Ebola Glycoprotein Research Articles

UV-Inactivated rVSV-M2e-Based Influenza Vaccine Protected against the H1N1 Influenza Challenge.

To investigate the immune responses and protection ability of ultraviolet light (UV)-inactivated recombinant vesicular stomatitis (rVSV)-based vectors that expressed a fusion protein consisting of four copies of the influenza matrix 2 protein ectodomain (tM2e) and the Dendritic Cell (DC)-targeting domain of the Ebola Glycoprotein (EΔM), (rVSV-EΔM-tM2e). In our previous study, we demonstrated the effectiveness of rVSV-EΔM-tM2e to induce robust immune responses against influenza M2e and protect against lethal challenges from H1N1 and H3N2 strains. Here, we used UV to inactivate rVSV-EΔM-tM2e and tested its immunogenicity and protection in BALB/c mice from a mouse-adapted H1N1 influenza challenge. Using Enzyme-Linked Immunosorbent Assay (ELISA) and Antibody-Dependent Cellular Cytotoxicity (ADCC), the influenza anti-M2e immune responses specific to human, avian and swine influenza strains induced were characterized. Likewise, the specificity of the anti-M2e immune responses induced in recognizing M2e antigen on the surface of the cell was investigated using Fluorescence-Activated Cell Sorting (FACS) analysis. Like the live attenuated rVSV-EΔM-tM2e, the UV-inactivated rVSV-EΔM-tM2e was highly immunogenic against different influenza M2e from strains of different hosts, including human, swine, and avian, and protected against influenza H1N1 challenge in mice. The FACS analysis demonstrated that the induced immune responses can recognize influenza M2 antigens from human, swine and avian influenza strains. Moreover, the rVSV-EΔM-tM2e also induced ADCC activity against influenza M2e from different host strains. These findings suggest that UV-inactivated rVSV-EΔM-tM2e could be used as an inactivated vaccine against influenza viruses.

The S1 spike protein of SARS-CoV-2 upregulates the ERK/MAPK signaling pathway in DC-SIGN-expressing THP-1 cells.

Dendritic cells, macrophages, neutrophils, and other antigen-presenting cells express various C-type lectin receptors that function to recognize the glycans associated with pathogens. The dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) binds various pathogens such as HIV glycoprotein 120, the Ebola glycoprotein, hemagglutinin, and the dengue virus glycoprotein in addition to the SARS-CoV-2 spike protein, and also triggers antigen-presenting cell endocytosis and immune escape from systemic infections. Many studies on the binding of SARS-CoV-2 spike protein with glycans have been published, but the underlying mechanism by which intracellular signaling occurs remains unclear. In this study, we report that the S1 spike protein of SARS-CoV-2 induces the phosphorylation of extracellular signal-regulated kinases (ERKs) in THP-1 cells, a DC-SIGN-expressing human monocytic leukemic cell line. On the other hand, the phosphorylation level of NF-κB remained unchanged under the same conditions. These data suggest that the major cell signaling pathway regulated by the S1 spike protein is the ERK pathway, which is superior to the NF-κB pathway in these DC-SIGN-expressing THP-1 cells and may contribute to immune hyperactivation in SARS-CoV-2 infections. Additionally, several glycans such as mannans, mannosylatedbovine serum albumin, the serum amyloid beta protein, andintracellular adhesion molecule 3 suppressed ERKphosphorylation,suggesting that these molecules are target molecules for SARS-CoV-2 infection by suppressing immune hyperactivation that occurs in the ERK signaling pathway.

Bivalent VSV Vectors Mediate Rapid and Potent Protection from Andes Virus Challenge in Hamsters.

Orthohantaviruses may cause hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. Andes virus (ANDV) is the only orthohantavirus associated with human-human transmission. Therefore, emergency vaccination would be a valuable public health measure to combat ANDV-derived infection clusters. Here, we utilized a promising vesicular stomatitis virus (VSV)-based vaccine to advance the approach for emergency applications. We compared monovalent and bivalent VSV vectors containing the Ebola virus (EBOV), glycoprotein (GP), and ANDV glycoprotein precursor (GPC) for protective efficacy in pre-, peri- and post-exposure immunization by the intraperitoneal and intranasal routes. Inclusion of the EBOV GP was based on its favorable immune cell targeting and the strong innate responses elicited by the VSV-EBOV vaccine. Our data indicates no difference of ANDV GPC expressing VSV vectors in pre-exposure immunization independent of route, but a potential benefit of the bivalent VSVs following peri- and post-exposure intraperitoneal vaccination.

