key: cord-0800688-vya52vv8
authors: Fluckiger, Anne-Catherine; Ontsouka, Barthelemy; Bozic, Jasminka; Diress, Abebaw; Ahmed, Tanvir; Berthoud, Tamara; Tran, Anh; Duque, Diane; Liao, Mingmin; McCluskie, Michael; Diaz-Mitoma, Francisco; Anderson, David E.; Soare, Catalina
title: An enveloped virus-like particle vaccine expressing a stabilized prefusion form of the SARS-CoV-2 spike protein elicits potent immunity after a single dose
date: 2021-04-29
journal: bioRxiv
DOI: 10.1101/2021.04.28.441832
sha: acb3be4d64c9367dcbf3335881dd1b3b0fe00288
doc_id: 800688
cord_uid: vya52vv8

Development of efficacious single dose vaccines would substantially aid efforts to stop the uncontrolled spread of the COVID-19 pandemic. We evaluated enveloped virus-like particles (eVLPs) expressing various forms of the SARS-CoV-2 spike protein and several adjuvants in an effort to identify a COVID-19 vaccine candidate efficacious after a single dose. The eVLPs expressing a modified prefusion form of SARS-CoV-2 spike protein were selected as they induced the highest antibody binding titers and neutralizing activity after a single injection in mice. Formulation of SARS-CoV-2 S eVLPs with aluminum phosphate resulted in balanced induction of IgG2 and IgG1 isotypes and antibody binding and neutralization titers were undiminished for more than 3 months after a single immunization. A single dose of this candidate, VBI-2902a (prefusion S eVLPs formulated with aluminum phosphate), protected Syrian golden hamsters from challenge with SARS-CoV-2 and supports the on-going clinical evaluation of VBI-2902a as a potential single dose vaccine against COVID-19. Highlights VBI-2902a is a VLP-based vaccine candidate against SARS-COV-2 VBI-2902a contains VLPs pseudotyped with a modified prefusion SARS-COV-2 S in Alum. VBI-2902a induces robust neutralization antibody response against SARS-COV-2 S VBI-2902a protects hamsters from SARS-CoV-2 induced lung inflammation A single dose of VBI-2902a provides protective benefit in hamsters

(n=12/group) in two independent experiments (Regimen II and Regimen I). Groups A placebo received 0.9%-saline buffer, Groups B received VBI-2902a. Each dose of VBI-2902a contained 1µg of SPG and 125 µg of Alum. Injection was performed by intramuscular (IM) route at one side of the thighs in a 100 µL volume. The schedule for immunization, challenge and sample collection was depicted on Fig. 6a . All animals were challenged intranasally via both nares with 50 μL/nare containing 1×10 5 TCID50 of SARS-CoV-2/Canada/ON/VIDO-01/2020/Vero'76 (Seq. available at GISAID EPI_ISL_413015) strain per animal. Body weights and body temperature were measured at immunization for 3 days and daily from the challenge day. General health conditions were observed daily through the entire study period. Blood samples and nasal washes were collected as indicated on Fig. 6a . Half of the animals (6/group) were euthanized at 3 days post-infection (dpi), and the remaining animals were euthanized at 14 dpi. The challenge experiments were performed in the animal biosafety level 3 (ABSL3) laboratory at VIDO (Saskatchewan, Canada).

Anti-SARS-CoV-2 specific IgG binding titers in mouse sera were measured by standard ELISA procedure described elsewhere [18] , using recombinant SARS-CoV-2 S (S1+S2) protein (Sinobiological). For total IgG binding titers, detection was performed using a goat anti-mouse IgG-Fc HRP (Bethyl) for mouse serum, or goat anti-human IgG heavy and light chain HRP-conjugated (Bethyl) for human serum. HRP-conjugated Goat anti-mouse IgG1 and HRP-conjugated goat antimouse IgG2b HRP (Bethyl) were used for the detection of isotype subtype. Determination of Ab binding titers to the RBD was performed using SARS-COV-2 RDB recombinant protein with 5% non-fat skim milk powder in PBS containing 0.05% Tween 20. Fourfold dilutions of serum were used. Goat anti-Hamster IgG HRP from ThermoFisher (PA1-29626) was used as the secondary antibody at 1:7000. Plates were developed with OPD peroxidase substrate (0.5 mg/ml) (Thermo Scientific Pierce). The reaction was stopped with 2.5 M sulfuric acid and absorbance was measured at 490 nm. Throughout the assay, plates were washed with PBS containing 0.05% Tween 20. The assay was performed in duplicate. The titres were reported as the end point of the dilutions.

