Subcellular Localizations of RIG-I, TRIM25, and MAVS Complexes.

The retinoic acid-inducible gene 1 (RIG-I) signaling pathway is essential for the recognition of viruses and the initiation of host interferon (IFN)-mediated antiviral responses. Once activated, RIG-I interacts with polyubiquitin chains generated by TRIM25 and binds mitochondrial antiviral signaling protein (MAVS), leading to the production of type I IFN. We now show specific interactions among these key partners in the RLR pathway through the use of bimolecular fluorescence complementation (BiFC) and super-resolution microscopy. Dimers of RIG-I, TRIM25, and MAVS localize into different compartments. Upon activation, we show that TRIM25 is redistributed into cytoplasmic dots associated with stress granules, while RIG-I associates with TRIM25/stress granules and with mitochondrial MAVS. In addition, MAVS competes with TRIM25 for RIG-I binding, and this suggests that upon TRIM25-mediated activation of RIG-I, RIG-I moves away from TRIM25 to interact with MAVS at the mitochondria. For the first time, the distribution of these three proteins was analyzed at the same time in virus-infected cells. We also investigated how specific viral proteins modify some of the protein complexes in the pathway. The protease NS3/4A from hepatitis C virus redistributes the complexes RIG-I/MAVS and MAVS/MAVS but not RIG-I/TRIM25. In contrast, the influenza A virus NS1 protein interacts with RIG-I and TRIM25 in specific areas in the cell cytoplasm and inhibits the formation of TRIM25 homocomplexes but not the formation of RIG-I/TRIM25 heterocomplexes, preventing the formation of RIG-I/MAVS complexes. Thus, we have localized spatially in the cell different complexes formed between RIG-I, TRIM25, and MAVS, in the presence or absence of two viral IFN antagonistic proteins. IMPORTANCE: The first line of defense against viral infections is the innate immune response. Viruses are recognized by pathogen recognition receptors, such as the RIG-I like receptor family, that activate a signaling cascade that induces IFN production. In the present study, we visualized, for the first time in cells, both in overexpression and endogenous levels, complexes formed among key proteins involved in this innate immune signaling pathway. Through different techniques we were able to analyze how these proteins are distributed and reorganized spatially within the cell in order to transmit the signal, leading to an efficient antiviral state. In addition, this work presents a new means by how, when, and where viral proteins can target these pathways and act against the host immune system in order to counteract the activation of the immune response.

A Sendai virus-derived RNA agonist of RIG-I as a virus vaccine adjuvant.

The innate immune system is responsible for recognizing invading pathogens and initiating a protective response. In particular, the retinoic acid-inducible gene 1 protein (RIG-I) participates in the recognition of single- and double-stranded RNA viruses. RIG-I activation leads to the production of an appropriate cytokine and chemokine cocktail that stimulates an antiviral state and drives the adaptive immune system toward an efficient and specific response against the ongoing infection. One of the best-characterized natural RIG-I agonists is the defective interfering (DI) RNA produced by Sendai virus strain Cantell. This 546-nucleotide RNA is a well-known activator of the innate immune system and an extremely potent inducer of type I interferon. We designed an in vitro-transcribed RNA that retains the type I interferon stimulatory properties, and the RIG-I affinity of the Sendai virus produced DI RNA both in vitro and in vivo. This in vitro-synthesized RNA is capable of enhancing the production of anti-influenza virus hemagglutinin (HA)-specific IgG after intramuscular or intranasal coadministration with inactivated H1N1 2009 pandemic vaccine. Furthermore, our adjuvant is equally effective at increasing the efficiency of an influenza A/Puerto Rico/8/34 virus inactivated vaccine as a poly(I·C)- or a squalene-based adjuvant. Our in vitro-transcribed DI RNA represents an excellent tool for the study of RIG-I agonists as vaccine adjuvants and a starting point in the development of such a vaccine.

Hemagglutinin stalk-based universal vaccine constructs protect against group 2 influenza A viruses.

