Lecture 3 - HIV & Vaccines

Question 1

What are the three main types of vaccine manufactured at present?

Outline them

Answer 1

1. Whole killed vaccine
   • Virion can not replicate / cause infection
   • Immune response launched
2. Live attenuated
   • Virion can not cause pathology
   • Immune response launched
3. VLPs
   • Specific structural component of virus administered
   • Immune response launched

Question 2

Describe the changes in viral load over a course of 10 years in HIV+ patients

Answer 2

Acute, initial phase: high viral load

Seroconversion: decrease in viral load

Over the years: slow increase in viral load

Question 3

What is the desired effect of an HIV-1 vaccine?

Answer 3

Viral load is kept very low, but not fully removed

This greatly reduces transmission

Individual never develops AIDS, despite some decrease in CD4+ T cell count

Question 4

Which molecule in HIV is the target for vaccines?

Answer 4

CD4 binding site on gp120 molecule

Question 5

Describe HIV entry into cells

Answer 5

1. gp120 binds to CD4
2. Conformational change in gp120, revealing CoR binding site
3. Binding of the co-receptor (CoR); CCR5 to newly exposed binding site on gp120
4. Fusion of membranes and insertion of contents into host cell

Question 6

What is the CoR?

Answer 6

Co-receptor

• CCR5

Question 7

Considering HIV entry into cells, what could be an approach for a vaccine?

Answer 7

VLP vaccination:
• Vaccination of Env: gp120/gp41
• Immune system launches a response against it
• When exposed to virus, Ab’s block the binding site, so the virion can not enter host cells

NB Adjuvant would also be required

Question 8

What are the three really large trials for an HIV vaccine?

What were the outcomes?

Answer 8

1. Vaxgen
   • No protection
2. STEP
   • No protection
   • Possible increased susceptibility to infection
3. RV144
   • Moderate efficacy: 20%

Question 9

Compare the genetic changes in HIV and Influenza

Answer 9

Influenza:
• Variability due to recombinant between different strains
• Once an individual is infected with a given strain, one can launch a sterilising immune response against it
• Linear antigenic drift;
• One predominate variant at a given time

HIV:
• Highly mutable
• Great number of variants circulating at a given time
• Mutating faster than the immune system can keep up with
• Thus, cannot launch a sterilising immune response

Question 10

Why is HIV highly mutable?

What does this mean for our immune system?

Answer 10

Error prone reverse transcriptase

The immune system may make antibodies against an antigen of the HIV, only for mutation in this antigen to render the antibodies useless.

Question 11

Outline the escape mechanisms of HIV

Answer 11

1. HIV is highly mutable
   • Due to error prone reverse transcriptase
   • Immune system can not keep up with the escape variants
   • Env can withstand much antigenic variation
2. Unstable association between gp120 and gp41
   • gp120 release to expose irrelevant Ag
3. N-glycan carbohydrate shielding
   • Immune system not good at making high affinity Ab against carbohydrates
   • Antigenic sites of gp120 hidden
4. Hidden CoR binging site on gp120

Question 12

Describe the unstable association between gp120 and gp41 and the implication

Answer 12

1. gp120 head can dissociate

2. Exposes irrelevant epitopes which can dominate the immune response

Question 13

Describe carbohydrate shielding and the effect

Answer 13

The trimeric head of Env can be glycosylated to form a ‘glycan shield’

This shields the antigenic sites on gp120

Question 14

Describe the function of the hidden CoR binding site on gp120

Answer 14

This binding site is only revealed once CD4 binds to gp120

The site is not available for neutralising antibodies

Question 15

What features of the HIV Env protein that render it difficult for the immune system to control?

Answer 15

Env can withstand many mutations and still be functional, thus ESCAPING from the immune system

Question 16

What is some evidence of HIV escape from immune surveillance?

Answer 16

In a given patient, the virus was recovered at different point in time (time of infection, 2 weeks, month, 100 days, 150 days)

Even after a few weeks there was accumulation of mutations in this individual

These ‘escape variants’ are selected for by the immune system

Question 17

What / who are elite controllers?

Answer 17

These are individuals who are HIV+, but the infection does not progress
They develop antibodies against multiple HIV isolates (broadly neutralising Ab)

Question 18

What are RSC3 and ΔRSC3?

