key: cord-0896623-m2h25u7h
authors: Yilmaz, Ismail Cem; Ipekoglu, Emre Mert; Bulbul, Artun; Turay, Nilsu; Yildirim, Muzaffer; Evcili, Irem; Yilmaz, Naz Surucu; Guvencli, Nese; Aydin, Yagmur; Gungor, Bilgi; Saraydar, Berfu; Bartan, Asli Gulce; Ibibik, Bilgehan; Bildik, Tugce; Baydemir, İlayda; Sanli, Hatice Asena; Kayaoglu, Basak; Ceylan, Yasemin; Yildirim, Tugce; Abras, Irem; Ayanoglu, Ihsan Cihan; Cam, Sefa Burak; Ciftci Dede, Eda; Gizer, Merve; Erganis, Osman; Sarac, Fahriye; Uzar, Serdar; Enul, Hakan; Adiay, Cumhur; Aykut, Gamze; Polat, Hivda; Yildirim, Ismail Selim; Tekin, Saban; Korukluoglu, Gulay; Zeytin, Hasan Ersin; Korkusuz, Petek; Gursel, Ihsan; Gursel, Mayda
title: Development and preclinical evaluation of virus‐like particle vaccine against COVID‐19 infection
date: 2021-09-21
journal: Allergy
DOI: 10.1111/all.15091
sha: 906feaa988c3c857f2728f9c4be8375cdd5ca963
doc_id: 896623
cord_uid: m2h25u7h

BACKGROUND: Vaccines that incorporate multiple SARS‐CoV‐2 antigens can further broaden the breadth of virus‐specific cellular and humoral immunity. This study describes the development and immunogenicity of SARS‐CoV‐2 VLP vaccine that incorporates the four structural proteins of SARS‐CoV‐2. METHODS: VLPs were generated in transiently transfected HEK293 cells, purified by multimodal chromatography, and characterized by tunable‐resistive pulse sensing, AFM, SEM, and TEM. Immunoblotting studies verified the protein identities of VLPs. Cellular and humoral immune responses of immunized animals demonstrated the immune potency of the formulated VLP vaccine. RESULTS: Transiently transfected HEK293 cells reproducibly generated vesicular VLPs that were similar in size to and expressing all four structural proteins of SARS‐CoV‐2. Alum adsorbed, K3‐CpG ODN‐adjuvanted VLPs elicited high titer anti‐S, anti‐RBD, anti‐N IgG, triggered multifunctional Th1‐biased T‐cell responses, reduced virus load, and prevented lung pathology upon live virus challenge in vaccinated animals. CONCLUSION: These data suggest that VLPs expressing all four structural protein antigens of SARS‐CoV‐2 are immunogenic and can protect animals from developing COVID‐19 infection following vaccination.

Rapid development of effective vaccines is indispensable in constraining the COVID-19 pandemic. Multiple highly effective COVID-19 vaccines have recently been approved for human use and several are still in clinical development. 1 The majority of current SARS-CoV-2 vaccines target only the Spike (S) antigen with the main intent of eliciting neutralizing antibodies against the receptorbinding domain (RBD) to neutralize infection. [2] [3] [4] [5] [6] However, the emergence of variants of concern Alpha (B. 

SARS-CoV-2 VLP vaccine that incorporates the four structural proteins of SARS-CoV-2 is reproducibly produced in suspension adapted HEK293 cells. Alum adsorbed, K3-CpG ODN-adjuvanted VLPs elicit high titer anti-S, anti-RBD, anti-N IgG, and neutralizing antibodies in mice, rats, and ferrets. The VLP vaccine supports multifunctional Th1-biased T-cell responses and demonstrate immunoprotective activity against live SARS-CoV-2 challenge in vaccinated mice.

neutralizing and spike-binding antibodies strongly correlate with protective immune mechanisms, 19 cellular immunity also likely contributes to virus clearance. [20] [21] [22] [23] In addition to spike, targeting of other SARS-CoV-2 antigens in vaccines, such as the membrane (M) and nucleocapsid (N), could hypothetically present an advantage over S-dependency of vaccines twofold: First, M and N harbor immunodominant CD4+ and CD8+ T-cell epitopes that can further broaden the breadth of cellular and humoral immunity 24 ; second, non-neutralizing anti-N antibodies can potentially contribute to cross-immunity between SARS-CoV-2 variants, akin to heterosubtypic immunity previously reported for influenza viruses. 25, 26 In this context, although an inactivated SARS-CoV-2 vaccine would harbor multiple virus antigens, the inactivation process alters the ratio of prefusion form of spike toward its postfusion form, impacting the ability of the vaccine to induce a neutralizing response. 27 To this end, herein, we describe the preclinical development of a viruslike particle (VLP) vaccine expressing the Hexaproline prefusionstabilized spike (S-6p). 28 To expand the spectrum of ensuing T-cell responses, the VLPs were designed to also express the N, M, and envelope (E) of SARS-CoV-2 structural proteins. To improve immunogenicity, S-6p VLPs were adsorbed to alhydrogel (alum) and formulated with a K-type CpG ODN (also referred to as B type) as a vaccine adjuvant to boost both humoral immunity and cellular (Th1 cells and CTL) immunity. 29 

