Abstract
Background
Waning immunity and emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlight the need for further research in vaccine development.
Methods
A recombinant fusion protein containing the receptor-binding domain (RBD) fused to the human IgG1 Fc (RBD-Fc) was produced in CHO-K1 cells. RBD-Fc was emulsified with four adjuvants to evaluate its immunogenicity. The RBD-specific humoral and cellular immune responses were assessed by ELISA. The virus neutralizing potency of the vaccine was investigated using four neutralization methods. Safety was studied in mice and rabbits, and Antibody-Dependent Enhancement (ADE) effects were investigated by flow cytometry.
Results
RBD-Fc emulsified in Alum induced a high titer of anti-RBD antibodies with remarkable efficacy in neutralizing both pseudotyped and live SARS-CoV-2 Delta variant. The neutralization potency dropped significantly in response to the Omicron variant. RBD-Fc induced both TH2 and particularly TH1 immune responses. Histopathologic examinations demonstrated no substantial pathologic changes in different organs. No changes in serum biochemical and hematologic parameters were observed. ADE effect was not observed following immunization with RBD-Fc.
Conclusion
RBD-Fc elicits highly robust neutralizing antibodies and cellular immune responses, with no adverse effects. Therefore, it could be considered a promising and safe subunit vaccine against SARS-CoV-2.
Introduction
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes pulmonary infections that result in coronavirus disease (COVID-19) [1]. The World Health Organization (WHO) declared COVID-19 outbreak a global pandemic on March 11, 2020. Until now, there have been more than 774 million confirmed cases of COVID-19 with over 7 million deaths reported worldwide [2]. Despite the announcement of the WHO that COVID-19 is no longer a global public health emergency, development of safe and effective COVID-19 vaccines is still essential [3]. Vaccination protects against severe disease, hospitalization, and death by COVID-19, as well as lessening the transmission risk of the virus [4]. According to WHO, herd immunity against COVID19 should be achieved through vaccination, not by exposing people to the pathogen. The evolution of new variants, some of which might be more contagious or resistant to the immunity conferred by existing vaccines, emphasizes the need to continue research on designing novel and more effective vaccines especially for eradication of these newly emerging mutant viruses [5].
Although several vaccines have been approved for emergency use, there is still a high demand for additional safe and effective vaccines [6]. Various platforms such as mRNA, DNA, live attenuated or killed, and recombinant subunit proteins have been employed to produce SARS-CoV-2 vaccines [7, 8]. Here, we have developed a recombinant fusion protein-based candidate vaccine against SARS-CoV-2. The vaccine contains the receptor-binding domain (RBD) of the viral spike protein, which is responsible for binding to the angiotensin-converting enzyme 2 (ACE2), the virus receptor on host cells [9, 10]. Fusing the IgG Fc domain to recombinant RBD improves the stability of RBD and also induces an efficient immune response by increasing antigen uptake through Fc-receptors on immune cells [11].
In our quest for a robust and adaptable vaccine, we further explore the critical aspect of adjuvants in enhancing the immunogenicity of the RBD-Fc fusion protein. The selection of an appropriate adjuvant is pivotal for optimizing the immune response and, consequently, the overall vaccine efficacy. The immunostimulatory effects of a variety of adjuvants have previously been investigated and compared in a number of RBD-Fc based vaccine formulations [12–15]. This study delves into the comparative analysis of four distinct adjuvants, namely Alum, CpG ODN 1826, Montanide ISA 206, and MPL, aiming to elucidate their immunostimulatory effects on RBD-Fc vaccines. Notably, the comprehensive investigation of CpG ODN 1826 and Montanide ISA 206 adjuvants in the context of RBD-Fc vaccines has not been previously undertaken, underscoring the novelty and significance of our approach.
Furthermore, we employed a combination of four different virus neutralization assays for the first time. This assessment method allows us to gauge the vaccine's neutralization potency against the SARS-CoV-2 Delta and Omicron (BA.1) variant in both mice and rabbits. The inclusion of this diverse set of assays provides a comprehensive evaluation of our vaccine candidate's effectiveness, offering insights into its ability to combat emerging variants and contribute to the ongoing efforts in the fight against COVID-19.
Materials and methods
Protein expression and purification
The gene encoding the Fc region of human IgG1 without hinge sequence (GenBank accession number: NC_000014.9) was fused at the N-terminal to the gene encoding SARS-CoV-2 RBD (Wuhan Hu1; GenBank accession number: MN908947) and then inserted into a homemade dual promoter expression plasmid that contains glutamine synthetase (GS) as a selection marker [16, 17]. The plasmid containing RBD-Fc was transfected into CHO-K1 cells using Lipofectamine 3000 reagent (Life Technologies, USA). Stable clones were then selected in the presence of methionine sulfoximine (MSX) (Sigma-Aldrich, USA). The final high-producing clone was selected and cultured in serum-free First CHOice® medium (UGA Biopharma GmbH, Germany), and the recombinant fusion protein was purified from culture supernatant using protein A affinity chromatography column (GE Healthcare, USA). The purified RBD-Fc was subjected to 10% SDS-PAGE under reducing and non-reducing conditions. Following electrophoresis, the gel was stained with Coomassie brilliant blue as previously described [18].
Immunogenicity studies in mice
Six to eight week-old female BALB/c mice (Pasteur Institute of Iran, Karaj, Iran) were randomly divided into different groups, each group contained 5 mice.
For “adjuvant selection study”, RBD-Fc (5 µg) was emulsified with the following adjuvants, including: Imject Alum (Thermo Fisher Scientific, USA), CpG ODN 1826 (Invivogen, USA), Montanide ISA 206 (Seppic, France), and MPL (Sigma-Aldrich, Germany). Two doses of each vaccine formulation were administered intramuscularly (i.m.) to each mice group. For “dose escalation study”, four doses of RBD-Fc (5, 10, 20, and 40 µg) were emulsified with Alum, and each mouse was immunized three times at 21 days intervals. Control animals were immunized with Alum without RBD-Fc with the same schedule. For assessment of the immune potentiating effect of Fc “RBD + IgG Fc immunogenicity study”, RBD-Fc, RBD as well as RBD plus human IgG Fc fragments were emulsified with Alum, and administered separately to three groups of mice as mentioned above. Human IgG Fc fragments were prepared from human IgG by papain cleavage and were purified using protein A affinity chromatography [19]. Half of the molecular weight of RBD-Fc fusion protein is made up of RBD and the other half is made up of Fc. Regarding the dose of antigen, RBD-Fc fusion protein was administered at 10 µg, whereas RBD alone was administered at 5 µg and mice receiving a combination of RBD plus human Fc fragment were administered with a dose of 5 µg each of RBD and Fc fragments. In all experiments, mice were i.m. injected and blood was collected via tail vein before each injection and three weeks after the last booster dose and sera were stored at −20 °C until use. In addition, mice were sacrificed and the splenocytes were isolated and stimulated with RBD to measure levels of IFN-γ, IL-13, IL-17, and TGF-β in culture supernatants by enzyme-linked immunosorbent assay (ELISA).
Immunogenicity studies in rabbits
White New Zealand female rabbits (Pasteur Institute of Iran) aged 3–4 months were randomly divided into three groups (6 rabbits each). In a dose escalation study, two doses of RBD-Fc (10 and 40 µg) with Alum were administered three times at 21 days intervals. The control group received Alum alone. All doses were administered intramuscularly. Prior to each injection and three weeks after the last immunization, blood samples were obtained by auricular vein puncture and the sera were stored at −20 °C until use.
Assessment of anti-RBD antibody level in sera of immunized mice and rabbits by ELISA
The level of anti-RBD antibody in serum of immunized mice and rabbits was determined by ELISA. Recombinant RBD (2 ug mL−1) was coated on Maxisorp flat-bottom 96 well plates (Nunc, Denmark).
