ABSTRACT
BACKGROUND: Bivalent RSV prefusion F subunit vaccine (RSVpreF), comprised of equal quantities of stabilized prefusion F antigens from the major circulating subgroups (RSV A, RSV B), is licensed for prevention of RSV-associated lower respiratory tract illness (LRTI) in older adults and for maternal vaccination for prevention of RSV-associated LRTI in infants. To support licensure and large-scale manufacturing, this lot consistency study was conducted to demonstrate equivalence in immunogenicity across 3 RSVpreF lots. METHODS: This phase 3, multicenter, parallel-group, placebo-controlled, randomized (1:1:1:1), double-blind study evaluated immunogenicity, safety, and tolerability of RSVpreF in healthy 18-49-year-old adults. Participants received a single 120-µg injection of 1 of 3RSVpreF lots or placebo. Geometric mean ratio (GMR) of RSV serum 50 % neutralizing geometric mean titers obtained 1 month after vaccination were compared between each vaccine lot for RSV A and RSV B, separately. Equivalence between lots was defined using a 1.5-fold criterion (GMR 95 % CIs for every lot pair within the 0.667-1.5 interval). Safety and tolerability were assessed. RESULTS: Of 992participants vaccinated, 948 were included in the evaluable immunogenicity population. All 3 RSVpreF lots elicited strong immune responses, meeting the 1.5-fold equivalence criterion for all between-lot comparisons for both RSV A and RSV B. Across the 3 lots, RSV A and RSV B 50 % neutralizing geometric mean titers substantially increased from baseline (RSV A, 1671-1795; RSV B 1358-1429) to 1 month after RSVpreF vaccination (RSV A, 24,131-25,238; RSV B, 19,238-21,702), corresponding to ≥14-fold increases in 50 % neutralizing titers for both RSV A and RSV B from before to 1 month after vaccination. Single doses of RSVpreF were safe and well tolerated, with similar safety profiles across the 3 RSVpreF lots. CONCLUSIONS: These findings support the reproducibility of RSVpreF vaccine manufacturing with similar safety and reactogenicity profiles (NCT05096208).
Subject(s)
Antibodies, Viral , Immunogenicity, Vaccine , Respiratory Syncytial Virus Infections , Respiratory Syncytial Virus Vaccines , Respiratory Syncytial Virus, Human , Humans , Respiratory Syncytial Virus Vaccines/immunology , Respiratory Syncytial Virus Vaccines/administration & dosage , Respiratory Syncytial Virus Vaccines/adverse effects , Female , Male , Adult , Double-Blind Method , Young Adult , Adolescent , Antibodies, Viral/blood , Middle Aged , Respiratory Syncytial Virus Infections/prevention & control , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus, Human/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Vaccines, Subunit/immunology , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/adverse effects , Healthy Volunteers , Vaccination/methods , Viral Fusion Proteins/immunologyABSTRACT
Human respiratory syncytial virus (RSV) continues to be the leading viral cause of severe acute lower respiratory tract disease in infants and children worldwide. A licensed vaccine or antiviral drug suitable for routine use remains unavailable. Like RSV, Murine pneumonia virus (MPV) is a member of the genus Orthopneumovirus, family Pneumoviridae Humans are not normally exposed to MPV, and MPV is not cross-protective with RSV. We evaluated MPV as an RSV vaccine vector expressing the RSV fusion (F) glycoprotein. The RSV F open reading frame (ORF) was codon optimized, and the encoded RSV F protein was made identical to an early passage of RSV strain A2. The RSV F ORF was placed under the control of MPV transcription signals and inserted at the first (rMPV-F1), third (rMPV-F3), or fourth (rMPV-F4) gene position of a version of the MPV genome that