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Molecular Evolution & Medicine• Systemic Diseases

(will discuss later inthe course)

• Infectious Diseasesa. Focus on disease

organism1. Origin and identity of

new infectious agents2. Monitoring

epidemiology of disease(including vectors)

3. Etiology of disease4. Treatment resistance5. Vaccine Design

b. Focus on geneticvariation in humans inresponse to agent

AIDS, An Emerging Disease

AIDS, An Emerging DiseaseOrigin and identity: HIV/SIV

Phylogeny (ML Pol)• HIV-1 Evolved From SIV

Strains in Chimps• HIV-2 Evolved From SIV

Strains in the Sooty Mangabey• There were multiple cross-

species transmission events,even within HIV-1(all originsAfrican, supports colonialhypothesis)

• A molecular clock implies HIV-1 (Group M) made the transitionfrom chimps to humans prior to1940, thereby falsifying the oralpolio vaccine hypothesis.

See Sharp et al. 2001. Phil Trans. Roy. Soc. Lond. B 356: 867-876

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Monitoring epidemiology ofdisease (including vectors)

Molecular evolution studieshelped establish that HIV is

transmitted via sexualintercourse, mother-to-fetus,sharing needles, and direct

contact of bodily fluids (recall“Dental Clade” paper)

Neighbor-joining trees of the gp120 and pol regions wereestimated in 51 HIV+ subjects.

In 28 subjects, the gp120 and pol trees were monophyleticby subject, indicating a single-source infection.

In 23 subjects, the trees were polyphyletic, and thesewere always highly significant using a Templeton test in

PAUP* against a tree of forced monophyly. Thisindicates multiple HIV infections of the same subject.The subjects were then categorized by whether or not

they were injection drug users, with the following results:

Monophyletic Polyphyletic Not an IDU: 22 13 IDU: 6 10

FET (2-tailed), p = 0.0233

SUBJECTS WITH A HISTORY OF IDU ARE MOREPRONE TO MULTIPLE INFECTIONS OF HIV.

Etiology of disease HIV Life Cycle

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Aids As An Evolutionary Disease

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V3 Region of the Envelope Gene• Specifies the Coreceptor Usage of HIV-1:

Therefore, nonsynonymous mutations in theV3 loop can alter cell tropism.

• Specifies the transition from non-synctium-inducing (NSI) to synctium-inducing (SI)HIV-1. (The NSI is the form that can infectand dominates the early phases of thedisease; the SI is an extremely virulentfrom)

• Presents one of the primary targets to theimmune system.

ALIVE Study In Baltimore, MD

• Cohort study that follows HIV-1 positive or at-riskinjection drug users at 6 month intervals.

• 15 subjects were chosen who became HIV +during the study and displayed a broad spectrumof clinical responses to HIV-1 infection.

• A sample of the HIV present in their blood istaken at each visit and CD4 & CD8 T cell countsare made.

• A 285 bp region of the envelope gene wassequenced that includes the V3 region.

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Evolutionary Success and Positionsin the Haplotype Tree

• Intravisit Tip Branches: recent and with noknown descendants, unproven evolutionarily orevolutionary dead-end.

• Intravisit Interior Branches: successfulevolutionarily because an internal node has twoor more descendant haplotypes.

• Intervisit Branches: successful evolutionarilybecause it has displayed temporal persistence.

Haplotype Tree of V3 Region In Subject 7

Recombination inV3 Region In

Subject 7

Most (but not all)Recombinants Are

Selected Against (noteshort branch lengths

of descendants)

Peeled Tree of V3 Region In Subject 7

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Evolutionary Success and Types ofMutation in the Haplotype Tree

• Replacement or nonsynonymous mutationsare more likely to alter protein structure, andtherefore cell tropism, SI vs. NSI, and targets forimmune system.

• Silent or synonymous mutations have no effecton amino acid sequence of protein.

