ANTIMICROBIALS AND VACCINES Flashcards
(125 cards)
1
Q
give an example of why we need antibiotics?
A
1941 mortality rate from S. aureus bacteraemia 82% and predominantly in young people
2009 mortality rate 21% and mostly old fucks
2
Q
what do antibiotics do?
A
inhibit bacterial growth by targeting molecular targets
interfere with specific bacterial enzymes
different toxicity for bacterial cells as target not present or sufficiently different in eukaryotic cells
3
Q
what are the three broad targets of antibiotics?
A
cell wall aka peptidoglycan synthesis
protein synthesis (ribosome)
nucleic acid synthesis
4
Q
outline how antibiotics target peptidoglycan synthesis?
A
peptidoglycan layer gets cross linked to form lattice structure in both gram+ and gram- and it provides strength against osmotic lysis of bacteria
peptidoglycan synthesis has a number of steps which can be inhibited
e.g. bacitracin inhibits lipid carrier recycling, beta lactams and glycopeptides inhibit peptidoglycan subunit cross-linking
5
Q
outline how beta lactams inhibit peptidoglycan cross-linking?
A
key molecule of beta lactams (e.g. penicillin) is beta-lactam ring as this responsible for its activity
cross linking of peptidoglycan key for providing its function; facilitated by transpeptidases called penicillin binding protein (PBP) and this has serine residue which crosslinks peptide side chains of peptidoglycan backbone
beta lactam ring reacts with serine residue on PBP forming covalent bond so PBP can no longer cross-link peptidoglycan chains
6
Q
why is cross-linking so important for peptidoglycan function?
A
autolysis are enzymes present in peptidoglycan layer and are responsible for degrading it when activated
this is normal part of turnover of peptidoglycan, growth and division
so when no more cross linking (cause beta lactic activity) this leads to bacterial lysis to osmotic pressure (hence b-lactams are bactericidal)
autolysins most active during exponential cell growth so beta lactams most effective during this
7
Q
compare and contrast the classes of beta-lactams?
A
there are multiple families of beta-lactams e.g penicillins, cephalosporins, carbapenem, monobactam
they have different spectra of activity and resistance to beta-lactamases which is influenced by side chain R group - can also affect pharmacokinetics (what body does to drug)
we can alter R-group to alter how drug works
all beta-lactams have beta-lactam ring tho and have all come from natural sources
8
Q
how do glycopeptides work?
A
glycopeptides e.g. vancomycin are beta lactams
they are big molecules so only effective against gram positive as cannot penetrate gram negative outer membrane (also means have to be given intravenously unless luminal infection e.g. C. difficile)
glycopeptides recognise the D-alanine-D-alanine portion of muramylpentapeptide
inhibits ability of PBP to come in and cross-link peptidoglycan
9
Q
outline how protein synthesis inhibiting antibiotics function?
A
bind either 16s rRNA in 30s subunit (e.g. aminoglycosides, tetracyclines) inhibiting mRNA from binding OR 23s rRNA in 50s subunit (macrolides, lincosamides, oxazolidinones) inhibiting tRNA from binding
all these drugs work by binding the ribosome thus inhibiting protein synthesis
reason they don’t inhibit our protein translation is difference between prokaryotic and eukaryotic ribosomes i.e. differential selectivity
10
Q
what are the three main things inhibited by antibiotics that target nucleic synthesis?
A
inhibition of DNA synthesis
inhibition of RNA synthesis
inhibition of folate metabolism
11
Q
how do antibiotics target folate synthesis and give examples?
A
folate synthesis is a bacterial pathway for production of tetrahydrofolic acid which is an essential co-factor for synthesis of nucleic acids
this pathway involves three key steps
sulphonamides (inhibits first step) are structurally similar to p-aminobenzoic acid (important precursor in folate synthesis) and thus is competitive inhibitor of dihydropteroate synthase
trimethoprim (inhibits final step) is structurally similar to dihydrofolic acid so is competitive inhibitor of dihydrofolate reductase
12
Q
how does metronidazole target nucleic acid synthesis?
A
makes breaks in DNA
doesn’t damage our DNA cause is a prodrug i.e. requires activation
activation involves reduction by ferredoxin or flavodoxin - these are electron acceptors in anaerobes and microaerophiles
aerobes and mitochondria use pyruvate dehydrogenase so we allg
13
Q
how do fluoroquinolones target nucleic acid synthesis?
A
inhibit type II topoisomerase (e.g. DNA gyrase, topoisomerase IV) which are very important for bacterial replication
they are important for replication as they unwind positively supercoiled DNA into a relaxed state, negatively supercoil it to be packaged into cell and also decatenation of daughter chromosomes at cell division
14
Q
how do rifamycins inhibit nucleic acid synthesis?
A
inhibit RNA synthesis by binding B subunit of DNA-dependent RNA polymerase inhibiting its function
resistance can occur due to a single point mutation - huge issue with rifampicin (important for TB) so almost always use in combination
15
Q
what are some different approaches we could take to find new antibiotics?