To eliminate host cell-derived nucleic acids, the harvest was treated with 200 U/ml of Denarase (c-LEcta) for 2 h at 37°C. VLPs were purified on a Hi-Screen Capto Core 400 (Cytiva) column using ÄKTA-GO fast protein liquid chromatography system (Cytiva). Flow-through fractions containing VLPs were pooled and subjected to ultrafiltration/diafiltration on a Sartocon ® Slice 200 Hydrosart ® 100 kDA (Sartorius) cassette.

For SDS-PAGE, the samples were mixed with reducing 4× Laemmli Buffer and denatured at 95°C for 5 min. 18 µl of sample was loaded into each well of 4-20% Mini-PROTEAN TGX Stain-Free Protein Gel (Bio-Rad). Following completion of SDS-PAGE, gels were transferred to a PVDF 0.2 µm membrane using the Mini Trans-Blot ® Cell System (Bio-Rad) for an hour at 100 V. As primary antibodies, HRP-conjugated 6xHis, His-Tag antibody (Proteintech), Spike-S1

and Nucleocapsid antibody (ProSci) were used. Anti-rabbit and antimouse secondary antibodies were used for anti-spike S1 and anti-N immunoblots. The HRP activity was detected with ECL™ Prime HRP Reagent (Cytiva) and imaged by an Amersham Imager 600 (Cytiva).

VLP content was quantified with the Pierce™ micro BCA protein assay kit (Thermo Fisher Scientific) according to manufacturer's instructions.

A 10 μl of the VLP solution was deposited onto a silica surface, air-dried and sputter-coated with 8 nm of Au/Pd alloy using a precision coating system prior to imaging on an environmental SEM (Technia; FEI).

Purified VLPs were diluted 1:100 in PBS and adsorbed onto mica sheets. The adsorbed samples were air-dried and micrometerscale AFM imaging was conducted in non-contact dynamic mode (NanoMagnetics Instruments) according to manufacturer's instructions. Scans were analyzed using the NMI Image Analyzer software.

VLP-producing HEK293 cells were processed for standard TEM.

Briefly, cells were fixed in 2% glutaraldehyde 30 min at RT, fixed in 1% osmium tetroxide, dehydrated through a graded series of ethanol (30-100%), and embedded in Epon 812 resin. Sections were stained with uranyl acetate and lead citrate. Imaging was performed at 80 kV using a JEOL-JEM 1400 transmission electron microscope. Digital images of the specimens were acquired using a CCD camera (Gatan Inc.). 

Carboxyl modified latex beads (2 mg of 4% (w/v), Thermo Fisher Scientific) were coated with 5 μg recombinant hACE2 (ProSci) or anti-IL-1β in PBS and blocked in 5% BSA in PBS. Beads were washed once and resuspended in 5% BSA/PBS/0.05% NaN3 (FACS buffer).

VLPs were loaded with 50 μM carboxyfluorescein succinimidyl ester (CFSE) for 30 min at 37°C, and free dye was removed using a HiTrap ® Desalting column (Cytiva). Recombinant hACE2 and anti-IL1β-coated beads were diluted 1:50. CFSE-labeled VLPs were serially diluted five times; each dilution was mixed with an equal volume of coated bead followed by overnight incubation at 4°C. Beads were washed three times and analyzed on a Novocyte 3000 flow cytometer. 

Lung samples were fixed in buffered formaldehyde solution. Tissues were dehydrated in an automated tissue processor (TP1020, Leica). Sections were obtained in a temperature-controlled paraffin station (LG1150H-C, Leica) on a sliding microtome (SM2000R, Leica), were deparaffinized at 60°C overnight and stained with Hematoxylin-Eosin and Gomori's Trichrome techniques. All sections were evaluated using a bright field microscope with a camera attachment using an image analysis program (DM6B, DFC7000T, LAS X, Leica). Inflammation was semi-quantitatively scored between 0 and 5 in the perivascular, peribronchiolar, subpleural regions and in the whole section. American Thoracic Society's acute lung injury scoring was followed to report total lung injury. 32 The parenchymal inflammation area was evaluated for each animal by combining the images obtained at 4× magnification using Tile Scanning feature of the analysis program and the area of inflammation was calculated quantitatively in µm 2 and then proportioned to the total lung area.

