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.

The zoonotic emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the ensuing coronavirus disease 2019 (COVID-19) pandemic have profoundly affected our society. The rapid spread and continuous evolution of new SARS-CoV-2 variants continue to threaten global public health. Recent scientific advances have dissected many of the molecular and cellular mechanisms involved in coronavirus infections, and large-scale screens have uncovered novel host-cell factors that are vitally important for the virus life cycle. In this Review, we provide an updated summary of the SARS-CoV-2 life cycle, gene function and virus–host interactions, including recent landmark findings on general aspects of coronavirus biology and newly discovered host factors necessary for virus replication.

Bovine dendritic-cell subsets can be readily identified in flow cytometry by staining for Flt3, CD4 and CD13. While this gating strategy could be confirmed by bulk RNA-seq of subsets sorted from peripheral blood mononuclear cells, it remains to be determined if the same gating strategy can be applied to gate on cDC1, cDC2 and pDC within lymphoid and non-lymphoid tissues. With this preprint we aim to inform on the current status of phenotyping experiments performed on Flt3+ cells in mesenteric lymph nodes, spleen and lung of cattle. Distinct DC subsets can be gated in all tissues based on CD4 and CD13, with high frequencies of Flt3+CD13+ cells (putative cDC1) detected in the lung. Further phenotyping performed on lymph node cells (in particular CADM1, CD26 and CD123) support the subset classification analogous to blood, with pDC being Flt3+CD4+CD13- and cDC1 being Flt3+CD4-CD13+. Heterogeneous expression of CADM1 and CD26 on Flt3+CD4-CD13- putative cDC2 warrants further investigations. ### Competing Interest Statement The authors have declared no competing interest.

Setting up a global SARS-CoV-2 surveillance system requires an understanding of how virus isolation and propagation practices, use of animal or human sera, and different neutralisation assay platforms influence assessment of SARS-CoV-2 antigenicity. In this study, with the contribution of 15 independent laboratories across all WHO regions, we carried out a controlled analysis of neutralisation assay platforms using the first WHO International Standard for antibodies to SARS-CoV-2 variants of concern (source: NIBSC). Live virus isolates (source: WHO BioHub or individual labs) or spike plasmids (individual labs) for pseudovirus production were used to perform neutralisation assays using the same serum panels. When comparing fold drops, excellent data consistency was observed across the labs using common reagents, including between pseudovirus and live virus neutralisation assays (RMSD of data from mean fold drop was 0.59). Utilising a Bayesian model, geometric mean titres and assay titre magnitudes (offsets) can describe the data efficiently. Titre magnitudes were seen to vary largely even for labs within the same assay group. We have observed that overall, live Microneutralisation assays tend to have the lowest titres, whereas Pseudovirus Neutralisation have the highest (with a mean difference of 3.2 log2 units between the two). These findings are relevant for laboratory networks, such as the WHO Coronavirus Laboratory Network (CoViNet), that seek to support a global surveillance system for evolution and antigenic characterisation of variants to support monitoring of population immunity and vaccine composition policy.

Vaccines are the most effective measure against COVID-19. However, novel and highly efficacious vaccines with simplified administration and broad immunogenicity, providing systemic and mucosal immunity are needed. Here, we show the development of live-attenuated vaccines (LAV) based on (i) recoding the SARS-CoV-2 genome to enrich for "one-to-stop" (OTS) codons, (ii) facilitating host responses by disabling non-structural-protein-1 (Nsp1) mediated translational repression, and deletion of open reading frames (ORF) 6, 7ab and 8, and (iii) deleting the spike polybasic cleavage site (PCS) to reduce LAV replication in the lung and prevent vaccine shedding. The OTS-modified SARS-CoV-2 LAV is adjustable regarding the level of attenuation, particularly vulnerable to mutagenic antiviral drugs, and protects efficiently against wild-type (WT) SARS-CoV-2 and recent variants of concern (VOC) in K18-hACE2 transgenic mice and Syrian hamsters. Furthermore, LAV immunization results in faster virus clearance after SARS-CoV-2 challenge compared to mRNA vaccines, a complete block of transmission of WT SARS-CoV-2, and significantly reduced transmission of Omicron BA.2 and BA.5 variants. Overall, the newly developed OTS-based LAVs represent a new generation of live vaccines that are intranasal administered at the natural site of infection, provide efficient and innovative infection control, and conceptionally are readily applicable to many other emerging viruses.