After overnight incubation at 4 °C, the plates were blocked with 3% w/v skimmed milk (Sigma Aldrich) in PBST (0.05% v/v Tween-20 in PBS, Sigma Aldrich). Serially diluted sera were then added and incubated at 37 °C for 1 h, followed by three washes with PBST. Subsequently, the plates were incubated with horse-radish peroxidase (HRP)-conjugated sheep anti-mouse or -rabbit immunoglobulin (SinaBiotech, Iran) at 1:2000 dilution in the blocking buffer and incubated at 37 °C for 1 h. After adding tetramethylbenzidine (TMB) substrate (Pishtaz Teb, Iran) the reaction was stopped with 1M H2SO4. Finally, ELISA microplate reader (BioTek, USA) was used to measure the optical density (OD) of the reactions at 450/630 nm. Since a commercial standard was unavailable in our laboratory, we established a homemade standard using serum of an immunized mice and rabbit containing high titer of anti-RBD antibody for assignment of arbitrary units.
Determination of cytokines levels in immunized mice by ELISA
In order to evaluate the cell-mediated immune response, mice immunized with RBD-Fc, RBD and control mice were sacrificed and the splenocytes were isolated and stimulated with RBD to measure levels of IFN-γ, IL-13, IL-17, and TGF-β in culture supernatants by ELISA. The splenocytes were cultured in RPMI-1640 medium containing 10% (v/v) FBS (fetal bovine serum), 100 U mL−1 penicillin, 100 μg mL−1 streptomycin, and 2.0 g L−1 sodium bicarbonate (all from Gibco, USA). Next, cells were stimulated by addition of 5 μg mL−1 of recombinant RBD protein. Briefly, splenocytes were incubated at a concentration of 1 × 106 cells/well for 72 h at 37 °C in the presence of 5% CO2 and 95% humidity. Supernatants collected from splenocytes cultured in the absence of RBD or in the presence of phytohaemagglutinin (PHA) were considered as negative and positive controls, respectively. The secreted cytokines in the supernatant were analyzed using ELISA kits (Invitrogen, USA). OD of the reactions was measured at 450/630 nm using BioTek microplate reader and the concentration of each cytokine was extrapolated based on the standard proteins provided in the kits.
SARS-CoV-2 neutralization assays
Surrogate virus neutralization test (sVNT)
Surrogate virus neutralization test (sVNT) (Pishtaz Teb Co., Iran) was used to measure the ability of the serum neutralizing antibodies to compete with HRP- conjugated ACE2 protein for binding to coated RBD protein.
Serially diluted serum samples or standard solutions (50 μL) and HRP-conjugated ACE2 (50 μL) were added to individual wells, mixed for 15 s, and then incubated for 30 min at 37 °C. Following washing, the microplate wells were treated with 100 μL of TMB chromogen solution and incubated for 15 min at room temperature. Next, 100 μL of a stop solution was added, and subsequently the ODs were measured at 450/630 nm. The percent inhibition value was determined using the formula: ((sample OD – negative control OD)/negative control OD) × 100.
Flow cytometry
To determine the neutralizing potency of immunized sera to inhibit binding of the virus to ACE2 receptor, we developed a flow cytometry assay using HEK293 cells expressing ACE2 receptor (HEK-ACE2) (a kind gift from Renap Therapeutics Co., Tehran, Iran). 25 µL of serially diluted serum samples were mixed with an equal volume of RBD-Fc (0.25 μg mL−1) and incubated at 37 °C for 2 h on a shaker incubator at 90 RPM. Next, the serum/RBD-Fc mixture was added to 1 × 105 HEK293T cells and incubated on ice for 45 min.
Human IgG was used as isotype control. After washing with PBS containing 2% FBS, 50 µL FITC-labeled sheep anti-human antibody (SinaBiotech, Iran) at a final dilution of 1:100 was added and incubated on ice for 45 min. After washing with PBS-2% FBS, cells were scanned by a Partec PAS Flow Cytometer (Partec GmbH, Germany) and data was analyzed using FlowJo V10 software.
Pseudotyped virus neutralization test (pVNT)
HEK-ACE2 cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin at 37 °C in the presence of 5% CO2 and 95% humidity. Diluted serum samples were mixed with an equal volume of eGFP-spike (Delta variant SARS-CoV-2) pseudotyped lentivirus (a kind gift from Renap Therapeutics Co., Tehran, Iran). After two hours incubation at 37 °C, the mixture was added to HEK-ACE2 cells and seeded in 96-well plates at a confluency of 14 × 103 cells/well. After 48 h incubation, the medium was removed and the pseudovirus-infected eGFP-positive cells were imaged and detected using fluorescence microscopy. Microscopic images were acquired from four or more fields, and then analyzed using the ImageJ software (NIH, USA) to determine the number of fluorescence labelled cells. The ID50 value stands for the serum dilution required to achieve 50% virus neutralization (50% decrease in the proportion of infected cells), was calculated as reported by Ferrara et al. [20].
Conventional virus neutralization test (cVNT)
The RBD-Fc vaccine efficacy against the SARS-CoV-2 Delta and Omicron (BA.1) variants was measured by conventional virus neutralization test (cVNT). The complement cascade proteins were initially inactivated by heating serum samples at 56 °C for 30 min in a water bath. Next, 50 μL of serially diluted sera were mixed with 50 μL of virus diluent containing 100 Tissue Culture Infectious Dose 50 (TCID50) SARS-CoV-2 in Dulbecco's Modified Eagle Medium (DMEM, Gibco, USA) and incubated for 60 min at 37 °C. The mixture was then added to 96-well plate containing monolayers of 1–2 × 104 Vero-E6 cells at 80% confluency and incubated for 1 h at 37˚C and 5% CO2. The infected cells were subsequently washed twice with DMEM and incubated at 37 °C in a 5% CO2 incubator. The virus-specific cytopathic effect (CPE) was determined 72 h post infection and the ID50, which is the dilution of serum that leads to 50% decrease in CPE formation, was finally determined. A neutralization antibody titer less than 1:4 was considered negative, while equal or greater than 1:4 was considered positive.
Toxicity studies in mice and rabbits
Forty-eight BALB/c mice (24 female and 24 male) 6–8 weeks age were randomly divided into three groups with equal distribution of female and male in each group (8 female and 8 male mice, totally 16, in each group). Four doses of 20 µg RBD-Fc with or without Alum were administered to the animals at 21 days intervals. The control group received Alum alone.
White New Zealand female and male rabbits aged 3–4 months were randomly divided into three groups (12 rabbits consisting of 6 females and 6 males). 40 µg RBD-Fc with or without Alum was administered to the animals at 21 days intervals. The control group received Alum alone.
A study was conducted to assess the toxicity of single and repeated doses of the administered candidate vaccine. Briefly, body temperature and body weight were measured both pre- and post-administrations. Furthermore, examination of hematologic and biochemical parameters, including white blood cell total and differential counts, red blood cell count, hemoglobin, hematocrit, platelet count, blood sugar, blood urea nitrogen, cholesterol, sodium, potassium, aspartate aminotransferase, total protein, albumin and globulin, along with fibrinogen as a coagulation factor, was carried out before first administration and two days after the initial and final immunizations, as well as ten days after the last boost.
To study the repeated-dose toxicity, half of the animals in each group containing equal number of male and female were sacrificed two days after the last injection and histopathologic investigation was carried out. The other half of each group was sacrificed ten days after the last dose to study the toxicity effects of candidate vaccine after vaccination recovery. Sections of different tissues and organs, including: muscular tissue at the site of injection, heart, brain, kidney, liver, lung, spleen, thymus, and inguinal lymph node were prepared for histopathological studies. The tissues were fixed in 10% formalin and subsequently embedded in paraffin blocks. Micro-sections were then prepared on glass slides, and histopathological examination was performed using hematoxylin and eosin (H&E) staining.
Antibody-Dependent Enhancement (ADE) assay
THP-1 and Raji cell lines (National Cell Bank of Iran (NCBI), Pasteur Institute, Tehran, Iran) expressing FcγRII were used to perform the ADE assay. Briefly, 100 ul of serial 10-fold dilutions of heat-inactivated immunized sera were mixed with 100 ul of SARS-CoV-2 pseudovirus and incubated at 37 °C for two hours. Subsequently, the mixture was added to THP-1 or Raji cells in a 96-well plate. Pseudovirus infected THP-1/Raji and HEK293T-ACE2 cells in the absence of immunized sera were used as negative and positive controls, respectively.
