Different Formulations of Inactivated SARS-CoV-2 Vaccine Candidates in Human Compatible Adjuvants: Potency Studies in Mice Showed Different Platforms of Immune Responses.

Several inactivated SARS-CoV-2 vaccines have been approved for human use, but are not highly potent. In this study, different formulations of the inactivated SARS-CoV-2 virus were developed in Alum, Montanide 51VG, and Montanide ISA720VG adjuvants, followed by assessment of immune responses. The SARS-CoV-2 virus was inactivated with formalin and formulated in the adjuvants. BALB/c mice were immunized subcutaneously with 4 μg of vaccines on days 0 and 14; (IL-4) and (IFN-g), cytotoxic T lymphocyte (CTL) activity, and specific immunoglobulin G (IgG) titer and IgG1, IgG2a, and IgG2a/IgG1 ratio, and anti-receptor-binding domain (RBD) IgG response were assessed 2 weeks after the final immunization. Immunization with SARS-CoV-2-Montanide ISA51VG showed a significant increase in the IFN-γ cytokine versus SARS-CoV-2-Alum, SARS-CoV-2-Montanide ISA720VG, and control groups (p < 0.0033). Cytokine IL-4 response in SARS-CoV-2-Alum group showed a significant increase compared with SARS-CoV-2-Montanide ISA51VG, SARS-CoV-2-Montanide ISA720VG, and control groups (p < 0.0206). In addition, SARS-CoV-2-Montanide ISA51VG vaccine induced the highest IFN-γ/IL-4 cytokine ratio versus other groups (p < 0.0004). CTL activity in SARS-CoV-2-Montanide ISA51VG and SARS-CoV-2-Montanide ISA720VG groups showed a significant increase compared with SARS-CoV-2-Alum and control groups (p < 0.0075). Specific IgG titer in SARS-CoV-2-Montanide ISA51 VG and SARS-CoV-2-Montanide ISA720VG showed a significant increase compared with SARS-CoV-2-Alum and control groups (p < 0.0143). Results from specific IgG1and IgG2a in SARS-CoV-2-Alum, SARS-CoV-2-Montanide ISA51VG, and SARS-CoV-2-Montanide ISA720VG vaccine showed a significant increase compared with phosphate buffer saline (PBS) group (p < 0.0001), but SARS-CoV-2-Montanide ISA51VG and SARS-CoV-2-Montanide ISA 720VG groups showed the highest IgG2a/IgG1 ratio and a significant increase compared with SARS-CoV-2-Alum group (p < 0.0379). Moreover, inactivated SARS-CoV-2+Alum and SARS-CoV-2-Montanide ISA 720VG groups demonstrated a significant increase in anti-RBD IgG response versus the SARS-CoV-2-Montanide ISA51VG group. It seems that the type of vaccine formulation is a critical parameter, influencing the immunologic pattern and vaccine potency and human-compatible oil-based adjuvants were more potent than Alum adjuvant in the vaccine formulation.


Introduction
The COVID-19 pandemic started in December 19 in Wuhan (China). The new SARS-CoV-2 is the seventh member of the Coronaviridae of the order Nidovirales with RNA genome that infects humans and mammals. The World Health Organization named the epidemic a Public Health Emergency of Global Importance in January 2020 and a pandemic in March 2020 (Jeyanathan, Afkhami et al. 2020).
Vaccination is the most effective way to prevent viral infections and it seems that it is important to have a global vaccination to prevent the pandemic (Mahdavi, Ebtekar et al. 2011). SARS CoV-2 transmission occurs with high effectiveness and infectivity, as with other respiratory viruses, predominantly along the respiratory pathway. The most known route is droplet delivery, while aerosols can constitute another signi cant route (Ciotti, Ciccozzi et al. 2020). As well, nancial problem, lost personal freedom and widespread emotional distress caused the increased of mental problems (Pfefferbaum and North 2020).
Thus, different countries and governments have been paid a high price to prevent the spread and treatment of this disease and also development an e cient vaccine (Wang, Shi et al. 2006). Analysis of the genomes and comparing with the previously identi ed genomes of coronaviruses show that SARS-CoV-2 has distinct characteristics that separate it from the other coronaviruses (Nao, Yamagishi et al. 2017, Andersen, Rambaut et al. 2020). It is believed that SARS-CoV-2 is able to transmit to another species (Shi, Wang et al. 2020). It is clear that both humoral and cellular immune responses are needed to combat and clear the viral infections, so these properties for a successful vaccine should be considered in the vaccine design and also vaccine formulation (Jamali, Mahdavi et al. 2007 In this study, we developed different formulations of inactivated SARS-CoV-2 viral particles with different classes of adjuvants and then cellular and humoral immune responses against each formulation of vaccine was assessed in the experimental mice. We have shown that different formulations of vaccine triggered different levels and platforms of immune responses.