contained a codon-pair-optimized polymerase (L) gene. The recovered viruses replicated in vitro as efficiently as the empty vector, with stable expression of RSV F protein. Replication and immunogenicity of rMPV-F1 and rMPV-F3 were evaluated in rhesus macaques following intranasal and intratracheal administration. Both viruses replicated at low levels in the upper and lower respiratory tracts, maintained stable RSV F expression, and induced RSV-neutralizing serum antibodies at high levels similar to those induced by wild-type RSV replicating to a 5- to 25-fold-higher titer. In conclusion, this study demonstrated that rMPV provides a highly attenuated yet immunogenic vector for the expression of RSV F protein, with potential application in RSV-naive and RSV-experienced populations.IMPORTANCE Human respiratory syncytial virus (RSV) is an important human pathogen that lacks a licensed vaccine or antiviral drug suitable for routine use. We describe here the evaluation of recombinant murine pneumonia virus (rMPV) as a live-attenuated vector that expresses the RSV F protein, the major RSV neutralization antigen, as an experimental RSV vaccine. The rMPV-RSV-F vectors expressing RSV F from the first, third, or fourth gene position were genetically stable and were not restricted for replication in vitro In contrast, the vectors exhibited highly attenuated replication in the respiratory tract of rhesus macaques, maintained stable RSV F expression, and induced RSV-neutralizing serum antibodies at high titers similar to those conferred by wild-type RSV. Given the lack of preexisting immunity to MPV in humans and the lack of cross-neutralization and cross-protection between MPV and RSV, an rMPV-vectored RSV vaccine should be immunogenic in both RSV-naive children and RSV-experienced adults.
Subject(s)
Murine pneumonia virus/genetics , Respiratory Syncytial Virus Vaccines/immunology , Respiratory Syncytial Virus, Human/genetics , Viral Fusion Proteins/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Antibodies, Viral/immunology , Chlorocebus aethiops , Genetic Vectors , Humans , Macaca mulatta , Mice , Murine pneumonia virus/immunology , Murine pneumonia virus/metabolism , Respiratory Syncytial Virus Vaccines/administration & dosage , Respiratory Syncytial Virus Vaccines/genetics , Respiratory Syncytial Virus, Human/immunology , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunology , Vero Cells , Viral Fusion Proteins/genetics , Virus ReplicationABSTRACT
MDA5 is a cytosolic sensor of double-stranded RNA (ds)RNA including viral byproducts and intermediates. We studied a child with life-threatening, recurrent respiratory tract infections, caused by viruses including human rhinovirus (HRV), influenza virus, and respiratory syncytial virus (RSV). We identified in her a homozygous missense mutation in IFIH1 that encodes MDA5. Mutant MDA5 was expressed but did not recognize the synthetic MDA5 agonist/(ds)RNA mimic polyinosinic-polycytidylic acid. When overexpressed, mutant MDA5 failed to drive luciferase activity from the IFNB1 promoter or promoters containing ISRE or NF-κB sequence motifs. In respiratory epithelial cells or fibroblasts, wild-type but not knockdown of MDA5 restricted HRV infection while increasing IFN-stimulated gene expression and IFN-ß/λ. However, wild-type MDA5 did not restrict influenza virus or RSV replication. Moreover, nasal epithelial cells from the patient, or fibroblasts gene-edited to express mutant MDA5, showed increased replication of HRV but not influenza or RSV. Thus, human MDA5 deficiency is a novel inborn error of innate and/or intrinsic immunity that causes impaired (ds)RNA sensing, reduced IFN induction, and susceptibility to the common cold virus.