Statistical analysis
All statistical analyses were performed using Graphpad Prism 9.0 software (GraphPad Software Inc., La Jolla, USA) and the data is presented as mean ± standard deviation (SD). One-way ANOVA followed by Tukey's multiple comparison post-test were used to compare multiple groups, while unpaired Student's t-test was used to compare two groups. For non-parametric variables, Kruskal-Wallis and Mann-Whitney tests were used. Statistically significant P values were presented as *0.05, **0.01, ***0.001, and ****0.0001. The ID50 values (inhibition dilution 50%) for the sera were calculated using non-linear regression, i.e., [inhibitor] vs. normalized response -- Variable slope. Correlation between neutralization results of flow cytometry, sVNT, pVNT, and cVNT was analyzed by Spearman test.
Ethics
This study was approved by the Research Ethics Committee of Tehran University of Medical Sciences (IR.TUMS.SPH.REC.1400.334).
Results
Characterization of RBD-Fc protein
The construct containing SARS-CoV-2 RBD gene fused to Fc domain of human IgG1 was successfully generated. The RBD-Fc protein was expressed in CHO cells and purified by protein A affinity chromatography. Structure of RBD-Fc was characterized by SDS-PAGE under reduced and non-reduced conditions. As Fig. 1 shows, a single band at approximately 60 kDa was observed under reduced condition, and one band at approximately 120 kDa was observed under non-reduced condition, which is the dimer form of RBD-Fc. These results indicate that RBD-Fc protein was highly pure.
SDS-PAGE analysis of RBD-Fc. Five µg of purified RBD-Fc was loaded on 10% gel at reduced (R) and non-reduced (NR) conditions. A band at approximately 120 kDa in the absence of 2 ME (NR) and approximately 60 kDa in the presence of 2 ME was detected
Citation: European Journal of Microbiology and Immunology 14, 3; 10.1556/1886.2024.00045
RBD-Fc candidate vaccine elicits strong humoral immune response in mice
Comparison of different adjuvants
The efficacy of different adjuvants such as Alum, CpG ODN 1826, Montanide ISA 206, and MPL in enhancing the immunogenicity of RBD-Fc was evaluated. Our results showed that mice immunized with RBD-Fc formulated with Alum showed a significantly higher titer of anti-RBD antibody compared to other adjuvants (Fig. 2A), suggesting that Alum is the most effective adjuvant for inducing antibody response against RBD-Fc. Therefore, Alum was selected for the rest of study. It is noteworthy that the other adjuvants also significantly improved anti-RBD specific antibody response as compared to RBD-Fc alone, but the stimulatory effect was lower than Alum.
RBD-Fc candidate vaccine elicited robust antibody response against RBD in mice and rabbits. (A) Adjuvant selection study: RBD-Fc (5 µg) was formulated in different adjuvants. Two doses of each vaccine formulation were administered to animals at 21 days intervals. (B) Dose escalation study in mice: Four doses of RBD-Fc (5, 10, 20, and 40 µg) were emulsified with Alum and administered three times at 21 days intervals. (C) Fc immunogenicity study: Different compositions, including RBD-Fc, RBD, and RBD plus human Fc fragment were emulsified with Alum, and each mice group was administered with one formulation three times. (D) Dose escalation study in rabbits: Two doses of RBD-Fc (10 and 40 µg) with Alum were administered three times. Alum was injected in control group. Data are presented as the mean ± SD. P-values are determined by one-way ANOVA
Citation: European Journal of Microbiology and Immunology 14, 3; 10.1556/1886.2024.00045
Dose escalation analysis
In order to determine the optimal vaccine dose in mice, dose-escalation study was conducted using different doses of RBD-Fc, including 5, 10, 20, and 40 µg formulated in Alum.
The results showed that the level of anti-RBD antibody was significantly higher in mice which received 10 µg of RBD-Fc compared to the other doses (Fig. 2B).
Assessment of the effect of Fc domain on immunogenicity of RBD
To evaluate the effect of Fc domain in enhancing the immunogenicity of RBD-Fc vaccine candidate, three groups of BALB/c mice were intramuscularly immunized with three doses of RBD-Fc, IgG Fc fragments plus RBD and RBD alone, all emulsified in Alum.
Serum samples were collected 21 days post immunization and analyzed for anti-RBD specific titers (Fig. 2C). The results showed that the fusion protein RBD-Fc elicited notably elevated RBD-specific antibody in comparison to RBD or RBD plus IgG Fc fragments (Fig. 2C). In addition, the level of anti-RBD specific antibody in mice immunized with IgG Fc plus RBD was also significantly higher than those receiving RBD alone, but less significant than the group immunized with the fusion protein RBD-Fc.
RBD-Fc elicits robust humoral immune response in rabbits
Based on the results obtained from the dose escalation study in mice, rabbits were immunized with two different doses, including 10 µg which is the optimized dose in mice and a four-fold dose (40 µg). The immunization schedule for rabbits was the same as the mice. The findings showed that the level of anti-RBD antibody was significantly higher in rabbits immunized with 40 µg of RBD-Fc compared to those receiving the lower dose (Fig. 2D).
RBD-Fc induces both TH2 and TH1 immune responses in mice
To investigate whether RBD-Fc vaccination could stimulate cellular immune response, mice immunized with RBD-Fc or RBD alone were euthanized 21 days after last vaccination and their splenocytes were isolated. Results of cytokine release assay showed that immunization with RBD-Fc formulated in Alum leads to significant elevation in the level of interferon-gamma (IFN-γ) and IL-13 compared to the control group (P < 0.0001). It is noteworthy that RBD-Fc without Alum, as well as RBD with Alum also induced IFN-ɣ and IL-13 secretion, but the levels were significantly lower compared to RBD-Fc (Fig. 3A and B). In contrast, the level of IL-17 was undetectable in all groups, including those receiving RBD-Fc with Alum (Fig. 3C). Furthermore, there were no statistical differences in TGF-β level among all groups (Fig. 3D).
RBD-Fc candidate vaccine is capable of eliciting both TH1 and TH2 responses. Mice immunized with RBD-Fc, RBD, or PBS were sacrificed and their splenocytes were isolated. After stimulation with 5 μg mL−1 RBD, the supernatants were collected after 72 h and the level of (A) IFN-γ, (B) IL-13, (C) TGF-β, and (D) IL-17 were detected by ELISA. Splenocytes cultured without RBD protein were considered as negative control, whereas phytohaemagglutinin (PHA) was used to stimulate cytokine response as a positive control. Data are presented as the mean±SD. P-values are determined by one-way ANOVA
Citation: European Journal of Microbiology and Immunology 14, 3; 10.1556/1886.2024.00045
RBD-Fc elicits potent neutralizing antibody response in both mice and rabbits
In order to determine the neutralizing efficacy of anti-RBD antibodies in the sera of immunized animals, we used a pseudovirus that expressed S protein of the Delta variant of SARS-CoV-2 and also the live SARS-COV2 of Delta and Omicron (BA.1) variants. Sera were obtained from mice that were immunized with 10 µg of RBD-Fc or RBD, and from rabbits immunized with 40 µg of RBD-Fc at day 21 post last immunization. Our results showed that these sera potently neutralized Delta variant of SARS-CoV-2 pseudovirus (PsV) infection in HEK293T cells expressing ACE2, and the ID50 values obtained for mice immunized with RBD-Fc and RBD as well as rabbits immunized with RBD-Fc were 1:379, 1:30, and 1:331, respectively (Fig. 4A). These results suggest that immunization of mice and rabbits with 10 and 40 µg RBD-Fc, respectively, induces potent neutralizing antibody responses.
