The Path Brightens for Vaccine Researchers: Luminescent Reporter Viruses Detect Neutralizing Antibodies

Developing a vaccine that is safe, effective, easily manufactured and distributed is a daunting task. Yet, that is exactly what is needed in response to the COVID-19 pandemic.

Computer generated 3D image of coronavirus

Vaccine development, safety and efficacy testing take time. The mumps vaccine is thought to be the quickest infectious disease vaccine ever produced, and its development required four years from sample collection to licensing (2). However, there are many reasons to anticipate quicker development for a COVID-19 vaccine: Researchers are collaborating in unprecedented ways, and most COVID-19 scientific publications are free for all to access and often available as preprints. As of August 11, 2020, researchers around the globe have more than 165 vaccine candidates in development, 30 of which are in some phase of human clinical trials (1). The range of vaccine formulations available to scientists has expanded to include RNA and DNA vaccines, replication-defective adenovirus vaccines, inactivated or killed vaccines and subunit protein vaccines. Equally important is that vaccine developers and researchers have greater access to powerful molecular biology tools like bioluminescent reporters that enable quicker testing and development.

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Using the Power of Technology for Viral Outbreaks

Artist’s rendition of a virus particle.

When the world is experiencing a viral pandemic, scientists and health officials quickly want data-driven answers to understand the situation and better formulate a public health response. Technology provides tools that researchers can use to develop a rapid sequencing protocol. With such a protocol, the data generated can help answer questions about disease epidemiology and understand the interaction between host and virus. Even better: If the protocol is freely available and based on cheap, mobile sequencing systems.

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Social Distancing: Taking a Lesson from Creatures Big and Small

For many of us, the current SARS-CoV-2 pandemic means working from home. For many, working from home means being away from human companionship that’s normally part of our work lives. While my four-legged office mates are quiet and do not require meetings, they are no substitute for human coworkers.

How about you? In our socially distanced world, do you find strength in the knowledge that others are also self-isolating to stay healthy?

What if I told you that numerous animal species, lobsters to mongoose, ants to mandrills, all practice social distancing to avoid infectious agents? Here are a few examples.

Image of banded mongoose family group.
Banded mongoose family group.
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Connecting and Collaborating: How Scientists Across the Globe are Supporting Each Other During The COVID-19 Pandemic

Many research labs around the world have temporarily closed their doors in response to the COVID-19 pandemic, while others are experiencing unprecedented need for reagents to perform viral testing. This urgency has led many scientists to make new connections and build creative, collaborative solutions.

“In labs that are still open for testing or other purposes, there’s certainly heightened anxiety,” says Tony Vanden Bush, Client Support Specialist. “I feel that right now, I need to help them deal with that stress however possible.”

Last week, Tony was contacted by a lab at the University of Minnesota that was preparing to serve as a secondary COVID-19 testing facility for a nearby hospital lab. The two labs needed to process up to 6,000 samples per day, and the university lab was far short of that capacity.

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Emergency Use Authorization: The What, the Why and the How

This blog is written by guest blogger, Heather Tomlinson, former Director of Clinical Diagnostics at Promega.

Finding safe and effective treatments for human diseases takes time.  Medication and diagnostic tests can take decades to discover, develop and prove safe and effective.  In the United States, the FDA stands as the gold-standard gatekeeper to ensure that treatments and tests are reliable and safe. The time we wait in review and clearance means less risk of ineffective or unsafe treatments.

And yet, in a pandemic, we are behind before we even start the race to develop diagnostic tests, so critical for understanding how an infectious disease is spreading. That is when processes like the FDA’s fast track of Emergency Use Authorization (EUA) are critical. Such authorization allows scientists and clinicians to be nimble and provide the best possible test protocol as quickly as possible, with the understanding that these protocols will continue to be evaluated and improved as new information becomes available. The EUA focuses resources and accelerates reviews that keep science at the fore and gets us our best chance at staying safe and healing.

The Maxwell 48 RSC Instrument and the Maxwell RSC Total Viral Nucleic Acid Isolation Kit are now listed as options within the CDC EUA protocol.

