Pharmacology: ID & Micro (Viruses)

Anti-HIV Drugs......................................................................................................................................................................... 2
Vaccines I ................................................................................................................................................................................ 5
Vaccines II ............................................................................................................................................................................... 7
Antivirals ................................................................................................................................................................................. 9

1
 Anti-HIV Drugs
Goals: how does chronic HIV cause disease? CD4+ T-cell depletion  immune suppression; direct consequence of HIV
replication, so inhibit HIV replication!
 Decrease HIV replication by as much as possible for as long as possible in every patient

HIV dynamics
 HIV replicates very rapidly (can be good: can shut off consequences of infection very quickly)
 Can only suppress chronically, not eradicate (latent reservoir)
 Need to get into brain, LN, genital tract, etc where infection is
 Can develop resistance extremely rapidly (esp. on monotherapy)

Nucleoside analog reverse transcriptase inhibitors

(NRTIs)

Zidovudine (azidothymidine, AZT)

 Also: ddI, ddC, d4T, 3TC, ABC, FTC, TDF
 thymidine analog; 5’ OH replaced with N3
 Synthesized in 1964 as anti-cancer; known
antiretroviral in ‘ 74, anti-HIV in ‘ 85, approved in ‘87
really quickly (single small trial)
 Still one of most potent anti-HIV drugs

zidovudine Mechanism of Action: NRTI, Anti-HIV antiretroviral agent. Thymidine analog. Triphosphate form inhibits
(azidothymidine, HIV reverse transcriptase, acts like chain terminator
AZT) Effects: Incorporated but not substrate for elongation in RNA-dep-DNA-pol activity of HIV RT
Selective Toxicity: poor, also inhibits mitochondrial DNApol
Indications: HIV
Administration: Short plasma half-life (1h) but much longer intracellular AZT-TP half-life (allows more
infrequent dosing, q12h).
Toxicity:
 Bone marrow suppression (common, mostly anemia, less commonly granulocytopenia).
 Rare: Myopathy, lactic acidosis/steatosis
 (steatosis = accumulation of fat in liver cells, fatal and class-wide for NRTIs albeit rare)
Resistance: Need 5+ AA changes. Slow to develop (only 1/3 on monotherapy resistant in 1 year), limited
cross-resistance with other NRTIs
Other:
 Phosphorylated by cellular enzymes to triphosphate (active form). Rapidly converted to AZT-MP,
accumulates in cell (-DP, -TP formation more slow)
 Well absorbed, eliminated by glucuronidation (Phase II).
 Commonly used in other countries (cheap generic) and sometimes for needle-stick prophy here,
although others probably work as well (only one studied)
Tenofovir (TDF) is most common in US now
tenofovir (TDF) NRTI antiretroviral; unlike AZT is a broad-spectrum antiviral (anti-HBV too),
more commonly used than AZT in USA

2
 Non-nucleoside reverse transcriptase inhibitors (NNRTIs)
 Not lots of structural homology in this group (unlike NRTIs) – binding to flexible binding pocket
Nevirapine (NVP)
 Also: delavirdine, etravirine, efavirenz
 Only one that doesn’t inhibit HIV-2, resistance develops rapidly (makes sense)

nevirapine Mechanism of Action: NNRTI Anti-HIV antiretroviral. Non-competitive reverse transcriptase inhibitor
(NVP) Effects: Binds to HIV RT distant from active site, causes conf. change to make RT less efficient
Selective Toxicity: No effect on human DNApols (incl. mitochondrial)
Indications: HIV
Administration: bid but could be qd (long half life)
Toxicity: mostly immune-mediated
 Rash, hypersensitivity (common), hepatitis (rare)
 Stevens-Johnson syndrome (rare, systemic attack of immune system against epithelium: full body
burn, slough off mucosa/epithelium)
 IMPORTANT: CYP450 3A4 INDUCER (drug interactions - like rifampin).
Resistance: FAST (very poor monotherapy) - needs single AA change (1000x resistance), days to weeks,
Cross-resistance to other NNRTIs (exception = etravirine)
Other: no intracellular activation required. Doesn't work against HIV-2 (doesn't bind RT). Well-absorbed,
eliminated by CYP450 3A4

