Researchers at UC Santa Cruz have developed a novel chip-based antigen test that can provide ultrasensitive detection of SARS-CoV-2 and influenza A, the viruses that cause COVID-19 and flu, respectively.
The test is sensitive enough to detect and identify individual viral antigens one by one in nasal swab samples. This ultrasensitive technique could eventually be developed as a molecular diagnostic tool for point-of-care use.
The researchers reported their findings in a paper published May 4 in Proceedings of the National Academy of Sciences.
“This is a chip-based biosensor capable of detecting individual proteins one at a time, and we show how it can be used to detect and identify the antigens for multiple diseases at the same time,” said senior author Holger Schmidt, professor of electrical and computer engineering at UC Santa Cruz.
“It’s a whole new way of looking for molecular biomarkers, not only for infectious diseases, but for any protein biomarkers used in medical testing,” added Schmidt, who holds the Kapany Chair in Optoelectronics and directs the W. M. Keck Center for Nanoscale Optofluidics at UCSC’s Baskin School of Engineering.
The current gold standard for diagnosing SARS-CoV-2 infections uses PCR technology to amplify small amounts of the virus’s genomic material, and samples are analyzed in centralized laboratories such as UCSC’s Colligan Clinical Diagnostic Laboratory.
Antigen tests, which detect viral proteins, are faster and easier to use and have been approved for testing at the point of care (e.g., doctor’s offices) and even for at-home use, but these tests are not considered accurate enough for clinical decision-making, and their results may require confirmation with a more reliable technique.
The new chip-based antigen test is not only highly sensitive, but also enables simultaneous testing for multiple viruses from one sample. This is important for diseases such as COVID-19 and flu which have similar symptoms. Measures implemented to control the COVID-19 pandemic have reduced the incidence of flu dramatically, but in the future doctors may need a rapid test that can tell them which respiratory virus a patient is infected with.
Schmidt’s lab, in collaboration with coauthor Aaron Hawkins’ group at Brigham Young University, has pioneered “optofluidic chip” technology for biomedical diagnostics, combining microfluidics (tiny channels for handling liquid samples on a chip) with integrated optics for optical analysis of single molecules.
To develop the new antigen test, Schmidt’s team designed a fluorescent probe bright enough that individual markers can be detected optically on the chip. “The ability to detect individual markers means there is no need for an amplification step, which removes some of the complexity of the processing,” he explained.
Schmidt’s lab had been developing tests for other infectious diseases when COVID-19 emerged as a global pandemic last year. At first, research ground to a halt as a statewide shutdown kept everyone at home.
But it was clear to Schmidt that the diagnostic technology his lab was developing for Zika virus and other infectious diseases could be adapted for COVID-19.
“Once we were allowed to come back to the lab for essential research, my students started coming in to work in the lab by themselves on a coronavirus test,” Schmidt said. “It was a heroic effort by my students to develop these tests from scratch. First we were shut down by the pandemic, and then the wildfires hit and we had to evacuate our samples to Stanford and shut down again. But they kept going.”
Graduate student Alexandra Stambaugh led the effort and is first author of the paper. The team worked with the campus diagnostic lab to obtain nasal swab samples for testing. They only used samples that had tested negative for the coronavirus, adding viral antigens to the samples at clinically relevant concentrations to validate the tests.
The test uses an “antibody sandwich” approach commonly used for immunoassays. In this case, antibodies specific for the target antigen are attached to magnetic microbeads, so that any target antigen present in the sample sticks to the beads. After washing, a second antibody with the fluorescent marker attached is added, and it binds to any target antigen present on the beads.
The fluorescent markers are attached to the antibodies by a spacer that can be cleaved by ultraviolet light, which releases the markers to flow through the detection chip where they are detected one by one. The researchers attached a green marker to the coronavirus antibody and a red marker to the influenza antibody to distinguish between the two viruses.
The past century has witnessed several pandemics disrupting the socio-economic harmony of humankind. The influenza pandemic of 1917 and the current novel coronavirus pandemic are examples of the devastation caused by newly evolved viruses.
The short replication time of viruses helps them acquire zoonotic potential through numerous mutations over a short period. Influenza is a substantial threat to public health as it caused multiple catastrophic pandemics killing millions of people around the world. It has a segmented genome and relatively high mutation rate, leading to better survivability(1) and evolvability(2).
