1. Introduction
The utilization of non-pathogenic or weakly pathogenic viruses for the generation of vaccine constructs is a promising direction in the prevention of human infectious diseases. Viral vector-based vaccines are safe and induce both innate and adaptive immune responses without the involvement of the entire dangerous pathogen. In addition, viral vectors have adjuvant properties through the expression of various pathogen-associated molecular patterns (PAMPs) and subsequent activation of innate immunity [
1].
As the respiratory tract is the main point of entry for SARS-CoV-2, intranasal immunization is of great importance to halt this infection. Compared to injectable vaccines, intranasal droplet vaccines provide additional levels of protection such as antigen-specific S-IgA, effector CD8+ T-cells, and resident memory T-/B-cells through direct delivery of the antigen to the site of infection, the respiratory mucosa. The most important advantage of intranasal droplet vaccines is safety in use due to the avoidance of needles [
2,
3].
The Sendai virus (mouse parainfluenza virus type I, genus Respirovirus, family
Paramyxoviridae) is well suited for these purposes, as it is a respiratory virus and is capable of limited replication in human bronchial epithelial cells [
4], as well as in certain categories of dendritic cells [
5], without causing disease. During this limited replication process, recombinant variants of the Sendai virus are capable of inducing a specific immune response to the foreign viral proteins they express within the human body [
6]. The use of the Sendai virus as a vaccine vector also provides non-specific antiviral protection, as it is one of the most potent natural inducers of interferon. The Sendai virus was previously used to produce human leukocyte interferon before the era of recombinant proteins [
7,
8].
An important advantage of using the Sendai virus as a vaccine vector is the high stability of its genome. This stability is associated with an unusual property of the Sendai virus genome, as well as other paramyxoviruses, known as the “rule of six”. This rule denotes a strict polyhexameric genome length (6n + 0, where n is one nucleotide). This property contributes to a particularly low frequency of homologous recombination of paramyxovirus genomic RNAs [
9]. Additionally, the replication of the Sendai virus occurs exclusively in the cytoplasm and not in the cell nucleus, which minimizes the risk of genetic integration of the viral genome into the host genome [
10].
One potential problem in testing of Sendai virus-vectored vaccines in a laboratory rodent model is the susceptibility of rodents to infection by the virus. Among rodents, mice are the most susceptible to Sendai virus infection, but some mouse lines, such as C57BL/6J and BALB/c, are resistant and tolerate a high infectious dose (10
5 EID
50) of the virus [
11,
12,
13]. It has also been shown that insertion of transgenes between the
P and
M genes in the Sendai virus genome results in significant attenuation of the virus [
13]. In our study, we used BALB/c mice, and the transgene insertion into the vaccine construct was performed exactly between the
P and
M genes of the Sendai virus genome.
The vaccine properties of the Sendai virus have been actively studied in global practice. Thus, intranasal Sendai virus-based vaccines have successfully completed clinical trials against human parainfluenza type I and respiratory syncytial virus [
14]. A number of recombinant Sendai viruses are undergoing preclinical studies as vaccines against human respiratory infections [
15], including COVID-19 [
10].
Despite the relative decline in the incidence of COVID-19, the need to develop vaccines, especially rapid response vaccines, remains due to the possibility of the emergence of variants of concern (VOCs) of SARS-CoV-2. In our work, we tested a prototype of a single-dose intranasal vaccine, which is an example of a rapid response vaccine against the delta and gamma VOCs of SARS-CoV-2.
According to currently available data, the most effective immunogen in the case of SARS-CoV-2 infection is a full-length copy of the spike S protein of the virus, in its native, non-optimized form [
16].