Subject(s)
Interferon-Induced Helicase, IFIH1/genetics , Mutation , Picornaviridae Infections/genetics , Picornaviridae Infections/virology , Rhinovirus/physiology , Antiviral Agents/pharmacology , Base Sequence , Cells, Cultured , Child, Preschool , DNA Mutational Analysis/methods , Female , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/virology , Gene Expression/drug effects , Genes, Recessive/genetics , Heterozygote , Homozygote , Host-Pathogen Interactions , Humans , Interferon-Induced Helicase, IFIH1/deficiency , Interferons/pharmacology , Male , PedigreeABSTRACT
Human respiratory syncytial virus (RSV) is an enveloped RNA virus that is the most important viral cause of acute pediatric lower respiratory tract illness worldwide, and lacks a vaccine or effective antiviral drug. The involvement of host factors in the RSV replicative cycle remains poorly characterized. A genome-wide siRNA screen in human lung epithelial A549 cells identified actin-related protein 2 (ARP2) as a host factor involved in RSV infection. ARP2 knockdown did not reduce RSV entry, and did not markedly reduce gene expression during the first 24 hr of infection, but decreased viral gene expression thereafter, an effect that appeared to be due to inhibition of viral spread to neighboring cells. Consistent with reduced spread, there was a 10-fold reduction in the release of infectious progeny virions in ARP2-depleted cells at 72 hr post-infection. In addition, we found that RSV infection induced filopodia formation and increased cell motility in A549 cells and that this phenotype was ARP2 dependent. Filopodia appeared to shuttle RSV to nearby uninfected cells, facilitating virus spread. Expression of the RSV F protein alone from a plasmid or heterologous viral vector in A549 cells induced filopodia, indicating a new role for the RSV F protein, driving filopodia induction and virus spread. Thus, this study identified roles for ARP2 and filopodia in RSV-induced cell motility, RSV production, and RSV cell-to-cell spread.
Subject(s)
Actin-Related Protein 2/metabolism , Pseudopodia/virology , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Viruses/pathogenicity , A549 Cells , Blotting, Western , Flow Cytometry , Gene Knockdown Techniques , Humans , Microscopy, Confocal , Microscopy, Electron, Transmission , Pseudopodia/ultrastructure , Real-Time Polymerase Chain Reaction , Virus InternalizationABSTRACT
Human respiratory syncytial virus (RSV) is the most important viral agent of serious pediatric respiratory-tract disease worldwide. A vaccine or generally effective antiviral drug is not yet available. We designed new live attenuated RSV vaccine candidates by codon-pair deoptimization (CPD). Specifically, viral ORFs were recoded by rearranging existing synonymous codons to increase the content of underrepresented codon pairs. Amino acid coding was completely unchanged. Four CPD RSV genomes were designed in which the indicated ORFs were recoded: Min A (NS1, NS2, N, P, M, and SH), Min B (G and F), Min L (L), and Min FLC (all ORFs except M2-1 and M2-2). Surprisingly, the recombinant CPD viruses were temperature-sensitive for replication in vitro (level of sensitivity: Min FLC > Min L > Min B > Min A). All of the CPD mutants grew less efficiently in vitro than recombinant wild-type (WT) RSV, even at the typically permissive temperature of 32 °C (growth efficiency: WT > Min L > Min A > Min FLC > Min B). CPD of the ORFs for the G and F surface glycoproteins provided the greatest restrictive effect. The CPD viruses exhibited a range of restriction in mice and African green monkeys comparable with that of two attenuated RSV strains presently in clinical trials. This study provided a new type of attenuated RSV and showed that CPD can rapidly generate vaccine candidates against nonsegmented negative-strand RNA viruses, a large and expanding group that includes numerous pathogens of humans and animals.
