The emergence of SARS-CoV-2 variants, like XBB.1.5, causing immune evasion and frequent breakthrough infections, emphasizes the need for vaccines that limit transmission and target newly emerging variants. Mucosal vaccines, particularly live attenuated vaccines (LAV), are promising candidates for inducing strong mucosal immune responses to prevent viral replication and transmission. Vaccination with the previously described "one-to-stop" codon-modified LAV OTS-228, carrying the ancestral spike protein, induced sterilizing immunity against ancestral SARS-CoV-2 but also broad protection against Omicron variants, including XBB.1.5, but transmission of XBB.1.5 to contacts could not be prevented completely. As a proof-of-concept, we updated OTS-228 by replacing the sequence coding for the ancestral SARS-CoV-2 spike protein with that of the XBB.1.5 variant. We applied flow cytometry to detect SARS-CoV-2-specific T cell responses, as well as ELISA and qPCR, to characterize systemic and mucosal immune responses in Syrian hamsters in detail. The new OTS construct designated as "OTS-300" exhibited an optimal safety profile in Syrian hamsters comparable to the original candidate vaccine. A single-dose intranasal (i.n.) vaccination with OTS-300 protects against disease, substantially limits XBB.1.5 replication, and reduces transmission in Syrian hamsters, showcasing the adaptability of the OTS platform for other emerging variants. OTS-300 induced accelerated mucosal and systemic antibody responses and reduced virus-mediated inflammation as compared with an intramuscularly delivered mRNA vaccine encoding the XBB.1.5 Spike.

An increasing number of mpox cases caused by the monkeypox virus (MPXV; Orthopoxvirus monkeypox ), are reported in West and Central Africa, driven by frequent zoonotic spillovers into human populations that sometimes give rise to epidemic lineages. Designing mitigation strategies to reduce spillover requires a better understanding of the ecology underlying the zoonotic emergence of MPXV. Here, we show that orthopoxviruses, including MPXV, infect duikers, which are popular bushmeat in many mpox endemic regions. In bushmeat markets from the Democratic Republic of the Congo (DRC), we detected orthopoxvirus DNA in a Weyns's duiker ( Cephalophus weynsi ) and a common duiker ( Sylvicapra grimmia ) using PCR. High throughput sequencing (HTS) detected a small number of reads assigned to MPXV, and 25% of the genome of a close relative to the taterapox virus (TATV; Orthopoxvirus taterapox ), respectively. In the Taï National Park (TNP), Côte d’Ivoire, tissue samples from a Maxwell’s duiker ( Philantomba maxwellii ) carcass were also MPXV PCR positive, and 50% of the viral genome could be recovered. Suspecting scavenging as a route of exposure, we ran metabarcoding analyses on duiker faeces from TNP, which revealed that more than 21% contained non-duiker mammal DNA, including species linked to previous MPXV infections in this ecosystem. Revisiting western chimpanzee ( Pan troglodytes verus ) diet data from an mpox outbreak in TNP in 2017, supported an epidemiological link to duiker consumption.

The highly pathogenic avian influenza viruses of subtype H5N1 represent a major threat to animal and public health. The current panzootic with H5 clade 2.3.4.4b has caused numerous, widespread outbreaks in various domestic and wild avian species with high mortalities, massive losses, and a high frequency of spillover events to unexpected novel mammalian hosts, such as dairy cows. The global H5N1 situation raises serious concerns about zoonotic risks due to effective mammal-to-mammal transmission. Therefore, it is critical to increase surveillance intensity of a broadened species range, particularly at the human-animal interface. For this purpose, reliable and cost-effective serological tools that are easy to perform and suitable for high-throughput screening are critically needed. The newly developed double-antigen enzyme-linked immunosorbent assay format employing a luminescence-based detection technology has demonstrated compliance with such prerequisites. The assay allowed sensitive and specific detection of antibodies directed against H5 hemagglutinin of clade 2.3.4.4b in a wide range of birds and mammals, including humans. Furthermore, it allowed differentiating H5 anti-head-specific from cross-reacting anti-stalk antibodies, which represents a valuable feature with regard to the monitoring of future vaccination programs with H5-specific vaccines. Thus, the assay is a significant contribution to existing serological diagnostic tests for a clade-optimized and species-independent detection of influenza A virus antibodies. IMPORTANCE:The ongoing highly pathogenic avian influenza virus H5N1 panzootic has caused numerous outbreaks in domestic and wild animals, with frequent spillover events to unexpected host species, which underscores the importance of intensified surveillance. However, sensitive and specific multi-species serological assays represent a major gap. For this purpose, we developed a novel double-antigen enzyme-linked immunosorbent assay that employs an innovative luminescence-based readout strategy. The test allowed a highly sensitive and specific detection of H5-specific antibodies in a wide range of avian and mammalian species, including humans. It therefore represents a valuable contribution to improving species-independent serological diagnostic tools for the detection of influenza A virus antibodies.