Recently, there has been a debate on the safety of using S protein as an immunogen due to its possible involvement in the development of coagulopathy, which is one of the known complications of severe COVID-19. Studies that have examined the direct effect of spike proteins from SARS-CoV-2 variants on platelet activity and blood clotting have reported conflicting results. Thus, the work of Kuhn et al. showed that the SARS-CoV-2 S protein, through the RGD (Arg-Gly-Asp) motif, could weakly interact with some integrins on the surface of human platelets and trigger their stochastic activation. The authors suggest that such activation may be associated with the pathogenesis of COVID-19 and the occurrence of coagulopathies [
17]. However, the work of Kusudo et al. showed that spike proteins from SARS-CoV-2 variants (alpha, beta, gamma, delta) had no effect on coagulation, activity, quantity, average volume platelets, and thromboelastography parameters in an ex vivo study [
18].
To date, much evidence has accumulated on the efficacy and safety of COVID-19 vaccines, including vector vaccines that utilized the SARS-CoV-2 spike protein as an immunogen. A meta-analysis of eight randomized controlled trials of four COVID-19 vaccines based on mRNA, adenovirus, whole virion inactivation, and subunit vaccines, comprising 195,196 participants, found no statistically significant risk of thromboembolism and bleeding after vaccination compared with placebo [
19]. A cumulative analysis of global data on the use of vaccines against COVID-19 (more than 300 million vaccinated individuals in Europe and USA) has shown that during infection with SARS-CoV-2 and the related disease (COVID-19), thrombosis occurs at least 100 times more often without vaccination than after vaccination [
20]. The experience with the Russian Sputnik V vaccine (a vector vaccine based on recombinant adenoviruses expressing the SARS-CoV-2 spike protein) has shown that it prevents the severe course of COVID-19 and the development of fatal outcomes, pulmonary embolism, and venous and arterial thrombosis [
21].
Based on the information provided, we used the full-length DNA sequence of the S gene from a SARS-CoV-2 natural isolate belonging to the B.1.617.2 lineage (delta VOC) as an immunogenic transgene in a recombinant variant of the Sendai virus.
In this paper, we present data on the engineering of a recombinant Sendai virus, Moscow strain, expressing the full-length spike (S) protein SARS-CoV-2 delta VOC, and the results of studying the immunogenic and protective properties of the recombinant virus in a single intranasal immunization of BALB/c mice and Syrian golden hamsters.
2. Materials and Methods
2.1. Cell Cultures
Rhesus macaque kidney cell culture LLC-MK2 (Flow Laboratories, London, UK) was used to titrate Sendai virus. Vero E6 African green monkey kidney cells (Cell Culture Collection of FBRI SRC VB “Vector”, Rospotrebnadzor) were used to titrate SARS-CoV-2 and determine the virus-neutralizing activity of animal blood sera. Recombinant 293-T7 cells stably expressing the T7 polymerase were kindly provided by Dr. Sergey V. Kulemzin (Institute of Molecular and Cellular Biology SB RAS, Novosibirsk, Russia).
All cells were maintained in DMEM nutrient medium with 4.5 g/L glucose (Invitrogen, Carlsbad, CA, USA), supplemented with GlutaMAX™ (Gibco, Miami, FL, USA), 10% FBS (HyClone, Logan, UT, USA), and antibiotic-antimycotic (Gibco, USA) at 37 °C and 5% CO2.
2.2. Chicken Embryos and Red Blood Cells of Animals
Chicken embryos of 11 days of age were obtained at Novo-Baryshevskaya Poultry Farm (Baryshevo village, Novosibirsk region, Russia).
The blood of a rooster and a guinea pig were obtained from the vivarium of FBRI SRC VB “Vector”, Rospotrebnadzor. Erythrocyte lavage and preparation of a working 1% suspension was carried out with 0.9% sodium chloride solution (saline solution).