A
improve existing antibiotics e.g. alter B-lactam ring
repurpose old drugs
discover untested new chemical diversity (either from natural or synthetic products; natural has proven most effective)
target based approach (finding novel targets then make drugs for them)
rediscover old antibiotics
16
Q
why has most antibiotic discovery been from natural products?
A
many have come from bacteria and fungi which produce antibiotics in response to competition or stress
this is also an issue as it means producer species have intrinsic mechanism of resistance and so resistance exists for so many antibiotics
17
Q
outline how teixobactin was discovered using iChip process?
A
potential new antibiotic for clinical use
discovered through growth in iChip in soil for a month, then move to agar (50% survive), bacterial extracts screened for activity against S. aureus
ones with anti-staph activity undergo purification into different compounds and are screened against a range of bacteria
assess compounds in vitro and work out mechanism (teixobactin related to cycling of lipids)
18
Q
what is the research and development process for antibiotics?
A
drug discovery and pre-clinical (3-6 years) (teixobactin only just finished this)
clinical trials (6-7 years):
phase I - potential adverse side effects and safety
phase II - efficacy and safety
phase III - efficacy and safety (larger group)
seperate phase I and II trials for carried out each indication
only then can it be FDA approved and throughout this process fuck loads of compounds narrowed down to only a few
19
Q
outline the financial issues with antibiotic research and development?
A
cost of bringing new drug to market >1 billion dollars and profits not made till a long way down the line (and not much)
so little incentive for pharmaceutical companies
this is fucked cause the antibiotics pipeline is running dry
20
Q
how is the mortality rate with MRSA changing in New Zealand?
A
mortality rate for MRSA bacteraemia has doubled since 2009
methicillin first line drug for S. aureus bactaemia
21
Q
outline the human and financial burden associated with increasing incidence of antimicrobial resistance?
A
increased cause of death - 10 million by 2050 at current rates
increased global loss of GDP - 100 trillion by 2050
22
Q
outline the three broad mechanisms that AMR can come under?
A
restricted access to target (decreased permeability, increased efflux)
inactivation of antibiotic
modification of drug target
23
Q
how do some bacteria restrict access to the antibiotic target by decreasing permeability?
A
beta lactams must cross outer membrane of gram negatives and many other antibiotics must access cytoplasm
gram negatives encode multiple porins in their outer membrane to allow selective diffusion of small molecules into periplasm - mutations can down regulate porin expression or restrict antibiotic access by narrowing channel (often affecting multiple antibiotics) - acquired resistance
gram negatives also intrinsically resistant to vancomycin cause it too big to cross outer membrane
24
Q
how do bacteria restrict antibiotic access to target through increasing efflux?
A
efflux pumps actively pump small molecules out out of cytoplasm such as metabolites and toxic substances
bacteria contain multiple efflux pumps, some are highly specific (e.g. tetracycline efflux pump) and others more broad - can give resistance to multiple classes
this common resistance mechanism now described for almost every antibiotic class
25
outline how inactivation of antibiotics occurs via beta lactamases?
beta lactamases are enzymes secreted into periplasmic space (gram neg) or extracellularly (gram pos)
especially common in gram negs and can be distinguished into class A, B and C beta lactamases
class A and C have serine residue at active site which forms covalent bond with beta lactam ring and cleaves it and then undergoes hydrolysis freeing beta lactase from inactivated beta lactam - some of these more narrow spectrum (e.g. TEM) and some more broad (e.g. AmpC, ESBLs) destroying lots of beta lactams
AmpC are class C while ESBLs are class A
class B beta lactamases have different mechanism of action; are zinc dependent (have zinc ion at active site) and are broad spectrum
26
what are beta lactamase inhibitors and how effective are they?
e.g. clavulanic acid
can inhibit class A beta lactamases (e.g. ESBL) by binding it and remaining bound thus removing it from action
does not inhibit class C beta-lactamases (AmpC) produced by ESCAPPM group organisms which express class C beta-lactamase via inducible production or have mutation which can allow constitutive production - so can become resistant to beta lactams during treatment
metallic-beta-lactamases (aka class B) are not inhibited by clavulanic acid but are by EDTA (can't use this clinically tho)
27
how do bacteria gain antibiotic resistance through modification or protection of antibiotic target- give some fuckiogn exampels?
mutations in target: such as gyrA/B and parC/E mutations for fluoroquinolones, rpoB point mutation for rifampicin, PBP mutations for beta lactams
enzymatic modification of target: stops antibiotic function but still allows target function e.g. glycopeptides; d-alanine-d-alanine to d-alanine-d-lactate
bypassing target: for example MRSA acquire different PBP (PBP2A encoded by mecA) which beta lactams don't work on
28
where has altered penicillin binding proteins via mutation conferred resistance?
common in gram-positives
mutation in chromosomal copies of PBPs can then be acquired by horizontal gene transfer
particularly a problem in viridans group streptococci e.g. S. pneumoniae
29
how does resistance to glycopeptides (e.g. vancomycin) occur through enzymatic modification of target?
