 1 h at 37°C. The media was removed and fresh media was added, the flasks were then incubated at 32°C for 72 h. The supernatant from infected cells was collected and clarified by high speed centrifugation, followed by a PEG 8,000 precipitation with 3.2% salt. The PEG mixture is spun at10K xG for 45 min. The pellet was resuspended overnight in 1 mL TNE buffer, then filtered using a 0.45 μm filter, aliquoted and stored at -70°C.

Plaque Assay. Approximately 200mg of lung tissue was homogenized in 1.0mL of cEMEM using a gentleMACS M tubes and a gentleMACS dissociator on the RNA setting. Tubes were centrifuged to pellet debris and supernatants collected. Ten-fold dilutions of the samples were adsorbed to Vero 76 monolayers (200µl of each dilution per well). Following a 1 hour adsorption in a 37°C, 5% CO2 incubator, cells were overlaid and stained identically as described for PRNT.

The limit of detection for this assay is 50 plaque forming units (PFU).

Hematology. Whole blood collected in EDTA tubes was analyzed on an HM5 hematology analyzer on the DOG2 setting.

Preparation of tissues for histology. Tissues were fixed in 10% neutral buffered formalin, trimmed, processed, embedded in paraffin, cut at 5 to 6µm, and stained with hematoxylin and eosin (H&E).

Mark II (mfr#9128B002AA) and Canon EF 100mm f/2.8L Macro (mfr#3554B002) lens. Slides were placed on a lightbox and photographed at 1:1 magnification with a shutter speed of 1/100sec, aperture of f8.0, ISO 400 and saved as Canon RAW files. Contrast was adjusted equally for all images with Photoshop Lightroom and then exported as PNG files.

In situ hybridization. To detect SARS-CoV-2 genomic RNA in FFPE tissues, in situ hybridization (ISH) was performed using the RNAscope 2.5 HD RED kit (Advanced Cell Diagnostics, Newark, CA, USA) as described previously 21 . Briefly, forty ZZ ISH probes targeting SARS-CoV-2 genomic RNA fragment 21571-25392 (GenBank #LC528233.1) were designed and synthesized by Advanced Cell Diagnostics (#854841). Tissue sections were deparaffinized with xylene, underwent a series of ethanol washes and peroxidase blocking, and were then heated in kit-provided antigen retrieval buffer and digested by kit-provided proteinase. Sections were exposed to ISH target probe pairs and incubated at 40°C in a hybridization oven for 2 h. After rinsing, ISH signal was amplified using kit-provided Pre-amplifier and Amplifier conjugated to alkaline phosphatase and incubated with a Fast Red substrate solution for 10 min at room temperature. Sections were then stained with hematoxylin, air-dried, and cover slipped.

Statistical analyses. Statistical analyses were completed using GraphPad Prism 8. Weight data was analyzed using a one-way ANOVA with multiple comparisons for experiments with ≥2 groups; unpaired t-tests were used to analyze weight data for experiments with 2 groups.

Comparisons of lymphocyte levels and lung viral load was assessed using a one-way ANOVA with multiple comparisons for experiments with ≥2 groups; unpaired t-tests were used to analyze weight data for experiments with 2 groups. Significance of survival data was assessed using logrank tests. In all analyses, P<0.05 is considered statistically significant.

Data and materials availability: All data is available in the main text.

Competing interests: Authors declare no competing interests. 

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Experimental design. Groups of 8 hamsters each were vaccinated (vacc) with the nCoV-S(JET) DNA vaccine or PBS, immunosuppressed with cyclophosphamide (dashed vertical lines in D) and E)), and then challenged with 1,000 PFU of SARS-CoV-2 virus by the intranasal route. B) PRNT50 and PsVNA50 titers from serum collected at indicated timepoints after 1 (open symbols) and 2 (closed symbols) vaccinations (LLOQ = 20, grey shade). C) Lymphopenia was confirmed by hematology. D) Average animal weights relative to starting weight. Viral RNA in E) pharyngeal swabs and F) lung homogenates (LLOQ = 50 copies, grey shade). G) Infectious virus as measured by plaque assay (LLOD = 50 PFU, grey shade). A single animal from the PBS group succumbed on Day Fig. 1 and Fig. 2) . Correlation was analyzed by Spearman with an r=-0.2588 and P=0.3319 with linear regression (black line) and 95% confidence intervals (shaded area) shown.