HIV Flashcards
1
Q
LO
A
- To know about the history, incidence and clinical progression of AIDS.
- To understand the biology of the AIDS virus and how it causes immunosuppression.
- To learn about the immune response to the AIDS virus
- Be aware of forms of chemotherapy and issues of vaccine development in AIDS.
2
Q
Key topics
A
- History of AIDS (Acquired Immunodeficiency Syndrome)
- The human immunodeficiency virus (HIV)
- Structure
- Replication cycle
- Clinical course of HIV infection
- Immunology of HIV infection
Effects of HIV on the immune system
Immune response to HIV
- Chemotherapy of AIDS
- HIV vaccines and microbiocides
3
Q
The story of AIDS began in 1981, when a cluster of unusual disease were observed in certain groups of people. What were the two main diseases and what were they caused by?
A
- pneumonia caused by a yeast, Pneumocystis carinii
- and an unusual tumour called Kaposi’s sarcoma caused by human herpes virus 8
4
Q
Initially AIDS was noticed in homosexual men before later symptoms appearing in who? What did this suggest about the nature of the virus?
A
- Initially in homosexual men but later the same symptoms appeared in intravenous drug users and haemophiliacs who were injecting blood-clotting factors.
- This pattern of disease occurrence suggested that a transmittable agent was responsible for the diseases
5
Q
What was an unusual aspect noticed about AIDS during its discovery?
A
. Unusual aspect of these diseases was they were only seen in immunosuppressed people and not in people with a fully functioning immune system.
6
Q
What were some observations made about the individuals who had the disease?
A
The observation that individuals with these diseases had low numbers of CD4 T cells was consistent with immunosuppression
7
Q
When was the term for the diseases used by the CDC?
A
1982 the term acquired immunodeficiency syndrome, or AIDS, was used by the Centres for Disease Control (CDC) in Atlanta to describe the disease
8
Q
In 1983, shortly after AIDS discovery, what was discovered?
A
1983 virus causing isolated from lymph node of an infected individual by Luc Montagnier’s group in Paris and was called the human immunodeficiency virus, or HIV for short.
9
Q
What was identified in 1986?
A
Second strain of HIV was identified in 1986; called HIV-2 and first strain was renamed HIV-1
10
Q
Tell me how HIV-1 and HIV-2 differ?
A
HIV-1 and HIV-2 differ in their virulence (severity and how harmful it is) and geographical location.
HIV-2 is less virulent than HIV-1 and is found primarily in western Africa.
11
Q
What have genetic studies shown about HIV-1 and HIV-2?
Tell me the origins of both HIV-1 and HIV-2
A
Genetic studies showed that both HIV-1 and HIV-2 are natural viruses of primates that have jumped species to infect humans.
HIV-1 came from chimpanzees
and HIV-2 from the sooty mangabey.
12
Q
How did we as humans get HIV from animals?
A
Both animals are killed for food and assumed that it was during this process that the virus initially infected humans.
13
Q
Tell me an effect we as humans had from HIV that the animals didnt?
A
HIV-1 and HIV-2 do not cause immunosuppression in chimpanzees or sooty mangabeys; only when virus crossed into humans did it cause the profound immunosuppression seen in AIDS.
14
Q
Estimated since 1981, how many worldwide have been infected, and died?
A
Estimated since 1981 over 75 million people worldwide were infected with HIV and over 32 million died (43% dead); 2 million died in 2007 and 2008.
15
Q
Where is the highest rate of increase in HIV infection seen?
How has this expected life-expectancy in this area?
A
The highest rate of increase in HIV infection is seen in sub-Saharan Africa; estimated 20-40% of young adults are infected.
Thus, life expectancy in sub-Saharan Africa almost halved; it is now in the 30s-40s instead of approaching 70, the estimated life expectancy if AIDS pandemic had not occurred.
16
Q
What is the pattern of spread of HIV in Africa? Compare this to that seen in more developed countries?
A
- Pattern of spread in Africa appears to be primarily by heterosexual contact and has a similar incidence in men and women.
- By contrast, spread in Europe, the USA and Oceania are still mostly among ‘high-risk’ groups such as homosexuals and intravenous drug users, much more prevalent in men than women.
- However, signs in USA that pattern of spread is changing, with more women being infected.
- End of 2008, women accounted for 50% of all adults living with HIV worldwide
17
Q
Sub-sahara stats
A
18
Q
Women can pass HIV onto their children, how do they do this?
A
Women can pass on HIV to their children via childbearing and breastfeeding
19
Q
What are the two 1 strains of HIV?
A
The two 1 strains of HIV are called HIV-1 and HIV-2.
Also, many different subtypes of HIV-,1 so vaccines needed to protect against all subtypes.
20
Q
Why are there different varients of HIV and why is this important?
A
- Also, HIV has very high mutation rate, giving rise to different forms of the virus known as variants.
