key: cord-0282318-yv9xizix
authors: Mai, Tran Thi Nhu; May, Bruce; Thuan, Ung Trong; Khoi, Nguyen Mai; Trang, Nguyen Thi Thuy; Van Long, Dinh; Chung, Doan Chinh; Vinh, Tran The; Hiep, Khong; Truc, Nguyen Thi Thanh; Huy, Hua Hoang Quoc; Anh, Nguyen Viet; Phat, Ha Tan; Luu, Phan Dang; An, Nguyen Truong; Ngoc, Bui Thi; My, Tu Tieu; Theo, Nguyen Thi; Hang, Le Thi Thuy; Lan, Dong Thi; Hieu, Huynh Trong; Huong, Ho Phien; Thao, Le Nguyen Thanh; Thao, Truong Cong; Phi, Pham Hoang; Luong Cong, Y; Lim, Nie; Ngoc, Cao Minh; Khanh, Nguyen Duy; Hung, Trinh Thanh; Si, Do Minh
title: PRE-CLINICAL IMMUNE RESPONSE AND SAFETY EVALUATION OF THE PROTEIN SUBUNIT VACCINE NANOCOVAX FOR COVID-19
date: 2021-07-21
journal: bioRxiv
DOI: 10.1101/2021.07.20.453162
sha: 02d6132a22879e8282d9d12fd41114f99fb584cd
doc_id: 282318
cord_uid: yv9xizix

The Coronavirus disease-2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), has become a dire global health concern. The development of vaccines with high immunogenicity and safety is crucial for control of the global COVID-19 pandemic and prevention of further illness and fatalities. Here, we report development of SARS-CoV-2 vaccine candidate, Nanocovax, based on recombinant protein production of the extracellular (soluble) portion of the S protein of SARS-CoV-2. The results showed that Nanocovax induced high levels of S protein-specific IgG, as well neutralizing antibody in three animal models including Balb/C mice, Syrian hamsters, and non-human primate (Macaca leonina). In addition, the viral challenge study using the hamster model showed that Nanocovax protected the upper respiratory tract from SARS-CoV-2 infection. No adverse effects were induced by Nanocovax in swiss mice (Musmusculus var. Albino), Rats (Rattus norvegicus), and New Zealand rabbits. These pre-clinical results indicated that Nanocovax is safe and effective.

different digestion strategies. The focus of the peptide mapping was sequence verification and analysis of N-and C-terminal modifications. The mass spectrometric analysis was performed with a Compact QTOF mass spectrometer (Bruker Daltonik GmbH, Bremen Germany). The recorded LC-ESI-MS and -MS/MS spectra were processed, annotated and searched against a customized sequence database using Mascot (Matrix Science). Modified peptides were identified by their exact mass and retention time and quantified by their mass spectrometric signal intensity.

Aluminum hydroxide gel adjuvant (alhydrogel ® 1.3%) in this study was obtained commercially from Croda (Denmark) (#21645-51-2) and re-autoclaved according to the manufacturer's instruction. The antigen S protein of SARS-CoV-2 was diluted in saline buffer with 20 mM of phosphate. For antigen formulation, 1 ml of S protein was mixed with aluminum hydroxide gel adjuvant and stirred at 200 RPM for 18 hours at cool temperature (2-8 0 C). Finally, one dose contained 25 µg; 50 µg; 75 µg; and 100 µg of S protein plus 0.5 mg of Al 3+ .

and non-human primate models

Balb/c mice of both sexes, 6-10 weeks old were used for immunological studies. Syrian hamsters (Mesocricetus auratus) of both sexes, 8-12 weeks old were used for immunological studies. Other model, northern pig-tailed macaques (Macaca leonina), male, 4-5 years old, 7-9 kg were also used for immunological study. They were housed in temperature-controlled rooms at the animal facilities with fixed light-dark cycles (12-hour shifts) . Upon arriving, animals were quarantined and let to acclimate to housing conditions for at least one week before being used. The Balb/c mice, Syrian hamsters, as well non-human primates were immunized intramuscularly with Nanocovax at doses of 25 µg, 50 µg, 75 µg, and 100 µg, their serum samples were collected quantification of spike protein specific IgG antibodies by ELISA. Blood samples were collected and let to clot at room temperature for 60 min, then centrifuged at 1000 x g for 15 min. Upper serum fraction was collected and heat-inactivated at 56 0 C in 30 minutes before use or kept at -20 0 C.

