module 3: HIV

Question 1

what are the potential barriers to HIV cure?

Answer 1

1) latent infected cells= makes elimination of virus difficult (latency reverse = shock & kill)
factors that maintain HIV latency = keep the virus hidden inside host cells
1. integration of virus into nuclear chromatin
2. defects in transcription of viral genome = delays latency
3. low level of TF expression in resting T eclls
4. defects in RNA export in resting T cell
5. limited translation needed to produce viral proteins
2) residual viral replication that forms reservoir of active virus
3) privileged sites: where infected host cells hide in protected areas (brain, lymphoid tissue, GIT)
- why HIV can persist on ART

Question 2

how does HIV evade the immune response

Answer 2

1) sequence variation
2) down regualation of MHC class 1 expression on infected cells by tat, vpu, nef
- nef = allows HIV to persist during immune response- when the cd8 t cells are trying to find viral infected host cells expressing mhc1 but cant
3) loss of effector immune cells
- loss of CD4 T cells
- clonal exhaustion (when cellls are dividing so much due to immune activation) that slowly they reach exhaustion due to REPLICATIVE SENESCENE (LIMIT IN NO. OF TIMES IMMUNE CELLS CAN DIVIDE)

4) LATENCY:
HIV virus is able to hide in host cell genome after it has successfully integrated (via intergrase ) its viral genome into the host cell.
- this prevents immune cells from detecting it and therfore makes virus infected cells hard to eliminate

latency is maintained by:

1. virus integrated into nuclear chromatin= hides and can stay here for long time if the virus has infected a RESTING T cell
   - if virus went into an activated CD4 T cell = it will cause death of virus
2. low levels of transcription factor expression = slows down pace of transcribing the now infected cell’s virus
3. defects in transcription of viral genome
4. limited translation to produce viral proteins
5. defects in RNA export in resting T cell

5) PRIVLEDGE SITES:
virus infected cells will move to sites where there is little immune detection 
- such as blood-brain barrier
- testes
- git 
- lymphoid tissue

Question 3

what are the important (2) components of a vaccine response

Answer 3

target B cells and T cells
1. generate antibodies from B cells that bind & neutralise HIV

2. develop better CD8 and CD4 T cell response that can recongise infecte T cells & eliminate them

Question 4

what are general vaccine approaches (3)?

Answer 4

1. recombinant proteins:
   - good antibody response, poor T cell response (gp160)
   - no protection
2. live attenuated viruses:
   - good T cell response, bad antibody response
   - may not be safe DNA vaccines
3. live vectors (ad26)
   - good T cell response, bad antibody response
   - eg. ad26, mva, pox virus

Question 5

describe the properties of broadly neutralising antibodies

Answer 5

(1) Properties:
1) Can “neutralise” a large no. of viruses
2) Are directed against highly conserved regions of envelope that are “hidden”
3) Detected after 1-2 years of infection & of limited help to the patient (make them too late- virus would have escaped being targeted by these bnabs)
- Delayed bNAB response = slow, antigen dependant affinity maturation process = undergoes more SHM + AM = carries more mutations

Other features of its use:
• Used in COMBINATION
- Mono in combination, bi- / tri-specific bNAbs= enhance efficacy and reduce chance of resistance
- Bi/tri = can target >1 epitope
• Modification of the Fc tail of bNAb to :
1) increase half life
2) activate Fc receptors
• serum concentration of bnabs required to provid full protection against HIV

Question 6

what are the roles of bnabs in treatment (describe the processs)

Answer 6

1. clone HIV specific B cells from long term non progressors (that produce bnabs)
2. there are many different types of anti-HIV bnabs
3. passive transfer of bnabs to provide:
   1) HIV prevention
   2) direct HIV therapy
   3) prolong supression of viral rebound after stopping ART