• Therefore, nonsynonymous mutations are morelikely to be under natural selection thansynonymous ones.

Mutations From All Subjects

0

100

200

300

400

500

600

700

800

900

Intravisit Tip Intravisit Interior Intervisit

Silent

Replacement

NeutralExpectations

p=2X10-5

p=0.13

p=6X10-8

χ = 17.27, 2 df, p = 0.00022

From the degeneracyOf the genetic code,Expect about a 2:1

Ratio of replacementTo silent substitutionsWith a Jukes-Cantor

Mutation Model.

These Results Show An Excessof Replacement Mutations On

the Branches With KnownEvolutionary Success.

Hence, Natural Selection IsOperating Upon the V3 Region toCause Change in its Amino Acid

Sequence During the Progression ofThe Disease.

These Simple Tests Can BeUsed To Test Many DetailedHypotheses About Natural

Selection.

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ALIVE Study In Baltimore, MD

• Each HIV-1 sequence was classified as syncytium-inducing (SI) if it had either arginine or lysine at aminoacid position 306

ORglycine at amino acid position 306 coupled with arginineor lysine at position 320.

• The actual presence of SI viruses was tested directly viaa tissue culture test for every visit. All such tests wereconcordant with the SI criteria given above.

Just Two Mutational Changes Are Necessary and SufficientFor the Evolutionary Transition From NSI to SI.

Mutations on The Transitional Branches* From the NSI to SIPhenotypes Versus All Other Branches

Replacement

Silent

Contingency Chi-Square = 4.05, Probability under the Null Hypothesis = 0.044

Transitional Branches Non-Transitional Branches

38*

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1004

377

*This excludes the replacement substitution(s) that changes the NSI/SI status

The transition from NSI to SI is marked by a burst of selectivelydriven evolutionary change in the V3 region beyond the twodefining changes. These changes differ in different subjects,

implying that there are many ways to fine-tune the SI phenotype.

Perplexing Question

Many Studies Show SI Form IsMuch More Virulent Than NSI.

Given That It Takes Only 2Mutational Changes For the NSI

Form to Evolve Into SI, Why Don’tSI Forms Evolve Early and Take

Over?

Disease Progression Categories

• Rapid Progressors: CD4 < 200within two years of seroconversion

• Moderate Progressors: CD4decline to 200-650 during periodof observation

• Non Progressors: CD4 > 650during period of observation

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Logit analysis (a maximum likelihood regression procedurefor categorical data with a 0/1 response variable, herenonsynonymous vs. synonymous mutations) of the NSI andSI portions of the haplotype trees as a function of theexplanatory variables of position in the tree topology (Top.),disease progression category (Prog.), CD4 category (CD4),CD8 category (CD8), receiving or not anti-viral drugs (AVD),and the interactions among these variables. The following arethe minimum parameter models that fit the data with p>0.05.

Virus Type Model Degrees of Freedom p-value

NSI Top. 1 0.6432

SI Top., Top. × CD4, Top. × CD4 × CD8 11 0.1134

A More Detailed Analysis RevealsThat The SI Form Is Under Strong

Selection When CD4 & CD8Levels Are High (Healthy Immune

System).As The Immune System Collapses,Selection Is At First Weakened on

SI, But With The CompleteCollapse, It Is Strengthened.

These Patterns of SI Selection AreConsistent With The Following:

•The V3 loop of NSI is more hidden from neutralizingantibody than the SI V3 loop, so SI is more “apparent” to theimmune system and subject to intense selection when theimmune system is healthy.

•This selective force declines with the collapse of theimmune system.

•As the immune system collapses, CD8 cells become moreabundant than CD4, and SI can better infect CD8 cells.Selection occurs on the SI forms to become more specializedon CD8 cells.