Regulation of Ebola GP conformation and membrane binding by the chemical environment of the late endosome.

Interaction between the Ebola virus envelope glycoprotein (GP) and the endosomal membrane is an essential step during virus entry into the cell. Acidic pH and Ca2+ have been implicated in mediating the GP-membrane interaction. However, the molecular mechanism by which these environmental factors regulate the conformational changes that enable engagement of GP with the target membrane is unknown. Here, we apply fluorescence correlation spectroscopy (FCS) and single-molecule Förster resonance energy transfer (smFRET) imaging to elucidate how the acidic pH, Ca2+ and anionic phospholipids in the late endosome promote GP-membrane interaction, thereby facilitating virus entry. We find that bis(monoacylglycero)phosphate (BMP), which is specific to the late endosome, is especially critical in determining the Ca2+-dependence of the GP-membrane interaction. Molecular dynamics (MD) simulations suggested residues in GP that sense pH and induce conformational changes that make the fusion loop available for insertion into the membrane. We similarly confirm residues in the fusion loop that mediate GP's interaction with Ca2+, which likely promotes local conformational changes in the fusion loop and mediates electrostatic interactions with the anionic phospholipids. Collectively, our results provide a mechanistic understanding of how the environment of the late endosome regulates the timing and efficiency of virus entry.

A novel in vitro system of supported planar endosomal membranes (SPEMs) reveals an enhancing role for cathepsin B in the final stage of Ebola virus fusion and entry.

Ebola virus (EBOV) causes a hemorrhagic fever with fatality rates up to 90%. The EBOV entry process is complex and incompletely understood. Following attachment to host cells, EBOV is trafficked to late endosomes/lysosomes where its glycoprotein (GP) is processed to a 19-kDa form, which binds to the EBOV intracellular receptor Niemann-Pick type C1. We previously showed that the cathepsin protease inhibitor, E-64d, blocks infection by pseudovirus particles bearing 19-kDa GP, suggesting that further cathepsin action is needed to trigger fusion. This, however, has not been demonstrated directly. Since 19-kDa Ebola GP fusion occurs in late endosomes, we devised a system in which enriched late endosomes are used to prepare supported planar endosomal membranes (SPEMs), and fusion of fluorescent (pseudo)virus particles is monitored by total internal reflection fluorescence microscopy. We validated the system by demonstrating the pH dependencies of influenza virus hemagglutinin (HA)-mediated and Lassa virus (LASV) GP-mediated fusion. Using SPEMs, we showed that fusion mediated by 19-kDa Ebola GP is dependent on low pH, enhanced by Ca2+, and augmented by the addition of cathepsins. Subsequently, we found that E-64d inhibits full fusion, but not lipid mixing, mediated by 19-kDa GP, which we corroborated with the reversible cathepsin inhibitor VBY-825. Hence, we provide both gain- and loss-of-function evidence that further cathepsin action enhances the fusion activity of 19-kDa Ebola GP. In addition to providing new insights into how Ebola GP mediates fusion, the approach we developed employing SPEMs can now be broadly used for studies of virus and toxin entry through endosomes. IMPORTANCE Ebola virus is the causative agent of Ebola virus disease, which is severe and frequently lethal. EBOV gains entry into cells via late endosomes/lysosomes. The events immediately preceding fusion of the viral and endosomal membranes are incompletely understood. In this study, we report a novel in vitro system for studying virus fusion with endosomal membranes. We validated the system by demonstrating the low pH dependencies of influenza and Lassa virus fusion. Moreover, we show that further cathepsin B action enhances the fusion activity of the primed Ebola virus glycoprotein. Finally, this model endosomal membrane system should be useful in studying the mechanisms of bilayer breaching by other enveloped viruses, by non-enveloped viruses, and by acid-activated bacterial toxins.

The Mucin-Like Domain of the Ebola Glycoprotein Does Not Impact Virulence or Pathogenicity in Ferrets.

