Live attenuated SARS-CoV-2 vaccine OTS-228 demonstrates efficacy, safety, and stability in preclinical model

Abstract

Live attenuated vaccines (LAV) have the potential to meet all the criteria for an efficacious vaccine. In addition to providing protection against the target disease, they offer the potential to prevent transmission, provide cross-protection by stimulating humoral and cellular immunity, and allow versatility in application routes. The SARS-CoV-2 LAV candidate, OTS-228, has demonstrated excellent safety and high efficacy in preclinical models, inducing transmission-blocking immunity and providing full protection, even against variants such as Omicron BA.2, BA.5, and XBB.1.5. However, to ensure that OTS-228 has no dose-dependent side effects and to evaluate potential risk of reversion to virulence-a known general issue with live vaccines-detailed characterization of LAV OTS-228 is essential. To address this, we conducted four different experiments using Syrian hamsters, a model for moderate to severe COVID-19. A maximum dose trial confirmed the vaccine's full attenuation and prevention of transmission, even at high doses. In addition, four intentional serial in vivo passages demonstrated the genomic stability of the vaccine and the non-infectivity of nasal washings. Furthermore, OTS-228 maintained its attenuation and immunogenicity even after 15 additional in vitro passages, providing full protection against lung infection with virulent SARS-CoV-2 strains. Finally, a low-dose experiment confirmed the high efficacy of the vaccine candidate, establishing the protective dose 50 (PD₅₀) at less than 100 TCID₅₀ per hamster. Our results provide strong evidence for the safety and efficacy of the LAV candidate OTS-228 and supports its potential as a safe and effective vaccine in a highly relevant preclinical model.

Fig. 1. Intranasal vaccination of Syrian hamsters with a maximum dose of OTS-228 confirms attenuation and immunogenicity.

a Experimental setup: Syrian hamsters (n = 14) were intranasally vaccinated with the maximum applicable dose of 10^6.1 TCID₅₀ of OTS-228 per animal. At 1 dpv, serologically naïve direct contact animals (n = 3) were co-housed with vaccinated animals in a 1:3 ratio to detect transmission events. b Survival rate and (c) body weight were monitored over 14 dpv, confirming no mortality and no weight loss. d Nasal wash samples were analyzed via RdRp (nsp12)-specific qPCR to determine genome copies per mL (gc/mL). Genome copy numbers were calculated based on a standard of known concentration. No viral genome shedding was detected in contact animals throughout the experiment. Bars indicate mean with SD. e Tissue sampling: Five vaccinated hamsters were euthanized at 5 dpv, and (f) the remaining nine vaccinated and three contact hamsters were euthanized at 14 dpv to assess genome copies in respiratory tract organ samples. At 5 dpv, virus genome was detected in nasal conchae and lung samples, but by 14 dpv, viral genome was completely cleared from lung samples and absent in contact animals. Mean is indicated by line. g Histopathology at 5 dpv showed no pneumonia-related atelectasis, shown as percentage of affected area. Representative hematoxylin-eosin stained sections showed the lack of atelectasis (whole slide image, scale bar 2.5 mm), along with interstitial macrophage infiltrates (green asterisk), perivascular (green arrow), and peribronchial immune cell infiltration (green arrowhead) (detailed images, scale bar 100 µm). h Antigen score and immunohistochemistry: Representative anti-SARS-CoV N-protein immunohistochemistry of lung sections showed multifocal virus antigen presence in type I pneumocytes (green arrowhead) and bronchial epithelial cells (green arrow) associated with the peribronchial and interstitial immune cell infiltration, shown in a consecutive slide of Fig. 1g, right lower image (scale bar 100 µm). i Serum antibodies specific to the SARS-CoV-2 RBD domain were detected in all vaccinated animals at 14 dpv, while absent in contact animals. j Virus neutralization test (VNT₁₀₀): The sera from vaccinated animals also exhibited neutralizing capacity in a virus neutralization test.

Fig. 2. Forced in vivo passaging confirms robust attenuation of OTS-228.

a Experimental setup: Intranasal inoculation of six index hamsters with OTS-228 (10^3.8 TCID₅₀/animal), followed by three consecutive passages using either nasal wash samples (“washing group”) or conchae samples (“tissue group”) as inoculum. b Body weight: None of the hamsters in the washing group or (c) tissue group experienced body weight loss, regardless of passage number. d Viral genome detection: Virus genome was found only in the nasal washing samples of the tissue group and in the (e) tissue samples, while the washing group remained negative throughout all passages. f, g Transmission: Direct contact animals did not become infected when co-housed with inoculated animals during passage 1. h Viral load: The viral quantity in the inoculum was determined by titration and RT-qPCR. i Histopathology: Examination of animals from passage 1 and passage 4 revealed no pneumonia-related atelectasis, shown as a percentage of the affected area. Representative hematoxylin-eosin stained sections showed no atelectasis (whole slide image, bar = 2.5 mm), no alveolar immune cell infiltration (green asterisk), but the presence of perivascular (green arrowhead) and peribronchial inflammatory infiltrates (green arrow), detailed images, bar = 100 µm. j Antigen detection: The same samples were screened for SARS-CoV N-protein antigen. The antigen score and representative immunohistochemistry lung sections illustrate multifocal viral antigen presence in type-I pneumocytes (green arrowhead) and bronchial epithelial cells (green arrow), bar = 100 µm.