Question 7

what are the challenges of bnabs

Answer 7

trying to generate bnabs in vivo from vaccination

Question 8

what is cmv

Answer 8

Cytomegalovirus
• HIV causes chronic immune activation, allows opportunistic pathogens (CMV) to infect
• CMV = opportunistic virus, often CO-INFECTS w HIV
• CMV contributes to immune activation of PLWH
• Seroprevalence of CMV is higher in PLWH (80-100%) than others (40-100%)
• PLWH often have ongoing CMV replication (persistent antigen exposure) in mucosal + tissue sites  causes major expansion of CMV-specific T cells  ongoing immune activation

Question 9

describe use of cmv vectors

Answer 9

• Induces broad range of HIV specific T cells
• peptides from vaccine are presented on HLA E (normally - presented in HLA A / B alleles)
- Not yet in clinical trials

Question 10

what are strategies that achieve remission of art

Answer 10

1. latency reversal = shock & kill
2. permanent silencing = block & lock
3. gene therapy

Question 11

describe the process of latency reversal shock & kill

Answer 11

aim = cause HIV transcription in latent cells so the immune system CD4 T cells can recognise it and kill it

uses latent reversing agent = TLR7 agonist + TLR7 receptor on dendritic cell
TLR7agonist binds TLR7 on DC –> activates DC & produces IFN –> activates CD4 T cells & increases HIV transcritpion –> becomes productive T cell (DNA positive, RNA positive, HIV protein positive)

1. immune mediated killing
2. use pro-apoptotic drugs
3. this has to be used whilst on ART therapy = to ensure other cells are not infected from productive cell
4. boost is given to kill off latenlty infected cells after shock (TLR agonist) is given = only has transient effects

Question 12

describe process of block & lock, permanent silencing

Answer 12

aim = to push the latency of a HIV infected cell even deeper so that it will never reactivate

initial infection –> HIV genome intergrates into host cell genome –> become latent cell (DNA postive, RNA negative, HIV protein negative) –> use drugs to push the latency even deeper so it will never reactivate

1. tat inhibitor or mTOR inhibitor
2. silencing RNAs

Question 13

describe the process of gene therapy in trying to acheive remission of ART

Answer 13

aim = to excise CCR5 chemokine coreceptor so that HIV is unable to fuse/enter host cells

Lukapharessi = process where you collect infected CD4 T cells (mix of CCR5 positive and negative) –> use ZFN z scissors in vivo to excise the CCR5 chemokine receptor –> expanded ex vivo –> reinfuse into patient

Question 14

what are the 2 important things that we want to achieve cure for HIV?

Answer 14

1. eliminate CCR5 = to prevent HIV viral entry/fusion with host cells
2. eliminate latent CD4 T cells = so that once person goes OFF ART treatment = they wont have viral load rebound

Question 15

what are the 2 possibilites of someone coming off ART treatment?

Answer 15

1. delay in viral rebound (months/years)
   - if person went on treatment very very early = HIV would have not gotten the chance to make latent T cells OR reservoir cells (in privileged sites)
   - going off ART = means there will be a delay in viral load rebound
2. Post treatment control (indefinite)
   - if person went on treatment within 6 months of infection
   - going off ART = means their viral load will rebound- but to LOW levels ( would not go as low if they did not go on treatment)

Question 16

what are the barriers to HIV cure?

Answer 16

1. latent cells
2. anatomical reservoirs= privledged sites= residual virus production
3. residual viral replication

Question 17

describe latent cells in regards to HIV

Answer 17

latent cells = a barrier to HIV cure

• DNA positive
• RNA negative
• HIV protein negative

1) initial infection = HIV infects resting LONG LIVED T cell
(memory or naive T cells)

2) virus can persist in cell until it activates–> upregualtes HIV transcription

3) latent T cell undergoes HOMEOSTATIC PROLIFERATION = to produce more latent T cells
- same integration centre = may have survival advantages

Question 18

why might HIV latent cells be invisible from the immune system

Answer 18

DNA positive
RNA negative
HIV protein negative

0) quickly recycle envelop proteins on surface of CD4 T cells (typically 14 protein spikes= disapear quickly)

1) Latent cells have minimal HIV transcription = dont produce enough viral proteins / RNA for it to be detected by the immune system
- low levels of TF expression
- defects in transcription of viral genome
- limited translation
- defects in RNA export