vancomycin binds D-alanine-D-alanine at end of peptide to inhibit PBP from coming in and cross linking it
bacteria can become vancomycin resistant through acquiring a new group/operon of genes which changes peptide to D-alanine-D-lactate
operon of genes has a two component regulatory system: VanS recognises presence of vancomycin and phosphorylates VanR which is transcriptional regulator of the system (i.e. turns on all the other genes) leading to synthesis of D-alanine-D-lactate via VanH and VanA and then destruction of D-alanine-D-alanine via VanX and VanY
30
how can altered-penicillin binding proteins (PBPs) allow bypassing of antibiotic target leading to resistance?
mecA in MRSA encodes PBP2A which isn't inhibited by methicillin
this replaces the normal transpeptidase allowing peptidoglycan cross-linking in presence of antibiotic
31
what are the two main drivers of antibiotic resistance?
antibiotic-mediated selection
horizontal gene transfer
32
what are the different ways resistance genes can undergo horizontal gene transfer?
conjugation - most common, especially between different species, occurs via pillus
transformation - uptake of DNA from external environment, integrates by homologous recombination so has to be closely related or same species
bacteriophage transduction - usually between same species or closely related cause need the same phage receptor
HGT much easier way to acquire resistance genes than through mutation, frequent exchanges of genetic material e.g. within animal guts
33
describe multiple antibiotic resistance?
most resistance mechanisms against single class of antibiotics; exceptions include multi-drug efflux pumps and macrolide-lincosamide-streptogramin B (MLSb) resistance (binds overlapping sites on ribosome)
HOWEVER multi drug resistance due to genetic linkage on MGEs (where resistance genes often accumulate) is more common
selection by one class of antibiotics can maintain resistance genes to other unrelated antibiotics (cross-selection) cause they genetically linked
antibiotic resistance genes can also be linked to disinfectant resistance genes i.e. disinfectants may also be selecting antibiotic resistant bacteria
34
what are integrons?
often encoded by mobile genetic elements
integrons encode integrase which integrates circular DNA gene cassettes (e.g. resistance gene) into the att site and creates operons by sequential integration of gene cassettes (they contain a promoter so they get expressed)
35
what is a transposon?
a mobile genetic element flanked by insertion sequences which encode transposase and are recognised by transposase meaning they can cut themselves out of DNA along with their cargo and insert somewhere else (e.g. plasmid, chromosome)
can be conjugative (chromosome to chromosome) and have a broader host range than most plasmids (e.g. can pass from gram positive to gram negative)
36
why do we need to think of the flux of resistance through the environment?
resistance genes in bacteria can travel through multiple hosts and locations due to their tendency to accumulate on MGEs
resistance bacteria can be transmitted between people, excreted into environment, accumulate in shellfish, the genes can get amplified in livestock and then back into people
37
how does New Zealand use antibiotics?
compared to other countries NZ has relatively low rates of antibiotic use in agriculture, but relatively high rates of antibiotic usage in medicine
38
broadly how did we come to the antibiotic resistance crisis?
inappropriate use of antibiotics (e.g. wrong use, wrong duration, over-prescription) applying selective pressure on bacteria with high genetic plasticity
this combined with increased spread as a result of socioeconomic issues, hospitals and increasing international travel and increasing susceptibility to infection and antibiotic usage as a result of poor nutrition, immunosuppression and invasive medical procedures
39
what are antiviral drugs?
inhibit viral replication by inhibiting specific viral proteins
they are quite specific (specific antivirals for specific viruses)
resistance develops by mutation of target protein
40
outline HIV virology?
retrovirus (subgroup lentivirus)
enveloped; important envelope glycoproteins gp120 and gp41
nucleocapsid (encoded by gag) main structural protein is p24
essential enzymes (encoded by pol) include integrate, protease and reverse transcriptase
41
how the fuck does HIV enter the host cell?
gp120 binds CD4 with high affinity
conformational change allows binding of gp120 to co-receptor which is CCR5 (or CCR4)
this exposes gp41 allowing membrane fusion and entry of nucleocapsid
42
how does maraviroc inhibit HIV attachment to host cell?
maraviroc is a CCR5 inhibitor
it binds CCR5 thus preventing gp120 binding
43
how does enfuvirtide inhibit HIV fusion host cell?
enfuvirtide is a peptide analogue of gp41 fusion domain
binds gp41 preventing fusion with host cell membrane
44
how can we inhibit HIV fusion and attachment to host cells?
maraviroc (attachment)
enfuvirtide (fusion)
45
how can monoclonal neutralising antibodies work as attachment inhibitors?
can be isolated from B cells encoding antibodies that neutralise SARS-CoV-2 and then be cloned and administered to patients
prevents virus binding to entry receptor (e.g. SARS-CoV-2 spike to ACE2)
already some covid drugs out there using monoclonal antibodies
46
how has viral resistance to monoclonal antibodies began to emerge?
