Introduction 

The Wuhan City Health Commission reported an increase in cases of pneumonia on December 31, 2019, which determined and named SARS-CoV-2 ¹. The virus spreads rapidly around the world with a high mutation rate, and emerging new mutants cause great health damage ². Several vaccines were approved and commercialized for COVID-19, but some vaccines were not highly effective, and in new waves of mutated ones, the vaccines could not neutralize new mutants, because some mutants of SARS-CoV-2 change amino acids that correlate with the neutralization activity of the humoral immune response ³. Therefore, develop-ment of new vaccines based on these variants is needed or change in the vaccine formulation may abrogate this

weakness. An adjuvant, as a component of vaccine formulation, has a critical role in immune response polarization and formation ⁴. Numerous different classes of compounds have been evaluated as vaccine adjuvants, including mineral salts, microbial items, emulsions, saponins, cytokines, polymers, microparticles, and liposomes based on their proposed components of activity and immunostimulatory adjuvants ^5-7. Particulate adjuvants have been designed to form a neighborhood pro-inflammatory environment to trigger expression of cytokines, chemokines, immune receptors, and some other biological molecules, which were commonly balanced by Alum, MF59, and other adjuvants at the location of vaccine injection ^8,9. The MF59 adjuvant up-regulates the expression of CCR2, a receptor for CCL2, which is involved in monocyte and other immune cell migration to the injection site ¹⁰. In addition, Alum, as the first human vaccine adjuvant, was demonstrated to promote Th2-type and humoral immune responses ¹¹. Several inactivated SARS-CoV-2 vaccines were formulated in Alum and various experiences have suggested that Alum generally favors humoral/Th2-biased responses in many formulations ^12,13.

However, immune response analysis in COVID-19 patients demonstrated the need for both Th1 and Th2 responses to efficiently clear viruses from the lung and body ^12,14. Furthermore, several waves of SARS-CoV-2 outbreak due to emerging new mutants of the virus led to the inefficacy of approved vaccines, especially inactivated vaccine platforms, and showed a huge challenge in the protection of societies ^15-17.

Therefore, the current vaccines must be optimized by changing either the antigen present in the vaccines, the adjuvant and/or the vaccine formulation to trigger both robust immune responses and cross-reactive immune responses against new variants. Studies show the potency of MF59 adjuvant in the induction of both humoral and cellular immune responses. Some studies have shown the superiority of MF59 adjuvant in the induction of immune responses and even protection in comparison to alum adjuvant ^18,19.

In the present study, the inactivated Wuhan-Hu-1 strain of SARS-CoV-2 virus was formulated in Alum and MF-59 adjuvants and the immune response and cross-binding activity of these vaccines were assessed in the experimental mice. In this study, we compared the immunogenicity and Alpha-binding IgG responses induced by inactivated SARS-CoV-2 vaccine formulations containing MF59 or Alum in mice. 

Materials and Methods 

Experimental animals

Six- to 8-week-old male inbred Balb/c mice were obtained from the Razi Vaccine and Serum Research Institute of Iran (Karaj, Iran). The mice were housed for 7 days prior to the experiments and given food and water ad libitum in standard conditions (light/dark cycle, 12 hr/12 hr, and 20±2°C temperature). 

Virus preparation and inactivation

The Wuhan-Hu-1 strain and Alpha variant (B.1.1.7) "English variant" were isolated from patients living in the Alborz province (Karaj, Iran) who had clinical symptoms of COVID-19 infection. The infection was confirmed by Real-time PCR and the isolated viruses were cultured on the VERO cell line (ATCC® CRL-1586™) using Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% heat-inactivated Fetal Bovine Serum (FBS). The viruses were inactivated and purified as reported previously ^12,20-22. The purified Wuhan-Hu-1 strain and Alpha variant (B.1.1.7) were dialyzed against Phosphate Bovine Serum (PBS) to remove undesired salts. Then, the virus samples were passed through a 0.22 filter, and the concentration of each virus was determined by Bradford. The viruses were stored at -70ºC until use. All procedures on the virus culture and treatments were performed in a BSL-III facility in compliance with the biosafety guidelines of the Razi Vaccine and Serum Research Institute of Iran. 