This eight segmented negative-sense single-stranded RNA virus from the Orthomyxoviridae family naturally infects diverse species, including birds and mammals. Mixing up or exchanging various genomic segments (antigenic shift) during co-infection in an intermediate host can result in the emergence of novel viral strains(3).
Apart from the pandemics, seasonal Influenza virus infections account for more than 5 million cases annually, severely affecting children and older adults(4, 5). An effective therapeutic strategy against emerging influenza virus strains is perplexing because it mutates very fast and subverts host immunity and cellular machinery. However, novel therapeutic approaches targeting host factors, essential for establishing viral infection, can prove to be more effective.
The RNA genome of Influenza A virus (IAV) in infected cells is sensed by evolutionarily preserved germline-encoded pathogen recognition receptors (PRRs), including Toll-like Receptors (TLR) 3 and 7(6), Retinoic acid-inducible gene I (RIG-I)(7), NOD-like receptor family member NOD-, Leucine-rich repeat (LRR)-and pyrin domain-containing 3 (NLRP3)(8), and the Z-DNA binding protein 1 (ZBP1)(9) in distinct cellular compartments. Upon sensing, the PRRs elicit an array of signaling pathways leading to the innate antiviral state through robust production of type I, III interferons and pro-inflammatory cytokines via different transcription factors like nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and interferon regulatory factors (IRFs).
Interferons further sensitize neighboring cells by inducing Interferon Stimulated Genes (ISGs) and collectively develop a potent antiviral state. In addition to this complex innate immune signaling cascade, interferons and pro-inflammatory cytokines also induce programmed cell death in virus-infected cells.
Programmed cell death is classified into various types based on the cues leading to cell death and macroscopic morphological variations. It has been observed in-vitro and in-vivo that the IAV induces apoptosis(10), primary necrosis(11), necroptosis(12), and pyroptosis(13, 14) in various cell types. Virus-associated programmed cell death was initially perceived as a host defense mechanism that limits viral replication by eliminating infected cells. However, recent studies indicate that IAV can manipulate host immunity to induce cell death, helping its propagation.
The IAV proteins NS1(14–16), M1(17), PB1-F2(10, 18), and NP(19) have been reported to activate apoptotic pathways to evade inflammatory responses and defend their replicative niche. The types of cell death pathways elicited upon IAV infection are mostly known. However, the innate immune sensing and signaling pathways deciding the fate of the cell upon IAV infection remain poorly understood.
This study reports a novel role of well-known PRR Interferon Gamma Inducible protein (IFI) 16 in eliciting cell death in alveolar epithelial cells. IFI16 is an intracellular DNA sensor mediating TBK-1-dependent IFNβ production via an adaptor STING. One of the AIM2-like Receptor (ALR) family member, IFI16, contains an N-terminal Pyrin domain (PYD) and two C-terminal HIN domains that bind to DNA in a sequence-independent manner(20). IFI16 was thought to be a cytosolic sensor, but recent studies show that it contains a multipartite nuclear localization signal (NLS) and senses nucleic acid in cytoplasm and nucleus in a PAMP-localization-dependent manner(21).
IFI16 plays a critical role during various DNA viruses (KSHV(22, 23), HSV-1(20), EBV(24), HCMV(25)), retrovirus (HIV(24)), and bacterial infections (Listeria Monocytogenes(26)) by restricting pathogens’ propagation. Several DNA virus proteins have evolved to inhibit or degrade IFI16, highlighting its vital role in defense against infections(25, 27). Recent studies show that IFI16 can also restrict various RNA virus infections (Sendai(28), EMCV(28), CHIKV(29)).
However, the mechanism by which IFI16 defends against RNA viruses remains elusive, especially about IAV infection. Through high-throughput transcriptomic analysis after IFI16 knockdown, we uncovered the mechanistic insights about how IFI16 protects the host against the IAV. Our study shows that IFI16 restricts the IAV infection by sensing viral RNA (predominantly in the nucleus) and stimulating cell death.
reference link : https://www.biorxiv.org/content/10.1101/2021.02.13.431067v1.full
More information: Alexandra Stambaugh et al, Optofluidic multiplex detection of single SARS-CoV-2 and influenza A antigens using a novel bright fluorescent probe assay, Proceedings of the National Academy of Sciences (2021). DOI: 10.1073/pnas.2103480118