Efavirenz is currently most used in US

efavirenz NNRTI like nevirapine
 Less toxicity, longer half life most common NNRTI in use in USA
 part of Atripla (1 pill qd);

Protease inhibitors (PIs)

HIV Protease: usually cleaves immature surface proteins (immature virion mature core/capsid structure, infectious)\
 Doesn’t prevent virion formation or release
 Inhibits maturation

3
Ritonavir
Ritonavir Mechanism of Action: PI, anti-HIV antiretroviral.
(r)  Competitive inhibitor of HIV protease (mimics transition state)
Effects: Inhibits HIV protease; prevents viral maturation (can make immature virus, but can't make it
infectious). 2-3 log reduction in VL (can't keep replicating + fast turnover of HIV = quick drop!), partially
restores CD4 count even on own
Selective Toxicity: No known human aspartyl proteases inhibited
Indications: HIV: PI but mostly used now as booster (increase concentrations of other HIV drugs)
Administration: bid (3-5h half life)
Toxicity:
 Inhibits CYP450 3A4 (also induces hepatic enzymes but net block). Drug interactions (but also used
for boosting).
 GI intolerance (nausea, vomiting, diarrhea), hyperlipidemia (elevated cholesterol & TGs, reversing
metabolic disturbance created by virus),
 First few weeks: common circumoral & extremity parasthesias (important for adherence)
 Rare: glucose intolerance, hepatic transaminitis (inceased AST/ALT).
Resistance: Weeks to months. Not necessarily in all subjects (unlike NRTIs)
 Primary resistance mutation: 1AA, 3-5x resistance.
 Secondary resistance changes accumulate, resistance keeps increasing, cross-resistance increases.
 If you can keep at high levels (e.g. boosting, drugs with high therapeutic index), 1st mutation will still
be suppressed (dose-response curve).
Other: no intracellular activation required. 99% protein bound. Variable bioavailability (first-pass metabolism,
autoinduction). Eliminated by CYP450 3A4 (oxidative)

Others
 Integrase inhibitors (e.g. raltegravir), inhibit HIV integrase (no chromosomal integration), very non-toxic (like
placebo!)
 Entry inhibitors
o Fusion inhibitors (e.g. enfuvirtide = T-20), interferes with membrane protein bundle formation needed
for fusion, only injectible BID & really expensive (salvage pts only)
o CCR5 antagonists (e.g. maraviroc) – inhibit host cell CCR5! Only effective if CCR5-trophic HIV, approved
for salvage pts only

HAART (highly active antiretroviral therapy)

 Combination therapy ONLY and for EVERY PATIENT
 Potent combinations possible for pretty much everybody
 Popular starting regimens:
o Efavirenz + 2 NRTIs (e.g. Atripla, 1 pill qd, 80% new HIV Rx in US
Atripla 1 pill qd for HIV! efavirenz (NNRTI) + emtricitabine (NRTI) + tenofovir (NRTI)
o Potent PI + 2 NRTIs (usually a boosted PI with ritonavir)

 Rationale: prevent drug resistance (probably need ~3 agents to prevent resistance emergence)
o Probably not synergy (3 NRTIs nearly as active as regimens with 2-3 different targets)
 Trends: less pills, co-formulated drugs, qd regimens, better tolerability, better resistance testing
o Make it easier since it’s for life

4
 Vaccines I
Vaccine: any formulation able to elicit antigen specific protective immunological memory

Classic vaccine principle: vaccine protection based on exposure of host to immunogenic agent followed by the natural
development of immunity (failed in HIV, malaria, cancer, etc)
 Adaptive immune response: only one that has memory, against certain antigens
 Primary response smaller,
 Secondary response faster & bigger to prevent infection
o Can prevent clinical disease!

