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To prevent infection by respiratory viruses and consequently limit virus circulation, vaccines need to promote mucosal immunity. The extent to which the currently used messenger RNA (mRNA)-based COVID-19 vaccines induce mucosal immunity remains poorly characterized. We evaluated mucosal neutralizing antibody responses in a cohort of 183 individuals. Participants were sampled at several time points after primary adenovirus vector-based or mRNA-based COVID-19 vaccination and after mRNA-based booster vaccinations. Our findings revealed that repeated vaccination with mRNA boosters promoted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies in nasal secretions. Nasal and serum neutralizing antibody titers of both IgG and IgA isotypes correlated to one another. We investigated the source of these mucosal antibodies in a mouse model wherein mice received repeated mRNA vaccines for SARS-CoV-2. These experiments indicated that neutralizing antibody-producing cells reside in the spleen and bone marrow, whereas no proof of tissue homing to the respiratory mucosa was observed, despite the detection of mucosal antibodies. Serum transfer experiments confirmed that circulating antibodies were able to migrate to the respiratory mucosa. Collectively, these results demonstrate that, especially upon repeated vaccination, the currently used COVID-19 mRNA vaccines can elicit mucosal neutralizing antibodies and that vaccination might also stimulate mucosal immunity induced by previous SARS-CoV-2 infection. Moreover, migration of circulating antibodies to the respiratory mucosa might be a main mechanism. These findings advance our understanding of mRNA vaccine-induced immunity and have implications for the design of vaccine strategies to combat respiratory infections.

Background: SARS-CoV-2-neutralizing antibodies (nABs) showed great promise in the early phases of the COVID-19 pandemic. The emergence of resistant strains, however, quickly rendered the majority of clinically approved nABs ineffective. This underscored the imperative to develop nAB cocktails targeting non-overlapping epitopes., Methods: Undertaking a nAB discovery program, we employed a classical workflow, while integrating artificial intelligence (AI)-based prediction to select non-competing nABs very early in the pipeline. We identified and in vivo validated (in female Syrian hamsters) two highly potent nABs., Findings: Despite the promising results, in depth cryo-EM structural analysis demonstrated that the AI-based prediction employed with the intention to ensure non-overlapping epitopes was inaccurate. The two nABs in fact bound to the same receptor-binding epitope in a remarkably similar manner., Interpretation: Our findings indicate that, even in the Alphafold era, AI-based predictions of paratope-epitope interactions are rough and experimental validation of epitopes remains an essential cornerstone of a successful nAB lead selection., Funding: Full list of funders is provided at the end of the manuscript., Competing Interests: Declaration of interests Ghent University has filed for patent protection on the antibody sequences described herein, and D.D.A., M.W., R.W., W.W., S.G. and L.V. are named as co-inventors on this patent (European Patent Application: 21186206.5). A.P. is employee of the MAbSilico, H.R. holds a patent regarding neutralizing VHH antibodies binding the Spike RBD (PCT/EP2021/052885) and has filed a priority application for neutralizing VHH antibodies binding Spike S2 (EP 23160838.1). X.S. is a recipient of FWO research project COVID-19 (G0G4920N) and FWO-FNRS project VIREOS (EOS ID: 30981113) grants., (Copyright © 2023. Published by Elsevier B.V.)

Addressing the elusive specificity of cysteine cathepsins, which in contrast to caspases and trypsin-like proteases lack strict specificity determining P1 pocket, calls for innovative approaches. Proteomic analysis of cell lysates with human cathepsins K, V, B, L, S, and F identified 30,000 cleavage sites, which we analyzed by software platform SAPS-ESI (Statistical Approach to Peptidyl Substrate-Enzyme Specific Interactions). SAPS-ESI is used to generate clusters and training sets for support vector machine learning. Cleavage site predictions on the SARS-CoV-2 S protein, confirmed experimentally, expose the most probable first cut under physiological conditions and suggested furin-like behavior of cathepsins. Crystal structure analysis of representative peptides in complex with cathepsin V reveals rigid and flexible sites consistent with analysis of proteomics data by SAPS-ESI that correspond to positions with heterogeneous and homogeneous distribution of residues. Thereby support for design of selective cleavable linkers of drug conjugates and drug discovery studies is provided., (© 2023. The Author(s).)