Formulation of inactivated SARS-CoV-2 in MF-59 and Alum adjuvants

Inactivated SARS-CoV-2 (Wuhan-Hu-1 strain) was formulated in MF-59 (SEPPIC, France), and/or Alum hydroxide (Brenntag Biosector, Denmark) adjuvants. To formulate in the MF-59 adjuvant, 4 μg of inactivated SARS-CoV-2 in 50 μl PBS buffer was mixed with 50 μl of MF-59 (at a v/v ratio of 50/50) (Vaccine Formulation Laboratory, Razi Vaccine and Serum Research Institute, Karaj, Iran) for 30 min using a vortex at 3000 rpm and a homogenizer until a homogeneous product developed. To formulate the inactivated SARS-CoV-2-Alum vaccine, according to our laboratory set up, 4 μg of the virus was admixed with 200 μg of Alum and shaken at 100 rpm for 60 min at room temperature to make adsorption of the viral particles on the surface of Alum gel ²³.  

Immunization and study design

The Balb/c mice were assigned to three experimental groups by simple randomization, each one consisting of 10 mice. Groups 1 to 3 were immunized subcutaneously with 100 µl of inactivated SARS-CoV-2-MF59 (4 μg), inactivated SARS-CoV-2-Alum and PBS, respectively. The experimental mice were immunized three times on days 0, 21, and 42, followed by evaluation of immune responses three weeks after the final immunization ¹². 

Spleen cell harvesting

Twenty-one days post the last immunization, the mice of all experimental groups were euthanized by cervical dislocation and the spleens were removed and put in cold PBS/FBS 2% under the laminar hood and dissected mechanically. The single cell suspension was provided by vigorously pipetting and centrifuging at 300 g/5min/4°C and the RBCs were lysed using ammonium chloride as mouse RBC lysis buffer. The single-cell suspension was centrifuged at 300 g/5min/4°C and then resuspended in RPMI-1640 (Gibco, Germany) which was supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acids, 100 µg/ml streptomycin and 100 IU/ml penicillin. The cell number and viability were checked by trypan blue dye exclusion ²². 

Lymphocyte proliferation assessment by the BrdU method

Three weeks after the third immunization, 3×10⁵ cells in 100 µl complete RPMI-1640 medium were dispensed into each well of 96-well flat-bottom culture plates (SPL, China). The cells were stimulated with 200 ng of inactivated SARS-CoV-2 in a total volume of 200 μl. Un-stimulated cells, Phytohemagglutinin-A (5 μg/ml, Gibco)-stimulated cells and culture medium were considered as negative control, positive control and blank, respectively. After three days of in vitro stimulation, 20 µl of BrdU (Roche, Germany) reagent was added to each well. After an 18-hr incubation, the plates were centrifuged at 300 g for 10 min, the supernatant was discarded, the plates were dried at 60ᵒC for 30 min, and 200 μl of fixation/denaturation buffer was added to each well at room temperature. After 30 min, the buffer was aspirated and 100 μl of the anti-BrdU-HRP conjugate was added to each well. After a 2-hr incubation, the plates were washed 5 times with PBS, and the TMB substrate was added to the wells and incubated for 10 min in the dark.   

Then, 100 μl of 2N H2SO4 was added to stop the reaction. The Optical Density (OD) of the samples was read at 450 nm by an ELISA plate reader (BioTek, USA). The lymphocyte proliferation response was reported as stimulation index (SI): OD₄₅₀ of the stimulated wells/OD₄₅₀ of the un-stimulated wells ²². 