RBD-Fc induced strong neutralizing antibody responses in mice and rabbits. The neutralizing potency of immunized sera was determined by different viral neutralization assays. (A) pseudotyped Virus Neutralization Test (pVNT): eGFP-pseudotyped lentivirus containing SARS-CoV-2 Delta variant spike protein was mixed with serial dilutions of sera. The mixture was added to HEK293T cells expressing ACE2. After 48 h, the pseudovirus-infected eGFP-positive cells were imaged and detected using fluorescence microscopy. (B) conventional Virus Neutralization Test (cVNT): Serum samples were heat-inactivated to destroy complement. The, 100 Tissue Culture Infectious Dose 50 (TCID50) of live Delta variant SARS-CoV-2 was mixed with serial dilutions of sera. The virus/serum mixtures were then added to Vero-E6 cells. After 1 h incubation, the supernatant was washed away, and the infected cells incubated in DMEM. The virus-specific CPE of each well was recorded under microscopes 72 h post-infection, and the ID50 was determined as the inhibitory dilution. (C) surrogate Virus Neutralization Test (sVNT): The ability of RBD-specific neutralizing antibodies to inhibit binding of RBD to ACE2 was investigated in competitive ELISA. After coating RBD in ELISA plate, serially diluted serum samples or standard solutions and HRP-conjugated ACE2 were added to individual wells. Following addition of chromogen solution and stop solution, the OD of the reactions were measured. (D) Flow cytometry: Serially diluted serum samples were mixed with an equal volume of RBD-Fc (0.25 μg mL−1). Serum/RBD-Fc mixture was then added to HEK293T cells that were expressing ACE2. Human immunoglobulin was used as an isotype control. Then, FITC-labeled sheep anti-human antibody was added at a final dilution of 1:100. Finally, data were acquired on a flow cytometer device and analyzed using FlowJo V10 software. Data are presented as the mean±SD. P-values are determined by one-way ANOVA. (E–J) The correlation of obtained ID50 values between flow cytometry, sVNT, pVNT, and cVNT was calculated by Spearman analysis
Citation: European Journal of Microbiology and Immunology 14, 3; 10.1556/1886.2024.00045
To further test the neutralization activity of the antisera collected from mice and rabbits, we conducted a CPE assay using live SARS-CoV-2 Delta and Omicron (BA.1) variants infection in Vero-E6 cells. The ID50 values obtained by cVNT on the same variant with pseudovirus (Delta) were 1:3242, 1:181 and 1:2657 for the same groups, respectively (Fig. 4B).
We additionally developed a competition ELISA and also a flow cytometry test to evaluate the virus neutralization capacity of the immunized sera. Accordingly, binding of SARS-CoV-2 RBD to hACE2 was more significantly inhibited by sera from mice and rabbits immunized with RBD-Fc, compared to the control sera (Fig. 4C and D), indicating that RBD-Fc induced antibodies were able to neutralize SARS-CoV-2 infection through blocking the binding of RBD on the surface of SARS-CoV-2 to hACE2. Highly significant correlations (P < 0.0001) were observed between all the four neutralization assays employed in this study (Fig. 4E–J).
Using the cVNT assay, we compared the neutralization potential of sera from immunized animals against the currently dominant Omicron (BA.1) variant and compared the data with our results on the Delta variant. Our findings revealed a substantial decrease in neutralization potency (Fig. 5). Specifically, the serum from immunized mice exhibited nearly a 19-fold decrease, while serum from immunized rabbits showed a 23-fold decrease in neutralizing power against the Omicron compared to the Delta variant.
Comparison of RBD-Fc induced neutralizing antibody responses against Omicron (BA.1) and Delta variants of live SARS-CoV-2 virus (cVNT) in mice and rabbits. Serum samples were heat-inactivated to destroy complement. The, 100 Tissue Culture Infectious Dose 50 (TCID50) of live Delta or Omicron (BA.1) variant SARS-CoV-2 was mixed with serial dilutions of sera. The virus/serum mixtures were then added to Vero-E6 cells. After 1 h incubation, the supernatant was washed away, and the infected cells incubated in DMEM. The virus-specific CPE of each well was recorded under microscopes 72 h post-infection, and the ID50 was determined as the inhibitory dilution
Citation: European Journal of Microbiology and Immunology 14, 3; 10.1556/1886.2024.00045
Toxicity study
Mice and rabbits received single or repeated doses of RBD-Fc vaccine candidate, and toxicity was evaluated. Histopathologic examinations indicated no significant toxicity in several organs, including the muscle at injection site, heart, brain, kidney, liver, lung, spleen, thymus, and inguinal lymph node. The results showed no vaccine-associated lesions in the immunized animals in comparison with the control group (Supplementary Fig. S1) The general health, body temperature, and body weight of the animals received RBD-Fc vaccine candidate were found to be within the normal range, and there were no remarkable differences in the blood hematological and biochemical parameters between the immunized and control groups (Supplementary Tables S1–S3).
Assessment of the Antibody-Dependent Enhancement of viral infection
Although anti-RBD antibodies can neutralize SARS-CoV-2 and clear the virus through binding to Fcɣ receptors on immune cells, these interactions could lead to antibody-dependent disease enhancement (ADE). Here, we investigated whether the RBD-Fc-induced antibody response in the sera of immunized mice and rabbits could lead to ADE. Accordingly, Fc-mediated viral entry of SARS-CoV-2 pseudovirus to FcɣR-expressing THP-1 and Raji human cell lines was studied. Both cell lines express FcγRI, FcγRII, and FcγRIII and have frequently been used in ADE research studies [21–24].
The sera of mice and rabbits immunized with RBD-Fc were serially diluted and incubated with SARS-CoV-2 pseudovirus and subsequently were added to the cells. The results showed that the entry of SARS-CoV-2 pseudovirus into THP-1 and Raji cells was negligible (less than 0.02%), whereas equal amount of SARS-CoV-2 pseudovirus leads to an infection rate of approximately 4% in HEK-ACE2 cells which were used as a control. Serially diluted control sera or immunized animal antisera, with dilutions ranging from 1:10 to 1:105, did not show significant impact on the entry of SARS-CoV-2 pseudovirus into the two cell lines (Fig. 6). Therefore, anti-RBD sera do not seem to enhance SARS-CoV-2 pseudovirus infection, indicating that anti-RBD antibodies in the sera of RBD-Fc immunized animals may not promote ADE.
Serum from immunized mice and rabbits did not induce ADE of SARS-CoV-2 in both THP-1 and Raji cell lines. Representative fluorescence images of cells infected with eGFP-pseudotyped lentiviruses were obtained in presence or absence of various dilutions of immunized sera. The entry of SARS-CoV-2 pseudovirus into FcγR-expressing cell lines (THP-1 and Raji) was investigated using serially diluted immunized or non-immunized (control) sera, ranging from 1:10 to 1:105. Treatment with the same quantity of SARS-CoV-2 pseudovirus resulted in an infection rate of approximately 4% in HEK-ACE2 cells
Citation: European Journal of Microbiology and Immunology 14, 3; 10.1556/1886.2024.00045
Discussion
Although WHO has recently declared that COVID-19 is no longer a global health emergency, it does not mean that it is no longer a global threat. Vaccination is one of the most effective tools to protect people against severe illness, hospitalization, and mortality [3]. Since the first report of COVID-19, emergence of new circulating variants of the COVID-19 virus harboring mutations in the spike molecule, as the main target of neutralizing antibodies, has raised significant concern about the efficacy of current vaccines [25].
Current COVID-19 vaccines provide high protection against severe disease and death. However, vaccine effectiveness has declined due to waning immunity and also emergence of novel variants. Accordingly, studies have shown that cross-protection and protective effect of neutralizing antibodies elicited by vaccination against the Wuhan spike-based vaccines significantly decline against some emerging variants such as Omicron [26]. Therefore, developing new vaccines with more efficient immunogenicity highlights the necessity of further research in this area. Besides inducing neutralizing antibody, recombinant subunit vaccines have several advantages including high safety and yield as well as easy storage and transportation [11]. Studies have shown that RBD comprises of several immunodominant epitopes and therefore is a major target for neutralizing antibodies, which blocks the interaction of RBD-ACE2 and inhibits virus entrance to the host cells [24, 27–29], however, the immunogenicity of RBD is not strong enough. Using Fc domain of human IgG as a fusion partner has several advantages, including interaction with FcRn, increasing the half-life and stability of the fusion protein, binding to Fc receptor on immune cells and enhancing their immunological functions, enhancing immunogenicity by increasing uptake via FcγR by antigen presenting cells, and also efficient purification of the fusion protein by protein A- or G-affinity chromatography [11, 30–32].