immune escape variants can escape monoclonal antibodies due to surface proteins (e.g. of SARS-CoV-2) rapidly mutating causing resistant strains
in vitro assessment of the ability of monoclonal antibodies to neutralise original and omicron variants of SARS-CoV-2 show increasingly high doses of antibody required
47
what does amantadine do?
binds influenza A M2 protein inhibiting uncoating
mutation in the M2 protein however confers resistance
48
what antivirals inhibit HIV reverse transcriptase?
once HIV enters the cytoplasm it needs to start reverse transcription to convert RNA genome to cDNA which is catalysed by viral enzyme reverse transcriptase
two drugs inhibiting this (reverse transcriptase inhibitors or RTI)
- nucleoside analogues (NRTI)
- non-nucleoside RT inhibitors (NNRTI)
49
how do nucleoside analogues (NRTI) function to inhibit reverse transcriptase?
e.g. zidovudine (thymidine analogue; 3' hydroxyl group replaced with nitrogens) - competes with the natural substrates (dNTPs) for HIV reverse transcriptase; gets incorporated into DNA leading to chain termination (as no 3' OH)
e.g. acyclovir is an analogue of the nucleoside deoxyguanasine and has activity against HSV-1 and 2 and also VSV. Requires activation by virally encoded thymidine kinase which phosphorylates it allowing its incorporation by DNA polymerase leading to chain termination - resistance can develop through mutations in thymidine kinase or DNA pol
50
how do non-nucleoside reverse transcriptase inhibitors (NNRTI) function?
bind hydrophobic pocket near the catalytic site of HIV reverse transcriptase leading to a structural change in RT so it can no longer function
51
how do integrase inhibitors prevent HIV integrating its cDNA into the host genome?
integrase is bound to long-terminal repeats at each end of the HIV cDNA - it cleaves host DNA allowing the cDNA to be inserted into host genome - this is an essential step for transcription (only occurs from integrated cDNA) and once cDNA inserted HIV forms a provirus
integrase inhibitors such as raltegravir prevent integration of HIV cDNA into host genome
52
how do protease inhibitors inhibit HIV maturation?
Gag and Pol genes require cleavage by protease for virus assembly
protease inhibitors are substrate analogues (like NRTIs) and bind the active site of protease inhibiting its function
53
what is an example of a protease inhibitor for SARS-CoV-2?
paxlovid inhibits Mpro, the main protease of SARS-CoV-2
is comprised of nirmatrelvir (blocks genome replication) and ritonavir (inhibits metabolism of nirmatrelvir keeping levels up in blood)
early treatment with paxlovid significantly reduces risk of hospitalisation or death
54
what is hepatitis C?
transmitted by blood, infects hepatocytes in the liver leading to chronic infection causing liver damage, cirrhosis and risk of developing cancer
binds receptors and uncoats, +ssRNA so directly translated by host ribosomes, makes polyprotein which cleaves itself
NS3B confers proteolytic function for polyprotein cleavage
NS5B encodes RdRp activity
NS5A important for formation of replication complex
55
what direct acting antivirals (DAAs) are used for hepatitis C?
protease inhibitors of NS3B
polymerase inhibitors of NS5B (both nucleotide inhibitors and non-nucleotide inhibitors)
NS5A inhibitors
these are used in combination to prevent resistance developing and have very high cure rates (>90%) as well as being much shorter than prior HepC treatment regimens
56
how do neuraminidase inhibitors prevent the release of influenza virus (A and B)?
influenza virus relies on neuraminidase (NA) enzyme which is involved in viral release
NA inhibitors e.g. oseltamivir (tamiflu) and zanamivir inhibit NA activity preventing virion release
significantly reduce mortality in serious influenza infections
57
what are the difficulties associated with anti fungal drugs?
fungi are eukaryotes so more metabolically similar to human cells than bacterial cells - makes it difficult to find drug targets
because of this many drugs which inhibit or kill fungi are quite toxic for humans i.e. have a low therapeutic index
differences of fungi cells with human cells are the composition of the cell membrane and the presence of a cell wall
58
what are key targets of anti fungal drugs?
ergosterol in fungal cell membrane is important for membrane fluidity (can target both it and its synthesis) (good target cause mammalian cell membranes have cholesterol instead)
can target beta glucan synthesis by targeting beta-1, 3-glucan synthase
can also target DNA/RNA synthesis
59
what are polyenes?
polyenes are compounds used as antifungals given their greater avidity for ergosterol than cholesterol
bind ergosterol in fungal cell membrane creating channels that cause depolarisation of cell membrane
however they still bind cholesterol so are dose limiting
60
what are azoles?
compound used as antifungals
inhibits 14alpha-demethylase which is crucial intermediate enzyme for ergosterol synthesis thus depleting ergosterol thus impairing membrane fluidity and also producing toxic 14alpha-methylated sterols which accumulate and cause membrane stress
fluconazole is an azole
resistance emerges through mutations in 14alpha-demethylase preventing azaleas from binding or from fungi over expressing efflux pumps
61
what are allylamines?