Ebola virus (EBOV) is considered among the most dangerous viruses with case fatality rates approaching 90% depending on the outbreak. While several viral proteins (VPs) including VP24, VP35, and the soluble glycoprotein are understood to contribute to virulence, less is known of the contribution of the highly variable mucin-like domain (MLD) of EBOV. Early studies have defined a potential role in immune evasion of the MLD by providing a glycan shield to critical glycoprotein residues tied to viral entry. Nonetheless, little is known as to what direct role the MLD plays in acute EBOV disease (EVD). We generated an infectious EBOV clone that lacks the MLD and assessed its virulence in ferrets compared with wild-type (WT) virus. No differences in growth kinetics were observed in vitro, nor were there any differences in time to death, viremia, or clinical picture in ferrets infected with recombinant EBOV (rEBOV)-WT or rEBOV-Δmucin. The EBOV MLD does not play a critical role in acute pathogenesis of EVD in ferrets.

Immune response of a two-dose heterologous Ebola vaccine regimen: summary of three African clinical trials using a single validated Filovirus Animal Nonclinical Group enzyme-linked immunosorbent assay in a single accredited laboratory

This analysis evaluated the immune response to the two-dose, heterologous Ad26.ZEBOV, MVA-BN-Filo Ebola virus vaccine regimen, administered 56-days apart, from multiple African sites based on results from one analytic laboratory. Immunogenicity across three trials (EBL2002, EBL2004/PREVAC, EBL3001) conducted in East and West Africa is summarised. Vaccine-induced Ebola glycoprotein-binding antibody concentrations were analysed by Q2 Solutions laboratory at baseline, 21 days (EBL2002 and EBL3001) or 28 days (EBL2004) post-dose 2 (regimen completion), and 12 months post-dose 1 using the validated Filovirus Animal Nonclinical Group Ebola glycoprotein enzyme-linked immunosorbent assay (ELISA). Responders were defined as those with a >2.5-fold increase from baseline or the lower limit of quantification (LLOQ) if<LLOQ at baseline. At 21 or 28 (21/28) days post-dose 2, the geometric mean concentration (GMC) range was 3810-7518 ELISA units (EU)/mL (percent responders: ≥98%) in adults, 9929-13532 EU/mL (≥98%) in adolescents aged 12-17 years, 10,212-17388 EU/mL (≥99%) in older children, and 22,568-25111 EU/mL (≥98%) in younger children. When stratified by country, GMCs at 21/28 days post-dose 2 were generally similar among adults and within paediatric cohorts (percent responders: 95%-100%). At month 12, GMC range was 259-437 EU/mL (percent responders: 49%-88%) in adults and 386-1139 EU/mL (70%-100%) in paediatric participants. Based on data from a single laboratory using a single validated assay, Ad26.ZEBOV, MVA-BN-Filo induced a strong humoral immune response, with ≥95% of participants across countries classified as responders at 21/28 days post-dose 2 (regimen completion), regardless of age. Janssen Vaccines & Prevention BV; Innovative Medicines Initiative.

Structurally Different Yet Functionally Similar: Aptamers Specific for the Ebola Virus Soluble Glycoprotein and GP1,2 and Their Application in Electrochemical Sensing.

The Ebola virus glycoprotein (GP) gene templates several mRNAs that produce either the virion-associated transmembrane protein or one of two secreted glycoproteins. Soluble glycoprotein (sGP) is the predominant product. GP1 and sGP share an amino terminal sequence of 295 amino acids but differ in quaternary structure, with GP1 being a heterohexamer with GP2 and sGP a homodimer. Two structurally different DNA aptamers were selected against sGP that also bound GP1,2. These DNA aptamers were compared with a 2'FY-RNA aptamer for their interactions with the Ebola GP gene products. The three aptamers have almost identical binding isotherms for sGP and GP1,2 in solution and on the virion. They demonstrated high affinity and selectivity for sGP and GP1,2. Furthermore, one aptamer, used as a sensing element in an electrochemical format, detected GP1,2 on pseudotyped virions and sGP with high sensitivity in the presence of serum, including from an Ebola-virus-infected monkey. Our results suggest that the aptamers interact with sGP across the interface between the monomers, which is different from the sites on the protein bound by most antibodies. The remarkable similarity in functional features of three structurally distinct aptamers suggests that aptamers, like antibodies, have preferred binding sites on proteins.