Combining an IgG Fc fragment with an antigen has been shown to be an effective approach for vaccination [33]. This method relies on either enhancing or inhibiting interactions with a specific group of Fc receptors (FcRs) [34]. Recently, this vaccination approach has been used to develop vaccines against various infectious agents, including herpes simplex virus (HSV) [33], influenza [35], Ebola [36], tuberculosis [37], and human immunodeficiency virus (HIV) [38]. Notably, the HIV gp120-Fc fusion protein vaccine demonstrated a strong antibody response in nonhuman primates [39]. In addition to these infectious vaccines, the Fc fusion strategy has been used in several studies to develop vaccine against SARS-CoV-2. In this regard, some investigators fused Fc portion to spike protein [40] and some others used RBD-Fc fusion protein [12, 32, 41–44]. The Fc fusion protein were able to induce potent immune responses that are biased towards Th1 with high levels of neutralizing antibodies. These responses and antibodies play a crucial role in the effectiveness of the SARS-CoV-2 vaccines, as was demonstrated in mice [15, 42–44], rabbits [15], and nonhuman primates [15, 42, 43]. It was also demonstrated that the antibody response induced against a mutated RBD protein from a given variant of SARS-CoV-2displays cross-reactivity with both the wild-type and other variants in preclinical studies [15, 45, 46]. As a result, we and some other investigators utilized this promising strategy for the design of SARS-CoV-2 vaccine.
In this study, Fc domain of human IgG1 was fused to RBD protein to improve the immunogenicity of RBD. The RBD-Fc gene construct was generated and transfected to CHO-K1 cells and RBD-Fc fusion protein was purified from the culture supernatant by affinity chromatography. Purified RBD-Fc was visualized by SDS-PAGE as main bands of approximately 120 and 60 kDa in non-reduced and reduced conditions, showing dimeric and monomeric forms of the recombinant protein, respectively (Fig. 1).
To compare the effect of different adjuvants on improving the immunogenicity of RBD-Fc, the fusion protein was formulated in Alum, CpG ODN 1826, Montanide ISA 206 and MPL and used for mice immunization (Fig. 2A). Although the most potent antibody response to RBD was induced by Alum, the other adjuvants substantially enhanced RBD-specific antibody response compared to the mice administered with RBD-Fc without adjuvant or adjuvant alone, but their effect was statistically less significant than Alum. This study represents the first investigation regarding the comparative assessment of these four different adjuvants on enhancing the immune response elicited by RBD-Fc. Two of these adjuvants (CpG ODN 1826 and Montanide ISA 206) had not been investigated in previous studies evaluating RBD-Fc SARS-CoV-2 vaccine formulations.
The effect of some adjuvants on enhancing the immunogenicity of this fusion protein has previously been compared in some studies. Laotee et al. [12] compared three different adjuvants including Montanide ISA51, poly(I:C) and MPLA/Quil-A in formulation with RBD-Fc. The results of PRNT and sVNT viral neutralization tests showed superiority of Montanide ISA51 over the other two adjuvants. Liu et al. [15] introduced a new STING agonist named CF501 as an adjuvant and compared it with Alum and four other adjuvants in mice immunized with RBD-Fc. They reported superiority of this adjuvant over the other adjuvants. In another study, Sun et al. [47] compared Alum with SCT-VA02B adjuvant and reported a better immunostimulatory response for SCT-VA02B. Zhang et al. [48] compared the effect of three different adjuvants including Alum, nanoparticle manganese and MF59 and showed that the nanoparticle manganese adjuvant produces the highest titers of IgG, IgG1 and IgG2a against SARS-CoV-2 RBD as well as neutralizing antibodies against the Delta variant. In another investigation Alum, MF59, MPL and poly(I:C) were compared. The results showed that Alum and MPL induced higher titer of neutralizing antibody than MF59 and poly(I:C). Shi et al. [14] explored the efficacy of four adjuvants—Alum + MPL, QS21 + MPL, AddaVax, and Imiquimod in the context of their RBD-Fc subunit vaccine. QS21 + MPL adjuvant demonstrated superior neutralization potency. All these findings underscore the nuanced effects of different adjuvants in shaping the immunogenicity of RBD-Fc subunit vaccines, emphasizing the importance of adjuvant selection in vaccine development. In several studies, only Alum adjuvant has been used to enhance immunogenicity of the RBD-Fc fusion protein [41, 42, 46, 49–52].
Next, we determined the minimum effective dosage, necessary to generate an optimal RBD-specific immune response. Mice were immunized with different doses of RBD-Fc (5, 10, 20, or 40 µg) to determine the optimal dose required to induce a high titer of RBD-specific antibody. Since immunization with 10 µg RBD-Fc resulted in the highest level of anti-RBD antibody, this dose was selected for the rest of the study (Fig. 2B).
To investigate how the Fc fragment could enhance the immunogenicity of the RBD protein, we immunized three groups of BALB/c mice separately with RBD-Fc fusion protein, RBD without Fc and RBD plus the Fc fragment of human IgG1. The RBD-Fc fusion protein induced significantly higher levels of RBD-specific antibody than RBD alone or RBD+Fc. The level of anti-RBD antibody was also higher when RBD was mixed with IgG1 Fc fragments, but far less than the level induced by the fusion protein (Fig. 2C). Our findings indicate that although mixing RBD with the isolated Fc fragments could moderately augment the antibody response to RBD, however to achieve the optimum immunogenicity, the Fc needs to be fused to the RBD protein. This allows more efficient attachment, uptake and processing of the RBD by the FcγR expressing antigen presenting cells (APC) leading to better presentation of the RBD peptides and more potent anti-RBD antibody response.
In accordance with our results, Alleva and colleagues have recently reported that RBD fused to either human IgG1 Fc or mouse IgG2a Fc fragments induced significantly higher levels of RBD-specific neutralizing antibody compared to RBD alone. Interestingly, both mouse and human Fc gave similar responses which implies that the enhanced immunogenicity is not linked to the heterologous nature of the human IgG Fc in immunized mice [43]. As a matter of fact human IgG1 could efficiently bind to the mouse FcγRI, II and III [53] leading to attachment of the RBD-human Fc fusion protein to mouse APC and enhancement of the antibody response to RBD.
The cellular immune response plays a crucial role in the control and elimination of viruses during acute infections [54–56]. Our results of cytokine release assay showed that splenocytes derived from RBD-Fc-immunized mice exhibited a heightened ability to secrete IFN-γ and IL-13 upon stimulation with the RBD protein, suggesting engagement of a strong TH cell responses. The elevated levels of IFN-γ and IL-13 are crucial for long-term immunity against viral infections [57]. However, IL-17 was not induced and the level of TGF-β was not different between studied groups (Fig. 3). Our results are in accordance with a previous report on RBD-Fc with Alum adjuvant showing elevated secretion of IFN-γ, IL-4 and IL-10 cytokines in the immunized mice [41]. In contrast with our results, a previous study on RBD-Fc with Alum vaccinated Macaca fascicularis macaques failed to detect any significant cellular immune response [42]. Various animal models may account for the observed differences. The secretion of IFN-γ is primarily attributed to TH1 cells, which are responsible for cell-mediated immunity. Conversely, TH2 cells predominantly secrete IL4 and IL-13, which can lead to substantial antibody production [58]. The high level of neutralizing antibodies observed in RBD-Fc immunized mice suggests that the recombinant fusion protein can elicit both humoral and cellular immune responses in mice. Similar findings have recently been reported using human IgG1 Fc-RBD fusion protein in mice [41]. The synergy between humoral and cellular immune responses may more effectively mitigate the severity of COVID-19 infection [59].