Antigen recall of lymphocytes and collecting culture supernatant

Spleen cells from the experimental mice, in a total number of 3×10⁶ in 1500 μl of complete RPMI-1640 medium, were seeded into each well of 24-well plates (SPL, China). The cells were stimulated by adding 1500 ng/well of inactivated SARS-CoV-2 at 37°C in 5% CO₂. After 60 hr of the antigen recall, the culture supernatant was harvested and stored at -70°C for IFN-γ and IL-4 cytokine assessment. 

Quantitative ELISA for IFN-γ and IL-4 cytokines

The quantity of IFN-γ and IL-4 cytokines in the culture supernatant of spleen cells was estimated by quantitative mouse IFN-γ and IL-4 cytokine ELISA Kits (Mabtech, Stockholm, Sweden) according to the manufacturer’s manual. The quantity of each cytokine was reported as pg/ml using a standard curve of each cytokine. In addition, the IFN-γ/IL-4 ratio was reported ²⁴. 

Mouse bleeding and serum separating

Three weeks after the last immunization, the mouse bleeding was performed by retro-orbital sinus puncturing. The blood samples were centrifuged at 10000 rpm/10 min and the serum samples were harvested and frozen at -20°C until specific IgG response assay. 

Specific total IgG response by an optimized indirect ELISA

An optimized indirect ELISA was used to assess the specific IgG response. Briefly, 500 ng of inactivated SARS-CoV-2 (Wuhan-Hu-1 strain and Alpha variant (B.1.1.7) in each ELISA) in 100 μl PBS was added into 96-well ELISA Maxisorp plates (Greiner, Germany) and incubated at 4°C overnight. The plates were washed with washing buffer (PBS plus 0.1% Tween 20) and blocked for 60 min at 37°C with blocking buffer (2% skimmed milk in washing buffer). The plates were washed and 100 µl of serial dilutions of serum samples from 1/500 up to 1/1124000 were added into each well. The plates were incubated at 37°C for 90 min and washed five times. Then, 100 µl of 1/10000 dilution of anti-mouse HRP-conjugate (Razirad, Iran) was added to each well and the plates were incubated for 2 hr. The plates were washed five times and 100 µl of TMB substrate was added to each well and kept in the dark. The colorimetric reaction was stopped with 100 µl of 2N H₂SO₄ and the optical density was measured at 450/630 nm with an ELISA plate reader (BioTek, USA) and IgG response was reported as endpoint titer of each individual mouse ²². 

Specific IgG1 and IgG2a isotypes assessment

The assessment of the specific IgG1 and IgG2a subclasses was carried out on the 1/1000 serum dilution of experimental mice. The ISO-2 kit (Sigma, USA) was used to assess these isotypes. Goat anti-mouse HRP-conjugate (Razirad, Iran) was used for the assessment, according to the manufacturer’s manual ²⁵. 

Statistical analysis

All experiments for each mouse were done in duplicate and/or triplicate. The data of all the experiments were presented as mean±standard deviation (SD). Test for normal distribution was done using Kolmogorov-Smirnov test. Data of BrdU, IFN-γ, IL-4, and IFN-γ/IL-4 ratio were analysed by One-way ANOVA followed by Tukey test (Graph Pad Prism 6.01 software, La Jolla, CA, USA) were used for statistical analysis. The data of total IgG titer, IgG1 and IgG2a isotypes and total IgG cross-binding IgG were analysed by Mann Whitney test considering to the distribution of raw data. In all of the cases, p values less than 0.05 were considered significantly different. A confidence intervals of 95% was considered for statistical analysis. 

Results 

Lymphocyte proliferation

Results from the lymphocyte proliferation response in the experimental groups showed that injection of the inactivated SARS-CoV-2-MF59 led to a significant increase as compared with the control group (p=0.0012). However, the vaccine formulated in Alum led to increased proliferative responses by 9.037% as compared with the control group, but was not statistically significant (p=0.8267). In addition, inactivated SARS-CoV-2-MF59 showed a significant increase in proliferation responses as compared with the inactivated SARS-CoV-2-Alum group (p=0.0042) (Figure 1). 

