key: cord-0822968-hp6s6ebh
authors: Petráš, Marek; Lesný, Petr; Musil, Jan; Limberková, Radomíra; Pátíková, Alžběta; Jirsa, Milan; Krsek, Daniel; Březovský, Pavel; Koladiya, Abhishek; Vaníková, Šárka; Macková, Barbora; Jírová, Dagmar; Krijt, Matyáš; Králová Lesná, Ivana; Adámková, Věra
title: Early immune response in mice immunized with a semi-split inactivated vaccine against SARS-CoV-2 containing S protein-free particles and subunit S protein
date: 2020-11-03
journal: bioRxiv
DOI: 10.1101/2020.11.03.366641
sha: f61613107fb8c0a7f89801d8734404316dc3ea63
doc_id: 822968
cord_uid: hp6s6ebh

The development of a vaccine against COVID-19 is a hot topic for many research laboratories all over the world. Our aim was to design a semi-split inactivated vaccine offering a wide range of multi-epitope determinants important for the immune system including not only the spike (S) protein but also the envelope, membrane and nucleocapsid proteins. We designed a semi-split vaccine prototype consisting of S protein-depleted viral particles and free S protein. Next, we investigated its immunogenic potential in BALB/c mice. The animals were immunized intradermally or intramuscularly with the dose adjusted with buffer or addition of aluminum hydroxide, respectively. The antibody response was evaluated by plasma analysis at 7 days after the first or second dose. The immune cell response was studied by flow cytometry analysis of splenocytes. The data showed a very early onset of both S protein-specific antibodies and virus-neutralizing antibodies at 90% inhibition regardless of the route of vaccine administration. However, significantly higher levels of neutralizing antibodies were detected in the intradermally (geometric mean titer - GMT of 7.8 ± 1.4) than in the intramuscularly immunized mice (GMT of 6.2 ± 1.5). In accordance with this, stimulation of cellular immunity by the semi-split vaccine was suggested by elevated levels of B and T lymphocyte subpopulations in the murine spleens. These responses were more predominant in the intradermally immunized mice compared with the intramuscular route of administration. The upward trend in the levels of plasmablasts, memory B cells, Th1 and Th2 lymphocytes, including follicular helper T cells, was confirmed even in mice receiving the vaccine intradermally at a dose of 0.5 μg. We demonstrated that the semi-split vaccine is capable of eliciting both humoral and cellular immunity early after vaccination. Our prototype thus represents a promising step toward the development of an efficient anti-COVID-19 vaccine for human use.

The COVID19 pandemic has made the development of a vaccine an emergency priority. 46

Hectic research was enabled by recent major advances in sequencing, protein structure 47 8 applied into the right caudal thigh muscle, the intradermal one was applied into the auricle 167 under the guidance of a microscope. 168

One week after the first or second dose, the mice were anesthetized with isoflurane and blood 169 was intracardially withdrawn using a 2 ml syringe, transferred to anticoagulant tubes 170 (K 2 EDTA), and mixed. Whole blood was centrifuged (3300 rpm, 10 minutes, 4°C), plasma 171 aliquoted into 100 µl microtubes and frozen at -80° C. The spleens were collected in chilled 172 RPMI medium on ice and transported to laboratory within 4 hours for flow cytometry 173 examination. 174 175

Anti-S specific IgG antibodies as well as virus-neutralizing antibodies were expressed as the 177 titer. The value had to be log-transformed to pass a normality test (D'Agostino & Pearson or 178 Shapiro-Wilk test) followed by a parametric t-test or analysis of variance (ANOVA). 179

As the lymphocyte populations were measured from pooled spleens of each group, the 180 traditional statistical approach could not be employed; hence, the change in lymphocyte 181 subsets using linear regression was determined. If the slope of lines exhibited values 182 significantly different from null, then the observed change was considered to be confirmed. 183

Merged lymphocyte subpopulations were analyzed with parametric tests after log-184 transformation to pass a normality test (Shapiro-Wilk test). The power of each test was 185 insufficient since the sample size was small. A >80% power of the test was only achieved 186 when comparing the geometric mean titers of antibodies between immunized and 187 unimmunized mice. 188

The correlation between IgG antibodies and virus-neutralizing antibodies was assessed with 189 the Pearson correlation coefficient after the log-transformation of their values.