Several reports have demonstrated a strong correlation between the serum anti-RBD titer and neutralizing antibody levels in the immunized subjects [32, 60]. In our study, the neutralizing potency of immunized mice and rabbits' sera was evaluated using four different viral neutralization methods, including sVNT, pVNT (GFP), cVNT (CPE) and inhibition flow cytometry. To our knowledge, there is no previous report that has used all these four methods to investigate SARS-CoV-2 vaccine neutralizing potency. We found that a 10 µg RBD-Fc dose in mice was sufficient to induce a high titer of RBD-specific antibodies capable of neutralizing SARS-CoV-2 PsV and live virus infection (Delta variant), with an ID50 of 1:379 and 1:3242, respectively (Fig. 4A and B). In rabbits, due to their larger size, we found that a 40 µg dose was suitable, achieving an ID50 of 1:301 and 1:2657 for PsV and live virus infection, respectively. In particular, RBD-Fc was more efficient than RBD in induction of protective neutralizing antibody. These findings have significant implications for designing an optimal RBD-based vaccine against SARS-CoV-2 infection. In addition, recognition and binding to host receptors are essential for the entry of SARS-CoV-2. In our study, the anti-RBD sera from both mouse and rabbit were found to inhibit the RBD binding to hACE2 in a dose-dependent manner (Fig. 4C and D), demonstrating that the major mechanism responsible for the observed neutralization by the anti-RBD sera is the blocking of RBD binding to ACE2. Notably, the ID50 values obtained by all four assays displayed highly significant correlations (Fig. 4E–J).
In addition to assessing the Delta variant, we performed a cVNT assay specifically targeting the Omicron (BA.1) variant. Our results revealed a notable decline in the neutralization potency of sera from immunized animals, as depicted in Fig. 5. This finding aligns with previous studies involving convalescent or vaccinated individuals, which have also reported a diminished neutralizing capability against the Omicron variant [61–63]. These collective observations underscore the immune evasion potential of Omicron, attributed to the extensive mutations present within the Omicron RBD sequence.
One of the primary challenges in development of vaccines for SARS-CoV-2 is the potential risk of ADE effect, which may increase the severity of COVID-19 [64–66]. The phenomenon of ADE has been previously noted in feline coronavirus [65, 67, 68] and SARS-CoV [69–72]. Several in vitro studies have indicated that ADE may be facilitated by non-neutralizing antibodies against S protein through FcR-mediated internalization/entry of virions into the immune cells bearing Fc receptors [21, 23, 73–77].
Moreover, evidence from dengue virus infection in mice showed that antiviral T cell-mediated response also plays a role in reducing ADE effect [78]. Thus, a vaccine approach that induce both high affinity SARS-CoV-2 neutralizing antibodies and also a strong T cell medicated immune response could prevent the infection and avoid the risk of ADE [79]. The ADE effect has not been extensively investigated in previously reported recombinant RBD-based vaccines. Our results obtained from two different human FcR expressing cell lines, RAJI and THP1, implies lack of ADE effects in the immunized animals (Fig. 6).
Conclusion
Our RBD-Fc vaccine candidate induced both humoral and cellular immune responses leading to efficient neutralization of the Delta variant of SARS-CoV-2 as evidenced by the four viral neutralization assays employed in this study. Substantial reduction of the neutralization potency in response to the Omicron variant was observed. No substantial toxicity or ADE effects were noticed in immunized animals suggesting safety of this vaccine formulation. Substantial reduction of the neutralization potency which we observed against the currently dominant Omicron has already been reported [18, 61–63, 80, 81] and is due to the extensive mutations within the Omicron RBD sequence.
Funding sources
This work was partially supported by Tehran University of Medical Sciences (TUMS) under Grant number 1401-1-99-57094.
Authors' contributions
Navid Dashti: Investigation, Formal analysis, Writing – original draft. Forough Golsaz-Shirazi, Amir-Hassan Zarnani and Mahmood Jeddi-Tehrani: Data curation, Validation, Writing – review & editing. Haleh Soltanghoraee: Investigation, Validation. Mehdi Mohammadi and Danyal Imani: Methodology. Mohammad Mehdi Amiri: Conceptualization, Methodology, Software, Supervision, Writing – review & editing, Data curation. Fazel Shokri: Conceptualization, Data curation, Funding acquisition, Project administration, Supervision, Validation, Writing – review & editing.
All authors had full access to all data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
We are grateful to Professor Vahid Salimi for scientific consultation. The authors gratefully acknowledge Renap Therapeutics Co., Iran, for generously providing the pseudotyped lentiviruses and the hACE2-HEK293T cell line used in this study.
Supplementary material
Supplementary data to this article can be found online at https://doi.org/10.1556/1886.2024.00045.
References
- 1.↑
Li C, Ye Z, Zhang AJX, Chan JFW, Song W, Liu F, et al. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by intranasal or intratesticular route induces testicular damage. Clin Infect Dis. 2022;75(1):e974–e90.
- 2.↑
World Health Organization. COVID-19 weekly epidemiological update. edition 164. 16 February 2024.
- 3.↑
Silk BJ, Scobie HM, Duck WM, Palmer T, Ahmad FB, Binder AM, et al. COVID-19 surveillance after expiration of the public health emergency declaration―United States, may 11, 2023. Morbidity Mortality Weekly Rep. 2023;72(19):523.
- 4.↑
Rubin R. COVID-19 vaccines vs variants—determining how much immunity is enough. Jama. 2021;325(13):1241–3.
- 5.↑
Bok K, Sitar S, Graham BS, Mascola JR. Accelerated COVID-19 vaccine development: milestones, lessons, and prospects. Immunity. 2021;54(8):1636–51.
- 6.↑
Marks PW, Gruppuso PA, Adashi EY. Urgent need for next-generation COVID-19 vaccines. JAMA. 2023;329(1):19–20.
- 8.↑
Röltgen K, Boyd SD. Antibody and B Cell responses to SARS-CoV-2 infection and vaccination: the end of the beginning. Annu Rev Pathol Mech Dis. 2024;19:69–97.
- 9.↑
Borkotoky S, Dey D, Hazarika Z. Interactions of angiotensin-converting enzyme-2 (ACE2) and SARS-CoV-2 spike receptor-binding domain (RBD): a structural perspective. Mol Biol Rep. 2023;50(3):2713–21.
- 10.↑
Maghsood F, Shokri M-R, Jeddi-Tehrani M, Torabi Rahvar M, Ghaderi A, Salimi V, et al. Identification of immunodominant epitopes on nucleocapsid and spike proteins of the SARS-CoV-2 in Iranian COVID-19 patients. Pathog Dis. 2022;80(1).
- 11.↑
Chen L, Qi X, Liang D, Li G, Peng X, Li X, et al. Human Fc-conjugated receptor binding domain-based recombinant subunit vaccines with short linker induce potent neutralizing antibodies against multiple SARS-CoV-2 variants. Vaccines. 2022;10(9):1502.
- 12.↑
Laotee S, Duangkaew M, Jivapetthai A, Tharakhet K, Kaewpang P, Prompetchara E, et al. CHO-produced RBD-Fc subunit vaccines with alternative adjuvants generate immune responses against SARS-CoV-2. Plos one. 2023;18(7):e0288486.
- 13.
Siriwattananon K, Manopwisedjaroen S, Shanmugaraj B, Prompetchara E, Ketloy C, Buranapraditkun S, et al. Immunogenicity studies of plant-produced SARS-CoV-2 receptor binding domain-based subunit vaccine candidate with different adjuvant formulations. Vaccines. 2021;9(7):744.
- 14.↑
Shi J, Zhao Y, Peng M, Zhu S, Wu Y, Ji R, et al. Screening of efficient adjuvants for the RBD-based subunit vaccine of SARS-CoV-2. Vaccines. 2023;11(4):713.
- 15.↑
Liu Z, Zhou J, Xu W, Deng W, Wang Y, Wang M, et al. A novel STING agonist-adjuvanted pan-sarbecovirus vaccine elicits potent and durable neutralizing antibody and T cell responses in mice, rabbits and NHPs. Cell Res. 2022;32(3):269–87.
- 16.↑
Mohammadi M, Jeddi-Tehrani M, Golsaz-Shirazi F, Arjmand M, Bahadori T, Judaki MA, et al. A novel anti-HER2 bispecific antibody with potent tumor inhibitory effects in vitro and in vivo. Front Immunol. 2021;11:600883.
