Molecular engineering and plant expression of an immunoglobulin heavy chain scaffold for delivery of a dengue vaccine candidate.

In order to enhance vaccine uptake by the immune cells in vivo, molecular engineering approach was employed to construct a polymeric immunoglobulin G scaffold (PIGS) that incorporates multiple copies of an antigen and targets the Fc gamma receptors on antigen-presenting cells. These self-adjuvanting immunogens were tested in the context of dengue infection, for which there is currently no globally licensed vaccine yet. Thus, the consensus domain III sequence (cEDIII) of dengue glycoprotein E was incorporated into PIGS and expressed in both tobacco plants and Chinese Ovary Hamster cells. Purified mouse and human cEDIII-PIGS were fractionated by HPLC into low and high molecular weight forms, corresponding to monomers, dimers and polymers. cEDIII-PIGS were shown to retain important Fc receptor functions associated with immunoglobulins, including binding to C1q component of the complement and the low affinity Fcγ receptor II, as well as to macrophage cells in vitro. These molecules were shown to be immunogenic in mice, with or without an adjuvant, inducing a high level IgG antibody response which showed a neutralizing potential against the dengue virus serotype 2. The cEDIII-PIGS also induced a significant cellular immune response, IFN-γ production and polyfunctional T cells in both the CD4+ and CD8+ compartments. This proof-of-principle study shows that the potent antibody Fc-mediated cellular functions can be harnessed to improve vaccine design, underscoring the potential of this technology to induce and modulate a broad-ranging immune response

Evolutionary Selection Against Short Nucleotide Sequences in Viruses and Their Related Hosts

Viruses are under constant evolutionary pressure to effectively interact with the host intracellular factors, while evading its immune system. Understanding how viruses co-evolve with their hosts is a fundamental topic in molecular evolution and may also aid in developing novel viral based applications such as vaccines, oncologic therapies, and anti-bacterial treatments. Here, based on a novel statistical framework and a large-scale genomic analysis of 2,625 viruses from all classes infecting 439 host organisms from all kingdoms of life, we identify short nucleotide sequences that are under-represented in the coding regions of viruses and their hosts. These sequences cannot be explained by the coding regions’ amino acid content, codon, and dinucleotide frequencies. We specifically show that short homooligonucleotide and palindromic sequences tend to be under-represented in many viruses probably due to their effect on gene expression regulation and the interaction with the host immune system. In addition, we show that more sequences tend to be under-represented in dsDNA viruses than in other viral groups. Finally, we demonstrate, based on in vitro and in vivo experiments, how under-represented sequences can be used to attenuated Zika virus strains

Multi-color fluorescent reporter dengue viruses with improved stability for analysis of a multi-virus infection.

Reporter virus is a versatile tool to visualize and to analyze virus infections. However, for flaviviruses, it is difficult to maintain the inserted reporter genes on the viral genome, limiting its use in several studies that require homogeneous virus particles and several rounds of virus replication. Here, we showed that flanking inserted GFP genes on both sides with ribosome-skipping 2A sequences improved the stability and the consistency of their fluorescent signals for dengue-virus-serotype 2 (DENV2) reporter viruses. The reporter viruses can infect known susceptible mammalian cell lines and primary CD14+ human monocytes. This design can accommodate several fluorescent protein genes, enabling the generation of multi-color DENV2-16681 reporter viruses with comparable replication capabilities, as demonstrated by their abilities to maintain their fluorescent intensities during co-infections and to exclude superinfections regardless of the fluorescent tags. The reported design of multi-color DENV2 should be useful for high-throughput analyses, single-cell analysis, and characterizations of interference and superinfection in animal models

Evolutionary selection against short nucleotide sequences in viruses and their related hosts

Abstract Viruses are under constant evolutionary pressure to effectively interact with the host intracellular factors, while evading its immune system. Understanding how viruses co-evolve with their hosts is a fundamental topic in molecular evolution and may also aid in developing novel viral based applications such as vaccines, oncologic therapies, and anti-bacterial treatments. Here, based on a novel statistical framework and a large-scale genomic analysis of 2,625 viruses from all classes infecting 439 host organisms from all kingdoms of life, we identify short nucleotide sequences that are under-represented in the coding regions of viruses and their hosts. These sequences cannot be explained by the coding regions’ amino acid content, codon, and dinucleotide frequencies. We specifically show that short homooligonucleotide and palindromic sequences tend to be under-represented in many viruses probably due to their effect on gene expression regulation and the interaction with the host immune system. In addition, we show that more sequences tend to be under-represented in dsDNA viruses than in other viral groups. Finally, we demonstrate, based on in vitro and in vivo experiments, how under-represented sequences can be used to attenuated Zika virus strains.</jats:p

Functional analysis of flavivirus replicase by deep mutational scanning of dengue NS5

AbstractFlavivirus NS5 is multi-functional viral protein that play critical roles in virus replication, evolution, and immune antagonism against the hosts. Its error-prone replicase activity copies viral RNA for progeny virus particles and shapes virus evolution. Its methyltransferase activity and STAT2-targeting activity compromise type-I interferon signalling, dampening protective immune response during infection. It interacts with several host factors to shape the host-cell environment for virus replication. Thus, NS5 represents a critical target for both vaccine and antiviral drug development. Here, we performed deep mutational scanning (DMS) on the NS5 of dengue virus serotype 2 in mammalian cells. In combination with available structural and biochemical data, the comprehensive single amino-acid mutational data corroborated key residues and interactions involved in enzymatic functions of the replicase and suggested potential plasticity in NS5 guanylyl transferase. Strikingly, we identified that a set of strictly conserved residues in the motifs lining the replicase active site could tolerate mutations, suggesting additional roles of the priming loop in viral RNA synthesis and possible strategies to modulate the error rate of viral replicase activity through active-site engineering. Our DMS dataset and NS5 libraries could provide a framework and a resource to investigate molecular, evolutionary, and immunological aspects of NS5 functions, with relevance to vaccine and antiviral drug development.</jats:p

Sequences of primers and protospacer sequence of gRNAs used in this work.

<p>Sequences of primers and protospacer sequence of gRNAs used in this work.</p