Dendritic and monocytic cells co-operate to initiate and shape adaptive immune responses in secondary lymphoid tissue. The complexity of this system is poorly understood, also because of the high phenotypic and functional plasticity of monocytic cells. We have sequenced mononuclear phagocytes in mesenteric lymph nodes (LN) of three adult cows at the single-cell level, revealing ten dendritic-cell (DC) clusters and seven monocyte/macrophage clusters with clearly distinct transcriptomic profiles. Among DC, we defined LN-resident subsets and their progenitors, as well as subsets of highly activated migratory DC differing in transcript levels for T-cell attracting chemokines. Our analyses also revealed a potential differentiation path for cDC2, resulting in a cluster of inflammatory cDC2 with close transcriptional similarity to putative DC3 and monocyte-derived DC. Monocytes and macrophages displayed sub-clustering mainly driven by pro- or anti-inflammatory expression signatures, including a small cluster of cycling, presumably self-renewing, macrophages. With this transcriptomic snapshot of LN-derived mononuclear phagocytes, we reveal functional properties and differentiation trajectories in a "command center of immunity", and identify elements that are conserved across species.

Variant of concern (VOC) Omicron-BA.1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and animal models are urgently needed. Here, we characterize Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in hamsters, ferrets and hACE2-expressing mice, and immunized hACE2-mice. We demonstrate a spike-mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In hamsters, Delta shows dominance over Omicron-BA.1, and in ferrets Omicron-BA.1 infection is abortive. In hACE2-knock-in mice, Delta and a Delta spike clone also show dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in naïve K18-hACE2 mice, we observe Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of replication and pathogenicity. Finally, the Omicron-BA.1 spike clone is less well-controlled by mRNA-vaccination in K18-hACE2-mice and becomes more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance.

Similar to human monocytes, bovine monocytes can be split into CD14 high CD16 - classical, CD14 high CD16 high intermediate and CD14 -/dim CD16 high nonclassical monocytes (cM, intM, and ncM, respectively). Here, we present an in-depth analysis of their steady-state bulk- and single-cell transcriptomes, highlighting both pronounced functional specializations and transcriptomic relatedness. Bulk gene transcription indicates pro-inflammatory and antibacterial roles of cM, while ncM and intM appear to be specialized in regulatory/anti-inflammatory functions and tissue repair, as well as antiviral responses and T-cell immunomodulation. Notably, intM stood out by high expression of several genes associated with antigen presentation. Anti-inflammatory and antiviral functions of ncM are further supported by dominant oxidative phosphorylation and selective strong responses to TLR7/8 ligands, respectively. Moreover, single-cell RNA-seq revealed previously unappreciated heterogeneity within cM and proposes intM as a transient differentiation intermediate between cM and ncM.

Two years into the COVID-19 pandemic there is still a need for vaccines to effectively control the spread of novel SARS-CoV-2 variants and associated cases of severe disease. Here we report a messenger RNA vaccine directly encoding for a nanoparticle displaying 60 receptor binding domains (RBDs) of SARS-CoV-2 that acts as a highly effective antigen. A construct encoding the RBD of the Delta variant elicits robust neutralizing antibody response, and also provides protective immunity against the Delta variant in a widely used transgenic mouse model. We ultimately find that the proposed mRNA RBD nanoparticle-based vaccine provides a flexible platform for rapid development and will likely be of great value in combatting current and future SARS-CoV-2 variants of concern.