- 17.↑
Ghaedi M, Golsaz-Shirazi F, Bahadori T, Khoshnoodi J, Mortezagholi S, Jeddi-Tehrani M, et al. Potent anti-tumor immune response and tumor growth inhibition induced by HER2 subdomain fusion protein in a mouse tumor model. J Cancer Res Clin Oncol. 2023;149(6):2437–50.
- 18.↑
Maghsood F, Amiri MM, Zarnani A-H, Salimi V, Kardar GA, Khoshnoodi J, et al. Epitope mapping of severe acute respiratory syndrome coronavirus 2 neutralizing receptor binding domain-specific monoclonal antibodies. Front Med. 2022;9:973036.
- 19.↑
Luo Q, Mao X, Kong L, Huang X, Zou H. High-performance affinity chromatography for characterization of human immunoglobulin G digestion with papain. J Chromatogr B. 2002;776(2):139–47.
- 20.↑
Ferrara F, Temperton N. Pseudotype neutralization assays: from laboratory bench to data analysis. Methods Protoc. 2018;1(1):8.
- 21.↑
Jaume M, Yip MS, Cheung CY, Leung HL, Li PH, Kien F, et al. Anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a pH-and cysteine protease-independent FcγR pathway. J Virol. 2011;85(20):10582–97.
- 22.
Kuzmina NA, Younan P, Gilchuk P, Santos RI, Flyak AI, Ilinykh PA, et al. Antibody-dependent enhancement of Ebola virus infection by human antibodies isolated from survivors. Cell Rep. 2018;24(7):1802–15. e5.
- 23.↑
Wan Y, Shang J, Sun S, Tai W, Chen J, Geng Q, et al. Molecular mechanism for antibody-dependent enhancement of coronavirus entry. J Virol. 2020;94(5):e02015–19.
- 24.↑
Zhou Y, Liu Z, Li S, Xu W, Zhang Q, Silva IT, et al. Enhancement versus neutralization by SARS-CoV-2 antibodies from a convalescent donor associates with distinct epitopes on the RBD. Cell Rep. 2021;34(5):108699.
- 25.↑
Noh JY, Jeong HW, Shin E-C. SARS-CoV-2 mutations, vaccines, and immunity: implication of variants of concern. Signal Transduction Targeted Ther. 2021;6(1):203.
- 26.↑
Patel RS, Agrawal B. Heterologous immunity induced by 1st generation COVID-19 vaccines and its role in developing a pan-coronavirus vaccine. Front Immunol. 2022;13:952229.
- 27.↑
Polyiam K, Phoolcharoen W, Butkhot N, Srisaowakarn C, Thitithanyanont A, Auewarakul P, et al. Immunodominant linear B cell epitopes in the spike and membrane proteins of SARS-CoV-2 identified by immunoinformatics prediction and immunoassay. Scientific Rep. 2021;11(1):20383.
- 28.
Mittal A, Khattri A, Verma V. Structural and antigenic variations in the spike protein of emerging SARS-CoV-2 variants. PLoS Pathog. 2022;18(2):e1010260.
- 29.
Du L, Yang Y, Zhang X. Neutralizing antibodies for the prevention and treatment of COVID-19. Cell & Mol Immunol. 2021;18(10):2293–306.
- 30.↑
Kruse RL. Therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in Wuhan, China. F1000Research. 2020;9.
- 31.
Li C-J, Jiang C-L, Chao T-L, Lin S-Y, Tsai Y-M, Chao C-S, et al. Elicitation of potent neutralizing antibodies in obese mice by ISA 51-adjuvanted SARS-CoV-2 spike RBD-Fc vaccine. Appl Microbiol Biotechnol. 2023;107(9):2983–95.
- 32.↑
Liu Z, Xu W, Xia S, Gu C, Wang X, Wang Q, et al. RBD-Fc-based COVID-19 vaccine candidate induces highly potent SARS-CoV-2 neutralizing antibody response. Signal Transduction Targeted Therapy. 2020;5(1):282.
- 33.↑
Ye L, Zeng R, Bai Y, Roopenian DC, Zhu X. Efficient mucosal vaccination mediated by the neonatal Fc receptor. Nat Biotechnol. 2011;29(2):158–63.
- 34.↑
Czajkowsky DM, Hu J, Shao Z, Pleass RJ. Fc‐fusion proteins: new developments and future perspectives. EMBO Mol Med. 2012;4(10):1015–28-28.
- 35.↑
Loureiro S, Ren J, Phapugrangkul P, Colaco CA, Bailey CR, Shelton H, et al. Adjuvant-free immunization with hemagglutinin-Fc fusion proteins as an approach to influenza vaccines. J Virol. 2011;85(6):3010–4.
- 36.↑
Konduru K, Bradfute SB, Jacques J, Manangeeswaran M, Nakamura S, Morshed S, et al. Ebola virus glycoprotein Fc fusion protein confers protection against lethal challenge in vaccinated mice. Vaccine. 2011;29(16):2968–77.
- 37.↑
Soleimanpour S, Farsiani H, Mosavat A, Ghazvini K, Eydgahi MRA, Sankian M, et al. APC targeting enhances immunogenicity of a novel multistage Fc-fusion tuberculosis vaccine in mice. Appl Microbiol Biotechnol. 2015;99:10467–80.
- 38.↑
Lu L, Palaniyandi S, Zeng R, Bai Y, Liu X, Wang Y, et al. A neonatal Fc receptor-targeted mucosal vaccine strategy effectively induces HIV-1 antigen-specific immunity to genital infection. J Virol. 2011;85(20):10542–53.
- 39.↑
Shubin Z, Li W, Poonia B, Ferrari G, LaBranche C, Montefiori D, et al. An HIV envelope gp120-Fc fusion protein elicits effector antibody responses in rhesus macaques. Clin Vaccin Immunol. 2017;24(6):e00028–17.
- 40.↑
Li W, Wang T, Rajendrakumar AM, Acharya G, Miao Z, Varghese BP, et al. An FcRn-targeted mucosal vaccine against SARS-CoV-2 infection and transmission. Nat Commun. 2023;14(1):7114.
- 41.↑
Sun Y-S, Zhou J-J, Zhu H-P, Xu F, Zhao W-B, Lu H-J, et al. Development of a recombinant RBD subunit vaccine for SARS-CoV-2. Viruses. 2021;13(10):1936.
- 42.↑
Sun S, He L, Zhao Z, Gu H, Fang X, Wang T, et al. Recombinant vaccine containing an RBD-Fc fusion induced protection against SARS-CoV-2 in nonhuman primates and mice. Cell Mol Immunol. 2021;18(4):1070–3.
- 43.↑
Alleva DG, Delpero AR, Scully MM, Murikipudi S, Ragupathy R, Greaves EK, et al. Development of an IgG-Fc fusion COVID-19 subunit vaccine, AKS-452. Vaccine. 2021;39(45):6601–13.
- 44.
Liu X, Drelich A, Li W, Chen C, Sun Z, Shi M, et al. Enhanced elicitation of potent neutralizing antibodies by the SARS-CoV-2 spike receptor binding domain Fc fusion protein in mice. Vaccine. 2020;38(46):7205–12.
- 45.↑
Shi J, Jin X, Ding Y, Liu X, Shen A, Wu Y, et al. Receptor-binding domain proteins of SARS-CoV-2 variants elicited robust antibody responses cross-reacting with wild-type and mutant viruses in mice. Vaccines 2021;9(12):1383.
- 46.↑
Khorattanakulchai N, Manopwisedjaroen S, Rattanapisit K, Panapitakkul C, Kemthong T, Suttisan N, et al. Receptor binding domain proteins of SARS‐CoV‐2 variants produced in Nicotiana benthamiana elicit neutralizing antibodies against variants of concern. J Med Virol. 2022;94(9):4265–76.
- 47.↑
Sun C, Kong D, Guo E, Zhao J, Jia J, Wang R, et al. A polysaccharide-RBD-Fc-conjugated COVID-19 vaccine, SCTV01A, showed high immunogenicity and low toxicity in animal models. Vaccines. 2023;11(3):526.
