Immunogenicity and efficacy of recombinant subunit SARS-CoV-2 vaccine candidate in the Syrian hamster model

Highlights

• •Efficacy of Baiya SARS-CoV-2 Vax 1 was tested in hamster model.
• •Baiya SARS-CoV-2 Vax 1 provided protection against SARS-CoV-2 challenge in animals.
• •Clinical trial of Baiya SARS-CoV-2 Vax 1 is currently ongoing.

Abstract

SARS-CoV-2 causes devastating impact on the human population and has become a major public health concern. The frequent emergence of SARS-CoV-2 variants of concern urges the development of safe and efficacious vaccine against SARS-CoV-2 variants. We developed a candidate vaccine Baiya SARS-CoV-2 Vax 1, based on SARS-CoV-2 receptor-binding domain (RBD) by fusing with the Fc region of human IgG. The RBD-Fc fusion was produced in Nicotiana benthamiana. Previously, we reported that this plant-produced vaccine is effective in inducing immune response in both mice and non-human primates. Here, the efficacy of our vaccine candidate was tested in Syrian hamster challenge model. Hamsters immunized with two intramuscular doses of Baiya SARS-CoV-2 Vax 1 induced neutralizing antibodies against SARS-CoV-2 and protected from SARS-CoV-2 challenge with reduced viral load in the lungs. These preliminary results demonstrate the ability of plant-produced subunit vaccine Baiya SARS-CoV-2 Vax 1 to provide protection against SARS-CoV-2 infection in hamsters.

Abbreviations

ACE2

Angiotensin-converting enzyme 2

Alum

Aluminum hydroxide

ANOVA

Analysis of variance

BSL

Biosafety level

COVID-19

Coronavirus disease 2019

CI

Confidence interval

Dpi

Days post infection

ELISA

Enzyme-linked immunosorbent assay

Fc region

Fragment crystallizable region

G

Gram

GMT

Geometric mean titer

IgG

Immunoglobulin G

LOD

Limit of detection

MN₅₀ titer

50% microneutralizing titer

PBS

Phosphate buffered saline

PFU

Plaque forming units

RBD

Receptor-binding domain

S

Spike or surface glycoprotein

SARS-CoV

Severe acute respiratory syndrome coronavirus

SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2

SD

Standard deviation

WHO

World Health Organization

1. Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the global pandemic coronavirus disease (COVID-19), that has led to millions of infections with >6 million deaths worldwide [1]. The virus outbreak has caused a drastic impact on human health and global economy. SARS-CoV-2 belongs to the family Coronaviridae and are enveloped, positive-sense, single-stranded RNA viruses. Their genome encodes for both structural and non-structural proteins [2,3]. The main antigenic targets for neutralizing antibodies are the surface-exposed spike (S) glycoprotein present on the viral surface. The receptor-binding domain (RBD) located in the S protein of SARS-CoV-2 interacts with cell surface receptor angiotensin-converting enzyme 2 (ACE2) mediating the viral entry into the host cell. Available reports in animal models showed that antibodies targeting against RBD or S protein correlate with the protection. Anti-spike or RBD antibodies have been shown to cross-neutralize recently evolved SARS-CoV-2 variants [4], [5], [6], [7] .

Vaccination is the currently available long-term solution to prevent the illness and mortality associated with COVID-19. A variety of technologies have been explored to produce effective COVID-19 vaccines [8,9] and several vaccines have been approved in many countries globally. Even though vaccines are available, the vaccination rate in many low-income countries are low due to the inequality in vaccine access and hence only a smaller number of people have received at least one dose of a vaccine in resource limited nations. In addition, the virus is emerging into new variants frequently with multiple spike mutations which may evade immunity acquired from past infection or vaccination. Hence safe, affordable, and effective vaccine that can protect against SARS-CoV-2 is highly essential to combat the pandemic.