Subject(s)
Codon/genetics , Genome, Viral/genetics , Respiratory Syncytial Virus, Human/genetics , Animals , Cells, Cultured , Chlorocebus aethiops , Humans , Mice, Inbred BALB C , Molecular Sequence Data , Nasopharynx/virology , RNA, Viral/metabolism , Recombination, Genetic/genetics , Respiratory Syncytial Virus, Human/immunology , Respiratory Syncytial Virus, Human/pathogenicity , Temperature , Vero Cells , Viral Proteins/metabolism , Virus ReplicationABSTRACT
UNLABELLED: The cytokine storm is an intensified, dysregulated, tissue-injurious inflammatory response driven by cytokine and immune cell components. The cytokine storm during influenza virus infection, whereby the amplified innate immune response is primarily responsible for pulmonary damage, has been well characterized. Now we describe a novel event where virus-specific T cells induce a cytokine storm. The paramyxovirus pneumonia virus of mice (PVM) is a model of human respiratory syncytial virus (hRSV). Unexpectedly, when C57BL/6 mice were infected with PVM, the innate inflammatory response was undetectable until day 5 postinfection, at which time CD8(+) T cells infiltrated into the lung, initiating a cytokine storm by their production of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Administration of an immunomodulatory sphingosine-1-phosphate (S1P) receptor 1 (S1P1R) agonist significantly inhibited PVM-elicited cytokine storm by blunting the PVM-specific CD8(+) T cell response, resulting in diminished pulmonary disease and enhanced survival. IMPORTANCE: A dysregulated overly exuberant immune response, termed a "cytokine storm," accompanies virus-induced acute respiratory diseases (VARV), is primarily responsible for the accompanying high morbidity and mortality, and can be controlled therapeutically in influenza virus infection of mice and ferrets by administration of sphingosine-1-phosphate 1 receptor (S1P1R) agonists. Here, two novel findings are recorded. First, in contrast to influenza infection, where the cytokine storm is initiated early by the innate immune system, for pneumonia virus of mice (PVM), a model of RSV, the cytokine storm is initiated late in infection by the adaptive immune response: specifically, by virus-specific CD8 T cells via their release of IFN-γ and TNF-α. Blockading these cytokines with neutralizing antibodies blunts the cytokine storm and protects the host. Second, PVM infection is controlled by administration of an S1P1R agonist.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cytokines/immunology , Disease Models, Animal , Murine pneumonia virus , Pneumovirus Infections/immunology , Respiratory Syncytial Viruses , Animals , Antibodies/administration & dosage , Cytokines/metabolism , Flow Cytometry , Immunoglobulin G , Interferon-gamma/immunology , Lung/immunology , Mice , Mice, Inbred C57BL , Receptors, Lysosphingolipid/agonists , Sphingosine-1-Phosphate Receptors , Tumor Necrosis Factor-alpha/immunologyABSTRACT
UNLABELLED: Human metapneumovirus (HMPV) is a major etiologic agent of respiratory disease worldwide. HMPV reinfections are common in healthy adults and children, suggesting that the protective immune response to HMPV is incomplete and short-lived. We used gene-deletion viruses to evaluate the role of the attachment G and small hydrophobic SH glycoproteins on virus uptake by primary human monocyte-derived dendritic cells (MDDC) in vitro and on subsequent MDDC maturation and activation of autologous T cells. HMPV with deletion of G and SH (ΔSHG) exhibited increased infectivity but had little effect on MDDC maturation. However, MDDC stimulated with ΔSHG induced increased proliferation of autologous Th1-polarized CD4(+) T cells. This effect was independent of virus replication. Increased T cell proliferation was strictly dependent on contact between virus-stimulated MDDC and CD4(+) T cells. Confocal microscopy revealed that deletion of SH and G was associated with an increased number of immunological synapses between memory CD4(+) T cells and virus-stimulated MDDC. Uptake of HMPV by MDDC was found to be primarily by macropinocytosis. Uptake of wild-type (WT) virus was reduced compared to that of ΔSHG, indicative of inhibition by the SH and G glycoproteins. In addition, DC-SIGN-mediated endocytosis provided a minor alternative pathway that depended on SH and/or G and thus operated only for WT. Altogether, our results show that SH and G glycoproteins reduce the ability of HMPV to be internalized by MDDC, resulting in a reduced ability of the HMPV-stimulated MDDC to activate CD4(+) T cells. This study describes a previously unknown mechanism of virus immune evasion. IMPORTANCE: Human metapneumovirus (HMPV) is a major etiologic agent of respiratory disease worldwide. HMPV reinfections are common in healthy adults and children, suggesting that the protective immune response to HMPV is incomplete and short-lived. We found that HMPV attachment G and small hydrophobic SH glycoproteins reduce the ability of HMPV to be internalized by macropinocytosis into human dendritic cells (DC). This results in a reduced ability of the HMPV-stimulated DC to activate Th1-polarized CD4(+) T cells. These results contribute to a better understanding of the nature of incomplete protection against this important human respiratory virus, provide new information on the entry of HMPV into human cells, and describe a new mechanism of virus immune evasion.