The method of quantifying SARS-CoV-2 spike protein-specific IgG antibody was adapted from Tan C. W. et al, [15] . Briefly, S protein was diluted in PBS to a final concentration of 1000 ng/mL. The 96 well plate was coated by adding 100 µL of coating buffer (1000 ng/mL of S protein) per well, sealed and incubated at 4 o C for overnight or at room temperature for 2h, then washed 3 times with wash buffer. After washing step, each well was blocked with 300 µL of blocking buffer (PBS + 2% BSA) and incubated at room temperature (RT) for 2 hours. After blocking step, wells were washed 3 times with washing buffer and loaded with 100 µL of standard solutions (SARS-CoV-2 Spike S1 subunit antibody (#1035206; R&D Systerms) and serum samples and incubated at RT for 2 hours. Wells were washed 3 times with washing buffer then 100 µL of diluted goat anti-mouse IgG (Fc specific)-Peroxidase antibody (#A0168; Sigma-aldrich) were added. After incubating 1 hour, 100 µL of TMB substrate was added. The plate was kept in dark at RT for 5 minutes for color development. Color development reaction was stopped by adding 100 µL of 1M H2SO4 solution. The absorbance was measured as the optical density 450 nm.

The virus neutralization ability of antibodies in sera of Balb/C mice, hamster, and nonhuman private were determined by the virus surrogate neutralization kit (cat # L00847, Genscript, Singapore). The percent of neutralizing virus in sera were determinded according to the manufacturer's protocol

The PRNT assay detects and quantifies neutralizing antibody SARS-CoV-2 in serum samples. Sera were 2-fold serially diluted in culture medium with a starting dilution of 1:20. The diluted sera were mixed with 100 plaque-forming units (PFU) of SARS-CoV-2 virus for 1 h at 37 °C. The virus-serum mixtures were added onto Vero E6 cell monolayers and incubated 1 h at 37 °C in 5% CO2 incubator. Then the plates were overlaid with 1% agarose in cell culture medium and incubated for 4 days when the plates were fixed and stained. Antibody titres were defined as the highest serum dilution that resulted in > 50% (PRNT50) reduction in the number of plaques.

PRNT was performed in duplicate using 24-well tissue culture plates in a biosafety level 3 facility (BSL3) in the National Institute of Hygiene and Epidemiology (NIHE), Hanoi, Vietnam, adapted from Okba N. et al,. [16] 2.5. Protective efficacy evaluation of Nanocovax vaccine in Syrian hamsters

Syrian hamsters (Mesocricetus auratus) of both sexes, 8-12 weeks old were used for the viral challenge study. They were housed in temperature-controlled rooms at the animal facilities with fixed light-dark cycles (12-hour shifts). Upon arriving, animals were quarantined and let to acclimate to housing conditions for at least one week before being used. Syrian hamsters were immunized intramuscularly with two doseages of Nanocovax 50 µg on day 0 and day 7.

All procedures were performed in BSL-3 facility for animals at NIHE (National Institute of Hygiene and Epidemiology) Vietnam. We used the hCoV-19/Vietnam/CM99/2020 isolate which was isolated in NIC-NIHE. The isolate was propagated in Vero E6 to prepare virus stocks.

The hamsters were assigned to the following groups: (1) vaccinated with Nanocovax on day 0 and day 7 and then challenged with the high level of the SARS-CoV-2 virus on day 14 by the intranasal route (TCID50 = 2x10 5 ) ; (2) vaccinated with Nanocovax on day 0 and day 7 and then challenged with the low level of the SARS-CoV-2 virus on day 14 by the intranasal route (TCID50 = 1x10 3 ), and (3) placebo injection with PBS and challenged with the high/low level of the SARS-CoV-2 virus on day 14 by the intranasal route (TCID50 = 2x10 5 and 1x10 3 ). Baseline body weights were measured before infection. Animals were monitored for signs of morbidity (such as weight loss, ruffled hair, sweating, etc.) for 14 days. On day 28, their lungs were collected for detection of SARS-CoV-2 by Real-time RT-PCR. Quantitative detection of SARS-CoV-2 on the lung samples was adapted from WHO's protocol [17] . Infection doses were chosen to base on Imai et al,. [18] .