Demonstration, using novel supported planar endosomal membranes (SPEM), that further cathepsin action augments the fusion activity of the primed (19-kDa) Ebola virus glycoprotein.

Ebola virus (EBOV) causes a hemorrhagic fever associated with fatality rates up to 90%. The EBOV entry process is complex and incompletely understood. Following attachment to host cells, EBOV is trafficked to late endosomes where its glycoprotein (GP) is processed to a 19-kDa form, allowing the glycoprotein to bind to its intracellular receptor Niemann Pick type C1. We previously showed that the cathepsin protease inhibitor, E64d, blocks infection by pseudovirus particles bearing 19-kDa GP, suggesting that further cathepsin action is needed to trigger fusion. This, however, has not been demonstrated directly. Since 19-kDa Ebola GP fusion occurs in late endosomes, we devised a system in which enriched late endosomes are used to prepare supported planar endosomal membranes (SPEMs) and fusion of fluorescent (pseudo)virus particles is monitored by TIRF microscopy. We validated the system by demonstrating the pH dependencies of influenza virus HA- and Lassa virus GP-mediated fusion. Using SPEMs, we next showed that fusion mediated by 19-kDa Ebola GP is dependent on low pH and enhanced by Ca2+, consistent with other studies. We further showed that addition of cathepsins (somewhat more prominently with cathepsin B than L) augments both hemi- and full fusion. Subsequently we found that SPEMs appear to retain cathepsin activity, and that E64d inhibits both hemi- and full fusion mediated by 19-kDa GP. Hence we provide both gain- and loss-of-function evidence that further cathepsin action enhances the fusion activity of pre-primed 19-kDa Ebola GP. Thus we have provided new evidence for how Ebola GP mediates fusion with endosomes, and we have developed a novel approach employing SPEMs that can now be used for studies of any virus that fuses in endosomes.

Ebola virus disrupts the inner blood-retinal barrier by induction of vascular endothelial growth factor in pericytes.

Ebola virus (EBOV) causes severe hemorrhagic fever in humans with high mortality. In Ebola virus disease (EVD) survivors, EBOV persistence in the eyes may break through the inner blood-retinal barrier (iBRB), leading to ocular complications and EVD recurrence. However, the mechanism by which EBOV affects the iBRB remains unclear. Here, we used the in vitro iBRB model to simulate EBOV in retinal tissue and found that Ebola virus-like particles (EBO-VLPs) could disrupt the iBRB. Cytokine screening revealed that EBO-VLPs stimulate pericytes to secrete vascular endothelial growth factor (VEGF) to cause iBRB breakdown. VEGF downregulates claudin-1 to disrupt the iBRB. Ebola glycoprotein is crucial for VEGF stimulation and iBRB breakdown. Furthermore, EBO-VLPs caused iBRB breakdown by stimulating VEGF in rats. This study provides a mechanistic insight into that EBOV disrupts the iBRB, which will assist in developing new strategies to treat EBOV persistence in EVD survivors.

Engineering recombinantly expressed lectin-based antiviral agents.

Cyanovirin-N (CV-N), a lectin from Nostoc ellipsosporum was found an infusion inhibitory protein for human immunodeficiency virus (HIV)-1. A tandem-repeat of the engineered domain-swapped dimer bound specific sites at hemagglutinin (HA), Ebola and HIV spike glycoproteins as well as dimannosylated HA peptide, N-acetyl-D-glucosamine and high-mannose containing oligosaccharides. Among these, CV-N bound the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein at a dissociation constant (KD) of 18.6 µM (and KD=260 µM to RBD), which was low-affinity carbohydrate-binding as compared with the recognition of the other viral spikes. Binding of dimannosylated peptide to homo-dimeric CVN2 and variants of CVN2 that were pairing Glu-Arg residues sterically located close to its high-affinity carbohydrate binding sites, was measured using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Binding affinity increased with polar interactions, when the mutated residues were used to substitute a single, or two disulfide bonds, in CVN2. Site-specific N-linked glycans on spikes were mediating the infection with influenza virus by broadly neutralizing antibodies to HA and lectin binding to HA was further investigated via modes of saturation transfer difference (STD)-NMR. Our findings showed that stoichiometry and the lectin’s binding affinity were revealed by an interaction of CVN2 with dimannose units and either the high- or low-affinity binding site. To understand how these binding mechanisms add to viral membrane fusion we compare our tested HA-derived peptides in affinity with SARS-CoV-2 glycoprotein and review lectins and their mechanisms of binding to enveloped viruses for a potential use to simulate neutralization ability.

