lecture 3

Question 1

Why make IgM and then change to IgG?

Answer 1

• Secreted IgM is pentameric
• IgM provides 10 binding sites thus greatly increasing avidity despite potentially low affinity
• Secreted IgG is dimeric
• Provides 2 binding sites, so binding reflects affinity rather than avidity (somatic mutation increases the affinity of the binding site over time)
• • changes effector functions: IgM is very limited in its effector function
• Pentameric IgM allows low affinity antibody to bind antigen
• As affinity increases, IgM can be replaced with IgG or IgA with their specialised effector functions

Question 2

What are the different effector functions of human Igs?

Answer 2

• Complement activation (G1/2/3, M)
• Placental transfer (G1/2/3/4)
• Mucosa (A1/2) (M - poorly)
• Extravascular transfer (G1/2/3/4, A1/2)
• Sensitising mast cells (E)

Question 2

How does Class Switch Recombination occur?

Answer 2

• Alters Ig class without affecting specificity
• Each has a switch (S) region upstream that is homologous to other S regions
• CSR is deletional recombination mediated by S-S recognition requiring double stranded breaks in the DNA
• Enzyme AID introduces nicks into S region DNA, providing a substrate for recombination
• VDJ segment is unaffected by CSR
• CSR occurs at the Heavy chain locus only and requires the enzyme AID

Question 3

What is the role of activation induced cytidine deaminase (AID) in CSR?

Answer 3

• discovered about 14 years ago
• Recognises a target sequence in DNA and it finds cytosines within that sequence and deaminates them which converts them into uracil
• Uracil should not be in DNA and there are various repair mechanisms in cells that will remove it
• this creates gaps/nicks in the DNA
• these gaps are close enough together that they actually represent a double stranded break in the DNA
• AID also does the same at another point in the DNA (before whichever constant region is required)
• the two staggered cuts are joined together, removing the DNA in between
• shifts the variable region in front of a new constant region

Question 3

What is the relationship between the antigen and the immunoglobulin isotype?

Answer 3

e.g. you have a worm
this worm has specific antigens your body has learnt to recognise as being part of a worm.
When those bits of worm are detected by antigen presenting cells e.g. Dendritic cells they are activated and go on to stimulate T cells. It will do it through cell surface markers and cytokines (soluble molecules) that programme the T cell in a particular way.
The immune system has evolved so that , in response to a worm, the T cell will produce IL4, IL5, IL13, IL25 (i.e. a set programme: worms always induce cytokines of this type in an immune response).

Simultaneously parts of the worm will be recognised by the B cell as antigens. It will recognise this as foreign and migrate to a point where it will meet up with the T helper cell that has the appropriate antigen from the same worm. The B cell receives the specific set of cytokines from the T cell and these tell it what to do. The B cell undergoes CSR specific for that antigen. –> IgE is the best isotype to make in response to worms because it invokes mast cells.

Mast cells degranulate on exposure and release toxins and compounds capable of expelling worms.

This process is not perfect but it works pretty well to induce the correct CSR differentiation.

Question 6

How and why are mutations introduced into GC B cell Ig V genes?

Answer 6

• mutations are introduced at ‘random’ = somatic
• this disversifies the binding properties of the expressed immunoglobulin
• amino acid replacements that improve affinity for Ag are selected for
• when B cells start they are exactly as they were in the bone marrow - germline
• if you take a B cell undergoing a response out you will notice a small number of changes at random - these are unique to that V gene undergoing the response i.e. somatic (e.g. day 7; low mut. freq.)
• introduced as random nucleotide changes along the gene
• as the response progresses, the frequency of these mutations, in any V gene, increases
• these mutations tend to be clustered into the regions of the antibody that interact with the antigen
• day 10; increasing
• day 21; plateau
• only a small number of these mutations will increase the binding affinity, but those that do will be very strongly selected for and will dominate the whole response
• the affinity can increase by up to 10000 fold just by changing a few nucleotides and selection for improved binding

Question 7

Which enzyme is used to create somatic mutations in the Ig V gene?

