Immunogenomics

Yana Safonova

University of California San Diego

University of Louisville School of Medicine

About the course

Contacts

Yana Safonova, instructor

• safonova.yana@gmail.com
• Telegram: @ysafonova

Anastasia Vinogradova, assistant

• Telegram: @vinogradovana

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• Course web-page: https://sites.google.com/view/yana-safonova/course_bi2020
• Thursdays, 7 pm Saint Petersburg / 8 am PST

Syllabus - 1

Week 1.

• History of immunology
• Introduction to immunology and adaptive immune repertoires (AIRR)

Week 2.

• Repertoire sequencing (Rep-Seq) data

Week 3.

• Processing Rep-Seq data

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Week 4.

• Clonal analysis of antibody repertoires

Syllabus - 2

Week 5.

• Somatic hypermutations and definitions of antigen-binding sites

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Week 6.

• Population analysis and assembly of IG and TR loci
• De novo inference of germline IG genes using Rep-seq data

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Week 7.

• Applications of repertoire analysis to biomedical applications: flu, HIV, measles, dengue

Syllabus - 3

Week 8.

• Types of antibodies. Comparative analysis of antibodies of vertebrate species.

Week 9.

• Design of antibody drugs. Vaccines. Proteomics identification of neutralizing antibodies.

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Week 10.

• Adaptive immunity of SARS-CoV-2

Homework assignments

• Week 3: Basic analysis of immunosequencing data
• Week 4: Clonal analysis of antibody repertoires
• Week 5: Analysis of Ab / Ag complexes
• Week 6: Population studies of germline IG genes

Requirements to the course

• Programming skills
• Confident work in command line
• Basic knowledge of algorithms
• Basic knowledge of English
• A laptop with Linux or MacOS system (needed for HW assignments)

History of immunology

History of immunology

• Over centuries, outbreaks of infectious diseases shaped history of humankind
• Although some diseases caused deaths of thousands of people in the past, we have almost forgotten about them now.

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History of immunology

• Over centuries, outbreaks of infectious diseases shaped history of humankind
• Although some diseases caused deaths of thousands of people in the past, we have almost forgotten about them now.
• Does it mean that we become invincible? Yes and no.
• New infections continue to emerge - the ongoing pandemic of SARS-CoV-2

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What is immunity?

• The concept of immunity from disease dates back at least to Greece in the 5th century BC

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• Notes say that individuals who recovered from the plague, which was raging in Athens at the time became “immune” or “exempt” to it

Spread of smallpox

• The earliest evidence of the disease dates back to

the 3rd century BCE in Egyptian mummies

• 6th Century – Increased trade with China and Korea

introduces smallpox into Japan.

• 7th Century – Arab expansion spreads smallpox into

northern Africa, Spain, and Portugal.

• 11th Century – Crusades further spread smallpox in Europe.
• 15th Century – Portuguese occupation introduces smallpox into part of western Africa.
• 16th Century – European colonization and the African slave trade import smallpox into the Caribbean and Central and South America.
• 17th Century – European colonization imports smallpox into North America.
• 18th Century – Exploration by Great Britain introduces

smallpox into Australia.

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Variolation against smallpox

• Death rate is 30%
• China, 6th century CE: epidemia of smallpox. Variolation as a preventive measure involving exposing healthy people to material from the lesions caused by the disease
• Since 1670, variolation was known and practiced in the Ottoman Empire

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• In the beginning of 18th century, variolation became popular in England.
• Two grandchildren of the king of England were variolated on April 17, 1722

From variolation to vaccination

• Cowpox is similar to smallpox virus still can infect people, but it is not so dangerous
• Milkmaids who had cowpox, never caught smallpox
• In the end of 18th century, Edward Jenner inoculated a boy with material obtained from a cowpox lesion that appeared on the hand of a dairymaid
• Six weeks later, he inoculated the boy with smallpox without producing disease

Why cowpox vaccination worked?