Main types of vaccines

Live Vaccines Inactive Vaccines
antigens encoded by a genetic material and synthesized in protein/polysaccharide antigen is directly injected into
host (e.g.live attenuated) host (inactivated vaccines, recombinant proteins, purified
polysaccharides, etc)

presented on MHC class I (made inside cells) and MHC II; injected antigens presented on MHC class II (internalized)
make CD8 (cytotoxic) T-cells and helper T-cells too! make mostly helper T-cells

infectious agent, locally deposited, distributed to regional lymphnodes

makes strong innate inflammatory responses, weak, innate inflammatory response
strong induction of all adaptive responses (B and T cells) (requires addition of adjuvant)
longer memory mainly induce antibody responses (weak CD8 responses)

Essential components of vaccine formation

1. Route of administration: Appropriate presentation to immune system (B cells, CD4/CD8 T-cells)
2. Adjuvant(danger signal): immune stimulatory signals
o Start innate immune responses, shape adaptive effector mechanism
o Only really need for inactive vaccines – live vaccines have enough “danger” on their own
3. Active principle: Antigenic epitopes correlated with protection!
o Exact region of molecule & pathogen recognized by T-cell / antibodies

Route of administration (oral, subcutaneous, intradermal, intramuscular)

 Changes how antigen is processed (which cells?), how it modulates immune system
 Want mucosal immunity? Have to expose to mucosa!
 Want B-cell epitope? Polysaccharides (IgM, CD4-independent Ab) or protein (IgG, CD4-dep Ab)
 Want T-cell epitope? CD4: needs class II presentation, CD8: needs to be intracellular, class I presented

5
Adjuvant: Immunology mini-review:
 Live vaccines naturally activate innate immune responses (retain PAMPs,
ability to have C’ fixed, opsonization, etc.) so they don’t need adjuvant T-cells: immune system
 Inactive vaccines: include some stuff that binds to innate immune receptors degrades pathogen protein,
(adjuvant) processes via Class I or Class II
o Aluminum compounds, liposomes, virosomes, viruslike-particles, etc. MHC, presented as small
o Can also conjugate your antigen to an immunogenic protein (older) fragments

Mechanism of vaccine action  T-cells recombine to

1. Activate B-cell response  make Ag-specific antibodies generate molecules;
some can perfectly bind
a. Neutralization: block biological function of antigen
to MHC-epitope
b. Opsonization: faster clearance of antigen combination

2. Activate T-cell response  CD4 helper & CD8 cytotoxic B-cells: antibody’s exact
a. CD4+: cytokine secretion, supports B-cell/CD8+ cytolytic cell conformation is important
activation, proliferation, maturation, memory differentiation (needs to bind actually virus,
b. CD8+: cytolytic (kill infected cells) not virus+MHC)

Vaccines against viruses

 neutralizing antibodies (target surface envelope glycoproteins / proteins)
o Can use plaque reduction neutralization assay (mix virus & ab on culture, if neutralizing no plaques form
in culture cause virus can’t get in; otherwise plaques form where cells infected)
 CD8+ cytotoxic action (target cytoplasmic non-structural proteins)

Vaccines against bacteria (if extracellular)

 Opsonizing antibodies for ↑phagocytosis (target surface polysaccharides, envelope glycoproteins/proteins)
 Antitoxin antibodies (bind & neutralize toxins) – may not even need to kill bacteria to neutralize disease

T-cell independent response (for vaccines against sugars, for instance)

 make mostly IgM because they don’t stimulate CD4+
 Go away relatively quickly – conjugate to protein to induce helper T-cells & get better response!

