Bio 307D Exam 2

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Origin of HIVs

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- cross-species transmission on SIV/HIV genomic sequence comparison
- HIV-1 from chimpanzees (SIVcpz)
- HIV-2 from sooty mangabeys (SIVsm)

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HIV-1 genetic subtypes

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- aka clades
- Groups M, N, O, P represent 4 independent primate-human transmission events
- Group M further classified into clades (A, B, C...); West-central Africa
- Group N: Cameroon
- Group O: West-central Africa
- Group P: Cameroon (?)

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Nucleocapsid contains

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- 2 copies of ssRNA - each strand a result of provirus
- viral enzymes (proteins that facilitate chemical reactions) - reverse transcriptase, integrase, protease

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HIV virus classification

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- retrovirus
- lentivirus subfamily

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"Simple" retrovirus genome

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- 7000-11000 ribonucleotides long
- encodes 3-4 viral genes: gag, pro, pol, env

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Gag gene

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- encodes capsid proteins

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Pro gene

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- encodes protease

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Pol gene

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- encodes reverse transcriptase and integrase

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Env gene

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- encodes envelope (spike) proteins

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LTR (Long Terminal Repeat)

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- proviral sequence created by integration of provirus
- 5' LTR contains promoter region where cellular RNA polymerase binds to begin transcription of viral RNA; 3' LTR contains transcription termination sequences

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Virion structure

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- enveloped virus
- capsid [p24]
- matrix [p17]
- spikes [gp120 + gp41]

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HIV attachment

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- host cells must display 2 receptors: CD4 molecule + one of 2 chemokine receptors (CCR5 or CXCR4)
1. gp120 initiates attachment to CD4 receptor
2. binding to chemokineR changes shape of gp120; reveals gp41
- gp41 inserts into cell membrane, triggers membrane fusion

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Chemokine receptors

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- small proteins synthesized and release by certain cells that communicate/activate other immune system cells

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Genetic mutation in CCR5 coreceptor

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- 1996: CCR5 coreceptor identified, CCR5 deletion mutant identified [CCR5Δ32] - Koup & Landau found mutant CCR5 allele in 2 homosexual, high-risk HIV- men
- genetic testing revealed mutation in Caucasian Americans (spec., Western European origin); <1% homozygous [both alleles mutated], ~20% heterozygous [one mutant allele, one wild-type (i.e., functional) allele]

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Allele

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- variant form of a gene

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Mutation

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- change in nucleotide sequence within genome
- change in the code, deletion of nucleotide(s), insertion of new nucleotide(s)
- can occur spontaneously (no outside influence)
- can be 'induced' by influences outside the cell (chemicals, radiation)

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Effect of mutation

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- no effect
- deleterious effect
- advantageous effect

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Potential effect of mutation on protein

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- change in DNA nucleotide sequence within a gene encoding a protein may:
- have no effect on amino acid sequence
- result in a different amino acid (may or may not change protein function)
- result in premature termination of protein synthesis which likely will produce a non-functional protein

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Cellular targes for HIV infection

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- T-helper cells: CD4 and CCR5 (or CXCR4)
- Macrophage: CD4, CCR5

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Inside an HIV-infected cell

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- after partial uncoating, RT (carried inside the infecting virus capsid) begins synthesizing a dsDNA copy of HIV genome; process occurs in pre-integration complex (PIC); RT does not 'proofread'; RT can 'switch' between template RNA strands
- dsDNA genome transported across nuclear membrane in a complex containing viral integrase
- integration of proviral DNA by action fo viral integrase & cellular DNA repair enzymes; IN chemically 'breaks' bond in each of the strands of chromosomal DNA; positions viral dsDNA into chromosome break; cellular DNA enzymes 'repair' the break, restoring integrity of host cell chromosome
- expression of proviral DNA by cellular RNA polymerase; host cell RNA polymerase binds to promoter region in proviral LTR; RNA polymerases do not 'proofread'; HIV-1 genome encodes 15 viral proteins

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Structural genes

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- gag (capsid & matrix proteins)
- pol (RT, RNase, IN, protease)
- env (gp120, gp41)

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Regulatory genes

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- tat (transcriptional activator)
- rev (nuclear export of viral RNAs)

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Accessory genes

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- vif, vpr, vpu, nef

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-Static drugs

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- stop replication

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-Cidal drugs

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- kill microbe

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Chemoprophylaxis

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- attempt to prevent disease
- administer pre-exposure

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Chemotherapy

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- attempt to treat disease
- administer after exposure (most often, symptomatic patient)

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Reverse transcriptase

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- copies ssRNA into strand of DNA, then replicates DNA into its complement (reverse transcription)

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Integrase

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- breaks chemical bonds that makes up strands of chromosomes

