Lecture 14. SARS-CoV-2 Diagnosis & Vaccination Flashcards
How can SARS-CoV-2 be diagnosed in a laboratory?
Detection of viral nucleic acid
Detection of viral antigens
Detection of viral antibodies
How can the viral nucleic acid of SARS-CoV-2 be detected?
RT-PCR (reverse transcriptase PCR)
What genes of SARS-CoV-2 could be the target for RT-PCR (as long as the correct primers are made for them)?
ORF1a/b
RdRp (RNA-dependent RNA polymerase)
S (spike protein)
E (envelope)
N (nucleocapsid)
Hel (RNA helicase)
What happens in the process of RT-PCR?
- Specimen collection
- RNA extraction/isolation
- Reverse transcription (needed because RNA virus)
- cDNA amplification
How are specimens collected for RT-PCR?
Nasopharyngeal (NP), oropharyngeal (OP) swabs
Global swab shortage during pandemic
Discomfort associated with NP collection
Trained healthcare personnel
Alternatives to NP specimens: Saliva
What is RNA highly susceptible to?
Degradation
Sample storage
Handling
What is RNA isolation and what must it follow?
Isolation of RNA is the initial step of the RT-PCR assay and critical for the assay’s reproducibility and biological relevance
Some important points to consider here is that unlike DNA, RNA is highly susceptible to degradation; sample storage, handling, and RNA isolation must follow optimised protocols to minimise degradation at each step
What happens in one-step RT-PCR?
In a one-step assay, reverse transcription and PCR amplification are consolidated into one reaction utilising a single tube and buffer for RT and PCR steps
What are the advantages of one-step RT-PCR?
Shorter time, reproducible results, this type of assay is suitable for high-throughput diagnosis, and may reduce risk of cross-contamination and human error by limiting sample management
What happens in two-step RT-PCR?
The reactions are done sequentially in separate tubes with independently optimised buffers
What are the advantages of two-step RT-PCR?
Although more time consuming is considered more sensitive and has lower detection limits
Which genes or regions of SAR-CoV-2 should be targeted by RT-PCR?
Genes of SARS-CoV-2 that are highly conserved and abundantly expressed genes should be targeted by the RT-PCR primers
What specific genes is it recommended for RT-PCR to target?
Assays should target and detect viral nucleocapsids genes N
What are the positive controls for RT-PCR?
Samples that have been previously validated as positive as positive control or you can spike a solution with synthetic SARS-CoV-2 RNA and use it as positive control]
Negative controls important too
Does the detection of Viral RNA by RT-PCR demonstrate the presence of infectious virus?
Not really
The presence of the nuclear acid doesn’t confirm the virus is present
It has been shown that patients who have recovered can be persistently PCR-positive but they are non-infectious, which is confusing for quarantine and control
What are immunodiagnostic tests based on?
Based on the specific interactions between Antigens and antibodies
Detection of viral antigens in clinical samples
Detection of antibodies against the viral antigens in the serum
What are features of immunodiagnostic tests?
Generate rapid results (in less than 1 hour)
Less complex than molecular tests
Seroconversion for SARS-CoV-2 occurs between 7 and 11 days after onset of symptoms
Why are immunodiagnostic tests impractical for diagnosis of acute (current) infection at the early stage?
Immunodiagnostic tests identify antibodies present, not the virus itself
What are examples of immunodiagnostic tests that detect antibodies?
ELISA
Chemiluminescence
Immunochromatrography (rapid-test lateral flow test)
What is an example of immunodiagnostic tests that detect antigens?
Immunochromatrography (rapid-test lateral flow test)
How do lateral flow tests work?
Antibodies are present in the conjugate pad of the lateral flow test
Analyte is added to the sample and pad and moves by capillary flow up the nitrocellulose membrane of the test
Tagged antibodies bind to the viral antigen on the nuclear capsid, forming antibody:antigen complexes
Accumulation of labelled antibody at test line (where different antibody binds to antigen) results in coloured line, control line also has αIG antibodies to show the complexes have travelled correctly
What are the drawbacks of using the viral antigen lateral flow assay/test?
Lower sensitivity (34% to 80%)
Lower specificity
Due to high nucleotide sequence similarity with SARS-CoV, which may lead cross-reactivity
How could viral antigen lateral flow tests be improved?
Targeting unique and conserved domains of proteins in SARS-CoV-2 (increase specificity)
Prior treatment to concentrate the targeted antigen and (ii)
Use of monoclonal antibodies to different epitopes of the antigen to be detected
How can antibodies be detected in the serum of a patient?
The test is based on the same principle as viral antigen lateral flow tests, but the antibodies in the serum are detected as opposed to the detection of antigens in the NA swab
How many vaccines have been approved for SARS-CoV-2?
Up to 50
BioNTech/Pfizer, Moderna - RNA-based
Oxford/AstraZeneca - Non-replicating viral vector
What do all SARS-CoV-2 vaccines use as a target (except inactivated, attenuated and AstraZeneca)?
Spike protein - responsible for binding to the cell
Creating antibodies for the spike protein stops the first stage of replication
What are the types of vaccination strategies used for SARS-CoV-2 vaccinations?
Inactivated vaccines contain SARS-CoV-2 that is grown in cell culture and then clinically inactivated
Live attenuated vaccines are mead of genetically weakened versions of SARS-CoV-2 that is grown in cell culture
Recombinant spike-protein-based vaccines
Recombinant RBD-based vaccines (RBD part of spike protein)
VLPs carry no genome but display the spike protein on their surface
Replication-incompetent vector vaccines cannot propagate in the cells of the vaccinated individuals but express the spike protein within them
Replication-competent vector vaccines can propagate in the cells to some extent in the vaccinated individuals and express the spike protein within them
What was the viral vector used to develop the AstraZeneca vaccine for SARS-CoV-2?
Chimp-Adeno-Vector vaccine (from chimpanzee)
How does the Pfizer vaccine function?
30μg of mRNA that encodes for the full length SARS-CoV-2 spike protein is encapsulated within lipid nanoparticle to facilitate the delivery of the nucleic acid and the fusion/delivery into the host and the cells
What are the active ingredients of the Pfizer vaccine?
Nucleoside-modified messenger RNA (modRNA) encoding spike glycoprotein (S) trimers of SARS-CoV-2 in a pre-fusion confirmation
Lipids
Salts (KCl, monobasic potassium phosphate, NaCl, basic sodium phosphate dihydrate)
Sucrose
What is the vaccine efficacy of the Pfizer vaccine?
95%