In our research, qRT-PCR was performed to quantify SARS-CoV-2. This The absolute copy number of viral loads was determined using serially diluted DNA control targeting the E gene of SARS-CoV-2.

Tissue samples from lung of hamster were processed overnight at room temperature for formalin fixation and dehydration following a established protocol. The fixed specimens were embedded directly in paraffin blocks and cut into sections with 3-5 μm thickness using a rotary microtome (Leica RM 2125RTS, Leica Biosystems Nussloch GmbH, Nussloch, Germany) and then stained with HE solution. The mounted specimens were observed and photomicrographs were taken using an optical microscope with DP27 digital camera (Olympus, Tokyo, Japan). The histopathologic examination was performed by a pathologist.

According to ICH/GLP guidelines with minor modification, single-dose toxicity study was conducted on adult male and female mice with minor modifications. The mice were fasted overnight but with free access to water prior to treatment. A total of 60 mice of both sexes were divided into 06 groups (n = 10, 5 females and 5 males) and received Nanocovax at single doses of 25 µg, 50 µg, 75 µg and 100 µg or placebo. Untreated mice were used as a control. All animals were regularly monitored continuously within the first 4 hours for behavioural and pathological signs and then daily for the next 14 days for mortality, abnormal behaviour and body weight. At the end of the study, all mice were sacrificed by cervical dislocation and some vital organs, like kidneys, liver and spleen were collected to examine morphological and histological characteristics and weights of the organs.

According to ICH/GLP guidelines with minor modification, repeat-dose toxicity study was performed on healthy male and female rats with a few modifications. The animals were carefully examined and weighed prior to starting the designed experiment. A total of 36 rats were divided into 6 groups (n = 6; 3 males and 3 females). Rats were intramuscular injected (I.M) Nanocovax daily doses of 25 µg, 50 µg, 75 µg and 100 µg or placebo for 28 days. Untreated rats were used as a control. The mortality and clinical signs was observed daily and the body weight was determined at the indicated time points during the experimental period. The water and food intake was also daily measured. At the end of treatment, all tested rats were anesthetized to collect blood samples for analysis of biochemical and hematological parameters. Following sacrifice of the animals, three vital organs (kidneys, spleen and liver) were immediately isolated, weighed individually and examined histologically.

According to ICH/GLP guidelines with minor modification, the local tolerance experiments were performed on New Zealand rabbits. The animals were carefully examined prior to starting the designed experiment. A total of 12 rabbits were divided into 2 groups (n = 6; 3 males and 3 females). The rabbits were intravenously injected (I.V) with a highest dose of Nanocovax (100 µg) and control rabbits were received placebo. Festering and inflammation at the injected position were observed.

This study was carried out in strict adherence to the animal laboratory of Nanogen pharmaceutical Company, the National Institute of drug quality control (NIDQC), and the laboratory of Hanoi Medical University (HMU). The processes were designed according to the guide of ICH/GCP, Drug administration of Vietnam, as well ACTD and approved by Ethics committee of Ministry of Health

The collected data were statistically analyzed using Grapthpad Prism, version 5 (Grapthpad Software). Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using two-way ANOVA analysis with Bonferroni post-tests and one-way ANOVA followed by the Newman-Keuls multiple comparision test to assess the difference between the various groups. Differences described as significant in the text correspond to *p < 0.05; **p < 0.01; ***p < 0.001.

To generate SAR-CoV-2 antigen for vaccine development, we designed an optimized DNA sequence encoding the extracellular domain sequence of the Spike protein which has some changes in (1) the S1/S2 Furin cleavage site to minimize the cleavage of S1/S2 during protein production, Figure 2D ). Besides that, this protein bound specifically to anti-S1 protein antibody ( Figure 2E ). The harvest sample was purified by conducting on AKTA Pilot 600R. The results of ELISA assay also indicated the purified protein concentration was 21.4 ± 0.18 g per batch with 96.56% of purity ( Figure 2H ). In addition, residual host cell protein and DNA were not detected in purified S protein (data was not showed).