We have previously reported that the plant-produced RBD-Fc based subunit vaccine adjuvanted with alum (Baiya SARS-CoV-2 Vax 1) induced robust anti-SARS-CoV-2 immune responses upon two intramuscular doses in mice and non-human primates [10]. Further, the vaccine has been shown to be safe, non-toxic in animal models and protects mice upon SARS-CoV-2 challenge [11]. In this study, we demonstrate the ability of Baiya SARS-CoV-2 Vax 1 to protect Syrian hamsters from SARS-CoV-2 challenge. We showed that the intramuscular vaccination induced neutralizing antibodies and there was a significant decrease in the level of infectious virus in the lungs of hamsters immunized with Baiya SARS-CoV-2 Vax 1. These results add to our preclinical data that the intramuscular injection of Baiya SARS-CoV-2 Vax 1 can protect hamsters against SARS-CoV-2 infection.

2. Materials and methods

2.1. Ethical statement for laboratory animal care and use

All the animal experiments were carried out in accordance with institutional and national guidelines. Animal procedures complied with all relevant ethical regulations for animal testing and research and were approved by the Faculty of Veterinary Sciences, Mahidol University, Institutional Animal Care and Use Committee (FVS-MU-IACUC-protocol number MUVS-2020–07–30). All procedures throughout the study were designed to minimize animal suffering. Animal experiments are reported in compliance with the PHS policy on human care and use of laboratory animals. Mahidol University Institutional Biosafety Committee approved work with infectious SARS-CoV-2 virus strains under BSL-3 and ABSL-3 conditions.

2.2. Adjuvants and excipients

Alhydrogel® adjuvant 2% (vac-alu-250) was procured from InvivoGen, USA. Vaccine excipients sucrose (107,651) was purchased from Merck, Germany and glycine (PR0608) was obtained from Vivantis Technologies, Malaysia.

2.3. Vaccine formulation

Codon optimized gene sequence encoding for RBD-Fc fusion protein was subcloned into the plant expression geminiviral vector and transiently expressed in N. benthamiana. The infiltrated plant leaves were harvested, and the recombinant RBD-Fc protein was extracted and purified using Protein A affinity column chromatography (Expedeon, Cambridge, United Kingdom). The protein profiles of the purified antigen were analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting analysis as described previously [10]. The candidate vaccine was prepared in phosphate-buffered saline (PBS) with alum adjuvant.

2.4. Challenge studies in Syrian hamsters

Female LVG golden Syrian hamsters (Mesocricetus auratus) approximately 6–8 weeks old were purchased from Beijing Vital River Laboratories (Beijing, China). Hamsters were housed in ABSL-3 and were supplied with a standard diet and ad libitum access to autoclaved water.

Twelve hamsters were randomly divided into 2 groups of 6 per group and received either PBS (control) or 10 µg of vaccine (Fig. 1). Animals were intramuscularly immunized at thigh muscle of hindleg with 50 µl of each test samples for 2 doses with 3-week interval (on day 0 and day 21). Blood samples were collected on day 14, and 35 prior to challenge for the quantification of RBD-specific IgG antibodies by ELISA. On day 42, hamsters were challenged by intranasal inoculation (50 µL) of SARS-CoV-2 (3.75 × 10⁴ plaque-forming units), 6 weeks post-initial immunization.

Fig. 1.

Schematic representation of Syrian hamster challenge study. Hamsters were divided into 2 groups (n = 6) ie., 10 µg-dose Baiya SARS-CoV-2 Vax 1 and control group. Hamsters were immunized on day 0 and 21 and were bled on day 14, 35 and 47. Hamsters were challenged intranasally with SARS-CoV-2 on day 42 and were euthanized on day 47 (5-days post infection, dpi).

2.5. Evaluation of immune responses and neutralizing antibody titer

ELISA was used to measure the antigen specific antibody titers of serum samples [10]. Further, Baiya SARS-CoV-2 Vax 1 specific neutralizing antibody was assessed using two-fold dilutions of heat-inactivated serum and positive control in a microneutralization assay performed in Vero E6 cells as reported previously [12,13].

