Antiviral activity of the EB peptide against zoonotic poxviruses

<p>Abstract</p> <p>Background</p> <p>The EB peptide is a 20-mer that was previously shown to have broad spectrum <it>in vitro </it>activity against several unrelated viruses, including highly pathogenic avian influenza, herpes simplex virus type I, and vaccinia, the prototypic orthopoxvirus. To expand on this work, we evaluated EB for <it>in vitro </it>activity against the zoonotic orthopoxviruses cowpox and monkeypox and for <it>in vivo </it>activity in mice against vaccinia and cowpox.</p> <p>Findings</p> <p>In yield reduction assays, EB had an EC₅₀of 26.7 μM against cowpox and 4.4 μM against monkeypox. The EC₅₀for plaque reduction was 26.3 μM against cowpox and 48.6 μM against monkeypox. A scrambled peptide had no inhibitory activity against either virus. EB inhibited cowpox <it>in vitro </it>by disrupting virus entry, as evidenced by a reduction of the release of virus cores into the cytoplasm. Monkeypox was also inhibited <it>in vitro </it>by EB, but at the attachment stage of infection. EB showed protective activity in mice infected intranasally with vaccinia when co-administered with the virus, but had no effect when administered prophylactically one day prior to infection or therapeutically one day post-infection. EB had no <it>in vivo </it>activity against cowpox in mice.</p> <p>Conclusions</p> <p>While EB did demonstrate some <it>in vivo </it>efficacy against vaccinia in mice, the limited conditions under which it was effective against vaccinia and lack of activity against cowpox suggest EB may be more useful for studying orthopoxvirus entry and attachment <it>in vitro </it>than as a therapeutic against orthopoxviruses <it>in vivo</it>.</p

Studies of a Lacustrine-Volcanic Mars Analog Field Site with Mars-2020-like Instruments

On the upcoming Mars‐2020 rover two remote sensing instruments, Mastcam‐Z and SuperCam, and two microscopic proximity science instruments, SHERLOC and PIXL, will collect compositional (mineralogy, chemistry, and organics) data essential for paleoenvironmental reconstruction. The synergies between and limitations of these instruments were evaluated via study of a Mars analog field site in the Mojave Desert, using instruments approximating the data that will be returned by Mars‐2020. A ground truth dataset was generated for comparison to validate the results. The site consists of a succession of clay‐rich mudstones of lacustrine origin, interbedded tuffs, a carbonate‐silica travertine deposit, and gypsiferous mudstone strata. The major geological units were mapped successfully using simulated Mars‐2020 data. Simulated Mastcam‐Z data identified unit boundaries and Fe‐bearing weathering products. Simulated SuperCam passive shortwave infrared and green Raman data were essential in identifying major mineralogical composition and changes in lacustrine facies at distance; this was possible even with spectrally downsampled passive IR data. LIBS and simulated PIXL data discriminated and mapped major element chemistry. Simulated PIXL revealed mm‐scale zones enriched in zirconium, of interest for age dating. SHERLOC‐like data mapped sulfate and carbonate at sub‐mm scale; silicates were identified with increased laser pulses/spot or by averaging of hundreds of spectra. Fluorescence scans detected and mapped varied classes of organics in all samples, characterized further with follow‐on spatially targeted deep‐UV Raman spectra. Development of dedicated organics spectral libraries is needed to aid interpretation. Given these observations, the important units in the outcrop would be sampled and cached for sample return

Vaccine candidates for dengue virus type 1 (DEN1) generated by replacement of the structural genes of rDEN4 and rDEN4Δ30 with those of DEN1