A micro-neutralization assay was carried out to detect SARS-CoV-2-neutralizing antibodies. 33 The virus used in the authentic Wuhan 

Statistical differences of all treatment groups were analyzed using Graph Pad Prism 9 statistical software. Groups were compared by one-way ANOVA with Dunnett's multiple comparisons test. Extra comparative statistical analyses were mentioned in the figure legends. In all analyses, a P value below .05 was considered to be statistically significant. Figure 1C ), scanning electron microscopy (SEM; Figure 1D ), atomic force microscopy (AFM; Figure 1E ), TRPS ( Figure 1F ), and immunoblotting for SARS-CoV-2 antigen content using anti-His-Tag, anti-N ( Figure 1G ), and anti-S1 antibodies ( Figure 1H ). HEK293 producer cells released intact VLPs into the culture supernatant ( Figure 1C ).

Purified VLPs were spherical, vesicular structures ( Figure 1D ,E) that were similar in size to SARS-CoV-2 virions (117 ± 38, 127 ± 41, and 119 ± 36 for the WT, 2p or 6p incorporating VLPs, respectively; Figure 1F ). Spike protein expression in 2p-VLPs was enhanced relative to WT spike displaying VLPs, whereas 6p-S base construct enabled maximal spike incorporation ( Figure 1G ). Membrane, envelope, and nucleocapsid expressions were relatively stable and did not change substantially among WT, 2p or 6p spike expressing VLPs ( Figure 1G ). Comparison of spike-specific immunoblots of 6p-VLPs with inactivated SARS-CoV-2 virions revealed that 6p-VLPs displayed intact full-length spike, whereas a substantial amount of the spike protein generated S1 fragments in the case of inactivated SARS-CoV-2 virions ( Figure 1H ). 6p-VLPs specifically bound to human ACE2 receptor coated beads but not to anti-IL-1β-coated control beads ( Figure S1 ), demonstrating the specificity of the VLPexpressed spike protein toward the host receptor. The yield of VLPs from one liter harvest was found to be 25 ± 3 mg/L based on mi-croBCA method. Moreover, VLPs retained their intact antigenic content even when incubated at 40°C up to 3 days ( Figure S2A) , and they retained their morphology after adsorption to alum and CpG adjuvantation ( Figure S2B ). Furthermore, formulated VLP vaccine was stable for 90 days following storage at 2-8°C and preserved its long-term in vivo immunopotency ( Figure S2C ). These results illustrate the feasibility of generating VLPs as a vaccine candidate, targeting the four structural proteins of SARS-CoV-2.

To assess the immunogenicity of the VLP vaccine Antigen-specific helper T-cell responses were also investigated in immunized mice. Following restimulation with recombinant spike or nucleocapsid, splenocytes from mice immunized with 6p-VLP or 6p-VLP plus Alum, secreted significant amounts of Th2 cytokines IL-4, IL-5, IL-13, and IL-10 ( Figures 2C,D and S4A,B) . In contrast, only the K3-CpG or CpG/Alum-adjuvanted VLPs induced a Th1-biased IFNγ response but no Th2-associated cytokines ( Figures 2C,D and S4A,B) , suggesting that 6p-VLP/Alum/K3-CpG vaccination would prevent Th2-biased immune responses and therefore avoid Th2-dependent vaccine-associated enhanced respiratory disease (VAERD). [42] [43] [44] To study the effect of the formulation dose on VLP immunogenicity, BALB/c mice were subcutaneously immunized with six different doses (ranging from 24 to 0.75 µg) of 6p-VLP/Alum/K3-CpG and IgG titers against the whole inactivated virus was determined by ELISA ( Figure 3A ).

