Vaccines (lecture 2)

Question 1

What is transcriptomics?

and its limitations?

Answer 1

Monitoring genes expressed during host-pathogen infection and detecting genes which are up-regulated.
This is high throughput

Limitations: no direct correlation between gene expressions and actual protein levels. RNA isolation in vivo is very difficult. Overlooks the effects of post-transcriptional modification on the regulation of gene expression

Question 2

Neisseria meningitidis profile and mechanism

Answer 2

Profile:
Gram negative
12 serogroups polysaccharide capsule (resist phagocytosis)

Mechanism:
Colonise asymptomatically upper respiratory tract (5-30%). It can traverse the epithelium and reach the blood stream causing septicemia. Can access meninges (membrane covering brain and spinal cord) causing meningitis.

500,000-1,000,000 cases a year by serogroups B and C
Affects infants between 6months-2 years mainly
Antibiotic are main treatments

Question 3

Vaccines for Neisseria meningitidis

Answer 3

Based on capsular polysaccharides
Not effective in young children, do not ellicit T-cell response hence no immunological memory.
Capsular pollysaccharides conjugated to a carrier protein elliciting T-cell based responses enhances vaccine efficacy.

Capsular polysaccharide serogroup B is NOT immunogenic
Very identical to human polysailic acid, hence can induce auto immunity

Serogroup B cause up to 80% meningococcal disease

Question 4

Identifying new Neisseria meningitidis vaccine candidates

Answer 4

Infect cells
N. Meningitidis have adherent and non-adherent componenets.
Wash away non adherent
Tyrpsin to remove adherent components
Centrifugation to seperate
Supernatant extracted
RNA extracted from supernatant
RNA is grown in absence of host cells AND with host cells
Labell cDNA synthesis with Cy3-dCTP and Cy5-dCTP
Compare fluorescence ratios to determine genes expressed

2152 genes on total

Predict genes which are surface exposed (40%)
12 genes encoding for adhesion-induced proteins

Question 5

How is microarray expressions confirmed?

Answer 5

Reverse transcription real time PCR (RT-PCR)

Question 6

How are antigenic properties confirmed?

Answer 6

Express antigenic genes in E.coli and isolate
Immunise mice with recombinant proteins in order to produce anti-sera
Sera containing antibodies against the selected proteins were tested for their bactericidal activity. Viable anti-sera should target cells that are killed by the complement system.

Question 7

Compare transcriptomics and proteomics

Answer 7

Transcriptomics require:
small starting material
Amplification steps

Proteomics require:
Protein function
Post transcriptional modifications
Spatial and temporal resolution
Large starting material

Question 8

Chlamydia pneumoniae life cycle

Answer 8

Elementary body enters cell by phagocytosis and becomes a phagosome.
Lysosomes do NOT interact/digest phagosome
Elementary body becomes and reticulate body (replicative form)
Reticulate body replicates
If under antibiotic stress becomes a persistent non-repliicating form. Otherwise becomes elementary body and excreted from cell.

Question 9

What is an elementary body?

Answer 9

A spore-like infectious form involved in cell adhesion and colonisation

Question 10

What is Reticulate body?

Answer 10

An intracellular replicative form of elementary body

Question 11

Identifying surface proteins (Chlymidia pneumoniae)

Answer 11

In silico selection of genes encoding surface-exposed proteins

Cloning 124 candidate genes and recombinant expression in E.coli

Proteins are purified and used to prepare immune sera in mice

Ability of sera to bind to surface of elementary bodies tested using flow cytometry (FACS)

53 putative surface exposed proteins

Can be further purified

Question 12

What is usually done after flow cytometry? FACS regarding protein identification

Answer 12

2D SDS PAGE, it shows isoforms of the same protein. This can be followed by mass spectrometry

Question 13

How can the sera efficacy be confirmed?

Answer 13

Inhibition assay

Comparing non-immunised and immunised mouse sera mixed with elementary body.

Chlamydia cells are fluorescently tagged

Immunised sera shows significantly reduced fluorescence.

Question 14

What is in-ivio expression technology (IVET)?

Answer 14

A high throughput method to identify candidate vaccines

It does not require genome sequence

It identifies genes activated during infection of host tissue (in vivo) by the pathogen

This is done by the PROMOTER TRAP method.

A promoterless essential gene is fused with random DNA fragments of the pathogen.

If the random DNA fragments have a promoter, they will be expressed.