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.
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.
Celebrating the art of science is something the University of Wisconsin-Madison Cool Science Image Contest has been doing since its inception 10 years ago as part of The Why Files. The 2020 winning images include entries as diverse as videos of neural stem cells, eye-ball licking geckos and yes, even a picture of rock: actually a thin section of tractolite, an igneous rock composed of feldspar and olivine collected near Duluth Minnesota form the Proterozoic Mid-continent Rift. This image was collected by Natalie Betz, PhD, Associate Director of the UW-Madison Master of Science in Biotechnology program and her daughter Anya Wolterman, a recent graduate of Macalester College with degrees in Geology and Physics. Natalie has a long-time connection with Promega and the BioPharmaceutical Technology Center Institute, so we reached out to her to get the perspective of a contest entrant. Natalie is answering for both her and her daughter while her daughter is away doing some trail maintenance in the Rockies and is not available for comment.
Promega Connections: Why did you decide to enter the UW Cool Science Image contest?
Photo 51 is the now-famous X-ray diffraction picture that allowed Watson and Crick to crystalize centuries work of scientific study (from Mendel to Chargaff) into a viable structural model that explained how DNA could serve as the material of the gene. The photo was painstakingly produced by Dr. Rosalind Franklin, a contemporary of Watson and Crick. Although she and her colleague R.G. Gosling did publish their work in the same issue of Nature as the Watson and Crick paper (1,2), their work did not receive the same public accolades of that of Watson and Crick.
Women scientists have been contributing to our understanding of the world around us throughout history. On this 100th anniversary of Dr. Rosalind Franklin’s birth, we want to take a little time to recognize the work that women scientists are doing at Promega.
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.
Monitoring the use of performance-enhancing substances among athletes is complex and the requirements for tests and assays that detect use of such substances have changed significantly over the last few decades.
The haematological (blood) module of Athlete Biological Passport was adopted December 1, 2009 (ABP) by the World Anti-Doping Agency. The module sets out standard protocols to monitor doping of professional athletes by looking at changes in biological parameters, without relying on the detection of illegal compounds in body fluids. Such biological methods eliminate the need to develop and validate a test to detect every new compound that can be used for doping. The current version of the ABP, adopted in 2014, also adds monitoring of certain steroid use indicators from urine samples.
Blood doping which aims at increasing red blood cells so that more oxygen can be transported to muscles to increase stamina or performance is particularly difficult to detect. There are typically three ways that it is accomplished: use of erythropoietin (EPO) or synthetic oxygen carriers and blood transfusions. While transfusions of large volumes of blood or use of EPO can be detected, microdosing EPO or transfusing smaller volumes of packed red blood cells is much harder to detect.
Nicolas Leuenberger and colleagues at the Swiss Laboratory for Doping Analysis have developed a method to detect blood doping. In addition to addressing the detection of blood doping, his laboratory is also concerned about easing the transport and storage requirements for samples and ensuring that sample collection does not adversely affect athlete performance.
Improving Collection and Storage of Blood Samples
Because sample collection and storage are so critical to accurate test results, any new assays developed to detect blood doping benefit from ease of collection and storage. The Leuenberger laboratory investigated the use of the TAP™ Push Button collection device, which is billed as a simple method for blood collection that is easy to use and eliminates the need for painful needle sticks or finger pricks that can affect athlete performance. After TAP collection, 20µl of blood from the device was placed on to filter paper and dried (dried blood samples; DBS), which are much easier to store and transport from collection site to laboratory.
An RNA Biomarker for Blood Doping
Blood withdrawal and autologous transfusion or recombinant human EPO injection stimulate erythropoiesis and immature red blood cells can be distinguished based on their gene expression profiles. One of the genes that is expressed by immature red blood cells is aminoleuvulinate synthase 2, a gene that encodes an enzyme ALAS2 involved in the synthesis of heme, a pathway active during RBC maturation. RNA transcripts are unstable and tend to degrade rapidly, so isolating linear RNA transcripts from a collected sample can be difficult. However circular RNAs (circRNAs) are a class of RNA molecule produced by the backsplicing of pre-mRNAs that are high in abundance, quite stable and maintain cell-type specific expression. The Leuenberger laboratory developed a method for measuring the linear and circular forms of ALAS2 RNA in DBS to monitor erythropoiesis.
