The document discusses host immune responses and diagnostic approaches for SARS-CoV-2. It outlines the innate and adaptive immune responses to the virus, including the roles of cytokines, dendritic cells, and T cells. It also describes several molecular diagnostic techniques for SARS-CoV-2, including RT-PCR, CRISPR, lateral flow immunoassays, ELISA, and CLIA tests that detect viral RNA or antibodies. The accuracy of diagnostic tests can be impacted by viral load and sample type.

1. HOST RESPONSES, AND DIAGNOSTIC APPROACHES
OF SARS-COV-2
IUG 2021
1
Prof.Abdelraouf Elmanama
by
Ahmed A. Abdallah

2. Outlines
• Introduction
• Immune responses to SARS-CoV-2
• Molecular and immunological diagnostic techniques.
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3. INTRODUCTION
■ Coronaviruses (CoVs) are enveloped, single-stranded RNA viruses (ssRNA), that belong to
the the Coronaviridae family, subfamily Orthocoronavirinae.
■ Currently, they are considered as the main groups of viruses that cause acute respiratory
infections, and one of them, SARS-CoV-2 is responsible for the pandemic that has affects
public health worldwide.
■ CoVs infect the respiratory tract and cause diseases varying from mild to moderate clinical
manifestations to the most serious forms that can be fatal.
■ The severity of the disease caused by SARS-CoV-2 can be modulated by several factors
related to the virus-host interaction, such as viral load, immune response, age, and
presence of comorbidities
■ The presence of comorbidities such as diabetes, obesity, heart and kidney failure, among
others, is also associated with severe disease.
■ Factors involved in virus-host interaction, such as viral load, viral evasion to host immune
responses, and the exacerbated acute inflammatory response, can play an important role
in defining the severity of the disease.
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4. INTRODUCTION
■ A characteristic of CoVs is their rapid viral replication. The virus can be detected
in respiratory tract secretions (bronchoalveolar aspirate, tracheal aspirate and
nasal secretions), feces, urine and blood (serum/plasma/whole blood).
■ Although elevated viral load may contribute to disease severity,
■ There are suggested studies suggesting that increasing secretion of pro-
inflammatory cytokines such as IL-2, IL-7, IL-10, G-CSF, IP-10, MCP1, MIP1A
and TNF-α, associate with severity of disease.
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5. Immune response
■ Innate immune response is essential for the control and resolution of the disease.
■ An exacerbated acute inflammatory response generates an imbalance of the innate immune
system with the massive production of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α and
other chemokines that can contribute to the severity of the disease, increasing morbidity and
mortality.
■ A major cellular entry point is the angiotensin-converting enzyme 2 (ACE2) which consider as
surface receptor for both SARS-CoV and SARS-CoV-2.
■ SARS-CoV-2 binds with its spike (S) protein to the angiotensin-converting enzyme-related
carboxypeptidase-2 (ACE-2) receptor on the host cell.
■ TheACE-2 receptor is widely expressed in pulmonary and cardiovascular tissues, hematopoietic cells,
including monocytes and macrophages which may explain the broad range of pulmonary and extra-
pulmonary effects of SARS-CoV-2 infection including cardiac, gastrointestinal organs, and kidney
affection.
■ The epithelial cells of the respiratory airways is mainly the infected site.
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6. Immune response
■ Following CoVs entry into human host cells, viral RNAs are released and act as
pathogen-associated molecular patterns (PAMPs), which are recognized by pattern
receptors (PRRs) as toll-like receptors (TLR3, TLR7 and TLR9) and retinoic acid-
inducible (RIG-I) type I receptors.
■ This recognition event leads to activation of the downstream signaling cascade, i.e.
NF-κB and IRF3, accompanied by their nuclear translocation
■ Expression of type I IFN and other pro-inflammatory cytokines initiate responses
comprise the first line defense against viral infection at the entry site.
■ A successful mounting of this type I IFN response should be able to suppress viral
replication and dissemination at an early stage.
■ the transcription of genes and expression of acute inflammatory response proteins,
such as C-reactive protein (CRP), pro-inflammatory cytokines and chemokines (like
IL-1, IL-6, TNF-α, MPC-1, IL-8 and IP-10), as well as secretion of soluble factors that
encodes interferons (IFNs), triggering an antiviral state.
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7. Immune response
■ Dendritic cells (DCs) constitute a heterogeneous family of cells involved in the
innate and adaptive immune response.
■ DCs are the main antigen presentation cells (APCs) involved in the capture,
processing and presentation of viral peptides associated with class II and class I
major histocompatibility complex (MHC) molecules for CD4 + and CD8 + cells.
■ pDCs, secrete high levels of both type I and type III IFNs. These cells also
express cellular PRR receptors (TLR7, TLR9 and RIG-1) that can detect CoV
nucleic acids (ssRNA) and rapidly induce high-level secretion of type I IFNs,
especially IFN-α, and high levels of type III IFNs.