VLPs elicit robust antibody and helper T cell responses in mice. BALB/c mice (n = 12/group) were immunized on days 0 and 14 with 0.4 µg (low dose; LD) or 4 µg (high dose; HD), 6p-S VLP or 2p-S VLPs without or with Alum (5 µg/mouse), without or with K3 CpG ODN (20 µg/mouse) or with Alum + CpG ODN. Control BALB/c mice were administered Alum or CpG ODN alone (black and gray). Sera were collected 2 weeks post-prime (A) and 2 weeks post-boost (B) and assessed for SARS-CoV-2 S-specific IgG, IgG1, and IgG2a by ELISA. Vaccinated groups were compared by one-way ANOVA with Dunnett's multiple comparisons test. *P < .05, **P < .01, ***P < .001, ****P < .0001. Data are presented as GMT ± geometric SEM. (C) Spleens were collected 2 weeks after booster (n = 6). 1×10 6 /250 µl splenocytes from naive or immunized mice were stimulated with recombinant spike (5 µg/ml) in the presence of 1 μg/ml anti-mouse CD28. T helper cell cytokine levels were assessed from 48 h culture supernatants using the LEGENDplex™ MU Th Cytokine Panel (12-plex). Groups were compared by one-way ANOVA with Dunnett's multiple comparisons test. *P < .05, **P < .01, ***P < .001, ****P < .0001. Data are presented as mean cytokine levels ± SEM. (D) Pie charts representing the proportions of individual secreted S-specific T helper cell cytokines are presented

The effective concentration at 50% (EC50) was then determined by a non-linear regression curve fit in GraphPad Prism ( Figure 3A ). The EC50

for the 6p-VLP/Alum/K3-CpG vaccine was determined to be 2.83 µg.

To test the HD 6p-VLP + Alum + K3-CpG immunogenicity in different animal species, rats were immunized subcutaneously with 40 µg of 6p-VLP/K3-CpG/Alum 2 weeks apart and live virusneutralizing antibody titers (VNT) were evaluated 2 weeks after booster injection ( Figure 3B) . Similarly, ferrets were vaccinated either with a 10 µg or a 40 µg dose of the 6p-VLP vaccine and live VNTs were determined 2 weeks after priming and booster injections ( Figure 3C ).

6p-VLP/Alum/K3-CpG combination induced robust neutralizing antibodies against live SARS-CoV-2 in rats and ferrets. These data indicate that 6p-VLP/Alum/K3-CpG formulation is a potent immunogen that can elicit virus-neutralizing activity in multiple species.

VLPs expressing either WT 6p-S or alpha variant 6p-S were also synthesized and then formulated with alum/CpG ODN to test their immunogenicity in C57BL/6 mice. In alpha 6p-S VLPs, spike protein expression was more enhanced compared to WT 6p-S VLPs ( Figure 3D ).

Consistently, alpha 6p-S VLPs elicited higher levels of anti-S and antiinactivated virus (Wuhan) IgG in comparison with WT 6p-S VLPs, whereas anti-N IgG levels remained similar ( Figure 3E ). Antibodies raised against WT, Alpha, Beta, and Gamma variant RBDs were also analyzed ( Figure 3F ). Alpha 6p-S VLPs elicited ~37.3-, 20.5-, 1.7-, and 11.9-fold more anti-WT, anti-alpha, anti-beta, and anti-gamma RBD IgG, respectively, when compared to WT 6p-S VLP immunized mice ( Figure 3F ). These results suggest that alpha 6p-S-expressing VLPs might be advantageous over their WT 6p-S VLP counterpart in eliciting a broader cross-protective response against variant RBDs. On day 21 after booster injection, mice were intranasally challenged with 10 5 pfu of live SARS-CoV-2 (Wuhan strain) on 3 consecutive days. One week after the last instillation, lungs were harvested for histopathological evaluation ( Figure 4D,E) . Histomorphometric evaluations were based on the following criteria: (i) Inflammation was semi-quantitatively scored between 0 and 5 in the perivascular, peribronchiolar, and subpleural regions and in the whole section. 45 (ii) Total lung injury was evaluated based on the American Thoracic Society's acute lung injury score. 32 The parenchymal inflammation area was quantitatively evaluated. 46 Untreated/unchallenged healthy K18-hACE2 transgenic mouse lung samples (negative control) exhibited low-grade local parenchymal inflammation at the periphery ( Figure 4D ). Alveolar integrity was preserved without interalveolar septum thickening, intra-alveolar inflammatory cell infiltration, or protein debris accumulation. High-dose vaccine prevented perivascular (P < .0001), peribronchiolar (P = .0002), subpleural (P < .0001), and total (P < .0001) lung parenchymal inflammation when compared to the placebo group ( Figure 4D,E) . Minimal inflammation scores equivalent to healthy animals were recorded in the high-dose vaccine group ( Figure 4E ).