Materials And Methods
Virus isolation, propagation SARS-CoV-2 virus was isolated from throat/nasal swab sample of a patient (positive in real time PCR) from Karaj, Iran (SARS-CoV-2/human/IRN/Alborz-IR/2020 surface glycoprotein (S) gene, complete cds 3,822 bp linear RNA MW709393.1 GI: 1998262910). The virus replication was performed on Vero cell line which were cultured in Dulbecco's Modi ed Eagle's Medium (DMEM) supplemented with 5% heatinactivated fetal bovine serum (FBS) and then con rmed by real-time PCR and gene sequencing (MW709393). For e cient growth in Vero cells, the virus strain was puri ed by plaque assay. The rst puri ed clone passaged three times on Vero cells to obtain an e cient virus stock. This stock virus shows more e ciency with more than 90% CPE in 48 hours post infection and a titer 6.5 to 7 TCID50/ml. The SARS-CoV-2 propagation carried out in biosafety level-III (BSL-III) facilities.

Virus inactivation and quanti cation
A SARS-CoV-2 strain was cultivated in Vero cell line for propagation, and the supernatant of the infected cells was inactivated with formalin 0.04% vol/vol at RT for 30 hours ( nally 4:10000). Following clari cation of cell debris concentration were performed by ultra ltration and 8% PEG-6000. Puri cation performed using column chromatography. The puri ed and inactivated viruses dialyzed versus PBS and passed through 0.22 lter and nally quanti ed using Bradford method. The quanti ed virus aliquots stored at -70 ºC until use.

Vaccines formulation
Inactivated SARS-COV-2 virus was formulated in Montanide ISA 720VG, Montanide ISA51VG (SEPPIC, France), and /or Alum hydroxide (Pasteur Institute of Iran) adjuvants. In order to formulate in oil-based adjuvants, inactivated SARS-COV-2 virus in PBS buffer was admixed with Montanide ISA 720VG (at the ratio of 30/70) and Montanide ISA 51VG (at the ratio of 50/50) by vigorously shaking to develop a milky white suspension and homogenized using homogenizer to develop a homogen suspension. In order to formulate Alum-based vaccine, 4µg of inactivated SARS-COV-2 virus in PBS buffer was mixed with 200µg of Alum hydroxide adjuvant (for each dose of vaccine formulation) and shaked at 100 RPM for 60 minutes at room temperature and allowed that the viral particles adsorb on the surface of Alum gel. All vaccine formulations were performed in the clean room of Department of FmD Vaccine formulation of Razi Vaccine and Serum Institute of Iran (Karaj, Iran). After nal vaccine formulation, each 200 µl of all vaccines contained 4µg of inactivated SARS-COV-2 virus that used for immunization.

Mice and immunization
Six-to eight-week-old male BALB/c mice (N=40) were purchased from Royan Institute of Iran (Tehran, Iran). The mice were housed for one week before the experiments, given free access to the food and water and maintained in a 12 hrs light\and 12 hrs dark cycles. The experimental mice were handled with an expert technician and in accordance with the Animal Care and Use Protocol of Razi Vaccine and Serum Research Institute of Iran. Experimental mice were randomly divided into four groups and each one consisting of 10 mice as below:

Group 4
Mice were immunized with PBS as control group.
Experimental groups of mice were immunized subcutaneously two times with14-day-intervals and two weeks after the nal immunization immunologic parameters were assessed.
In vitro antigen recall of spleen cells Two weeks after the second immunization, the spleen of the immunized mice was removed under sterile conditions and mechanically dissected and suspended in the sterile cold PBS containing penicillin and streptomycin. The cell suspension centrifuged at 300 g/ 5min and RBCs were lysed using lysis buffer and single-cell suspension was washed three times and adjusted to 3×10 6 cells/ml in RPMI-1640 (Gibco, Germany) supplemented with 10% FBS, 4 mM L-glutamine, 1 mM sodium pyruvate, 100 µg/ml streptomycin and 100 IU/ml penicillin. One milliliter of cell suspension containing a total number of 3 × 10 6 spleen cells were seeded in each well of 24-well plates. The spleen cells were then stimulated in vitro with adding 1 µg/ml of inactivated SARS-COV-2 virus and the plates incubated at 37°C in 5% CO2. After 48 hrs post antigen recall, the culture supernatant were centrifuged at 5000 RPM/10 min and the supernatants were harvested and stored at -70ºC for cytokines assay.

IFN-γ and IL-4 cytokines ELISA
The quantity of IFN-γ and IL-4 cytokines was estimated by quantitative mouse IFN-γ and IL-4 cytokine ELISA Kits (Mabtech, Stockholm, Sweden) according to the manufacturer's instruction. The ELISA of IFNγ and IL-4 cytokines were performed in the present of cytokines standards and using the standard curve of each cytokine. The pg/ml of each sample was reported and also for calculating the IFN-γ/IL-4 ratio, the quantity of IFN-γ of each mouse was divided into that one of IL-4 cytokine.

CTL assay
The CTL activity of experimental groups was assessed by Granzyme B (Gr-B) release. Brie y, 1.5×10 6 spleen cells of experimental mice were cultured in 96-well plate and stimulated with 1 µg/ml of inactivated SARS-COV-2 virus. As negative control for each mouse, some wells were cultured without antigen stimulation. The plates were incubated at 37°C in 5% CO 2 and after 48 hrs of antigen stimulation; culture supernatants were harvested and were used for Gr-B assay by ELISA according to the manufacturer's instruction (eBioscience, USA). The quantity of Gr-B of stimulated wells was subtracted with unstimulated one of each mouse and used as net Gr-B release as a criterion of CTL activity.

ELISA of speci c IgG antibodies titer and their isotypes
Speci c antibodies were determined by an optimized indirect ELISA method. Brie y, 100µl of 5µg/ml of inactivated SARS-COV-2 in PBS were added into 96-well ELISA Maxisorp plates (Greiner, Germany) and incubated overnight at 4°C. The wells washed with PBS containing 0.1% Tween 20 (washing buffer) and blocked 1 hr at 37°C with 2% skimmed milk in PBS + 0.1% Tween 20 (blocking buffer). The plates were washed with washing buffer and 100 µl of 1/25 diluted sera up to 16 serial dilutions were added into each wells and incubated at 37°C for 90 min. The wells washed ve times with washing buffer and incubated for 2 hrs with 100 µl of 1/8000 dilution of anti-mouse conjugated to HRP (Razirad, Iran). The wells washed ve times and incubated 30 min with 100 µl of TMB substrate in the dark and the reaction was stopped with 100 µl of 2N H2SO4 and colour density was measured at 450 nm with ELISA plate reader. The cut off of ELISA for each titer was calculated on 10 sera samples of sham group by formula: Mean+3SD of these samples and based on the cut off the titer of each mouse of the experiments was earned. The nal results of antibody titer for each mouse presented as Log10 of antibody titer. In addition, detection of speci c IgG1 and IgG2a subclasses were carried out using goat anti-mouse IgG1and IgG2a secondary antibodies (Sigma, USA) according to the manufacture's instruction.