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8. Immune response
■ mDCs, are also capable of secreting pro-inflammatory cytokines, such as IL-6,
which is a potent cytokine that stimulates the synthesis of acute phase
inflammation proteins.
– IL-6 also induces activation of the alternative macrophage (M2) pathway,
inducing fibrosis in lung tissue.
– It induces a cellular immune response mediated by CD4 + Th cells which
generates an immune response rich in neutrophils and monocytes.
– Thus, DCs through IL-6 secretion also contribute to exacerbating the acute
inflammatory response and disease severity.
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9. Molecular and immunological diagnostic
techniques
■ Two main detection strategies are currently available for the diagnosis of SARS-CoV-2
either via the detection of the viral RNA or the antibody produced upon exposure to
the infection.
■ The SARS-CoV-2 viral RNA is usually detected by polymerase chain reaction (PCR) or
nucleic acid hybridization techniques.
■ The virus antibody or antigens can be detected using immunological and serological
assays.
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10. ■ The virus is enveloped and spherical (~120 nm in diameter), with petal-shaped surface
spikes (~20 nm long).
■ Key structural proteins, spike (S), envelope (E), and membrane (M), are anchored on
the viral envelop.
■ The nucleocapsid (N) protein, together with the genomic RNA, forms a helical
nucleocapsid inside the envelop.
■ The virus enters host cells through the binding of S protein to angiotensin-converting
enzyme 2 (ACE2) on the cell surface.
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11. Diagnostic Approaches for COVID-19
■ 1.Virological/molecular tests
– RT-PCR
– Point-of-care RT-PCR
– LAMP, CRISPR
■ 2. Immunologic tests
– LFIA (for antibodies/antigens)
– Traditional ELISA
– CLIA
– Neutralization assay
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12. 1. RT-PCRTest
■ Reverse transcription polymerase chain reaction (RT-PCR) was the first method developed for COVID-
19 detection and is the current gold standard.
■ There are two essential steps in the process: (1) viral RNA extraction and (2) PCR amplification and
probe-based detection.
■ In this technique, reverse transcriptase (RT) is used to convert viral RNA targets into complementary
DNA (cDNA), and then the resulting cDNA is amplified by conventional PCR.
■ Conventional RT-PCR demonstrated overall sensitivity ranging from 73% to 100% and specificity
ranging from 99% to 100% in the detection of viral infection
■ false-negative results due to low sample volumes, variable sampling techniques and sampling
locations, sample degradation during transportation,
and/or improper nucleic acid extraction are a concern.
■ Given the high expression of ACE2 on alveolar epithelial cells and negative expression on nasal, oral,
and nasopharynx cells, it would be prudent to perform Broncho alveolar lavage on patients to rule out
false-negative results from swabs of upper respiratory tract samples.
■ the method is not used commonly in clinical specimens owing to its high cost and time-consuming
process
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13. 1. RT-PCRTest
■ DNA polymerase enzyme, extracted DNA Samples, primers and deoxynucleoside
triphosphates are the essential components of a PCR test kit.
■ Reverse transcription PCR (RT-PCR) is a type of PCR methods that uses reverse
transcriptase enzyme to convert RNA molecules to cDNA molecules.
■ Then cDNA works as a template sequence for the PCR reaction.
■ Quantitative PCR determines a DNA molecule with the help of fluorescent dye
■ A typical RT-PCR method includes four steps.
1- RNA isolation followed by cDNA synthesis with reverse transcription kit.
2- Mixing buffer, DNA polymerase enzyme, primers of a target gene, deoxynucleoside
triphosphate, cDNA template and fluorescent dye.
3- Incubation of the mixture at different temperatures to perform thermal cycling in PCR
instrument and fluorescence measurements for calculatingCycle threshold (Ct) data.
4- Relative expression estimation based on Ct data of control
and experimental samples
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14. 1. RT-PCRTest
■ Analytical accuracy of COVID-19 RT-PCR relies primarily on the primer design. Due to high genomic
similarity among different coronavirus species, identifying unique gene sequences is important to eliminate
cross-reactivity.
■ Viral targets are selected from E, N, S, and Orf1ab regions of SARS-CoV-2 genome.
■ RT-PCR offers both high accuracy and throughput.The limit of detection (LOD) was reportedly down to 4–8
copies of the virus upon amplification of Orf1ab, E, and N genes at 95% confidence intervals.
■ Specificity of a test is enhanced by targeting multiple loci.
■ Indeed, the US-CDC diagnostic recommends the use of two targets (N1, N2) in N gene and human RNAse P
(RNP) gene as a control.
■ The metric for COVID-19 diagnosis is the cycle threshold (Ct).
■ A Ct value less than 40 is clinically reported as PCR positive.
■ Viral RNA loads become detectable as early as day 1 of symptom onset and peak within a week.
■ The positivity declines by week 3 and subsequently becomes undetectable.
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15. 15

