Lecture 4 Flashcards
1
Q
How do we enrich for antibodies that recognise the CD4 binding site of HIV, gp120?
A
- create a probe that mimics the CD4 binding site (RSC3)
- create a variant that has a single amino acid change within the CD4 binding site (Delta RSC3)
- Select for antibodies that bind the probe (b12), but not the variant – i.e. retain CD4 binding-site recognition
- antibodies that bind both (2G12) are not specific for CD4 binding site
- use RSC3 and delta RSC3 to isolate B cells (specific markers sorted via FACS) from peripheral blood of HIV patients that encode CD4-binding site specific antibodies
2
Q
What are markers for B cells ?
A
- CD20 and CD19 are only expressed on B cells
3
Q
What does the expression of IgG mean for a B cell?
A
- It means they have been through an immune response
- they’ve received T cell help
- they’ve proliferated
- probably been into a GC
- undergone CSR
4
Q
What is the method for large-scale isolation of bNt Mabs from Memory B cells?
A
- Sort single IgG B cells that bind a modified gp160 trimer called 2CC
- Recover the IgV genes from each cell
- Re-express as a recombinant, complete IgG in a test tube
- you have a probe that is part of the HIV virus
- you find B cells which are isotype switched and that recognise this prove
- isolate them
- recover their V genes, from each individual cell, Heavy and Light chain V region genes
- Re-express them in another cell type in a test tube
- can make that cell now produce that antibody
- so you’ve gone from a circulating B cell in the blood of a patient to an Ab factory in your laboratory that is making an anti-HIV antibody that may have the properties of neutralisation because it binds to a site that you think is important to that process
- you can also get info about that V region genes for example the number of somatic mutations they have
5
Q
What are some more strategies to isolate and characterise HIV-specific antibodies from HIV-infected individuals?
A
Firstly you screen their serum to see if they have the desired Abs or not. (binding assay)
- take WBCs –> high throughput sequencing - don’t try to isolate anything just whack the whole stuff through a HTS machine and see which Ig sequences come back most often – represent the B cells mounting a response at the time you have isolated the blood. Compare them in an attempt to work out what the mutational history is and what they came from.
- Try to identify the individual rare B cells that are recognising the virus by using parts of the virus as a bait to fish them out. If you do it through this process of cell sorting you can attach other attributes e.g. it has to be a B cell, isotype switched etc - enrich for the kind of cell that you are interested in
- alternatively you can take everything - recover the V genes from these cells, remake the antibody and re-express it in a cell type in a laboratory dish - you can then analyse what it looks like, what it binds to and does it work in neutralisation
you’ve then gone from a patient with serum Abs to actually purifying those Abs that have desirable properties
6
Q
What is VRC01? What has been done with it?
A
- an antibody that blanks out completely the ability of HIV to bind to CD4 and so prevents infection
- highly mutated V gene
- idea was that if we could find out what it recognised at the beginning before it went into the GC we’d know what we had to immunise people with in order to trigger that response and guide it through the process of affinity maturation to make these good high-affinity Abs
- Took the V gene sequences and identify all the somatic mutations and change them back to what they were in the very beginning at the onset of the immune response
- it actually no longer bound to HIV
- only acquired the capacity to bind to HIV by virtue of these mutations
- turns out this is not uncommon
- if so, then how did they get to react to the virus in the first place? if in these sorts of assays they don’t recognise it
- turns out the assay is wrong rather than the immunology - tested the wrong form of the virus against their capacity to bind
7
Q
How do response, escape and counter-response shape the Ab repertoire to HIV?
A
- antibodies acquire increasing numbers of SHM in GC
- rapid excape mutation in Env
- Increasing breadth and potency of neutralisation
- successive emergence of broadly neutralising Abs in patient
- First Abs are strain specific
- Then V2 specific bNAbs to emerge
- Followed by a 3rd wave to as yet unknown epitope
- possible late bNAbs do not recognise initiating virus
- Each HIV escape is into an increasingly limited ‘space’
- important to remember that the virus has parts of it that can change - and it does - these also happens to be the bits that your immune system most likes to recognise, other parts of the virus are unchangeable - change would mean that it lost its capacity to infect - this is the most difficult to make a response to and usually happens much later
8
Q
How can we rationally design Anti-HIV Mabs with increased and improved neutralising activity?
