lecture 12 immunology and disease: parasite antigenic diversity and phylogenetic trees

Lecture 12

Immunology and disease: parasite antigenic diversity

and

Phylogenetic trees

Benefits of antigenic variationBenefits of antigenic variation

2. Infect hosts with prior exposure

• Hosts often maintain memory against prior infections, generating a selective pressure for parasites to vary

• Cross-reaction occurs when the host can use its specific recognition from a prior exposure to fight against a later, slightly different antigenic variant

• Good vaccines are ones that have excellent cross-reactivity (e.g. measles virus)

Figure 11-1 part 1 of 3In the simplest case, each antigenic variant acts like a separate parasite that doesn’t cross-react with other variants

Figure 11-1 part 2 of 3



• A more dynamic mechanism of antigenic variation is seen in influenza virus

• Antigenic drift is caused by point mutations in the genes encoding surface proteins

• Antigenic shift is caused by reassortments leading to novel surface proteins



• Antigenic drift is caused by point mutations in the hemagglutinin and neuraminidase genes, which code for surface proteins

• Every 2-3 years a variant arises that can evade neutralization by antibodies in the population

• Previously immune individuals become susceptible

• Most individuals still have some cross-reactivity and the ensuing epidemic tends to be relatively mild (but still kills 100s of thousands per year!)



• Antigenic shift brings in an all-new hemagglutinin or neuraminidase gene to a naïve population

• Can lead to severe infections and massive pandemics like the Spanish flu of 1918.

Phylogenetics

Today:Today:

• What is a phylogenetic tree?

• How are trees inferred using molecular data?

• How do you assess confidence in trees and clades on trees?

• Why do trees for different data sets sometimes conflict?

• What can you do with trees beyond simply inferring relatedness?

Molecular epidemiologyMolecular epidemiology

•The study of HIV ushered in a new way to think about pathogen variation where the nucleotide sequence became the primary source of information

•Phylogenetic trees have become very important analytical tools for tracking epidemics, understanding where “new” pathogens came from, testing forensic hypotheses, and reconstructing demographic changes

•Often termed molecular epidemiology since it answers many of the same questions as traditional epidemiology

Where did HIV come from?• Divine retribution• Doesn’t matter--it doesn’t cause AIDS• Conspiracy theories - e.g. the CIA did it• Voodoo rituals• Ritualistic use of monkey blood• Contamination of vaccines• Zoonosis (a disease communicable from animals to humans under

natural conditions)

How can we discriminate between these hypotheses?

Origins of HIV/AIDSOrigins of HIV/AIDS

Where did HIV come from?• Divine retribution

“The poor homosexuals--they have declared war upon nature, and now nature is exacting an awful retribution” -Pat Buchanan

"With 80,000 dead of AIDS, our promiscuous homosexuals appear literally hell-bent on Satanism and suicide” -Pat Buchanan

"AIDS is not just God's punishment for homosexuals; it is God's punishment for the society that tolerates homosexuals.” -Jerry Falwell

“Grown men should not be having sex with prostitutes, unless they are married to them” -Jerry Falwell

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are needed to see this picture.


Where did HIV come from?• Doesn’t matter--it doesn’t cause AIDS


Where did HIV come from?

• Doesn’t matter--it doesn’t cause AIDS


• Divine retribution • Doesn’t matter--it doesn’t cause AIDS• Conspiracy theories - e.g. the CIA did it• Ritualistic use of monkey blood• Zoonosis (a disease communicable from animals to man

under natural conditions)• Contamination of vaccines

• THE PLAUSIBLE HYPOTHESES ALL HAVE IN COMMON THE INCRIMINATION OF SIMIAN IMMUNODEFICIENCY VIRUSES (SIVcpz) FROM CHIMPANZEES

• THE KEY DISCOVERY WAS THE FINDING THAT AFRICAN PRIMATES ARE INFECTED WITH SIMILAR VIRUSES…


What is this thing and why is

it on all these slides?