SDS-PAGE analysis showed that the molecular weight of purified S protein is about 180 kDa, ( Figure 2F ). Consistent with SDS-PAGE results, anti-S1 antibodies bound specifically to predicted S protein as assayed by Western Blot analysis ( Figure 2G ). The data of intact mass analysis by MALDI-MS also confirmed that our S protein mass was determined to be 185668 ±1849 Da (Supplementary data S1). On the other hand, the N-and C-terminal sequence and peptide mapping of recombinant S protein were suggested a truncation of N-terminal serine and complete pyroglutamate formation of the N-terminal glutamine. Besides, the C-terminus showed a high heterogeneity with a C-terminal peptide with truncation of AA 1215-1222 as the most abundant variant (Supplementary data S2). Furthermore, the conservation of recombinant S protein was evaluated by comparing N-and C-terminal peptide sequences to complete nonredundant database of protein sequences storage at NCBI, using the BLASTP computer program. The analysis data showed that N-and C-terminal peptide sequences matched 100% to published SARS-CoV-2 Spike protein sequences ( Figure 2I ).

The high purity of S protein-based vaccines requires adjuvant co-administration to enhance immunogenicity [19] . Therefore, SARS-CoV-2 Spike antigen was absorbed into the various concentration of Al 3+ in Aluminum hydroxide adjuvant. To further evaluate the immunogenicity of Nanocovax, Balb/c mice received intramuscular (IM) injection of PBS (Placebo) or two dose of Nanocovax one week intervally with or without Alhydrogel. On day 14 post-priming injection, blood was collected from the mice to measure humoral immune response by ELISA. As shown in For further studies, 0.5 mg Al 3+ was applied to evaluate the immunogenicity of Nanocovax vaccine.

To assess the immunogenicity of Nanocovax, Balb/c mice were injected with various dosages (25 µg; 50 µg; 75 µg; 100 µg) of vaccine absorbed with 0.5 mg Al 3+ (aluminum hydroxide adjuvant). The priming and boosting injection were performed on day 0 and day 7, respectively. 

All procedures were performed in BSL-3 facility at NIHE (National Institute of Hygiene and Epidemiology) Vietnam. The hamsters were assigned to the following groups: (1) 

In the single-dose toxicity test, the mice were injected intramuscularly with Nanocovax at dosages of 25 µg, 50 µg, 75 µg and 100 µg and were observed up to 14 days. Control mice received placebo. The result of the study showed no mortality and no drug-related toxicity signs in the tested mice at all tested doses within 14 days. Moreover, there were no significant differences between groups for body weights and relative organ weights ( Fig. 7A; 7B) . Macroscopic examination showed that there were no abnormalities in physical appearance of liver, heart, kidneys, spleen, lungs and intestines between the treated groups and the control group (Fig. 7C) . Histological analysis shows no sign of congestion or necrosis in the liver, kidneys, and spleen (Fig. 7D ).

In the repeat-dose study, the male and female rats were injected intramuscularly once a day for 28 consecutive days with with Nanocovax at dosages of 25 µg, 50 µg, 75 µg and 100 µg.

Control rats received placebo. The treated rats and the controls appeared uniformly healthy and no lethality was recorded in tested rats during the 28-day treatment period. There were no clinically abnormal symptoms in general behaviour between treatment and control groups. In comparison with control rats, the body weight of female and male rats gradually increased during the test period. However, there was no statistically significant difference in body weight gain between the treated groups and the control group (Fig. 8A) . Similarly, no significant difference was observed in organ weights of rats, both males and females between treated groups and control group (Fig.   8B ). The results showed that during the treatment, vital organs such as the liver and kidney were not adversely affected. Macroscopic observations showed that there were no lesions and no abnormalities of physical appearance of liver, kidneys and spleen observed in all groups. As illustrated in figure 8C , no significant macroscopic changes, such as colour, size, shape and texture, in liver, spleen, and kidneys were observed between groups. The indicators of the hematological parameters and biochemical parameters of rats are shown in Fig 9A; 9B , respectively that had no significant difference in vaccinated groups and control groups. Similarly, urinalysis of rats also had no significant difference in vaccinated groups and control groups (data not shown). The rerults from histopathological analysis revealed that none of kidney, liver and spleen tissues of both male and female rats that received Nanocovax daily at all doses showed any damage or pathological alterations under microscopic observation ( Figure 9C ).