One of the greatest challenges in developing this protocol was achieving efficient RNA extraction from only 20ul of dried blood. Leuenberger and his colleagues adopted a two-step purification; beginning with a phenol:chloroform extraction on the DBS followed by a further purification on the Maxwell® RSC automated instrument, using the Maxwell RSC miRNA Serum and Plasma kit. Switching from a manual to an automated method for the second step was crucial. It reduced chances of contamination as well reduced pipetting errors, without compromising good quality and yield of RNA therefore contributing to assay reproducibility. To normalize volumes within the blood spot, the protocol uses RNA produced by housekeeping genes. The work to automate the assay has been published in Bioanalysis.
This protocol is being tested to see if microdosing of EPO or small transfusions can also be detected by monitoring ALAS2 RNA expression in DBS. The Swiss laboratory of Doping Analysis is also in the process of developing a method to detect gene doping by isolating plasmid DNA from whole blood samples, using the Maxwell RSC.
Additionally, the collection and storage methods used have implications for the clinic, especially for patients that need routine blood monitoring. The ability to isolate circular RNAs shows promise in forensic applications to identify body fluids.
Laboratories can be crowded places. We are used to working around other people, tossing ideas back and forth. Dark rooms, cold rooms and large equipment spaces are often shared by several labs. Some labs have shut down completely in response to the COVID-19 pandemic; others, especially those labs doing research around coronavirus biology, testing and detection and drug development are running continually. For those labs, maintaining the recommended 6-foot (2m) distance to help stem the coronavirus pandemic isn’t easy.
At Promega our operations, quality assurance, applications and research and development labs are up and running—focused on providing as much support as possible to our partners who are studying, diagnosing and developing treatments for COVID-19. At the same time, we are maximizing the safety of our employees. Here are a few ways we have found to maintain critical distances in our laboratory that might help your lab group stay productive and safe too.
This blog was written with much guidance from Jennifer Romanin, Senior Director IVD Operations and Global Service and Support, and Ron Wheeler, Senior Director, Quality Assurance and Regulatory Affairs at Promega.
A Trip Down Memory Lane
Back in the day when we all walked two miles uphill in the snow to get to our laboratories, RNA and DNA extraction was a home-brew experience. You made your own buffers, prepped your own columns and spent hours lysing cells, centrifuging samples, and collecting that fluorescing, ethidium bromide-stained band of RNA in the dark room from a tube suspended over a UV box. Just like master beer brewers tweak their protocols to produce better brews, you could tweak your methodology and become a “master isolater” of RNA. You might get mostly consistent results, but there was no guarantee that your protocol would work as well in the hands of a novice.
Enter the biotechnology companies with RNA and DNA isolation kits—kits and columns manufactured under highly controlled conditions delivering higher quality and reproducibility than your home-brew method. These systems have enabled us to design ever more sensitive downstream assays–assays that rely on high-quality input DNA and RNA, like RT-qPCR assays that can detect the presence of a specific RNA molecule on a swab containing only a few hundred cells. With these assays, contaminants from a home-brew isolation could result in false positives or false negatives or simply confused results. Reagents manufactured with pre-approved standard protocols in a highly controlled environment are critical for ultra sensitive tests and assays like the ones used to detect SARS-CoV-2 (the virus that causes COVID-19).
The Science of Manufacturing Tools for Scientists
There are several criteria that must be met if you are
producing systems that will be sent to different laboratories, used by
different people with variable skill sets, yet yield results that can be
compared from lab to lab.
Chris had extreme leg pain off and on for about a month. Pain that came and went, creeping in slowly but sometimes with extreme intensity. Based on x-rays an orthopedist diagnosed a torn hamstring that was on the mend. We were sent home to rest and ice his muscles.