Treatment resistance

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Initially, Treatments Began With Onset of AIDS and Used Drugs Sequentially

HIV Rapidly Evolved Resistance To Each Drug, TherebyExtending the Life of the Patient Only By a Few Years. The Failure of This Treatment Regime

Is Not Surprising In Light of HIV’sEvolutionary Potential:

1. High Mutation Rates2. Generation Time of 2.6 Days During “Latent”

Phase3. Production of 1010 Virons Per Day During

“Latent” Phase4. Putting 1, 2 and 3 Together, Every Single

Possible Point Mutation Occurs Every Day5. HIV Is Also Highly Recombinagenic Once

Viral Loads Are High, Which Greatly IncreasesThe Rate of Selectively Driven Evolution

Now, Treatments Began As Soon As A Person Is HIV+ and Uses A Cocktail ofThree Drugs Simultaneously. This Has Proven Far More Effective.

The New Treatment WasExplicitly Designed to Prevent

or At Least Slow HIV Evolution• By Starting Treatment Early, the HIV Gene Pool

Has Not Already Amassed Large Levels ofGenetic Diversity

• By Keeping Viral Loads Low, The Amount ofRecombination Is Minimized.

• By Targeting 3 Loci Simultaneously &Minimizing Recombination, It Is Difficult ForHIV to Evolve Effective Drug Resistance

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An Application of AncestralReconstruction: Vaccine Development

Ancestor Preserves MostConserved and UniversalAspects of Protein Folding andFunction

The Center of Tree (An Ancestorto Part of the Tree, Descendantof Other Parts) Has SameAdvantages and MinimizesImmunological Distances

Consensus (the traditional norm)does neither

HIV INFECTION VERSUS RESISTANCEHIV INFECTION VERSUS RESISTANCE

Genetic variation in humans in response to agent

Candidate Genes For Resistance to HIVHIV and Humans

For HIV To Have An Impact onHuman Evolution, This VariationMust be Inherited (& Heritable)

There is much variation in the rate at which AIDSdevelops after HIV infection in humans, with some

people never develope AIDS even decades after theiroriginal infection.

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Statistically Significant InferencesFor Resistance To HIV Using

Genetic Markers Scattered OverThe Human Genome

Data From Dean et al. (Science 273: 1856-1862, 1997).

HIV and Humans

CCR5 As A Candidate Gene For Resistance to HIV

HIV-1 initially interacts with a cell-surfacereceptor, primarily CD4

Conformational changes in both the viralenvelope and the CD4 receptor permit thebinding of gp120 to another cell-surface

receptor, such as CCR5. HIV then fuses withthe cell.

People bearing a frameshift mutation in the CCR5locus are resistant to HIV.

Other Genetic Variation Associated WithResistance to HIV

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Other Loci Have Been Identified For HIV Resistance viaGenome Scanning and Candidate Loci Approaches, And

Some Display Epistasis and Heterozygous Effects and HighAllele Frequencies (hence, they produce heritable variation).

SDF codes for a type of protein called achemokine that binds to other receptors

used by HIV such as CXCR4, Causing theReceptor To Be Taken Into the Cell and No

Longer on the Cell Surface

CurrentStatisticsFromSouthAfrica

These differences inresistance translate into

heritable fitnessdifferences, so HIV is

affecting human evolution

Under Isolation By Distance and Coalescent Theory, A NewMutant Is Usually Most Frequent Near Its Center of Origin.

The CCR5 Frameshift Polymorphism

Coalescent Analysis Also Indicates Frameshift MutantOriginated 275-1,875 Years Ago.

The Current Allele Frequencies In Europe Are Too HighTo Have Evolved In This Time Period Under GeneticDrift (drift theory implies it would take about 127,000

years to explain its current European frequencies)Hypothesis:

•The black plague bacillus produces aneffector protein that binds CCR5 thatleads to diminished immune response

•Europe has been subjected to severalwaves of black plague in the last 2000years, including one that killed 25-33% ofthe population in 1346-1352

•CCR5Δ32 arose in Europe and couldhave been selected if it provides resistanceto the black plague