The experiments were performed on New Zealand rabbits. The rabbits (n=6) were intravenously injected (I.V) with a sigle-dose of Nanocovax 75 µg and other rabbits group (n=6) were received PBS as a placebo. Before injection, rabbit ears were normal, and the injection sites, body temperatures, and bodyweights of all animals were monitored for 7 days. 2 hours after injection, 1 of 6 rabbits in each of the experiment groups was slightly swollen and light red at the injection site. 4 hours later, 3/6 rabbits in the experiment group and 4/6 rabbits in the placebo group were slightly swollen and light red at the injection site. After 24 hours, 2/6 rabbits in the experiment group were slightly swollen and light red at the injection site; 3/6 rabbits in the experiment group were swollen and red at the injection site. For the placebo group, 1/6 rabbits was slightly swollen and light red; 5/6 rabbits were swollen and red at the injected site. After 48 hours, 3/6 rabbits of the experiment group were swollen and red compared with the placebo group in which 4/6 rabbits were slightly swollen and light red and 2/6 rabbits were swollen and red at site of injection. 72 hours later, 3/6 rabbits were slightly swollen and light red at the site of injection in each group. 96 hours later, only one rabbit was slightly swollen and light red at the site of injection in the two groups. After 144 hours, all adverse events were fully recovered at the site of injection on the ear (data not shown). Furthermore, the bodyweights of rabbits were monitored on day -1, day 1, day 4, and day 7 after challenging with high dose of Nanocovax as well as PBS. The result from fig.   10 showed that the bodyweight of rabbits in all experiments slightly increased but the increase of bodyweight before versus after injection was not significantly different and there was no significant difference among all animal groups.

The development of vaccines with high immunogenicity and safety are the major concerns for control of the global COVID-19 pandemic and protection of further illness and fatalities.

Vaccine candidates are currently under development using different platforms, such as inactivated vaccines, live-attenuated vaccines, viral vector (adenovirus) vaccines, DNA vaccines, and mRNA vaccines. In other platforms, recombinant protein vaccines were used together with adjuvants to enhance adaptive immunity. Aluminum salt-based adjuvants are approved for many human vaccines such as Hepatitis A; Hepatitis B; Haemophilus influenza type b (Hib); Diphtheriatetanus-pertusis (DtaP, Tdap) [20] . Currently, Aluminum adjuvants have been applied in SARS-CoV-2 vaccines such as RBD vaccine; spike protein vaccine; inactivated virus vaccine; VLP vaccine [21] . For Nanocovax vaccine, spike protein was designed (Fig. 1) and firstly harvested at final concentration (21.06 g per batch) after purificattion with 96.56% purity (Fig. 2) . Secondly, spike protein was formulated with aluminum hydroxide adjuvant for pre-clinical research. Here, the results from fig. 3 showed that spike protein absorbed on aluminum hydroxide adjuvantinduced significantly higher IgG levels compared to mice group vaccinated with spike protein only, as well control group. Based on these results, aluminum hydroxide adjuvant was suggested for further experiments.

To evaluate the immunogenicity of Nanocovax vaccine, three animal models including The neutralizing antibodies to SARS-CoV-2 are urgently needed to determine not only the infection rate, immunity, but also vaccine efficacy during clinical trials [15] . Sera were collected from vaccinated animal models, as well non-human primates to measured the percent of neutralizing antibodies by surrogate virus neutralization test. The results from fig 5 showed that the antibody in sera can neutralize SARS-CoV-2 virus with a high percent of inhibition. Agreement with result from Bošnjak [22] , a strong positive correlation between surrogate virus neutralization test and the levels of S-specific IgG. Our animal models showed that the IgG levels in sera from vaccinated animals with Nanocovax increased high affinity viral neutralizing antibodies.

