Micro Final

Vaccine efficacy_______ vaccine effectiveness

Define vaccine efficacy and vaccine effectiveness

Does not equal

Efficacy-clinical trials, phases

Effectiveness-does it work, human error, different strains

Vaccine effectiveness is what? Why?

Dynamic

Changes as the virus changes

Vaccine series interval

Would have been altered if it were produced pre-pandemic

8 weeks between shots would’ve been best

Are all mRNA vaccines equal

No

Myocarditis decreases when

Spacing of the doses is increased

Infectious viral load

Is significantly less in boosted individuals compared to unvaccinated/fully vaccinated

Other than neutralization, what do antibodies do?

Enhance immunity through engagement of the Fc receptor and complement activation

Dose and timing: Pfizer vs Moderna

30 micrograms vs 100 micrograms

3 weeks vs 4 weeks

mRNA and BNT162b2 vaccines

Induce more robust Fc-functional antibodies compared to infection

Activating innate and complement

What produces differences in quality of humoral response

Vaccine dose

Interval b/w doses

mRNA modifications

Lipid nanoparticle formulation

New technologies in vaccines

Silk (thermostable)

Microneedle patch

Why do we need new/better antiviral drugs? (4)

Vaccines against some things but not others

Some vaccines do not eliminate the need for therapeutics

Established treatments are not always effective or well tolerable

Vaccines typically only work on patient if given before they are infected

Entry inhibitors

Pre-entry

Uncoating (no uncoating no genome release)

Nucleic acid synthesis and processing

Reverse transcriptase/RdRp

Integrase

Transcription

Translation

Replication

Other target for antivirals

Protease inhibitors

Virus assembly/release

Immune system stimulation

Blocking attachment

Evusheld (combination of two monoclonal antibodies)

Pre-exposure

Spike attachment inhibitor

Amino acid substitutions to extend half-life

Interfering with viral protein production

Paxlovid:

Nirmatrelvir-mimics Mpro, binds directly to the Mpro active site, preventing proteolytic processing of viral polypeptides

Ritonavir-inhibits metabolism on nirmatrelvir

Interfering with viral replication

Molnupiravir and remdesivir

prodrug that is phosphorylated in cells to give rise to its active form

Viral RNA polymerase incorporates molnupiravir/remdesivir into the newly synthesized viral genome

Accumulation of errors in the viral genome

Blocking viral release

Neuraminidase (NA) inhibitors (Tamiflu)

Drugs target the enzyme active site-conserved region

Sialic acid analog, 4’-OH replaced with an amino or guanidinyl group

No effect on non-influenza neuraminidases-different substrate specificity

Both influenza A and B blocked

Must be taken early

Highly Active Anti-Retroviral Therapy (HAART)

Drug combinations to improve the reduction in viral load-demanding on physician and patient (2 RT inhibitors and 1 PI or 3 RT inhibitors, impractical for the developing world)

Increased side effects but higher efficacy

“Drug holidays” allow a reprieve from side effects

Timeline of antimicrobial agents

Took a long time to farm penicillin

War time-pen for soldiers

40s-60s golden age of antibiotics

35,000 deaths from MDR bacteria

Microbial Targets

Best ones our cells lack

Cell wall synthesis

Attachment/entry into host

DNA or RNA synthesis

Metabolism

Protein synthesis

Disruption of cytoplasmic membranes

Inhibition of cell wall synthesis

The cell wall protects a cell from osmotic pressure (osmotic flux → lysis)

Bacteria and fungi have cell walls (mammalian cells lack)

Peptidoglycan is composed of polysaccharide chains of alternating NAG and NAM

Constantly produced/repaired

Terminal D-Alanine released

Cross linked-brings sugars together to be structurally sound

DPA to position 4 alanine -> terminal alanine is released

Beta-lactams

Penicillin

Functional portion is a beta-lactam ring

Inhibit peptidoglycan formation by irreversibly binding to the enzymes that cross-link NAM subunits

Not effective on organisms such as mycoplasma

Resistant strains have enzymes (beta-lactamase) that catalyze the hydrolysis of the beta-lactam bond, inactivating the drugs

Inhibition of protein synthesis

Cells use proteins for structure and regulation, enzymes in metabolism, and as channels and pumps to move materials across membranes

Bacterial ribosomes (70s) are distinct from eukaryotic ribosomes (80s)

Mostly bacteriostatic (exception is aminoglycosides)

Erythromycin

Binds 50s ribosome → blocks translocation of the ribosome

Large structures with 14-16 membered rings

Gram positive cocci, alternative to penicillin

Bacteriostatic

Disruption of cytoplasmic membranes

Some form a channel in the membrane, damaging its integrity (poking holes): Polymyxin B (effective against Bacillus polymyxa), Bacitracin (effective against B subtilis)

Some disrupt transport across the cytoplasmic membrane: Pyrazinamide (effective against M tuberculosis, most effective against intracellular nonreplicating bacterial cells)

Inhibition of metabolic pathways

Whenever differences exist between the metabolic processes of a pathogen and its host, antimetabolic agents can be effective