Mpox, caused by the monkeypox virus (MPXV; Orthopoxvirus monkeypox ), is on the rise in West and Central Africa 1–3 . African rodents, especially squirrels, are suspected to be involved in MPXV emergence, but no evidence of a direct transmission to humans or non-human primates has been established 4–9 . Here we describe an outbreak of MPXV in a group of wild sooty mangabeys ( Cercocebus atys ) in Taï National Park (Côte d’Ivoire). The outbreak affected one-third of the group, killing four infants. To track its origin, we analysed rodents and wildlife carcasses from the region. We identified a MPXV-infected fire-footed rope squirrel ( Funisciurus pyrropus ), found dead 3 km from the mangabey territory 12 weeks before the outbreak. MPXV genomes from the squirrel and the mangabey were nearly identical. A video record from 2014 showed a mangabey from this group eating the same squirrel species and diet metabarcoding of faecal samples collected from mangabeys before the outbreak identified two samples containing fire-footed rope squirrel DNA. One of these samples was also the first positive for MPXV. This represents a rare case of direct detection of interspecies transmission. Our findings indicate that rope squirrels were the source of the MPXV outbreak in mangabeys. Because squirrels and non-human primates are hunted, traded and consumed by humans in West and Central Africa 10,11 , exposure to these animals probably represents risk for zoonotic transmission of MPXV.

Abstract MERS-CoV poses a constant pandemic risk, as its viral lineages continue evolving, and zoonotic spillover events could lead to random viral polymorphisms that might lead to human adapted variants. Currently, no small animal model reliably recapitulates both disease progression and transmission dynamics, which are critical aspects for counter-viral measures like vaccine development. Although the Syrian hamster is an optimal animal model for SARS-CoV-2 infection and transmission, it is naturally resistant to MERS-CoV infection. Dipeptidyl peptidase-4 (DPP4) is the functional receptor for MERS-CoV infection, and is highly expressed in human kidney, intestine, liver, and lung tissues. Here, we evaluated the suitability of a human DPP4 (hDPP4) transgenic Syrian hamster model for MERS-CoV research. We used two different MERS-CoV strains (EMC/2012 and D10540/2023) for intranasal inoculation of hamsters. Both strains replicated efficiently, led to comparable severe clinical outcomes, and had similar viral transmission efficiencies. MERS-CoV RNA and nucleoprotein antigen were mainly detected in the brain and the respiratory tract. In summary, we validated a novel hDPP4-transgenic hamster as a suitable model for MERS-CoV infection enabling vaccine and transmission research.

Diagnostics and prevention of COVID-19 are essential for controlling the spread of the virus and reducing mortality rates. As SARS-CoV-2 surface proteins are susceptible to mutations, the nucleocapsid protein (NP) with its highly conserved gene sequence is an attractive target for studying virus-host interactions. NP plays a key role in the coronavirus life cycle, modulating viral RNA packaging, transcription, and assembly. In addition, its abundant expression during infection makes it a valuable diagnostic marker. NP is involved in modulating the host's innate immunity; however, the cellular mechanisms of its pathogenicity are not yet fully understood. This study developed and characterized murine monoclonal antibodies (MAbs) specific to the SARS-CoV-2 NP to investigate its antigenic regions and utilize the MAbs in virus-detecting systems or cellular NP blocking assays. The MAbs showed cross-reactivity with Omicron NP, recognizing epitopes within functionally active domains. They also identified NP in SARS-CoV-2-infected cells, supporting their feasibility in future immunoassays. Additionally, the ability to inhibit NP-cell interaction was assessed, with MAbs 4B3, 7F10, 16D9, and 18A8 found to reduce NP internalization. Overall, this study provides well-characterized tools for investigating SARS-CoV-2 antigenicity and pathogenicity and demonstrates the functional potential of the generated MAbs in studying NP-mediated host cell interactions.