Lung-resident (LR) mesenchymal stem and stromal cells (MSCs) are key elements of the alveolar niche and fundamental regulators of homeostasis and regeneration. We interrogated their function during virus-induced lung injury using the highly prevalent respiratory syncytial virus (RSV) which causes severe outcomes in infants. We applied complementary approaches with primary pediatric LR-MSCs and a state-of-the-art model of human RSV infection in lamb. Remarkably, RSV-infection of pediatric LR-MSCs led to a robust activation, characterized by a strong antiviral and pro-inflammatory phenotype combined with mediators related to T cell function. In line with this, following in vivo infection, RSV invades and activates LR-MSCs, resulting in the expansion of the pulmonary MSC pool. Moreover, the global transcriptional response of LR-MSCs appears to follow RSV disease, switching from an early antiviral signature to repair mechanisms including differentiation, tissue remodeling, and angiogenesis. These findings demonstrate the involvement of LR-MSCs during virus-mediated acute lung injury and may have therapeutic implications.

Similar to human monocytes, bovine monocytes can be split into CD14 + CD16 − classical and CD14 − CD16 + nonclassical monocytes (cM and ncM, respectively). Here, we present an in-depth analysis of their steady-state transcriptomes, highlighting pronounced functional specializations. Gene transcription indicates that pro-inflammatory and antibacterial processes are associated with cM, while ncM appear to be specialized in regulatory/anti-inflammatory functions and tissue repair, as well as antiviral responses and T-cell immunomodulation. In support of these functional differences, we found that oxidative phosphorylation prevails in ncM, whereas cM are clearly biased towards aerobic glycolysis. Furthermore, bovine monocyte subsets differed in their responsiveness to TLR ligands, supporting an antiviral role of ncM. Taken together, these data clearly indicate a variety of subset-specific functions in cM and ncM that are likely to be transferable to monocyte subsets of other species, including humans.

Dendritic cells (DC) and monocytes are vital for the initiation of innate and adaptive immune responses. Recently, we identifiedbona fideDC subsets in blood of cattle, revealing subset- and species-specific transcription of toll-like receptors (TLR). In the present study, we analyzed phenotypic and transcriptional responses of bovine DC subsets and monocytes to in vitro stimulation with four to six different TLR ligands. Bovine DC subsets, especially plasmacytoid DC (pDC), showed a clear increase of CCR7, CD25, CD40, CD80, CD86, and MHC-II expression both on mRNA and protein level. Flow cytometric detection of p38 MAPK phosphorylation 15 min after stimulation confirmed activation of DC subsets and monocytes in accordance with TLR gene expression. Whole-transcriptome sequencing of sorted and TLR-stimulated subsets revealed potential ligand- and subset-specific regulation of genes associated with inflammation, T-cell co-stimulation, migration, metabolic reprogramming, and antiviral activity. Gardiquimod was found to evoke strong responses both in DC subsets and monocytes, while Poly(I:C) and CpG preferentially triggered responses in cDC1 and pDC, respectively. This in-depth analysis of ligand responsiveness is essential for the rational design of vaccine adjuvants in cattle, and provides a solid basis for comparative studies on DC and monocyte biology across species.

New vaccine formulations that include novel strains of Mycoplasma hyopneumoniae and innovative adjuvants designed to induce cellular immunity could improve vaccine efficacy against this pathogen. The aim of this experimental study was to assess the efficacy of three experimental bacterin formulations based on M. hyopneumoniae field strain F7.2C which were able to induce cellular immunity. The formulations included a cationic liposome formulation with the Mincle receptor ligand trehalose 6,6-dibehenate (Lipo_DDA:TDB), a squalene-in-water emulsion with Toll-like receptor (TLR) ligands targeting TLR1/2, TLR7/8 and TLR9 (SWE_TLR), and a poly(lactic-co-glycolic acid) micro-particle formulation with the same TLR ligands (PLGA_TLR). Four groups of 12 M. hyopneumoniae-free piglets were primo- (day (D) 0; 39 days of age) and booster vaccinated (D14) intramuscularly with either one of the three experimental bacterin formulations or PBS. The pigs were endotracheally inoculated with a highly and low virulent M. hyopneumoniae strain on D28 and D29, respectively, and euthanized on D56. The main efficacy parameters were: respiratory disease score (RDS; daily), macroscopic lung lesion score (D56) and log copies M. hyopneumoniae DNA determined with qPCR on bronchoalveolar lavage (BAL) fluid (D42, D56). All formulations were able to reduce clinical symptoms, lung lesions and the M. hyopneumoniae DNA load in the lung, with formulation SWE_TLR being the most effective (RDSD28-D56 -61.90%, macroscopic lung lesions -88.38%, M. hyopneumoniae DNA load in BAL fluid (D42) -67.28%). Further experiments raised under field conditions are needed to confirm these results and to assess the effect of the vaccines on performance parameters.