Human respiratory syncytial virus (RSV) is the leading cause of severe acute lower respiratory tract infections in infants worldwide. Nonstructural protein NS1 of RSV modulates the host innate immune response by acting as an antagonist of type I and type III interferon (IFN) production and signaling in multiple ways. Likely, NS1 performs this function by interacting with different host proteins. In order to obtain a comprehensive overview of the NS1 interaction partners, we performed three complementary protein-protein interaction screens, i.e., BioID, MAPPIT, and KISS. To closely mimic a natural infection, the BioID proximity screen was performed using a recombinant RSV in which the NS1 protein is fused to a biotin ligase. Remarkably, MED25, a subunit of the Mediator complex, was identified in all three performed screening methods as a potential NS1-interacting protein. We confirmed the interaction between MED25 and RSV NS1 by coimmunoprecipitation, not only upon overexpression of NS1 but also with endogenous NS1 during RSV infection. We also demonstrate that the replication of RSV can be enhanced in MED25 knockout A549 cells, suggesting a potential antiviral role of MED25 during RSV infection. Mediator subunits function as transcriptional coactivators and are involved in transcriptional regulation of their target genes. Therefore, the interaction between RSV NS1 and cellular MED25 might be beneficial for RSV during infection by affecting host transcription and the host immune response to infection. IMPORTANCE Innate immune responses, including the production of type I and III interferons, play a crucial role in the first line of defense against RSV infection. However, only a poor induction of type I IFNs is observed during RSV infection, suggesting that RSV has evolved mechanisms to prevent type I IFN expression by the infected host cell. A unique RSV protein, NS1, is largely responsible for this effect, probably through interaction with multiple host proteins. A better understanding of the interactions that occur between RSV NS1 and host proteins may help to identify targets for an effective antiviral therapy. We addressed this question by performing three complementary protein-protein interaction screens and identified MED25 as an RSV NS1-interacting protein. We propose a role in innate anti-RSV defense for this Mediator complex subunit.

Respiratory syncytial virus (RSV) is the leading cause of severe acute lower respiratory tract infections in infants worldwide. Although several pattern recognition receptors (PRRs) can sense RSV-derived pathogen-associated molecular patterns (PAMPs), infection with RSV is typically associated with low to undetectable levels of type I interferons (IFNs). Multiple RSV proteins can hinder the host's innate immune response. The main players are NS1 and NS2 which suppress type I IFN production and signalling in multiple ways. The recruitment of innate immune cells and the production of several cytokines are reduced by RSV G. Next, RSV N can sequester immunostimulatory proteins to inclusion bodies (IBs). N might also facilitate the assembly of a multiprotein complex that is responsible for the negative regulation of innate immune pathways. Furthermore, RSV M modulates the host's innate immune response. The nuclear accumulation of RSV M has been linked to an impaired host gene transcription, in particular for nuclear-encoded mitochondrial proteins. In addition, RSV M might also directly target mitochondrial proteins which results in a reduced mitochondrion-mediated innate immune recognition of RSV. Lastly, RSV SH might prolong the viral replication in infected cells and influence cytokine production.