The transmission of influenza A virus H5N1 clade 2.3.4.4b from cattle to humans highlights the risk of an H5N1 pandemic. Pre-existing immunity strongly impacts the course and severity of viral infections, making detailed knowledge of antibodies against the spilled-over strain crucial. Here, we assessed humoral immunity against H5N1 A/Texas/37/2024 in H5N1-naive individuals. We performed complementary binding and neutralization assays on 66 individuals and ranked activities among a panel of 76 influenza A virus isolates. We detected low but distinct cross-neutralizing titers against A/Texas/37/2024, with a 3.9- to 15.6-fold reduction compared with selected H1N1 or H3N2 strains. By cloning and characterizing 136 memory B cell-derived monoclonal antibodies, we identified potent A/Texas/37/2024-neutralizing antibodies in five out of six individuals we investigated. These antibodies cross-neutralized H1, competed with antibodies targeting the hemagglutinin (HA) stem, and protected mice from lethal H5N1 challenge. Our findings demonstrate partial pre-existing humoral immunity to A/Texas/37/2024 in H5N1-naive individuals.

The repeated spill-over of Influenza A virus H5N1 clade 2.3.4.4b from cattle to humans highlights the risk of a human H5N1 pandemic. Given the impact of pre-existing immunity on the course and severity of viral infections, we assessed in detail the humoral immunity against the H5N1 A/Texas/37/2024 isolate in H5N1-naive individuals. To this end, we performed complementary binding and neutralization assays on 66 subjects and ranked activities among a panel of 76 influenza A virus isolates. We detected low but distinct cross-neutralizing titers against A/Texas/37/2024 with a 3.9 to 15.6-fold reduction compared to selected H1N1 or H3N2 strains. Moreover, by cloning and evaluating 136 monoclonal antibodies from single memory B cells, we identified potent A/Texas/37/2024-neutralizing monoclonal antibodies in five out of six investigated individuals. These antibodies predominantly utilize VH1-69 gene segments, cross-neutralize H1, and compete with antibodies targeting the HA stem. Our findings demonstrate partial pre-existing humoral immunity to A/Texas/37/2024 in H5N1-naive individuals. ### Competing Interest Statement DR, MM, CK, FK, and ML are members of the non-profit Center for Predictive Analysis of Viral Evolution (Previr). LG, HG, CK and FK are inventors on patent applications on virus neutralizing antibodies filed by the University of Cologne and have received payments from the University of Cologne for licensed patents.

Wild rodents are important reservoirs and vectors of various pathogens, and play a crucial role in the spread of zoonotic pathogens. The garden dormouse (Eliomys quercinus), an arboreal nocturnal rodent species native to Europe, has declined throughout much of its natural range in recent decades. The reason for this ongoing decline is not yet fully understood, but infectious diseases may play a role. This study aimed to review the diversity of pathogens associated with the garden dormouse. For this purpose, a comprehensive review of the existing literature on garden dormouse-associated viruses, bacteria and protozoa was conducted. In parallel, we analysed samples from 294 garden dormice, that were found dead in Germany, for rodent-associated and zoonotic pathogens. The scientific literature currently, comprising 53 references, covers 73 years and primarily addresses Borrelia spp. and Eimeria myoxi. In the literature, a total of eight pathogens have been detected in garden dormice. In our own investigations, we were able to detect four different pathogens as well as antibodies against hepatitis E virus. The most prevalent pathogen found in our study was Staphylococcus aureus (29

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.

Coronavirus disease 2019 (COVID-19) mRNA vaccines that have contributed to controlling the SARS-CoV-2 pandemic induce specific serum antibodies, which correlate with protection. However, the neutralizing capacity of antibodies for emerging SARS-CoV-2 variants is altered. Suboptimal antibody responses are observed in patients with humoral immunodeficiency diseases, ongoing B cell depletion therapy, and aging. Common experimental mouse models with altered B cell compartments, such as B cell depletion or deficiency, do not fully recapitulate scenarios of declining or suboptimal antibody levels as observed in humans. We report on SARS-CoV-2 immunity in a transgenic mouse model with restricted virus-specific antibodies. Vaccination of C57BL/6-Tg(IghelMD4)4Ccg/J mice with unmodified or N1mΨ-modified mRNA encoding for ancestral spike (S) protein and subsequent challenge with mouse-adapted SARS-CoV-2 provided insights into antibody-independent immunity and the impact of antibody titers on mucosal immunity. Protection against fatal disease was independent of seroconversion following mRNA vaccination, suggesting that virus-specific T cells can compensate for suboptimal antibody levels. In contrast, mRNA-induced IgG in the nasal conchae limited the local viral load and disease progression. Our results indicate that parenteral mRNA immunization can elicit nasal IgG antibodies that effectively suppress local viral replication, highlighting the potential of vaccines in controlling SARS-CoV-2 transmission and epidemiology.