2.6. Clinical observations

Hamsters were observed daily for overall health conditions during the study. Body weights were recorded at the time of vaccination, and then daily after challenge. The baseline body weights of all the animals were measured before virus infection. After challenge, animals were monitored for clinical signs such as weight loss, lethargy, anorexia, moribund and ruffled hair for 5 consecutive days.

2.7. Virus detection in hamster lung tissue

Virus titration was performed on oropharyngeal swabs and lung tissue samples. The left lobe of the lung collected from euthanized hamsters were weighed and homogenized in 1 mL serum-free MEM using pestle. Tissue homogenates were clarified by centrifugation at 13,000 rpm for 10 min at 4 °C. Plaque assay was performed as described with some modifications [14]. Briefly, Vero cells (ATCC: CCL-81) grown in minimum essential medium (MEM, 61,100–061, (Thermo Fisher Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum were seeded at a concentration of 3.5 × 10⁵ cells/well on the day before the assay. Serial dilutions of each sample were added to the wells. The plate was incubated at 37 °C, 5% CO₂ for 1 h and shaking the plates every 15 min. Then, the cells were overlaid with 1.5 mL/well of overlay medium containing MEM supplemented with 2% FBS and 1.5% carboxymethylcellulose (C4888, Sigma Aldrich, St. Louis, MO, USA). The culture was incubated at 37 °C, 5% CO₂ for three days for plaque development. Then the overlay was removed, cell monolayers were fixed with 10% formaldehyde, and stained with 1% crystal violet. Viral titers were reported as PFU/g of sample.

2.8. Statistical analysis

Statistical analyses were performed using GraphPad Prism version 9 software (GraphPad Software, Inc., CA, USA). Each specific test used for the analysis was indicated in the respective figure.

3. Results

3.1. Baiya SARS-CoV-2 Vax 1 induced the production of neutralizing antibodies

To assess the immunogenicity of the Baiya SARS-CoV-2 Vax 1, hamsters were administered twice by intramuscular injection with 10 µg of vaccine or with PBS as a negative control (Fig. 1). Intramuscular immunization with subunit vaccine candidate, Baiya SARS-CoV-2 Vax 1 elicited significant serum IgG responses against the plant-produced RBD in hamsters on day 35 (GMT = 635), after 2 doses (Fig. 2a). Next, the neutralizing antibody titers were evaluated by using microneutralization assay. Neutralizing antibodies were elicited by our vaccine candidate in hamsters. Sera collected from Baiya SARS-CoV-2 immunized hamsters could neutralize SARS-CoV-2 virus in vitro (GMT = 160) (Fig. 2b).

Fig. 2.

Two intramuscular dose of Baiya SARS-CoV-2 Vax 1 generates high titer neutralizing antibodies. SARS-CoV-2 RBD-specific total IgG in the serum at day 14 and 35 was measured by ELISA (a) and 50% microneutralizing (MN50) titer (b) of the sera collected from immunized hamsters. Data presented as GMT ± 95% CI of the endpoint titer in each group (n = 6). The total IgG titers lower than cut-off are plotted as 100 (a). Values smaller than the limit of detection (LOD) are plotted as 0.5*LOD (b). Two-way ANOVA, Šídák test, was used. (**: p < 0.01, ****: p < 0.0001).

3.2. Baiya SARS-CoV-2 Vax 1 reduced viral load in the lungs of vaccinated hamsters

The ability of our vaccine candidate to protect against SARS-CoV-2 challenge was evaluated in Syrian hamsters by intramuscular injection at day 0 and 21 before intranasal challenge with SARS-CoV-2. As illustrated in Fig. 1, all hamsters were challenged by intranasal infection with SARS-CoV-2 virus, at 6 weeks post-prime immunization in order to assess the protective efficacy of the vaccine. Clinical signs were observed daily for five consecutive days. All animals survived the challenge with SARS-CoV-2 until scheduled necropsy. Hamsters were weighed and observed daily for weight loss after the virus challenge. There were no adverse clinical signs and no significant differences in the body weight and body temperature between two groups (Fig. 3).