- Variants important because, as described later, they differ in which cell types they can infect.
- Although different strains, subtypes and variants of HIV, very similar in structure and replication; therefore, will be described together and referred to collectively as HIV.
21
Q
What type of virus is HIV? What does this mean?
A
- HIV retrovirus (lentivirus family)
- contains RNA as genetic material
- Retrovirus: any of a group of RNA viruses which insert a DNA copy of their genome into the host cell in order to replicate, e.g., HIV.
22
Q
What does the HIV genome contains and what is this bound by?
A
HIV genome contains…
- 2 molecules of single-stranded RNA;
- each bound by a molecule of reverse transcriptase
- Within genome are also a p10 protease and a p32 integrase
23
Q
What is the HIV genome surrounded by?
A
Genome is surrounded by nucleocapsid consisting of inner layer of protein p24 and outer layer of protein p17
(both part of Gag (group specific antigen) polyprotein complex)
24
Q
What does the outer portion of the virus consist of?
A
Lipid envelope derived from host cell membrane
25
What does the outer portion of the HIV contain?
Contains two viral proteins, **gp120 and gp41**, which collectively are called **viral envelope proteins.**
26
Label this HIV structure
inner circle= p24
outer circle= p17
(of my arrows drawn onto diagram)
27
This is the HIV genome, explain what each section is associated with or encodes?
* **LTR**-long terminal repeats; repetitive sequence of bases
* **gag**-group specific antigen gene, encodes viral nucleopcapsid proteins: p24, a nucleoid shell protein, MW=24000; several internal proteins, p7, p15, p17 and p55.
* **pol**-polymerase gene; encodes the viral enzyme, protease (p10), reverse transcriptase (p66/55; alpha and beta subunits) and integrase (p32).
* **env**-envelope gene; encodes the viral envelope glyocproteins gp120 (extracellular glycoprotein, MW=120 000) and gp41 (transmembrane glycoprotein, MW=41000).
* **tat**: encodes transactivator protein
* **rev**: encodes a regulator of expression of viral protein
* **vif**: associated with viral infectivity
* **vpu**: encodes viral protein U
* **vpr**: encode viral protein R
* **nef**: encodes a 'so-called' negative regulator protein
28
What does HIV **GAG** encode?
HIV GAG encodes structural capsid proteins, produced as a GAG precursor polyprotein, which is processed by viral protease.
29
What does the 'p' represent?
P number refer to their molecular size. The larger the number to larger the number of AA in the protein
30
What does membrane associated GAG attract?
Membrane-associated Gag attracts two copies of viral RNA along with cellular and viral proteins that trigger budding of virion from surface of cell.
31
Tell me about the HIV particle, its main structures and proteins
* HIV is an enveloped **retrovirus** and has two single-stranded **RNA** molecules as its genetic material. The RNA is associated with **reverse transcriptase**, **integrase** and **polymerase** enzymes, which are necessary for viral DNA and RNA synthesis.
* Surrounding this is the nucleocapsid, which consists of an inner layer of **p24** protein and an
* outer layer of **p17** protein. The outer portion of the virus consists of a lipid layer derived from the host cell into which is inserted the viral **gp41** envelope protein. Each gp41 protein molecule is associated with a molecule of the **gp120** envelope protein.
* Many Glycan side chains on **gp120**
32
Tell me the replication cycle of HIV?
* Like any other virus, HIV must infect a host cell before replicating, with the viral progeny leaving the cell to infect others
* Retrovirus: any of a group of RNA viruses which insert a DNA copy of their genome into the host cell in order to replicate, e.g., HIV.
33
Tell me how HIV infects the host?
Whats infected and what is initially bound to?
* **HIV infects glycoprotein-CD4+ cells** (helper T cells recognize antigens on the surface of a virus-infected cell and secrete lymphokines that stimulate B cells and killer T cells; BUT helper T cells are infected and killed by the AIDS virus).
* Initial binding to host cell involves gp120 protein on surface of HIV particle binding to CD4 on host cell surface.
34
In addition ot CD4 T cells, what else expresses CD4?
In addition to CD4 T cells, monocytes and dendritic cells also express CD4, although at lower levels than T cells
35
What are the two stages to viral infection?
Two stages to viral infection:
1. binding to the host cell,
2. and fusion with the cell membrane to allow the virus to enter the cell
36
What is CD4?
CD4 is a co-receptor assisting T cell receptor (TCR) in communicating with an antigen presenting cell (APC)
37
What does CD4 bind to?