Neutralizing activity in mouse serum samples was measured by standard plaque reduction neutralization test (PRNT) on Vero cells at the NRC (Ottawa, Canada) using 100 PFU of SARS-CoV-2/Canada/ON/VIDO-01/2020. Results were represented as PRNT90, PRNT80, or PRNT50 end point titer, corresponding to the lowest dilution inhibiting respectively 90% or 80% or 50% of plaque formation in Vero cell culture.

Virus neutralization assays against the challenge SARS-CoV-2 virus were performed at VIDO, Saskatchewan on the hamster serum samples collected at pre-challenge and at the end day; 3 days post-challenge or 14 days post-challenge. The study was conducted using the cell line Vero E6. The serum samples were heat-inactivated for 30 min at 56°C. The serum samples were initially diluted 1:10 and then serially diluted (2-fold serial dilutions). The virus was diluted in medium for a final concentration of 3×10 2 TCID50/mL. Initially 60 μL of the virus solution was mixed with 60 μL serially diluted serum samples. The mixture was incubated for 1hr at 37°C, with 5% CO2. The preincubated virus-serum mixtures (100 μL/well) were transferred to the wells of the 96-well flatbottom plates containing 90% confluent pre-seeded VeroE6 cells. The plates were incubated at 37°C, with 5% CO2 for 5 days. The plates were observed using a microscope on day 1 postinfection for contamination and on days 3 and 5 post-infection for cytopathic effect (CPE). The serum dilution factor for the last well with no CPE at 5 dpi was defined as the serum neutralization titer. The initial serum dilution factor was 1:20.

RNA was extracted using QIAamp Viral RNA Mini Kit (Qiagen). Briefly, 140 μL of hamster nasal wash was added into 560 μL viral lysis buffer (Buffer AVL). The mixture was incubated at room temperature for 10 min. After brief centrifugation, the solution was transferred to a fresh tube containing 600 μL of 100% ethanol, and the tube was incubated at room temperature for 10 min.

RNA was then purified using QiaAmp Viral RNA Mini Kit and eluted with 60 μL of RNase Free water containing 0.04% sodium azide (elution buffer AVE). Extraction of RNA from lung lobes and nasal turbinates was completed using approximately 100 μg of tissue. The tissues were homogenized in 600 μL of lysis buffer (RLT Qiagen) with a sterile stainless steel bead in the TissueLyserII (Qiagen) for 6 min, at 30 Hz. The solution was centrifuged at 5000 x g for 5 min.

Supernatant was transferred to a fresh tube containing 600 μL of 70% ethanol, and the tube was incubated at room temperature for 10 min. Viral RNA was then purified using Qiagen Rneasy Mini Kit (Cat No /ID: 74106) and eluted with 50 μL elution buffer.

The qRT-PCR assays were performed on RNA from samples of nasal washes, lung tissues and nasal turbinates using SARS-CoV-2 specific primers targeting the E gene ( 

IFN-γ ELISPOT analyses to measure Th1 T cell responses were performed as follows. One day before the spleens were removed, ELISpot plates (Millipore) were coated with IFN-γ capture antibody at a concentration of 15 µg/mL (Mabtech). The following day, mice were sacrificed and spleens were removed. Spleens from individual mice were processed to produce single cell suspensions. Erythrocytes were lysed using a commercially available RBC lysis buffer (BioLegend). Fifty microliters containing 2x10 6 splenocytes were then to each well of a pre-blocked ELISPOT plate. Then, fifty microliters of stimulant pepmixes (JPT peptides) resuspended in RPMI+10%FBS (R10) with recombinant mouse IL-2 (rmIL-2) (R&D Systems) were added to each well. The final concentration of each peptide in the assay was 1µg/mL/peptide, and the final concentration of rmIL-2 was 0.1 ng/mL. R10 alone was used as a negative control and PMA+Ionomycin as a positive control. The ELISPOT plates were then placed into a humid 37°C with 5% CO2 incubator for 40-48 hours. After incubation, the plates were washed and IFN-γ capture antibody was added, followed by streptomycin horseradish peroxidase (strep-HRP). The plates were developed with commercially available 3-Amino-9-ethylcarbazole (AEC) substrate (Sigma-Aldrich). The observed spots were counted using an ELISPOT plate reader by ZellNet and the final data was reported as spot forming cells (SFC) per one million splenocytes.