Continuous data were summarized using standard descriptive statistics, i.e., median including 191 range or geometric mean with standard deviation or 95% confidence interval. All tests were 192 two-tailed, and the level of significance was set at 0.05. Statistical analyses were performed 193 using Prism 8 (GraphPad Software, Inc., San Diego, CA) and STATA version 16 software 194 (StatCorp, College Station, TX). 195 196 Results 197 198 Course of the culture, inactivation and purification process 199

To obtain a viral stock for vaccine production, the virus strain 951 was first purified by 4 200 passages in VERO E6 cells. Subsequently, another 8 passages were performed to generate a 201 sufficient stock of virus for the production of the master seed as the source of a virus bank for 202 vaccine production through the seed lot system. Growth kinetics analysis of the second 203 passage showed a sufficient virus replication rate reaching a titer of 6.0-6.5 log 10 TCID 50 /mL 204 in the supernatant within 2-8 days ( Figure 1 ). The peak titers of 6.5 and 7.5 log 10 TCID 50 /mL 205 from the supernatant and lysate, respectively, were at 56 hours post-infection. Moreover, 206 multiplicities of infection (MOI) of 0.001-0.0001 at a culture temperature of 37°C and 5% 207 CO 2 were confirmed. 208 Confluent monolayers of Vero E6 cells, grown in OptiPro serum-free medium (Life 209 Technologies Europe BV, the Netherlands) with 10% fetal bovine serum (FBS), were infected 210 by the strain from the second passage of the stock and incubated in a serum-free medium at 211 37°C and 5% CO 2 for 56 hours. The first viral suspension of 225 mL was obtained by 212 centrifugation of the supernatant harvest at 300 g for 5 minutes. The second suspension was 213 harvested from infected cells by adding an approx. 90 mL of medium, overnight freezing and 214 thawing at -20°C followed by centrifugation under the same conditions. Both live viral 215 suspensions containing viral particles with typical corona spikes as documented by electron-216 microscopic inspection ( Figure 2A ) were stored at 4°C for at least 11 days. 217

The suspensions were subsequently inactivated by adding beta-propiolactone diluted 1:2,000, 218 and continuously shaken up at a temperature of 4°C for 48 hours. The beta-propiolactone 219 hydrolysis at 37°C lasted 2 hours. Inactivated suspensions were centrifuged and the 220 supernatants were stored at 4°C for one week. The second inactivation was performed with 221 beta-propiolactone diluted 1:1,000 at 4°C for 66 hours. After that, the pellets were centrifuged 222 at 1,800 g for 10 minutes and both inactivated suspensions were subsequently placed in water 223 baths to hydrolyze beta-propiolactone with continuous stirring at 37°C for 2 hours with the 224 temperature gradually decreasing to 27°C overnight. 225

The inactivated viral suspension contained of 0.5 μ g/mL of the S protein for the supernatant 226 harvest and 4.9 μ g/mL for the lysate harvest. While the first inactivation did not influence the 227 virion structure, the second one showed partial changes, i.e., complete and incomplete 228 particles with disrupted S protein ( Figure 2B and 2C). 229

The inactivation including hydrolysis decreased the pH to 6.0-6.5 that disrupted S protein 230 binding to the viral envelope as documented by a record from the electron microscope ( Figure  231 2C). Both suspensions were stored at 4°C for 3 days to be followed by vaccine purification 232 and thickening to obtain 3 μ g/10 μ L the for harvested supernatant and 1 μ g/10 μ L for the 233 harvested lysate. This was achieved by a multiple ultra/diafiltration process utilizing Amicon ® 234 Ultra centrifugal filter units (Amicon Ultracel) with a volume of 15 mL and a membrane 235 molecular weight cutoff (MWCO) of 100 kDa (Amicon ® Ultra-15 Centrifugal Filter Unit -236 100 kDa cutoff, Merck Millipore Ltd., Darmstadt, Germany). The suspensions were 237 centrifuged several times in Amicon Ultracel at 1,000 g for at least 5 minutes to exchange the 238 culture medium and to remove cellular debris and low-molecular substances. The suspension 239 was washed several times with phosphate buffer saline (PBS) of pH 7.4. 240

within a dilution range of 10 -1 to 10 -4 . 242

The result of purification and thickening were suspensions containing both virion particles 243 free of the spike and separated S protein as documented by electron microscopy ( Figure 2D ). 244