High-dose vaccine significantly reduced acute lung injury score consisting of inflammatory cell infiltration in the alveolar lumen and interstitial space, hyaline membrane formation, protein debris in the airways, and thickening of the interalveolar septum compared to that of the placebo group ( Figure 4D ). Lung specimens from animals vaccinated with high-dose VLP had low injury scores similar to healthy lung specimens ( Figure 4E ). Placebo and low-dose vaccine failed to prevent acute lung injury and presented with parenchymal consolidation with diffuse infiltration of mononuclear cells and macrophages, thickened interalveolar septa to varying degrees, and hyaline membranes at the alveolar walls facing the lumen ( Figure 4D ). The high-dose vaccine group generally exhibited a limited and mild parenchymal infiltration at peribronchiolar regions.

These results indicate that 6p-VLP vaccination confers immunoprotective activity against SARS-CoV-2 challenge and a suboptimal vaccine dose does not exacerbate virus-induced immunopathology.

Several highly effective and safe SARS-CoV-2 vaccines have been approved and are widely administered to the populations of several countries as an indispensable measure in controlling the current pandemic. Almost all of these vaccines are based on the spike antigen and elicit neutralizing antibodies especially against the receptor-binding motif, the least conserved region of the spike antigen. With the emergence of new SARS-CoV-2 variants of concern and in light of evidence of reduced neutralization activity against some of the VOCs, vaccines that incorporate multiple antigens that are not under selective antibody pressure, could in theory contribute to long-term protective immunity through expanding the breadth of virus-specific T-cell responses.

In this respect, herein, we described the development and immunogenicity of SARS-CoV-2 VLP vaccine that incorporates the four structural proteins of the virus, all of which possess T-cell epitopes. [24] [25] [26] 47, 48 Our results showed that HEK-293 cells transfected with SARS-CoV-2 structural proteins reproducibly generated VLPs that were sim- 50 Differently, following injection, CpG ODN adjuvants locate less efficiently to draining lymph nodes in species larger than mice. 51 This drawback can be overcome through formulating the antigen and K3-CpG ODN together with alum to facilitate their delivery to lymph nodes. 52 Our data also indicate that VLPs expressing hexaproline stabilized alpha variant spike elicited a more potent response against WT and variant RBDs compared to 6p-WT S incorporating VLPs. Whether this is due to enhanced spike expression in VLPs or a change in immunogenicity of the variant spike, remains to be determined. 

There is no financial conflict of interest to declare by the Authors.

Immunoprotective activity of the VLP vaccine in K18-hACE2 transgenic mice. K18-hACE2 transgenic mice (n = 10/group) were subcutaneously immunized with 2 µg (low dose; LD) or 8 µg (high dose; HD) of the VLP vaccine on days 0 and 14. Two weeks after booster injection, (A) RBD-specific IgG, IgG1, IgG2c antibody titers were determined by ELISA and (B) neutralizing antibody titers against the authentic Wuhan strain and the B.1.1.7. Alpha variant were determined. Groups were compared by one-way ANOVA with Dunnett's multiple comparisons test. *P < .05, **P < .01, ***P < .001, ****P < .0001. On day 21 after booster, mice were challenged intranasally on 3 consecutive days with 50 µl of 1 × 10 5 pfu/mouse of SARS-CoV-2 (Wuhan strain). Lungs were collected 7 days after last virus instillation. (C) Infectious virus loads in lung homogenates were assessed by qRT-PCR against the nucleocapsid (NC1 and NC2). Bars represent the mean virus load (n = 10/group) as 1/ct values. Comparisons were performed by unpaired Student's t test; *P < .05, **P < .01, ***P < .001, ****P < .0001. (D) Histological micrographs showing healthy (first column), placebo (second column), low-dose vaccine (third column), and high-dose vaccine (fourth column) groups. (A-D) Hematoxylin-eosin (H&E), areas marked green shows inflamed parts of the lungs; (E-L) H&E, 20×; M-P, Gomori Trichrome (GT), 40×. a, alveoli; b, bronchiole; v, blood vessel; blue arrow, protein debris; red arrow, hyaline membrane. (E) Histomorphometric measurements. The descriptive statistics were presented as median and interquartile range in all graphs except for inflamed area percent (mean ± SD). Statistical significance (P < .05): a, compared to healthy group; b, compared to placebo group; c, compared to low-dose vaccine group, d, compared to high-dose vaccine group. Nonparametric variables were compared between groups using Kruskal-Wallis test. Pairwise comparisons were made with Dunn's test. Parametric variables were compared in multiple groups using one-way analysis of variance. Pairwise comparisons were performed with the Tukey test

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ICY, MG, and IG conceptualized the project. MG and IG led the study. These authors acted as co-second authors: EMI, AB, NT, NSY,   NG, MY, and IE. ICY designed protocols and carried out experiments   together with BG, BS, TB, YA, IB, AGB, BI, HAS, BK, YC, TY, IA,