T-cell dependent response (vaccines against proteins)

 Have antigen and stimulate T-helper cells (make IgG after IgM, differentiate into memory phenotypes)

Vaccines require very high standards

 High safety: Giving to large numbers of healthy people / especially babies!
 High benefit / efficacy:
o High individual protection levels (reduces risk greatly in individual)
o Herd immunity (reduce contagion in community, disease in unvaccinated population)

Herd Immunity

6
 Vaccines II
Lots of vaccine preventable diseases; these are hard to make though! US health care is expensive! Yada yada yada…

Main classes of antigen formation in clinical use

Host attenuated (mutant)
Whole organism
“Live” e.g. BCG; adapt pathogen to non-human host & back into person
(weakened agent)
Recombinant mutant
Purified fraction from organism (part of toxin, antigen, etc)
Sub-unit
“Inactivated” Recombinant antigen
“Whole organism”

Vaccines with live-replicating organisms

 Need to attenuate pathogenicity but preserve immunogenicity
Immune response:
 activate innate immune system, don’t need adjuvant
 presented on MHC I/II, whole shebang: T-cell/CD4-Th/CD8-CTL/B-cells/memory/neutralizing Ab
Can sometimes cause disease if immunocompromised (risk/benefit; depends on disease burden, etc.)

Examples:
1. Attenuated virus: e.g. varicella virus vaccine (varivax)
 Changing hosts can cause virus to adapt in ways that make it less pathogenic to humans
 This vaccine: from child with varicella to human lung cell cx  guinea pig cell cx (gets adapted to different host)  back to
human cell cx (ensure immunogenicity); given sub-q
Oral vaccines:
2. Recombinant/Reassortment virus: e.g. rotavirus vaccine (RotaTeq) can provide mucosal
 Reassorted with different animal strain, using reverse genetics, to immunity (IgA)
attenuate pathogenicity
 Cover multiple strains if there are various pathogenic strains of virus
 This vaccine: human + bovine rotavirus reassorted, covers 5 rotavirus strains, given orally

Vaccines that are inactive/non-replicating

Immune response
 presented on MHC class II (CD4, not CTL CD8s, good memory response)
 often need to give adjuvant to better stimulate innate immune system (often Aluminum)


Examples:
1. Whole organism e.g. hepatitis A vaccine
o Need inactivation of pathogenic properties but preservation of immunogenicity
o Need to give with adjuvant (aluminum)
o This vaccine: given IM; formalin-inactivated whole virus vaccine from attenuated HAV in cell culture (fibroblasts);

2. Subunit: Polysaccharide e.g. meningococcal polysaccharide vaccine (Menomune)

o Need to purify antigen & inactivate toxicity (if applicable); no epitope mapping needed
o Stimulates innate immune system via TLR receptors
o Produces CD4-independent B-cell response (IgMs produced, short-lived)
o This vaccine: sub-q, from several groups of N. meningitides polysaccharides

3. Subunit: Protein-conjugated polysaccharide e.g. conjugated meningococcal polysaccharide vaccine (Menactra)

o Same basic idea as above; still stimulates innate immune system, need to purify / remove toxicity, etc.
o Conjugate polysaccharide to something immunogenic (inactivated diphtheria protein in this case)
7
 o CD4-dependent B-cell response (IgGs, better memory response)

4. Subunit: Recombinant protein e.g. Hepatitis B vaccine

o Express antigen in genetically modified heterologous organism
o Requires adjuvant; produces CD4-dep B-cell response, IgGs, etc. (longer-lived)
o This vaccine: included in childhood to prevent perinatal transmission & cause it’s hard to diagnose

Vaccine uses:
 Active immunity: protection from person’s own immune system after vaccine, long-lasting
 Passive immunity: transferred from another person or animal as antibody; temporary, wanes with time
o Transplacental IgGs passed from mother to child: need to schedule childhood vaccinations accordingly
(or else mom’s IgGs will neutralize the vaccine antigen!)
o Results from: all blood/blood products, homologous pooled human Abs / Igs, antitoxins (pooled serum)

Immunizing during pregnancy

 Protects both mother and infant; transplacental Abs are cheaper & safer than Ig therapy
 Factors that influence Ab transfer in pregnancy
o Time from vax to delivery
o IgG level/subclass (IgM, IgA, IgE: DO NOT CROSS PLACENTA)
o Gestational age: @ 33wks IgGmaternal = IgGfetal, then fetal Abs more prevalent
 Influence on neonatal immunization
o Protect the infant
o Neutralize live attenuated vaccines; influence immunization schedule