IFN-γ cytokine

Results from the IFN-γ cytokine response in the experimental groups showed that injection of the inactivated SARS-CoV-2-MF59 vaccine led to a significant increase in the IFN-γ cytokine as compared with the control group (p=0.0017). In addition, the inactivated SARS-CoV-2-Alum group showed a 24.39% increase the control group but was not statistically significant (p=0.5985). Furthermore, injection of the inactivated SARS-CoV-2-MF59 group showed a significant increase in the IFN-γ cytokine response as compared with the vaccine formulated in Alum (p=0.0089) (Figure 2). 

IL-4 cytokine

Results from the IL-4 cytokine response in the inactivated SARS-CoV-2-MF59 and inactivated SARS-CoV-2-Alum groups revealed a significant increase as compared with the control group (p=0.0462 and p=0.0113, respectively). However, no significant difference was observed between the inactivated SARS-CoV-2-MF59 and inactivated SARS-CoV-2-Alum groups in the IL-4 cytokine response (p=0.7662) (Figure 3). 

IFN-γ/IL-4 cytokine ratio

The analysis of the IFN-γ/IL-4 ratio in the inactivated SARS-CoV-2-MF59 group showed a 49.55% increase as compared with the control group (p=0.0741). However, immunization with the inactivated SARS-CoV-2-Alum group did not show a significant difference as compared with the control group (p=0.7707). In addition, the inactivated SARS-CoV-2-MF59 group showed a significant increase in the IFN-γ/IL-4 ratio the inactivated SARS-CoV-2-Alum group (p=0.0068) (Figure 4). 

The results from the specific IgG titer against the Wuhan strain of SARS-CoV-2

Results from the specific IgG titer against the Wuhan strain virus showed that immunization with the inactivated SARS-CoV-2-MF59 and inactivated SARS-CoV-2-Alum groups resulted in a significant increase the control group (p=0.0001). In addition, immunization with inactivated SARS-CoV-2-MF59 vaccine led to a significant increase in the specific IgG titer the inactivated SARS-CoV-2-Alum group (p= 0.0270) (Figure 5). 

Specific IgG1 response

Results from the specific IgG1 isotype antibody in the inactivated SARS-CoV-2-MF59 and inactivated SARS-CoV-2-Alum groups showed a significant increase the control group (p=0.0001). In addition, immunization with the inactivated SARS-CoV-2-MF59 vaccine revealed a significant increase in the specific IgG1 response the inactivated SARS-CoV-2-Alum group (p=0.0121) (Figure 6A). 

Specific IgG2a response

Results from the specific IgG2a isotype antibody in the inactivated SARS-CoV-2-MF59 showed a significant increase the control group (p=0.0079), while the inactivated SARS-CoV-2-Alum vaccine showed no significant difference as compared with the negative control group (p=0.2456). In addition, immunization with the inactivated SARS-CoV-2-MF59 vaccine led to a significant increase in the specific IgG2a response as compared with the inactivated SARS-CoV-2-Alum vaccine (p=0.0493) (Figure 6B). 

Results from cross-binding IgG titer against Alpha variant (B.1.1.7) of SARS-CoV-2

Results from the specific IgG titer against the heterologous Alpha variant virus in the inactivated SARS-CoV-2-MF59 and inactivated SARS-CoV-2-Alum groups showed a significant increase the control group (p=0.0001). In addition, immunization with the inactivated SARS-CoV-2-MF59 vaccine showed a numerically higher (70.83%), but not statistically significant (p=0.1989), Alpha-binding IgG response compared with Alum (Figure 7). 