2.3. Viruses
A recombinant variant of the Sendai virus expressing SARS-CoV-2 full S protein (Sen-Sdelta(M)) was generated based on the Moscow strain (GenBank: KP717417.1) [
22] using a set of recombinant plasmid DNA described in [
23]. A transgene corresponding to the full-size S protein of the SARS-CoV-2 delta VOC (Sdelta transgene) was obtained by RT-PCR using the primers presented in
Table 1 and genomic RNA obtained from the SARS-CoV-2 HCoV-19/Russia/MOS-2406/2021 strain belonging to the lineage B.1.617.2 (delta). The HCoV-19/Russia/MOS-2406/2021 strain was isolated from an adult patient in June 2021, GISAD ID: [EPI_ISL_7338789].
The amplicon of the Sdelta transgene was inserted into the polylinker region of the genomic plasmid pSen2-MCS(M) using the restriction–ligation method at the BsiWI and BssHII restriction sites (
Figure 1) to obtain the genomic plasmid DNA pSen2-CoVSpike2(M). A complete nucleotide sequences of pSen2-CoVSpike2(M) was verified by Sanger sequencing.
The recombinant Sen-Sdelta(M) was rescued as a result of transfecting 293-T7 cells with a set of four plasmid DNAs: a genomic plasmid (pSen2-CoVSpike2(M)) and three helper plasmids expressing the N, P, and L genes of the Sendai virus. Transfection was performed using Lipofectamine Plus Reagent (Invitrogen, USA) according to the manufacturer’s instructions. The 293-T7 cells together with the culture medium were frozen/thawed once 48 h post-transfection and incubation at 37 °C with 5% CO2. The transfection material was then transferred into the allantois cavity of 11-day-old chicken embryos (200 μL per egg), and the recombinant virus was isolated from the allantoic fluid after 72 h of incubation at 37 °C. The presence of the rescued virus was determined by a hemagglutination (HA) assay using 1% chicken red blood cell solution. For this purpose, 50 µL of allantois fluid from each egg was placed in a well of a round-bottom 96-well plate. Then, 50 μL of 1% chicken red blood cell solution was added to each well and the plate was incubated for 1 h at 4 °C. Allantois fluid from the eggs whose samples elicited a hemagglutination reaction was pooled and used to grow and purify recombinant virus.
The Sendai virus, both the original vector and recombinant, was grown in the allantoic fluid of 11-day-old chicken embryos for 72 h at 37 °C. The HA-positive samples were combined. The debris was pelleted by centrifugation at 1150× g for 10 min, and the supernatant was frozen at −40 °C. After thawing, the samples were centrifuged again at 12,000× g for 30 min at 6 °C, and the virus in the supernatant was pelleted by centrifugation at 100,000× g for 45 min at 6 °C. The supernatants were aspirated, and the pellets were resuspended in PBS, pH 7.4 (1/10 the initial volume), with the addition of magnesium chloride to a final concentration of 1 mM. The resulting viral suspension was sonicated three times using a cup horn sonicator. Each sonication was performed at 4 °C with ice for 1 min at 160 W, followed by vortexing for 30 s. The ice water was replaced between each sonication cycle. The product was then packaged and stored at −80 °C.
Titration of the Sendai virus was performed by plaque method on LLC-MK
2 cell culture using guinea pig erythrocytes for virus visualization as described in [
24]. The titer of the virus was expressed in the number of plaque-forming units (PFU) per 1 mL of suspension. The titer of the purified concentrated preparation of the Sendai virus was 1.5 × 10
9 PFU/mL.
The following SARS-CoV-2 strains, including various variants of concern (VOCs), were used to evaluate the immunogenicity and protective efficacy of the vaccine: delta (HCoV-19/Russia/MOS-2406/2021, GISAID ID: [EPI_ISL_7338789]) and gamma (HCoV-19/Russia/SA-17620-080521/2021, GISAID ID: [EPI_ISL_6565014]). Additionally, the B.1.1 strain (hCoV-19/Russia/Omsk202118_1707/2020, GISAID ID: [EPI_ISL_1242008]), which shares homology with the Wuhan strain, was also included in the assessment. All strains were obtained from the State collection of causative agents of viral infections and rickettsioses FBRI SRC VB Vector, Rospotrebnadzor.