antifungals which inhibit squalene monoxygenase which is a key intermediate in ergosterol synthesis
e.g. terbinafine which concentrates in skin and nail beds and has relatively low bloodstream concentration - use restricted to onchyomyosis and cuteness fungal infections
works by depleting cell membrane ergosterol impairing membrane fluidity and leading to accumulation of toxic sterols in cell membrane
62
what are echinocandins?
target fungal cell wall
bind beta-1, 3-D-glucan synthase enzyme complex inhibiting synthesis of 1, 3-D-glucan polymers which are key cross-linking structural components of cell wall in some fungi
glucan-depeleted cell wall susceptible to osmotic lysis
63
what is flucytosine (5-FC)?
antifungal which get selectively uptakes by fungus-specific enzymes cytosine permease (gets it into cell) and cytosine deaminase (activates it)
gets converted to cytostatic 5-fluorouracil (5-FU) in fungal cell which inhibits thymidylate synthase and causes RNA miscoding
so only gets converted to 5-FU in fungi
mutations in the enzymes confers resistance
64
how do vaccines work?
they stimulate immune memory as the secondary immune response is faster and of greater magnitude due to T and B cell memory
65
what is boosting?
sequential administration of multiple doses of a vaccine to stimulate memory cells
increases the size of the response
important for infections where a certain protective antibody level must be maintained
66
what are immune correlates of protection?
most vaccines act by stimulating antibodies which are effective at protecting as shown by passive immunisation and also transplacental antibody
the function of the antibody is important - what type of antibody is required depends on the pathogen, what disease it causes and how quickly it causes disease
e.g. viruses require neutralising antibody, N. meningitidis requires bactericidal antibodies
sometimes circulating antibody (through the like of a booster) is required for protection or sometimes the memory (anamnestic) response may suffice
so immune correlates of protection are aspects of immune response shown to correlate with protection against a particular infection
66
what type of immunity should a vaccine elicit for cytopathic viral infections?
kills the cells its infecting so important to have antibodies which can prevent infection from circulating viruses
67
what type of immunity should a vaccine elicit for a toxin-producing bacteria?
antibodies that neutralise the toxin cause this protects against disease
68
what type of immunity should a vaccine elicit for non-cytopathic viral infection?
need to induce both antibodies and cell-mediated immunity
antibodies prevent further infection and cellular response kills off infected cells so they don't continue producing virus (cause virus not killing cells)
69
what type of immunity should a vaccine elicit for non-invasive mucosal infections?
IgA antibodies as need a mucosal immune response
70
why are T cell responses important for vaccine mediated immunity?
T cells provide help to B cell responses
71
how is the site of the response relevant to vaccines?
most infections occur at mucosal surfaces but systemic vaccines given IM or SC don't induce mucosal immune response
e.g. polio vaccine; live attenuated polio vax (Sabin) prevents infection and disease (given orally and causes mucosal response) - inactivated polio vax (Salk) prevents disease but not infection (given IM)
we need to stimulate immune response at mucosal surfaces
72
why is it important to consider the inductive site when designing a vaccine?
inductive site is where immune response is induced
so mucosal vaccination leads to mucosal immune response - this is cause lymphocytes at that site will be primed to express site-specific integrins and chemokine receptors which allow them to recognise vascular adhesion molecules on endothelial cells and chemokines so they can home to effector site
73
why is vaccine coverage important to consider when designing a vaccine?
vaccines provide sufficient coverage through herd immunity; when unvaccinated individuals are protected by high levels of vaccine coverage
threshold for herd immunity depends on infectivity of the pathogen i.e. the R0 (number of secondary cases generated by an infectious individual in susceptible population)
so higher R0 (basic reproduction number) requires more vaccine coverage to achieve herd immunity
74
what makes an ideal vaccine?
it is safe with minimal side effects but it elicits a strong, protective and long-lasting immune response
targets protective epitopes that don't vary
heat and dryness stable
long shelf life
low cost
easy to administer (ideally not multiple boosters or incompatibility for co-administration with other vaccines) no needles)
75
what types of vaccine are there?
live attenuated vaccine
inactivated organisms
virus-like particles (VLPs)
subunit vaccines
gene-based
76
outline the advantages and disadvantages of live vaccines?
advantages:
- most closely mimic natural infection (replicate at anatomical site of infection, stimulate mucosal immunity, replicate intracellularly stimulating MHCI presentation and thus good CD8 response
- longer duration of immunity (cause of above)
- dose sparing as can replicate so easier to make
disadvantages:
- reactogenecity (e.g. rash/fever with MMR)
- inadequate attenuation
- reversion to virulence (e.g. sabin)
- risk to immunocompromised
77
what are the three main types of live vaccines?
host range mutants
attenuated
recombinant bacterial vectors (bacteria encoding a transgene which is antigen of interest - e.g. salmonella, recombinant BCG)
78
what are host range mutants?
virus that comes from natural infection in non-human mammalian species and has attenuated characteristics in humans meaning it doesn't cause severe infection but provides protection from closely related pathogens e.g. vaccinia for variola virus
79
what are attenuated vaccines?
