This document discusses serological approaches for detecting COVID-19 through immune-based detection of antibodies. It provides an overview of the SARS-CoV-2 virus, current diagnostic methods including RT-PCR and immunoassays, considerations around specificity and sensitivity of different viral targets for immunoassays, applications of immunoassays for surveillance and developing treatments, and the need for point-of-care tests. Moving forward, it emphasizes that collating immunoassay data and developing rapid decentralized tests can help address testing shortages, while ensuring rigorous evaluation of new tests.

1. Serological Approaches for COVID-19: Epidemiologic Perspective on Surveillance
and Control
By Mesele Tilahun Belete
University of Science and Technology,
Korean Research Institute of Bioscience and Biotechnology (KRIBB) Campus
Dec 21, 2022
KRISS
Course Instructor: 최 준혁 (Ph.D.)

3. Contents
1. Introduction to the Virus
2. Diagnosis SARSCoV-2
3. Current immune-based detection approaches against sars-cov-2
4. Specificity and Sensitivity of Immunoassays Against SARS-CoV-2
5. Identification of B-Cell Epitopes Against SARS-CoV-2 on Immunogenic Proteins
6. Applications of immunoassays to control sars-cov-2 transmission
7. Drawbacks of Serological Studies
8. The Way Forward
9. Conclusion

4. 1. Introduction to the Virus
 Ongoing pandemic and the most serious new health threats
 In March 2020, the WHO declared COVID-19 as pandemic,
- issued guidelines about clinical and epidemiological findings
 caused by SARS-CoV-2, a Beta-coronavirus closely related to the SARS virus.
 There after, 2019-nCOV was named SARS-CoV-2.
COVID-19
 family / genus Coronaviridae , Betacoronavirus,
Enveloped, spherical, Monopartite, linear ssRNA(+) genome
 Capped, and polyadenylated (guanosine nucleotide @5'-end of mRNAs and then, methylating the guanosine).
The leader RNA (65-89 bp) at the 5' end of the genome is also present at the end of each subgenomic RNAs.
• 10 or more nonstructural proteins (nsp proteins)
• structural proteins spike (S)
• envelope (E)
• membrane (M) and
• nucleocapsid(N)
 Genome size of ~30kb,
encodes for multiple structural proteins comprising

5. GENE EXPRESSION
gRNA encodes ORF1a, as for ORF1b, it is translated
by ribosomal frameshifting. Resulting polyproteins pp1a and
pp1ab are processed into the viral polymerase (RdRp) and
other non-structural proteins involved in RNA synthesis.
Structural proteins are expressed as subgenomic RNAs.
https://viralzone.expasy.org/764
Genome and proteins
-two-third of 5’genome occupied by the NS proteins
-one-third of 3’genome occupied by major structural proteins (S,E,M and N)
-Spike glycoprotein (S) key protein
-S-protein composed of two subunits, S1 and S2
-S1 much more variable while the S2 conserved
others
-lipid bilayer envelop, membrane proteins, and nucleocapsid protect the virus when it is outside the host cell
-lipid bilayer in which the M,E, and S structural proteins are anchored
-M protein crucial during the assembly, budding, envelop formation, and pathogenesis stages of the virus lifecycle.

6.  The clinical manifestations of COVID-19 form a spectrum, from being asymptomatic to fever with mild respiratory illness,
to acute respiratory distress syndrome, and death from respiratory failure or associated complications.
2. Diagnosis SARSCoV-2
 Incubation period varies among different patient cohorts,
 Difficult to ascertain the actual day of onset,
 Asymptomatic (pre-symptomatic) may go undetected
 Early detection: crucial interventions to control virus transmission
With the discovery of the virus, numerous diagnostic assays were developed.
 qRT-PCR: gold standard, high sensitivity & accuracy in the acute phase.
 viral RNA has been detected in both throat and nasal swabs
 which becomes almost undetectable by 14 days post-illness onset (pio)
(symptom onset)
 Costly and time consuming to perform,
 False negative results due to
-improper handling of nucleic acid samples,
-inadequate & variable sampling insufficient viral genetic material
 biological variation
 With the limitations of qRT-PCR, immunoassays may
offer another avenue to reduce undiagnosed cases,
with the advantage that rapid test formats may deliver
results in a relatively shorter time and lower cost

7.  SARS-CoV-2 viral RNA (pink) in throat or nasal
swab of patients are typically undetectable by 14 day
post illness onset (pio).
 IgM
-detectable as early as 3 days pio, and
-peaks between 2 and 3 weeks pio.
-it’s response was still detectable after >1 month pio
 Both IgA and IgG are
-present as early as 4 days pio, and
-peaks after 2 weeks pio in serum samples
 No reports on the presence of SARS-CoV-2-specific
antibodies in the later phase pio, indicated dotted lines.
 This depicts the importance of serological studies to
identify individuals with current or prior exposure
to SARS-CoV-2 that went undetected, by testing for
either IgM, IgG, or IgA antibodies against SARS-
CoV-2.
Fig.2 Schematic illustration on the window period of detection for either
viral RNA or antibodies in SARS-CoV-2-infected individuals.
SARS-CoV-2-specific antibodies (blue):

8.  Immunoassays: alternative diagnostic approach,
-provide information on active viral infections and past exposures (Fig. 2).
 To date, many serological assays have developed to detect SARSCoV-2 AB from patient serum/plasma samples.
3. Current immune-based detection approaches against sars-cov-2
• S protein: the most exposed viral protein, and
• N protein: abundantly expressed during infection.
• Receptor-binding domain (RBD): located along the S protein, is also
a target of interest to detect the presence of SARS-CoV-2-specific
antibodies
Main target immunogenic coronavirus proteins:
• Molecular targets with in their positive sense , ss RNA genome
• Include structural proteins: S, E, M, Hel, N,
• RdRp, HE (hemagglutinin-esterase), ORF1 and ORF1b
• CDC recommends two nucleocapsid targets (N1 and N2)
• WHO recommends initial screening with E followed by confirmation using the RdRp
• Ideal design would include at least one conserved region and
• one specific region to mitigate against the effect of genetic drift.