A Recombinant VSV-Based Bivalent Vaccine Effectively Protects against Both SARS-CoV-2 and Influenza A Virus Infection.

ABSTRACTCOVID-19 and influenza are both highly contagious respiratory diseases that have been serious threats to global public health. It is necessary to develop a bivalent vaccine to control these two infectious diseases simultaneously. In this study, we generated three attenuated replicating recombinant vesicular stomatitis virus (rVSV)-based vaccine candidates against both SARS-CoV-2 and influenza viruses. These rVSV-based vaccines coexpress SARS-CoV-2 Delta spike protein (SP) bearing the C-terminal 17 amino acid (aa) deletion (SPΔC) and I742A point mutation, or the SPΔC with a deletion of S2 domain, or the RBD domain, and a tandem repeat harboring four copies of the highly conserved influenza M2 ectodomain (M2e) that fused with the Ebola glycoprotein DC-targeting/activation domain. Animal immunization studies have shown that these rVSV bivalent vaccines induced efficient humoral and cellular immune responses against both SARS-CoV-2 SP and influenza M2 protein, including high levels of neutralizing antibodies against SARS-CoV-2 Delta and other variant SP-pseudovirus infections. Importantly, immunization of the rVSV bivalent vaccines effectively protected hamsters or mice against the challenges of SARS-CoV-2 Delta variant and lethal H1N1 and H3N2 influenza viruses and significantly reduced respiratory viral loads. Overall, this study provides convincing evidence for the high efficacy of this bivalent vaccine platform to be used and/or easily adapted to produce new vaccines against new or reemerging SARS-CoV-2 variants and influenza A virus infections.IMPORTANCE Given that both COVID-19 and influenza are preferably transmitted through respiratory droplets during the same seasons, it is highly advantageous to develop a bivalent vaccine that could simultaneously protect against both COVID-19 and influenza. In this study, we generated the attenuated replicating recombinant vesicular stomatitis virus (rVSV)-based vaccine candidates that target both spike protein of SARS-Cov-2 Delta variant and the conserved influenza M2 domain. Importantly, these vaccine candidates effectively protected hamsters or mice against the challenges of SARS-CoV-2 Delta variant and lethal H1N1 and H3N2 influenza viruses and significantly reduced respiratory viral loads.

Development and characterization of influenza M2 ectodomain and/or hemagglutinin stalk-based dendritic cell-targeting vaccines.

A universal influenza vaccine is required for broad protection against influenza infection. Here, we revealed the efficacy of novel influenza vaccine candidates based on Ebola glycoprotein dendritic cell (DC)-targeting domain (EΔM) fusion protein technology. The four copies of ectodomain matrix protein of influenza (tM2e) or M2e hemagglutinin stalk (HA stalk) peptides (HM2e) were fused with EΔM to generate EΔM-tM2e or EΔM-HM2e, respectively. We demonstrated that EΔM-HM2e- or EΔM-tM2e-pseudotyped viral particles can efficiently target DC/macrophages in vitro and induced significantly high titers of anti-HA and/or anti-M2e antibodies in mice. Significantly, the recombinant vesicular stomatitis virus (rVSV)-EΔM-tM2e and rVSV-EΔM-HM2e vaccines mediated rapid and potent induction of M2 or/and HA antibodies in mice sera and mucosa. Importantly, vaccination of rVSV-EΔM-tM2e or rVSV-EΔM-HM2e protected mice from influenza H1N1 and H3N2 challenges. Taken together, our study suggests that rVSV-EΔM-tM2e and rVSV-EΔM-HM2e are promising candidates that may lead to the development of a universal vaccine against different influenza strains.

Engineering Antiviral Agents via Surface Plasmon Resonance.

