RSV and HMPV are cytoplasmic enveloped RNA viruses of the paramyxovirus family. Their genomes are single strands of negative-sense RNA of 15.2 kb (RSV) and 13.3 kb (HMPV) that encode 10 mRNAs and 11 unique proteins (RSV) or 8 mRNAs and 9 unique proteins (HMPV). Each virus encodes a nucleoprotein N, phosphoprotein P, matrix protein M, small hydrophobic protein SH, major glycoprotein G, fusion glycoprotein F, M2-1 and M2-2 polymerase factors, and polymerase L. In addition, RSV encodes two nonstructural proteins NS1 and NS2. 
We have been working to develop attenuating mutations in RSV that have improved genetic and phenotypic stability, for the purpose of producing live-attenuated vaccine strains with increased stability. In one approach, we worked to create attenuating mutations by deleting one or several codons at a given locus in coding sequence in the viral genome, using known missense mutations as a guide. Most of the attempted codon-deletions proved to be lethal to the virus, but we found that deletion of codon 1313 in the L polymerase gene (del1313) resulted in a virus that replicated with wt-like efficiency at the permissive temperature of 32C but was restricted at 37C. In addition, it was restricted 50-fold and 150-fold in the upper and lower respiratory tract, respectively, of mice. We combined this del1313 mutation with the previously described attenuating NS2 gene deletion (delNS2) to produce the recombinant live-attenuated RSV vaccine candidate delNS2/del1313. During in vitro stress tests involving serial passage at incrementally increasing temperatures to evaluate genetic stability, a second-site compensatory mutation was detected in close proximity of del1313, namely I1314T. This site was genetically and phenotypically stabilized by an I1314L substitution. Combination of I1314L with delNS2/del1313 yielded genetic stability at physiological temperature. This stabilized vaccine candidate was moderately temperature-sensitive and had a level of restriction in experimental animals comparable to that of MEDI-559 and RSV cps2, two promising RSV vaccine candidate that presently are in or entering clinical trials. The level of attenuation and the genetic stability of delNS2/del1313 indicate that it is a promising candidate suitable for evaluation in pediatric phase I studies. This virus has been manufactured into a clinical lot and has been approved by the FDA for studies beginning this year in seropositive children 6-59 months of age. 
 We evaluated the strategy of codon-pair deoptimization (CPD) as a means of developing genetically and phenotypically stable attenuated RSV strains. It is well known that there is a bias in codon-pair usage in nature. Specifically, any given pair of amino acids has the possibility to be encoded by a variety of different combinations of synonymous codons due to the degeneracy of the genetic code, but the observed usage of codon-pairs typically is biased to favor a subset of the possible combinations. One factor in this bias is thought to be translational efficiency and accuracy, because certain combinations of tRNA pairs are favored at the A and P sites in the ribosome due to tRNA geometry and other factors. CPD involves the deliberate introduction of under-represented synonymous codon-pairs into numerous sites in protein-coding sequence to achieve sub-optimal expression. These substitutions only involve the ORFS, and thus non-protein-coding genome regions are not affected. Also, CPD involves only synonymous codon substitutions, and thus amino acid coding is unaffected and the antigens remain unchanged. In addition, CPD applied to one or several genes typically involves hundreds or thousands of nucleotide changes, and thus should be highly refractory to de-attenuation. Recently, CPD was applied to poliovirus and influenza virus and was shown to result in attenuated strains. We designed the following set of four CPD RSV genomes in which the indicated ORFs were recoded: (i) Min A;NS1, NS2, N, P, M, and SH (i.e., the left-hand third of the genome);(ii) Min B;G and F (located in the middle of the genome);(iii) Min L;L (located at the right-hand end of the genome);and (iv) Min FLC;all ORFs except M2-1 and M2-2. The recoded genome regions were synthesized commercially and the four CPD viruses were constructed and recovered by reverse genetics. All of the CPD viruses were temperature-sensitive (level of sensitivity: Min FLC>Min L>Min B>Min A) for replication in vitro. This was unexpected given the lack of change in amino acid coding, which was confirmed by re-sequencing. All of the CPD mutants grew less efficiently in vitro than wild type (wt) RSV, even at the permissive temperature of 32C (growth efficiency: wt>Min L>Min A>Min FLC>Min B). Thus, CDP of G and F ORFs provided the greatest effect. The CPD viruses exhibited a range of restriction in mice and African Green Monkeys (AGM) and induced immunity against wt RSV. This study identified new vaccine candidates for RSV and showed that CPD of a nonsegmented