• Survivals of an infection become immune to it

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• The goal of variolation and later vaccination was the development of the disease in small and weak form

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• Since cowpox virus is similar to smallpox virus, the immunity against cowpox also works against smallpox

• Why vaccination is better than variolation?

The beginning of anti-vaccination movement

Vaccine against rabies

Rabies: death rate > 95%. Transmitted by infected animals (mostly dogs)

• Infected individual develops hydrophobia, muscle weakness, and paralysis
• Incubation period takes 1-3 months

Discovery of rabies vaccine - 1

• No safe replacement from animal viruses

as cowpox for smallpox. Vaccine should be

made of the real virus

• Viruses and mechanisms of their work had not been discovered yet!
• It was important to identify the source of the disease
• Tongues of sick dogs were covered by foam. Pasteur injected saliva from a sick dog into a healthy rabbit that caused rabbit death.
• Then he repeated the experiment and injected saliva of a healthy human into a healthy rabbit. This also caused the death of the rabbit.

Discovery of rabies vaccine - 2

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• Thus, Pasteur decided to develop vaccine based on brains of infected dogs
• A rabbit inoculated with a fresh brain immediately died
• A brain that was kept dry for 14 days did not lead to development of rabies!
• A series of injections 14 days after drying + 13 days after drying + 12 days after drying + … + infection of a fresh brain also did not lead to development of rabies

• Analyzing symptoms, Pasteur suggested that rabies affects the central nervous system

Vaccine against cholera

• Cholera: death rate varies from 5 to 50%

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• Developed by Louis Pasteur in the middle of 19th century

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• Pasteur inadvertently left a flask with cholera on the bench over the summer and inoculated 8 chickens with this “old but viable” stock of chicken cholera bacillus. None of inoculated chickens developed the disease

Vibrio cholerae

Elimination of polio

Eradication of smallpox

Other vaccines

Stanley Plotkin. History of Vaccination. PNAS. 2014

Science behind vaccine discoveries

• Germ theory: Robert Koch and Louis Pasteur suggested that microorganisms are responsible for development of diseases
• Early explanation of immunity: Pasteur thought that microbes could produce chemical substances toxic to themselves that circulated throughout the body, thus pointing to the use of toxins and antitoxins in vaccines
• Later explanation of immunity: Élie Metchnikoff discovered phagocytosis and established the first concept of cell-mediated immunity

The concept of cell-mediated immunity

• Metchnikoff suggested that white blood cells attack invaders from outside the body
• He observed that certain cells in starfish larvae engulfed and digested carmine dye particles that he had introduced into the bodies of the larvae
• He called this process phagocytosis

Élie Metchnikoff

The concept of

humoral immunity

• Koch and colleagues found that cell-free serum extracted from blood is still able to fight bacteria. They called it humoral immunity
• Paul Ehrlich introduced term antibody: a body directed against something
• Antibodies are substances produced by the host organism for fighting antigens (something directed against gene)

Robert Koch

Paul Ehrlich

Side-chain theory

• The cell is equipped with side-chains or receptors to capture specific nutrients.
• A receptor can be blocked by a matching toxin (antigen); the cell then heals itself by shedding the blocked receptors and producing an excess of new ones.
• Some of the new side-chains are freed into the serum and constitute antibodies specific to the toxin.

Antibody-antigen binding

Fundamentals of Immunology, 1943

Later discoveries

• 1948 – Antibody production in plasma B cells
• 1957 – Clonal selection theory (Frank Macfarlane Burnet)
• 1959 – 1962 – Discovery of antibody structure (independently elucidated by Gerald Edelman and Rodney Porter). Antibodies became also known as immunoglobulins
• 1975 – Generation of monoclonal antibodies (Georges Köhler) and (César Milstein)
• 1976 – Identification of somatic recombination of immunoglobulin genes (Susumu Tonegawa)

References

• S. J. Holmes. Louis Pasteur. 1961.
• Pauline M. H. Mazumdar. Fundamental of Immunology (online version)
• Paul Ehrlich lecture at the Royal Society in London, transcript
• WHO: World Health Organization: https://www.who.int/
• The Immunisation Advisory Centre: http://www.immune.org.nz/
• https://en.wikipedia.org/wiki/Timeline_of_immunology
• Janeway’s Immunobiology. 8th edition: https://www.mta.ca/pshl/docs/janewayimmunobiology8.pdf

