Intranasal administration of a maximum dose of OTS-228 in Syrian hamsters confirms attenuation and absence of transmission

To determine whether OTS-228 has an optimal safety profile even when administered at a maximum dose, we intranasally vaccinated Syrian hamsters (n = 14) at the highest technically feasible dose, equivalent to an infectious titer of 10^6.1 TCID₅₀ per animal, as confirmed by back titration of the inoculum. The study included the co-housing of serologically naïve direct contact animals (n = 3) one day post-vaccination (dpv), at a ratio of one naïve contact animal to three vaccinated animals (Fig. 1a). Neither the vaccinated nor the contact animals showed mortality (Fig. 1b) or significant weight loss; only 4 out of 14 vaccinated hamsters showed a slight decrease in body weight at 3 dpv compared to baseline (Fig. 1c).

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.

Viral genome was detected in the nasal washings of vaccinated hamsters up to 12 dpv, peaking at 3 dpv. Although two 3 dpv nasal washing were infectious in cell culture (Supplementary Figure 3), the vaccine was not shed to any of the direct contact animals (Fig. 1d). At 5 dpv, the vaccine virus genome was detectable in respiratory tissues of both the upper (conchae and trachea) and lower respiratory tract (LRT, including cranial, medial, and caudal right lung lobes) (Fig. 1e). However, by 14 dpv, analysis of samples from the trachea and LRT revealed almost complete clearance of vaccine virus RNA, with detectable viral genome only in conchal samples from vaccinated animals (Fig. 1f). Importantly, none of the organ samples from contact animals tested positive for vaccine virus genome, reinforcing the absence of viral shedding to contact animals (Fig. 1f).

Histopathologic examination of the lungs at 5 dpv revealed that vaccination with the maximum dose of OTS-228 did not result in pneumonia-induced atelectasis, SARS-CoV-2 characteristic vascular lesions, or necrotizing bronchitis. However, peribronchial (5/5) and perivascular (5/5) inflammatory infiltrates were consistently observed, with some immune cells adhering to the vascular endothelium (3/5) (Fig. 1g). The detection of viral antigen (5/5) was associated with a slight expansion of the pulmonary interstitium primarily due to macrophages (Fig. 1h).

A humoral immune response was confirmed in the vaccinated animals by the SARS-CoV-2 WT RBD-specific ELISA (Fig. 1i) at 14 dpv, along with live virus neutralizing capacity (Fig. 1j). The absence of a serologic response in direct-contact animals corroborates the lack of transmission of the OTS-228 vaccine virus (Fig. 1i and j).

OTS-228 is genetically stable and resistant to forced transfer via nasal wash specimens

To assess the genomic stability of OTS-228 and the preservation of its attenuated phenotype, we intentionally passaged OTS-228 four times from hamster to hamster. For the initial inoculation, an OTS-228 cell culture-derived stock was used. Since the OTS-228 vaccine virus does not naturally transmit to contact animals, subsequent passages were based either on nasal washing samples (“washing group”) or on the supernatant of homogenized nasal conchae tissue material (“tissue group”). The experimental design is shown in Fig. 2a. No sustained body weight loss was observed in any group, regardless of the passage number (Fig. 2b, c).

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.

Nasal washings failed to induce infection. Only in the tissue group successful infection could be demonstrated by evaluation of nasal washing (Fig. 2d) and tissue samples (Fig. 2e) that were found positive for viral genome. Thus, for the nasal washing group, inoculation relied on the positive nasal washing samples from the tissue group, as indicated by circles in Fig. 2d, e. The natural block of transmission was confirmed by the absence of viral genome in nasal washing and tissue samples from direct contact animals at passage 1 (P.1) (Fig. 2f, g).

Although the inocula from the tissue and washing groups were comparable in terms of viral genome copies, no in vitro infectivity was detected in the washing samples (Fig. 2h). Histopathological examination of the lungs from animals in the initial (P.1) and final (P.4) passages did not reveal pneumonia-induced atelectasis, SARS-CoV-2-typical vascular lesions, necrotizing bronchitis, or alveolar immune cell infiltrates (Fig. 2i). Only focal perivascular (8/12 in P.1 and 3/6 in P.4) and peribronchial (4/12 in P.1 and 0/6 in P.4) inflammatory infiltrates were observed (Fig. 2i). Focal to multifocal viral antigen was found in all groups except the washing group from passage 4, and this was associated with mild expansion of the pulmonary interstitium, mainly by macrophages (Fig. 2j).