Current influenza virus vaccines contain H1N1 (phylogenetic group 1 hemagglutinin), H3N2 (phylogenetic group 2 hemagglutinin), and influenza B virus components. These vaccines induce good protection against closely matched strains by predominantly eliciting antibodies against the membrane distal globular head domain of their respective viral hemagglutinins. This domain, however, undergoes rapid antigenic drift, allowing the virus to escape neutralizing antibody responses. The membrane proximal stalk domain of the hemagglutinin is much more conserved compared to the head domain. In recent years, a growing collection of antibodies that neutralize a broad range of influenza virus strains and subtypes by binding to this domain has been isolated. Here, we demonstrate that a vaccination strategy based on the stalk domain of the H3 hemagglutinin (group 2) induces in mice broadly neutralizing anti-stalk antibodies that are highly cross-reactive to heterologous H3, H10, H14, H15, and H7 (derived from the novel Chinese H7N9 virus) hemagglutinins. Furthermore, we demonstrate that these antibodies confer broad protection against influenza viruses expressing various group 2 hemagglutinins, including an H7 subtype. Through passive transfer experiments, we show that the protection is mediated mainly by neutralizing antibodies against the stalk domain. Our data suggest that, in mice, a vaccine strategy based on the hemagglutinin stalk domain can protect against viruses expressing divergent group 2 hemagglutinins.

Model of influenza A virus infection: dynamics of viral antagonism and innate immune response.

Viral antagonism of host responses is an essential component of virus pathogenicity. The study of the interplay between immune response and viral antagonism is challenging due to the involvement of many processes acting at multiple time scales. Here we develop an ordinary differential equation model to investigate the early, experimentally measured, responses of human monocyte-derived dendritic cells to infection by two H1N1 influenza A viruses of different clinical outcomes: pandemic A/California/4/2009 and seasonal A/New Caledonia/20/1999. Our results reveal how the strength of virus antagonism, and the time scale over which it acts to thwart the innate immune response, differs significantly between the two viruses, as is made clear by their impact on the temporal behavior of a number of measured genes. The model thus sheds light on the mechanisms that underlie the variability of innate immune responses to different H1N1 viruses.

COVA1-18 neutralizing antibody protects against SARS-CoV-2 in three preclinical models.

One year into the Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), effective treatments are still needed 1-3 . Monoclonal antibodies, given alone or as part of a therapeutic cocktail, have shown promising results in patients, raising the hope that they could play an important role in preventing clinical deterioration in severely ill or in exposed, high risk individuals 4-6 . Here, we evaluated the prophylactic and therapeutic effect of COVA1-18 in vivo , a neutralizing antibody isolated from a convalescent patient 7 and highly potent against the B.1.1.7. isolate 8,9 . In both prophylactic and therapeutic settings, SARS-CoV-2 remained undetectable in the lungs of COVA1-18 treated hACE2 mice. Therapeutic treatment also caused a dramatic reduction in viral loads in the lungs of Syrian hamsters. When administered at 10 mg kg - 1 one day prior to a high dose SARS-CoV-2 challenge in cynomolgus macaques, COVA1-18 had a very strong antiviral activity in the upper respiratory compartments with an estimated reduction in viral infectivity of more than 95%, and prevented lymphopenia and extensive lung lesions. Modelling and experimental findings demonstrate that COVA1-18 has a strong antiviral activity in three different preclinical models and could be a valuable candidate for further clinical evaluation.

Extrapulmonary tissue responses in cynomolgus macaques (Macaca fascicularis) infected with highly pathogenic avian influenza A (H5N1) virus.

The mechanisms responsible for virulence of influenza viruses in humans remain poorly understood. A prevailing hypothesis is that the highly pathogenic virus isolates cause a severe cytokinemia precipitating acute respiratory distress syndrome and multiple organ dysfunction syndrome. Cynomolgus macaques (Macaca fascicularis) infected with a human highly pathogenic avian influenza (HPAI) H5N1 virus isolate (A/Vietnam/1203/2004) or reassortants of human influenza virus A/Texas/36/91 (H1N1) containing genes from the 1918 pandemic influenza A (H1N1) virus developed severe pneumonia within 24 h postinfection. However, virus spread beyond the lungs was only detected in the H5N1 group, and signs of extrapulmonary tissue reactions, including microglia activation and sustained up-regulation of inflammatory markers, most notably hypoxia inducible factor-1alpha (HIF-1alpha), were largely limited to this group. Extrapulmonary pathology may thus contribute to the morbidities induced by H5N1 viruses.