Introduction to modern immunology

The concept of cell-mediated immunity

• Metchnikoff suggested that white blood cells attack invaders from outside the body
• He observed that certain cells in starfish larvae engulfed and digested carmine dye particles that he had introduced into the bodies of the larvae
• He called this process phagocytosis

Élie Metchnikoff

The concept of humoral immunity: side-chain theory

• The cell is equipped with side-chains or receptors to capture specific nutrients.
• A receptor can be blocked by a matching toxin (antigen); the cell then heals itself by shedding the blocked receptors and producing an excess of new ones.
• Some of the new side-chains are freed into the serum and constitute antibodies specific to the toxin.

Innate and adaptive immune system

https://microbiologyinfo.com/difference-between-innate-and-adaptive-immunity/

Generation of antibody repertoire

Adaptive immune system

• Variety of threats to human body is huge and unpredictable

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• Genome is too small to encode defences against all these threats

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• Immune system has an ability to adapt to various threats using agents (e.g., antibodies) that are not encoded in the genome.

Antibodies

• Antibodies are proteins that bind to a specific treat (called antigen) and cause its neutralization

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• Immune system generates millions of different antibodies (repertoire) to neutralize various antigens

Specificity rule:

one antibody – one antigen

(not necessarily true)

Antibody

Antigen

Generation of antibodies

Before recombination, the genome of an antibody-producing cell (B cell) looks exactly like genomes of all other cells:

Immunoglobulin locus (Chr 14), length ~1.25 Mb

V

165-305 nt

avg. 291 nt

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D

11-37 nt

avg. 24 nt

J

48-63 nt

avg. 54 nt

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Selection of J segment...

Left cleavage of J segment...

Selection of D segment...

Right cleavage of D segment...

Concatenation of D and J segments...

Newly created unique genomic region

Left cleavage of DJ fragment...

Selection of V segment...

Right cleavage of V segment...

VDJ concatenation (variable region of antibody)

360 nt of VDJ + 1000 nt of constant region

instead of original 1.25 Mb

Constant region

Variable region of antibodies contains antigen binding sites

Constant region

360 nt of VDJ + 1000 nt of constant region

instead of original 1.25 Mb

Human IGH locus

Generation of antibody repertoire - II

Variable region of antibodies contains antigen binding sites

Constant region

360 nt of VDJ + 1000 nt of constant region

instead of original 1.25 Mb

Human IGH locus

Why are antibodies so diverse if there are only 55×23×6 VDJ recombinations?

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Why are antibodies so diverse if there are only 55×23×6 VDJ recombinations?

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Recombination process is imperfect and includes many random processes:

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• Palindromic insertions

Why are antibodies so diverse if there are only 55×23×6 VDJ recombinations?

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Recombination process is imperfect and includes many random processes:

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• Palindromic insertions
• Segment cleavage

Why are antibodies so diverse if there are only 55×23×6 VDJ recombinations?

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Recombination process is imperfect and includes many random processes:

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• Palindromic insertions
• Segment cleavage

Why are antibodies so diverse if there are only 55×23×6 VDJ recombinations?

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Recombination process is imperfect and includes many random processes:

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• Palindromic insertions
• Segment cleavage
• Non-genomic insertions

Three types of antibody chains

Human antibody have one type of heavy chain (IGH) and two types of light chains:

• κ encoded by IGK locus (chr 2)
• λ encoded by IGL locus (chr 22)

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VDJ recombination randomly selects between IGK and IGL

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Clonal development of antibodies

Antibodies are subjects of fast evolution

Immune system mutates and amplifies a binding antibody

Mutation rate in antibody genes is 3-4 order of magnitude higher than in other genome

One antibody = one antigen

Antibody repertoire is a set of clonal lineages

Antibody repertoire is a set of unknown clonal lineages