The vaccine dose for the test of immunogenicity in mice was adjusted in PBS to 245 concentrations of 0. significantly increasing proportions not only of follicular Th lymphocytes but also Th1 and 302

Th2 lymphocytes with an increasing number of doses ( Figure 7A ). In addition, the same 303 effect was observed in these mice for subsets of plasmablasts and memory B cells. 304 305 Discussion 306

The above laboratory procedure generated a semi-split inactivated vaccine, i.e., a vaccine with 307 the S protein separated from the viral particle exhibiting an early, both humoral and cellular, 308 immune response. 309

The design of this prototype vaccine was based on the usual procedures [Tang 2003 , Spruth 310 2005 , Gao 2020 ]. We selected a strain showing higher infectivity with lower virulence, i.e., 311 one that showed signs of attenuation. Overall, we have tested several candidate strains for 312 both immunogenicity through convalescent plasma and propagation capacity by viral titration. 313 14 Viral growth kinetics analysis was in line with the published data [Gao 2020 , Manenti 2020 . 314 Furthermore, we devised a technique to increase the virus concentration in suspension about 315 100 to 1,000-fold (note: this finding requires validation). 316

To inactivate the virus properly, beta-propiolactone was chosen because it can alkylate the 317 viral genome and keeps the virus capable of inducing a protective immune response [Delrue 318 2012] . The process of viral suspension double inactivation was designed in accordance with 319 the procedures of other vaccine manufacturers [Pu 2020 , Xia 2020 . 

Design of a Multiepitope-Based Peptide Vaccine 370 against the E Protein of Human COVID-19: An Immunoinformatics Approach

Guidelines for the use of flow cytometry and cell sorting in 375 immunological studies (second edition)

Inactivated virus vaccines from 378 chemistry to prophylaxis: merits, risks and challenges

Safety and immunogenicity of the ChAdOx1 nCoV-386 19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, 387 randomised controlled trial

Development of an inactivated vaccine candidate for SARS-CoV-2

An in-silico 397 approach to develop of a multi-epitope vaccine candidate against SARS-CoV-2 398 envelope (E) protein. Res Sq

CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a 403

Beigel JH; mRNA-1273 Study Group

Vaccine against SARS-CoV-2 -Preliminary Report

Evaluation of SARS-CoV-2 neutralizing antibodies using a CPE-447 based colorimetric live virus micro-neutralization assay in human serum samples

Epub ahead 449 of print

RNA vaccine BNT162b1 in adults

Immune and bioinformatics identification of T cell and B cell epitopes in the 458 protein structure of SARS-CoV-2: A systematic review

An in-depth investigation of the safety and immunogenicity of an 468 inactivated SARS-CoV-2 vaccine

Determination of 50% endpoint titer using a simple formula

Convergent antibody responses to SARS-CoV-2 in 481 convalescent individuals

Concurrent human antibody and TH1 type 491

T-cell responses elicited by a COVID-19 RNA vaccine

Structural insight into the role of novel SARS-CoV-2 E protein: 494 A potential target for vaccine development and other therapeutic strategies

A double-inactivated 499 whole virus candidate SARS coronavirus vaccine stimulates neutralising and 500 protective antibody responses. Vaccine

Preparation, characterization and 505 preliminary in vivo studies of inactivated SARS-CoV vaccine

Immunization with SARS coronavirus vaccines leads to 510 pulmonary immunopathology on challenge with the SARS virus

RNA-Based COVID-19 Vaccine 518

BNT162b2 Selected for a Pivotal Efficacy Study. medRxiv

Inactivated Vaccine Against SARS-CoV-2 on Safety and Immunogenicity 526

Outcomes: Interim Analysis of 2 Randomized Clinical Trials

Immunogenicity and Safety of a SARS-CoV-2 533

Double-blind, and Placebo-controlled Phase 2 Clinical Trial

Augmentation of immune 537 responses to SARS coronavirus by a combination of DNA and whole killed virus 538 vaccines. Vaccine

Immunogenicity and safety of a recombinant adenovirus type-544 5-vectored COVID-19 vaccine in healthy adults

Safety, tolerability, and immunogenicity of a recombinant 551 adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation

non-randomised, first-in-human trial