Genetically engineered Newcastle disease virus for malignant melanoma therapy.

Despite the advances in cancer therapies in the past century, malignant melanoma continues to present a significant clinical challenge due to lack of chemotherapeutic response. Systemic therapy with immunostimulatory agents such as interferon and interleukin-2 (IL-2) has shown some promise, though each is associated with significant side effects. Over the past 50 years, oncolytic Newcastle disease virus (NDV) has emerged as an alternative candidate for cancer therapy. The establishment of reverse-genetics systems for the virus has allowed us to further manipulate the virus to enhance its oncolytic activity. Introduction of immunomodulatory molecules, especially IL-2, into the NDV genome was shown to enhance the oncolytic potential of the virus in a murine syngeneic colon carcinoma model. We hypothesize that a recombinant NDV expressing IL-2 would be an effective agent for therapy of malignant melanoma. We show that recombinant NDV possesses a strong cytolytic activity against multiple melanoma cell lines, and is effective in clearing established syngeneic melanoma tumors in mice. Moreover, introduction of murine IL-2 into NDV significantly enhanced its activity against syngeneic melanomas, resulting in increased overall animal survival and generation of antitumor immunity. These findings warrant further investigations of IL-2-expressing NDV as an antimelanoma agent in humans.

Influenza A viruses with truncated NS1 as modified live virus vaccines: pilot studies of safety and efficacy in horses.

REASONS FOR PERFORMING STUDY: Three previously described NS1 mutant equine influenza viruses encoding carboxy-terminally truncated NS1 proteins are impaired in their ability to inhibit type I IFN production in vitro and are replication attenuated, and thus are candidates for use as a modified live influenza virus vaccine in the horse. HYPOTHESIS: One or more of these mutant viruses is safe when administered to horses, and recipient horses when challenged with wild-type influenza have reduced physiological and virological correlates of disease. METHODS: Vaccination and challenge studies were done in horses, with measurement of pyrexia, clinical signs, virus shedding and systemic proinflammatory cytokines. RESULTS: Aerosol or intranasal inoculation of horses with the viruses produced no adverse effects. Seronegative horses inoculated with the NS1-73 and NS1-126 viruses, but not the NS1-99 virus, shed detectable virus and generated significant levels of antibodies. Following challenge with wild-type influenza, horses vaccinated with NS1-126 virus did not develop fever (>38.5 degrees C), had significantly fewer clinical signs of illness and significantly reduced quantities of virus excreted for a shorter duration post challenge compared to unvaccinated controls. Mean levels of proinflammatory cytokines IL-1beta and IL-6 were significantly higher in control animals, and were positively correlated with peak viral shedding and pyrexia on Day +2 post challenge. CONCLUSION AND CLINICAL RELEVANCE: These data suggest that the recombinant NS1 viruses are safe and effective as modified live virus vaccines against equine influenza. This type of reverse genetics-based vaccine can be easily updated by exchanging viral surface antigens to combat the problem of antigenic drift in influenza viruses.

The virus battles: IFN induction of the antiviral state and mechanisms of viral evasion.

Response to IFN involves a rapid and direct signal transduction mechanism that quickly reports that presence of extracellular cytokine to the cell nucleus, preserving the specificity inherent in cytokine-receptor interactions to transcriptionally induce expression of a set of genes encoding important antiviral proteins. Establishment of the resulting antiviral state provides a crucial initial line of defense against viral infection. Studies of IFN-deficient cells and animals derived by gene targeting have demonstrated the essential nature of IFN-mediated innate immunity. The long co-evolutionary history of viruses with their hosts has seen the development of a variety of evasive adaptations that allow viruses to circumvent or inactivate host antiviral mechanisms. Further understanding of both host and viral components of this battle may provide important new strategies for vaccine development and creation of novel antiviral compounds.

Antitumor properties of influenza virus vectors.