Answer 7

• Activation-Induced Deaminase (AID)
• initiates somatic mutation by converting cytosines to uracils (U=T) in Ig genes
• This induces error-prone DNA repair -> these constitute the mutations
• doesn’t result in DNA ds breaks because the number of nicks is much smaller
• many of the mutations will be deleterious, leading to a defective antibody or no improvement with affinity
• many cells in the GC will die in the process because the mutation leads to poorer binding
• very few selected but this is okay because we make so many lymphocytes in such a short amount of time that wasting 90% of them is irrelevant
• the 10% you are left with do a fantastic job - they have the high affinity antibody to fight of the infection

Question 8

What is affinity maturation? How does it occur?

Answer 8

The selective survival of Ag-binding variants, generated by V gene SHM, with improved affinity for antigen.

1. In the early GC there is T cell driven proliferation CSR from IgM to IgG/A - no SHM
2. Onset of SHM diversifies V genes by mutation leading to a range of antigen binding affinities including improvement, diminution or complete loss of Ig expression
3. Selective expansion of B cell clones with improved binding to antigen and death of remainder increases affinity of the population. Repeat process.

Question 9

What are the interactions between B and T cells when responding to an antigen?

Answer 9

• The B cell and T cell meet at the edge of the B/T areas and decide they need to do something about the antigen
• B cells migrate in and start proliferating to form the GC
• T cell enters in as well
• B cell and T cell communicate with each other through cell surface protein interactions and cytokines (soluble molecules)
• Whole series of ligands and receptors on the B cells and the T cells that are communicating with each other throughout the response: intermittently but repetitively
• this is to ensure that only those cells involved in the response are initiate proliferation and differentiation.
• B cells have to be activated and in that activation they will express several of these molecules
• T cells also have to be activated, and in that activation they express the counter molecules
• So you need to have two activated cells that express the right molecules on the surface in order to tell the other that they are the activated cell, therefore stopping the activation of bystander cells
• Surrounding B cells are not expressing the correct molecules and are therefore ignored by the T cells

Question 10

How do TH cells induce CSR and SHM of B cells?

Answer 10

• activated T cells express CD40L and ICOS
• B cells express the receptors for these: CD40 and ICOSL
• It is signalling through both of these which is what driving proliferation and SHM and CSR
• there is a signalling cascade downstream of both of these receptors
• CD40 runs through a relatively complex pathway involving NF-kappaB - these are crucial for activating the AID genes
• AID will then activate CSR and SHM
• CSR targeted by the cytokines secreted by the T cell
• mutations in each of these steps are causative of immune deficiency

Question 11

What is the enzyme that removes Uracil from DNA?

Answer 11

UNG (Uracil DNA deglycosylase)

Question 12

What deficiencies are associated with the induction in B cells of CSR and SHM by T cells?

Answer 12

• XL-CD40L deficiency
• AR-CD40 deficiency
• XL-HIGM-ED (x linked hyper IgM with anhydrotic ectodermal displasia) (ass. with NF-kappaB)
• AR-AID deficiency
• AR-HIGM
• AR-UNG
• mutations in the cascade from CD40 onwards result in the same phenotype: people who are able to make IgM but unable to activate class switch recombination or affinity maturation

Question 13

What is the molecular basis of Hyper-IgM?

Answer 13

• CD40L – X-linked, combined immune deficiency with cellular, humoral and innate defects. Defective B cell proliferation, no GC, no memory, defective DC activation
• CD40 – humoral immune deficiency, no GC, no memory (autosomal recessive)
• AID - GC form but no CSR, no switched memory, no SHM (autosomal recessive)
• AID-Cterm - GC form, no CSR, but SHM normal (autosomal dominant)
• UNG - Acts downstream of AID, GC, no CSR, no SHM (AR)
• NFkappaB signalling - abrogates signals from CD40, no GC, no CSR, no SHM (X-linked and AD)

Question 14

Therapy for people with HIgM?

