CMRF-56(+) blood dendritic cells loaded with mRNA induce effective antigen-specific cytotoxic T-lymphocyte responses.

There are numerous transcriptional, proteomic and functional differences between monocyte-derived dendritic cells (Mo-DC) and primary blood dendritic cells (BDC). The CMRF-56 monoclonal antibody (mAb) recognizes a cell surface marker, which is upregulated on BDC following overnight culture. Given its unique ability to select a heterogeneous population of BDC, we engineered a human chimeric (h)CMRF-56 IgG4 mAb to isolate primary BDC for potential therapeutic vaccination. The ability to select multiple primary BDC subsets from patients and load them with in vitro transcribed (IVT) mRNA encoding tumor antigen might circumvent the issues limiting the efficacy of Mo-DC. After optimizing and validating the purification of hCMRF-56(+) BDC, we showed that transfection of hCMRF-56(+) BDC with mRNA resulted in efficient mRNA translation and antigen presentation by myeloid BDC subsets, while preserving superior DC functions compared to Mo-DC. Immune selected and transfected hCMRF-56(+) BDC migrated very efficiently in vitro and as effectively as cytokine matured Mo-DC in vivo. Compared to Mo-DC, hCMRF-56(+) BDC transfected with influenza matrix protein M1 displayed superior MHC peptide presentation and generated potent antigen specific CD8(+) T-cell recall responses, while Wilms tumor 1 (WT1) transfected CMRF-56(+) BDC generated effective primary autologous cytotoxic T-cell responses. The ability of the combined DC subsets within hCMRF-56(+) BDC to present mRNA delivered tumor antigens merits phase I evaluation as a reproducible generic platform for the next generation of active DC immune therapies.

Programmed ribosomal frameshift alters expression of west nile virus genes and facilitates virus replication in birds and mosquitoes.

West Nile virus (WNV) is a human pathogen of significant medical importance with close to 40,000 cases of encephalitis and more than 1,600 deaths reported in the US alone since its first emergence in New York in 1999. Previous studies identified a motif in the beginning of non-structural gene NS2A of encephalitic flaviviruses including WNV which induces programmed -1 ribosomal frameshift (PRF) resulting in production of an additional NS protein NS1'. We have previously demonstrated that mutant WNV with abolished PRF was attenuated in mice. Here we have extended our previous observations by showing that PRF does not appear to have a significant role in virus replication, virion formation, and viral spread in several cell lines in vitro. However, we have also shown that PRF induces an over production of structural proteins over non-structural proteins in virus-infected cells and that mutation abolishing PRF is present in ∼11% of the wild type virus population. In vivo experiments in house sparrows using wild type and PRF mutant of New York 99 strain of WNV viruses showed some attenuation for the PRF mutant virus. Moreover, PRF mutant of Kunjin strain of WNV showed significant decrease compared to wild type virus infection in dissemination of the virus from the midgut through the haemocoel, and ultimately the capacity of infected mosquitoes to transmit virus. Thus our results demonstrate an important role for PRF in regulating expression of viral genes and consequently virus replication in avian and mosquito hosts.

West Nile virus NS2A protein facilitates virus-induced apoptosis independently of interferon response.

The flavivirus NS2A protein is a small, multifunctional protein, involved in replication, virion formation and regulation of the innate immune response. Using the Kunjin strain of West Nile virus (WNV(KUN)) we previously demonstrated that a single amino acid change from alanine to proline at position 30 of the NS2A protein (A30P) reduced viral cytopathicity in cells and virulence in mice. To further investigate functions of the NS2A protein we have substituted alanine at position 30 with different amino acids (A30 mutants) in a WNV(KUN) infectious clone. The virulence of mutant viruses in wild-type (WT) and IRF3/IRF7 double-knockout mice was influenced by the amino acid change and ranged from high to low in the order of WT>A30L>A30E>A30P/A30G. Moreover, infection of beta interferon (IFN-ß)-deficient Vero cells with A30P virus showed less pronounced chromosomal DNA degradation and lower percentage of cells with positive TUNEL labelling than in WT virus infection, indicating a role for the WT NS2A protein in IFN-independent apoptotic cell death.

RNA structures required for production of subgenomic flavivirus RNA.

Flaviviruses are a group of single-stranded, positive-sense RNA viruses causing ∼100 million infections per year. We have recently shown that flaviviruses produce a unique, small, noncoding RNA (∼0.5 kb) derived from the 3' untranslated region (UTR) of the genomic RNA (gRNA), which is required for flavivirus-induced cytopathicity and pathogenicity (G. P. Pijlman et al., Cell Host Microbe, 4: 579-591, 2008). This RNA (subgenomic flavivirus RNA [sfRNA]) is a product of incomplete degradation of gRNA presumably by the cellular 5'-3' exoribonuclease XRN1, which stalls on the rigid secondary structure stem-loop II (SL-II) located at the beginning of the 3' UTR. Mutations or deletions of various secondary structures in the 3' UTR resulted in the loss of full-length sfRNA (sfRNA1) and production of smaller and less abundant sfRNAs (sfRNA2 and sfRNA3). Here, we investigated in detail the importance of West Nile virus Kunjin (WNV(KUN)) 3' UTR secondary structures as well as tertiary interactions for sfRNA formation. We show that secondary structures SL-IV and dumbbell 1 (DB1) downstream of SL-II are able to prevent further degradation of gRNA when the SL-II structure is deleted, leading to production of sfRNA2 and sfRNA3, respectively. We also show that a number of pseudoknot (PK) interactions, in particular PK1 stabilizing SL-II and PK3 stabilizing DB1, are required for protection of gRNA from nuclease degradation and production of sfRNA. Our results show that PK interactions play a vital role in the production of nuclease-resistant sfRNA, which is essential for viral cytopathicity in cells and pathogenicity in mice.

NS1' of flaviviruses in the Japanese encephalitis virus serogroup is a product of ribosomal frameshifting and plays a role in viral neuroinvasiveness.

Flavivirus NS1 is a nonstructural protein involved in virus replication and regulation of the innate immune response. Interestingly, a larger NS1-related protein, NS1', is often detected during infection with the members of the Japanese encephalitis virus serogroup of flaviviruses. However, how NS1' is made and what role it performs in the viral life cycle have not been determined. Here we provide experimental evidence that NS1' is the product of a -1 ribosomal frameshift event that occurs at a conserved slippery heptanucleotide motif located near the beginning of the NS2A gene and is stimulated by a downstream RNA pseudoknot structure. Using site-directed mutagenesis of these sequence elements in an infectious clone of the Kunjin subtype of West Nile virus, we demonstrate that NS1' plays a role in viral neuroinvasiveness.