less pathogenic than wild type virus for some reason e.g. using old cultures of chicken cholera or dried virus for rabies
genetic basis of attenuation often not understood, just know that it gives host protection
80
what are some methods of attenuation?
serial passage in vivo or in vitro in non-natural host (polio)
altered culture media (Mtb - BCG; cultured for over ten years so it acquires mutations attenuating it)
selection of cold adapted mutants (e.g. influenza, can't replicate in lungs)
genetic recombination or reassortment in vitro (influenza, rotavirus)
identify virulence genes and delete by genetic engineering (requires us to know the basis of these mutations)
81
outline the use of killed organisms for vaccines?
not as effective as live vaccines as they don't replicate in hosts and so require higher and repeated doses making them more expensive
however they are safer
inactivated with either formaldehyde or beta-propiolactone
whole organism e.g. inactivated poliovirus vaccine (Salk)
subunit: influenza virus vaccine (attenuated donor master strain crossed with new virulent antigenic variant strain to create attenuated vaccine strain - HA and NA then purified from this for vaccine)
82
what are virus-like particles (VLPs) and other nanoparticles?
VLPs - carry empty virus particles presenting several copies of the same antigen on their surface; allows self-assembly of recombinant viral capsid proteins
e.g. HPV VLP contains L1 capsid proteins from 9 HPV subtypes
can have complex enveloped VLPs
other nanoparticle antigen carriers include self-assembling protein nanoparticles (non-viral proteins carrying antigen) which could allow for multimeric antigen presentation which strongly stimulates B cells
83
how do we make the HepB vaccine using recombinant DNA technology?
take surface antigen from hepB and express it in plasmids adapted for yeast
grow up plasmids in yeast in fermenter to make lots of copies of surface antigen
surface antigen can self assemble into VLPs which can be purified and put into vaccine
84
what are subunit vaccines?
use protein or polysaccharide from pathogen to induce predominantly antibody responses
important to know which antigen provides a protective response to make these vaccines
four types: destroy pathogen and then purify subunit (influenza), recombinant proteins, polysaccharide and polysaccharide-conjugate vaccines, experimental e.g. peptide epitope
low immunogenicity means subunit vaccines often need adjuvants
85
what are polysaccharide vaccines?
capsular polysaccharides recognised through T cell independent mechanisms (BCR cross linking)
stimulate short-lived B cell responses with no memory and so no protective, long-lasting antibody responses (cause no protein so no T cell stimulation)
poor response in young children
I think these are subunit vaccines
86
what are conjugate vaccines?
capsular polysaccharide conjugated to protein antigen (e.g. tetanus or diphtheria toxoids)
protein processed via MHCII inducing CD4 T cell response
strong long lasting humoral response
87
what are gene-based vaccines?
encode antigen of interest which gets expressed by host cells
three types:
viral vectors; replication competent (can be replicated in host), replication deficient (can infect cell but not replicate) and replicon (virus can replicate but not pass on to another cell)
RNA (get translated into protein); mRNA vaccines, self-amplifying RNA vaccines
DNA; plasmid that gets introduced into cell to produce gene of interest
88
what are recombinant viral vectors?
virus encoding a transgene (antigen)
can be replication competent, replication deficient or replicons
infection of cells with it leads to production of foreign antigen
e.g. vaccinia, adenovirus, alphavirus
benefits: achieve good antigen expression, stimulates both T and B cell immunity, can express multiple antigens simultaneously
negatives: high production cost, difficult storage (cold), moderate side effects, risk of disease in immunocompromised if replication competent
89
outline how replication competent recombinant viral vectors work?
whole viral genome plus antigen of interest (so have both non-structural and structural genes)
infect cells in vitro we can produce the virus that can then infect cells in vivo and those infected cells will make more of the protein of interest and more of the virus (which is pretty much the vaccine) which will go on to infect more cells
90
outline how replication deficient recombinant viral vectors are made?
removed a lot of the essential genes and provided transgene of interest and seperate plasmid expressing necessary genes for viral reproduction into producing cell line
when virus introduced in vivo we no longer have reproduction genes so can't make any more virus but can make antigen of interest
91
outline how replicon recombinant viral vectors are made?
removed structural proteins but left in essential non-structural proteins (in particular RdRp)
we put structural proteins in in trans in vitro so we can make the virus
virus can then infect cells in vivo and can't make any more virus (cause no structural genes) but do have RdRp so can replicate viral genome
92
how do we make RNA vaccines?
mRNA - synthesise mRNA by in vitro transcription and protect it in lipid nanoparticles which get taken up in host cell and protein expressed on MHC to stimulate both T and B cell responses (PRR in endosome also important for stimulating immune response)
self-amplifying - have the replicase but given as lipid nanoparticle rather than virus leading to amplification within cell
93
what are adjuvants?
enhance immunogenicity as they are immune potentiators (do so by activating innate immunity through APC recruitment, activating PRRs or inflammasome activation)
have different delivery systems which concentrate antigens, target them to APCs or allow slow release (depot effect)
94
what are prime boost strategies?