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 µM as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-µ-molar range.

Interferon-Induced HERC5 Inhibits Ebola Virus Particle Production and Is Antagonized by Ebola Glycoprotein

Survival following Ebola virus (EBOV) infection correlates with the ability to mount an early and robust interferon (IFN) response. The host IFN-induced proteins that contribute to controlling EBOV replication are not fully known. Among the top genes with the strongest early increases in expression after infection in vivo is IFN-induced HERC5. Using a transcription- and replication-competent VLP system, we showed that HERC5 inhibits EBOV virus-like particle (VLP) replication by depleting EBOV mRNAs. The HERC5 RCC1-like domain was necessary and sufficient for this inhibition and did not require zinc finger antiviral protein (ZAP). Moreover, we showed that EBOV (Zaire) glycoprotein (GP) but not Marburg virus GP antagonized HERC5 early during infection. Our data identify a novel ‘protagonist–antagonistic’ relationship between HERC5 and GP in the early stages of EBOV infection that could be exploited for the development of novel antiviral therapeutics.

Conserved immunogenic peptides of Ebola glycoprotein elicit immune response in human peripheral blood mononuclear cells.

In the past 45 years, ebolaviruses have periodically caused epidemics on the African continent. In December 2019, approval of a recombinant vector-based EBOV vaccine, named Ervebo, came as encouraging news; still, there is a long way to go in the development of an accessible, global, and pan-ebolavirus vaccine. The current study expanded our previous in silico work which was conducted on ebolavirus glycoprotein and this resulted in the identification of three potentially immunogenic peptides (P1 - FKRTSFFLWVIILFQRTFSIPL, P2 - LANETTQALQLF, and P3 - RATTELRTFSILNRKAIDF). An analysis to estimate the number of expected human leukocyte antigen (HLA) responders revealed that P1, P2, and P3 can potentially interact with 2540, 2150, and 2802 HLA alleles, respectively. Further, these peptides were subject to in vitro analysis wherein the human peripheral blood mononuclear cell proliferation and interferon-gamma (IFN-γ) production by peptide stimulated cells was studied in 10 healthy human blood samples with the help of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and a sandwich enzyme-linked immunosorbent assay (ELISA) respectively. P3 presented the best results, a significant (P < 0.05) peptide induced cell proliferation and IFN-γ stimulation for 8 and 10 samples, respectively, followed by P1 (5 and 6) and P2 (5 and 7). The in silico and in vitro results obtained in this study indicate the immunogenic potential of these peptides and warrant exploration of the effects on other cytokines as well as in vivo experimental validation.

A Protein Subunit Filovirus Vaccine Facilitates Maternal Immunization in Mice

Abstract Ebola virus disease (EVD) is highly pathogenic and virulent and has caused large scale outbreaks in Central and West Africa. To date there are no known correlates of protection however, recently the FDA and EMA have approved a vaccine, Ervebo. This vaccine utilizes a replication competent viral vector which may be contraindicated in immunocompromised individuals, pregnant women, and infants. Our lab has developed a recombinant protein subunit vaccine, a platform that is generally considered safe for immunocompromised, pregnant women, and infants. Our vaccine, when properly adjuvanted, demonstrates protection in non-human primates and may also induce maternal immunization. Maternal Immunization is the phenomena of inducing passive immunity via maternal antibody. This occurs via transfer of IgG using the FcRn. This receptor binds the Fc portion of IgG in a pH dependent manner which facilitates transcytosis of IgG across the placental, mammary tissue, as well as gut epithelial layer. In rodents, maternal IgG is transferred across the gut epithelia throughout the nursing period. This project demonstrates in mice the immunogenicity of a protein subunit vaccine comprised of Ebola glycoprotein and adjuvant administered during gestation as well as the transfer of antigen specific maternal antibody to offspring through milk. To investigate maternal immunization we vaccinated female Swiss Webster mice during gestation and measured antigen specific IgG levels in both the mothers and pups as well as in the milk. Antigen specific maternal antibody titers found in pup sera mimic those found in fully immunized adult females weeks after weaning, indicating the potential for maintaining an antigen specific humoral response in neonates.

The shape of pleomorphic virions determines resistance to cell-entry pressure.

