Lecture 8 - strategies and challenges in prevention and intervention

Question 1

What are vaccine targets?

Answer 1

targets that result in a specific antibody response which in turn facilitates bacterial clearance (serum bactericidal activity)

Question 2

Give some examples of vaccine targets

Answer 2

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

Question 3

What are the features of a live attenuated vaccine?

Answer 3

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

Question 4

What are the different types of vaccine?

Answer 4

• Dead pathogen
• Live attenuated strain
• Antigenetic components of the pathogen

Question 5

What is antigenicity?

Answer 5

when a host recognises and can induce an immune response

Question 6

How can you get around the low immune antigenicity of carbohydrates for the development on antibodies? 2 Methods, examples

Answer 6

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

Question 7

What are the three main targets of current successful antibiotics? And what is the basic categories of antibiotics?

Answer 7

1. Cell wall synthesis
2. Ribsosome
3. DNA gyrase/topisomerase

important, remember

Can be either bacteriostatic (when the antibiotic is withdrawn, the pathogen may grow again) or bacteriocidal

Question 8

How can you assess vaccines/drugs using live imaging (Xenogen)?

Answer 8

• 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)

Question 9

What are the features of Neisseria meningitidis?

Answer 9

• 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

Question 10

What is the reverse vacccinology technique that was used to creat the 4CMenB vaccine?

Answer 10

1. Genome sequence of the N. meningitidis virulent strain MC58
2. 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
3. Cloned and recombinantly expressed these proteins in e.coli, purified them and tested for their ability to induce bactericidal antibodies by immunising mice
4. Antibody response was analysed by western blot analysis, ELISA, flow cytometery
5. 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
6. priotitised proteins based on their ability to induce protection against a diverse collection of N.meningitidis strains
7. Identified 5 proteins: 1 adhesin, H binding protein, Heptarin binding protein, 2 additional antigens

Question 11

Where are the precursors of peptidoglycan made? And how is peptidoglycan formed?

Answer 11

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).

Question 12

What is the target of the penicillin antibiotic?

Answer 12

Cell wall

Question 13

What is the activity of glycopeptide and beta-lactans antibiotics?

Answer 13

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

Question 14

What resistance mechanisms can bacteria have to beta-lactams?

Answer 14

• 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

Question 15

What resistance mechaisms can bacteria have to vancomycin (glycopeptide)?

Answer 15

• 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)

Question 16

What is the human interpretation of antibiotic resistance?

Answer 16

Measuring bactericidal/static activity as a function of antibiotic concentration

All bacteria will be sensitive if given enough antibiotics (w/in reason) but the key is whether it is sensitive at levels that are appropriate to use in clinics because of side effects

Question 17

How can the resistance of bacteria to antibiotics be measures?

Answer 17

E-test: strip containing dilutions of a drug is placed on a lawn of cells and MIC (minimum inhibitory concentration) is read based on the lowest concentration of the clearing

Used for defined strictly controlled bacterial growth condidtions

Question 18

What different mechanisms might bacteria have to attain resistance to an antibiotic?

Answer 18

1. Modifiying the antibiotic target
2. Bypass pathway by developing different metabolic pathways
3. Limit antibiotic in the cell
4. Enzymatic modification of an antibiotic

Question 19

Outline TB as an example of antibiotic resistance development

Answer 19

in 2012 more than 7% of UK tuberculosis cases were resistant to at least one first-line drug

Resistance to any first line drug has increased by 84%, multidrug resistance has increased by 189% between 200-2012

Question 20

How does a strain acquire resistance?

Answer 20

1. Horizontal gene transfer
2. Mutations

Question 21

By what mechanism are potential antibiotic resistance mechanisms aquired?

Answer 21

1. Modifiying the antibiotic target in the cell
   
   • mutations
2. Limiting access of the antibiotic -
   
   • reducing penetration: innate, mutations;
   • high efflux: muations, innate (psuedomonas has high innate efflux rate)
3. Enzymatic inactivation of an antibiotic
   
   • horizontal gene transfer
4. Bypass pathway
   
   • horizontal gene transfer

Question 22

How is multidrug resistance developed?

Answer 22

• some mechanisms (mainly increased expression of efflux pumps) confer resistance to more than one type of drug
• one strain can have several mechanisms conferring resistance to certain drugs

Question 23

Give an example of how resistance has been understood through genomics?

Answer 23

iNTS (invasive non-typhoidal salmonella) - able to live in the blood

• Sequenced the isolate
• Phylogenetic analysis
• see how the resistance strains relate to each other by changes in sequence
• identifying the location of the differences
• identified two changes from a deletion and aquisition event from two gastrenteritis strains (two lineages identified): pSLT (virulence plasmid) and cat (chloramphenicol resistance
• Lineage I: two composite Tn21 like transposon elements encoding MDR determinants on pSLT
• Lineage II: Tn21 aquired cat, clonal replacementand expansion

Question 24

What type of resistance do persisters show?