Answer 14

• IVIg (intragram)
• or for XL-HIGM bone marrow transplantation: often used for people who have the combined immune deficiencies, they get someone else’s hematopoietic stem cells and that reconstitutes their immune system with now fully functional immune cells - has a lot of its own issues but is a relatively effective immune therapy

Question 15

What is the largest class of primary antibody deficiency?

Answer 15

CVID: common variable immunodeficiency

Question 16

What are Common Variable Immune Deficiencies?

Answer 16

• Characterised by hypogammaglobulinaemia: IgG < 3g/L and IgA < 0.05g/L
• Recurrent pyogenic infections from polysaccharide encapsulated organisms
• genetic basis is identified for less than 20% of cases
• they don’t have severe deficiencies like X-linked lymphoproliferative disease (XLP), Hyper IgM Syndrome (HIGM), or X-linked agammaglobulinemia (XLA)
• may be unable to make one or two isotypes
• much more difficult to work out exactly what the basis is

Question 17

What are common mutations for CVID?

Answer 17

ICOS: disruption of GC, loss of switched and unswitched memory; 1% of cases
CD19: defective B cell activation, loss of switched and unswitched memory
STAT3: Defective B cell response to cytokines, poor differentiation to PC, Also constitutive activation of CD4 T cells leading to hyper-IgE
TACI: expressed by B cells, involved in CSR and PC differentiation and survival. Most common genetic change in CVID (10-15% of individuals)

Question 18

What is the therapy for patients with CVID?

Answer 18

IVIg (Intragram)

Question 19

What is the frequency of CVID?

Answer 19

• 1 in 25 000

- cf 1 in 250 000 for XID

Question 20

What is the onset age for CVID?

Answer 20

• onset age is usually young adult

Question 21

What kinds of cells do germinal centres produce?

Answer 21

• memory B cells
• plasma cells
• both can survive for years

Question 22

What mutations will prevent germinal centres from forming?

Answer 22

• CD40L
• CD40
• ICOS
• CD19

Question 23

What mutations will allow the formation of GCs but prevent isotype switching therefore preventing the development of the appropriate kinds of memory/plasma cells?

Answer 23

• AID
• UNG
• TACI (helps to keep B cells alive)

Question 24

What are different types of vaccines?

Answer 24

• Live attenuated virus: polio “sabin”, measles, rubella, mumps, varicella, yellow fever
• • you go through the good parts of the immune response without the deleterious parts of the infection: virus has been made so no longer pathogenic
• Inactivated virus: Polio “salk”, Tick borne Enceph.
• Protein subunit: HepB, HPV (rec. protein/VLP), seasonal Flu (“split” virus)
• • works really well if you know the exact part of the virus to make an immune response against to stop it being infectious
• Plasmid DNA (none yet)
• Recombinant viruses (none yet)

Question 25

What would be the goal of a vaccine against HIV?

Answer 25

- To create a vaccine that prevents the viremia from reaching the very high levels they do in unvaccinated individuals and to then control them at a very low level so that over the course of a normal lifespan the levels of CD4+ T cells would not deplete to a level deemed immune deficient

Question 26

What is the HIV entry process?

Answer 26

- Upon HIV binding to the primary receptor, CD4, the envelope glycoproteins undergo conformational changes that expose the co-receptor (CoR) binding site on gp120 - Further conformational changes are induced after CoR interactions, which allows gp41 to insert its fusion peptide into the target cell membrane to promote virus-cell fusion 1. Native Trimer 2. CD4 binding - T20 binding site exposure 3. CoR binding - FUsion peptide insertion? 4. 6-Helix Bundle Formation - membrane fusion

Question 27

How do recombinant proteins provide opportunities to tailor vaccine responses?

Answer 27

- individual antigens can be chosen - antigens can be modified through protein engineering to change their properties in favourable ways - antigens can be made as soluble subunit vaccines, as virus-like particles (VLPs e.g. HPV vaccine) or as fusion proteins - well-characterised and highly pure antigen preparations can be made - co-administration with an adjuvant (something that enhances an immune response) is usually required

Question 28

Which recombinant antigens have been made in response to HIV-1?