The OTS-228 genome was sequenced from a nasal washing sample obtained from a P.4 hamster (tissue group) at 5 days post-inoculation (dpi) and compared to the OTS-228 reference sequence (Supplementary Figure 1a). This analysis revealed only two nonsynonymous mutations—one in ORF1a nsp3 (N1922Y) and one in the spike gene (S514Y) (Supplementary Figure 1b). All 325 modified leucine/serine codons (OTS codons) and the nsp1 modifications remained unchanged. In summary, these results demonstrate remarkable genomic stability of the modified genomic regions under forced in vivo transmission conditions, and that nasal wash samples from OTS-228-vaccinated hamsters were not able to induce infection after intranasal inoculation.

In vitro passaging did not impair the safety profile or efficacy of OTS-228

To assess the genetic stability of vaccine candidate OTS-228 and the retention of its attenuation and protective efficacy, we intranasally inoculated Syrian hamsters (n = 12) with the maximum possible dose of OTS-228 after 15 in vitro passages (OTS-228 P.15). The in vitro passages were conducted in triplicate (rep.1–3) using Vero E6 cells. Sequencing results (Supplementary Figure 1c, upper panel) revealed a total of 15 cell culture-adaptive mutations (rep.1: 4, rep.2: 7, rep.3: 8), as previously reported⁴. The three replicates were pooled in equal amounts and used as inoculum (10^6.0 TCID₅₀/animal) (Fig. 3a). No mortality or weight loss was observed in either the vaccinated or contact hamsters (Fig. 3b, c). High genome copy numbers (10^5.9–10^7.7 gc/mL) were detected in the nasal washing samples of the vaccinated animals at 3 dpv, with only one of the six contact animals tested positive for viral RNA (10^4.1 gc/mL) (Fig. 3d). Sequencing of the OTS-228 genome from the 7 dpv nasal washing sample of the contact animal revealed a change in 1 of the 325 modified Leucine/Serine OTS codons (S3732L) located in nsp6 (OTS fragment 5). Four additional consensus mutations were detected: one synonymous mutation (ORF1a: nucleotide exchange A7729C) and three nonsynonymous mutations (two in the spike gene: D253V, L1197P, and one in the M gene: T7I). Three of these mutations were present in the OTS-228 P.15 inoculum (Schön et al. ⁴, Supplementary Table 5), while two emerged during hamster infection (or were present at undetectable levels in the inoculum), and 12 cell culture-related mutations were lost (Supplementary Figure 1c, lower panel). Additionally, a new minor synonymous variant was found in the N gene (nucleotide exchange A29467G, 42% variant frequency) (Supplementary Figure 1c, lower panel). Seroconversion was confirmed in the vaccinated animals at 19 dpv by ELISA (Fig. 3e) and the development of neutralizing antibodies (Fig. 3f), with the only PCR-positive contact animal also showing seroconversion (Fig. 3e).

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.

To evaluate the protective efficacy of OTS-228 P.15, we challenged the vaccinated hamsters with an equal mixture of SARS-CoV-2 Alpha and Delta variants (in total 10^4.8 TCID₅₀/animal) (Fig. 3a). The contact hamsters, which were introduced 24 h after vaccination, were separated again for 24 hours just before challenge infection (Fig. 3a). Post-challenge, none of the vaccinated animals experienced mortality or weight loss (Fig. 3g, h). However, two contact animals started losing weight from 4 days post-challenge (dpc) and succumbed to disease at 9 and 10 dpc (Fig. 3g), while two others lost weight between 8 and 9 dpc. The remaining two contact animals, including the one that tested positive for OTS-228 P.15 genome and seroconversion prior to challenge infection, did not lose weight (Fig. 3h).

All challenged animals tested positive for challenge virus genome in nasal wash samples, with a gradual decrease over time (Fig. 3i). Three contact animals tested positive at 2 dpc, one day post co-housing, while at later time points 5 out of 6 were tested positive (Fig. 3i). Evaluation of upper and lower respiratory tract (URT and LRT) organ samples at 5 dpc showed full protection from lung infection and limited replication in the URT in vaccinated animals (Fig. 3j). No infectious virus was detectable for nasal washing samples 3 dpc (Supplementary Figure 4a) and organ samples 5 dpc (Supplementary Figure 4b) by in vitro titration. By 14 dpc, organ samples confirmed that four out of six contact animals had become infected with the challenge virus (Fig. 3k). The vaccinated animals showed only low viral genome loads in conchae samples, while lung samples tested almost negative (Fig. 3k). Histopathologic examination of the lungs at 5 dpc revealed that the SARS-CoV-2 Alpha/Delta challenge infection after OTS-228 P.15 vaccination did not lead to pneumonia-related atelectasis, SARS-CoV-2-typical vascular lesions, or necrotizing bronchitis. Multifocal perivascular and peribronchial inflammatory infiltrates were observed, likely remnants from virus clearance (Fig. 3l). No virus antigen was found in the alveolar or bronchial epithelium (Fig. 3m). The absence of pulmonary atelectasis and viral antigen at 5 dpc confirmed the high level of protection. Serological analysis also showed that one contact animal remained seronegative, indicating reduced transmission (Fig. 3n). Additionally, vaccinated and challenged animals exhibited high levels of neutralizing antibodies (Fig. 3o).

