Respiratory syncytial virus nonstructural proteins 1 and 2 : exceptional disrupters of innate immune responses

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

Potent Single-Domain Antibodies that Arrest Respiratory Syncytial Virus Fusion Protein in its Prefusion State

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

Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state

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

Protection and mechanism of action of a novel human respiratory syncytial virus vaccine candidate based on the extracellular domain of small hydrophobic protein

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 FcRI and FcRIII. 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

New antibody-based prevention and treatment options for influenza

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

How RSV proteins join forces to overcome the host innate immune response

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

A vulnerable, membrane-proximal site in human respiratory syncytial virus F revealed by a prefusion-specific single-domain antibody

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 sites III and IV. This new VHH reveals a previously underappreciated membrane-proximal region sensitive to 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 has been 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

Recombinant influenza virus carrying the respiratory syncytial virus (RSV) F85-93 CTL epitope reduces RSV replication in mice

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-F85-93, that carries the RSV CD8(+) T cell epitope F85-93 in its neuraminidase stalk. F85-93-specific cytotoxic T lymphocytes (CTLs) were induced in mice after a single intranasal immunization with PR8/NA-F85-93 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-F85-93 virus infection. Treatment with anti-M2e antibodies reduced the infiltration of immune cells in the lungs upon PR8/NA-F85-93 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 F85-93 epitope

Protective efficacy of recombinant influenza hemagglutinin ectodomain fusions

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