2.4. Western Blot Analysis
LLC-MK2 cells infected by recombinant or original vector strains of the Sendai virus were lysed in buffer: 50 mM Tris (pH 8.0), 5 mM EDTA, 150 mM NaCl, 0.1% SDS, 1 mM PMSF, and complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Samples were separated by 12% SDS–PAGE and transferred to a trans-blot nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) by a wet blotting procedure (100 V, 500 mA, 90 min, 15 °C). The membrane was blocked with 5% dry milk (Fisher scientific, Waltham, MA, USA) in TBS for 1 h at RT on a shaker. Then, membrane was washed with TBS on a shaker 3 times (10 min at RT each time) and incubated with primary antibodies diluted in TBST containing 5% dry milk overnight at 5 °C on a shaker. To detect the spike protein of SARS-CoV-2, a human blood serum of COVID-19 convalescent (1:200) was used. The membranes were then washed with TBST on a shaker 3 times (10 min at RT each time) and incubated with goat anti-human-alkaline phosphatase-conjugated polyclonal IgG (1:3000) (Sigma-Aldrich, St. Louis, MO, USA) in TBST containing 5% dry milk for 1 h at RT. The secondary antibody was discarded and the membranes were washed with TBST on a shaker 3 times (10 min at RT each time). The immune complex was visualized by adding BCIP/NBT-Purple liquid substrate (Sigma-Aldrich, USA). The reaction was stopped by washing the membrane in distilled water. A GelDoc Go scanner (Bio-Rad, USA) was used for detection.
2.5. Laboratory Animals and Immunization Procedures
Six-week-old female golden Syrian hamsters (n = 16) and eight-week-old female BALB/c mice (n = 28) were obtained from the nursery of laboratory animals of the FBRI SRC VB “Vector”, Rospotrebnadzor. The hamsters and mice were housed in groups of 4 and 6–8 animals, respectively, under standard conditions, and had free access to food and water at all times. All manipulations with the animals, including immunization, infection, and tissue collection, were carried out after premedication with a combination of Zoletil 100 (Valdepharm, Val-de-Reuil, France) and Xyla (Interchemie, Harju maakond, Estonia).
The animals were divided into two equal groups—immunized and control. Immunization with the recombinant virus Sen-Sdelta(M) was carried out once intranasally at a dose of 105 PFU per mouse and 106 PFU per hamster. The volume of each inoculum was 100 μL for hamsters (50 μL in each nostril) and 10 μL for mice. The control groups received a similar amount of PBS, pH 7.4, in which the virus was diluted. Blood was collected from animals 28 days after vaccination, incubated for 1 h at 37 °C and 2 h at 4 °C, and then centrifuged at 7000× g for 10 min. The sera were deactivated by heating for 30 min at 56 °C and stored at −20 °C. The sera samples were used to detect total and virus-neutralizing antibodies to SARS-CoV-2. To assess the cellular immune response, 6 mice were used from the vaccinated and control groups, were euthanized on the 28th day after vaccination, and the spleens were collected and subjected to an analysis for the specific immune cell populations.
2.6. ELISA and Neutralization Assay for SARS-CoV-2
The determination of total IgG class antibodies to coronavirus was carried out using the commercial test system “SARS-CoV-2-ELISA-Vector” (FBRI SRC VB “Vector”, Rospotrebnadzor, Moscow, Russia). To detect IgG, HRP-conjugated goat anti-Syrian hamster IgG (Invitrogen, USA) or goat anti-mouse IgG (Biomedicals, Santa Ana, CA, USA) was used. The neutralizing activity of blood serum was determined on Vero E6 cell culture as described in [
25] against three variants of SARS-CoV-2: hCoV-19/Russia/MOS-2406/2021 (delta VOC), HCoV-19/Russia/SA -17620-080521/2021 (gamma VOC), and Wuhan.