16. 16

17. The sensitivity of RT-PCR tests may be affected by particular issues:
■ variable viral loads depending on sample types,
■ time of infection,
■ sample collection (i.e. nasopharyngeal vs. oropharyngeal, upper vs. lower respiratory
tract),
■ conservation and transport,
■ And different gene targets.
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18. 2. CRISPR-Based Detection
■ Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)
■ CRISPR is another promising isothermal amplification method for the detection of viruses.
■ CRISPR represents a family of nucleic acid sequences found in prokaryotic organisms, such as bacteria.
■ These sequences can be recognized and cut by a set of bacterial enzymes, called CRISPR-associated
enzymes, exemplified by Cas9, Cas12, and Cas13.
■ Certain enzymes in the Cas12 and Cas13 families can be programmed to target and cut viral RNA sequences
■ In this method, CRISPR-Cas12 and lateral flow technology were coupled for the rapid detection of SARS-
CoV-2 from nasopharyngeal or oropharyngeal swabs.
■ Most advanced form of these assays use Cas12a (CRISPR-associated protein 12a) or Cas13a (CRISPR-
associated protein 13a) enzymes, exploiting collateral cleavage of single-stranded DNA (Cas12a) or RNA
(Cas13a) by these nucleases.
■ Two companies, Mammoth Biosciences and Sherlock Biosciences, are independently exploring the
possibility of using the gene editing CRISPR methodology for detection of SARS-CoV-2.
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19. ■ The SHERLOCK method developed by Sherlock Biosciences uses Cas13 that is capable of
excising reporter RNA sequences in response to activation by SARS-CoV-2-specific guide
RNA.
■ The DETECTR assay by Mammoth Biosciences relies on the cleavage of reporter RNA by
Cas12a to specifically detect viral RNA sequences of the E and N genes, followed by
isothermal amplification of the target, resulting in a visual readout with a fluorophore.
■ Cas12 targets ssDNA, and dsDNA.While cas13 targets ssRNA only.
■ This method showed high sensitivity and selectivity in RNA extracts compared to RT-PCR.
■ This assay is low-cost, relatively rapid, can be performed in as little as 1 h. and thus, has
great potential for POC diagnosis of COVID 19.
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20. ■ Viral RNA is extracted and subject to isothermal amplification by primers that target the E and the N2
gene of SARS-CoV-2.
■ If the Cas12a-sgRNA complex binds to the target sequences, Cas12a activity gets activated.
■ This unleashes indiscriminate ssDNA cleavage activity which cleaves the supplied fluorescently
labelled ssDNA substrates.
■ Upon cleavage, the fluorescence is no longer quenched and can be visually detected using lateral flow
strips.
■ Since the probes are tagged with streptavidin, the lateral flow strip first captures the streptavidin
portion of the probe via biotin.
■ The fluorescent portion of the probe that is cut will continue to flow and be captured by an antibody
further along on the strip.
■ Sensitivity:10-100 copies of RNA per microliter
■ Time: ~40 minutes
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22. 22