Fifty percent protective dose of OTS-228 vaccination is below 100 TCID₅₀ in the Syrian hamster model

To evaluate whether a low dosage of OTS-228 vaccination is sufficient to elicit a broad immune response and provide protection against viral challenge infection, we intranasally vaccinated Syrian hamsters (n = 12) with a very low dose of OTS-228, using less than 100 TCID₅₀ per animal in a single vaccine shot. At 21 days post-vaccination, we challenged the vaccinated animals with SARS-CoV-2 WT (Fig. 4a).

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.

Following the low-dose vaccination, 6 out of 12 vaccinated hamsters showed positive reactions for SARS-CoV-2-specific antibodies at 19 dpv. The remaining six animals did not seroconvert. The seroconverted animals were subsequently designated as “responders,” while the six animals without seroconversion were designated as “non-responders” (Fig. 4b). Correspondingly, five of the responder hamsters were tested positive for the vaccine genome in nasal wash samples at 7 dpv (Fig. 4c).

After challenge infection with SARS-CoV-2 WT, the six responder hamsters did not exhibit weight loss. However, one animal died unrelated to the challenge infection under short-term anesthesia during the sampling procedure. The histological examination of this animal confirmed that there is no evidence of a correlation with the infection (Supplementary Figure 2). In addition, two of the non-responders succumbing to the infection at 7 dpc or reaching humane endpoint criteria by 8 dpc (Fig. 4d). In contrast, the six non-responders lost more than 10% of their body weight with in the first 5 dpc, which is significant (p < 0.001) more than the responders (Fig. 4e). In addition, the contact animals housed with the non-responders began to lose body weight from 1 dpc, whereas the single contact animal housed with the responder did not (Fig. 4d, e).

Virus shedding was significantly lower after challenge infection (p < 0.034, 1 dpc) in samples from the responder animals compared to the non-responders and only the contact animals to the non-responders began to shed from 2 dpc on (Fig. 4f). Furthermore, a high level of lung protection was observed in the responder animals, evidenced by the low abundance of viral genome in lung samples (Fig. 4g, h). Infectious virus was detected in nasal wash samples at 3 days post-challenge (dpc) in one of six responders, two of six non-responders, and three of five contact animals exposed to non-responders (Supplement Figure 5a). Organ samples were largely negative, with the exception of two low-level titers: one in a conchae sample from a responder and one in a caudal lung sample from a non-responder (Supplement Figure 5b).

Histopathological examination of the lungs of the responders showed no evidence of pneumonia-induced atelectasis or SARS-CoV-2-specific lesions (Fig. 4i, j, m). However, perivascular and peribronchial immune cell infiltrates and mild alveolar macrophage infiltration were observed in the majority of animals. In contrast, the non-responders exhibited moderate to severe atelectasis, SARS-CoV-2-characteristic vascular lesions, and necrotizing bronchitis during the acute phase of infection (Fig. 4k, l), accompanied by mild to moderate hyperplasia and hypertrophy of type II pneumocytes in the chronic, regenerative stage (Fig. 4n, o). Intralesional, multifocal to coalescing viral antigen was detected only in three out of six non-responders during the acute phase of infection, while focal sparse antigen was observed in one out of six responders. (Fig. 4p–r).

Challenge virus infection in the non-responders and virus transmission to the co-housed contact animals were confirmed via serological analysis. In contrast, transmission of the challenge virus to contact animals co-housed with the responders was blocked (Fig. 4s). The responders showed high neutralizing antibody titers as early as 5 dpc, while the non-responders remained negative (Fig. 4t). Although only one responder animal was used in a transmission setup, our data suggest that even a low-dose immunization can induce transmission-blocking immunity in principle (Fig. 4u).

Together, the data indicate a strong correlation between OTS-228 virus replication, seroconversion, and subsequent protection against viral challenge infection.