2.7. IFN-γ ELISpot and ICS
Splenocytes were isolated by pressing individual spleen through 70 and 40 μm cell filters (Jet BioFIL, Guangzhou, China). After removal of erythrocytes using a buffer for erythrocyte lysis (Sigma, USA), splenocytes were washed twice and resuspended in RPMI 1640 nutrient medium supplemented with gentamicin (50 μg/mL) and
L-glutamine. Cell viability and concentration were determined by the trypan blue dye test (Bio-Rad, USA) on an automatic cell counter TC20 (Bio-Rad, USA). A pool of peptides from the SARS-CoV-2 S protein sequence restricted by MHC class I and II molecules of BALB/c mice at a concentration of 20 μg/mL each was used to stimulate cells (
Table 2). Peptides were calculated using IEDB Analysis Resource tools and synthesized by AtaGenix Laboratories (Wuhan, China), the purity of peptides was >80%.
The intensity of T-cell immune response in immunized mice was determined by the number of IFN-γ-producing splenocytes using the IFN-γ ELISpot method. The analysis was performed using a Murine IFNγ ELISPOT Kit (with precoated plates) (Abcam, Waltham, MA, USA) according to the manufacturer’s instructions. Splenocytes were plated at 2.5 × 105 cells per well and stimulated with a mixture of the peptides mentioned above. The cells were incubated for 18 h at 37 °C in the presence of 5% CO2. The number of IFN-γ-producing cells was counted using an ELISpot reader from Carl Zeiss (Germany).
Intracellular cytokine staining (ICS) was performed according to a standard protocol using flow cytometry. Splenocytes of 0.7 × 10
6 cells were incubated with the peptide mixture (
Table 2) or without it for 3 h at 37 °C and 5% CO
2 followed by Brefeldin A (1 μg/mL) adding and 15 h incubation. Monoclonal antibodies produced by Biolegend (San Diego, CA, USA) against CD3 (clone 500A2), CD4 (clone GK1.5), and CD8 (clone 53-6.7) conjugated to A.F 700, BV 785, and FITC, respectively, were used for staining of surface markers. After fixation with 1% paraformaldehyde in PBS and permeabilization with 0.5% Tween 20 in PBS, the cells were stained to detect intracellular cytokines by anti-IFN-γ APC (clone XMG1.2) and anti-IL-2 BV 421 (clone JES6-5H4) (Biolegend, USA).
The samples were analyzed by using a ZE5 flow cytometer (Bio-Rad, USA) and Everest software 5.50.2100.
2.8. Virus Challenge
A SARS-CoV-2 challenge study was conducted in an animal biosafety level 3 (BSL-3) facility in compliance with the Russian Sanitary Rules and Regulations.
Thirty-five days after immunization, animals were infected by intranasal inoculation of 3.8 Log10TCID50 of SARS-CoV-2 gamma variant per mouse and 3.3 Log10TCID50 of delta variant per hamster. The animals were monitored daily after infection. The endpoint of the study on viral load in target organ tissues (nasal cavity and lungs) of mice and hamsters was set based on the timing of the peak of viral replication, according to our previous studies. After 5 days of infection for mice and 6 days of infection for hamsters, the animals were humanely euthanized with CO2. The lungs and nasal turbinates were collected, and 10% homogenates (v/v in PBS) were prepared to analyze the viral load.
2.9. Viral RNA Quantification
The viral load was assessed by RT-qPCR in the lungs and nasal turbinates as described in [
26]. Briefly, the total RNA was isolated using the Riboprep kit (ILS, Russia), and the copy number of viral genomes was measured using a TaqMan real-time PCR reaction (qPCR) with the following primers:
5′-GTTGCAACTGAGGGAGCCTTG-3′ (forward),
5′-GAGAAGAGGCTTGACTGCCG-3′ (reverse), and
5′-FAM-TACACCAAAAGATCACATTGGCACCCG-BHQ1-3′ (probe).