Many enveloped animal viruses produce a variety of particle shapes, ranging from small spherical to long filamentous types. Characterization of how the shape of the virion affects infectivity has been difficult because the shape is only partially genetically encoded, and most pleomorphic virus structures have no selective advantage in vitro. Here, we apply virus fractionation using low-force sedimentation, as well as antibody neutralization coupled with RNAScope, single-particle membrane fusion experiments and stochastic simulations to evaluate the effects of differently shaped influenza A viruses and influenza viruses pseudotyped with Ebola glycoprotein on the infection of cells. Our results reveal that the shape of the virus particles determines the probability of both virus attachment and membrane fusion when viral glycoprotein activity is compromised. The larger contact interface between a cell and a larger particle offers a greater probability that several active glycoproteins are adjacent to each other and can cooperate to induce membrane merger. Particles with a length of tens of micrometres can fuse even when 95% of the glycoproteins are inactivated. We hypothesize that non-genetically encoded variable particle shapes enable pleomorphic viruses to overcome selective pressure and may enable adaptation to infection of cells by emerging viruses such as Ebola. Our results suggest that therapeutics targeting filamentous virus particles could overcome antiviral drug resistance and immune evasion in pleomorphic viruses.

Immunization and Drug Metabolizing Enzymes: Focus on Hepatic Cytochrome P450 3A

ABSTRACT Objective Infectious disease emergencies like the 2013–2016 Ebola epidemic and the 2009 influenza and current SARS-CoV-2 pandemics illustrate that vaccines are now given to diverse populations with preexisting pathologies requiring pharmacological management. Many natural biomolecules (steroid hormones, fatty acids, vitamins) and ~60% of prescribed medications are processed by hepatic cytochrome P450 (CYP) 3A4. The objective of this work was to determine the impact of infection and vaccines on drug metabolism. Methods The impact of an adenovirus-based vaccine expressing Ebola glycoprotein (AdEBO) and H1N1 and H3N2 influenza viruses on hepatic CYP 3A4 and associated nuclear receptors was evaluated in human hepatocytes (HC-04 cells) and in mice. Results CYP3A activity was suppressed by 55% in mice 24 h after administration of mouse-adapted H1N1, while ˂10% activity remained in HC-04 cells after infection with H1N1 and H3N2 due to global suppression of cellular translation capacity, indicated by reduction (70%, H1N1, 56%, H3N2) of phosphorylated eukaryotic translation initiation factor 4e (eIF4E). AdEBO suppressed CYP3A activity in vivo (44%) and in vitro (26%) 24 hours after infection. Conclusion As the clinical evaluation of vaccines for SARS-CoV-2 and other global pathogens rise, studies to evaluate the impact of new vaccines and emerging pathogens on CYP3A4 and other metabolic enzymes are warranted to avoid therapeutic failures that could further compromise the public health during infectious disease emergencies.

Durable natural killer cell responses after heterologous two-dose Ebola vaccination

Natural killer (NK) cells are implicated among immune effectors after vaccination against viral pathogens, including Ebola virus. The two-dose heterologous Ebola virus vaccine regimen, adenovirus type 26.ZEBOV followed by modified vaccinia Ankara-BN-Filo (EBOVAC2 consortium, EU Innovative Medicines Initiative), induces NK cell activation and anti-Ebola glycoprotein (GP) antibody-dependent NK cell activation post-dose 1, which is further elevated post-dose 2. Here, in a multicentre, phase 2 clinical trial (EBL2001), we demonstrate durable ex vivo NK cell activation 180 days after dose 2, with responses enriched in CD56bright NK cells. In vitro antibody-dependent responses to immobilised Ebola GP increased after dose 1, and remained elevated compared to pre-vaccination levels in serum collected 180 days later. Peak NK cell responses were observed post-dose 2 and NK cell IFN-γ responses remained significantly elevated at 180 days post-dose 2. Individual variation in NK cell responses were influenced by both anti-Ebola GP antibody concentrations and intrinsic interindividual differences in NK cell functional capacity. In summary, this study demonstrates durable NK cell responses after Ad26.ZEBOV, MVA-BN-Filo Ebola virus vaccination and could inform the immunological evaluation of future iterations of the vaccine regimen and vaccination schedules.