At necropsy the left lung of hamsters was perfused with neutral-buffered formalin immediately after collection. Tissues were fixed in neutral-buffered formalin for a week, then placed into fresh neutral-buffered formalin before being transferred from containment level 3 to containment level 2 

All statistical analyses were performed using GraphPad Prism 9 software (La Jolla, CA). Unless indicated, multiple comparison was done with Kruskall-Wallis test. The data were considered significant if p < 0.05. Geometric means with standard deviation are represented on graphs. No samples or animals were excluded from the analysis. Randomization was performed for the animal studies.

Four constructs were designed based on the spike protein sequence of the SARS-CoV-2 Wuhan-Hu1 isolate and subcloned into expression plasmids for the production of eVLPs as described in Methods (Fig. 1a ). To obtain a stabilized prefusion form of S (SP), the furin cleavage site of S, RRAR, was inhibited by mutation of the 3 arginines into a glycine and 2 serine (GSAS) and 2 proline substitutions were introduced at successive residues K986 and V987. Our previous work has demonstrated that the swap of the transmembrane cytoplasmic terminal domain (TMCTD) of CMV gB resulted in enhanced yields and immunogenicity of the gB glycoprotein presented on eVLPs [18] . Based on this data, two additional constructs, Native-VSVg (SG) and Stabilized Prefusion-VSVg (SPG) were designed by swapping the TMCTD of S with that of VSV-G.

Western blot analysis of eVLPs using a polyclonal Ab directed against the SARS-CoV-2 S receptor binding domain confirmed the processing of SARS-CoV-2 S during biosynthesis in HEK-293 cells as expected by the presence of the furin cleavage site in S1/S2 [6] Overexpression of S in the prefusion forms showed a major band at 180 kDa, the size commonly described for uncleaved S180 kDa and an additional band around 150 kDa. The additional band around 150 kDa is reproducibly seen upon overexpression of uncleaved S, and most likely represents the S protein deprived of N-Glycosylation [21] that would occur because of overloading of the host cell machinery. Similar results were obtained after blotting with human convalescent sera.

Quantitative analysis of protein content in eVLP preparations showed that for a similar number of particles and comparable amounts of Gag protein, the amount of SARS-CoV-2 S protein was increased substantially with replacement of the TMCTD and by use of the stabilized prefusion construct, suggesting that the density of the S protein was enhanced using the VSV-G constructs (Table 1 ). The best yield was reproducibly obtained when producing the eVLPs expressing the prefusion-VSV-G form of S, with up to a 40-fold increase relative to eVLPs expressing native S.

Comparison to convalescent serum is commonly used as a benchmark to help evaluate immunogenicity and potential efficacy of Covid-19 candidate vaccines. However, a wide spectrum of Ab responses can be observed in recovering patients, ranging from barely detectable to very high levels, likely influenced by time since infection and severity of disease. To enable comparison across experiments, we obtained a cohort of 20 sera from COVID-19 confirmed convalescent patients with moderate COVID-19 symptoms who all recovered without specific treatment intervention or hospitalization. The cohort was separated into two groups of 10 samples according which to assess the immunogenicity of the vaccine candidates, only the high titer pooled sera was used to assess vaccine-induced responses in animals.

Humoral responses of the various types of SARS-CoV-2 eVLPs were evaluated in C57BL/6 mice that received 2 intraperitoneal injections at 3 week intervals (Fig. 3) . The first injection of unmodified S presented on eVLPs induced levels of anti-SARS-CoV-2 S Ab binding titers similar to those in mice that received a recombinant trimerized prefusion S protein, but they were not associated with significant (90% or greater) neutralization activity as measured in a plaque reduction neutralization test (PRNT) (Fig. 3a-b) . In contrast, a significant nAb response was induced by a single injection of eVLPs expressing prefusion SP or SPG, with PRNT90 end-point titers (EPTs) of 80 and 160 respectively. These values were higher than those observed with the human convalescent control pool (PRNT90 EPT of 50). All nAb responses were greatly enhanced by the second injection and reflected the responses that were observed prior to the boosting dose.

Notably, all forms of SARS-CoV-2 S presented on eVLPs induced higher antibody titers than recombinant prefusion S protein, both in the levels of total IgG and neutralization activity, after one or two injections.

Individual mice sera obtained 14 days after the second injection of eVLPs were evaluated for the specificity of the Ab responses against the whole S1+S2 protein or the RBD (Fig. 3c-d) . All immunized mice that received eVLPs showed robust anti-SARS-CoV-2 Ab responses either against a full length S1+S2 protein (Fig. 3c ) or against the RBD protein (Fig. 3d) . A more homogenous response was observed in mice that received the SPG eVLPs, with all Ab EPTs above 400,000 against S (5.6 Log 10), and above 650,000 against RBD (5.8 Log10).