We are investigating the potential use of influenza virus vectors expressing selected tumor-associated antigens (TAAs) as therapeutic agents in anticancer strategies. Previously, we have shown that recombinant influenza viruses expressing a model TAA mediated the regression of established pulmonary metastases in mice through the induction of cytotoxic T-cell responses (N. P. Restifo et al., Virology, 249: 89-97, 1998). We have now expanded these observations in the mouse model using survival as the end point of the assay. Animals with a high tumor burden showed extended survival times when treated with a recombinant influenza virus expressing a TAA, but they finally succumbed to death. Death was associated with the presence of a small number of large tumors in lungs. Interestingly, these tumors were found to express undetectable levels of the TAAs because of a down-regulation in the TAA-specific mRNA levels. On the other hand, mice with five times lower tumor burden showed complete tumor regression and survival for >6 six months when treated with the recombinant virus. These animals showed protection against a tumor challenge 6 months after treatment. Our results suggest that recombinant influenza viruses may be useful as therapeutic agents for the prevention and treatment of cancers with known TAAs.

A genetically engineered influenza A virus with ras-dependent oncolytic properties.

The NS1 protein of influenza virus is a virulence factor that counteracts the PKR-mediated antiviral response by the host. As a consequence, influenza NS1 gene knockout virus delNS1 (an influenza A virus lacking the NS1 open reading frame) fails to replicate in normal cells but produces infectious particles in PKR-deficient cells. Because it is known that oncogenic ras induces an inhibitor of PKR, we addressed the question of whether the delNS1 virus selectively replicates in cells expressing oncogenic ras. We show that upon transfection and expression of oncogenic N-ras, cells become permissive for productive delNS1 virus replication, suggesting that the delNS1 virus has specific oncolytic properties. Viral growth in the oncogenic ras-transfected cells is associated with a reduction of PKR activation during infection. Moreover, treatment of s.c. established N-ras-expressing melanomas in severe combined immunodeficiency mice with the delNS1 virus revealed that this virus has tumor-ablative potentials. The delNS1 virus does not replicate in nonmalignant cell lines such as melanocytes, keratinocytes, or endothelial cells. The apathogenic nature of the delNS1 virus combined with the selective replication properties of this virus in oncogenic ras-expressing cells renders this virus an attractive candidate for the therapy of tumors with an activated ras-signaling pathway.

Proteins of Newcastle disease virus envelope: interaction between the outer hemagglutinin-neuraminidase glycoprotein and the inner non-glycosylated matrix protein.

Using linear sucrose-density ultracentrifugation analysis of Triton-solubilized Newcastle Disease Virus envelopes, we have evidenced, for the first time, the existence of interactions between the outer hemagglutinin-neuraminidase transmembrane glycoprotein and the inner non-glycosylated peripheral matrix protein. Such interactions seem to be electrostatic. These conclusions are based on the behavior of both proteins at different ionic strengths. When in low ionic strength buffer, hemagglutinin-neuraminidase and matrix proteins band together in the sucrose gradient, whereas at high ionic strength both proteins band at different rates in the gradient. The behavior of the inner matrix protein in our conditions was the expected one for a peripheral protein. The results of these 'in vitro' studies are also discussed in terms of the possible 'in vivo' role of such interactions.

Identification and characterization of viral antagonists of type I interferon in negative-strand RNA viruses.

Interferons are cytokines secreted in response to viral infections with potent antiviral activity, and they represent a critical component of the innate immune response against viruses. It has now become apparent that many viruses have evolved different mechanisms to counteract the interferon response, allowing their efficient replication and propagation in their hosts. This review discusses how the development of reverse genetics techniques and the increase in our knowledge of the interferon response have led to the discovery of interferon-antagonistic functions of different genes of viruses belonging to the negative-strand RNA virus group. In many cases, these viral genes encode accessory pro- teins that are not required for viral infectivity but are critical for optimal replication and for virulence in the host.

Negative-strand RNA viruses: applications to biotechnology.

The establishment of reverse-genetics techniques to manipulate the genome of negative-strand RNA viruses has contributed enormously to a better understanding of the replication mechanisms and pathogenicity of this group of viruses. The generation of recombinant viruses bearing specific mutations in the coding and noncoding regions of their genomic RNAs now allows the functions in the replicative cycle of specific RNA regions and protein domains of these viruses to be studied. In addition, recombinant negative-strand RNA viruses can now be designed to have specific properties that make them attractive biotechnological tools.

