Zika virus protection by a single low dose nucleoside modified mRNA vaccination

Zika virus (ZIKV) has recently emerged as an explosive pandemic associated with severe neuropathology in newborns and adults1. There are no ZIKV-specific treatments or preventatives; thus, development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins2,3. Here, we demonstrate that a single low-dose intradermal immunization with lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) encoding the pre-membrane and envelope (prM-E) glycoproteins of a 2013 ZIKV outbreak strain elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 μg of nucleoside-modified ZIKV mRNA-LNPs protected mice from ZIKV challenges at 2 weeks or 5 months post-vaccination, and a single dose of 50 μg was sufficient to protect non-human primates from a challenge at 5 weeks post-vaccination. These data demonstrate that nucleoside-modified mRNA-LNPs elicit rapid and durable protective immunity and thus represent a new and promising vaccine candidate for the global fight against ZIKV

Zika Virus Is Not Uniquely Stable at Physiological Temperatures Compared to Other Flaviviruses

Zika virus (ZIKV) is a flavivirus that has emerged as a global health threat due in part to its association with congenital abnormalities. Other globally relevant flaviviruses include dengue virus (DENV) and West Nile virus (WNV). High-resolution structures of ZIKV reveal many similarities to DENV and suggest some differences, including an extended glycan loop (D. Sirohi, Z. Chen, L. Sun, T. Klose, T. C. Pierson, et al., 352:467–470, 2016, http://dx.doi.org/10.1126/science.aaf5316) and unique interactions among envelope (E) protein residues that were proposed to confer increased virion stability and contribute mechanistically to the distinctive pathobiology of ZIKV (V. A. Kostyuchenko, E. X. Lim, S. Zhang, G. Fibriansah, T. S. Ng, et al., Nature 533:425–428, 2016, http://dx.doi.org/10.1038/nature17994). However, in the latter study, virus stability was inferred by measuring the loss of infectivity following a short incubation period. Here, we rigorously assessed the relative stability of ZIKV, DENV, and WNV by measuring changes in infectivity following prolonged incubation at physiological temperatures. At 37°C, the half-life of ZIKV was approximately twice as long as the half-life of DENV (11.8 and 5.2 h, respectively) but shorter than that of WNV (17.7 h). Incubation at 40°C accelerated the loss of ZIKV infectivity. Increasing virion maturation efficiency modestly increased ZIKV stability, as observed previously with WNV and DENV. Finally, mutations at E residues predicted to confer increased stability to ZIKV did not affect virion half-life. Our results demonstrate that ZIKV is not uniquely stable relative to other flaviviruses, suggesting that its unique pathobiology is explained by an alternative mechanism

Disrupting protein expression with Peptide Nucleic Acids reduces infection by obligate intracellular Rickettsia.

Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria's ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria

PNA is specific for designed target.

<p>A) <i>rickA</i> PNA target with putative Shine Dalgarno region in red and start codon in bold B) Western blot of <i>in vitro</i> translation assay supplemented with PNA complementary to the cloned region of <i>rickA</i>, no PNA, or non-targeting control-1 PNA. <i>In vitro</i> translation reactions contained vectors coding both truncated RickA and the control CALML3, or CALML3 alone. C) <i>rOmpB</i> PNA target with start codon in bold D) Dot blot confirming biotinylation of <i>rOmpB</i> and non-targeting-2 PNA. E) Nylon membrane probed to detect biotinylated PNA-ssRNA pairs following <i>rOmpB</i> PNA target RNA denaturation and hybridization to biotinylated PNA, incubated with increasing ratios of unlabeled PNA for competitive binding assay. Incubations with either no PNA or biotinylated non-targeting-2 PNA serve as a control.</p

PNA decreases rOmpB protein production and <i>in vitro</i> infection by TG <i>R</i>. <i>typhi</i>.