Fig. 3.

Body weight (a), percentage weight change (b) and body temperature (c) of the animals were monitored for 5 days after challenge (n = 6). Data were plotted as mean ± SD. Two-way ANOVA, Šídák test, was performed among the groups. Dpi: Days post infection.

At 5 days post challenge, all animals were euthanized, oropharyngeal swab samples and lung tissue samples were collected for determination of viral titers. In plaque assay, which gives a measure of the amount of infectious virus in the lung and oropharyngeal swab, there was a significant reduction (p < 0.01) in infectious virus in the lung of Baiya SARS-CoV-2 Vax 1 treated group compared to PBS treated control group. Indeed, the PFU/g tissue for all Baiya SARS-CoV-2 Vax 1 treated hamsters (1.7 $\times$ 10³ ± 3.4 $\times$ 10³ PFU/g) were lower than all measured PFU/g values for the PBS control group (8.5 $\times$ 10⁵ ± 4.0 $\times$ 10⁵ PFU/g). There were no PFU measured in the oropharyngeal swab of either the PBS control or the Baiya SARS-CoV-2 Vax 1 treated animals (Fig. 4). These findings suggested that the intramuscular immunization of hamsters with Baiya SARS-CoV-2 Vax 1 provided protection against the lethal SARS-CoV-2 infection.

Fig. 4.

Baiya SARS-CoV-2 Vax 1 immunized hamsters showed decrease in the viral load in the lung. PFU/g in lung tissue collected from SARS-CoV-2 challenged Syrian hamsters (n = 6). Data expressed as mean ± SD. Unpaired t-test, Mann-Whitney test, was used (**: p < 0.01).

4. Discussion

Development of safe and effective vaccine against SARS-CoV-2 is highly essential to control the virus spread and end the global pandemic. The scientific efforts in last two years have resulted in promising SARS-CoV-2 vaccines, in which some are available for human use and multiple candidates are currently in clinical trials. There are several vaccines in development by employing different approaches, including protein subunit, inactivated, viral-vectored, and nucleic acid-based [8]. Each approach has distinct benefits and limitations. The efficacy of different vaccines was reported to vary differently against SARS-CoV-2 and its newly emerged variants.

The plant-produced subunit vaccine used here combines the power and speed of plant transient expression for rapid vaccine development. Plant expression platform has several advantages compared to other available expression platforms. Plant-based vaccines have the potential to address limitations associated with the other platforms especially response time and scalability during pandemic situation [15]. The proteins produced using plant expression system can be easily scalable with substantial yields [16,17]. Using plant expression platform based on transient expression of SARS-CoV-2 antigens in N. benthamiana, we have recently demonstrated that a candidate subunit vaccine can be produced within two weeks of gene construct delivery [18]. We have also demonstrated that the plant-produced RBD-Fc with alum adjuvant induced antibody responses in mice and non-human primates which showed that this antigen could be considered as a promising vaccine candidate against SARS-CoV-2 [10]. In the present study, we continued to study the efficacy of our plant-produced subunit vaccine candidate, Baiya SARS-CoV-2 Vax 1 in hamster model and the results demonstrated that it can elicit a protective immune response when intramuscularly administered.

Although there are several proof-of-concept studies showed the expression of recombinant vaccine antigens in plants, very few candidates reached the clinical stage. Subunit vaccine antigens produced in plants have proved to be immunogenic in animal models [19], [20], [21], [22], [23], [24]. It is well known that the addition of appropriate adjuvants with subunit vaccine may enhance the production of neutralizing antibodies that confers protective immunity against the infection. Notably, clinical trials with plant-derived vaccine against diseases such as influenza (18–64 study NCT03301051; 65-plus study NCT03739112) [25], Rotavirus (NCT03507738) [26], COVID-19 vaccine (NCT04450004) [27] have demonstrated their safety and immunogenicity in humans. Clinical trials of other plant derived COVID-19 vaccine developed by Kentucky Bioprocessing (NCT04473690) and Baiya Phytopharm Co., Ltd (NCT04953078, NCT05197712) are currently ongoing [28].

