Molecular Approaches to Bacterial Vaccine Design: Problems and Solutions Flashcards
What is the preclinical development of vaccines
Research is carried out in lab assays and on animals
- Identification of relevant antigens
- Creation of vaccine concept
- Evaluation of vaccine efficacy in test tubes and animals
- Manufacture of the vaccine to Good Manufacturing Practice standards
What does the clinical development of vaccines include
This is when the vaccine is first tested in humans
This stage consists of 4 phases
What is Phase I of Clinical Development
Tests the safety, side effects, best dose, and timing of a new treatment.
It may also test the best way to give a new treatment (for example, by mouth, infusion into a vein, or injection) and how the treatment affects the body.
What is Phase II of the Clinical Development
Looks mainly to assess the efficacy of the vaccine against artificial infection and clinical disease. Vaccine safety, side effects and the immune response are also studied
What is Phase III of the Clinical Trial
Vaccines that progress onto the 3rd stage are studied on a large scale of many hundreds of subjects across several sites to evaluate efficacy under natural disease conditions
If the vaccine retains safety and efficacy over a defined period, the manufacturer is able to apply to the regulatory authorities for a licence to market the product for human use
What is Phase IV of Clinical Development
Happens after the vaccine has been licensed and introduced into use.
Also called post-marketing surveillance, this stage aims to detect rare adverse effects as well as to assess long term efficacy.
What are the challenges faced when
1) Difficulty in which gene products are required and expressed during natural infection
2) How do gene products change over time and space
3) How to determine what goes on globally inside a host during infection
Why do what gene products are produced during natural infection matter
Bacteria only express certain genes when they’re inside the host - these include virulence factors, toxins, or proteins that help evade the immune system
A good vaccine targets proteins that are actually made during infection
Why is it important to take into account how Gene Products of bacteria change over time and space
Bacteria can change their gene expression depending on:
The stage of infection (early vs. late)
The site of infection (blood vs. lungs vs. gut)
Host immune pressure
Effective vaccines must take dynamic expression into account
What is STM and what are its limitations
Signature Tagged Mutagenesis
- Method: Mutate many genes in parallel, infect a host, and see which mutants are missing
- Strength: Identifies essential genes for survival in a host
- Limitation: Doesn’t show changes in expression - just presence and absence
What is IVET
Method: Identifies genes that are turned on during infection
- Strength: Helps find in-vivo expressed genes
- Limitation: Not genome-wide or fully quantitative
What are the modern approaches to vaccine development
1) Transcriptomics - RNA-seq, measures which genes are turned on, and how much, across the whole genome
2) Proteomics - Measures actual proteins produced
3) Genomics - Looks at all the genetic material, often to find variability or mutations
How has bacterial expression analysis improved over time
Moved from single-gene studies to whole genome approaches like RNA-seq and proteomics, allowing researchers to track gene and protein expression globally during infection
What is the significance of gene expression in Microarray and RNA-seq
Key assumption is that the level of expression of the gene = level of mRNA
Measurement of RNA gives the expression profile of the cell and thus also defines cell properties and functions
no mRNA = no protein
What is transcriptomics
The study of the transcriptome, the complete set of RNA transcripts produced from the genome and plasmids at any one time
The regulation of gene expression is a key process for adaptation to changes in environmental conditions and thus for survival.
Transcriptomics describes this process on a genome-wide scale
What is a transcriptome
The subset of genes transcribed in a given organism - the dynamic link between:
- The genome
- The proteome
- The cellular phenotype
What do technologies like DNA-microarrays and RNA-seq show
Determination of the mRNA expression level of practically every gene of an organism
How do transcriptomics work
Typically involves comparing gene expression between two conditions to identify differentially expressed genes
Examples:
- Bacteria grown in broth vs. inside host cells
- Iron-starved vs. iron rich
- Antibiotic-treated vs. untreated samples
What are the two main transcriptomic technologies
1) Microarrays
2) RNA-seq
How do microarrays work
A microarray is a glass slide or membrane with thousands of DNA probes attached to it, each corresponding to a known gene.
You isolate RNA from two conditions, convert it to fluorescently labeled cDNA, and hybridize it to the array.
The amount of fluorescent signal at each spot tells you how much RNA (i.e., gene expression) there was for that gene in each condition.
What is the key point of DNA - microarrays
Microarrays rely on prior knowledge of the genome - you need to know the gene sequences to design the array.
What are the two types of microarrays
Spotted arrays: PCR-amplified DNA fragments printed on the array.
Oligonucleotide arrays (e.g., Affymetrix): Short DNA sequences synthesized directly onto the slide.
How does RNA-seq work
You isolate total RNA → convert to cDNA → sequence it using next-generation sequencing (NGS).
No need for pre-designed arrays or prior knowledge of gene sequences.
Align reads to the genome or assemble transcripts de novo.
Quantify how many reads map to each gene → gives expression levels.