CD4 binding to MHC (Major Histocompatibility Complex) non-polymorphic region
38
Tell me about CD4 binding to the MHC moleucle during antigen presentation
* During antigen presentation, TCR complex and CD4 recruited to bind to different regions of the MHCII molecule (α1/β1 and β2, respectively)
* Close proximity between the TCR complex and CD4 means **Lck kinase** bound to CD4 cytoplasmic tail able to tyrosine-phosphorylate the Immunoreceptor tyrosine activation motifs (ITAM) present on the cytoplasmic domains of **CD3**
* Phosphorylated **ITAM motifs** on CD3 recruits and activates SH2 domain-containing protein tyrosine kinases (**PTK**) e.g. Zap70 to further mediate downstream signal transduction via tyrosine phosphorylation, leads to transcription factor activation including **NF-κB and consequent T cell activation.**
39
Tell me the stages to the life cycle of HIV?
1. Binds to **CD4 +ve cells** through **gp120 to CD4**; interactions between virus and chemokine co-receptors.
2. **Nucleocapsid enters the cell**, unfolds, releasing viral RNA, which is reverse transcribed to DS DNA.
3. The **viral DNA integrates in the host genome**, where it lies dormant as a provirus.
4. Following cell activation, viral DNA directs the **transcription of viral RNA**.
5. **Viral proteins are translated** from the RNA.
6. Viral proteins and single-stranded viral RNA assemble to **form new viral particles.**
7. Virus buds from the cell, picking up some of cell membrane, **complete viral particles can infect other cells.**
40
HIV life cycle diagram
41
Image to show budding of HIV particle
42
Tell me about Hijacked immune cell- HIV circumvents normal dendritic cell- T-cell interaction
An HIV-infected dendritic cell shoots out thin projections called filopodia (red) with virus particles (white) at their ends. Actin filament rearrangement projects filopodia in a waving arc towards T-cells at μm/sec, facilitating 800 dendritic cell - T-cells interactions/hour.
Anupriya Aggarwai and Stuart Turville, PLOS Pathogens, doi:10.1371/journal.ppat.1002762
43
Binding of g120 to CD4 is not enough for HIV to infect cells.
What was originally thought?
* Originally thought **gp41 component of envelope protein binds to a second protein on cell surface**. Second protein differs between variants of HIV.
* In some variants originally considered that gp41 binds to the β-chemokine receptor CCR-5, which is on the surface of CD4 T cells, monocytes and dendritic cells.
* These variants can therefore infect all of these cell types and have been called **M-tropic.**
* Other HIV variants originally thought to bind to another α-chemokine receptor, CXCR-4, present on CD4 T cells but not on monocytes or dendritic cells.
* These variants only infect T cells and are called **T-tropic.**
44
In the old binding model, what was an **M-tropic** **variant?**
In some variants originally considered that gp41 binds to the β-chemokine receptor CCR-5, which is on the surface of CD4 T cells, monocytes and dendritic cells.
These variants can therefore infect all of these cell types and have been called M-tropic.
45
In the old binding model, what were **T-tropic variants?**
Other HIV variants originally thought to bind to another α-chemokine receptor, CXCR-4, present on CD4 T cells but not on monocytes or dendritic cells.
These variants only infect T cells and are called T-tropic.
46
In the new binding model what is now thought to help binding?
Tell me about this protein type, role and structure
**gp160**, is a fusion glycoprotein to **overcome the energy barrier associated with the fusion of two membranes**. **gp160 is in trimer** formation, meaning three molecules of gp160 are arranged together, often called "spikes".
47
What often happens during the early fusion process of biding?
* During early fusion process, **gp160 cleaves** into **gp41** (considered the **transmembrane glycoprotein**) and **gp120** (considered the **surface glycoprotein**)
* However, **gp41 and gp120 remain in trimer formation and noncovalently attached to each other**. In this configuration, gp41 is thought to be in high energy state, with its fusion peptide buried inwards.
* Fusion peptide is molecule composed of linked amino acids.
48
What is the function of gp120?
gp120 binds onto host cell surface to the receptor CD4
Causes a conformational or structural change of gp120, which in turn, allows gp120 to bind again, this time to a chemokine coreceptor, usually CXCR4 or CCR5.
49
Tell me what happens to gp41?
gp41 is released from its high energy state and the previously buried fusion peptide springs out towards the host cell membrane, bridging the divide between the virion and the host cell membrane.
50
What happens to gp120 after chemokine coreceptor binding?
After chemokine coreceptor binding, gp120 is thought to disassociate away.
At this point, glycoprotein 41 transiently becomes an integral component of two membranes: the viral membrane in which it is anchored, and the cellular membrane that it has gaffed.
51
How does HIV enter cells?
HIV enters cells by binding of the HIV glycoprotein gp120 to the host CD4 molecule; this process requires chemokine receptors as obligate accessory proteins or ‘co-receptors’.
52
What are chemokine receptors?
Chemokine receptors are G-protein-coupled and have seven membrane-spanning domains
53
How do chemokine receptors normally function?
Chemokine receptors normally function by binding to chemokines in order to direct leukocytes to migrate to sites of inflammation.