For scientists working around the clock, the FDA’s EUA process is ready to review and respond. Getting an EUA  gives clinical labs a very specific and tested resource to guide them to the tools and tests to use in a crisis.

Typically the Centers for Disease Control (CDC) will develop the first test or protocol that receives FDA EUA in response to a crisis like a pandemic.  For COVID-19 the CDC 2019-Novel Coronavirus Real-Time RT-PCR Diagnostic Panel received FDA EUA clearance in early February. This is the test protocol used by the public health labs that work with the CDC and test manufacturers around the world.

Throughout a crisis such as the current pandemic, scientists continually work to improve the testing protocols and add options to the EUA protocols. This enables more flexibility in the test protocols. Promega is fortunate to play a part of the CDC EUA equation for diagnostic testing. Our GoTaq® Probe 1-Step PRT-qPCR System is one of a few approved options for master mixes in the  CDC qPCR diagnostic test,  and now our medium-throughput Maxwell 48 Instrument and Maxwell Viral Total Nucleic Acid Purification Kit  were added to the CDC protocol as an option for the RNA isolation step as well. These additions to the CDC EUA means that laboratories have more resources at their disposal for the diagnostic testing which is so critical to effective pandemic response.

The Emergency Use Authorization provides the FDA guidance to strengthen our nation’s public health during emergencies, such as the current COVID-19 pandemic. The EUA allows continual improvement of an authorized protocol through the collaborative efforts scientists in all academia, government and industry to identify and qualify the most reliable technologies and systems, giving labs more flexibility as new products are added as options.

Dr. Tomlinson was the Director for the Global Clinical Diagnostics Strategic Business Unit at Promega Corporation bringing over 15 years of experience in clinical diagnostic test development. She was responsible for leading the team that drives strategy in the clinical market for Promega. Her background was in infectious disease diagnostic testing, with a focus on HIV drug resistance and evolution. Her last work focused on oncology companion diagnostic test development.  Heather was an accomplished international presenter, delivering conference presentations in the United States, Europe, Asia, and Africa. Heather passed away in 2023.

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Antibodies, Immunity and Vaccines: A Short Primer on the Adaptive Immune Response

MERS-CoV credit: NIAID

Our skin, respiratory system and gastrointestinal tract are continually bombarded by environmental challenges from potential pathogens like SARS-CoV-2. Yet, these exposures do not often cause illness because our immune system protects us. The human immune system is complex. It has both rapid, non-specific responses to injury and disease as well as long-term, pathogen-specific responses. Understanding how the immune response works helps us understand how some pathogens get past it and how to stop that from happening. It also provides key information to help us develop safe and effective vaccines.

The immune response involves two complementary pathways: Innate Immunity and Adaptive Immunity. Innate immunity is non-specific, rapid and occurs quickly after an injury or infection. As a result of the innate immune response, cytokines (small signaling molecules) are secreted to recruit immune cells to an injury or infection site.  Innate immunity does not develop “memory” of an antigen or confer long-term immunity.

The immune response involves to complementary pathways: Innate Immunity and Adaptive Immunity.

Unlike innate immunity, adaptive immunity is both antigen-dependent and antigen-specific, meaning that adaptive immune response requires the presence of a triggering antigen—something like a spike protein on the surface of a virus. The adaptive immune response is also specific to the antigen that triggers the response. The adaptive immune response takes longer to develop, but it has the capacity for memory in the form of memory B and T cells. This memory is what enables a fast, specific immune response (immunity) upon subsequent exposure to the antigen.

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Go fISH! Using in situ Hybridization to Search for Expression of a SARS-CoV-2 Viral Entry Protein

Loss of smell (olfaction) is a commonly reported symptom of COVID-19 infection. Recently, Bilinska, et al. set out to better understand the underlying mechanisms for loss of smell resulting from SARS-CoV-2 infection. In their research, they used in situ hybridization to investigate the expression of TMPRSS2, a SARS-CoV-2 viral entry protein in olfactory epithelium tissues of mice.