A
- can one improve Ab binding to gp120 by changing individual amino acids?
- examine the binding site at the amino acid level of Vh and CD4
- make an AA exchange to make Vh more like CD4 - Swap G for Phe?
- Swapping Vh G for an aromatic amino acid (Y, W, F) greatly improves affinity - 10 fold improvement by a single aa change
9
Q
What has injecting mice with broadly neutralising Mabs show us?
A
- mice transplanted with human CD34+ stem cells - humanised
- this reconstitutes the human immune system in the mice (B, T macrophage etc)
- mice infected with HIV1 generate stable viraemia for > 60d (i.e. HIV replication model)
- Test if neutralising Mabs provide long term protection
- Mono-therapy with bnMabs is ineffective –> 5 different Mabs and 1 HIV strain, neither on their own effective
- tri-therapy is partly effective - injecting three different Mabs gave some mice protective levels
- Penta-therapy with bnMabs is effective - most/all mice showed controlled, low levels of HIV-1 when treated with all 5 Mabs
- But there is a viral rebound as Abs disappear - as they slip below a particular level due to not being constitutively made the virus is eventually able to start replicating once more - this means that the Mabs are not curative, merely protective - once below a certain threshold the virus is able to escape from that suppression and start infecting things
10
Q
What is important about showing that penta-therapy with broadly neutralising Mabs is effective?
A
- Shows that it is possible to contain HIV with sufficient range of neutralising specificities, which might inform vaccine design
- reveals a therapeutic approach to suppress HIV-1 viraemia, possibly helping those who are unable to take HART or in conjunction with HART
- Possible for long-term production of Abs in people using Adenovirus vectors
- Also shows what is required for prophylaxis and a way of stopping vertical transmission
11
Q
What are the (original 6) hallmarks of cancer?
A
- sustaining proliferation
- evading growth suppression
- metastasis - dissemination
- immortality of replication - senescence
- blood supply / inducing angiogenesis
- blocking apoptosis (cell death)
12
Q
How does a cell become a cancer cell?
A
- a cancer is the uncontrolled growth of a cell
- unrestrained in its proliferation and in its location
- its those properties of expanding number and distribution which ultimately cause pathology and death
- cells have a lot of inbuilt mechanisms to prevent cancer from developing and each of these have to be overcome for a normal cell to be transformed into a cancerous cell
- these properties that a cell has to acquire are not normal - they require a change in the normal expression pattern either by enforced expression of a gene which shouldn’t be expressed or by mutations in a gene to change its properties so it can no longer be regulated or it gains additional features
- mutation is a key driver of cancer
12
Q
What must the molecules that promote B-cell cancer do?
A
- promote cell division
- extend cell life span by enhancing survival
- enable DNA mutation
- metastasis not necessary for these circulating cells
- special properties of B cels that can predispose them to malignancy
12
Q
How are B cells in GCs similar to cancer cells?
A
- they are proliferating like crazy
- not responsive to signals which stop their growth
- able to circulate throughout the body
- have processes to prevent their death and accumulate DNA damage while doing that
- i.e. proliferating B cells in germinal centres are already a reasonable distance along the path to becoming a cancer cell
12
Q
How do you find translocations?
A
- cytogenetic techniques where you look for banding patterns - low resolution
- last 30/40 years people have developed probes that label whole stretches of chromosome, you can ‘paint’ the chromosome, can see using colour the translocations. could combine this with a probe for an oncogene e.g. Myc - a very potent growth promoting gene (older - 3/4 years ago but still widely used in clinic)
- more sophisticated and global assessment is to actually just sequence everything
- sequencing the genome has actually become a relatively trivial thing, can be done for less than $1000, in only a couple of hours - a day at the most
- possible to take tumours from people, to sequence them, and to sequence all of the chromosomes.
- if you have little tiny fragments and you sequence them from both ends, and then map those sequences back onto the genome you can find in some cases that instead of just being a continuous sequence (e.g. myc or heavy chain locus) it actually swaps from one to the other
- in this way, at a nucleotide level, you can actually see that there is a translocation by comparing the tumour DNA with the DNA of a healthy cell - exact
- this helps to produce a map of recurrent chromosomal translocations in B-cell cancers
