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Principal Investigator: Ursula Buchholz
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2020
Award: $1,258,386
Funding agency: National Institute of Allergy and Infectious Diseases
We previously performed codon-pair deoptimization (CPD) of various open reading frames (ORFs) of RSV. This is done by rearranging codons using computer algorithms and de novo gene synthesis to increase the content of normally-underrepresented codon-pairs without changing amino acid coding or overall codon usage. CPD is still a new technique and is incompletely understood. It typically has the effect of attenuation. This is thought to be due primarily to reduced efficiency of translation, although this is controversial and other mechanisms may also contribute. In previous years, we produced four RSV mutants by CPD of various combinations of ORFs, and showed that these viruses indeed were attenuated and, unexpectedly, were temperature-sensitive. We also evaluated the genetic stability of two of these viruses under restrictive temperatures, identified potential de-attenuating mutations, and used this information to make a promising vaccine candidate bearing a CPD L polymerase ORF that had desirable feature of attenuation, immunogenicity, and genetic stability. CPD RSV strains have been licensed for development to Codagenix, Inc.
While CPD has been used mostly to generate live-attenuated vaccine candidates, the effects of the converse approach of generating codon pair optimized viruses are still largely unknown. To evaluate effects of codon pair optimization (CPO), we subjected various open reading frames (ORFs) in the RSV genome to CPO by increasing the content of codon pairs that are overrepresented in the human genome without changing overall codon usage and amino acid sequences. This has the potential to increase the expression of the encoded protein(s) and antigenic determinants, and could be useful to generate vaccine candidates with increased immunogenicity. Four viruses were made: Max A (with CPO of NS1, NS2, N, P, M, and SH ORFs), Max B (with CPO of G and F), Max L (with CPO of L), and Max FLC (with CPO of all ORFs except M2-1 and M2-2). Because of the possibility of increased viral replication, each CPO virus was attenuated by the inclusion of a codon deletion mutation (del1313) and a missense mutation (I1314L) in the L polymerase. We found that CPO had no effect on multicycle virus replication in vitro, temperature sensitivity, or specific infectivity. Max A and L, which in common had CPO of one or more ORFs of proteins of the polymerase complex, exhibited global increases in viral protein synthesis. Max B (with CPO of the major antigenic determinants of RSV, the G and F glycoproteins) exhibited decreased protein synthesis, and it alone had reduced single-cycle virus replication in vitro. All CPO RSVs exhibited marginal reductions in replication in mice and hamsters. Surprisingly, the CPO RSVs induced lower levels of serum RSV-neutralizing antibodies in hamsters. This reduced immunogenicity might reflect reduced viral replication and possibly also the decrease in CpG and UpA dinucleotides as immune stimulators. Overall, our study describes paradoxical effects of CPO of an RNA virus on viral replication and the adaptive humoral immune response.
Respiratory syncytial virus (RSV) infects and causes disease in infants and reinfects with reduced disease throughout life without significant antigenic change. In contrast, reinfection by influenza A virus (IAV) largely requires antigenic change. The adaptive immune response depends on antigen presentation by dendritic cells (DC), which may be too immature in young infants to induce a fully protective immune response against RSV reinfections. We therefore compared the ability of RSV and IAV to activate primary human cord blood (CB) and adult blood (AB) myeloid DC (mDC). While RSV and IAV infected with similar efficiencies, RSV poorly induced maturation and cytokine production in CB and AB mDC. This difference between RSV and IAV was more profound in CB mDC. While IAV activated CB mDC to some extent, RSV did not induce CB mDC to increase the maturation markers CD38 and CD86 or CCR7, which directs DC migration to lymphatic tissue. Low CCR7 surface expression was associated with high expression of CCR5, which keeps DC in inflamed peripheral tissues. To evaluate a possible inhibition by RSV, we subjected RSV-inoculated AB mDC to secondary IAV inoculation. While RSV-inoculated AB mDC responded to secondary IAV inoculation by efficiently upregulating activation markers and cytokine production, IAV-induced CCR5 downregulation was slightly inhibited in cells exhibiting robust RSV infection. Thus, suboptimal stimulation and weak and mostly reversible inhibition seem to be responsible for inefficient mDC activation by RSV. The inefficient mDC stimulation and immunological immaturity in young infants may contribute to reduced immune responses and incomplete protection against RSV reinfection.