The plasmid pJet1.2_SARS, containing a fragment of the SARS-CoV-2 genome (strain MN997409.1, nucleotide positions 28670-28826), was used as a reference for qPCR normalization. The number of copies of the viral genome was calculated using the DNA Copy Number and Dilution Calculator (Thermo Fisher).
2.10. Determination of Infectious Virus Titer in Tissue Homogenates
A 50% tissue culture infectious dose (TCID
50) assay was used to quantify SARS-CoV-2 titers in lung and nasal turbinate homogenates. Vero E6 cells were seeded into 96-well plates and cultured for 24 h to form monolayers. Serial tenfold dilutions of 10% tissue homogenates were added to Vero E6 cell monolayers in eight replicates each. The plates were incubated for 4 days at 37 °C, and then the monolayers were stained with 0.2% Gentian violet solution. The presence of a specific cytopathic effect (CPE) was assessed visually by microscopic examination of the cell monolayer. Homogenate dilutions causing CPE in 50% of wells (endpoint dilution) were calculated according to the Reed–Muench method [
27].
2.11. Statistics
Statistical data processing was performed using GraphPad Prism 9.0 software (Graph-Pad Software, Inc., San Diego, CA, USA). Quantitative data are provided as geometric mean titer or median with range and analyzed using nonparametric tests. Differences between groups (p-value) were obtained in pairwise comparison using the Mann–Whitney U criterion. A p-value less than 0.05 was considered statistically significant.
4. Discussion
The SARS-CoV-2 variants of concern (VOCs) pose the greatest risk to public health due to their increased transmissibility, more severe illness (resulting in higher hospitalization or mortality rates), reduced effectiveness of antibodies created from previous infection or vaccination, and lower efficacy of treatments. The World Health Organization (WHO) has currently identified five VOCs: alpha, beta, gamma, delta, and omicron. These VOCs have the ability to evade the protective effects of most registered COVID-19 vaccines. Therefore, the WHO encourages the development and initiation of clinical trials for variant-specific candidate vaccines targeting these designated VOCs [
31,
32]. There is particular emphasis on vaccines that can generate mucosal immunity, as this is considered a critical area to address in the development of the next generation of COVID-19 vaccines [
3].
Here, we report the construction and characterization of a mucosal vaccine based on a recombinant Sendai virus, Sen-Sdelta(M), which encodes the transgene of full-length SARS-CoV-2 spike (S) protein of the delta VOC. We have shown that the S protein of the SARS-CoV-2 delta VOC was expressed as a transgene in cells infected with the Sen-Sdelta(M) and was also incorporated into the purified recombinant Sendai virus particles obtained from the allantois fluid of embryonated chicken eggs. The phenomenon of incorporating recombinant SARS-CoV-2 spike protein into recombinant virus particles had been demonstrated earlier using another paramyxovirus, the Newcastle disease virus [
29]. In both cases, recombinant S protein is presented predominantly as an S1/S2 cleaved form. This is due to the fact that the allantois fluid of chicken embryos contains a large number of active proteolytic enzymes involved in the processes of digestion, morphogenesis, and hemostasis of embryos [
33]. Although SARS-CoV-2 predominantly utilizes the cellular protease Furin for S1/S2 processing [
34], other proteases have been shown to be capable of such processing [
35]. Apparently, one or more proteases contained in the allantois fluid of chicken embryos also have the ability to efficiently cleave the S protein of SARS-CoV-2.
To evaluate the immunogenicity and protectiveness of the Sen-Sdelta(M) vaccine construct against SARS-CoV-2 infection, we used two types of laboratory animals: golden Syrian hamsters, highly sensitive to coronavirus infection, and BALB/c mice, selectively sensitive to some strains of SARS-CoV-2. In both cases, we used a single intranasal delivery of Sen-Sdelta(M), as this route of vaccination is the best in terms of ease of use, as well as the least traumatic and safe compared to parenteral administration.