54
What members of the chemokine receptor familt are used in macrophages(M)-tropic and T-cell(T)-tropic viruses?
What can this entry be inhibited by?
CCR5 for M-tropic HIV and CXCR4 for T-tropic HIV
This entry can be inhibited by downregulating the expression of the co-receptors or by saturating them with their ligands.
55
Tell me about **M-tropic HIV ...**
* caused by?
* Found in what populations?
**M-tropic HIV**
* A nonfunctional mutant allele of **CCR5** with an internal **deletion of 32 bp (CCR5Δ32)** is found with high frequency in **European and North American** populations, and this effectively protects individuals against M-tropic virions.
* This mutant allele is **rarely found in Asian Indians**
* In addition, insertions in the promoter region of macrophage inflammatory protein
* 1α (MIP-1α), a ligand for CCR5, have been reported in 1 in 5 Indians.
56
Tell me about **T-tropic HIV...**
* Caused by?
* Population found in?
**T-tropic HIV**
* A **guanine to adenine point mutation** (3′UTR801G/A) in stromal-cell-derived factor 1 (SDF-1), a ligand for CXCR4, has been reported in **40% of healthy Asian Indians**
57
The CCR-5 gene of the M-tropic variant has two alleles,
one is mutated in 10% of Northern Europeans and results in a non-functional form of the CCR-5 protein which delays progression to AIDS.
Approximately 1% of Caucasians are homozygous for the null allele and therefore express no functional CCR-5 protein. These individuals are resistant to infection with HIV.
What is thought that initial infection is due to?
Whats thought to be a sign to rapid progression to AIDS?
What allows the viral nucleocapsid to enter the cell?
* Since their expression of CXCR-4 is normal, suggests that i**nitial infection is by M-tropic variants using the CCR-5 receptor**
* Indeed, isolates of HIV in recently infected individuals are predominantly M-tropic.
* **Emergence of T-tropic variants is often a sign of rapid progression to AIDS.**
* Following binding of Gp120 to CD4 and the relevant chemokine receptor, gp41 mediates fusion between the viral envelope and host cell membrane, allowing viral nucleocapsid to enter the cell
58
What happens once the viral nucleocapsid enters the host cell?
Once inside the cell virus nucleocapsid is removed and **reverse transcriptase copies the RNA into double-stranded DNA**
59
Why do so many mutations arise during the replication process?
the reverse transcriptase has poor fidelity, with no proof-reading ability, ~10 mistakes per replication round; so many mutations arise
60
Once the viral DNA integrates into the host cell DNA, what is it now known as?
What stage is this?
Viral DNA integrates into the host cell DNA, where it is known as a **provirus** (**RNase H** degrades the copied RNA template).
This stage of viral infection is known as **latent stage** and virus can lie dormant for a long time.
61
What is virus production initiated by?
Virus production is initiated by cellular transcription factors e.g. **NF-κB** which is upregulated when T-cells become activated
62
What is viral RNA transcribed from?
Viral RNA is transcribed from proviral DNA and viral proteins are translated using host cell protein synthesis machinery.
63
What do viral protein and RNA assemble into?
Viral proteins and RNA assemble into particles that bud from the cell, taking some of the host cell membrane to form the viral envelope.
These particles can now infect other cells.
64
Individual steps in HIV’s invasion and replication, in schematic form, projected into an infected helper T-cell.
65
What is the ER important for?
Endoplasmic reticulum is important for protein glycosylation e.g. **gp120**
66
The clinical progression of HIV infection can be divided into three stages. What are these stages?
1. Infection
2. the “latent” stage
3. development of AIDS
67
Most people show no symptoms immediately after infection. But about 15% demonstrate symptoms reminiscent of influenza, what do these include?
* fever
* malaise
* aching muscles
* sore throat
* swollen lymph nodes
Some people also develop swollen lymph nodes without any other clinical symptoms
68
Following infection what occurs and what is this process called?
Following infection, antibodies to HIV antigens are produced: a process called **seroconversion**
69
What is used to test for infection?
Detection of antibodies to HIV is used to test for infection
70
Tell me about the Latency period of HIV infection?
* This period is **generally asymptomatic** although about 33% of infected people will have swollen lymph nodes.
* The **average time from infection to development of AIDS is about 10 years**
* But length of the “latency” period is extremely variable, lasting less than a year in some people and upwards of 15 years in others.
* Not clear whether everyone infected with HIV will inevitably develop AIDS.
71
What is the end of 'latency' period of HIV accompanied with?
End of “latency” period is accompanied by emergence of various symptoms that indicate progression to AIDS without treatment.
72
What are some symptoms of development of AIDS?
Symptoms include **weight loss, night sweats, fever and diarrhoea.**
Also, **infections** such as oral candidiasis, herpes simplex and shingles caused by minor opportunistic infectious agents.
73
How is AIDS defined clinically?