when we give more than one dose
homologous prime boost - use same vector or same vaccine type for both prime and boost
heterologous - use two different sorts of vaccines to stimulate immune response
95
what are platform technologies?
a common backbone for vaccine production which can be adapted for use against different pathogens by inserting new genetic/protein sequences
allows for rapid manufacturing for rapid use against novel pathogens - could allow development in weeks to months
examples include RNA, DNA and viral vectors
96
have we got enough vaccines?
vaccines have proved hugely beneficial to public health and disease eradication
however there are still many diseases which require vaccines killing millions of people every year and putting huge burdens on the health sector
97
why aren't vaccines available against all diseases?
pathogen biology (N. gonorrhoeae; antigenic variation, HIV; envelope structure/integration/latency, Mtb; dormancy/intracellular survival)
difficulty in virus isolation/propogation (Ebola, HCV)
evolution of vaccine escape mutants (HIV, HCV, universal influenza)
lack of immune correlate of protection (HIV, HCV, malaria, Mtb)
safety concerns (RSV; formaldehyde-inactivated vaccine worsened disease, dengue virus; potential for immune potentiation)
98
what considerations need to be made for the development of new vaccines?
basic research considerations
commercial considerations
funding considerations
98
what are the basic research considerations for development of new vaccines?
pathogen-host interactions (how does pathogen cause disease, virulence factors)
what kind of immunological response is requires e.g. immune correlates of protection (antibody vs cellular)
whereabouts is a response required (mucosal surface, serum)
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what are the commercial considerations for the development of new vaccines?
will it make a profit (production costs, dose requirements)
who will buy it (children or adults, travellers, developed vs developing world)
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what are the funding considerations for the development of new vaccines?
disease prevalence
is it effective (protective)
will it save money
is it safe
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what does pre-clinical vaccine development involve?
immunogenicity studies in animals - involves assessing relevant immune responses (antibodies, cell mediated). Must provide proof of concept by demonstrating protective immune responses, this step guides selection of doses, schedules and route of administration
quality controlled manufacturing process - good manufacturing practice (GMP) outlines the guidelines for this, consider composition, potency, stability
safety testing in animals - challenge with high dose in toxicology assessments
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what does clinical phase vaccine development involve?
phase I - assess safety and immunogenicity using 20-100 healthy young adults and varying doses of antigen and adjuvant
phase II - assess safety and immunogenicity with randomised controlled trial with 100-300 participants representative of intended target population
phase III - assess safety and efficacy with randomised controlled trial with 300-3000 participants
regulatory review and approval
post-licensure evaluation of efficacy and safety (e.g. rare side effects)
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what is HIV?
retrovirus
enveloped, RNA genome
key envelope glycoproteins are gp120 and gp41, CD4 primary host receptor
infects CD4+ T cells and integrates into genome (latent reservoir)
causes progressive depletion of CD4+ T cells leading to immunosuppression
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how does HIV infection progress?
the first few weeks of infection result in acute HIV syndrome which involves acute decline in CD4+ cell count and wide dissemination of virus in lymphoid organs
immune system brings it under control and clinical latency begins involving progressive decline in CD4+ cell count as viral replication increases
constitutional symptoms occur after about 7 years, increasing incidence of opportunistic infection and eventually death
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how does HIV infection start?
virus crosses epithelial layer via micro abrasions and gets picked up by APCs which take it to draining lymph node. Lots of CD4 T cells here so becomes hotspot for HIV replication and this spills over into blood and other lymphoid organs
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how would we theoretically prevent HIV infection and spread and why can't we?
need neutralising antibodies to prevent infection and also need cytotoxic T cells to kill infected cells once infection occurred
problem is that there are no known immune correlates of protection i.e. we don't see anyone with natural immunity
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outline the diversity of HIV?
massive diversity with a number of different clades circulating in different regions
also massive intra-individual diversity over time of infection due to HIV exhibiting a high rate of mutation
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why does HIV exhibit a high mutation rate?
reverse transcriptase and RNA polymerase II both lack proof reading ability - approx every 1/4 replication cycle has a mutation
high rate of viral turnover due to generation of 10^10 visions per day
HIV within-host phylogeny often very different to founder virus due to immune selection as well - leads to mutation of gp120 and CD8 epitopes (stuff recognised by CD8 T cells) in other proteins
this leads to huge intra-individual diversity
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what success have studies had at inducing adequate immune response to prevent HIV with a vaccine?
one study on high-risk individuals (injection drug users) in Thailand performed a randomised controlled trial using recombinant gp120 subunit vaccine with alum adjuvant
induced good levels of antibodies against gp120 including neutralising antibodies against a laboratory HIV strain - so was immunogenic
however failed to protect against infection despite stimulating neutralising antibodies
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what is the evidence that CD8+ T cell responses are associated with slower progression of HIV infection?