The cytoplasmic tail of the neuraminidase protein of influenza A virus does not play an important role in the packaging of this protein into viral envelopes.

We have rescued a transfectant influenza virus, NA/TAIL(-), whose neuraminidase (NA) protein lacks the predicted cytoplasmic tail. The virus was attenuated (one log10 reduction) both in tissue culture and in mouse lungs. Attenuation correlated with a 50% reduction of the level of NA in infected cells and levels of incorporation of the tail-less NA protein into viral particles paralleled that in infected cells. This result indicates that the signal for packaging of the NA protein into the viral envelope is not located in its cytoplasmic domain.

Increased influenza A virus sialidase activity with N-acetyl-9-O-acetylneuraminic acid-containing substrates resulting from influenza C virus O-acetylesterase action.

Influenza virus type C (Johannesburg/1/66) was used as a source for the enzyme O-acetylesterase (EC 3.1.1.53) with several natural sialoglycoconjugates as substrates. The resulting products were immediately employed as substrates using influenza virus type A [(Singapore/6/86) (H1N1) or Shanghai/11/87 (H3N2)] as a source for sialidase (neuraminidase, EC 3.2.1.18). A significant increase in the percentage of sialic acid released was found when the O-acetyl group was cleaved by O-acetylesterase activity from certain substrates (bovine submandibular gland mucin, rat serum glycoproteins, human saliva glycoproteins, mouse erythrocyte stroma, chick embryonic brain gangliosides and bovine brain gangliosides). A common feature of all these substrates is that they contain N-acetyl-9-O-acetylneuraminic acid residues. By contrast, no significant increase in the release of sialic acid was detected when certain other substrates could not be de-O-acetylated by the action of influenza C esterase, either because they lacked O-acetylsialic acid (human glycophorin A, alpha 1-acid glycoprotein from human serum, fetuin and porcine submandibular gland mucin) or because the 4-O-acetyl group was scarcely cleaved by the viral O-acetylesterase (equine submandibular gland mucin). The biological significance of these facts is discussed, relative to the infective capacity of influenza C virus.

DNA immunization of newborn mice with a plasmid-expressing nucleoprotein of influenza virus.

Lower responsiveness and higher susceptibility to tolerance are the two main characteristics of neonatal immunity that limit the efficacy of conventional vaccines administered during this period. Based on the fact that DNA immunization of adult organisms is able to generate protective immune responses, we investigated the ability of a plasmid-(NPV1) encoding nucleoprotein (NP) of A/PR8/34 strain of influenza virus to generate a cellular immune response following intramuscular delivery in neonates. Newborn mice immunized with NPV1 plasmid developed significant cytotoxic immunity, comparable to the immune response displayed by adult mice injected with the same dose of plasmid. Furthermore, mice infected with influenza virus 1 month after completion of immunization showed a significant decrease of virus lung titer between day 3 and 7 after challenge, consistent with the protectivity conferred by specific cytotoxic immunity. Thus, mice immunized as neonates with NPV1 plasmid developed a protective cellular immune response, like the adult mice. Therefore, the strategy of DNA immunization may be considered for the purpose of human vaccination to prevent horizontally and vertically transmitted life-threatening infections in infants or children.

Learning from our foes: a novel vaccine concept for influenza virus.

Concerted efforts to study the molecular biology of influenza viruses and the ability to genetically engineer them have dramatically advanced our understanding of the functions of influenza viral genes and gene products. The only nonstructural protein (NS1) coded for by the influenza virus was shown to possess interferon antagonist activity and thus to play an important role in countering the interferon (antiviral) response of the host following infection. Influenza A and B virus mutants with "weak" anti-interferon activity are highly attenuated because the host is able to mount an effective interferon response. It is suggested that these NS1-modified attenuated influenza viruses can induce a protective immune response and that they are ideal live virus vaccine candidates against influenza.

Chimeric animal and plant viruses expressing epitopes of outer membrane protein F as a combined vaccine against Pseudomonas aeruginosa lung infection.