Though mice are commonly used lab animal for biomedical research, mice cannot be efficiently infected with wild type viruses. Hamsters are used as a potential infection model in the research of SARS-CoV-2 infection and other infections caused by respiratory viruses. Hamsters are known to develop similar clinical manifestations in the lungs exposed to SARS-CoV-2 like humans [29]. There have been multiple reports available for other vaccination studies performed in hamsters which shown protection against virus challenge [30], [31], [32]. Hence, the protective efficacy of our vaccine was evaluated in Syrian hamster challenge model. Here, we employed a 10-µg dose of our vaccine, which was selected based on the immunogenicity of our vaccine tested earlier in mice and monkeys. Remarkably, 14 days following the second immunization, Baiya SARS-CoV-2 Vax 1 induced high titers of IgG and neutralizing antibodies in hamsters. The neutralizing antibody titer measured by microneutralization assay showed that the antibodies induced by Baiya SARS-CoV-2 Vax 1 could neutralize the SARS CoV-2. Even though there were no difference in the level of SARS-CoV-2 RNA in the lungs of the vaccinated hamsters (not shown), there was a significant reduction in the viral load of the Baiya SARS-CoV-2 Vax 1 vaccinated hamsters, suggesting the reduction in the level of infectious virus in lungs post-vaccination. Another study reported that the administration of one or two doses of ChAdOx1 nCov-19 in rhesus macaques significantly reduced the viral loads in lungs, but there was no significant difference in SARS-CoV-2 RNA loads in nasal swabs between control or vaccinated animals was observed [33]. The preliminary findings from this study showed that, our vaccine can induce neutralizing antibodies and protected animals against a lethal SARS-CoV-2 challenge.

Additionally, stability profile of our vaccine candidate Baiya SARS-CoV-2 Vax 1 has also been assessed. Further, the safety pharmacology and toxicity studies performed in non-human primates and Wistar rats respectively showed that the vaccine is safe, and no unanticipated findings were observed throughout the study. Baiya SARS-CoV-2 Vax 1 was evaluated in different animal models and has demonstrated ability to elicit neutralizing antibody response against SARS-CoV-2 [11]. The study presented here has few obvious limitations, the most important of which were the durability of immune responses needs to be evaluated, protective efficacy of the vaccine against variants of concern at different doses were not tested, the cellular immune response of the vaccine was not measured and the effect of vaccine dose with and without adjuvant on the immunogenicity and efficacy in hamsters also not assessed.

In summary, we demonstrated that the two immunizations with plant-produced vaccine candidate, Baiya SARS-CoV-2 Vax 1 elicits anti-RBD antibodies and provides protection from SARS-CoV-2 infection in Syrian hamster. This recombinant subunit vaccine produced in plants could make a safe and effective vaccine against SARS-CoV-2 which can be well suited for the deployment of large-scale manufacturing of low-cost vaccine candidates. These preliminary results present the evidence of preclinical immunogenicity and efficacy of the Baiya SARS-CoV-2 Vax 1 in animal model.

Data availability

All data supporting the findings of this study are available within the paper and are also available from the corresponding author upon request.

Author contributions

W.P conceived the project. B.S, N.K performed the plant experiments and prepared the vaccine formulations. W.Pa and Y.A performed the hamster challenge experiments. S.M and A.T performed the virus neutralization assay. B.S drafted and revised the manuscript. All authors analyzed the data and approved the submitted version.

Funding

This research was funded by National Vaccine Institute, Thailand and Baiya Phytopharm Co., Ltd. Thailand.

Declaration of Competing Interest

Waranyoo Phoolcharoen (WP) from Chulalongkorn University is a founder/shareholder of Baiya Phytopharm Co., Ltd. Thailand.

Data availability

• Data will be made available on request.