General questions for thesis defense Flashcards
Vaccine WHO definition
A biological preparation that improves immunity against certain diseases
What is a Virus
An infectious, obligate intracellular parasite comprising genetic material (DNA or RNA), surrounded by a protein coat, sometimes a
membrane
Why do coronaviruses have high mutation/evolution rates compared to other viruses?
Coronaviruses, such as SARS-CoV-2, exhibit mutation rates that are generally lower than those of many other RNA viruses.
This reduced mutation rate is primarily due to the presence of a proofreading mechanism within their replication machinery, specifically an exonuclease enzyme nsp14 (nonstructural protein 14) ExoN in the RNA-dependent RNA polymerase (RdRp) complex that corrects errors during genome replication.
Coronaviruses, including SARS-CoV-2, have an estimated mutation rate of…?
1 to 2 mutations per million nucleotides (1 × 10⁻⁶ to 2 × 10⁻⁶) per replication cycle.
or 0.03 to 0.06 mutations per replication cycle per virus particle on average.
Explain the Maximum likelihood (ML): A statistical method
It is used to estimate which phylogenetic tree is most likely to have produced a given set of genetic sequences. It evaluates different trees and selects the one with the highest probability.
Explain the Approximate heuristics method
These are computational shortcuts or algorithms that quickly search for a solution that’s close enough to the best possible answer, rather than exhaustively checking every possibility, which would be computationally very expensive.
Explain the fasttree approximate maximum likelihood heuristic
It to quickly produce a good approximation of the most probable evolutionary relationships (phylogenetic tree) between coronavirus sequences from your alignment. Once you have this tree, you can infer ancestral sequences, meaning you reconstruct what genetic sequences might have looked like at earlier points in evolutionary history.
How do proline substitutions increase protein expression in a spike protein?
Enhance protein expression and stability in spike proteins by stabilizing the prefusion conformation.
Stabilization of Prefusion Conformation: Proline substitutions introduce rigidity and structural constraints
Reduction of Misfolding and Aggregation:
Prolines introduce backbone rigidity by restricting rotation around peptide bonds.
Enhanced Protein Stability and Half-life:
Proline residues reduce susceptibility to proteolytic cleavage by altering local structure, preventing degradation in host cells.
Why does BALB/c species is the first choice for vaccine testing over other species?
Consistent Genetic Background:As an inbred strain, BALB/c mice have a uniform genetic makeup.
Defined Immune Response: BALB/c mice exhibit a well-characterized immune system
Reproducibility: The combination of genetic uniformity and comprehensive baseline data ensures that experiments using BALB/c mice can be replicated with high fidelity across different laboratories,
Why do they usually pick up only female mice?
In vaccine research, female mice are often preferred over males due to differences in immune responses influenced by sex hormones. Studies have shown that females typically exhibit higher antibody levels compared to males across various strains. This enhanced immune response is partly attributed to the effects of estrogen, which can bind to estrogen response elements within immunoglobulin heavy chain gene enhancers, thereby influencing antibody production.
They fight less in the cages as well
What is the main immune cell in humans, and what is the main immune cell in mice. How do these differ, and do you need to consider that in your results?
Humans:
-Neutrophils (50-70% of circulating WBCs)
-Higher frequency of CD4+ T cells
-Balanced Th1/Th2 responses; strong Th1-skewed responses typical
Mice:
-Lymphocytes (70-90% of circulating WBCs)
-Higher frequency of CD8+ T cells
-Often biased toward Th2 immune responses (especially BALB/c mice)
Difference between MHC from Mice and Humans
Humans: Highly polymorphic (hundreds-thousands of alleles) and High genetic diversity within human populations, increasing antigen diversity.
Mice: Limited polymorphism (due to inbreeding) & Genetically uniform within inbred strains, limited antigen diversity
Vaccine Development Implications when unisng mice BALB/c
Successful vaccine candidates identified in BALB/c mice often require further validation in genetically diverse mouse strains, or even better, humanized MHC (HLA) mouse models.
HLA-transgenic mice (expressing human MHC genes) are often employed to better predict human immune responses.
How does the age of mice affect their immune response?
Young Mice (3–8 weeks old):
Immune system maturity:
Still developing; lower numbers of memory T and B cells. Primarily naïve immune cells with limited antigen exposure.
Adult Mice (8–20 weeks old):
Immune system maturity:
Fully mature immune system; balanced naïve and memory compartments. Optimal immune responses and peak cellular activity.
How many years laboratory healthy Balb/c mice lives?