chromium release assays using target cells infected with viral vector expressing antigen showed higher cytotoxic T cell response slowed disease progression
other studies showed CD8+ T cell responses associated with lower viral load as depleting CD8+ T cells in rhesus macaques increased viral load
vaccination of rhesus macaques with adenovirus vaccine expressing SIVgag (SIV v similar to HIV) was successfully immunogenic in stimulating CD8 T cell response - challenge showed those with vaccine had lower lower viral set point and prevented CD4+ T cell loss
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what studies have tried to replicate successful results of vaccines inducing cell-mediated immunity for SIV in rhesus macaques for HIV humans?
one study performed randomised controlled trial using adenovirus vaccine on high-risk participants (e.g. IV drug users) not aiming to prevent infection but to suppress viral load and CD4 T cell decline
However showed no difference in rate of infection or viral load set point, also showed people with vaccine more likely to get infected cause more CD4 cells for HIV to infect
so this worked in closest animal model but not humans
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why are adenovirus vectors a good option for designing HIV vaccine to induce cell mediated immunity?
adenovirus vectors are good at stimulating CTL responses
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what vaccine trial has been most successful so far at preventing HIV infection?
RV144 vaccine trial in Thailand tested prime boost combination of two vaccines (ALVAC-HIV which is replication deficient canarypox vector expressing env, gag and pol and also AIDSVAX B/E)
showed 31.2% vaccine efficacy against acquiring infection but no effect on viral loading infected
so not great outcome but best yet
they also looked for immune correlates of protection during peak vaccine-induced immune responses using many assays and identified IgA binding env was associated with increased risk of infection while gp70-V1V2 binding antibodies associated with reduced risk
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did follow on studies from RV144 manage to replicate its efficacy?
nah
HVTN 702 vaccine trial using similar vaccine and prime-boost regimen in Southern Africa interim analysis met pre-specified criteria for non-efficacy and trial discontinued
other trials e.g. HVTN 705 and HVTN 706 used similar methods and either produced statistically insignificant results or were stopped early for futility
so one trial has shown statistically significant (but modest) protection but it hasn't been successfully replicated
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what are broadly neutralising antibodies (bNAbs)?
these are conserved between HIV variants and broadly neutralising
10-25% of HIV-1 infected patients develop these cross reactive neutralising antibodies, usually after several years by when the virus is resistant due to addition of glycans hiding epitopes
four highly conserved targets have been identified and multiple potent monoclonal bNAbs have been isolated and shown in vitro to broadly neutralise virus
potential use for passive immunisation
passive immunisation has been shown in rhesus macaques using infusion of neutralising bNAbs prior and during high dose intarectal challenge with chimeric SHIV
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have any successful human studies using bNAbs occurred?
two randomised trials of bNAbs targeting CD4 binding site didn't show statistically significant protection but there was in sensitive strains
this provides proof of concept that bNAbs can prevent HIV infection
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what is the main issue with using bNAbs?
they show high level of somatic mutation during germinal centre response making them difficult to develop in vivo - these mutations are also essential from germline
40-100 in bNAbs while 15-20 in most other monoclonal antibodies
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how to stimulate bNAbs with a vaccine?
need to develop an immunogenicity that induces bNAbs production
could use a series of immunogens to prime and then 'shepherd' B cells to mature from germline to the point at which they produce potent bNAbs (germline targeting)
mRNA technology could accelerate iterative testing of HIV vaccines
HOWEVER will have to induce sustained production of high antibody levels and more than one bNAb will prob be needed to protect against all circulating viruses
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why would we need to induce effector response in tissues rather than Tcm response?
conventional prime-boost regimens using adenovirus and poxvirus vectors induce lymphoid tissue-based memory (central memory/Tcm)
Tcm's need antigen driven expansion for peak effector response and HIV rapid replication in mucosa means we need earlier response
rapid replication and spread of HIV means T cell effector responses likely to be more effective in hours to days after mucosal infection (effector memory/Tem)
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how might we better stimulate Tem response for HIV?
one study used novel CMV vector vaccine and compared with adenovirus vector vaccine and used homologous intrarectal challenge with SIV infection
CMV good at initiating tissue response
showed that those animals vaccinated with CMV vector vaccine were protected against HIV infection and could control it in rectal mucosa (long term control with no loss of CD4+ T cells)
this control correlated with SIV-specific CD8 cells (not CD4 or neutralising Ab)
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what are the stages of primary HIV/SIV infection?
stage I - mucosal translocation and initial infection
stage II - viral replication at portal of entry and recruitment of target cells
stage III - early viral spread to lymphatics and blood
stage IV - generalised systematic viral replication with immune activation and dysfunction
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at what stages of HIV infection could we intercept the virus based on current evidence?
could intercept early on (stage I and II) using neutralising antibodies and possibly non-neutralising as shown by RV144 trial which could prevent infection
could intercept at stage II or III using pre-positioned Tem in rectal mucosa which could arrest viral replication and clear it
T cell responses have been shown to be ineffective at providing protection later on (stage IV) (hence why pre-position Tem response better than Tcm response which occurs later on)
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