One Sunday Chris played in the pool for 5 hours straight and didn’t wince once. The following week he was fine so he went to soccer practice on Wednesday and swim team practice the next day. At 11:30 that night he woke up screaming in pain. Same leg. Same spot. Back again.
Late June 2016
We were on vacation in Greece. The pain started again, severe and intense and scary, so bad he couldn’t sleep lying down in a bed. Desperate, we ended up in a Greek hospital… the local pediatrician was wonderful and recommended we fly home and see an orthopedic doctor as soon as possible…a terrifying flight home: No answers and a pit in our stomachs. Chris was in a wheelchair.
We finally got the orthopedist to order the MRI. The MRI results were what every parent fears: “leukemia or lymphoma” and a referral to an oncologist. After many invasive tests, the oncologist said it was probably not cancer. We felt such relief, but we were left with no answers for all his pain. We moved on to infectious disease.
The infectious disease specialist said they could not culture
anything so they didn’t believe that Chris had an infection. Again, incomplete
answers. We were then passed off to
rheumatology. The frustration of not
having any answers and our child still in pain was heart-breaking, isolating,
Based on the bone biopsy and MRIs the rheumatologists
finally gave Chris a diagnosis: Chronic Recurrent Multifocal Osteomyelitis
(CRMO often pronounced “chromo” for short).
The good news: it was not cancer; the bad news: very little is known about CRMO because it is a rare disease.
The Virology lab at the Universidade Federal da Bahia (UFBA), led by Dr. Gúbio Soares, has developed a fast and specific real-time PCR assay using GoTaq 1-Step RT-qPCR for detection of SARS-2-CoV (the coronavirus previously named 2019-nCoV), which causes the respiratory disease COVID-19. The Maxwell RSC instrument is used for automated extraction of RNA from oral-pharyngeal secretion collected by swab or bronchial wash prior to the assay. This coronavirus-specific assay can shorten the time to identify SARS-2-CoV from 48 hours to 3 hours (1), providing critical information to public health officials in a timely fashion.
“Promega has been providing all our reagents for standard and real-time PCR and also for nucleic acid extraction. It’s a company I can rely on the relationship; they are our partners and have provided excellent support both technically and financially. Promega is the base of all our assays.” Dr. Gúbio Soares.
Dr. Soares’ laboratory has experience developing assays to identify and detect emerging viral pathogens. Their laboratory first identified the Zika virus as the etiologic agent in the large outbreaks of acute exanthematous illness (AEI) in northeast Brazil in April 2015 (2). Zika was eventually declared a public health emergency of international importance by the World Health Organization in February 2016, after increased incidence of microcephaly was detected in the infants of women infected during pregnancy. Many of the lessons learned in the management of the Zika crisis are informing how scientists are addressing SARS-2-CoV. The Zika response was characterized by a collaborative spirit to share data, samples and resources among scientific labs across the globe.
Once the purview of virology researchers, the word “coronavirus” is now part of the vernacular in the mainstream media as reports of quarantined cruise ships (1) and makeshift hospitals (2) fill our online news feeds. While there is currently no approved anti-viral treatment for coronavirus infection (3), a team led by researchers from Vanderbilt University recently published work characterizing the anti-CoV activity of a compound, which they now plan to test against 2019-nCoV (4).
Developing New Therapeutics Against Coronaviruses
Coronaviruses (CoVs) are enveloped, single-stranded RNA viruses that exhibit cross-species transmission—the ability to spread quickly from one host (e.g., civet) to another (e.g., human). Scientists classify CoVs into four groups based on the nature of the spikes on their surface: alpha (α), beta (ß), gamma (γ) and delta (δ, 1). Only the alpha- and beta-CoVs can infect humans. Four coronaviruses commonly circulate within human populations: Human CoV 229E (HCoV229E), HCoVNL63, HCoVOC43, and HCoVHKU1. Three other CoVs have emerged as infectious agents, jumping from their normal animal host species to humans: SARS-CoV, MERS-CoV and most recently, 2019-nCoV (5).