Atovaquone interferes with electron transport in fungi and protozoa

Drugs that block entry/replication of viruses

Sulfanilamide and trimethoprim structural analog of substrates in metabolic pathways-produced entirely by chemical synthesis

Structural analogs

Sulfanilamide and PABA

Trimethoprim and folic acid

Inhibiting the THFA pathway

Inhibition at any step results in no THFA synthesized (no new nucelotides)

Unlike human cells, many bacteria can synthesize THFA

Critical for making new nucleotides

Sulfonamides

Structural analog of PABA

Competes with PABA for the active site of dihydropteroate synthetase

Resistance is widespread (enzyme has decreased affinity for sulfonamideds)

Trimethoprim

Structural analog of folic acid

Inhibits dihydrofolate reductase

Drugs are often given in combination

Resistance

Synergistic, bactericidal

Inhibition of nucleic acid synthesis

Toxicity-only slight differences exist between the DNA of prokaryotes and eukaryottes

Not recommended during pregnancy/lactation, children

Research

Cancer

Bactericidal

Quinolones

Active against bacterial DNA by inhibiting DNA gyrase

Off target effects include mitochondrial DNA replication

Rifampin

Bind to and inhibit the function of RNA polymerase during transcription

Affinity for bacterial polymerase greater than eukaryotic polymerase

Resistant genes…

Are ancient

Antibiotics are produced…

By microbes in sub-therapeutic levels

Overuse/improper use

Medical-2010-2015 abx use increased 65%, 40% of patients don’t finish their antibiotics

Agricultural

Medical usage of antibiotics

Preventative (surgery)

Therapeutic

Agricultural usage of antibiotics

Recognized growth potential when given sub-therapeutic levels

Abx usage at various stages of production

Growth potential

Disease prevention/husbandry

Post harvest

Implications for human transmission of resistance genes and resistant organisms

Greater than 25% of all abx…

Have been used in animals (not to treat disease)

Increase in agricultural abx use

67%

63K tons in 2010 to 105K tons in 2030

How resistance develops

Mutation within a gene that conveys resistance

Selection-individuals that are resistant

What should be maintained in patients body to inhibit the pathogen

Sufficiently high concentrations of a drug for long enough time

Antimicrobial agents can be used in ______ to do what?

Combination

So that pathogen resistant drugs will be killed by other drugs (synergism too)

When should abx be used

In necessary cases

Should be limited otherwise

What should be developed for abx?

Semi-synthetic drugs which add/modify side chains to the original molecule (second and third generation drugs

Search for…

New antimicrobials

Diverse habitats, bacteriocins

Drug inactivation

Resistant cells may produce an enzyme that destroys or deactivates the drug

Altered uptake

Resistant cells may slow or prevent entry of the drug into the cell

Resistant cells may pump the antimicrobial out of the cell before the drug can act (porins)

Bacteria within biofilms are protected (reduced fusion/slower metabolic activity)

Altered target

Resistant cells may alter the target of the drug so that the drug can’t attach to it or binds it less effectively

Resistant cells may alter their metabolic chemistry or abandon the sensitive metabolic step

Resistant cells may produce decoy protein to protect the actual target of an antimicrobial drug

Resistance to quinolones

Ex: Ciprofloxacin

Effective against G- and intracellular bacteria

Drug inactivation

Altered uptake

Altered target

Disruption of the cytoplasmic membrane by colistin

LPS modification/ downregulation of LPS (prevents binding, altered target)

Efflux pumps (altered uptake)

Increase capsule (prevents uptake)

Porins and efflux pumps

Take porins out of membranes

Narrow the channel

Decrease expression (100-10)

BONUS: Which of the following is NOT an action that can lead to antimicrobial resistance

Taking two antibiotics simultaneously

BONUS: Never discontinue antimicrobials as soon as you feel better, always finish the bottle

Resistance to glycopeptides

Ex: Vacomycin

Effective against G+

Altered target

Prevent recognition

vanRSHAX

vanHAX genes encode enzymes that modify peptidoglycan precursors to terminate in a D-alanyl-D-lactate rather than a D-alanyll-D-alanine thereby altering the key drug-binding hydrogen bond

Expression is under the genetic control of regulator VanR and the receptor VanS

Results in 1,000-fold decrease in vancomycin binding and high-level resistance

VanS

Vancomycin binds VanS, causing self-phosphorylation and dimerization

VanS phosphorylates VanR

Sensing

VanR

Activates transcription of vanHAX

Regulator

VanH

Reduces pyruvate to d-lactate, which is then used as a substrate VanA

VanA

ATP dependent d-Ala-d-lactate depsipeptide ligase

VanX

Hydrolyzes the existing d-Ala-d-Ala peptide pool, resulting in the incorporation of peptidoglycan precursor terminating in d-Ala-d-Lac rather than d-Ala-d-Ala at the growing cell wall

Incidence

Number of new cases of a disease in a given area or population during a given time period