Fig. 3. Intranasal vaccination of Syrian hamsters with a maximum dose of OTS-228 passage 15 and subsequent challenge infection.

a Experimental setup: Intranasal vaccination of 12 index hamsters with 10^6.0 TCID₅₀/animal of OTS-228 P.15. Direct contact animals (n = 6) were co-housed 1 dpv. b Survival and (c) body weight were monitored daily, confirming no mortality or weight loss post-vaccination. d Shedding of OTS-228 vaccine virus genome was confirmed by RdRp (nsp12)-specific RT-qPCR of nasal wash samples, determining genome copies per mL (gc/mL). One of six direct contact animals tested positive on days 3 and 7. e Sera from 19 dpv were evaluated for SARS-CoV-2 RBD-specific antibodies by ELISA, confirming a humoral immune response in vaccinated animals and the genome-positive contact animal. f Virus neutralization test (VNT₁₀₀): Some sera were tested for neutralizing antibodies against different VOCs (threshold > 1:128 starting dilution due to limited sample volume), showing positive results for WT (3/12), Alpha (4/12), and Delta (4/12). Three weeks post-vaccination, the vaccinated animals were challenged intranasally with a mixture of Alpha/Delta SARS-CoV-2 variants, and naïve direct contact animals were co-housed again one day post-challenge in a 1:1 setup. g Survival and (h) body weight were tracked until 14 dpc. None of the vaccinated animals showed mortality or weight loss, while two contact animals did not survived the infection. i Virus shedding: Viral genome copy numbers in nasal wash samples showed relatively high shedding on day 1 (mean 10^8.2gc/mL) but a significant decline by day 5 (mean 10^4.7 gc/mL, a 3017-fold reduction). j Viral genome load was also determined in organ samples at 5 dpc and (k) 14 dpc, with only residual viral genome detectable in lung samples at both time points. l Lung evaluation for pneumonia-related atelectasis at 5 dpc showed no significant damage (Hematoxylin-eosin stained whole lung slide images (bar 2.5 mm), with detailed images showing perivascular (green arrow) and peribronchial inflammatory infiltrates (green arrowhead) (bar 100 µm)), and (m) the absence of SARS-CoV-2 N-protein antigen indicated a high level of lung protection. n Humoral immune response: ELISA measurements confirmed transmission of infection in three of the four remaining contact animals, based on binding of the SARS-CoV-2 RBD-domain of the spike protein. o Neutralizing antibodies: At 14 dpc, the neutralizing humoral immune response, tested in a VNT₁₀₀, confirmed high titers of biologically relevant cross-reacting neutralizing antibodies in the vaccinated hamsters.

Fig. 4. Intranasal vaccination of Syrian hamsters with low-dose OTS-228 and subsequent challenge infection.

a Experimental Setup: Vaccination of 12 hamsters with low-dose OTS-228 (10¹–10² TCID₅₀/animal). Six contact animals were co-housed one day post-vaccination to monitor potential transmission. b Antibody Response: Sera from 19 dpv were analyzed by RBD-specific ELISA. Six vaccinated hamsters showed a specific immune response (“responders”), while the others were designated “non-responders.” c Viral Genome Detection Post-Vaccination: Viral genome copies in nasal wash samples were quantified by RT-qPCR (orf1ab-specific), confirming virus presence in five of 12 vaccinated animals. d Survival Post-Challenge: Vaccinated animals were challenged with SARS-CoV-2 WT at day 21 post-vaccination. One responder died during sampling, while two non-responders succumbed to infection by day 7. e Body Weight Changes Post-Challenge: Responders maintained body weight, while non-responders exhibited significant weight loss by day 5 post-challenge. Statistical analysis was performed using a mixed-effects model with Geisser-Greenhouse correction and Sidak’s multiple comparisons test, with individual variances computed for each comparison between responder and non-responder groups. f Viral Shedding Post-Challenge: RT-qPCR analysis of nasal wash samples revealed significantly lower viral genome copies in responders compared to non-responders. Statistical analysis was performed using a mixed-effects model with Geisser-Greenhouse correction and Tukey´s multiple comparisons test, with individual variances computed for each comparison between responder and non-responder groups. g, h Viral Genome in Respiratory Organs: At 5 dpc, lung samples from non-responders contained high viral loads, while responders showed minimal virus. By 14 dpc, viral genomes were still detectable in non-responders but not in responders. i Histopathology, pneumonia-related atelectasis given in % affected area. Representative hematoxylin-eosin stained lung sections (bar 2.5 mm) are shown: (j) Lack of atelectasis in all responders, (k) moderate atelectasis (53%) in the non-responder at 5 dpc, (l) severe atelectasis (76–77%) in the two non-responding hamsters of 7/8 dpc and (m) lack of atelectasis for responders and (n) non-responders at 14 dpc. o Moderate hyperplasia and hypertrophy of type II pneumocytes (green arrow) for the non-responders at 14 dpc, bar 100 µm. p–r Antigen Detection: Immunohistochemistry revealed (q) minimal viral antigen in one of five responders (5 dpc) but (r) extensive antigen presence in three of six non-responders examined at 5/7/8 dpc, particularly in type I pneumocytes. s, t Seroconversion and Neutralizing Antibodies: All non-vaccinated hamsters seroconverted post-challenge, while neutralizing antibodies were present in responders, correlating with protection against lung infection. u Overall Conclusion: OTS-228 vaccination led to a robust humoral immune response in responders, correlating with protection against severe lung infection and virus replication. Non-responders experienced more severe outcomes, with higher viral loads and lung damage.