Broadly neutralizing antibodies are an important treatment for individuals with coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Antibody-based therapeutics are also essential for pandemic preparedness against future Sarbecovirus outbreaks. Camelid-derived single domain antibodies (VHHs) exhibit potent antimicrobial activity and are being developed as SARS-CoV-2–neutralizing antibody-like therapeutics. Here, we identified VHHs that neutralize both SARS-CoV-1 and SARS-CoV-2, including now circulating variants. We observed that the VHHs bound to a highly conserved epitope in the receptor binding domain of the viral spike protein that is difficult to access for human antibodies. Structure-guided molecular modeling, combined with rapid yeast-based prototyping, resulted in an affinity enhanced VHH-human immunoglobulin G1 Fc fusion molecule with subnanomolar neutralizing activity. This VHH-Fc fusion protein, produced in and purified from cultured Chinese hamster ovary cells, controlled SARS-CoV-2 replication in prophylactic and therapeutic settings in mice expressing human angiotensin converting enzyme 2 and in hamsters infected with SARS-CoV-2. These data led to affinity-enhanced selection of the VHH, XVR011, a stable anti–COVID-19 biologic that is now being evaluated in the clinic.

ISG15 is an interferon-stimulated, ubiquitin-like protein that can conjugate to substrate proteins (ISGylation) to counteract microbial infection, but the underlying mechanisms remain elusive. Here, we use a virus-like particle trapping technology to identify ISG15-binding proteins and discover Ring Finger Protein 213 (RNF213) as an ISG15 interactor and cellular sensor of ISGylated proteins. RNF213 is a poorly characterized, interferon-induced megaprotein that is frequently mutated in Moyamoya disease, a rare cerebrovascular disorder. We report that interferon induces ISGylation and oligomerization of RNF213 on lipid droplets, where it acts as a sensor for ISGylated proteins. We show that RNF213 has broad antimicrobial activity in vitro and in vivo, counteracting infection with Listeria monocytogenes, herpes simplex virus 1, human respiratory syncytial virus and coxsackievirus B3, and we observe a striking co-localization of RNF213 with intracellular bacteria. Together, our findings provide molecular insights into the ISGylation pathway and reveal RNF213 as a key antimicrobial effector., (© 2021. The Author(s).)

In current seasonal influenza vaccines, neutralizing antibody titers directed against the hemagglutinin surface protein are the primary correlate of protection. These vaccines are, therefore, quantitated in terms of their hemagglutinin content. Adding other influenza surface proteins, such as neuraminidase and M2e, to current quadrivalent influenza vaccines would likely enhance vaccine efficacy. However, this would come with increased manufacturing complexity and cost. To address this issue, as a proof of principle, we have designed genetic fusions of hemagglutinin ectodomains from H3 and H1 influenza A subtypes. These recombinant H1-H3 hemagglutinin ectodomain fusions could be transiently expressed at high yield in mammalian cell culture using Expi293F suspension cells. Fusions were trimeric, and as stable in solution as their individual trimeric counterparts. Furthermore, the H1-H3 fusion constructs were antigenically intact based on their reactivity with a set of conformation-specific monoclonal antibodies. H1-H3 hemagglutinin ectodomain fusion immunogens, when formulated with the MF59 equivalent adjuvant squalene-in-water emulsion (SWE), induced H1 and H3-specific humoral immune responses equivalent to those induced with an equimolar mixture of individually expressed H1 and H3 ectodomains. Mice immunized with these ectodomain fusions were protected against challenge with heterologous H1N1 (Bel/09) and H3N2 (X-31) mouse-adapted viruses with higher neutralizing antibody titers against the H1N1 virus. Use of such ectodomain-fused immunogens would reduce the number of components in a vaccine formulation and allow for the inclusion of other protective antigens to increase influenza vaccine efficacy.

Human respiratory syncytial virus (RSV) is a major cause of lower respiratory tract disease, especially in young children and the elderly. The fusion protein (F) exists in a pre- and postfusion conformation and is the main target of RSV-neutralizing antibodies. Highly potent RSV-neutralizing antibodies typically bind sites that are unique to the prefusion conformation of F. In this study we screened a single-domain antibody (VHH) library derived from a llama immunized with prefusion-stabilized F and identified a prefusion F-specific VHH that can neutralize RSV A at subnanomolar concentrations. Structural analysis revealed that this VHH primarily binds to antigenic site I while also making contacts with residues in antigenic site III and IV. This new VHH reveals a previously underappreciated membrane-proximal region sensitive for neutralization. Importance RSV is an important respiratory pathogen. This study describes a prefusion F-specific VHH that primarily binds to antigenic site I of RSV F. This is the first time that a prefusion F-specific antibody that binds this site is reported. In general, antibodies that bind to site I are poorly neutralizing, whereas the VHH described here neutralizes RSV A at subnanomolar concentrations. Our findings contribute to insights into the RSV F antigenic map., (Copyright © 2021 American Society for Microbiology.)