= doesnt actually present viral rna/ proteins on surface

2) HIV accessory proteins (nef, vpu) down-regulate MHC 1 markers on host cell = cant be detected by CD8 T cells
3) detection of RNA is via Toll like receptors (thats why we use TLR agonists as a latent reversing agent to activate the dendritic cell - produce IFN which activates CD4 T cell & increase HIV transcription so that it becomes a productive cell = can be destroyed)

Question 19

describe anatomical reservoirs and their purpose

Answer 19

aim = to hide in privledged sites that have little immune cells so to not be detected and destroyed by immune T cells

• undergo viral replication
• still dribble out low doses of HIV viral load

privledged sites = blood brain barrier, testis, git, lymphoid tissue (B cell follicle= a sanctuary where cytotoxic CD8 T cells and ART cant enter)

Question 20

how does HIV evade immune response (6)

Answer 20

1. genetic diversity = sequence variation
   - HIV constantly mutating due to high error prone reverse transcriptase = rapid evolution
2. altered antigen presentation
   - HIV accessory protiens vpu, nef down regulate MHC class 1 molecule, especially of latent cells = avoid recognition by CD8 T cells
3. loss of effector cells
   1) CLonal exhaustion (so many T cells divide until they finallly exhaust) due to
   replicative senescence (there is a limit to the no. of times T cells can replicate

2) loss of CD4 T cell help= everything needs an initial signal from CD4 T cells. but since CD4 T cells deplete- the signal is gone
4) latent cells
5) anatomical resevoir/ privledged sites
6) causes immune activation

Question 21

what is the major effect of HIV on the immune system

Answer 21

decline in CD4 T CELLS

• clonal exhaustion
• lakc of CD4 t cell help= cant acitvate other pathways

when T cells <200 cells/mm3 = viral load increases = get constitutional symptoms –> opportunistic idiseases–> death (if untreated)

Question 22

what causes CD4+ T cell decline

Answer 22

increase destruction(GI-SAIL)& impaired production

increase destrcution

1) direct:
- GIT»> Blood (HIV directly infects CD4 T cells)
- Impaired reverse transcription in naive T cells
2) indirect:
- Synctium formation (HIV viral proteins coat CD4 T cell surface- attract unaffected CD4 T cells= more cells infected)
- apoprosis
- immune activation
- lymph node fibrosis (overactive immune = thickening of lymph node tissue=. cytokines cant function properly = limits T cell proliferation)

imparied prodcution
- direct effect on thymus & cd34+ progenitor cells

Question 23

how does HLA type impact immune response?

Answer 23

HLA TYPE IMPORTANT FOR IMMUNE RESPONSE
Certain HLA molecules present HIV epitopes more effectively  enhances adaptive immune response

B13, B27, B51, B57 = good prognosis (elite controllers)
A23, B37, B49 = rapid disease progression

Question 24

what immune cells does HIV deplete / cause dysfunction to

Answer 24

1. CTL
   - initial increase (immune activation)
   - clonal exhaustion = depletes
2. natural killer cells
   - impaired number & function

3. monocytes/macrophages
defects in:
- chemotaxis
- inability to promote T cell prolifeation
- Fc effector functions

4. B cells
   - no CD4 T cell help= no class switching
   - increase IgG + IgA = but ineffective
   - can lead to B cell malignancies

Question 25

what causes immune activation

Answer 25

1) Deplete CD4 T cells in mucosa, causes:
• ^ microbial translocation a
• LPS (bacterial products ) to activate of TLR4

2) Activation of innate immune response (pDCs- plasma dendritic cells)
• HIV RNA is a TLR7/8 ligand
- TLR receptors = the body’s way to detect foreign RNA: HIV RNA binds TLR = activates immune response
• Increased plasma IFN-a

3) Cytomegalovirus (CMV)-specific response
• Expansion of CMV-specific activated CD4+ and CD8+ T-cells
• Shows dysregulated imnune response to other pathogens

4) Loss of T regulatory cells (normally express CD4) = important to dampen immune response