Prevalence

The total number of cases, both new and already existing in a given area or population during a given time period

Endemic

A disease that normally occurs continually at a relatively stable incidence within a given population or geographical area

Sporadic

Only a few scattered cases within an area or population

Epidemic

A disease occurs at a greater frequency that is usual for an area or population

Methods of disease transmission

Direct (person to person, droplet)

Indirect (airborne, fecal-oral, fomites)

Vector (Insect bite)

C diff pathogen characteristics

Gram-positive bacilli, spore-forming, anaerobe

C diff disease transmission

Direct-fecal-oral, person to person

Indirect persists on fomites

C diff characteristics

Natural member of gut microbiome

Nosocomial infection

Risk factors-antibiotics, >65, white male

C diff infection

Typically follows antibiotic exposure

Microbiota of 1-3% adults and 15-20% infants

Probiotics (saccharomyces boulardii) may be beneficial

Toxins of C diff

Enterotoxin and Cytotoxin (TcdA and TcdB)-degrade gut mucoe, attract inflammatory cells, pseduomembranous colitis

C diff treatment

Vancomycin-gets rid of vegetative cells

FMT (poop transplant)

Chlamydia trachomatis pathogen characteristics

Small gram-negative rods spore forming

Chlamydia trachomatis transmission

Direct: sexually, perinatal

Indirect: fomites

Chlamydia characteristics

Strict intracellular parasite: no cell wall, two membranes, can’t make its own ATP

Infectious elementary bodies, non infectious reticulate bodies

Risk factors: poor sanitation (trachoma), sexual intercourse (chlamydia)

Chlamydia pathogenesis

EB protect bacterium from harsh environmental factors

It has enzymes that inhibit the host inflammatory response

Type III secretion apparatus inhibits lysosome fusion

Steals lipids from golgi

Chlamydia clinical disease I

Serovars of chlamydia are responsible for different disease pathology

Gets in the eye causes blindness

Chlamydia clinical disease II

People with a penis: urethritis, epididymitis, proctitis

People with a vagina: cervicitis, dysuria-pyuria syndrome, pelvic inflammatory disease

Chlamydia-facts

Most frequently reported STI globally and in the US

85-90% of infected individuals are asymptomatic “silent disease”

Males complain of painful urination and watery discharge

Females may complain of UTI symptoms

Spreading to the fallopian tubes can cause salpingitis/ectopic pregnancy

Left untreated, it can cause pelvic inflammatory disease (PID)

Increases the danger of infertility in both males and females

Chlamydia management

Have protected sex

Get tested regularly: 2/3 of infections in people 15-24, detection 2-3 weeks after exposure, unprotected sex with new or multiple partners, at home tests

Treatable with antibiotics (azithromycin, doxycycline)

Transmission-vectors

The spread of pathogens via arthropods that transmit diseases

Biological-transmit pathogens but also serve as hosts for the multiplication of a pathogen during some stage of the pathogen’s life cycle

Mechanical-not required as hosts by the pathogens they transmit; passively carry pathogens to new hosts

Borrelia burgdorferi characteristcs

Spirochete

Vector (deer tick)

Most common arthropod borne illness in the US

Named for Willy Burdorfer (Old Lyme CT)

Risk: spending time outdoors

Borrelia burgdorferi-commonality

Most commonly transmitted by the deer tick: smaller than American dog tick, reservoir is the white-footed mouse, lives and mates in the fur of white-tailed deer, additional hosts include dogs, rodents and humans

Spirochetes are transmitted during the…

Blood meal

While sucking blood, physiological changes in the gut of the tick facilitate transmission of the spirochete

Borrelia burgdorferi prevention

Remove ticks with forceps or tweezers

Thoroughly clean area with soap and water

Apply an antiseptic

Bites can occur year round

Physiological shifts from feeding

Serve as cues for transmission

Migration to salivary glands (increase in temp, decrease in pH)

SALP15

Binds to B. burgdorferi OspC to facilitate migration

Binds OspC and shields it from antibody

Binds CD4+ T cells (Th) and suppresses their function

Knockdown of SALP15, reduces transmission overexpression enhances transmission

Stages of lyme disease

Variable incubation period of 3-31 days

Early localized stage

Slow, expanding red rash at site of bite-erythema migrans

Can reach sizes of 10-15 inches

Bulls eye rash

Fever, aches, and pains

Doxycycline

Early disseminated stage

Begins weeks to months later

Spirochete spreads to the skin, heart, nervous system, and joints

Late stage

Months to years later

10% of patients develop chronic arthritis

Lyme disease diagnosis

Blood test (serological analysis via ELISA)

Lyme disease vaccine

Dogs-LymeVax, routinely used

Humans-Lymerix, removed from market in 2002 due to low demand

mRNA based vaccine has been generated at Yale (Nov 21) and tested in guinea pigs, awaiting human clinical trials 19 proteins

Indirect fomite

Agents are transferred from a fomite to the individual

Fomite-soilborne diseases

Found in the soil

Withstand environmental extremes

Often produce endospores