A Th2-type response has been suggested to contribute to the "cytokine storm" associated with vaccine-induced severe lung pathologies [23, 24] . In light of these results, we tested a variety of adjuvants that might enhance neutralizing Ab production while also promoting a balanced Th1/Th2 response. For this purpose, we compared formulation of eVLPs with Alum against a panel of adjuvants including MF59 and the adjuvant systems AS03 and AS04. We used SARS-CoV-2 native S eVLPs as they were less immunogenic than eVLPs expressing the prefusion form of the S protein and might better enable differences in the adjuvants to be observed. The various adjuvanted formulation of S eVLPs were compared to recombinant stabilized prefusion S protein (r-SP) formulated in Alum adjuvant, which was expected to induce a Th2-biased response [25] .

Mice received two IP injections and Ab and T cell responses were measured 14 days after the second injection (Fig. 4) . MF59 enhanced IFN- T cell responses compared to Alum (Fig. 4a ) but induced similar Ab responses (Fig. 4b, c) and a comparable, balanced IgG2/IgG1 ratio (Fig. 4d) .

The AS03 and AS04 adjuvants also skewed responses towards a Th1-type T cell response. Most remarkably, while r-SP in Alum preferentially induced IgG1 Ab representative of a Th2 response, S-eVLPs induced balanced production of IgG1 and IgG2b indicating a balanced Th1/Th2 response (Fig. 4d) .

Based on the results described above, we chose to evaluate the immunogenicity and potential efficacy of eVLPs expressing SPG protein formulated with Alum, named VBI-2902a, after one or two injections 21 days apart. Fourteen days after a single injection, sera from mouse immunized with VBI-2902a contained total anti-Spike IgG EPTs reaching geometric means of (4.8 Log 10) 54,891 that were associated with neutralizing PRNT90 titers of 365 (2.6 Log10). A second injection boosted Ab binding titers to 228,374 (5.4 Log10) with nAb titers of 1,079 (3.0 Log10) (Fig. 5a-b) .

Levels of nAb response were higher than those observed in sera from convalescent patients. Abs were preferentially directed against the RBD and S1 with only low binding to S2 (Fig. 5c) .

Mouse splenocytes collected 2 weeks after each immunization were stimulated ex vivo using two different peptide pools preferentially covering the S1 domain (pepmix 1) or the S2 domain (pepmix 2) respectively. Numbers of IFN- spot forming cells (Fig. 5d ) suggested preferential T cell responses against the S1 domain of the spike protein rather than against the S2 domain. No major increases in T cell responses were observed after the second injection of VBI-2902a. Additionally, we observed that a single dose of VBI-2902a induced a sustained Ab response for at least 15 weeks without any drop in neutralization titers (Fig. 5e ).

The protective efficacy of VBI-2902a was examined in Syrian Gold hamsters. SARS-CoV-2 infection in Syrian Gold hamsters resembles features found in humans with moderate COVID-19 and is characterized by a rapid weight loss starting 2 days post infection (dpi) [26, 27] . Two immunization regimens were compared. Regimen II consisted of two IM injections of VBI-2902a or saline at 3 weeks interval whereas Regimen I consisted of a single dose injection of VBI-2902a or saline (Fig. 6a) . Three weeks after the last injection (day 42 in Regimen II and day 21 in Regimen I), all animals were inoculated intranasally with 1×10 5 TCID50 of SARS-CoV-2 per animal and monitored daily for weight change, general health and behavior.

After a single injection of VBI-2902a the levels of anti-S IgG rapidly increased in the serum of immunized animals with EPTs reaching 1-2x10 3 (Fig. 6b) . The second injection enhanced these levels approximately 10-fold to reach EPTs of 2-3x10 4 at day 35, which translated into robust neutralization titers of over 10 3 EPT as measured by PRNT90 (Fig. 6c) . In the single dose regimen, the neutralization activity (GeoMean 69), was increased 250-fold to 1725 within 3 days after exposure to the virus. Animals in all groups lost 2-4% of body weight 2 days post infection (2dpi). Animals in the saline control groups continued to lost weight until an average 15% loss at 7dpi, before gradually regaining weight (Fig. 7a-b) . In marked contrast, none of the hamsters immunized with two doses of VBI-2902a lost any further weight after 2dpi, regaining normal weight by 7dpi, demonstrating robust protection against SARS-CoV-2 disease. In the single dose regimen, the majority of the animals regained body weight after 3dpi instead of 2dpi, suggesting slightly delayed but significant protection against disease.