Assessment of speci c IgG anti-RBD protein
The speci c IgG antibody response against RBD protein was determined by a laboratory-optimized indirect ELISA. At the rst for antigen coating, 100 µl of 2µg/ml of RBD protein (The Native Antigen Company, UK) in carbonate-bicarbonate buffer with pH 9.6 was added into 96-well ELISA Maxisorp plates (Greiner, Germany) and incubated overnight at 4°C. The wells were washed with PBS containing 0.1% tween 20 (washing buffer) and then blocked 1 hr at 37°C with 1.5% BSA in PBS + 0.05% tween 20 (blocking buffer). The plates were washed 5 times with washing buffer and 100 µl of 1/50 diluted sera of experimental vaccinated and sham group of mice were added into each well and incubated at 37°C for 90 min. The wells were washed 5 times with washing buffer and incubated for 90 min with 100 µl of 1/8000 dilution of anti-mouse HRP-conjugate (Razirad, Iran). The wells washed 5 times and incubated 10min with 100 µl of TMB substrate in the dark and the reaction was stopped by adding 100 µl of 2N HCL and colour density was measured at 450 nm with ELISA plate reader. The cutoff of RBD-ELISA was calculated on the sera samples of sham group by formula: Mean+3SD and then IgG response to RBD for individual mouse was reported by formula; OD of RBD ELISA of individual mouse/cutoff.

Statistical analysis
The data were presented as mean ± standard deviation (SD) of each experiment. The statistical signi cance of differences was analysed using two-way ANOVA followed by Tukey test (Graph Pad Prism 6.01 software, Graph Pad Software, La Jolla, CA). Statistical analysis of antibody titers and IgG isotypes were carried out by the Mann-Whitney U test. In all of the cases, P values less than 0.05 was considered signi cant difference.

IFN-γ cytokine
The results of IFN-γ cytokine in the experimental groups show that only the injection of SARS-COV-2 vaccine formulated in Montanide ISA51VG adjuvant resulted to a signi cant difference as compared with the control group (P= 0.0003). In addition, immunization with vaccine-Montanide ISA51VG showed a signi cant increase in IFN-γ response as compared with the vaccines that formulated in Alum and Montanide ISA 720VG adjuvants (P=0.0004 and P=0.0033, respectively) (Fig. 1).

Speci c IgG2a antibody
The results of speci c IgG2a isotype antibody in the experimental groups demonstrated that immunization with SARS-COV-2-Montanide ISA51VG, SARS-COV-2-Alum and SARS-COV-2-Montanide ISA 720VG vaccines showed a small increase versus control group but no signi cant differences was observed among experimental groups (P>0.1550) (Fig. 7). In this study, we also used the secretion of Granzyme B as a marker of CTL activity. The results showed that inactivated SARS-COV-2 vaccine based on Montanide ISA-51 VG formulation showed the highest Gr-B release meaning the highest CTL activity among the experimental groups. Gr-B triggers a cascade of events within the target cells that infected with virus that lead to induction the apoptosis and cell death (Lord, Rajotte et al. 2003) as well as clearance of viral infection from the lungs (Doherty, Topham et al. 1997). It is well-known that early decrease in TCD8+ population is an important factor in the pathogenesis of COVID-19 infection and improvement of TCD8+ lymphocyte response in the vaccination would be an important mechanism for resistance against COVID-19 infection (Elsaesser, Sauer et al. nding shows that Montanide-based vaccine is able to trigger anti-RBD response like as Alum-based vaccine but oil-based vaccines were superior to Alum-based vaccine in the induction of Th1 cytokine and CTL activity which is critical in the controlling SARS-COV-2 infection.

Conclusion
In conclusion the results of the present study provide evidences that the type of adjuvant in the vaccine formulation can modi es the type of immune responses and also potency of humoral immune responses. This nding encourages the SARS-COV-2 vaccinologists to focus on the different formulations of vaccine to achieve a more potent and safe vaccine especially for inactivated SARS-COV-2 based vaccines which has fewer e cacies than mRNA-based vaccines. Figure 1 The results of IFN-γ cytokine in the experimental groups. Immunization with SARS-COV-2-Montanide ISA-51VG shows a signi cant increase versus control group (P= 0.0003). In addition, immunization with SARS-COV-2-Montanide ISA-51VG showed a signi cant increase in IFN-γ response as compared with SARS-COV-2-Alum and SARS-COV-2Montanide ISA-720VG groups (P=0.0004 and P=0.0033, respectively).        Results from speci c IgG antibodies against RBD protein which reported as ratio of OD of RBD ELISA of individual mouse/cutoff. Immunization with inactivated SARS-COV-2+Alum and SARS-COV-2-Montanide ISA 720VG vaccines showed a signi cant increase versus SARS-COV-2-Montanide ISA51VG group