Why are these useful?

Answer 18

RSC3: A probe that mimics the CD4 binding site on gp120

ΔRSC3: Identical to RSC3, except it is missing the CD4 binding site

Using these two probes we can sort out B cells whose antibodies bind RSC3 and not ΔRSC3, indicating that their Ab binds to the CD4 portion of RSC3 (and thus are broadly neutralising)

Question 19

What are broadly neutralising Abs?

Answer 19

Antibodies that neutralise HIV not just from themselves, but other people

bnAbs recognise core, invariant, components of the HIV virion

Question 20

Describe the various methods of enrichment of bnAbs from elite controllers

Answer 20

– FACS using RSC3 –

1. FACS using and RSC3 ΔRSC3
2. Enrichment of antibodies that only recognise the CD4 binding site on RSC3

• Ignore those that bind both RSC3 and ΔRSC3, because these must be binding elsewhere
• Same goes for those only binding ΔRSC3

– 2CC probe isolation –

1. Sorting of cells that bind to 2CC probe
2. Recovery of genes from these B cells
3. Re-expression of these genes in cells in test tube

– High throughput sequencing –

1. Isolation of lymphocytes during HIV immune response in elite controllers
2. Sequencing of Ig genes in these B cells
3. Identification of common sequences present in elite controllers

– Binding assay –

1. Abs isolated from a patient
2. Binding tested in culture

Question 21

Describe the VRC01 story

Answer 21

• VRC01 is a bnAb (binds the CD4 binding site on gp120)
• VRC01 is highly mutated from its original form in the naive B cell
• ‘Germ line’ Ab, i.e. before it underwent somatic hypermutation, did not bind gp120 and thus was not neutralising

Question: how did it ever come to be neutralising?

• ‘Arms race’ between HIV and host response
• During the response, the Abs become no longer neutralising
• B cells re-enter the GC, are exposed to AID, and the variable regions are again mutated
• Some of the new Abs will be neutralising

Question 22

How did these germline Abs ever become specific for HIV antigen, if they don’t react at all?

Answer 22

Remember, the HIV virus mutates rapidly due to the ‘selective pressure’ of the immune system
These Abs were specific for the strain of the virus at an early stage

Question 23

Describe the ‘arms race’ between HIV and the host’s immune system

Answer 23

Once the host launches an immune response against HIV, there will be certain virions that have mutations and will not be affected.
These are selected for
Thus, over time, the virus changes strain and a new immune response must be launched

Question 24

Describe rational design of anti-HIV Ab

Answer 24

Using high-resolution imaging, we can see the CD4-gp120 binding site at a molecular level.

Observation:
• CD4 bind gp120 binding site with a Phe aromatic ring

By observing how CD4 binds to the CD4 binding site, we can create our Ab to mimic this

Do this by changing single amino acid bases

Question 25

Describe how we can infect mice with HIV in the lab

Answer 25

• Mice with mutation in RAG, and thus, defective immune systems
• Transplant lymphocytes from humans into the mice
• The HIV will now infect these human lymphocytes in the mice

Question 26

Describe the results of the bnmAb therapy study in mice

Answer 26

1. Mice with human lymphocytes able to be infected by HIV
2. Monotherapy:
   • 5 bNt mAbs individually were not neutralising
   • Mice were infected
3. Tri-therapy:
   • Some mice controlled the infection
   • Others didn’t
4. Penta-therapy:
   • All mice controlled the therapy
   • Low viral load
5. Removal of the therapy
   • Decay of bnmAbs
   • Infection comes back

Question 27

Are there any down sides to penta-therapy?

Answer 27

Once administration of the antibodies ceases, the viral load rebounds

Question 28

What is the generalised method of vaccine development for HIV?

Answer 28

Identification of the structures in the HIV virions against which we want to direct the immune response

(eg. CD4 binding site on gp120)

Question 29

What were the conclusions made about penta-therapy with bnmAbs?

Answer 29

1. Proof of principle
   • bnmAbs can be effective at suppressing the viral load
2. Adenoviral vectors
   • Containing genes for bnmAbs
   • These genes become expressed in muscle cells
   • Long term production of bnmAbs
3. Prophylaxis for vertical transmission