Futhermore, SARS-CoV-2-neutralizing antibodies can be measure by PRNT50. Here, sera from Balb/C and Hamster models had neutralizing antibodies titer from 80 to 640, and 20 from 320, respectively.

Next, the protective efficacy of Nanocovax vaccine on hamster was used in study. After challenging with SARS-CoV-2 virus, no symptoms were observed such as shortness of breath, ruffled fur, or lethargy as well as weight loss in all vaccinated mice verse control hamster groups.

On day 28, SARS-CoV-2 virus-specific RNA was detected in the lung samples of non-vaccinated groups by Real-time RT-PCR ( fig. 6) The safety of the Nanocovax vaccine was investigated based on studies of single-dose toxicity; repeat-dose toxicity; as well local tolerance test. The results from fig. 7 and fig. 8 showed that Nanocovax has no single-dose toxicity effect testing on Mus musculus var. Albino mice.

Similarly, the results from fig. 9 ; fig. 10 showed that Nanocovax vaccine has no repeated-dose toxicity effect testing on Rat (Rattus norvegicus) with three dosages (25 µg; 50 µg; 75 µg). The Balb/c mice (n group = 20 ) were immunized with various dosages of Nanocovax vaccine (25 µg; 50 µg; 75 µg; and 100 µg). The sera were collected on day 14 and the levels of total specific IgG were determined by ELISA (A). Syrian hamsters (n group =20) were vaccinated with were immunized with various dosages of Nanocovax vaccine (25 µg; 50 µg; 75 µg; and 100 µg). The sera were collected on D28; D45 and the levels of total specific IgG were determined by ELISA (B). The northern pig-tailed macaques (Macaca leonina) (n group =3) were administrated 02 doses of Nanocovax by I.M injection with 04 dosages: 25 µg; 50 µg; 75 µg; and 100 µg. The sera were collected on day 14; day 28; day 45 and the levels of total specific IgG were determined by ELISA (C). The data represent the mean ± SD and P-values were determined by one-way ANOVA analysis with Newman-Keuls multiple comparision test and two-way ANOVA analysis with Bonferroni post-tests (**P < 0.01; ***P < 0.001, ns = not significant) Figure 5 : The SARS-CoV-2-neutralizing antibodies: The Balb/c mice (n group = 20 ) were immunized with various dosages of Nanocovax vaccine (25 µg; 50 µg; 75 µg; and 100 µg) and the sera were collected on day 14. The levels of total specific IgG were determined by ELISA (A). The SARS-CoV-2-neutralizing antibodies in sera were measured by PRNT 50 (D). Syrian hamsters (n group =20) were vaccinated with were immunized with various dosages of Nanocovax vaccine (25 µg; 50 µg; 75 µg; and 100 µg). The sera were collected on D28; D45 and the levels of total specific IgG were determined by ELISA (B). The SARS-CoV-2-neutralizing antibodies in sera were measured by PRNT 50 (E). The northern pig-tailed macaques (Macaca leonina) (n group =3) were administrated 02 doses of Nanocovax by I.M injection with 04 dosages: 25 µg; 50 µg; 75 µg; and 100 µg. The sera were collected on day 14; day 28; day 45 and the levels of total specific IgG were determined by ELISA (C). The data represent the mean ± SD and Pvalues were determined by one-way ANOVA analysis with Newman-Keuls multiple comparision test and two-way ANOVA analysis with Bonferroni post-tests (**P < 0.01; ***P < 0.001, ns = not significant) 

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Diagnostic detection of 2019-nCoV by real-time RT-RCR

Syrian hamsters as a small animal model for SARS-CoV-2 infection and countermeasure development

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Aluminum Vaccine Adjuvants: Are they Safe?

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This study was supported by Ministry of Science and Technology, Vietnam under grant number: ĐTĐL.CN.51/20.

The authors contributed equally to this work

The authors declare that they have no competing interests.