- 48.↑
Zhang N, Ji Q, Liu Z, Tang K, Xie Y, Li K, et al. Effect of different adjuvants on immune responses elicited by protein-based subunit vaccines against SARS-CoV-2 and its delta variant. Viruses. 2022;14(3):501.
- 49.↑
Luo D, Yang X, Li T, Ning N, Jin S, Shi Z, et al. An updated RBD-Fc fusion vaccine booster increases neutralization of SARS-CoV-2 Omicron variants. Signal Transduction Targeted Therapy. 2022;7(1):327.
- 50.
Shanmugaraj B, Khorattanakulchai N, Paungpin W, Akkhawattanangkul Y, Manopwisedjaroen S, Thitithanyanont A, et al. Immunogenicity and efficacy of recombinant subunit SARS-CoV-2 vaccine candidate in the Syrian hamster model. Biotechnol Rep. 2023;37:e00779.
- 51.
Shanmugaraj B, Khorattanakulchai N, Panapitakkul C, Malla A, Im-Erbsin R, Inthawong M, et al. Preclinical evaluation of a plant-derived SARS-CoV-2 subunit vaccine: protective efficacy, immunogenicity, safety, and toxicity. Vaccine. 2022;40(32):4440–52.
- 52.
Siriwattananon K, Manopwisedjaroen S, Shanmugaraj B, Buranapraditkun S, Wijagkanalan W, Leetanasaksakul K, et al. Plant-produced receptor-binding domain of SARS-CoV-2 elicits potent neutralizing responses in mice and non-human primates. Front Plant Sci. 2021;12:682953.
- 53.↑
Dekkers G, Bentlage AE, Stegmann TC, Howie HL, Lissenberg-Thunnissen S, Zimring J, et al., editors. Affinity of human IgG subclasses to mouse Fc gamma receptors. MAbs: Taylor & Francis; 2017.
- 54.↑
Muñoz-Carrillo JL, Contreras-Cordero JF, Gutiérrez-Coronado O, Villalobos-Gutiérrez PT, Ramos-Gracia LG, Hernández-Reyes VE. Cytokine profiling plays a crucial role in activating immune system to clear infectious pathogens. Immune response activation and immunomodulation: IntechOpen; 2018.
- 55.
Yang J, Wang W, Chen Z, Lu S, Yang F, Bi Z, et al. A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity. Nature. 2020;586(7830):572–7.
- 56.
Moss P. The T cell immune response against SARS-CoV-2. Nat Immunol. 2022;23(2):186–93.
- 57.↑
Bailer RT, Holloway A, Sun J, Margolick JB, Martin M, Kostman J, et al. IL-13 and IFN-γ secretion by activated T cells in HIV-1 infection associated with viral suppression and a lack of disease progression. The J Immunol. 1999;162(12):7534–42.
- 59.↑
Moderbacher CR, Ramirez SI, Dan JM, Grifoni A, Hastie KM, Weiskopf D, et al. Antigen-specific adaptive immunity to SARS-CoV-2 in acute COVID-19 and associations with age and disease severity. Cell. 2020;183(4):996–1012. e19.
- 60.↑
Sahin U, Muik A, Derhovanessian E, Vogler I, Kranz LM, Vormehr M, et al. COVID-19 vaccine BNT162b1 elicits human antibody and TH1 T cell responses. Nature. 2020;586(7830):594–9.
- 61.↑
Liu L, Iketani S, Guo Y, Chan JFW, Wang M, Liu L, et al. Striking antibody evasion manifested by the Omicron variant of SARS-CoV-2. Nature. 2022;602(7898):676–81.
- 62.
Cele S, Jackson L, Khoury DS, Khan K, Moyo-Gwete T, Tegally H, et al. Omicron extensively but incompletely escapes Pfizer BNT162b2 neutralization. Nature. 2022;602(7898):654–6.
- 63.
Cameroni E, Bowen JE, Rosen LE, Saliba C, Zepeda SK, Culap K, et al. Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift. Nature. 2022;602(7898):664–70.
- 64.↑
Hotez PJ, Corry DB, Bottazzi ME. COVID-19 vaccine design: the Janus face of immune enhancement. Nat Rev Immunol. 2020;20(6):347–8.
- 65.↑
Huisman W, Martina B, Rimmelzwaan G, Gruters R, Osterhaus A. Vaccine-induced enhancement of viral infections. Vaccine. 2009;27(4):505–12.
- 66.
Haque A, Pant AB. Efforts at COVID-19 vaccine development: challenges and successes. Vaccines. 2020;8(4):739.
- 67.↑
Huisman W, Karlas JA, Siebelink KH, Huisman RC, de Ronde A, Francis MJ, et al. Feline immunodeficiency virus subunit vaccines that induce virus neutralising antibodies but no protection against challenge infection. Vaccine. 1998;16(2–3):181–7.
- 68.↑
Weiss RC, Scott FW. Antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever. Comp Immunol Microbiol Infect Dis. 1981;4(2):175–89.
- 69.↑
Bolles M, Deming D, Long K, Agnihothram S, Whitmore A, Ferris M, et al. A double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge. J Virol. 2011;85(23):12201–15.
- 70.
Luo F, Liao F-L, Wang H, Tang H-B, Yang Z-Q, Hou W. Evaluation of antibody-dependent enhancement of SARS-CoV infection in rhesus macaques immunized with an inactivated SARS-CoV vaccine. Virologica Sinica. 2018;33:201–4.
- 71.
Tseng C-T, Sbrana E, Iwata-Yoshikawa N, Newman PC, Garron T, Atmar RL, et al. Immunization with SARS coronavirus vaccines leads to pulmonary immunopathology on challenge with the SARS virus. PloS one. 2012;7(4):e35421.
- 72.
Wang Q, Zhang L, Kuwahara K, Li L, Liu Z, Li T, et al. Immunodominant SARS coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates. ACS Infect Dis. 2016;2(5):361–76.
- 73.↑
Corapi WV, Olsen C, Scott FW. Monoclonal antibody analysis of neutralization and antibody-dependent enhancement of feline infectious peritonitis virus. J Virol. 1992;66(11):6695–705.
- 74.
Olsen CW, Corapi W, Ngichabe C, Baines J, Scott F. Monoclonal antibodies to the spike protein of feline infectious peritonitis virus mediate antibody-dependent enhancement of infection of feline macrophages. J Virol. 1992;66(2):956–65.
- 75.
Wang S-F, Tseng S-P, Yen C-H, Yang J-Y, Tsao C-H, Shen C-W, et al. Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins. Biochem biophysical Res Commun. 2014;451(2):208–14.
- 76.
Yip MS, Leung NHL, Cheung CY, Li PH, Lee HHY, Daëron M, et al. Antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus. Virol J. 2014;11(1):1–11.
- 77.
Liu L, Wei Q, Lin Q, Fang J, Wang H, Kwok H, et al. Anti–spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection. JCI insight. 2019;4(4).
- 78.↑
Diamond MS, Pierson TC. The challenges of vaccine development against a new virus during a pandemic. Cell Host & Microbe. 2020;27(5):699–703.
- 79.↑
Jeyanathan M, Afkhami S, Smaill F, Miller MS, Lichty BD, Xing Z. Immunological considerations for COVID-19 vaccine strategies. Nat Rev Immunol. 2020;20(10):615–32.
- 80.↑
Ghotloo S, Maghsood F, Golsaz‐Shirazi F, Amiri MM, Moog C, Shokri F. Epitope mapping of neutralising anti‐SARS‐CoV‐2 monoclonal antibodies: implications for immunotherapy and vaccine design. Rev Med Virol. 2022;32(5):e2347.
- 81.↑
Chang MR, Ke H, Coherd CD, Wang Y, Mashima K, Kastrunes GM, et al. Analysis of a SARS-CoV-2 convalescent cohort identified a common strategy for escape of vaccine-induced anti-RBD antibodies by Beta and Omicron variants. EBioMedicine. 2022;80.
