Development of a Cell-Based Reporter Potency Assay for Live Virus Vaccines.

The rapid development of potency assays is critical in the development of life-saving vaccines. The traditional plaque assay or fifty percent tissue culture infectious dose (TCID) assay used to measure the potency of live virus vaccines is time consuming, labor intensive, low throughput and with high variability. Described here is the development and qualification of a cell-based reporter potency assay for two vaccines for respiratory viral infection, one based on the recombinant vesicular stomatitis virus (rVSV) backbone, termed Vaccine 1 in this paper, and the other based on the measles virus vector, termed Vaccine 2.

Development and scale-up of rVSV-SARS-CoV-2 vaccine process using single use bioreactor.

The outbreak of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the Coronavirus Disease 2019 (COVID-19) has spread through the globe at an alarming speed. The disease has become a global pandemic affecting millions of people and created public health crises worldwide. Among many efforts to urgently develop a vaccine against this disease, we developed an industrial-scale closed, single use manufacturing process for V590, a vaccine candidate for SARS-CoV-2.

Virus reduction neutralization test and LI-COR microneutralization assay bridging and WHO international standard calibration studies for respiratory syncytial virus.

Respiratory syncytial virus (RSV) vaccine is an unmet medical need. The virus reduction neutralization test (VRNT) was developed to replace the LI-COR microneutralization assay to measure RSV neutralization titers. A bridging study using selected V171 phase I samples and calibration studies using the WHO international standard antiserum to RSV were performed to compare VRNT and LI-COR.

Development and comparison of three cell-based potency assays for anti-respiratory syncytial virus monoclonal antibody.

There is an increasing demand for monoclonal antibody (mAb) therapies to confer passive immunity against viral diseases. Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis, lower respiratory tract infections, and hospitalization in infants. Currently, there is no RSV vaccine but a humanized mAb available for high risk infants.

Development and qualification of a fast, high-throughput and robust imaging-based neutralization assay for respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a common pathogen causing severe respiratory illness in infants and elder adults. The development of an effective RSV vaccine is an important unmet medical need and an area of active research. The traditional method for testing neutralizing antibodies against RSV in clinical trials is the plaque reduction neutralization test (PRNT), which uses 24-well plates and needs several days post infection to develop viral plaques.

Virus Reduction Neutralization Test: A Single-Cell Imaging High-Throughput Virus Neutralization Assay for Dengue.

Vaccine immunogenicity and clinical efficacy are often assessed by the measure of serum-neutralizing antibodies. The present gold standard for detecting neutralizing antibodies against many viruses, including dengue, is the plaque/focus reduction neutralization test (P/FRNT). The FRNT is a cell-based assay that inherits high variability, resulting in poor precision and has lengthy turnaround times.

Dysregulation of Toll-Like Receptor 7 Compromises Innate and Adaptive T Cell Responses and Host Resistance to an Attenuated West Nile Virus Infection in Old Mice.

Unlabelled: The elderly are known to have enhanced susceptibility to infections and an impaired capacity to respond to vaccination. West Nile virus (WNV), a mosquito-borne flavivirus, has induced severe neurological symptoms, mostly in the elderly population. No vaccines are available for human use.

In vitro analysis of MyD88-mediated cellular immune response to West Nile virus mutant strain infection.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods - an in vitro T cell priming assay and an intracellular cytokine staining (ICS) - were utilized to assess dendritic cells (DCs) and T cell functions.

Attenuated West Nile virus mutant NS1130-132QQA/175A/207A exhibits virus-induced ultrastructural changes and accumulation of protein in the endoplasmic reticulum.

We have previously shown that ablation of the three N-linked glycosylation sites in the West Nile virus NS1 protein completely attenuates mouse neuroinvasiveness (≥1,000,000 PFU). Here, we compared the replication of the NS1130-132QQA/175A/207A mutant to that of the parental NY99 strain in monkey kidney Vero cells. The results suggest that the mechanism of attenuation is a lack of NS1 glycosylation, which blocks efficient replication, maturation, and NS1 secretion from the endoplasmic reticulum and results in changes to the virus-induced ultrastructure.

A West Nile virus NS4B-P38G mutant strain induces adaptive immunity via TLR7-MyD88-dependent and independent signaling pathways.

Prior work shows that an attenuated West Nile virus (WNV), the nonstructural (NS)4B-P38G mutant infection in mice induced strong immune responses and protected host from subsequent lethal wild-type WNV infection. Here, we investigated NS4B-P38G mutant infection in myeloid differentiation factor 88-deficient (MyD88(-/-)) and Toll-like receptor 7-deficient (TLR7(-/-)) mice and found they had enhanced susceptibility compared to wild-type mice. Both groups had lower WNV-specific IgM response and reduced effector T cell functions.

Vertebrate attenuated West Nile virus mutants have differing effects on vector competence in Culex tarsalis mosquitoes.

Previous mutational analyses of naturally occurring West Nile virus (WNV) strains and engineered mutant WNV strains have identified locations in the viral genome that can have profound phenotypic effect on viral infectivity, temperature sensitivity and neuroinvasiveness. We chose six mutant WNV strains to evaluate for vector competence in the natural WNV vector Culex tarsalis, two of which contain multiple ablations of glycosylation sites in the envelope and NS1 proteins; three of which contain mutations in the NS4B protein and an attenuated natural bird isolate (Bird 1153) harbouring an NS4B mutation. Despite vertebrate attenuation, all NS4B mutant viruses displayed enhanced vector competence by Cx.