AIDS defined clinically by **appearance of major opportunistic infections or by a drop in the CD4 T cell count to below 200 cells/µl of blood**
74
Without treatment, what does AIDS lead to?
Tell me some causes for this in Europe/USA/ Oceania and Africe/Asia
**Without treatment AIDS invariably leads to death**. Death is caused by combinations of the infections although some infections are more prominent according to geographical location.
In Europe, the USA and Oceania the commonest infection is **pneumonia caused by Pneumocystis carinii.**
In Africa and Asia, infections such as **Cryptosporidium**, a protozoan that causes severe diarrhoea and weight loss, and **Mycobacterium tuberculosis** are more common.
75
Opportunistic infections in HIV
76
So does HIV kill you?
HIV does not kill you but destroys your immune system which leads to other organisms causing death
77
Tell me the immunological events associated with HIV infection
* HIV has one of the most complicated relationships with the immune system of any infectious agent.
* Because of the nature of the cells it infects and the molecules that it binds to, HIV has a dramatic effect on the immune system, eventually causing profound **immunosuppression.**
* Before that stage, and contrary to earlier beliefs, the immune system, far from failing to mount a significant response against the virus, generates a very powerful response against HIV.
* With almost any other virus this response would be enough to eliminate the pathogen but because of the **special features of HIV the virus is able to survive and eventually destroy the immune system.**
78
What does HIV cause a gradual decrease in?
CD4 T cell numbers
However, a number of other changes to immune system occur following HIV infection.
79
What do many HIV+ individuals develop during initial infection?
swollen lymph nodes
80
Can the intial swollen lymph nodes continue past intial infection with HIV?
Can persist even during the clinically latent phase.
81
What does historical examination of lymph nodes show?
* increasing disruption of normal lymph node architecture,
* influx of CD8 T cells
* eventual loss of germinal centres.
82
Tell me about lympth node inflammation?
* Dendritic cells patrol tissues, on the lookout for microbial invaders.
* When encounter a pathogen chop it up into tiny pieces and carry samples of it to local lymph nodes.
* There, they display their finds to the CD4 T cell, which then mounts full-fledged immune response against the invader.
* Lymph node inflammation.
83
Tell me how HIV exploits the dendritic cell surveillance network to its own advantage
Unfortunately, HIV exploits the DC surveillance network to its own advantage: HIV is picked up by DCs that patrol mucosal tissues, but avoids being killed by them, and instead hitches a ride to lymph nodes before transfer to the CD4 T cell. Until now, not clear how DCs recognize HIV for uptake—a mystery that's now been solved.
84
What is it that is exposed on HIV membrane that is recognised by dendritic cell and virus is taken in?
**Sialyllactose head on GM3 ganglioside** (glycosphingolipid) exposed on HIV membrane is recognised by DC and virus is taken in.
Future – how does virus escape destruction in DC?
85
Sialylactose head on GM3 ganglioside
86
Tell me about the loss of CD4 T cell function in regard to HIV infection?
Why is it lost?
* As infection progresses HIV+ individuals show loss of CD4 T cell function that cannot be explained simply on basis of reduced CD4 T cell numbers and is somehow associated with the effects of the virus.
* Transplantation recipients taking immunosuppressive drugs can show drops in CD4 T cell numbers similar to those seen in HIV+ individuals but the transplant recipients demonstrate much better CD4 T cell function.
* Reasons for loss of CD4 T cell function are not clear. One consequence of loss of Th activity is reduced ability to mount a delayed-type hypersensitivity reaction
* As infection progresses ability to generate antibody responses is also lost.
87
What can HIV+ individuals demonstrate increased total levels of?
* Somewhat paradoxically, HIV+ individuals can demonstrate **increased total levels of serum Ig** despite the impaired ability to generate specific antibody responses.
* Again the basis for this is not clear but thought to reflect abnormalities in normal regulation of immune responses caused by the virus.
88
Possibly also related to generalised immune dysfunction, HIV+ individuals can show increased production of autoantibodies to antigens such as?
* red blood cells,
* spermatozoa or
* myelin (a component of nerve sheaths)
* And may suffer from flare-ups of allergies such as eczema.
89
What happens immediately after infection with HIV?
Immediately following infection with HIV there is a period of rapid viral replication resulting in high levels of virus in the blood **(viraemia)**
90
HIV is an intracellular pathogen and as such would be expected to what...?
What stage would the disease then enter?
* HIV is an intracellular pathogen and as such would be expected to **stimulate a strong CD8 cytotoxic T cell response** and possibly a delayed type hypersensitivity response, as well as production of antibody.
* In fact **strong antibody responses to gp120 envelope protein and the p24 protein of the nucleocapsid** (also known as a Gag) are seen soon after viral infection
* Also a powerful CD8 cytotoxic T cell against gp120, p24 and some of the proteins that make up reverse transcriptase (p01 antigens).