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Protease

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- left over from when virus was released from its previous host cell

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Unique features of retrovirus replication

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- only way to kill viral DNA in the cell is to kill the cell itself
- viral info can be in a cell but not active
- cellular enzyme copies provirus DNA

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Tat

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- regulatory gene
- transcriptional activator

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Rev

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- regulatory gene
- nuclear export of viral RNAs

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Accessory genes

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- vif, vpr, vpu, nef

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From nonproductive to productive infection

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- activation of T-cell triggers HIV replications

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Synthetic drugs

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- manufactured from individual chemicals (chemists study reactions, components)

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Antibiotics

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- chemical compounds made by living microorganisms (usually by soil microorganisms)

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Semi-synthetic antibiotics

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- comes from naturally occurring compounds from microorganisms at its base, like antibiotics
- these naturally-occurring compounds are altered in a lab

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Broad spectrum

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- used to treat a variety of infections
- usually antibiotics

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Narrow spectrum

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- used to treat specific disease

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Selective toxicity

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- kill the pathogen, not the host
- least amount of damage to diseased person, most amount of damage to pathogen

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In vivo drug stability

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- how stable is the drug in the body

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In vivo drug distribution

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- where does the drug go in the body
- can it go where it needs to go

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Drug interactions

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- with other prescription drugs
- with OTC drugs
- with herbal ("natural") drugs

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Antagonism

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- one drug nullifies another's effect

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Synergism

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- two drugs work better together than separately

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Greatest selectivity found in antibacterial drugs

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- easiest to see differences between prokaryotic cells and human cells
- can stop synthesis of peptidoglycan/repairing cell wall (beta-lactam antibiotics - penicillins & vancomycin)
- can target assembly of ribosomes - different ribosomes from humans (have 50S & 30S; humans have 60S & 40S) - chloramphenicol & tetracyclines

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Selective toxicity in fungi & protozoa

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- much more problematic; must kill a eukaryotic pathogen inside a eukaryotic organism
- drugs with fewest side effects target ergosterol in plasma membranes of fungi
- cyst stage in protozoans not susceptible to drugs (metabolically 'dormant') - pathogen must be actively growing in order to treat it
- pathogens hard to treat once they are inside cells of the body

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Antiviral synthetic drugs

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- nucleotide must have a phosphate group
- deoxyguanosine
- ex. acyclovir: when acyclovir triphosphate is added into a growing DNA strand, no additional nucleotides can be added. No -OH for DNA polymerase to add on to; stops synthesis of DNA strand

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Physician's Desk Reference

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- source for information on prescription, OTC, and herbal drugs
- also has basic information on a wide range of human diseases and conditions

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Microbial drug resistance

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- mutations passed on to progeny (allows them to continue replicating) - bacteria, eukaryotic, viruses

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Drug resistance in bacteria through...

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- conjugation: direct contact between cells (DNA transferred)
- transduction: DNA carried into cell by a virus
- transformation: extracellular DNA taken up by cell

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Mechanisms of drug resistance

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- drug inactivation: inactivation of drug like penicillin by penicillinase, an enzyme that cleaves a portion of the molecule and renders it inactive
- decreased permeability: the receptor that transports the drug is altered, so that the drug cannot enter the cell
- activation of drug pumps: specialized membrane proteins are activated and continually pump the drug out of the cell
- use of alternate metabolic pathway: the drug has blocked the usual metabolic pathway, so the microbe circumvents it by using an alternate, unblocked pathway that achieves the required outcome

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4 sectors of the pharmaceutical industry

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- research intensive pharmaceutical firms (Pfizer, Lilly, Merck, Glaxo Smith Kline)
- Generic drug manufacturing firms (Sandoz, Teva)
- biotechnology firms (Amgen)
- drug delivery firms (Alza, Inhale)

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Pre-clinical drug development

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- lab and animal tests to establish pharmacology and toxicology in animal models
- pharmacokinetics (ADME) - provides data for dosing, drug formulation
- pharmacodynamics - drug's positive and adverse effects in animals
- toxicology - collect date on drug's safety in both high and low doses, including 24-month carcinogenicity of the product, impact on reproductive health
- human tissue cultures are sometimes used

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Goal of pre-clinical drug work

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- to provide data to support an Investigational New Drug Application (IND)
- IND provides evidence that human testing of the drug should be allowed
- FDA has 30 days to request more info, deny, or approve IND

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Contents of an IND

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- results of pre-clinical studies
- drug's chemical structure and how it is thought to work/mechanism of action
- listing of adverse effects
- manufacturing info
- detailed clinical testing plan which lists how the drug will be tested, patient types, and where the testing will take place and by whom

