scholarly journals An increase in glycoprotein concentration on extracellular virions dramatically alters vaccinia virus infectivity and pathogenesis without impacting immunogenicity

2021 ◽  
Vol 17 (12) ◽  
pp. e1010177
Author(s):  
Stephanie R. Monticelli ◽  
Peter Bryk ◽  
Matthew G. Brewer ◽  
Hector C. Aguilar ◽  
Christopher C. Norbury ◽  
...  
Keyword(s):  
Immune Response ◽  
Vaccinia Virus ◽  
Neutralizing Antibodies ◽  
Protective Immune Response ◽  
Virus Infectivity ◽  
Molluscum Contagiosum Virus ◽  
Marked Diminution ◽  
Animal Infection ◽  
Plaque Phenotype ◽  
Double Envelope

The extracellular virion (EV) form of Orthopoxviruses is required for cell-to-cell spread and pathogenesis, and is the target of neutralizing antibodies in the protective immune response. EV have a double envelope that contains several unique proteins that are involved in its intracellular envelopment and/or subsequent infectivity. One of these, F13, is involved in both EV formation and infectivity. Here, we report that replacement of vaccinia virus F13L with the molluscum contagiosum virus homolog, MC021L results in the production of EV particles with significantly increased levels of EV glycoproteins, which correlate with a small plaque phenotype. Using a novel fluorescence-activated virion sorting assay to isolate EV populations based on glycoprotein content we determine that EV containing either higher or lower levels of glycoproteins are less infectious, suggesting that there is an optimal concentration of glycoproteins in the outer envelope that is required for maximal infectivity of EV. This optimal glycoprotein concentration was required for lethality and induction of pathology in a cutaneous model of animal infection, but was not required for induction of a protective immune response. Therefore, our results demonstrate that there is a sensitive balance between glycoprotein incorporation, infectivity, and pathogenesis, and that manipulation of EV glycoprotein levels can produce vaccine vectors in which pathologic side effects are attenuated without a marked diminution in induction of protective immunity.

scholarly journals Neutralization Assay Using a Modified Vaccinia Virus Ankara Vector Expressing the Green Fluorescent Protein Is a High-Throughput Method To Monitor the Humoral Immune Response against Vaccinia Virus

2004 ◽  
Vol 11 (2) ◽  
pp. 406-410 ◽  
Author(s):  
Antonio Cosma ◽  
Silja Bühler ◽  
Rashmi Nagaraj ◽  
Caroline Staib ◽  
Anna-Lena Hammarin ◽  
...  
Keyword(s):  
Immune Response ◽  
Green Fluorescent Protein ◽  
Vaccinia Virus ◽  
High Throughput ◽  
Neutralizing Antibodies ◽  
Fluorescent Protein ◽  
Safety Concern ◽  
Neutralization Assay ◽  
Modified Vaccinia Virus Ankara ◽  
Green Fluorescent

ABSTRACT Vaccination against smallpox is again considered in order to face a possible bioterrorist threat, but the nature and the level of the immune response needed to protect a person from smallpox after vaccination are not totally understood. Therefore, simple, rapid, and accurate assays to evaluate the immune response to vaccinia virus need to be developed. Neutralization assays are usually considered good predictors of vaccine efficacy and more informative with regard to protection than binding assays. Currently, the presence of neutralizing antibodies to vaccinia virus is measured using a plaque reduction neutralization test, but this method is time-consuming and labor-intensive and has a subjective readout. Here, we describe an innovative neutralization assay based on a modified vaccinia virus Ankara (MVA) vector expressing the green fluorescent protein (MVA-gfp). This MVA-gfp neutralization assay is rapid and sensitive and has a high-throughput potential. Thus, it is suitable to monitor the immune response and eventually the efficacy of a large campaign of vaccination against smallpox and to study the vector-specific immune response in clinical trials that use genetically engineered vaccinia viruses. Most importantly, application of the highly attenuated MVA eliminates the safety concern in using the replication-competent vaccinia virus in the standard clinical laboratory.

scholarly journals The N-terminal domain of the vaccinia virus E3L-protein is required for neurovirulence, but not induction of a protective immune response

Virology ◽  
2005 ◽  
Vol 333 (2) ◽  
pp. 263-270 ◽  
Author(s):  
Teresa Brandt ◽  
Michael C. Heck ◽  
Sangeetha Vijaysri ◽  
Garilyn M. Jentarra ◽  
Jason M. Cameron ◽  
...  
Keyword(s):  
Immune Response ◽  
Vaccinia Virus ◽  
Protective Immune Response ◽  
Terminal Domain

scholarly journals CTIM-25. ONCOLYTIC VIRUS FOR DIPG: THE CLINICAL EXPERIENCE WITH DNX-2401

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii38-ii38
Author(s):  
Marta M Alonso ◽  
Ignacio Iñigo-Marco ◽  
Marc Garcia-Moure ◽  
Marisol González-Huarriz ◽  
Chris Jones ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cell ◽  
Neutralizing Antibodies ◽  
Immune Cell ◽  
Combined Treatment ◽  
Intratumoral Injection ◽  
Peripheral Blood Lymphocytes ◽  
Post Treatment ◽  
Virus Infectivity ◽  
Tumor Biopsy