* Combined antibody and cytotoxic T cell response is extremely effective and eliminates more than 99% of the virus.
* Following this strong response the disease usually **enters the latent stage.**
91
Initially what was thought about the latent stage?
* Initially thought that latent stage of disease was one in which virus was largely in proviral stage and therefore inactive; and that very few CD4 T cells were being destroyed as a consequence of being infected by the virus.
* These conclusions were based primarily on studies examining the number of productively infected (meaning that whole virus could be cultured from the cells) CD4 T cells and overall CD4 T cell numbers in the blood of HIV-infected individuals.
* Recent studies suggest that this picture obtained from blood was not accurate.
92
Newer techniques to measure viral RNA, a sign that the virus is replicating, have shown what?
* Newer techniques to measure viral RNA, a sign that the virus is replicating, have shown a **persistent level of active viral replication** in the blood even during the latent period.
* The **level of this viraemia** is a good prognostic **indicator of the progression** of the disease: **individuals with a low level of viraemia progress more slowly to AIDS than those with a higher level of viraemia.**
93
What does examination of lymph nodes show?
* Examination of lymph nodes have shown 10-100 times more productively infected CD4 T cells than seen in blood.
* Furthermore, lymph nodes of HIV+ people in the latent period show structural abnormalities that become more severe as the disease progresses.
94
What happens to the relative proportion of CD4 T cells in the blood when some one is HIV+?
Also during HIV infection the relative proportion of the **total number of CD4 T cells in blood increases**, so actual loss of CD4 T cells is greater than that reflected in the blood
95
What did treatment of infected individuals with drugs that prevented HIV from infecting new cells show?
**(i)** the immune svstem was eliminating up to 30% of the total viral load every day but that in the absence of these drugs the virus replicated at an enormous rate and the eliminated virus was replaced
**(ii)** up to 2 x 109 CD4 T cells were destroyed every day but most of these were replaced by the immune system so that the overall CD4 levels in the blood declined only very slowly.
96
It now appears that the latent stage of HIV infection may be clinically latent but what does it actually represent?
a very dynamic situation with high viral destruction and replication and extensive CD4 T cell destruction and replacement.
97
In terms of why the immune system does not clear the virus, HIV has several features that enable it to survive in the face of a powerful immunological onslaught. Tell me some of these features?
* Has a very **high replication rate** - main reason why the virus is not totally cleared.
* **Can hide** as provirus where not detectable by the immune system.
* Has very **high mutation rate**, so that antigens to which antibody and CD8 T cells have been made mutate and are no longer recognised by the immune system. Antibodies and CD8 Tcs are made against the mutated antigen but this can mutate again and so the process of evading the immune response goes on and on.
98
The cause of CD4 T cells loss is thought to be a combination of what?
Cause of CD4 T cell loss is thought to be a combination of direct killing by the virus and destruction of virally infected cells by the immune system.
99
What do CD4 cells that are infected by the virus express?
What could these be?
CD4 cells that are infected by the virus **express viral antigens on their surface**. These may be **viral peptides** presented by class I MHC or may be **soluble gp120** bound to CD4 on the T cells.
100
What do HIV+ indivdiduals have large amounts of that will bind to CD4
HIV+ individuals can have large amounts of soluble gp120 in their blood and lymph, which will bind to CD4.
101
Because of the expression of viral antigens, CD4 T cells can be killed in a number of ways. What are these ways?
102
Tell me the ways to kill a CD4 T cell
* HIV may directly cause lysis of CD4 T cells.
* Infected cells bearing HIV antigens may be killed by antibody and complement or killed by antibody-dependent cell-mediated cytoxicity
* HIV-infected cells presenting HIV peptides on their class I MHC molecules may be killed by CD8 T cells.
103
What are the CD4 T cell killing mechanisms and tell me what happens in each and the components involved?
* **Antibody + complement:** Anti-gpl20 binds to the gp120 bound to CD4, resulting in complement fixation and activation and cell lysis.
* **Antibody-dependent cell-mediated cytotoxicity:** Macrophages and NK cells have Fc receptors on their surface. This enables them to bind to the Fc portion of the anti-gpl20 antibody bound to the CD4 T cell and kill the cell.
* **CD8 cytotoxic T cells:** kill virally infected cells expressing HIV antigenic peptides in association with their class I MHC.
104
When HIV productively infects the few permissive CD4 T cells present in spleen, how does death occur and what is this done by?