Discussion 

It is clear that some properties of a vaccine are driven by the adjuvant used in the vaccine formulation. It has been shown that the adjuvant nature has an important role in vaccine immunogenicity, and this part is less studied regarding the potency of an adjuvant on the cross-reactive response for an inactivated SARS-CoV-2 vaccine ²⁶. Here, we hypothesized that vaccine formulation may be involved in the cross-binding response of vaccines. Therefore, the present study used MF59 and Alum adjuvants in the inactivated SARS-CoV-2 vaccine model to compare the immunogenicity as well as cross-binding properties of these vaccines. Results from this study showed a significant increase in lymphocyte proliferation response of the MF59-inactivated SARS-CoV-2 group as compared with the Alum-formulated one. Considering that lymphocyte proliferation is a marker of cell-mediated immune response ²⁴, this finding showed more potency of the MF59 adjuvant in the induction of cellular immune response as compared with the Alum-based vaccine. A study on recombinant H1N1 hemagglutinin formulated in MF59 and Alum adjuvants showed a stronger T cell response in the MF59-adjuvanted vaccine as compared with the Alum-based vaccine ²³. In another study, Wack et al showed that the influenza vaccine formulated in the MF59 adjuvant led to a better T cell response than the Alum-based vaccine ²⁷. These findings are parallel to our findings and confirmed that a better T cell response can be achieved when the vaccine is formulated in the MF59 adjuvant. Results from IFN-γ and IFN-γ/IL-4 ratio showed a significant increase in the MF59 adjuvant group vs. the Alum adjuvant. Nevertheless, IL-4 cytokine response in the MF59 adjuvant group showed no significant difference vs. the Alum group. This finding may be related to the nature of alum and MF59 adjuvants, because both adjuvants are able to induce IL-4 response. While, the MF59-inactivated SARS-CoV-2 vaccine was associated with a more IFN-γ-dominant cytokine profile.

A study on a recombinant H1N1 hemagglutinin vaccine showed a higher IFN-γ cytokine response in the MF59-formulated vaccine vs. the Alum-adjuvanted vaccine ²³. In addition, a study on the split influenza A/H7N9 vaccine formulated with the MF59 adjuvant showed strong IFN-γ and Th1 responses in comparison with the Alum vaccine ²⁸. These findings confirmed our results and showed the capability of the MF59-formulated vaccine vs. the Alum vaccine in the induction of the IFN-γ response. It is clear that IFN-γ through differentiation and stimulation of T cell response leads to a cellular immune response which in turn kills virus-infected cells through the function of CD8+T cells and finally leads to virus clearance ^29,30. However, interpretation about the pattern of immune response is based on a limited cytokine panel and does not directly involve phenotypic characterization of T-cell subsets or measurement of Th1-associated transcription factors and this can be considered a limitation for this study.

Next, the specific IgG titer was assessed in the experimental groups vs. the vaccine strain (Wuhan-Hu-1 strain) as well as a non-vaccine strain (Alpha variant (B.1.1.7) "English variant"). Results from the specific IgG response vs. the vaccine strain (Wuhan-Hu-1 strain) showed a significant increase in the MF-59 vaccine group vs. the Alum vaccine. A study carried out by Boudreau et al on the H5N1 avian influenza vaccine administered with MF59 and/or with alum showed a strong IgG response when MF59 was used as an adjuvant in comparison with the Alum vaccine ³¹. In addition, a study on healthy and diabetic mice showed that immunization with inactivated whole-virion H5N1-MF59 vaccine enhanced the antibody responses in both healthy and diabetic mice, but the MF59-adjuvanted vaccine group showed a significant increase in IgG responses as compared with the Alum-adjuvanted group ³².

In a study conducted by Bai et al., the inactivated SARS-CoV-2-MF59 vaccine was demonstrated to induce more robust humoral immune responses as compared with the Alum adjuvant ³³.

Results from these studies confirm our findings about a higher IgG response in the MF59-adjuvanted vaccine. In addition, results from IgG1 and IgG2a isotypes showed a significant increase in the SARS-CoV-2-MF59 vaccine vs. the Alum vaccine. It is proven that each isotype of IgG has a critical role on the bioactivity of the humoral immune response and sometimes one special isotype of IgG is correlated with protection in a host. Considering that each isotype of IgG has a distinct function, it is acceptable that poly-isotypic response is an advantage in the humoral immune response and higher IgG1 and IgG2a responses in the SARS-CoV-2-MF59 vaccine group may reflect a more potent humoral immune response occurring in this group ^25,31. An antibody response is one of the key mechanisms in the protection and prevention of infections in the body through several immunologic mechanisms. Antibodies are able to bind to the virus particles and neutralize and block the viral entry. In addition, antibodies are able to trigger viral elimination through activation of NK cells, macrophages and complement cascade.