Human respiratory syncytial virus (RSV) is the most important cause of acute lower respiratory tract disease in infants worldwide. As a first line of defense against respiratory infections, innate immune responses, including the production of type I and III interferons (IFNs), play an important role. Upon infection with RSV, multiple pattern recognition receptors (PRRs) can recognize RSV-derived pathogen-associated molecular patterns (PAMPs) and mount innate immune responses. Retinoic-acid-inducible gene-I (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) have been identified as important innate receptors to mount type I IFNs during RSV infection. However, type I IFN levels remain surprisingly low during RSV infection despite strong viral replication. The poor induction of type I IFNs can be attributed to the cooperative activity of 2 unique, nonstructural (NS) proteins of RSV, i.e., NS1 and NS2. These viral proteins have been shown to suppress both the production and signaling of type I and III IFNs by counteracting a plethora of key host innate signaling proteins. Moreover, increasing numbers of IFN-stimulated genes (ISGs) are being identified as targets of the NS proteins in recent years, highlighting an underexplored protein family in the identification of NS target proteins. To understand the diverse effector functions of NS1 and NS2, Goswami and colleagues proposed the hypothesis of the NS degradasome (NSD) complex, a multiprotein complex made up of, at least, NS1 and NS2. Furthermore, the crystal structure of NS1 was resolved recently and, remarkably, identified NS1 as a structural paralogue of the RSV matrix protein. Unfortunately, no structural data on NS2 have been published so far. In this review, we briefly describe the PRRs that mount innate immune responses upon RSV infection and provide an overview of the various effector functions of NS1 and NS2. Furthermore, we discuss the ubiquitination effector functions of NS1 and NS2, which are in line with the hypothesis that the NSD shares features with the canonical 26S proteasome., Competing Interests: The authors have declared that no competing interests exist.

The antigenic diversity of human influenza viruses represents a challenge to the development of vaccines with durable immune protection. In addition, small molecule anti-influenza viral drugs can bring clinical relief to influenza patients but the emergence of drug resistant viruses can rapidly limit the effectiveness of such drugs. In the past decade, a number of human monoclonal antibodies have been described that can bind to and neutralize a broad range of influenza A and B viruses. Most of these monoclonal antibodies are directed against the viral hemagglutinin (HA) stalk and some have now been evaluated in early to mid-stage clinical trials. An important conclusion from these clinical studies is that hemagglutinin stalk-specific antibodies are safe and can reduce influenza symptoms. In addition, examples of bi- and multi-specific anti-influenza antibodies are discussed, although such antibodies have not yet progressed into clinical testing. In the future, antibody-based therapies might become part of our arsenal to prevent and treat influenza., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This corrects the article DOI: 10.1038/ncomms14158.

Human respiratory syncytial virus (RSV) is the main cause of lower respiratory tract infections in young children. The RSV fusion protein (F) is highly conserved and is the only viral membrane protein that is essential for infection. The prefusion conformation of RSV F is considered the most relevant target for antiviral strategies because it is the fusion-competent form of the protein and the primary target of neutralizing activity present in human serum. Here, we describe two llama-derived single-domain antibodies (VHHs) that have potent RSV-neutralizing activity and bind selectively to prefusion RSV F with picomolar affinity. Crystal structures of these VHHs in complex with prefusion F show that they recognize a conserved cavity formed by two F protomers. In addition, the VHHs prevent RSV replication and lung infiltration of inflammatory monocytes and T cells in RSV-challenged mice. These prefusion F-specific VHHs represent promising antiviral agents against RSV.