BACKGROUND: Antigenic chimeric viruses have previously been generated in which the structural genes of recombinant dengue virus type 4 (rDEN4) have been replaced with those derived from DEN2 or DEN3. Two vaccine candidates were identified, rDEN2/4Δ30(ME) and rDEN3/4Δ30(ME), which contain the membrane (M) precursor and envelope (E) genes of DEN2 and DEN3, respectively, and a 30 nucleotide deletion (Δ30) in the 3' untranslated region of the DEN4 backbone. Based on the promising preclinical phenotypes of these viruses and the safety and immunogenicity of rDEN2/4Δ30(ME) in humans, we now describe the generation of a panel of four antigenic chimeric DEN4 viruses using either the capsid (C), M, and E (CME) or ME structural genes of DEN1 Puerto Rico/94 strain. RESULTS: Four antigenic chimeric viruses were generated and found to replicate efficiently in Vero cells: rDEN1/4(CME), rDEN1/4Δ30(CME), rDEN1/4(ME), and rDEN1/4Δ30(ME). With the exception of rDEN1/4(ME), each chimeric virus was significantly attenuated in a SCID-HuH-7 mouse xenograft model with a 25-fold or greater reduction in replication compared to wild type DEN1. In rhesus monkeys, only chimeric viruses with the Δ30 mutation appeared to be attenuated as measured by duration and magnitude of viremia. rDEN1/4Δ30(CME) appeared over-attenuated since it failed to induce detectable neutralizing antibody and did not confer protection from wild type DEN1 challenge. In contrast, rDEN1/4Δ30(ME) induced 66% seroconversion and protection from DEN1 challenge. Presence of the Δ30 mutation conferred a significant restriction in mosquito infectivity upon rDEN1/4Δ30(ME) which was shown to be non-infectious for Aedes aegypti fed an infectious bloodmeal. CONCLUSION: The attenuation phenotype in SCID-HuH-7 mice, rhesus monkeys, and mosquitoes and the protective immunity observed in rhesus monkeys suggest that rDEN1/4Δ30(ME) should be considered for evaluation in a clinical trial

A trade-off in replication in mosquito versus mammalian systems conferred by a point mutation in the NS4B protein of dengue virus type 4

AbstractAn acceptable live-attenuated dengue virus vaccine candidate should have low potential for transmission by mosquitoes. We have identified and characterized a mutation in dengue virus type 4 (DEN4) that decreases the ability of the virus to infect mosquitoes. A panel of 1248 mutagenized virus clones generated previously by chemical mutagenesis was screened for decreased replication in mosquito C6/36 cells but efficient replication in simian Vero cells. One virus met these criteria and contained a single coding mutation: a C-to-U mutation at nucleotide 7129 resulting in a Pro-to-Leu change in amino acid 101 of the nonstructural 4B gene (NS4B P101L). This mutation results in decreased replication in C6/36 cells relative to wild-type DEN4, decreased infectivity for mosquitoes, enhanced replication in Vero and human HuH-7 cells, and enhanced replication in SCID mice implanted with HuH-7 cells (SCID-HuH-7 mice). A recombinant DEN4 virus (rDEN4) bearing this mutation exhibited the same set of phenotypes. Addition of the NS4B P101L mutation to rDEN4 bearing a 30 nucleotide deletion (Δ30) decreased the ability of the double-mutant virus to infect mosquitoes but increased its ability to replicate in SCID-HuH-7 mice. Although the NS4B P101L mutation decreases infectivity of DEN4 for mosquitoes, its ability to enhance replication in SCID-HuH-7 mice suggests that it might not be advantageous to include this specific mutation in an rDEN4 vaccine. The opposing effects of the NS4B P101L mutation in mosquito and vertebrate systems suggest that the NS4B protein is involved in maintaining the balance between efficient replication in the mosquito vector and the human host

A replication-deficient rabies virus vaccine expressing Ebola virus glycoprotein is highly attenuated for neurovirulence

AbstractWe are developing inactivated and live-attenuated rabies virus (RABV) vaccines expressing Ebola virus (EBOV) glycoprotein for use in humans and endangered wildlife, respectively. Here, we further characterize the pathogenesis of the live-attenuated RABV/EBOV vaccine candidates in mice in an effort to define their growth properties and potential for safety. RABV vaccines expressing GP (RV-GP) or a replication-deficient derivative with a deletion of the RABV G gene (RVΔG-GP) are both avirulent after intracerebral inoculation of adult mice. Furthermore, RVΔG-GP is completely avirulent upon intracerebral inoculation of suckling mice unlike parental RABV vaccine or RV-GP. Analysis of RVΔG-GP in the brain by quantitative PCR, determination of virus titer, and immunohistochemistry indicated greatly restricted virus replication. In summary, our findings indicate that RV-GP retains the attenuation phenotype of the live-attenuated RABV vaccine, and RVΔG-GP would appear to be an even safer alternative for use in wildlife or consideration for human use

Antibody quality and protection from lethal ebola virus challenge in nonhuman primates immunized with rabies virus based bivalent vaccine.