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Dendritic Cells/virology , Glycoproteins/immunology , Immune Evasion/immunology , Metapneumovirus/immunology , Pinocytosis/immunology , Retroviridae Proteins, Oncogenic/immunology , Viral Proteins/immunology , Analysis of Variance , Cell Adhesion Molecules/immunology , Dendritic Cells/immunology , Flow Cytometry , Humans , Immunological Synapses/immunology , Lectins, C-Type/immunology , Lymphocyte Activation/immunology , Metapneumovirus/genetics , Microscopy, Confocal , Receptors, Cell Surface/immunology , Virus InternalizationABSTRACT
BACKGROUND: Patients diagnosed with fulminant hepatic failure face high mortality rates. A potential therapeutic approach for these patients is the use of extracorporeal porcine liver perfusion, to serve as a form of "liver dialysis." Previously, our laboratory has shown that, during a 72-hour extracorporeal perfusion with human blood, porcine Kupffer cells bind to and phagocytose human erythrocytes causing the hematocrit to fall to 2.5% of the original value. Subsequently, erythrocyte binding has been shown to involve N-acetylneuraminic acid (Neu5Ac) on the surface of human erythrocytes and sialoadhesin on the surface of the porcine Kupffer cells. METHODS: Given that no primate other than the human is known to express the majority of its sialic acid as Neu5Ac, we evaluated whether nonhuman primates would provide adequate evaluation of the loss of erythrocytes that might be expected in a clinical trial of extracorporeal porcine liver perfusion. RESULTS: We found that while porcine macrophages readily bound human erythrocytes, binding of nonhuman primate erythrocytes was significantly reduced (P<0.001). CONCLUSIONS: This study suggests that nonhuman primates may fail to serve as an adequate model for studying extracorporeal porcine liver perfusion because of the fact that porcine macrophages do not bind nonhuman primate erythrocytes.
Subject(s)
Cell Membrane/metabolism , Disease Models, Animal , Mutation , N-Acetylneuraminic Acid/genetics , Transplantation, Heterologous/methods , Animals , Erythrocytes/cytology , Gene Expression , Humans , Kupffer Cells/cytology , Liver/metabolism , Macrophages/cytology , N-Acetylneuraminic Acid/chemistry , N-Acetylneuraminic Acid/metabolism , Oxygen/chemistry , Pan troglodytes , Perfusion , Phagocytosis , Primates , Sialic Acid Binding Ig-like Lectin 1/chemistry , Species Specificity , SwineABSTRACT
Pneumonia virus of mice (PVM), a relative of human respiratory syncytial virus (RSV), causes respiratory disease in mice. There is serologic evidence suggesting widespread exposure of humans to PVM. To investigate replication in primates, African green monkeys (AGM) and rhesus macaques (n = 4) were inoculated with PVM by the respiratory route. Virus was shed intermittently at low levels by a subset of animals, suggesting poor permissiveness. PVM efficiently replicated in cultured human cells and inhibited the type I interferon (IFN) response in these cells. This suggests that poor replication in nonhuman primates was not due to a general nonpermissiveness of primate cells or poor control of the IFN response. Seroprevalence in humans was examined by screening sera from 30 adults and 17 young children for PVM-neutralizing activity. Sera from a single child (6%) and 40% of adults had low neutralizing activity against PVM, which could be consistent with increasing incidence of exposure following early childhood. There was no cross-reaction of human or AGM sera between RSV and PVM and no cross-protection in the mouse model. In native Western blots, human sera reacted with RSV but not PVM proteins under conditions in which AGM immune sera reacted strongly. Serum reactivity was further evaluated by flow cytometry using unfixed Vero cells infected with PVM or RSV expressing green fluorescent protein (GFP) as a measure of viral gene expression. The reactivity of human sera against RSV-infected cells correlated with GFP expression, whereas reactivity against PVM-infected cells was low and uncorrelated with GFP expression. Thus, PVM specificity was not evident. Our results indicate that the PVM-neutralizing activity of human sera is not due to RSV- or PVM-specific antibodies but may be due to low-affinity, polyreactive natural antibodies of the IgG subclass. The absence of PVM-specific antibodies and restriction in nonhuman primates makes PVM unlikely to be a human pathogen.