negative-strand RNA virus can rapidly generate vaccine candidates with a range of attenuation phenotypes. RSV infection results in the formation of viral inclusion bodies (IBs) that appear as large, prominent, electron-dense structures in the cytoplasm. IBs are thought to be sites of nucleocapsid accumulation and viral RNA synthesis. We found that, during RSV infection, the IBs also were the sites of major sequestration of two proteins involved in cellular signaling pathways. These are phosphorylated p38 mitogen-activated protein kinase (MAPK) (p38-P), a key regulator of cellular inflammatory and stress responses, and O-linked N-acetylglucosamine (OGN) transferase (OGT), an enzyme that catalyzes the post-translational addition of OGN to protein targets to regulate cellular processes including signal transduction, transcription, translation, proteasomal degradation, and the stress response. The virus-induced sequestration of p38-P in viral IBs resulted in a substantial reduction in the accumulation of a downstream signaling substrate, MAPK-activated protein kinase 2 (MK2). Sequestration of OGT in IBs was associated with suppression of stress granule (SG) formation. Thus, while the RSV IBs are thought to play an essential role in viral replication, the present results show that they also play a role in suppressing the cellular response to viral infection. The sequestration of p38-P and OGT in IBs appeared to be reversible: severe oxidative stress resulting from a brief arsenite treatment transformed large IBs into a scattering of smaller bodies, suggestive of partial disassembly, and this was associated with MK2 phosphorylation and OGN-addition. Unexpectedly, the RSV M2-1 protein was found to localize in SGs that formed during oxidative stress. This protein was previously shown to be a viral transcription elongation factor, and the present findings provide the first evidence of possible involvement in SG activities during RSV infection. This involvement is intriguing because the M2-1 protein has some structural similarity - including the presence of a CCCH zinc-binding motif - with the cellular protein tristetraprolin. Tristetraprolin is a SG-associated protein that helps regulate the stability of certain cellular mRNAs including a number encoding pro-inflammatory and antiviral proteins. This suggests a possible role for the viral M2-1 mRNA in mRNA stability.

Project Start
Project End
Budget Start
Budget End
Support Year
31
Fiscal Year
2013
Total Cost
$1,916,770
Indirect Cost
City
State
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Zip Code
Walsh, Kevin B; Teijaro, John R; Brock, Linda G et al. (2014) Animal model of respiratory syncytial virus: CD8+ T cells cause a cytokine storm that is chemically tractable by sphingosine-1-phosphate 1 receptor agonist therapy. J Virol 88:6281-93
Liesman, Rachael M; Buchholz, Ursula J; Luongo, Cindy L et al. (2014) RSV-encoded NS2 promotes epithelial cell shedding and distal airway obstruction. J Clin Invest 124:2219-33
Le Nouen, Cyril; Hillyer, Philippa; Brock, Linda G et al. (2014) Human metapneumovirus SH and G glycoproteins inhibit macropinocytosis-mediated entry into human dendritic cells and reduce CD4+ T cell activation. J Virol 88:6453-69
Luongo, Cindy; Winter, Christine C; Collins, Peter L et al. (2013) Respiratory syncytial virus modified by deletions of the NS2 gene and amino acid S1313 of the L polymerase protein is a temperature-sensitive, live-attenuated vaccine candidate that is phenotypically stable at physiological temperature. J Virol 87:1985-96
Stokes, Kate L; Currier, Michael G; Sakamoto, Kaori et al. (2013) The respiratory syncytial virus fusion protein and neutrophils mediate the airway mucin response to pathogenic respiratory syncytial virus infection. J Virol 87:10070-82
Tregoning, John S; Wang, Belinda Lei; McDonald, Jacqueline U et al. (2013) Neonatal antibody responses are attenuated by interferon-ýý produced by NK and T cells during RSV infection. Proc Natl Acad Sci U S A 110:5576-81
Fricke, Jens; Koo, Lily Y; Brown, Charles R et al. (2013) p38 and OGT sequestration into viral inclusion bodies in cells infected with human respiratory syncytial virus suppresses MK2 activities and stress granule assembly. J Virol 87:1333-47
Zhang, Liqun; Button, Brian; Gabriel, Sherif E et al. (2009) CFTR delivery to 25% of surface epithelial cells restores normal rates of mucus transport to human cystic fibrosis airway epithelium. PLoS Biol 7:e1000155
Luongo, Cindy; Yang, Lijuan; Winter, Christine C et al. (2009) Codon stabilization analysis of the "248" temperature sensitive mutation for increased phenotypic stability of respiratory syncytial virus vaccine candidates. Vaccine 27:5667-76
Buchholz, Ursula J; Ward, Jerrold M; Lamirande, Elaine W et al. (2009) Deletion of nonstructural proteins NS1 and NS2 from pneumonia virus of mice attenuates viral replication and reduces pulmonary cytokine expression and disease. J Virol 83:1969-80

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