Terms: <21+ years old><Adult><Adult Human><Age><Amino Acid Sequence><Amino Acids><Animal Model><Animal Models and Related Studies><Antigen Presentation><Antigenic Determinants><Attenuated><Attenuated Live Virus Vaccine><Back><Basic Research><Basic Science><Binding Determinants><Blood><Blood Reticuloendothelial System><Blood Serum><Body Tissues><C-C CKR-5><C-C CKR-5 Gene><C-C Chemokine Receptor Type 5><C-C Chemokine Receptor Type 5 Gene><CC Chemokine Receptor 5><CC-CKR-5><CC-CKR-5 Gene><CC-CKR5><CCCKR5><CCCKR5 Gene><CCR-5><CCR-5 Gene><CCR5><CCR5 Protein><CCR5 Receptors><CCR5 gene><CD195 Antigen><CD195 Antigen Gene><CD86><CD86 gene><CHEMR13><CHEMR13 Gene><CKR-5><CKR-5 Gene><CKR5><CKR5 Gene><CKR5 Receptors><CMKBR5><CMKBR5 Gene><CRADA><Cardiopulmonary><Cell Body><Cell Locomotion><Cell Migration><Cell Movement><Cells><Cellular Migration><Cellular Motility><Chemokine (C-C Motif) Receptor 5><Chemokine (C-C) Receptor 5><Chemokine (C-C) Receptor 5 Gene><Childhood><Clinical Research><Clinical Study><Code><Coding System><Codon><Codon Nucleotides><Complex><Computational algorithm><Cooperative Research and Development Agreement><Cord Blood><Cricetinae><DNA Molecular Biology><Deletion Mutation><Dendritic Cells><Development><Dinucleoside Phosphates><Disease><Disorder><Dorsum><Down-Regulation><Downregulation><Elderly><Epitopes><Evaluation><Exhibits><Family member><Genetic><Genetic Alteration><Genetic Change><Genetic defect><Genome><Glycoproteins><Goals><HIV-1 Fusion Co-Receptor><HIV-1 Fusion Co-Receptor Gene><Hamsters><Hamsters Mammals><Human><Human Genome><Human Metapneumovirus><Hydrophobicity><Immune><Immune response><Immunes><Immunobiology><Immunochemical Immunologic><Immunologic><Immunological><Immunological response><Immunologically><Immunologics><Immunophysiology><In Vitro><Individual><Infant><Influenza A><Influenza A virus><Influenza Viruses Type A><Influenzavirus A><Laboratory Study><Life><Live-attenuated Vaccine><Lymphatic Tissue><Lymphoid Tissue><M protein><Messenger RNA><Mice><Mice Mammals><Missense Mutation><Modern Man><Molecular Biology><Molecular Genetics><Morbidity><Morbidity - disease rate><Motility><Murine><Murine pneumonia virus><Mus><Mutation><Myelogenous><Myeloid><Non-Polyadenylated RNA><Non-structural Protein><Nonstructural Protein><Nucleoproteins><ORFs><Open Reading Frames><Orthomyxovirus Type A><Peripheral><Phosphoproteins><Play><Pneumonia Virus of Mice><Pneumovirus><Polymerase><Population><Primary Protein Structure><Production><Protein Biosynthesis><Protein Coding Region><Proteins><RNA><RNA Gene Products><RNA Viruses><RSV Vaccines><RSV infection><Reagent><Respiratory Syncytial Virus Infections><Respiratory Syncytial Virus Vaccines><Respiratory Tract Diseases><Respiratory syncytial virus><Ribonucleic Acid><Ribosomal Peptide Biosynthesis><Ribosomal Protein Biosynthesis><Ribosomal Protein Synthesis><Rodent><Rodentia><Rodents Mammals><Role><Secondary to><Serum><Surface><System><Techniques><Temperature><Tissues><Translations><Type A Influenza><Umbilical Cord Blood><Vaccine Clinical Trial><Vaccines><Variant><Variation><Veiled Cells><Viral><Viral Gene Products><Viral Gene Proteins><Viral Genome><Viral Proteins><Virus><Virus Replication><Work><adaptive immune response><adulthood><advanced age><ages><aminoacid><attenuation><base><cell motility><computer algorithm><cytokine><design><designing><developmental><dinucleotide><elders><fetal cord blood><gene synthesis><genome mutation><genome scale><genome-wide><genomewide><geriatric><glycoprotein G><host response><human whole genome><immunogenicity><immunoresponse><late life><later life><mRNA><member><model of animal><model organism><mortality><multiple myeloma M Protein><mutant><neutralizing antibody><older adult><older person><pathogen><pediatric><pre-clinical study><preclinical study><programs><protein sequence><protein synthesis><reverse genetics><senior citizen><social role><vaccine candidate><viral fitness><viral multiplication><viral replication><virus genome><virus multiplication><virus protein>