When HIV productively infects the few permissive CD4 T cells present in spleen, death occurs through **apoptosis** mediated by the enzyme **caspase-3**
105
When HIV abortively infects nonpermissive CD4 T cells, (fail to complete reverse transcription i.e. accumulation of incomplete HIV transcripts triggers cell death) how does cell death occur and what is this done by?
when HIV abortively infects nonpermissive CD4 T cells, (fail to complete reverse transcription i.e. accumulation of incomplete HIV transcripts triggers cell death) death occurs by **pyroptosis**, which depends on activation of **caspase-1**
**_Pyroptosis_ is a highly inflammatory form of lytic programmed cell death that occurs most frequently upon infection with intracellular pathogens and is likely to form part of the antimicrobial response.**
106
Approximately what % of CD4 T cell death in lymphoid tissues is driven by caspase-1-mediated pyroptosis?
107
approx. 95%
108
So, most CD4 T cells in lymphoid tissues, despite their ability to resist full infection by HIV, respond to presence of viral DNA by what?
So, most CD4 T cells in lymphoid tissues, despite ability to resist full infection by HIV, respond to presence of viral DNA by **sacrificing themselves via pyroptosis**—a highly inflammatory form of cell death that lures more CD4 T cells to the area, thereby creating a vicious cycle that ultimately wreaks havoc on the immune system.
109
Compare apoptosis and pyroptosis
**Apoptosis** is a form of caspase-mediated cell death with particular morphological features and an anti-inflammatory outcome
**Pyroptosis** is a pathway of cell death that inherently results in inflammation. Many techniques have been used to measure specific characteristics associated with cell death.
110
Targeting caspase 1 and pyroptosis of lymphoid CD4 T-cells via orally bioavailable and safe drug prevents lymphoid CD4 T-cell death by HIV-1
**a, b,** VX-765 efficiently blocks CD4 T-cell death in HIV-infected tonsillar and splenic lymphoid tissues. No toxicity was observed at any of these drug concentrations. Error bars represent s.e.m. from three independent experiments using tonsil or spleen cells from three different donors.
**c,** Pyroptosis in HIV-infected lymphoid tissues may establish a chronic cycle of CD4 T-cell death and inflammation, which attracts new CD4 T cells and ultimately contributes to disease progression and tissue damage. Inhibitors of caspase 1 such as VX-765 may inhibit pyroptosis in a manner that both preserves CD4 T cells and reduces inflammation.
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Progression to AIDS is driven by what?
What does this involve?
Progression to AIDS driven by CD4 T-cell depletion, mostly involving pyroptosis elicited by abortive HIV infection of CD4 T cells in lymphoid tissues.
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In the progession to AIDS driven by CD4 T-cell depletion, inefficient reverse transcription can lead to what and what is the result of this?
Inefficient reverse transcription in these cells leads to cytoplasmic accumulation of viral DNAs that are detected by the DNA sensor IFI16, resulting in inflammasome assembly, caspase-1 activation, and pyroptosis.
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What cells resist pyroptosis and why?
Peripheral blood-derived CD4 T-cells naturally resist pyroptosis, partly due to their deeper resting state, resulting in fewer HIV-1 reverse transcripts and lower IFI16 expression.
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What changes when blood-derived CD4 T-cells become co-cultured with lymphoid-derived cells?
when co-cultured with lymphoid-derived cells, blood-derived CD4 T cells become sensitized to pyroptosis, likely recapitulating interactions occurring within lymphoid tissues.
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What does sensitisation correlate with?
Sensitization correlates with higher levels of activated NF-κB, IFI16 expression, and reverse transcription.
Thus, lymphoid tissue microenvironment encountered by trafficking CD4 T lymphocytes dynamically shapes their biological response to HIV.
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Pyroptosis CD4 T-Cell location
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Tell me about the main cells that HIV effects and how this leads to immunosuppression?
* By generating these responses, the immune system is actually doing what it is supposed to; it is generating effector mechanisms that eliminate virally infected cells and thereby the virus.
* Unfortunately, the main type of infected cell is the CD4 T cell and therefore the virus directly and indirectly kills the very cells needed to generate an immune response against it.
* Although the immune system makes a great effort to replace the CD4 T cells that are destroyed every day, the production of CD4 T cells never quite matches the destruction and there is a gradual drop in CD4 T cell numbers.
* Eventually the CD4 T cell numbers become too low to maintain normal immune responsiveness and immunosuppression ensues.
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What is the main issue involved with trying to treat HIV+ individuals with antiviral drugs?
HIV has a high mutation rate
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What was the first anti-HIV drug developed, what was its function?
Why wasn't it successful?
First anti-HIV drug was zidovudine (AZT), a reverse transcriptase inhibitor introduced in 1987.
Although it inhibited replication of HIV, virus very quickly mutated and became resistant to the drug.
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Tell me about the drugs around today for HIV+ individuals
Now **three types of anti-HIV drug** in use clinically.
Combinations of two, or preferably three, drugs are of longer clinical benefit because there is less chance of the virus mutating to become resistant to three drugs at once.
Estimate 2.9 million lives saved by **Anti-Retroviral Therapy (ART)**
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What are the three categories for HIV inhibitors?