Answer 28

- (in particular) gp120 and gp41 which are the attachment molecules - been used as the basis for vaccine trials

Question 29

What are some of the completed HIV vaccine clinical trials?

Answer 29

- VAX04 (Vaxgen): recombinant monomeric Env protein in Alum - no protection observed - STEP: recombinant adenovirus type 5 vector with HIV-1 T cell antigens (no Env) - no protection observed, possible enhancement observed - RV144: recombinant poxvirus vector followed by a boost with recombinant Env protein - modest level of protection observed - -> clinical trials for HIV are very expensive and time consuming and take a very long time - -> all very disappointing, none have worked to the point where you would actually use the vaccine

Question 30

Why don't these vaccines against HIV work?

Answer 30

- HIV is changing faster than your immune system is able to deal with

Question 31

Compare the genetic variability of the influenza and HIV-1 surface antigens.

Answer 31

Influenza (RNA virus) - a predominant viral variant circulates at a given time - flu changes only episodically - it has 8 chromosomes - changes by resorting those - we get bad flu infections/pandemics when a new form of the virus occurs by mixing 2 previous forms by transmission through another host - kind of a viral recombination - once it's in you and infecting you, it doesn't change - so you can make an immune response which will protect you against that - vaccines against flu are reasonably effective - we predict what the change is likely to be, make vaccines based on that predicted change and vaccinate people - generally you are protected HIV-1 (Retrovirus) - a very large number of genetic variants circulate at the same time - the genetic variability of HIV-1 within the space of only a couple of years from a single starting point is massive - incredibly mutable: changes very frequently and within one person - your immune system finds it very hard to keep up - makes it difficult to predict what the change is going to be so hard to get ahead of the virus in terms of a vaccine

Question 32

What are some of HIV-1's immune escape mechanisms?

Answer 32

HIV Env: - Contains regions that tolerate an extreme degree of genetic variation (V1-5) - has an extensive glycan shield - has evolved to have the variable (immunodominant) and glycans (immunosilent) regions exposed on the outside of the Env trimer

Question 32

How does HIV escape your immune system?

Answer 32

1. High mutation rate - selection for new variants mutations introduced during viral replication change immune determinants on the virus, rendering existing (and previous) immune responses irrelevant. mutations are a feature of low fidelity of HIV RNA polymerase in replication. 2. Unstable association between gp120 and gp41 resulting in shedding of gp120 and exposure of irrelevant (immunogenic) antibody epitopes - immune responses do not occur to all antigens equally, some dominate at the expense of others - increasing the number of irrelevant targets. increases the likelihood of irrelevant antibodies dominating the response 3. Each gp120 molecule carries between 20 and 30 N-linked glycans, which together result in an efficient shield of antigenic sites on the trimeric spike - difficult to make 'good' neutralising antibodies against carbohydrates. Also coating the virus in sugar shields the region of HIV where neutralising antibodies should bind 4. The highly conserved co-receptor binding site on gp120 is only exposed after binding to CD4, thus this site is not accessible for neutralising antibodies Simultaneously to all these virus is killing your CD4 T cells and thus limiting your capacity to make an effective immune response.

Question 33

In what way is your immune response fighting a 'losing war' against HIV?

Answer 33

- if you track the development of HIV in a patient and regularly sequence the virus it becomes clear how rapidly the virus mutates - it is like the process of natural selection - whatever mutations keep the virus alive are selected for - so as you make your immune response, escape variants arise and repopulate even as your immune response thought it had things under control

Question 33

What about the immune system of some individuals makes them 'Elite Controllers'?

Answer 33

- the IS in some individuals can generate neutralising abs that work against multiple HIV isolates - these people get sick to some degree but never progress to the point of having full blown HIV - if one recovers antibodies from their blood (serum) and tests it in the laboratory for whether they can block infection of HIV they all contain that antibody - they have actually made an Ab that is neutralising the virus