Lecture 8 - strategies and challenges in prevention and intervention Flashcards
What are vaccine targets?
targets that result in a specific antibody response which in turn facilitates bacterial clearance (serum bactericidal activity)
Give some examples of vaccine targets
Proteins:
Disadvantages: those antigeneic proteins expressed in lab may not be expressed in humans (gene regulation)
- outermembrane proteins, fimbriae
- virulence factors, inactivated toxins (toxoids)
Carbohydrates (generally poorly immunoantigenic)
- O-antigen (highly immunogenic, host recognises sugar combinations that make up the O antigen. LPS (Gram-)
- capsule
What are the features of a live attenuated vaccine?
has an introduced mutation that doesn’t allow to be fully virulent. e.g. salmonella with a gene removed so that it is unable to self synthesise aromatic amino acids, without which it grows for a while and then stops
Bad: severe immune response
Good: allows the vaccine to be present for a longer period of time in a human host, and can transfer to other parts of the body
What are the different types of vaccine?
- Dead pathogen
- Live attenuated strain
- Antigenetic components of the pathogen
What is antigenicity?
when a host recognises and can induce an immune response
How can you get around the low immune antigenicity of carbohydrates for the development on antibodies? 2 Methods, examples
Method 1
Express antigen on the surface on a non-pathogenic bacteria
Express O antigen of psuedomonas in E.coli = non-pathogenic e.coli but surface mimics that of pseudomonas. If given to mice, the immune system makes antibodies against the heterologous O antigen. When challenged with the original pathogen, antibodies recognise and clear away the infection.
Live imaging Igen system allows a visualisation of the same animal multiple times to view the bacteria which have been manipulated to produce light, seen through the skin of the mouse. Heat map used.
Niave mouse given strain and get a high colonisation level and spread within 12 hours. Serum incubated w/ vector w/o O antigen then after 12 hours give pathogen, still see spread and colonisation. If mouse given vaccine first and then challenged with the pathogen, don’t see colonisation and spread. Also swabbed nasal cavity and quantified the number of CFUs
Method 2
Make a glycoconjugate, take a higly immunogenic carbohydrate and tag on a low immunogenic carbohydrate. Presented by B/T cells to produce antibodies e.g. Neisseria meningitidis
What are the three main targets of current successful antibiotics? And what is the basic categories of antibiotics?
- Cell wall synthesis
- Ribsosome
- DNA gyrase/topisomerase
important, remember
Can be either bacteriostatic (when the antibiotic is withdrawn, the pathogen may grow again) or bacteriocidal
How can you assess vaccines/drugs using live imaging (Xenogen)?
- Uses bacterial strains that have been engineered to emit light.
- Advantages: can view same animal multiple times
- Real-time monitoring of pathogen expressing lux in the acute pneumonia model. Luminescence is observed eminating from the nasopharynx, lungs, liver and gastrointestinal area, following immunisation with heterologously expressed polysaccharide (O-antigen P.aeruginosa)
What are the features of Neisseria meningitidis?
- Five main serogroups distinguished by different carbohydrate groups and structures on their surface
- 1992 first conjugate vaccie against meningococcus C
- then conjugate vaccines about A, C, Y and W (against the capular polysaccharide)
- Serotype B capsule: a polysialic acid, host like, poorly antiogenic, risk of autoimmunity
- New vaccine 4CMenB 2015, protein based using reverse vaccinology
What is the reverse vacccinology technique that was used to creat the 4CMenB vaccine?
- Genome sequence of the N. meningitidis virulent strain MC58
- looked at open reading frame that encoded proteins, identified approximately 2000 proteins, 570 with a potential surface localisation. Looked for hydrophobicity because expressed to that environment or looked for a signal sequence that direct the protein to the membrane
- Cloned and recombinantly expressed these proteins in e.coli, purified them and tested for their ability to induce bactericidal antibodies by immunising mice
- Antibody response was analysed by western blot analysis, ELISA, flow cytometery
- SBA (serum bacteriocidal assay) was used to evaluate the complement-mediated killing activity (does the protein induce killing of the bacteria, recruitment of host complement compounds) identified 28 proteins
- priotitised proteins based on their ability to induce protection against a diverse collection of N.meningitidis strains
- Identified 5 proteins: 1 adhesin, H binding protein, Heptarin binding protein, 2 additional antigens
Where are the precursors of peptidoglycan made? And how is peptidoglycan formed?
In the cytoplasm as active components with UDP
NAG (N-acteylglucosamine) and NAM (N-acetyl muramic acid) are linked by the loss of UDP (uridine diphosphate). In the membrane linked to a lipid that delivers the unit to the growing chain and combined into a network peptidoglycan via cross linking(transpeptidoglycan).
What is the target of the penicillin antibiotic?
Cell wall
What is the activity of glycopeptide and beta-lactans antibiotics?
Glycopeptides
bind to cell wall subunit (D-ala-D-ala) and prevent incorperation of NAM-NAG cell wall precursors
Beta-lactams
inhibit enzymes (penecillin binding proteins) required for the last step of cell wall synthesis - transpeptidation
What resistance mechanisms can bacteria have to beta-lactams?
- produce and secrete beta-lactamases to degrade the antimicrobial
- alter the PBP that are required for the transpeptidation step so that beta-lactam cannot interact with it yet retain function
What resistance mechaisms can bacteria have to vancomycin (glycopeptide)?
- G- bacteria have intrinsic resistance due to the OM pore size e.g. e.coli
- aquired resistance: alter target (peptidoglycan e.g. D-ala-D-ala changed to D-ala-D-lactate)