Infections with human respiratory syncytial virus (HRSV) occur globally in all age groups and can have devastating consequences in young infants. We demonstrate that a vaccine based on the extracellular domain (SHe) of the small hydrophobic (SH) protein of HRSV, reduced viral replication in challenged laboratory mice and in cotton rats. We show that this suppression of viral replication can be transferred by serum and depends on a functional IgG receptor compartment with a major contribution of FcγRI and FcγRIII. Using a conditional cell depletion method, we provide evidence that alveolar macrophages are involved in the protection by SHe-specific antibodies. HRSV-infected cells abundantly express SH on the cell surface and are likely the prime target of the humoral immune response elicited by SHe-based vaccination. Finally, natural infection of humans and experimental infection of mice or cotton rats does not induce a strong immune response against HRSV SHe. Using SHe as a vaccine antigen induces immune protection against HRSV by a mechanism that differs from the natural immune response and from other HRSV vaccination strategies explored to date. Hence, HRSV vaccine candidates that aim at inducing protective neutralizing antibodies or T-cell responses could be complemented with a SHe-based antigen to further improve immune protection., (© 2014 The Authors. Published under the terms of the CC BY 4.0 license.)

Human respiratory syncytial virus (HRSV) was first discovered in the 1950s, but despite decades of research, a licensed vaccine against it is not available. Epidemiological studies indicate that antibodies directed against the fusion protein (F) partially correlate with protection. In addition, an F-specific monoclonal antibody is licensed as a prophylactic treatment in children who are at high risk of developing complications following HRSV infection. Therefore, most HRSV-oriented vaccination strategies focus on inducing a humoral immune response against F. In the quest for the development of a safe HRSV vaccine, the induction of a T cell immune response has received a lot less attention. T cell immunity directed against HRSV has not been associated unequivocally with protection against HRSV and CD4(+) T helper cell responses may even worsen disease due to HRSV. However, many studies support a protective role for CD8(+) T cells in clearance of HRSV from the lungs. In this review we highlight the clinical and experimental evidence in favor of a CD8(+) T lymphocyte-based vaccination strategy to protect against HRSV. First, we describe how T cell responses and T cell memory are induced in the lungs upon respiratory viral infection. HRSV has evolved mechanisms that hamper CD8(+) T cell priming and effector functions. We appraise the information on HRSV-specific CD8(+) T cell immunity gained from laboratory mouse studies, taking into account the advantages and limitations of this animal model and, where possible, the accordance with clinical evidence. Finally, we focus on recent efforts to develop T cell based vaccines against HRSV., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants worldwide. Despite decades of research, there is still no registered vaccine available for this major pathogen. We investigated the protective efficacy of a recombinant influenza virus, PR8/NA-F(85-93), that carries the RSV CD8(+) T cell epitope F(85-93) in its neuraminidase stalk. F(85-93)-specific cytotoxic T lymphocytes (CTLs) were induced in mice after a single intranasal immunization with PR8/NA-F(85-93) virus, and these CTLs provided a significant reduction in the lung viral load upon a subsequent challenge with RSV. To avoid influenza-induced morbidity, we treated mice with matrix protein 2 (M2e)-specific monoclonal antibodies before PR8/NA-F(85-93) virus infection. Treatment with anti-M2e antibodies reduced the infiltration of immune cells in the lungs upon PR8/NA-F(85-93) infection, whereas the formation of inducible bronchus-associated lymphoid tissue was not affected. Moreover, this treatment prevented body weight loss yet still permitted the induction of RSV F-specific T cell responses and significantly reduced RSV replication upon challenge. These results demonstrate that it is possible to take advantage of the infection-permissive protection of M2e-specific antibodies against influenza A virus to induce heterologous CD8(+) T cell-mediated immunity by an influenza A virus vector expressing the RSV F(85-93) epitope.