We have previously described the generation of a novel Ebola virus (EBOV) vaccine platform based on (a) replication-competent rabies virus (RABV), (b) replication-deficient RABV, or (c) chemically inactivated RABV expressing EBOV glycoprotein (GP). Mouse studies demonstrated safety, immunogenicity, and protective efficacy of these live or inactivated RABV/EBOV vaccines. Here, we evaluated these vaccines in nonhuman primates. Our results indicate that all three vaccines do induce potent immune responses against both RABV and EBOV, while the protection of immunized animals against EBOV was largely dependent on the quality of humoral immune response against EBOV GP. We also determined if the induced antibodies against EBOV GP differ in their target, affinity, or the isotype. Our results show that IgG1-biased humoral responses as well as high levels of GP-specific antibodies were beneficial for the control of EBOV infection after immunization. These results further support the concept that a successful EBOV vaccine needs to induce strong antibodies against EBOV. We also showed that a dual vaccine against RABV and filoviruses is achievable; therefore addressing concerns for the marketability of this urgently needed vaccine

Vaccine candidates derived from a novel infectious cDNA clone of an American genotype dengue virus type 2

BACKGROUND: A dengue virus type 2 (DEN-2 Tonga/74) isolated from a 1974 epidemic was characterized by mild illness and belongs to the American genotype of DEN-2 viruses. To prepare a vaccine candidate, a previously described 30 nucleotide deletion (Δ30) in the 3' untranslated region of DEN-4 has been engineered into the DEN-2 isolate. METHODS: A full-length cDNA clone was generated from the DEN-2 virus and used to produce recombinant DEN-2 (rDEN-2) and rDEN2Δ30. Viruses were evaluated for replication in SCID mice transplanted with human hepatoma cells (SCID-HuH-7 mice), in mosquitoes, and in rhesus monkeys. Neutralizing antibody induction and protective efficacy were also assessed in rhesus monkeys. RESULTS: The rDEN2Δ30 virus was ten-fold reduced in replication in SCID-HuH-7 mice when compared to the parent virus. The rDEN-2 viruses were not infectious for Aedes mosquitoes, but both readily infected Toxorynchites mosquitoes. In rhesus monkeys, rDEN2Δ30 appeared to be slightly attenuated when compared to the parent virus as measured by duration and peak of viremia and neutralizing antibody induction. A derivative of rDEN2Δ30, designated rDEN2Δ30-4995, was generated by incorporation of a point mutation previously identified in the NS3 gene of DEN-4 and was found to be more attenuated than rDEN2Δ30 in SCID-HuH-7 mice. CONCLUSIONS: The rDEN2Δ30 and rDEN2Δ30-4995 viruses can be considered for evaluation in humans and for inclusion in a tetravalent dengue vaccine

A Single Dose of the DENV-1 Candidate Vaccine rDEN1Δ30 Is Strongly Immunogenic and Induces Resistance to a Second Dose in a Randomized Trial

Dengue is an emerging infectious disease that has become the most important arboviral infection worldwide. There are four serotypes of dengue virus, DENV-1, DENV-2, DENV-3, and DENV-4, each capable of causing the full spectrum of disease. rDEN1Δ30 is a live attenuated investigational vaccine for the prevention of DENV-1 illness and is also a component of an investigational tetravalent DENV vaccine currently in Phase I evaluation. A single subcutaneous dose of rDEN1Δ30 was previously shown to be safe and immunogenic in healthy adults. In the current randomized placebo-controlled trial, 60 healthy flavivirus-naive adults were randomized to receive 2 doses of rDEN1Δ30 (N = 50) or placebo (N = 10), either on study days 0 and 120 (cohort 1) or 0 and 180 (cohort 2). We sought to evaluate the safety and immunogenicity of this candidate vaccine in 50 additional vaccinees and to test whether the humoral immune response could be boosted by a second dose administered 4 or 6 months after the first dose. The first dose of vaccine was well tolerated, infected 47/50 vaccinees and induced seroconversion in 46/50 vaccinees. Irrespective of dosing interval, the second dose of vaccine was also well tolerated but did not induce any detectable viremia or ≥4-fold rise in serum neutralizing antibody titer.Only five subjects had an anamnestic antibody response detectable by ELISA following a second dose of vaccine, demonstrating that the vaccine induced sterilizing humoral immunity in most vaccinees for at least six months following primary vaccination.The promising safety and immunogenicity profile of this vaccine confirms its suitability for inclusion in a tetravalent dengue vaccine