Abstract Despite our increased understanding of Diffuse Intrinsic Pontine Glioma (DIPG) the outcome remains dismal. Recently we showed that the virus Delta-24-RGD (DNX-2401 in the clinic) was effective in preclinical models of DIPG and had the ability to trigger an antitumor immune response. These data allowed us to propel a phase I clinical trial for newly diagnosed DIPGs (NCT03178032) where the patients received an intratumoral viral injection followed by standard radiotherapy. The main objective is to determine the safety, tolerability, and toxicity of DNX-2401. Secondary endpoints are overall survival at 12 months, percentage of responses and induced immune response against tumor. Tumor biopsy was performed through the cerebellar peduncle, followed by intratumoral injection of DNX-2401 (N=12). Three patients were treated with the D1=1x1010vp and because the lack of toxicity we escalated to the D2= 5x1010vp. The procedure was well tolerated and safe. All patients displayed a reduced tumor volume after combined treatment. We performed molecular studies in 9 out of the 12 patients. The immune cell composition of the biopsies was assessed using multiplexed quantitative immunofluorescence. T cells were hardly noticeable in these tumors while macrophages were abundant. We detected increased clonal T cell diversity following treatment with virus in peripheral blood lymphocytes when compared paired pre- and post-treatment samples from the trial. In addition, we measure pre and post treatment neutralizing antibodies and its relationship with survival. Finally, we performed functional studies using 2 cell lines isolated from patients included in this trial to assess the response to the virus (infectivity, viability, T-cell recognition). Overall, the administration of DNX2401 was safe, feasible and therapeutically beneficial in a subgroup of patients. This trial constitutes a proof of principle that aids to understand the response of DIPGs to viral therapies allowing to set the bases to improve this strategy for DIPG.

scholarly journals Vaccinia virus gene F3L encodes an intracellular protein that affects the innate immune response

2007 ◽  
Vol 88 (7) ◽  
pp. 1917-1921 ◽  
Author(s):  
Graham C. Froggatt ◽  
Geoffrey L. Smith ◽  
Philippa M. Beard
Keyword(s):  
Immune Response ◽  
Vaccinia Virus ◽  
Innate Immune Response ◽  
Cytopathic Effect ◽  
Innate Immune ◽  
Intracellular Protein ◽  
Virus Growth ◽  
Plaque Phenotype

The Vaccinia virus BTB/kelch protein F3 has been characterized and its effects on virus replication in vitro and virus virulence in vivo have been determined. The loss of the F3L gene had no effect on virus growth, plaque phenotype or cytopathic effect in cell culture under the conditions tested. However, the virulence of a virus lacking F3L in an intradermal model was reduced compared with controls, and this was demonstrated by a significantly smaller lesion and alterations to the innate immune response to infection. The predicted molecular mass of the F3 protein is 56 kDa; however, immunoblotting of infected cell lysates using an antibody directed against recombinant F3 revealed two proteins of estimated sizes 37 and 25 kDa.

scholarly journals The Molluscum Contagiosum Gene MC021L Partially Compensates for the Loss of Its Vaccinia Virus Homolog, F13L

2020 ◽  
Vol 94 (20) ◽  
Author(s):  
Stephanie R. Monticelli ◽  
Peter Bryk ◽  
Brian M. Ward
Keyword(s):  
Vaccinia Virus ◽  
Molluscum Contagiosum ◽  
Molluscum Contagiosum Virus ◽  
Virion Protein ◽  
Small Plaque ◽  
Reading Frame ◽  
Extracellular Virus ◽  
Specific Proteins ◽  
Plaque Phenotype ◽  
Golgi Network

ABSTRACT Orthopoxviruses produce two antigenically distinct infectious enveloped virions termed intracellular mature virions and extracellular virions (EV). EV have an additional membrane compared to intracellular mature virions due to a wrapping process at the trans-Golgi network and are required for cell-to-cell spread and pathogenesis. Specific to the EV membrane are a number of proteins highly conserved among orthopoxviruses, including F13, which is required for the efficient wrapping of intracellular mature virions to produce EV and which plays a role in EV entry. The distantly related molluscipoxvirus, molluscum contagiosum virus, is predicted to encode several vaccinia virus homologs of EV-specific proteins, including the homolog of F13L, MC021L. To study the function of MC021, we replaced the F13L open reading frame in vaccinia virus with an epitope-tagged version of MC021L. The resulting virus (vMC021L-HA) had a small-plaque phenotype compared to vF13L-HA but larger than vΔF13L. The localization of MC021-HA was markedly different from that of F13-HA in infected cells, but MC021-HA was still incorporated in the EV membrane. Similar to F13-HA, MC021-HA was capable of interacting with both A33 and B5. Although MC021-HA expression did not fully restore plaque size, vMC021L-HA produced amounts of EV similar to those produced by vF13L-HA, suggesting that MC021 retained some of the functionality of F13. Further analysis revealed that EV produced from vMC021L-HA exhibit a marked reduction in target cell binding and an increase in dissolution, both of which correlated with a small-plaque phenotype. IMPORTANCE The vaccinia virus extracellular virion protein F13 is required for the production and release of infectious extracellular virus, which in turn is essential for the subsequent spread and pathogenesis of orthopoxviruses. Molluscum contagiosum virus infects millions of people worldwide each year, but it is unknown whether EV are produced during infection for spread. Molluscum contagiosum virus contains a homolog of F13L termed MC021L. To study the potential function of this homolog during infection, we utilized vaccinia virus as a surrogate and showed that a vaccinia virus expressing MC021L-HA in place of F13L-HA exhibits a small-plaque phenotype but produces similar levels of EV. These results suggest that MC021-HA can compensate for the loss of F13-HA by facilitating wrapping to produce EV and further delineates the dual role of F13 during infection.

Sign in / Sign up

Export Citation Format

Share Document