Phylogenetics interludePhylogenetics interlude

• It’s all about ancestors and offspring, lineages branching

• The ancestor could be distant great grandmother or a human immunodeficiency virus

• The ancestral form of some gene (a “marker”) is inherited in two offspring lineages

• Let’s assume that we’re looking at virus from a “patient 0” who then infects two others

All the phylogenetics you need to know, in

5 minutes…

patient 0patient 0

patient 1patient 1

patient 2patient 2


• Mutations happen when genetic material is copied

• Changes accumulate independently along each branch (within each new infectee)

• If one of these patients now infects two new victims, they inherit those changes


• Eventually, a series of branching events, plus mutations along each branch, lead to 4 current HIV infected patients

• Their viruses display genetic diversity that reflects their evolutionary history

patient 6patient 6

patient 5patient 5

patient 4patient 4

patient 3patient 3

patient 2patient 2

patient 0patient 0


• Unfortunately, we almost never have access to that history

• What we can do, is go out into nature and sample genetic markers

• Then we work backwards to infer the most likely series of events that gave rise to what we observe


• In this case, we would infer a tree that correctly recapitulated the chain of infections…


TRUE TRANSMISSION HISTORYAND SAMPLING TIMES

INFERRED TREE FROM GENE SEQUENCES


• Sequences recovered from the victim

• Sequences recovered from the patient

• Sequences also recovered from other HIV-positive individuals from the same city


• The evolutionary pattern in this HIV phylogeny is just like the pattern in human mtDNA

• In both, we see a subpopulation that has recently emerged from a more diverse “source” population

• A few years of HIV evolution = 1 million years of human mtDNA evolution

HIV

Human


Molecular phylogenetics fundamentalsAll of life is related by common ancestry. Recovering this pattern, the "Tree of Life",

is one of the primary goals of evolutionary biology. Even at the population level, the phylogenetic tree is indispensable as a tool for estimating parameters of interest. Likewise at the among species level, it is indispensable for examining patterns of diversification over time. First, you need to be familiar with some tree terminology.

Tree terminology

Internal nodes represent hypothetical ancestors; the ancestor of all the sequences that comprise the tree is the root of the tree. Edges can also be classified as internal (leading to an internal node) or external (leading to an external node). Most methods try to estimate the amount of evolution that takes place between each node on the tree, which can be represented as branch length. The branching pattern of the tree is its topology.

Inspect branch lengths; they speak volumes

Kinshasa 1959

Manchester 1959

??

?

Inspect branch lengths; they speak volumes

Tree styles

There are many different ways of drawing trees, so it is important to know whether these different ways actually reflect differences in the kind of tree, or whether they are simply stylistic conventions. Think of the tree as a mobile:

polytomies

These polytomies can represent two different situations; first they may represent simultaneous divergence- all the descendants evolved at the same time (a 'hard' polytomy); alternatively, they may indicate uncertainty about phylogenetic relationships (a 'soft' polytomy).

Rooted and unrooted treesCladograms and additive trees can either be rooted

or unrooted. A rooted tree has a node identified as the root from which ultimately all other nodes descend, hence a rooted tree has direction. This direction corresponds to evolutionary time. Unrooted trees lack a root, and therefore do not specify evolutionary relationships in quite the same way. They do not allow the determination of ancestors and descendants.

Here we have an unrooted tree for human, chimpanzee, gorilla, orang, and gibbon (B). The rooted tree (above) corresponds to the placement of the root on the branch leading to gibbon.

consensus trees

monophyletic clades

Inferring phylogenies

• All phylogeny reconstruction methods assume you start with a set of aligned sequences.

• The alignment is the statement of homology, that is shared ancestry from which historical inferences are made. The alignment, then, becomes critical to reconstructing phylogenies.

• In some cases, the alignment is trivial. In many cases it is not.