23. SerologicTests
■ While RT-PCR-based viral RNA detection has been widely used in diagnosis of COVID-19, it
cannot be used to monitor the progress of the disease stages and cannot be applied to
broad identification of past infection and immunity.
■ Serological test are methods that rely on the detection or quantitation of antigen/antibody
interactions.
■ Serology plays a key role in contact tracing, epidemiological studies, identification of
convalescent plasma donors, vaccine development, assessment of both short-term (days
to weeks) and long-term (years or permanence) trajectories of antibody response, as well
as antibody abundance and diversity.
■ In some of those “high clinical-suspicion-RT-PCR-negative cases”, antibodies detection
could be a helpful tool in COVID- 19 diagnosis.
■ IgM first becomes detectable in serum after a few days and lasts a couple of weeks upon
infection and is followed by a switch to IgG.
■ Thus, IgM can be an indicator of early stage infection, and IgG can be an indicator of
current or prior infection. IgG may also be used to suggest the presence of post-infection
immunity.
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24. SerologicTests
■ For SARS-CoV-2, the detection of both antibodies IgM and IgG could be detected 3–4 days after
premorbid, respectively.
■ However, some studies have shown that the number of positive tests for IgG was higher than IgM
after symptom onset.
■ Some immunological assays have been developed to detect theCOVID-19 virus.
■ Twenty synthetic peptides from ORF1ab, spike glycoprotein (S1 and S2 subunits, receptor-
binding domain) and nucleocapsid (N) proteins were chosen as antigens.
■ One peptide from S protein has shown the best results.
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25. Lateral Flow Immunoassay (LFIA)
■ This test is typically a qualitative (positive or negative) chromatographic assay.
■ LFIA is small, portable, and used at the point-of-care.
■ The test is a type of rapid diagnostic test (RDT) as the result can be obtained in 10-30 min.
■ In practice, Samples move via capillary flow on the nitrocellulose membrane.
■ When anti-SARS-CoV-2 antibodies are present, they bind to the labeled antigen and continue to
move until they are captured by the immobilized antihuman antibodies.
■ The presence of the captured antibody-antigen complex is visualized as a colored test band.
■ In data submitted to the FDA from a clinical study conducted byAbbott with several leading U.S.
research universities, the BinaxNOW COVID-19Ag Card demonstrated sensitivity of 97.1% and
specificity of 98.5% in patients suspected of COVID-19 by their healthcare provider within the first
seven days of symptom onset.
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26. 26

27. CLIA
■ An automated chemiluminescent immunoassay was developed to evaluate serum IgM and
IgG.
■ The magnetic particles were modified with SARS-CoV-2 antigens; S and N.
■ CLIA involves chemiluminescence and fluorescence.
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28. ELISA
■ Enzyme-linked immunosorbent assay (ELISA) is another developed method for the
detection of COVID-19.
■ ELISA is a sensitive method to detect the antigen or antibody and can be performed using
direct or indirect formats.
■ the plate wells are typically coated with a viral protein. If present, antiviral antibodies in the
patient samples will bind specifically, and the bound
antibody-protein complex can be detected with an additional tracer antibody to produce a
colorimetric or fluorescent-based readout.
■ ELISA is speedy, has the ability to test multiple sample, suitable for point-of-care
determinations, but the sensitivity can be variable.
■ The sensitivity of the ELISA was 87.3%
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29. 29

30. Neutralization assay
■ Neutralization assays determine the ability of an antibody to inhibit virus infection of
cultured cells and the resulting cytopathic effects of viral replication.
■ There are two types of antibody tests that aim for detecting COVID-19 infection with
sufficient specificity and sensitivity.The first type is the virus neutralization test (VNT)
which detects neutralizing antibodies (NAbs) in a patient's blood.
■ All other assays, such as ELISA and lateral flow rapid tests, represent the second assay type
which detect only binding antibodies, and not Nab.
■ Using purified receptor binding domain (RBD) protein from the viral spike (S) protein and
the host cell receptor ACE2, the test is designed to mimic the virus-host interaction by
direct protein-protein interaction in a test tube or an ELISA plate well.This highly specific
interaction can then be neutralized, i.e., blocked by highly specific Nabs in patient or
animal sera in the same manner as in a conventionalVNT.
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31. ■ The time to results for neutralization assays is typically 3-5 days, but recent advances have
reduced this to hours.
■ This type of testing requires cell culture facilities, and in the case of SARS coronavirus,
Biosafety Level 3 (BSL3) laboratories.
■ Despite these limitations, determination of neutralizing antibodies is important in the short
term for the therapeutic application of convalescent plasma and, in the long term, for
vaccine development.
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32. Ref.
■ Immune response in SARS-CoV-2 infection: the role of interferons type I and type III,
Caciane Portela Sousaa, and Carlos Britesb, Braz J Infect Dis. 2020 September-
October; 24(5): 428–433.
■ AssayTechniques andTest Development for COVID-19 Diagnosis, Linda J. Carter,
LindaV. Garner, Jeffrey W. Smoot,Yingzhu Li, Qiongqiong Zhou,Catherine J. Saveson,
Janet M. Sasso,Anne C. Gregg, Divya J. Soares,Tiffany R. Beskid, Susan R. Jervey and
Cynthia Liu.
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