At 3dpi, hamsters vaccinated with either one or two doses of VBI-2902a had greatly decreased viral RNA copy numbers in lungs (Fig. 8a) . Two doses of VBI-2902a resulted in a 5 Log decrease (Fig. c-d) . More viral RNA was found in nasal turbinates, which may have included residual viral inoculum as suggested previously [27, 28] . Data from prior studies also suggested an extended persistence of the virus in nasal turbinates while bearly detectable in the lung [26] . Both vaccine regimens protected against the development of lung pathology as indicated by reductions of the lung to body weight ratio (Fig. 9a-b) and histological analysis of the lungs (Fig. 9c-d) .

The unprecedented urgency for a safe COVID-19 vaccine that can confer protection as quickly as possible with as few doses as possible is evident as regulatory agencies and vaccine manufactures have discussed the risks and benefits of delaying planned second doses of currently available COVID-19 vaccines to enable immunization of a greater number of individuals as quickly as possible [29, 30] . We have previously demonstrated that expression of proteins on the surface of eVLPs dramatically enriches for neutralizing antibody, the presumed correlate of protection against SARS-CoV-2, relative to recombinant proteins [18] . Accordingly, we evaluated both conformations of the SARS-CoV-2 S protein as well as a variety of adjuvants in an effort to identify a COVID-19 vaccine candidate with the potential to confer protection after a single dose.

The eVLPs particles were pseudotyped with SARS-CoV-2 unmodified S protein but expressed low amounts of S that were not suitable for upscaled production. We therefore designed a modified prefusion form of S that resulted in both dramatic increases in yields and enhancement of the nAb response compared to native S. SPG eVLPs induced high titers of RBD Ab binding titers associated with robust neutralizing responses in mice at levels that were much higher than those The value of eVLP expression of the modified SP protein is consistent with prior reports which demonstrated that an anchored version of a stabilized prefusion S antigen provided optimal induction of protective nAbs in Rhesus macaques [13] . Our construct differed from the previously described S-2P [13, 31] by using the VSV-G transmembrane cytoplasmic domain to replace that of S, instead of a C-terminal T4 fibritin trimerization domain. Based on previous experience and published data [18, 19] , we hypothesized that the use of VSV-G tail and expression in the phospholipid membrane of eVLPs would result in natural trimerization of the spike ectodomains providing optimal presentation of neutralization epitopes. The use of the VSV-G tail has been shown to enhance expression and localization of viral glycoproteins at the phospholipid envelop of Moreover, compared to a clear Th2-biased profile observed in response to recombinant prefusion stabilized S protein in Alum, the similar prefusion S construct induced a balanced Th1/Th2 response when presented by eVLPs (Fig. 4d) . The balanced production of IgG2/IgG1 antibody isotypes after VBI-2902a immunization was comparable with those described in response to the recently emergency use authorized vaccine Ad26.COV2.S [25] . These results emphasize an important difference in the quality of the antibody response when immunizing with soluble, recombinant versus particulate forms of vaccine antigens.

The VBI-2902a vaccine candidate addresses several issues that have thus far hindered the speed and extent of vaccination with currently available COVID-19 vaccines. This includes the need to administer multiple doses and the need for storage, transport, and distribution of the vaccine at freezing temperatures not typically required for prophylactic vaccines. VBI-2902a received approval from Health Canada to initiate its ongoing Phase I/II clinical study (NCT04773665) to assess its potential for one dose immunogenicity and potential efficacy. Three weeks after the last injection corresponding to day 42 in regimen II and day 21 in regimen I, hamsters were exposed to SARS-CoV-2 at 1x10 5 TCID50 per animal via both nares. At 3 days post infection (dpi), 6 animals per groups were sacrificed for viral load analysis. The remaining animals were clinically evaluated daily until end of study at 14dpi. (b) Anti-SARS-CoV-2 S(S1+S2) total IgG EPT measured by ELISA 2 weeks after each immunization. (c) Neutralization activity was measured by PRNT90 in immunized groups; results are represented as PRNT90 EPT. 

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The authors want to thank Adam Asselin, Matthew Yorke, Teresa Daoud, Lanjian (Isabel) Yang, Rebecca Wang, Gillian Lampkin (VBI vaccines) for outstanding technical support;Traian Sulea