Reporter Virus Particles to Study Virus Neutralization Using Safe and High-Throughput Assays
Tech Snapshot® captures today’s cutting-edge tools and technologies that will help drive drug discovery tomorrow. This installment was written by Integral Molecular, a research-driven biotechnology company creating innovative technologies and therapeutic antibodies for under-exploited membrane protein targets, including GPCRs, ion channels, transporters, and viral envelopes.
Research on live viruses can often be dangerous, requiring the use of BSL-3 or BSL-4 labs, which are limited in availability. To improve accessibility to viral research, Integral Molecular developed reporter virus particles (RVPs), which are primarily replication-incompetent pseudotyped virus particles that enable safe (BSL-2), easy and high-throughput assessment of viral infectivity and neutralization using standard detection instrumentation. Scientists at Integral Molecular have deep expertise in virology and support customers in the use of RVPs as well as in developing RVPs for specific customer needs. They have been trusted by over 100 companies to support their R&D and clinical programs and have co-authored over 70 peer-reviewed virology publications.
Pseudotyped RVPs are made up of a virus-specific envelope protein on a heterologous virus core with a modified genome that expresses a convenient optical reporter gene (GFP or luciferase) within 24 hours of cellular infection. They are antigenically similar to live virus and produce comparable results in neutralization assays, among other experiments. RVPs are produced from transfected plasmids with confirmed sequences, so they are not subject to antigenic shift or drift like virus particles that are produced from live virus stocks. Different RVP production lots are also highly comparable, and results are reproducible across different RVP inputs for a given experiment. RVPs are subject to rigorous QC and are a critical reagent for FDA submissions. Available as ready-to-use reagents, RVPs can be used in many applications, including antibody neutralization, serum screening and high-throughput assays.
Over the past 10 years, the scientists at Integral Molecular have been responding to prevalent viral threats and researching immune responses for SARS-CoV-1, Zika and Ebola viruses, in addition to working on Marburg, dengue, MERS and over 70 SARS-CoV-2 variant viruses. Recently, Integral Molecular has added both pandemic and endemic influenza RVPs, as well as filovirus RVPs, to their extensive offerings. The breadth of the RVP catalog for highly pathogenic viruses enables researchers to safely study current viral threats and provide the basis of tools for future pandemic preparedness. All RVPs are developed under the guidance of Integral Molecular’s virology experts and manufactured with rigorous quality control to help meet FDA guidances for research and development programs. Additionally, the team at Integral Molecular can produce custom RVPs to accelerate vaccine or antibody development projects for a unique virus of interest or variants of RVPs currently available in the catalog.
RVPs Enable Immunogenicity Assays for Testing Vaccines Using Patient Sera
Immunogenicity assays are a vital part of screening patient serum samples in vaccine clinical trials. The researchers at PPD used Integral Molecular’s RVPs when developing high-throughput immunogenicity assays that were safe, quantitative and reliable to test vaccine efficacy against SARS-CoV-2 in accordance with WHO and FDA guidances (Bonhomme et al., 2022). The RVPs were a safe alternative to using live virus in the development of this assay that looked at the neutralizing effects of SARS-CoV-2 antibodies that are produced in response to vaccination. The RVP assay met all of PPD’s acceptance criteria for precision, dilutional linearity, selectivity and specificity.
Takeda Used RVPs to Assess Zika and Dengue Immunogenicity Data for Phase 1, 2 and 3 Vaccine Clinical Trials
Takeda is a global biopharmaceutical company developing vaccines against dengue and Zika viruses. They have relied on RVPs to assess serum responses to vaccine candidates in both their Zika and dengue programs. Plaque Reduction Neutralization Tests (PRNT assays) are traditionally used to study neutralization titers of sera; however, Takeda notes that these assays are imprecise, largely manual, labor-intensive and expensive to use in large clinical trials where hundreds or thousands of samples need to be tested (Bohning et al., 2021).
As an alternative to using ‘live’ virus, scientists at Takeda developed a high-throughput RVP 384-well microneutralization assay using Zika RVPs to quantitatively assess neutralizing antibodies in their patient serum samples. They validated their RVP neutralization assay as having high levels of precision, reproducibility and good correlation with live virus assays (Bohning et al., 2021). Takeda scientists then used the assay to study the immunogenicity of their purified inactivated Zika virus vaccine candidate TAK-426 in a phase 1 clinical trial (NCT03343626) that enrolled 271 patients (Han et al., 2021). In this clinical trial setting with a large number of patient samples, the RVP assay provided similar results with PRNT and showed increasing antibody titers following a second dose of the vaccine.
Human sera from a Zika vaccine clinical trial (ZIK-101) were tested in RPNT and RVP assays, with a strong correlation of 0.94 (Bohning et al., 2021).
Takeda also employed dengue virus (DENV) RVPs to characterize serum responses in their phase 2 and phase 3 clinical trials for their now approved dengue vaccine. Takeda’s live-attenuated tetravalent vaccine TAK-003 consists of an attenuated DENV-2 virus, which also provides the backbone for the structural components of DENV-1, -3, and -4 (DeMaso et al., 2022). In these trials patient sera showed high serum titers of antibodies against DENV-2, and Takeda needed to show that neutralizing antibodies were also produced against the other dengue serotypes. They were able to accomplish this by depleting patient sera samples of DENV-2 and cross-reactive antibodies before testing for neutralization of DENV-1, -3, and -4 RVPs. The scientists at Takeda were able to successfully show neutralization of all dengue RVP serotypes and thus that their vaccine induced a broad tetravalent immune response. TAK-003 has been now approved for use in Indonesia, marketed under the name QDENGA and is under evaluation in additional dengue-endemic countries.
Immunome Used a Large Panel of SARS-CoV-2 RVP Variants to Test Their Therapeutic Antibody Cocktail
Monoclonal antibodies can be an effective treatment for viruses. However, the mutation of viruses over time can lead to viral escape. The researchers at Immunome sought to develop a multi-antibody cocktail as a SARS-CoV-2 therapy that would remain effective against the quickly emerging variants. Their goal was to provide broad neutralization potency and sustained efficacy as SARS-CoV-2 mutated. Integral Molecular’s large panel of SARS-CoV-2 variant RVPs allowed the scientists at Immunome to test their cocktail for broad efficacy in a rapid, safe manner.
Molecular biologists at Integral Molecular keep close surveillance over emerging variants to produce new RVPs as quickly as possible. Immunome used a large panel of SARS-CoV-2 variant RVPs to show that their antibody cocktail neutralized all variants tested including Alpha, Gamma and Delta. They also demonstrated that their RVP neutralization assay provided comparable results to other methods, including live virus assays. The availability of a comprehensive panel of SARS-CoV-2 RVPs enabled Immunome to meet the FDA guidance requiring developers to routinely monitor genomic databases for emerging SARS-CoV-2 variants and report the neutralizing activity of their antibody against circulating variants. Immunome’s three-antibody cocktail IMM-BCP-01 is presently in a Phase 1 clinical trial (NCT05429021) to test safety and tolerability in patients with mild to moderate COVID-19.
The antibody cocktail was tested against 12 viral variants and the original viral strain in a neutralization assay. All of the variants tested were successfully neutralized by the antibody cocktail, showing its potential efficacy in treating the quickly mutating SAR-CoV-2 virus (Nikitin et al., 2022).
Integral Molecular’s RVPs are a safe and effective tool for virology research. They are non-replicating, are not subject to antigenic shift or drift and produce comparable results to live virus in many applications, including neutralization assays. As a critical reagent for FDA submissions, they are essential in enabling research programs targeting diverse viral targets.