Inferring phylogenies• There are two fundamental ways of treating data; as distances or as

discrete characters.• Distance methods first convert aligned sequences into a pairwise distance

matrix, then input that matrix into a tree building method• Discrete methods consider each nucleotide site (or some function of each

site) directly. Consider the following example:

Inferring phylogenies

Clustering methods versus optimality methods

• There are also two fundamental ways of finding the “best” phylogenetic tree

• Clustering methods use some algorithm to cobble together a single tree

• Optimality methods survey all possible trees and compare how well they fit the data

Phylogeny reconstruction: maximum parsimonyThe data for maximum parsimony

comprise individual nucleotide sites. For each site the goal is to reconstruct the evolution of that site on a tree subject to the constraint of invoking the fewest possible evolutionary changes.

In parsimony we are optimizing the total number of evolutionary changes on the tree or tree length. The tree length, then, is the sum of the number of changes at each site. So, if we have k sites, each with a length of l, then the length L of the tree is given by

Phylogeny reconstruction: maximum likelihood

The method of maximum likelihood is a contribution of RA Fisher, who first investigated its properties in 1922.

Principle: evaluate all possible trees (topology and branch lengths) and substitution model parameters (TS/TV, base freq, rate heterogeneity etc.). These are the hypotheses. Choose the one that maximizes the likelihood of your data (the alignment)

Likelihood: Given that the coin you’re tossing just gave you 15 heads out of 100 tosses, the likelihood that it is fair is very small.

Given the nature of molecular evolutionary data, where evolution has run just once, yielding one data set, maximum likelihood is a powerful framework--evaluate a bunch of different hypotheses to find the one most likely to have generated the observed data!

A non-biological example: coin tossing

If the probability of an event X dependent on model parameters p is written

P ( X | p )

then we would talk about the likelihood

L ( p | X )

that is, the likelihood of the parameters given the data.


Say we toss a coin 100 times and observe 56 heads and 44 tails. Instead of assuming that p is 0.5, we want to find the MLE for p. Then we want to ask whether or not this value differs significantly from 0.50.

How do we do this? We find the value for p that makes the observed data most likely.

p L -------------- 0.48 0.0222 0.50 0.0389 0.52 0.0581 0.54 0.0739 0.56 0.0801 0.58 0.0738 0.60 0.0576 0.62 0.0378


So why did we waste our time with the maximum likelihood method? In such a simple case as this, nobody would use maximum likelihood estimation to evaluate p. But not all problems are this simple!

Traditional versus

Bayesian phylogenetics

Traditional versus Bayesian phylogenetics

Estimating confidence: Bootstrapping trees

What can you do with trees beyond simply inferring relatedness?


•Chang et al. (2002) used maximum likelihood phylogenetic ancestral reconstruction methods to recreate a putative ancestral archosaur visual pigment (ca. 240 mya)


Chang et al. Fig 1


•To determine if these ancestral pigments would be functionally active, the corresponding genes were chemically synthesized and then expressed in tissue culture


Chang et al. Fig 2


•The expressed artificial genes were all found to yield stable photoactive pigments with max values of about 508 nm, which is slightly redshifted relative to that of extant vertebrate pigments.


Chang et al. Fig 3

•What might you speculate about the behavior of the ancestral archosaur based on these results?

Pan troglodytesPan troglodytes

Cercocebus Cercocebus atysatys

Cercopithecus Cercopithecus lhoestilhoesti

CercopithecusCercopithecusalbogularisalbogularis

Colobus guerezaColobus guereza

Chlorocebus aethiopsChlorocebus aethiops

• Back to HIV and chimpanzees• SIV was discovered in 1985 in a captive Asian monkey• SIVs are found naturally only in African primates

SIVcol

SIVsun

SIVlhoest

SIVmnd

SIVsm

HIV-2/A

HIV-2/B

SIVsyk

SIVagmVER

SIVagmGRI

SIVagmTAN

HIV-1/M

HIV-1/N

SIVcpz

HIV-1/O

SIVcpz

0.1

x

x

x

x

x



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are needed to see this picture.