Mutational analysis of the West Nile virus NS4B protein.

West Nile virus NS4B is a small hydrophobic nonstructural protein approximately 27 kDa in size whose function is poorly understood. Amino acid substitutions were introduced into the NS4B protein primarily targeting two distinct regions; the N-terminal domain (residues 35 through 60) and the central hydrophobic domain (residues 95 through 120). Only the NS4B P38G substitution was associated with both temperature-sensitive and small-plaque phenotypes.

Multiple amino acid changes at the first glycosylation motif in NS1 protein of West Nile virus are necessary for complete attenuation for mouse neuroinvasiveness.

West Nile virus (WNV), like all members of the Japanese encephalitis (JE) serogroup except JE virus, contains three N-linked glycosylation (N-X-S/T) sites in the NS1 protein at asparagine residues NS1(130), NS1(175) and NS1(207). Previously we showed that the ablation of these glycosylation sites in WNV, by substitution of asparagine for alanine, attenuated mouse neuroinvasiveness; however, full attenuation was not achieved and the virus retained a neurovirulence phenotype. Sequence of viral RNA extracted from mouse brains revealed a reversion at the NS1(130) site in some mice that succumbed to the attenuated NS1(130A/175A/207A) strain.

Immune responses to an attenuated West Nile virus NS4B-P38G mutant strain.

The nonstructural (NS) proteins of West Nile virus (WNV) have been associated with participation in evasion of host innate immune defenses. In the present study, we characterized immune response to an attenuated WNV strain, which has a P38G substitution in the NS4B protein. The WNV NS4B-P38G mutant induced a lower level of viremia and no lethality in C57BL/6 (B6) mice following a systemic infection.

Characterization of TEM1/endosialin in human and murine brain tumors.

Background: TEM1/endosialin is an emerging microvascular marker of tumor angiogenesis. We characterized the expression pattern of TEM1/endosialin in astrocytic and metastatic brain tumors and investigated its role as a therapeutic target in human endothelial cells and mouse xenograft models.

Methods: In situ hybridization (ISH), immunohistochemistry (IH) and immunofluorescence (IF) were used to localize TEM1/endosialin expression in grade II-IV astrocytomas and metastatic brain tumors on tissue microarrays.

Development and characterization of non-glycosylated E and NS1 mutant viruses as a potential candidate vaccine for West Nile virus.

West Nile virus is an arthropod-borne flavivirus that has caused substantial morbidity and mortality to animals as well as humans since its introduction in to the New York area in 1999. Given that there are no antiviral drugs available for treatment of the disease, vaccines provide an efficacious alternative to control this disease. Herein we describe an attenuated WNV strain developed by the ablation of the glycosylation sites in the envelope (E) and non-structural 1 (NS1) proteins.

A combination of naturally occurring mutations in North American West Nile virus nonstructural protein genes and in the 3' untranslated region alters virus phenotype.

We previously reported mutations in North American West Nile viruses (WNVs) with a small-plaque (sp), temperature-sensitive (ts), and/or mouse-attenuated (att) phenotype. Using an infectious clone, site-directed mutations and 3' untranslated region (3'UTR) exchanges were introduced into the WNV NY99 genome. Characterization of mutants demonstrated that a combination of mutations involving the NS4B protein (E249G) together with either a mutation in the NS5 protein (A804V) or three mutations in the 3'UTR (A10596G, C10774U, A10799G) produced sp, ts, and/or att variants.

Avian virulence and thermostable replication of the North American strain of West Nile virus.

The NY99 genotype of West Nile virus (WNV) introduced into North America has demonstrated high virulence for American crows (AMCRs), whilst a closely related WNV strain (KEN-3829) from Kenya exhibits substantially reduced virulence in AMCRs [Brault, A. C., Langevin, S.

A single amino acid substitution in the central portion of the West Nile virus NS4B protein confers a highly attenuated phenotype in mice.

West Nile virus (WNV) NS4B is a small hydrophobic nonstructural protein that is hypothesized to participate both in viral replication and evasion of host innate immune defenses. The protein has four cysteine residues (residues 102, 120, 227, and 237). Since cysteines are often critical for the function of proteins, each of the four cysteine residues found in WNV NS4B was mutated to serine by site-directed mutagenesis.

Envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage 1 West Nile virus strains.

The introduction of West Nile virus (WNV) into North America has been associated with relatively high rates of neurological disease and death in humans, birds, horses, and some other animals. Previous studies identified strains in both genetic lineage 1 and genetic lineage 2, including North American isolates of lineage 1, that were highly virulent in a mouse neuroinvasion model, while other strains were avirulent or significantly attenuated (D. W.

Chimeric dengue 2 PDK-53/West Nile NY99 viruses retain the phenotypic attenuation markers of the candidate PDK-53 vaccine virus and protect mice against lethal challenge with West Nile virus.

Chimeric dengue serotype 2/West Nile (D2/WN) viruses expressing prM-E of WN NY99 virus in the genetic background of wild-type D2 16681 virus and two candidate D2 PDK-53 vaccine variants (PDK53-E and PDK53-V) were engineered. The viability of the D2/WN viruses required incorporation of the WN virus-specific signal sequence for prM. Introduction of two mutations at M-58 and E-191 in the chimeric cDNA clones further improved the viability of the chimeras constructed in all three D2 carriers.