Answer 24

transient resistance

not metabolically active

role of stochasticity of gene expression

Question 25

How is popuation level resistance aquired?

Answer 25

**Biofilm resistance** must overcome * penetration of the layers of the biofilm * altered metabolic state * altered gene expression (targets might not be expressed) e.g. P.aeroginosa in vitro resistance to Tobramycin, surface layer of the biofilm killed but middle still alive

Question 26

How might antibiotic resistance be avoided by thinking differently about the approach to treating bacterial infections?

Answer 26

**Anti-pathogenic drugs**: reduce selection pressure on the pathogen by not trying to kill, instead reduce virulence factors

Question 27

give an example of anti-pathogenic treatments

Answer 27

Working on fimbrial adhesins in E.coli * multiple fimbrial operons, type I operon, pap, sfa, type I associated to adhearance to kidney cells = can adhesion of E.coli to a cell be blcoked * vaccination with FimH adhesin protected cynomolgus monkeys from colonisation and infection with uropathogenic e.coli * inhibited the action of chaperones ushers * pilicide ec240 is a compound that interferes with the assembly of all fimbrial chaperone assembly proteins therefore can't adhear or be pathogenic - affects type I Pili (FimH) - recognises mannose groups * shown that adhearence is decreased but growth is not affected - no need to develop resistance * Pillicide e240 alters CUP piliation - used a haemaglutanin assay (not mannose resistant - type I)

Question 28

What is the action of ec240?

Answer 28

prevents type I, P and S piliation UTI's and other E.coli infections -designed to interfere with CUP pilus assembly BUT found to affect type I phase variation, locked to OFF multiple effects linked to fim OFF - other fimbriae production, motility as fim link broken

Question 29

What are the challenges to developing a ec240 therapeutic?

Answer 29

* strain variations in fimbrial repetoire, regulatory circuits * pharmokinetics (bladder) * is the colonisation of beneficial e.coli affected * microbiome affect - and other strains with the CUP pili

Question 30

How can a disease be caused by antibiotics?

Answer 30

Clostridium difficile * antibiotic related diarrhoea casued by clostridium difficile uncontrlled growth (toxin produced) * c.d is inherently resistant to a lot of antibiotics * following antibiotic treatment c.d. colonises the gut * work with/towards a healthy microbiome * healthy microbiome has competition for nutrients * interaction between bacteria of the healthy microbiome * Normal microflora degrade primary and scondary bile salts (secreted to help degrade fatty acids), primary bile salts necessary for the germination of c.difficile spore, secondary bile salts necessary for the vegetative growth of c.diff and production of toxin [metabolic state of the microbiome is important] * probiotics/beneficial bacteria/microbiome replacement

Question 31

How can the use of animal models be avoided? (reduce, refine, replace)

Answer 31

Uses other experimental systems: * cell lines (good for basic assay, but cells taken out of context of disease) * droposphila, c.elegans, zebrafish (hosts that are not natural but can be used to work out mechanism of infection) * plants (toxins may work on plants as well as humans) * computational models: infection and immune response

Question 32

What is the process of drug discovery?

Answer 32

* simple clinical trial cost 200 million dollar, longer term cost up to 1 billlion * patents only last 15 years * 16 new approved antimicrobials between 1883-1987, 1 since 2008 * pharma has reduced efforts * lab studies (several years - preclinical), human safety (days or weeks - tens of people - phase I), expanded safety (weeks/months - hundreds of peple - phase I/II), efficiency and safety (several years) - thousands of people, phase III (looking for signs of positive host response , short term/long term, small subset of population may be sensitive

Question 33

Where will next solutions to antimicrobial resistance?

Answer 33

Interdisciplinery approaches

Question 34

How might bacteria modify the antibiotic target to get resistance?

Answer 34

e.g. in peptidoglycan synthesis, alter the PBP so that beta-lactam cannot interact with it but retain function

Question 35

How might bacteria limit the level of antibiotic the cell to aquire resistance?

Answer 35

* reduced penetration (intrinsic type of resistance E.coli (gram - ) vancomycin resistance) * increased efflux (increase expression of transporters naturally present to deal with biosalts)

Question 36

What do bacteria utilise to attain enzymatic modification of an antibacterial target for resistance?

Answer 36

e.g. AmpR betalamases

Question 37

How does a strain acquire resistance via horizontal gene transfer?

Answer 37

- aquire genes that mediate resistance, plasmids or transposons e.g. beta-lacatamases

Question 38

How does a strain acquire resistance via mutation?

Answer 38

Mutations: in target or regulatory elements, harder to transfer horizontally but will be passed on vertically and establish a clonal population creating a new clinical isolate resistant to a certain antibiotic e.g. alter gyrase (target for quinolones) increase expression of an efflux pump (multidrug)