•HIV-2 introduced at least 8 times from mangabeys

•HIV-1 introduced at least 3 times from chimps

•Two of these “groups” endemic to Cameroon

•Group M is pandemic

• HIV-1 group M causes >99.9% of HIV HIV-1 group M causes >99.9% of HIV infections worldwideinfections worldwide

• Slightly harder to pin down its geographical Slightly harder to pin down its geographical origins because of spreadorigins because of spread

• Various clues place it at the same seen Various clues place it at the same seen (probably Cameroon)(probably Cameroon)

• It’s a close relative to other AIDS viruses It’s a close relative to other AIDS viruses clearly linked to Cameroonclearly linked to Cameroon

• Chimpanzees themselves acquired their virus Chimpanzees themselves acquired their virus from preying on other primatesfrom preying on other primates

• How did the virus get into humans?How did the virus get into humans?


• Divine retribution • Doesn’t matter--it doesn’t cause AIDS• Conspiracy theories - e.g. the CIA did it• Ritualistic use of monkey blood• Zoonosis (a disease communicable from animals to man

under natural conditions)• Contamination of vaccines

• THE PLAUSIBLE HYPOTHESES ALL HAVE IN COMMON THE INCRIMINATION OF SIMIAN IMMUNODEFICIENCY VIRUSES (SIVcpz) FROM CHIMPANZEES

• THE KEY DISCOVERY WAS THE FINDING THAT AFRICAN PRIMATES ARE INFECTED WITH SIMILAR VIRUSES…


• There’s an apparent correlation between There’s an apparent correlation between oral polio vaccine (OPV) sites (1957-1960) oral polio vaccine (OPV) sites (1957-1960) and earliest instances of HIV-1 in and earliest instances of HIV-1 in Democratic Republic of Congo (DRC, ex-Democratic Republic of Congo (DRC, ex-Zaire).Zaire).

• 350/400 chimps sacrificed in experiments 350/400 chimps sacrificed in experiments at Lindi camp near Kisangani, DRC, and at Lindi camp near Kisangani, DRC, and allegedly OPV cultured in their kidneys allegedly OPV cultured in their kidneys (Hooper 1999). (Hooper 1999).

• This culturing process is suggested to have This culturing process is suggested to have facilitated the transfer to humans of facilitated the transfer to humans of chimpanzee simian immunodeficiency chimpanzee simian immunodeficiency virus (SIVcpz).virus (SIVcpz).

• There’s a precedent: early polio vaccines There’s a precedent: early polio vaccines are known to have been contaminated with are known to have been contaminated with the simian virus SV40.the simian virus SV40.

“The River: A Journey Back to the

Source of HIV and AIDS” by Edward

Hooper.


Non-invasive sampling of SIVcpz from the supposed “source” (and a big blank space on the map of SIVcpz distribution)


Western blot analysis of Kisangani chimpanzee urine samples

6 15 19 21 25 28 36 40 48 51

gp160gp120

p66

p51

gp41

p31

p24

p17


Worobey et al. (2004): Nature

Phylogenetic position Expected for source population

Phylogenetic position of Kisangani SIV


Vaccines used after 1957

Ancestor estimatedAt 1930ish


Summary

• The SIVcpz from the alleged HIV-1 group M source region (according to OPV/AIDS theory) is not the sister lineage to group M

• Confirms other lines of evidence (dating, ZR59, archival vaccine testing, group N recombination, genetic diversity in Kinshasa)

• A new region where we can expect possible emergence of HIV-1

• Sampling is continuing to study natural history of SIVcpz in wild chimps


lecture 12 immunology and disease: parasite antigenic diversity and phylogenetic trees

Documents

phylogenetics slide

traditional epidemiology

prior exposure antigenic

prior exposure antigenic

measles virus slide

origins of hivaids slide

antigenic variant acts

prior exposure hosts