Outer membrane protein F of Pseudomonas aeruginosa has vaccine efficacy against infection by P. aeruginosa as demonstrated in a variety of animal models. Through the use of synthetic peptides, three surface-exposed epitopes have been identified. These are called peptides 9 (aa 261-274 in the mature F protein, TDAYNQKLSERRAN), 10 (aa 305-318, NATAEGRAINRRVE), and 18 (aa 282-295, NEYGVEGGRVNAVG). Both the peptide 9 and 10 epitopes are protective when administered as a vaccine. In order to develop a vaccine that is suitable for use in humans, including infants with cystic fibrosis, the use of viral vector systems to present the protective epitopes has been investigated. An 11-amino acid portion of epitope 10 (AEGRAINRRVE) was successfully inserted into the antigenic B site of the hemagglutinin on the surface of influenza virus. This chimeric influenza virus protects against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Attempts to derive a chimeric influenza virus carrying epitope 9 have been unsuccessful. A chimeric plant virus, cowpea mosaic virus (CPMV), with epitopes 18 and 10 expressed in tandem on the large coat protein subunit (CPMV-PAE5) was found to elicit antibodies that reacted exclusively with the 10 epitope and not with epitope 18. Use of this chimeric virus as a vaccine afforded protection against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Chimeric CPMVs with a single peptide containing epitopes 9 and 18 expressed on either of the coat proteins are in the process of being evaluated. Epitope 9 was successfully expressed on the coat protein of tobacco mosaic virus (TMV), and this chimeric virus is protective when used as a vaccine in the mouse model of chronic pulmonary infection. However, initial attempts to express epitope 10 on the coat protein of TMV have been unsuccessful. Efforts are continuing to construct chimeric viruses that express both the 9 and 10 epitopes in the same virus vector system. Ideally, the use of a vaccine containing two epitopes of protein F is desirable in order to greatly reduce the likelihood of selecting a variant of P. aeruginosa that escapes protective antibodies in immunized humans via a mutation in a single epitope within protein F. When the chimeric influenza virus containing epitope 10 and the chimeric TMV containing epitope 9 were given together as a combined vaccine, the immunized mice produced antibodies directed toward both epitopes 9 and 10. The combined vaccine afforded protection against challenge with P. aeruginosa in the chronic pulmonary infection model at approximately the same level of efficacy as provided by the individual chimeric virus vaccines. These results prove in principle that a combined chimeric viral vaccine presenting both epitopes 9 and 10 of protein F has vaccine potential warranting continued development into a vaccine for use in humans.

Recombinant paramyxovirus type 1-avian influenza-H7 virus as a vaccine for protection of chickens against influenza and Newcastle disease.

Current vaccines to prevent avian influenza rely upon labor-intensive parenteral injection. A more advantageous vaccine would be capable of administration by mass immunization methods such as spray or water vaccination. A recombinant vaccine (rNDV-AIV-H7) was constructed by using a lentogenic paramyxovirus type 1 vector (Newcastle disease virus [NDV] B1 strain) with insertion of the hemagglutinin (HA) gene from avian influenza virus (AIV) A/chicken/NY/13142-5/94 (H7N2). The recombinant virus had stable insertion and expression of the H7 AIV HA gene as evident by detection of HA expression via immunofluorescence in infected Vero cells. The rNDV-AIV-H7 replicated in 9-10 day embryonating chicken eggs and exhibited hemagglutinating activity from both NDV and AI proteins that was inhibited by antisera against both NDV and AIV H7. Groups of 2-week-old white Leghorn chickens were vaccinated with transfectant NDV vector (tNDV), rNDV-AIV-H7, or sterile allantoic fluid and were challenged 2 weeks later with viscerotropic velogenic NDV (vvNDV) or highly pathogenic (HP) AIV. The sham-vaccinated birds were not protected from vvNDV or HP AIV challenge. The transfectant NDV vaccine provided 70% protection for NDV challenge but did not protect against AIV challenge. The rNDV-AIV-H7 vaccine provided partial protection (40%) from vvNDV and HP AIV challenge. The serologic response was examined in chickens that received one or two immunizations of the rNDV-AIV-H7 vaccine. Based on hemagglutination inhibition and enzyme-linked immunosorbent assay (ELISA) tests, chickens that received a vaccine boost seroconverted to AIV H7, but the serologic response was weak in birds that received only one vaccination. This demonstrates the potential for NDV for use as a vaccine vector in expressing AIV proteins.