In the BALB/c mouse model, we have shown that a single intranasal immunization with the Sen-Sdelta(M) effectively induces a humoral response with the formation of neutralizing IgG antibodies to delta and gamma VOCs of SARS-CoV-2, as well as, although to a lesser extent, to the analog of the original Wuhan strain. Thus, the Sen-Sdelta(M) vaccine has sufficiently broad cross-reactivity against different SARS-CoV-2 variants and could possibly, even without any further modifications, be used for vaccination against newly emerging virus variants after appropriate validation.
T-cell response was assessed using the ELISpot-IFN-γ and ICS methods. ELISpot-IFN-γ analysis showed that the number of IFN-γ-producing splenocytes in Sen-Sdelta(M) immunized mice was significantly higher than that in a control group. ICS analysis revealed that both CD4+ and CD8+ lymphocytes producing IFN-γ and IL-2 in response to stimulation with peptide fragments of the SARS-CoV-2 spike (S) protein were generated in the Sen-Sdelta(M) vaccinated mouse group. The data obtained indicate the formation of a pronounced virus-specific T-cell response.
Mice vaccinated with Sen-Sdelta(M) were protected well against the challenge of the SARS-CoV-2 gamma VOC, as evidenced by an 11-fold and more than 200-fold decrease in the concentration of vRNA SARS-CoV-2 in the lungs and nasal turbinates, respectively, compared with the control. No detectable infectious virus was revealed in more than half of the lung (62%) and nasal turbinate (85%) samples, which also indicates that SARS-CoV-2 infection had been stopped in the mice.
When the protectiveness of the Sen-Sdelta(M) was evaluated in the golden Syrian hamster model (
Figure 6), it was found that 100% of lung and nasal turbinate samples from vaccinated hamsters did not contain detectable level of infectious SARS-CoV-2 at the peak of infection after delta VOC challenge. The significant decrease in median infectious titer in the Sen-Sdelta(M) group compared to controls was 4 log
10 in both organs (
p < 0.001). These data are consistent with the findings of Ilinykh et al., who used a different paramyxovirus, human parainfluenza virus type 3, to generate a single-dose intranasal vaccine construct expressing the full-spike (S) protein of SARS-CoV-2 [
16].
As follows from the presented data, effectiveness of the single intranasal Sen-Sdelta(M) vaccine in reducing the risk of developing SARS-CoV-2 infection varies between mice and hamsters. The immunogenicity of Sen-Sdelta(M) for both animal species does not differ significantly: the level of specific IgG and virus-neutralizing antibodies in mice is only 2 and 2.5 times lower than the corresponding indices in hamsters. However, the protective effect in the hamster model is more pronounced than in mice, which is obviously due to differences in the pathogenesis of SARS-CoV-2 infection in these animal species. The delta variant of SARS-CoV-2 does not induce an infectious process in mice [
36], so we used a gamma variant adapted to mice [
37] for SARS-CoV-2 challenge, which allowed us to obtain a high viral load in nasal cavity and lung tissues at the peak of infection (day 5) and thus model the disease process. Syrian hamsters are highly susceptible to infection with SARS-CoV-2, without the need for prior adaptation, and develop severe pneumonia similar to COVID-19 patients [
38]. In this case, for SARS-CoV-2 challenge, we used the delta variant, which is the target of the Sen-Sdelta(M) vaccine, which obviously contributed to a more effective protection of the animals. Despite the differences identified, both experimental animal models demonstrated statistically significant immunogenicity and protectiveness of the Sen-Sdelta(M) vaccine.
The data obtained in our study indicate that recombinant Sen-Sdelta(M) is a promising vaccine candidate with protective properties against SARS-CoV-2 variants of concern already at a single intranasal administration. We also believe that Sen-Sdelta(M) or novel recombinants derived from our Sendai virus vector platform can be used for booster intranasal vaccination following parenteral administration of currently approved COVID-19 vaccines to induce a balanced systemic and local mucosal immune response in the respiratory tract.