9. 4. Specificity and Sensitivity of Immunoassays Against SARS-CoV-2
Human population has prior exposure to endemic coronavirus infections including
 alphacoronaviruses (229E and NL63),
 betacoronaviruses (OC42 and HKU1),
 Sever acute respiratory syndrome-related coronavirus (SARCOV) identified in 2003)
 Middle East Respiratory Syndrome coronavirus (MERS-CoV) identified in 2012
 Sever acute respiratory syndrome-related coronavirus (SARCOV-2) identified in 2019)
 S protein antigen: cross-reactivity was observed with MERS-CoV protein when samples tested against SARS-CoV-S
 Interestingly, there was no cross-reactivity with the S1 subunit of MERS-CoV.
 The high level of cross reactivity b/n SARS-CoV and SARS-CoV-2 (attributed to high degree of genetic homology).
 S2 subunit domain: cross-reactivity observed with only the S protein of MERS-CoV, and not with the S1 subunit.
 These data suggest that using an S1 subunit-based immunoassay may be more specific than the entire S antigen
 The RBD: lies along the S protein is usually the target of many neutralizing antibodies against SARS-CoV.
 Due to high level of similarity (90%) bn SARSCoV & SARS-CoV-2 N proteins, the N antigen of SARS-CoV was
also used for serological detection of SARS-CoV-2-specific antibodies.
 These N-based assays were reported to be more sensitive than S1 subunit-based tests.

10. TABLE 1. Immune-based assays developed against different SARS-CoV-2 viral protein

11.  conformational B-cell epitopes mapped onto the S1 subunit  in few identical residues within SARS-CoV & SARS-
CoV-2.
 These indicate that SARS-CoV-specific AB targeting these discontinuous regions may not be able to cross-react with
SARS-CoV-2.
 To identify other SARS-CoV AB that may recognize the conformational epitopes of SARS-CoV-2 S protein, which can
greatly reduce the amount of time needed to develop novel neutralizing antibodies.
5. Identification of B-Cell Epitopes Against SARS-CoV-2 on Immunogenic Proteins
 Apart from immunoassays early detection of SARSCoV-2, is critical to determine the regions where SARS-CoV-2-
specific AB bind to help guide vaccine designs.
 Using SARS-CoV-derived B-cell epitopes that have been experimentally identified from positive B-cell assays,
 49 out of 298 linear B-cell epitopes have an identical match with SARS-CoV-2 protein sequences without any mutations.
• Majority of these matches were located at both S & N viral antigens,
• Only 4 from the M protein, and
• None in the E protein
 6 conformational B-cell epitopes identified from the same database were located on the S antigen.
 However, unlike the linear epitopes, none of these mapped identically to the SARS-CoV-2 protein.

12. 6. Applications of immunoassays to control sars-cov-2 transmission
 To support diagnosis, treatment, and prevention of SARS-CoV-2 infections.
 Determining the optimal time points to collect serum or plasma samples for immunoassay screening
 Generation of therapeutic MoAB from peripheral blood B cells
• CR3022 recognizes an epitope along the RBD of SARS-CoV-2
• MoAB 47D11: enabling cross-neutralization against SARS-CoV-2 infection.
• Reports on antibodies against the coronavirus E protein are scarce, possibly due to it being the smallest protein
 Todate, two human SARS-CoV-specific antibodies (CR3022 & 47D11), have been shown to recognize SARS-CoV-2
 Able to detect mildly infected cases, which is important to ascertain the extent of community spread.

13.  Inability to detect early stage infection
-antibodies take several days to be generated after exposure to foreign material.
-A recent infection may provide false negative results.
instead, RT-PCR may be more suitable to diagnose an early acute SARS-CoV-2 infection.
 Inherent variability of the antibody response
-due to the unique genetic makeup of each individual.
-This could possibly explain the d/ce in AB profiles elicited among individuals infected with SARS-CoV-2 .
 Cross-reactivity could potentially be a limitation of immunoassays as
-it severely impacts the specificity and sensitivity of the test.
7. Drawbacks of Serological Studies
While it is fast, robust and easy to perform, there are several limitations to serological assays.

14.  Rapid development of diagnostic tools & immune-based assays are important against the ongoing pandemic.
 Serological assays: that target a diverse range of viral antigen has no doubt assisted in accurate diagnosis.
 data generated through serological studies can greatly aid in supplementing the results from qRT-PCR,
 Contribute to sero-epidemiology, which has been shown to help in the design of virus elimination programs.
 Moving forward, extensive collation of the current immunoassays against SARS-CoV-2 will provide insights toward
MoAB discovery & characterization for dev’t of a SARS-CoV-2 vaccine.
 Rapid increase in number of confirmed COVID-19 cases coupled with the shortage in test kits to meet rising demands,
 Decentralized point-of-care tests (POCT): alternative to facilitate SARS-CoV-2 diagnosis.
lateral flow assay (LFA): a paper-based platform for detection & quantification of analytes in complex mixtures.
designed to be rapid, sensitive, highly accessible, and easily performed, requiring small amount of sample.
 However, POCTs can't replace RT-PCR, it is crucial that these developing tests are rigorously assessed prior to use.
 wrong use and interpretation could lead to disastrous public health consequences.
9. Conclusions
8. The Way Forward