Neonates’ Immunological Responses: immunize as early as possible; boost as needed

 Immature lymphoid organs; need 8+ wks post-natal age, adjust for premies
 Limited responses: CD8, innate system
 Immature dendritic cells (less CD4 activation, fewer germinal centers formed)
 Maternal Abs: don’t affect T-cell priming; epitope specific

The Elderly
 Thymus regresses
 Less naïve T-cells, mostly memory population: if you introduce antigen, it might be half-recognized by some cells
that are already around, and a less specific/effective immune response is mounted
 CD4 impaired (TRC/MHC signaling impaired)
 CD8 cells  senescence; Mϕ impaired

Immunocompromised: don’t give most of these vaccines!

 HIV patients can often mount immune responses if CD4+ are OK, check recs for vaccine

8
 Antivirals
Background
Viruses: obligate intracellular parasites.
 Initial thoughts: kill viruses, kill the cell (bad), so develop ways to Need for therapeutic antivirals:
stimulate the immune system (vaccines).  more immunosuppression (chemo, transplants)
 Amantadine: selectively target influenza without killing cells, made  pathological / genetic immunodeficiency (AIDS)
antivirals seem plausible  greater potential for rapid spread of infection
 Current strategies: use basic science discoveries about viruses, (higher population density, greater global
biological screening / high throughput screening, rational drug mobility, emergence of new antivirals)
design
Antivirals against HBV
HBV: 300M+ worldwide, major cause of chronic hepatitis, cirrhosis, hepatocellular carcinoma
 Tiny genome (dsDNA, circular, single-stranded region) copied from RNA template by viral reverse transcriptase
after incorporation into virion

α-IFN Mechanism of Action: Anti-HBV agent. Stimulates Jak/STAT pathways leading to transcription of genes with
"interferon-specific response element (ISRE)"
Effects: ISRE genes interfere with pretty much all aspects of viral life cycle (especially protein synthesis)
Selective Toxicity: IFN is part of normal human antiviral response
Indications: HBV (chronic active HBV)
Administration: Subcutaneous or IM (poor oral bioavailability)
Toxicity: flu-like symptoms and sometimes neuropsychiatric problems
Resistance: tolerance develops in most patients; HBV terminal protein blocks signal transduction
Other: very short-lived effects

lamivudine Mechanism of Action: nucleoside analog (NRTI), inhibits both HIV and HBV reverse transcriptase
(3TC) (similarities in enzymes)
Effects: converted to triphosphate by cellular enzymes; competitive inhibitors / chain terminator of HBV
DNApol (no 3' OH)
Selective Toxicity: humans don't have RT
Indications: HIV, HBV
Administration: po (good oral bioavailability)
Toxicity: negligible
Resistance: mutations in viral RT, some mutants less fit in vitro, others 3TC-dependent. Discontinuing
leads to rebound of viremia.

Antivirals against Influenza (A, B, avian)

Influenza: (-) sense ssRNA genome, segmented, synthesis relies on viral RNA-dep-RNA-pol

M2 inhibitors
amantadine Mechanism of Action: Anti-influenza agent. Inhibits M2 protein
Effects: M2 protein = ion channel used to pump protons into virion compartment & reduce pH, which is
rimantadine required for uncoating.
1. Primary effect: Drug binds inside M2 channel & blocks.
2. Secondary effect: decreases pH in Golgi, causing premature HA conf change, decreasing release.
Selective Toxicity: Humans don't have M2 protein
Indications: Influenza; not used as much anymore
Administration: Oral (rimantadine is methylated deriviative of amantadine, better oral bioavailability)
Toxicity: CNS side effects (rimantadine can't cross BBB as well; less side effects)
Resistance: Rapid (30% in 5 days) via mutations in M2 AA's lining channel. Same mutations overcome
early & late effects. Mutants retain fitness.