<p>A) Western blot of Opti-prep purified, serially diluted <i>rOmpB</i> PNA-treated <i>R</i>. <i>typhi</i> and quantitative densitometry of the rOmpB expression as a percent of undiluted rOmpB. Adherence percentages of Vero cells by <i>rOmpB</i> or non-targetng control PNA-treated <i>R</i>. <i>typhi</i>. No statistical significance found between treatment groups. C) Infection percentages of Vero cells by <i>rOmpB</i> or non-targeting control PNA-treated <i>R</i>. <i>typhi</i>. Infection by <i>rOmpB</i> PNA treated <i>R</i>. <i>typhi</i> is reduced 56% (p = 0.02) compared to non-targeting control PNA. <i>In vitro</i> experiments were repeated twice and performed in duplicate. Error bars represent standard deviation.</p

PNA decreases protein production and <i>in vitro</i> and <i>in vivo</i> infection by SFG <i>R</i>. <i>montanensis</i>.

<p>A) Western blot of Opti-prep purified, serially diluted <i>R</i>. <i>montanensis</i> treated with <i>rickA</i> PNA and quantitative densitometry of the RickA expression as a percent of undiluted RickA. B) Adherence percentages and C) Infection percentages of L929 cells by <i>R</i>. <i>montanensis</i> treated with PNA designed to <i>rickA</i> at 24 and 48 hours. Infection by <i>rickA</i> PNA-treated <i>R</i>. <i>montanensis</i> is reduced 88% (p = 0.00006) at 24 hours and 80% (p = 0.005) at 48-hours post-infection. There is no statistically significant change in adherence with PNA treatment. <i>In vitro</i> experiments were repeated twice and performed in duplicate. Error bars represent standard deviation. D) Unfed <i>D</i>. <i>variabilis</i> adult ticks were injected with <i>rickA</i> PNA-treated (n = 11) or non-targeting control PNA-2-treated (n = 10) rickettsia. Rickettsial burden was measured using qPCR. Genomic copies for <i>gltA</i> were normalized to genomic copies for <i>actin</i>. Closed circles represent individual ticks and the closed horizontal bars represent the mean. Rickettsial burden in ticks infected with <i>rickA</i> PNA-treated <i>R</i>. <i>montanensis</i> is reduced 90% compared to the control (p = 0.004). <i>In vivo</i> experiments were repeated twice with 10 biological replicates per treatment.</p

Broadly Neutralizing Activity of Zika Virus-Immune Sera Identifies a Single Viral Serotype

Recent epidemics of Zika virus (ZIKV) have been associated with congenital malformation during pregnancy and Guillain-Barré syndrome. There are two ZIKV lineages (African and Asian) that share >95% amino acid identity. Little is known regarding the ability of neutralizing antibodies elicited against one lineage to protect against the other. We investigated the breadth of the neutralizing antibody response following ZIKV infection by measuring the sensitivity of six ZIKV strains to neutralization by ZIKV-confirmed convalescent human serum or plasma samples. Contemporary Asian and early African ZIKV strains were similarly sensitive to neutralization regardless of the cellular source of virus. Furthermore, mouse immune serum generated after infection with African or Asian ZIKV strains was capable of neutralizing homologous and heterologous ZIKV strains equivalently. Because our study only defines a single ZIKV serotype, vaccine candidates eliciting robust neutralizing antibody responses should inhibit infection of both ZIKV lineages, including strains circulating in the Americas

Characterizing virtual slide exploration through the use of 'search maps'

Currently very little is known about the process by which pathologists arrive at a diagnosis on a case. This process is an integration of the pathologist's slide exploration strategy, perceptual information gathering and cognitive decision making. We have developed a methodology to statically represent the pathologists' dynamic visual search of digital slides by creating a representation of visual sampling called 'search maps'. In these maps slide exploration is divided into three parts, according to the magnification range used. In other words, areas explored at low magnification (4x-10x) and high magnification (>10x-20x) are represented separately. Moreover, representation using the 'search maps' allows for quantitative analysis and pairwise comparison of slide exploration strategy. In this paper we have compared the search maps of experienced pathologists and those of Pathology residents. Our goal was to understand how search differs between the experts and the trainees. © 2011 SPIE