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NanoBRET™ Assays to Analyze Virus:Host Protein:Protein Interactions in Detail

Recently, Gordon et al. published an atlas of protein:protein interactions of all proposed SARS-CoV-2 proteins expressed individually in HEK 293 cells (Table 1). The study tagged each of the viral proteins with an epitope tag and performed a pull-down of the expressed protein followed by trypsin digestion and mass spec analysis, a process referred to as affinity purification–mass spec analysis. The group identified 332 human proteins interacting with 27 SARS-CoV-2 proteins.

The interactions identified in the HEK 293 cells helped Appelberg et al. analyze interactions over time in SARS-CoV-2-infected Huh7 cells. Gordon et al. used the PPI data to identify FDA-approved drugs, drugs in clinical trials, and pre-clinical compounds that bound to the identified human proteins and labs in New York and Paris tested some of these drugs for antiviral effects.   

Table 1. The general functional area of human proteins identified to interact with individually expressed SARS-CoV-2 proteins as reported by Gordon, et al. (1). The SARS-CoV-2 proteins are classified as non-structural proteins (nsp#), structural proteins (E, M, and N) and accessory proteins (orf#).  
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Using Wastewater Surveillance to Track COVID-19 Outbreaks

Today’s blog is written by guest blogger, Sameer Moorji, Director, Applied Markets.

Even as countries are now gradually starting to reopen after lockdown, the COVID-19 pandemic is far from over. Researchers around the world continue to find new ways to monitor, prevent and treat the disease. One new way of monitoring COVID-19 outbreaks relies on a somewhat unexpected source: sewage water.

In March 2020, researchers at the KWR Water Research Institute found the presence of SARS CoV-2 RNA in wastewater samples collected near Schiphol airport in Amsterdam and several other sites in Netherlands. The result came within a week after the first case of COVID-19 in the country was confirmed. This study opened the door to the possibility of using wastewater-based epidemiology to determine population-wide infections of COVID-19.

What is Wastewater-based epidemiology?

Wastewater based epidemiology (WBE), or sewershed surveillance, is an approach using analysis of wastewater to identify presence of biologicals or chemicals relevant for public health monitoring. WBE is not new, as wastewater has previously been used to detect the presence of pharmaceutical or industrial waste, drug entities (including opioid abuse), viruses and potential emergence of super bugs. In fact, several countries have been successful in containing Polio and Hepatitis A outbreaks within their geographic locations.

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Choices for Measuring Luciferase-Tagged Reporter Pseudotyped Viral Particles in Coronavirus Research

Coronavirus (CoV) researchers are working quickly to understand the entry of SARS-CoV-2 into cells. The Spike or S proteins on the surface of a CoV is trimer. The monomer is composed of an S1 and S2 domain. The division of S1 and S2 happens in the virus producing cell through a furin cleavage site between the two domains. The trimer binds to cell surface proteins. In the case of the SARS-CoV, the receptor is angiotensin converting enzyme 2. (ACE2). The MERS-CoV utilizes the cell-surface dipeptidyl peptidase IV protein. SARS-CoV-2 uses ACE2 as well. Internalized S protein goes though a second cleavage by a host cell protease, near the S1/S2 cleavage site called S2′, which leads to a drastic change in conformation thought to facilitate membrane fusion and entry of the virus into the cell (1).  

CDC / Alissa Eckert, MS; Dan Higgins, MAMS

Rather than work directly with the virus, researchers have chosen to make pseudotyped viral particles. Pseudotyped viral particles contain the envelope proteins of a well-known parent virus (e.g., vesicular stomatitis virus) with the native host cell binding protein (e.g., glycoprotein G) exchanged for the host cell binding protein (S protein) of the virus under investigation. The pseudotyped viral particle typically carries a reporter plasmid, most commonly firefly luciferase (FLuc), with the necessary genetic elements to be packaged in the particle. 

To create the pseudotyped viral particle, plasmids or RNA alone are transfected into cells and the pseudotyped viruses work their way through the endoplasmic reticulum and golgi to bud from the cells into the culture medium. The pseudoviruses are used to study the process of viral entry via the exchanged protein from the virus of interest. Entry is monitored through assay of the reporter. The reporter could be a luciferase or a fluorescent protein.   

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