9
 Neuraminidase inhibitors
zanamivir Mechanism of Action: Anti-influenza agent. Competitive, reversible inhibitor of viral neuraminindase (NA)
Effects: NA cleaves terminal sialic acids from glycoproteins, glycolipids, proteoglycans, promoting effective
oseltamivir spread of virus throughout respiratory epithelium. Drug is sialic acid analog with larger, positively-charged
(Tamiflu) guanadine to interact more strongly with negative AA in active-site cleft. Oseltamivir also has a
hydrophobic region to bind to enzyme hydrophobic pocket
Selective Toxicity: humans don't have NA
Indications: prophylaxis and treatment of influenza
Administration:
 Zanamivir: poor oral bioavailability (IV or aerosol spray) - CAN'T USE IN PTS WITH RESP PROBS
 Oseltamivir: prodrug, better oral bioavailability, can give PO
Toxicity: Minimal (some nausea)
Resistance: inefficient in vitro; no cross-reactivity between zanamivir/oseltamivir, mutants have reduced
fitness

Antivirals against herpes viruses

Herpes: large, complex virus, linear, dsDNA genome, famous for latent infection
 Tons of interesting targets (tons of proteins) but worst choice - viral DNApol – is most common target

acyclovir Mechanism of Action: Nucleoside analog antiviral agent. Inhibits DNA synthesis
Effects: Chain terminator (no 3' OH) and competitive inhibitor of viral DNApol
valacyclovir Selective Toxicity: Two mechanisms:
1. For initial phosphorylation (ACV to ACV-MP) viral TK >cellular TK in affinity, so drug accumulates
in infected cells. (Next two P-lations via cellular TKs.)
2. ACV-3P inhibits viral DNApol much more than cellular DNApol.
Indications: HSV-1 (facial), half as active against HSV-2 (genital), not useful against CMV or HHV6
Administration:
 Acyclovir: 10-30% orally bioavailable
 Valcyclovir: prodrug with more bioavailability (substrate for intestinal/renal peptide
transporters; rapidly converted to ACV by intestinal/hepatic enzymes after oral administration)
Toxicity: Well tolerated: some nausea, diarrhea, rash, headache; rare renal/neural toxicity
Resistance: Mutation in viral TK (can't P-late ACV); causes cross-resistance to analog. Less frequent:
mutations in viral DNApol (less incorporation)

ganciclovir Similar to ACV but active against CMV

Similarities: converted to monophosphate by viral kinase, then -2P, -3P by cellular enzymes; competitive
inhibitor of viral DNApol
Differences:
 single hydroxymethyl group on sugar side chain
 no viral TK (p-lated by UL97, a protein kinase)
 not an absolute chain terminator (competitive inhibitor)
 accumulates to higher concentrations in CMV-infected cells (although ACV is better viral DNApol
substrate)
Toxicity: SERIOUS. affects bone marrow progenitor cells (low therapeutic index); inhibits lymphocyte
blastogenic responses
Resistance: mostly kinase mutations (like ACV)

10
foscarnet Mechanism of Action: antiviral (anti-herpes) agent. Pyrophosphate analog (inhibits all herpesviruses)
Effects: reversibly blocks pyrophosphate binding site on DNApol, inhibiting cleavage of pyrophosphate from
nucleoside-3P during elongation (pushing reaction backwards)
Selective Toxicity: viral DNApol is 100x more sensitive than cellular DNApol
Indications: only for life-threatening infections with no other treatment available (mechanism of action
different, so often works against ACV/GCV -resistant mutants of HSV/HZV/CMV)
Administration: Oral
Toxicity: SERIOUS. accumulates in bone, causes kidney toxicity
Resistance: Mutations in viral DNApol

fomvirisen Mechanism of Action: anti-CMV agent. Anti-sense RNA complementary to a viral mRNA
Effects: Binds, inhibits translation of a CMV mRNA encoding a protein essential for viral replication
Selective Toxicity: Doesn't bind human mRNAs
Indications: ganciclovir-resistant or -contraindicated CMV retinitis
Administration: injected into eye (completely unstable)
Toxicity: to your wallet
Resistance: has been reported
Other: VERY EXPENSIVE, only FDA-approved antisense drug

11