Therefore, monitoring antibody response is an important aspect of understanding the scope of immunity. It is also one of the key factors for a successful vaccine development and also cross-protection against new variants of a virus strain ^34-37. Therefore, we assessed cross-binding response of IgG vs. non-vaccine strain. ELISA for cross-binding IgG response vs. non-vaccine strain [Alpha variant (B.1.1.7) "English variant"] showed a numerically higher in the specific IgG titer for the SARS-CoV-2-MF59 vaccine group vs. inactivated SARS-CoV-2-Alum group. This finding may show the potency of the MF59 adjuvant in the induction of higher cross-binding IgG response. However, testing additional variants would be necessary before making broader claims about heterologous breadth. We could not do this because of limitations in access to other variants, and this is a limitation of this study.

In addition, we couldn’t confirm cross-neutralization of humoral immune response and this is another limitation of the study. A study performed by Khurana et al showed that the MF59-H5 influenza vaccine in a prime-boost vaccination promotes a broad H5N1 cross-clade neutralization antibody and it seems affinity maturation of the humoral response has a critical role in this effect ³⁸. Bai et al in a study in mice showed that the MF59-like adjuvanted vaccine induced higher cross-reactive T cell responses against the SARS-CoV-2 prototype and new variants than the Alum adjuvant. They concluded that vaccine adjuvant strategy is a rational strategy against SARS-CoV-2 and variants of concern in older adults ³³. This judgment is based on the cross-reactive property of their vaccine when it was formulated in the MF59 adjuvant, suggesting the potency of MF59 adjuvant in the induction of cross-reactive response ³³. Frey et al in a study showed the potential of an adjuvanted vaccine to provide cross-protection to novel viruses. They used MF59 in the inactivated H5N1 influenza vaccine as a model and the results revealed the potential of the inactivated H5N1 influenza-MF59 vaccine in providing protection against other H5N1 strains ³⁹. These reports support the claim that MF59 adjuvant could potentially be the best insurance against strain diversity of pathogens and this can be achieved by induction of cross-binding immune response ⁴⁰. This property of an adjuvant is very important in the induction of cross-reactive immune response and thereby mitigating the new wave of infections, which can arise from newly mutated variants of a virus. However, we believe that more studies are needed to prove this claim, especially the cross-neutralization test, which is a serious limitation of this study. 

Conclusion

In this mouse model, the MF59-formulated vaccine enhanced selected immunogenicity endpoints, including lymphocyte proliferation, IFN-γ-associated responses, and IgG1, and IgG2a antibody responses, compared with the Alum formulation. Both formulations induced binding IgG responses to the Alpha variant; however, inactivated SARS-CoV-2 formulated in MF59 adjuvant led to a numerically higher cross-binding IgG response for Alpha variant. These findings showed that vaccine formulation may be important in inducing cross-binding humoral responses. Because neutralization and challenge assays were not performed, these findings cannot be interpreted as evidence of cross-neutralization or protection. 

Acknowledgement 

We thank Dr. Louiza Chitour, former Regional Sales & Marketing Manager from the Business Unit of SEPPIC Company for providing MF59 adjuvant as a gift. We also thank the staffs of Vaccine Formulation Laboratory of Razi Vaccine and Serum Research Institute of Iran for vaccine formulation. All the experiments on the mice were carried out in accordance with the Creature Care and Use Convention of Razi Vaccine and Serum Research Institute of Iran and also the Ethics Committee of Semnan University of Medical Sciences of Iran (Ethics code: IR.SEMUMS.REC. 1400.338). 

Conflict of Interest 

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.