Laboratory-raised BALB/c mice typically have an average lifespan of about 2–2.5 years (24–30 months)
Describe the codon optimisation process of a plasmid for being replicated in a bacteria but being expressed in a mammalian human cell
Reason: Different organisms preferentially use certain codons to encode the same amino acid. Bacteria and humans differ significantly in codon usage. Also for Removal of Rare Codons. Reason: Rare codons can cause ribosome stalling or reduce translation efficiency in human cells.
Why is important to measure the cellular immunologic response as well?
While T-cell responses may appear less specific due to their ability to recognize conserved epitopes across different variants, this characteristic enables them to provide a more versatile and enduring defense against SARS-CoV-2 variants.
T cells often target viral protein regions that are essential for the virus’s function and are less prone to mutations. This targeting ensures that T-cell responses remain effective even as the virus acquires mutations elsewhere.
Epitope Breadth: A diverse T-cell response targeting multiple epitopes reduces the likelihood that mutations in the virus will enable it to escape immune detection entirely. Studies have shown that individuals recovering from COVID-19 maintain a broad repertoire of memory T cells, which contributes to sustained immunity.
How an Elispot works?
Enzyme-Linked ImmunoSpot) assay is a highly sensitive immunoassay used to detect and quantify individual cells that secrete a specific protein, such as a cytokine or antibody.
You stimulate with an antigen the Splenocytes after adding them to the wells, so that they secrete the target protein like IL-2. As individual cells secrete the protein, the immediately adjacent capture antibody binds it. After incubation, the cells are washed away, leaving only the captured proteins on the membrane. A detection antibody—usually biotinylated—that recognizes a different epitope of the secreted protein is then added. Next, a streptavidin-enzyme conjugate (commonly linked to alkaline phosphatase or horseradish peroxidase) is introduced. This conjugate binds to the biotin on the detection antibody.
How does Promega Fugene work for transfection of mammalian cells?
Promega Fugene is a non‐liposomal cathionic polymer transfection reagent that works by forming complexes between its proprietary formulation and the DNA (or other nucleic acids) intended for delivery.
Fugene contains components that interact electrostatically with the negatively charged phosphate backbone of DNA. This results in the formation of stable DNA–Fugene complexes (often termed “lipoplexes”). these complexes are internalized primarily through endocytic pathways.
The transfected plasmids must be delivered to the nucleus so that the host cell’s transcription machinery can transcribe the viral genes. Once transcribed, the mRNA is exported to the cytoplasm where it is translated into the proteins (e.g., gag, pol, env) that assemble into lentiviral particles.
Could you please explain how the “ Lenti-x p24 sandwich ELISA from Takara” works? and Which is the specific gene/protein that is targeted?
The Lenti‑X p24 sandwich ELISA is designed to quickly quantify the titer of HIV‑1‑based lentiviral vectors by measuring the amount of p24 protein present on the Lentivirus ( gag gene)
The microtiter plate comes pre‑coated with an anti‑HIV‑1 p24 capture antibody. After washing away unbound material, a biotin‑conjugated anti‑p24 detection antibody is added. This antibody binds to a different epitope on the p24 protein, ensuring a “sandwich” is formed.
Streptavidin conjugated to horseradish peroxidase (HRP) is then introduced, which binds to the biotin on the detection antibody. A colorimetric substrate (typically TMB) is added next; HRP catalyzes a reaction that produces a color change. The intensity of the color, measured at 450 nm, is directly proportional to the amount of p24—and thus the viral titer—in your sample. A standard curve generated using known concentrations of p24 allows for precise quantification
Size of one Armstrong?
0.1nm
example: in water molecule the H–H distance ≈ 1.5Å.
Advantages of Viral Vector Vaccines?
Strong Immune Response due to the natural infection-like behavior
No Need for Adjuvants
Rapid Development and easy to switch antigens
Stability and better without cold storage
Ease of Large-Scale Production
Function of ACE2
ACE2 cleaves angiotensin II into angiotensin-(1–7), regulating blood pressure and inflammation. It serves as the receptor for SARS-CoV and SARS-CoV-2 entry. Highly expressed in lung, heart, kidney tissues, mediating viral tropism.
Function of TMPRSS2
It regulates sodium transport and fluid balance in respiratory epithelium by activating epithelial sodium channels (ENaC), helping maintain airway surface hydration and normal mucus clearance.
For CoV infections. Priming coronavirus spike proteins. Cleaves spike at the S1/S2 boundary and S2’, facilitating membrane fusion. Enhances viral entry efficiency; inhibition of TMPRSS2 blocks virus infection.