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Clinical development

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- process of testing a drug in humans
- goal = show the drug is safe and effective in treating specific conditions in certain patients
- prior to 1962 only the safety of the drug needed to be proven, but with the passage of 1962 FDA amendments effectiveness is required

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Phase 1 Trials

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- establishes safety of administration in humans and a range of safe doses
- trials conducted on young healthy male humans, who are put in a carefully controlled environment & are under observation for hours or day
- # of subjects 20 to 100

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Phase 2 trials

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- first clinical test of drug on people with the disease
- 100-500 patients
- determines if drug actually works ("proof of concept")
- helps establish dosing (min & max), safety (adverse effects), and efficacy testing, or endpoints

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Phase 3 trials

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- large scale randomized, blinded, placebo controlled trial (sometimes comparative product used instead of placebo)
- can take 3-6 years & cost millions
- 1000-5000 patients with targeted disease or condition
- goal = collect efficacy info about product to support New Drug Application to FDA (collect end-oint effectiveness data)
- many times conducted by contract research organizations (CROs)

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Placebo group

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- group of patients who are receiving the inactive, fake, or "sugar pill," but do not know it.

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Randomization

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- subjects are randomly assigned to either the placebo or test group
- being assigned to the test group is by chance

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Single blinded

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- subjects do not know if they are receiving the placebo or test drug, however the investigator knows

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Double blinded

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- subjects and caregiver or investigator do not know who is receiving test drug
- most common study

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NDA (New Drug Application)

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- submitted to the FDA for review following completion of phase 3 & when company believes that sufficient efficacy and safety data were collected
- non-biological agents - NDA cent to FDA's Center for Drug Evaluation and Research (CDER)
- biologicals: a BLA (Biological Application) is submitted to FDA's Center for Biological Evaluation and Research (CBER)

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Draft package insert included in NDA

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- proposed package insert
- specifies: indications, explains dosages and dosing administration, includes key statement as to safety, risks, mechanism of action, adverse effects

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FDA classifies NDAs

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- puts NDA into 2 categories based on: novelty of active ingredient, clinical improvement
- if product satisfies an unmet need or represents a significant therapeutic advancement, FDA may grant a priority review which takes 6 months or less
- if it doesn't fit the above, then the drug fits the normal time frame of about 12 to 15 months for FDA review

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Phase 4

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- "post-marketing surveillance" or "post-approval" studies - after drug has been approved and product is on the market
- not all products undergo phase 4 studies
- used to monitor adverse effects & check on safety of drug product

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Patents

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- granted anywhere along line of drug development, but normally occur early
- patent life is 20 years after patent is filed and approved
- pharmaceutical companies file for drug patent once they think they have an innovative molecule; normally this occurs before preclinical studies

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Exclusivity

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- exclusive marketing rights granted by FDA upon approval of the drug
- can run concurrently with patent time

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Exclusivity times

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- orphan drugs: 7 years
- new chemical entity: 5 years
- pediatric exclusivity: 6 months added to existing exclusivity or patent
- patent challenge: 180 days (ANDAs only)

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sterilization

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- process in which the end result is a sterile product - all infectious microorganisms that can replicate have been eliminated from that material
- only absolute term

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disinfection

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- inanimate objects
- adding or using a chemical that will kill microorganisms but does not guarantee total elimination of microbes

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sanitization

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- inanimate objects
- any process prescribed by a state health department designed to reduce the number of microbes on/in a specific material

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antisepsis/asepsis

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- living tissue
- chemical safe enough to use on living tissue that reduces number of microbes

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pasteurization

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- treating liquids - applying heat to kill microbes within that product thereby lessening the likelihood that there are harmful microbes in that product

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scale of most to least resistant microorganisms

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- bacterial endospores, protozoan cysts, gram-negative bacteria, viruses without envelopes, gram-positive bacteria, viruses with lipid envelopes

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factors affecting microbial inactivation

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- number of microbes
- nature of microbe
- inhibition vs. death
- pH
- temperature (generally, cold doesn't kill)
- presence of organic matter (it competes for the chemical that is supposed to be killing the microbe)

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approaches to killing microbes

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- physical agents: temperature (heat kills, cold storage preserves, heat changes shape of protein); desiccation (drying); radiation (ionizing = high energy, non-ionizing (UV) = lower energy)
- mechanical removal: filtration (membrane filtration removes microbes, used for liquids & gases)
- chemical methods: soaps, alcohols, halogens, hydrogen peroxide

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microbes & hands

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- normal microflora (what is already on your body) vs. transient microflor (what you pick up during the day)
- ease of microbial transfer: from non-porous surface (no liquid absorbed), from porous or absorbant surface - lower ability for transfer of microbes to you, from finger to lip
- hand washing: highly effective in preventing microbial speed