In March 2024, highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4b H5N1 infections in dairy cows were first reported from Texas, USA. Rapid dissemination to more than 190 farms in 13 states followed. Here, we provide results of two independent clade 2.3.4.4b experimental infection studies evaluating (i) oronasal susceptibility and transmission in calves to a US H5N1 bovine isolate genotype B3.13 (H5N1 B3.13) and (ii) susceptibility of lactating cows following direct mammary gland inoculation of either H5N1 B3.13 or a current EU H5N1 wild bird isolate genotype euDG (H5N1 euDG). Inoculation of the calves resulted in moderate nasal replication and shedding with no severe clinical signs or transmission to sentinel calves. In dairy cows, infection resulted in no nasal shedding, but severe acute mammary gland infection with necrotizing mastitis and high fever was observed for both H5N1 genotypes/strains. Milk production was rapidly and drastically reduced and the physical condition of the cows was severely compromised. Virus titers in milk rapidly peaked at 108 TCID50/mL, but systemic infection did not ensue. Notably, adaptive mutation PB2 E627K emerged after intramammary replication of H5N1 euDG. Our data suggest that in addition to H5N1 B3.13, other HPAIV H5N1 strains have the potential to replicate in the udder of cows and that milk and milking procedures, rather than respiratory spread, are likely the primary routes of H5N1 transmission between cattle.

Approved vaccines are effective against severe COVID-19, but broader immunity is needed against new variants and transmission. Therefore, we developed genome-modified live-attenuated vaccines (LAV) by recoding the SARS-CoV-2 genome, including ‘one-to-stop’ (OTS) codons, disabling Nsp1 translational repression and removing ORF6, 7ab and 8 to boost host immune responses, as well as the spike polybasic cleavage site to optimize the safety profile. The resulting OTS-modified SARS-CoV-2 LAVs, designated as OTS-206 and OTS-228, are genetically stable and can be intranasally administered, while being adjustable and sustainable regarding the level of attenuation. OTS-228 exhibits an optimal safety profile in preclinical animal models, with no side effects or detectable transmission. A single-dose vaccination induces a sterilizing immunity in vivo against homologous WT SARS-CoV-2 challenge infection and a broad protection against Omicron BA.2, BA.5 and XBB.1.5, with reduced transmission. Finally, this promising LAV approach could be applicable to other emerging viruses.

Influenza A viruses (IAVs) of subtype H9N2 have reached an endemic stage in poultry farms in the Middle East and Asia. As a result, human infections with avian H9N2 viruses have been increasingly reported. In 2017, an H9N2 virus was isolated for the first time from Egyptian fruit bats (Rousettus aegyptiacus). Phylogenetic analyses revealed that bat H9N2 is descended from a common ancestor dating back centuries ago. However, the H9 and N2 sequences appear to be genetically similar to current avian IAVs, suggesting recent reassortment events. These observations raise the question of the zoonotic potential of the mammal-adapted bat H9N2. Here, we investigate the infection and transmission potential of bat H9N2 in vitro and in vivo, the ability to overcome the antiviral activity of the human MxA protein, and the presence of N2-specific cross-reactive antibodies in human sera. We show that bat H9N2 has high replication and transmission potential in ferrets, efficiently infects human lung explant cultures, and is able to evade antiviral inhibition by MxA in transgenic B6 mice. Together with its low antigenic similarity to the N2 of seasonal human strains, bat H9N2 fulfils key criteria for pre-pandemic IAVs. In this study, the authors report that bat H9N2 influenza A virus replicates and transmits in ferrets, efficiently infects human lung explant cultures, evades MxA antiviral activity in mice, and has low antigenic similarity to seasonal N2, meeting pre-pandemic criteria.

Vaccines have played a central role in combating the COVID-19 pandemic, but newly emerging SARS-CoV-2 variants are increasingly evading first-generation vaccine protection. To address this challenge, we designed “single-cycle infection SARS-CoV-2 viruses” (SCVs) that lack essential viral genes, possess distinctive immune-modulatory features, and exhibit an excellent safety profile in the Syrian hamster model. Animals intranasally vaccinated with an Envelope-gene-deleted vaccine candidate were fully protected against an autologous challenge with the SARS-CoV-2 virus through systemic and mucosal humoral immune responses. Additionally, the deletion of immune-downregulating viral genes in the vaccine construct prevented challenge virus transmission to contact animals. Moreover, vaccinated animals displayed neither tissue inflammation nor lung damage. Consequently, SCVs hold promising potential to induce potent protection against COVID-19, surpassing the immunity conferred by natural infection, as demonstrated in human immune cells.