We are developing a live-attenuated, intranasal, pediatric vaccine against human respiratory syncytial virus (RSV). Building on years of molecular and biologic studies, we use reverse genetics to produce highly defined vaccine candidates from cloned cDNAs. This provides well-defined vaccine viruses that can have improved properties. This also provides virus with a short, well-defined passage history that greatly diminishes the chance of adventitious agents. It addition, the DNA intermediate provides a stable vaccine seed as well as the means to modify the virus as necessary in response to clinical data. Five cDNA-derived viruses have been evaluated to date in clinical studies, as described in previous years. The most promising virus, called rA2cp248/404/1030/delSH, was safe and immunogenic in young infants. However, there was evidence of loss of the 248 or 1030 attenuating point mutation (Tyr-1321-Asn or Gln-831-Leu, respectively, in the L protein) in some isolates. We are continuing to work to increase the genetic stability of this virus, and expect to have an improved candidate to enter phase 1 studies in 2011 or 2012. This ongoing work will be described in next years report. We also have identified new combinations of mutations that appear to have promising properties of attenuation and stability, and which also will be reported next year. As noted in an accompanying report, another new vaccine candidate, Medi delM2-2, entered phase 1 clinical studies in August, 2011. We also have been studying RSV immunobiology. One of the signal features of RSV is that it can readily re-infect symptomatically during infancy and throughout life without the need for significant antigenic change (in contrast, for example, with influenza A virus, for which significant antigenic change is needed for re-infection). This is frequently interpreted as evidence that RSV inhibits or subverts protective immunity. This is of potential importance for vaccine development. Previously, we showed that re-infection may be aided by a viral immune evasion mechanism involving a secreted form of the attachment G protein. As another approach to this issue, we previously initiated studies of the effects of RSV and other respiratory viruses on human dendritic cells (DC) in vitro. These are potent antigen presenting cells that play a major role in initiating and modulating the adaptive immune response. We compared the effects of RSV on human monocyte-derived DC (MDDC) in a side-by-side comparison with human metapneumovirus (HMPV) and human parainfluenza virus 3 (HPIV3) using viruses that expressed green fluorescent protein (GFP) in order to monitor infection. We also included influenza A virus (Flu) in the comparison. We analyzed MDDC that were exposed to these viruses in vitro for the expression of 62 genes pertinent to maturation. One of these was CCR7, which normally is up-regulated during maturation and directs migration of antigen-bearing DC to T cell-rich zones in lymphatic tissue. We found that DCs infected with RSV or HMPV did not efficiently up-regulate CCR7, in contrast to HPIV3 and, especially, Flu. This was confirmed at the level of cell surface protein expression. In addition, HMPV and RSV did not efficiently down-regulate surface expression of CCR1, 2 and 5, which maintain DC residence in peripheral tissues and normally are down-regulated during maturation. In an in vitro migration assay, RSV- and HMPV-treated DC migrated less efficiently to the CCR7 ligand CCL19, which directs DC chemotaxis to lymphatic tissue. Secondary stimulation with lipopolysaccharide reversed this phenotype, suggesting that it is due to suboptimal stimulation rather than irreversible inhibition. This phenotype appeared to be partly due to reduced expression of pro-inflammatory cytokines by DC treated with HMPV and RSV. Inefficient migration of DC in response to RSV and HMPV infection could contribute to a dampening of the adaptive response to these viruses. We investigated whether these viruses differentially affect the activation of CD4 T cells by virus-treated DC, as has been widely hypothesized. We infected human MDDC with RSV, HMPV, HPIV3, or Flu, and compared their ability to induce proliferation of autologous CD4+ T cells in vitro. We investigated both virus-specific memory responses as well as superantigen-induced responses. In general, there was little evidence of virus-specific inhibition. There was a trend of increasing memory responses, HMPV

Project Start
Project End
Budget Start
Budget End
Support Year
29
Fiscal Year
2011
Total Cost
$1,272,696
Indirect Cost
City
State
Country
Zip Code
Amarasinghe, Gaya K; Bào, Y?míng; Basler, Christopher F et al. (2017) Taxonomy of the order Mononegavirales: update 2017. Arch Virol 162:2493-2504
Le Nouën, Cyril; McCarty, Thomas; Brown, Michael et al. (2017) Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure. Proc Natl Acad Sci U S A 114:E386-E395
Postler, Thomas S; Clawson, Anna N; Amarasinghe, Gaya K et al. (2017) Possibility and Challenges of Conversion of Current Virus Species Names to Linnaean Binomials. Syst Biol 66:463-473
Hillyer, Philippa; Mane, Viraj P; Chen, Aaron et al. (2017) Respiratory syncytial virus infection induces a subset of types I and III interferons in human dendritic cells. Virology 504:63-72
Mehedi, Masfique; Collins, Peter L; Buchholz, Ursula J (2017) A novel host factor for human respiratory syncytial virus. Commun Integr Biol 10:e1319025
Norris, Emma L; Headlam, Madeleine J; Dave, Keyur A et al. (2016) Proteoform-specific insights into cellular proteome regulation. Mol Cell Proteomics :
Mehedi, Masfique; McCarty, Thomas; Martin, Scott E et al. (2016) Actin-Related Protein 2 (ARP2) and Virus-Induced Filopodia Facilitate Human Respiratory Syncytial Virus Spread. PLoS Pathog 12:e1006062
Caballero, Mauricio T; Serra, M Elina; Acosta, Patricio L et al. (2015) TLR4 genotype and environmental LPS mediate RSV bronchiolitis through Th2 polarization. J Clin Invest 125:571-82
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
Dave, Keyur A; Norris, Emma L; Bukreyev, Alexander A et al. (2014) A comprehensive proteomic view of responses of A549 type II alveolar epithelial cells to human respiratory syncytial virus infection. Mol Cell Proteomics 13:3250-69

Showing the most recent 10 out of 46 publications