Imaging Viral Entry and Replication for SARS-CoV-2
Understanding and assessing immune memory is crucial for protective immunity
Part of the global response to manage and contain the pandemic involved the generation of research data to guide evidence-based responses to the prevention of transmission and containment of the virus. As SARS-CoV-2 is spread via respiratory droplets and physical contact, major efforts were made toward precautionary measures surrounding hand hygiene and personal protective equipment both for the public and those on the frontlines. A gradual systematic shift toward data collection, contact tracing, testing, and, most importantly, the approval and widespread distribution of vaccines along with other public health measures have helped control the pandemic. However, the arrival of novel variants of SARS-CoV-2 and their alarming spread means the pandemic is here to stay for the foreseeable future.
Various strategies have been utilized in the development of vaccines for SARS-CoV-2 including attenuated live pathogen, inactivated pathogen, protein subunit, polysaccharide, conjugate, virus-like particles viral-vectored, and nucleic acid vaccines. The primary goal of vaccinations has been to protect from severe disease, provide long-term memory immune responses after a minimal number of booster shots, and prevent infection among vaccinated populations and immunocompromised individuals. mRNA vaccines such as Pfizer and Moderna, have been federally approved and have shown great efficacy for protection against the virus. These vaccinations administer viral antigens that can induce strong, protective neutralizing antibodies against the target pathogen or viral spike protein. The goal is to establish sterilizing immunity for vaccinated individuals.
Sterilizing immunity is when virus infection in a person is completely inhibited, and transmission of the virus is prevented. Unlike innate, B-cell, and T-cell mediated immunity that allow infection, sterilizing immunity effectively controls and destroys the pathogen. Yet, evidence has also suggested that T-cell-mediated responses to SARS-CoV-2 are equally important and provide longer-lasting immunity compared to B-cells. Thus, strategies for vaccine-induced strong cellular responses are also being considered. Researchers are now focused on the development of vaccines that can trigger sustained long-term antibody responses to prevent reinfections. In charting a course through the pandemic, the development of multiple vaccination strategies employing diverse vaccine delivery systems is crucial to thwarting the emergence of novel viral variants, minimizing viral loads, preventing infection, and generating long-term immune memory responses.
Advancing SARS-CoV-2 research the Thermo Fisher Scientific way
Untangling the stages of the viral life cycle and pathogen biology is key to developing treatments for the virus and its variants while also helping researchers understand the cell conditions that are conducive to virus production. Thermo Fisher Scientific is at the forefront of vaccine development by offering a complete portfolio of tools and technologies to enable research on SARS-CoV-2, thus supporting collaborative efforts and accelerating lifesaving discoveries. Elucidating the viral lifecycle is essential to studying how an infection progresses. Through its provision of an array of technologies, Thermo Fisher Scientific has aided in the investigation of the biology of SARS-CoV-2 through the major stages of viral replication: attachment, entry and uncoating, replication, assembly, and release.
High-content imaging from Thermo Fisher Scientific systems and reagents assist researchers in fully characterizing the molecules and signaling pathways implicated in SARS-CoV-2 entry. This is key to understanding viral infectivity, tropism, pathogenesis, immune evasion, and neutralization. Viral attachment and entry involve viruses accessing intracellular sites of replication. Cellular membranes present the first line of defense for entry against the virus into the host cell. Viral-encoded capsid or envelope proteins overcome this barrier using appropriate binding partners for host cell receptors. The specificity of this binding process not only defines the cell but also the disease. For the SARS-CoV-2 virus, this is facilitated by the Spike (S) protein. Neutralizing antibody assays are essential in this aspect as they enable the testing of antibodies to neutralize and prevent virions from infecting cultured cells.
Alongside the provision of antibodies for SARS-CoV-2 detection and research, for viral and host targets alike, and real-time PCR assays, a broad platform involving high-resolution and high-throughput single-cell imaging products and technologies from Thermo Fisher Scientific means researchers can track the internalization or entry mechanisms of the SARS-CoV-2 viral particle. All the way from large-scale genomics and proteomics to target analysis, the extensive portfolio of solutions from Thermo Fisher Scientific allows researchers to study host-pathogen interactions and understand disease progression at a deeper level than ever before.
Assessing immunological memory to SARS-CoV-2
These efforts are evident in recent studies concerning the assessment of immune memory for protective immunity. Immune memory is fundamental for protective immunity after infection or vaccination and consists of memory B cells, antibodies, memory CD4+ T cells, and/or memory CD8+ T cells. Knowledge of the dynamics and interrelationships between these four types of memory cells can help in the development of protective immunity against SARS-CoV-2 and assess the course of the pandemic.
Two such studies consider the use of neutralizing antibody assays to assess general immune system memory and tissue-specific immune memory post-infection. The former measured antigen-specific antibodies, memory B cells, CD4+ T, and CD8+ T cells in blood from subjects who had recovered from SARS-CoV-2 up to 8 months after infection, with substantial immune memory being discovered in all four major types of immune memory. The latter study related the specific sites of memory generation in response to infection in the bone marrow, spleen, lung, and multiple lymph nodes with results indicating local tissue coordination of circulating and tissue-resident memory T and B cells against SARS-CoV-2 for site-specific protection against future infections. A third study would identify and characterize a small molecule compound N-0385 that provided a high level of prophylactic and therapeutic benefit in early treatment options against SARS-CoV-2 and emerging variants.
All three studies feature the use of multiple assets from Thermo Fisher Scientific including consumables, imaging dyes and reagents, and high-content screening platforms. These tools proved instrumental for extensive cell analysis, characterization, and imaging to understand the dynamics of SARS-CoV-2 viral entry into cell nuclei and devise means to impede it, moderate viral lifecycles, and identify biomolecules with the potential for therapeutic application. Stain kits and reagents from Thermo Fisher Scientific, such as the Alexa Fluor dyes, provided a wide range of applicability and inherent versatility in their application to analyze disease progression and associated biomarkers. Coupled with imaging enabled by the CellInsight CX5 and CX7 platforms from Thermo Fisher Scientific, researchers were able to quantitate the total number of cells, based on nuclei staining using stain kits from Thermo Fisher Scientific, and the number of virus-infected cells, using nucleocapsid staining, and determine the percentage of infection in their assays. These assays were further supplemented by HCS Studio Cell Analysis Software from Thermo Fisher Scientific which measures sample cell fluorescence, quantifies cell loss, and verifies viral infection.
Conclusion
Repeated emergence and global transmission of novel coronaviruses have been a major cause for concern. SARS-CoV-2 has resulted in a significant number of deaths and has emphasized the importance of establishing integrated early warning and response systems to duly raise the alarm on novel and re-emerging pathogens. Technologies such as high-content screening, single-cell analysis, characterization, and imaging are critical to enabling pre-emptive and concurrent studies of such diseases.
The broad platform of tools including staining kits, assays, reagents, fluorescent dyes, and high-resolution and high-throughput imaging systems from Thermo Fisher Scientific allow researchers to study the intricate biologies of infectious pathogens, collect relevant data on viral structure and viral-host interactions, and develop vaccines and therapeutics, as well as improve existing and new strategies to protect against continually evolving viral strains.
Take advantage of our 40+ years of experience and expertise with end-to-end cell images products and resources, including relevant tools, protocols, selection guides, inspiration, and more at your fingertips. To learn more about cellular imaging and related technologies, visit the Thermo Fisher Scientific imaging resource center: www.thermofisher.com/cellularimaging.