Monkeypox Virus Infection of Rhesus Macaques Induces Massive Expansion of Natural Killer Cells but Suppresses Natural Killer Cell Functions

Natural killer (NK) cells play critical roles in innate immunity and in bridging innate and adaptive immune responses against viral infection. However, the response of NK cells to monkeypox virus (MPXV) infection is not well characterized. In this intravenous challenge study of MPXV infection in rhesus macaques (Macaca mulatta), we analyzed blood and lymph node NK cell changes in absolute cell numbers, cell proliferation, chemokine receptor expression, and cellular functions. Our results showed that the absolute number of total NK cells in the blood increased in response to MPXV infection at a magnitude of 23-fold, manifested by increases in CD56+, CD16+, CD16-CD56- double negative, and CD16+CD56+ double positive NK cell subsets. Similarly, the frequency and NK cell numbers in the lymph nodes also largely increased with the total NK cell number increasing 46.1-fold. NK cells both in the blood and lymph nodes massively proliferated in response to MPXV infection as measured by Ki67 expression. Chemokine receptor analysis revealed reduced expression of CXCR3, CCR7, and CCR6 on NK cells at early time points (days 2 and 4 after virus inoculation), followed by an increased expression of CXCR3 and CCR5 at later time points (days 7-8) of infection. In addition, MPXV infection impaired NK cell degranulation and ablated secretion of interferon-γ and tumor necrosis factor-α. Our data suggest a dynamic model by which NK cells respond to MPXV infection of rhesus macaques. Upon virus infection, NK cells proliferated robustly, resulting in massive increases in NK cell numbers. However, the migrating capacity of NK cells to tissues at early time points might be reduced, and the functions of cytotoxicity and cytokine secretion were largely compromised. Collectively, the data may explain, at least partially, the pathogenesis of MPXV infection in rhesus macaques

Studies of a Lacustrine-Volcanic Mars Analog Field Site with Mars-2020-like Instruments

On the upcoming Mars‐2020 rover two remote sensing instruments, Mastcam‐Z and SuperCam, and two microscopic proximity science instruments, SHERLOC and PIXL, will collect compositional (mineralogy, chemistry, and organics) data essential for paleoenvironmental reconstruction. The synergies between and limitations of these instruments were evaluated via study of a Mars analog field site in the Mojave Desert, using instruments approximating the data that will be returned by Mars‐2020. A ground truth dataset was generated for comparison to validate the results. The site consists of a succession of clay‐rich mudstones of lacustrine origin, interbedded tuffs, a carbonate‐silica travertine deposit, and gypsiferous mudstone strata. The major geological units were mapped successfully using simulated Mars‐2020 data. Simulated Mastcam‐Z data identified unit boundaries and Fe‐bearing weathering products. Simulated SuperCam passive shortwave infrared and green Raman data were essential in identifying major mineralogical composition and changes in lacustrine facies at distance; this was possible even with spectrally downsampled passive IR data. LIBS and simulated PIXL data discriminated and mapped major element chemistry. Simulated PIXL revealed mm‐scale zones enriched in zirconium, of interest for age dating. SHERLOC‐like data mapped sulfate and carbonate at sub‐mm scale; silicates were identified with increased laser pulses/spot or by averaging of hundreds of spectra. Fluorescence scans detected and mapped varied classes of organics in all samples, characterized further with follow‐on spatially targeted deep‐UV Raman spectra. Development of dedicated organics spectral libraries is needed to aid interpretation. Given these observations, the important units in the outcrop would be sampled and cached for sample return