Regulatory B cells (Bregs) play a crucial role in immunological tolerance primarily through the production of IL-10 in many diseases including autoimmune disorders, allergy, infectious diseases, and cancer. To date, various Breg subsets with overlapping phenotypes have been identified. However, the roles of Bregs in Helicobacter infection are largely unknown. In the present study, we investigate the phenotype and function of Helicobacter - stimulated B cells. Our results demonstrate that Helicobacter fells - stimulated IL-10-producing B cells (Hf-stim-IL-10(+) B) are composed of B10 and Transitional 2 Marginal Zone Precursor (T2-MZP) cells with expression of CD9, Tim-1, and programmed death 1 (PD-1). On the other hand, Helicobacter fells-stimulated IL-10- non-producing B B (Hf-stim- IL-10(-) B) cells are mainly marginal zone (MZ) B cells that express PD-L1 and secrete TGF-beta, IL-6, and TNF-alpha, and IgM and IgG2b. Furthermore, we show that both Hf-stim-IL-10(+) B cells and Hf-stim-IL-10(-) B cells induce CD49b(+) LAG-3(+) Tr1 cells. Here, we describe a novel mechanism for PD-1/PD-L1- driven B cell-dependent Tr1 cell differentiation. Finally, we explore the capability of Hf-stim-IL-10(-) B cells to induce Th17 cell differentiation, which we find to be dependent on TGF-beta. Taken together, the current study demonstrates that Hf-stim- B cells induce Tr1 cells through the PD-1/PD-L1 axis and Th17 cells by secreting TGF-beta.

A clear-cut delineation of bovine bona fide dendritic cells (DC) from monocytes has proved challenging, given the high phenotypic and functional plasticity of these innate immune cells and the marked phenotypic differences between species. Here, we demonstrate that, based on expression of Flt3, CD172a, CD13, and CD4, a precise identification of bovine blood conventional DC type 1 and 2 (cDC1, cDC2), plasmacytoid DC (pDC), and monocytes is possible with cDC1 being Flt3+CD172adimCD13+CD4-, cDC2 being Flt3+CD172a+CD13-CD4-, pDC being Flt3+CD172adimCD13-CD4+, and monocytes being Flt3-CD172ahighCD13-CD4-. The phenotype of these subsets was characterized in further detail, and a subset-specific differential expression of CD2, CD5, CD11b, CD11c, CD14, CD16, CD26, CD62L, CD71, CD163, and CD205 was found. Subset identity was confirmed by transcriptomic analysis and subset-specific transcription of conserved key genes. We also sorted monocyte subsets based on their differential expression of CD14 and CD16. Classical monocytes (CD14+CD16-) clustered clearly apart from the two CD16+ monocyte subsets probably representing intermediate and non-classical monocytes described in human. The transcriptomic data also revealed differential gene transcription for molecules involved in antigen presentation, pathogen sensing, and migration, and therefore gives insights into functional differences between bovine DC and monocyte subsets. The identification of cell-type- and subset-specific gene transcription will assist in the quest for "marker molecules" that-when targeted by flow cytometry-will greatly facilitate research on bovine DC and monocytes. Overall, species comparisons will elucidate basic principles of DC and monocyte biology and will help to translate experimental findings from one species to another.