1. nucleoside analogue reverse transcriptase inhibitors,
2. non-nucleoside analogue reverse transcriptase inhibitors
3. HIV protease inhibitors
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Two types of reverse transcriptase inhibitor act in different ways to inhibit the reverse transcriptase; therefore complement each other's action. Tell me about this
* **Nucleoside/Nucleotide analogues** lack a 3'-hydroxyl group on the deoxyribose moiety. Following incorporation the next incoming deoxynucleotide cannot form the next 5'-3‘phosphodiester bond needed to extend the DNA chain. Thus, viral DNA synthesis is halted, a process known as chain termination. All are classified as **competitive substrate inhibitors.**
* **Non-nucleoside analogues** block reverse transcriptase by binding at a different site on the enzyme, compared to NRTIs and NtRTIs. NNRTIs are not incorporated into the viral DNA but instead inhibit the movement of protein domains of reverse transcriptase that are needed to carry out the process of DNA synthesis. Classified as **non-competitive inhibitors.**
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What do protease inhibitors do?
What are they used for?
Protease inhibitors **inhibit the active site of the HIV aspartic protease**, used to cleave a number of viral precursor polyproteins to produce many HIV proteins and enzymes. Peptide linkage –NH-CO- is replaced by uncleavable hydroxyethylene group (-CH2-CH(OH)-) Without protease activity the virus cannot replicate.
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Anti-HIV drugs
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What does combination therapy usually consist of?
Combination chemotherapy usually consists of **two reverse transcriptase inhibitors** and a **protease inhibitor**
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Tell me about the effectiveness of Combination chemotherapy
Can be very effective at lowering levels of virus and raising CD4 T cell levels and results in significant clinical improvement so that many AIDS patients are able to leave the AIDS clinic and return home.
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New therapy is also thought to be responsible for the sharp drop in death from AIDS seen in the USA since 1996. However, there are problems with combination therapy. Tell me about this?
1. There is considerable **toxicity**, especially to the bone marrow and gut, which means some people cannot take the drugs.
2. **Regime** for taking the drugs is complicated and intrusive to a normal lifestyle. Some of the drugs have to be taken with food and some without, and some drugs cannot be taken within a certain time period of each other.
3. Finally, the drug treatment is **very expensive** - about $15,000 a year - so that it is not readily available in the countries that need it the most.
Therapy successfully suppresses HIV but is not cleared from the body. WHY?
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HIV and JAK/STAT pathway...
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New antiviral strategy
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Attempts to generate vaccines for HIV have fluctuated in intensity over the years for a variety of different reasons, both economic and scientific.
Initially two type of vaccines were considered. What are these two?
1. **Prophylactic vaccines**
2. **Therapeutic vaccines**
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Tell me about **Prophylactic vaccines**
**Prophylactic vaccines**
* Aim of these vaccines was to provide protection to individuals who were not infected with the virus.
* Thus, the aim was the same as for vaccines against other infectious agents such as measles, mumps, polio, smallpox, etc.
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Tell me about **Therapeutic vaccines**
**Therapeutic vaccines**
* AIDS was initially considered a disease of immunosuppression where HIV-infected people did not make a very good response against the virus.
* Therefore thought it might be possible to boost the immune response of HIV-infected people so that they could mount a response and clear the virus.
* With the current appreciation that most HIV-infected people make a very strong response against the virus it is not so clear how effective this approach might be.
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Tell me the main **immunological issues** concerning development of an HIV vaccine like the prophylactic vaccine
Development of prophylactic vaccines against HIV has considerable difficulties of both an immunological and a logistical nature.
Main immunological issues concerning development of an HIV vaccine are:
* which of the many different current approaches to vaccine development to use, given the many subtypes and variants and the mutation rate of the virus?
* what type of immune response to try and stimulate?
* additionally, there are no good animal models of HIV.
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What are the main logistical problems in testing the prophylactic vaccines?
* Traditionally vaccines were developed against acute diseases that often occurred in waves or epidemics (e.g. measles or mumps) and had a low mortality rate.
* Therefore comparatively straightforward to vaccinate a section of the population and see whether these people were protected from the disease compared with non-vaccinated individuals.
* Possible this way to get an answer to the effectiveness of the vaccine in a relatively short time.
* Because AIDS is a chronically progressive disease the conventional approach would take many years to give an answer - too long for most people to wait.
* A number of vaccines in clinical trials at present using DNA, recombinant and antigen/peptide approaches, although none has demonstrated convincing protection so far.
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As there are some issues with HIV vaccines, what was the new strategy developed?
* **Found:** naturally occuring, broadly neutralising HIV antibodies which mimic the CD4 binding site on the envelope spike that the virus uses to enter and infect its host cells.
* Cloned 576 new HIV antibodies from four individuals whose immune systems naturally developed antibodies with broad serum activity. Sequencing showed antibodies have similar sequence and originate from two independent genes.
* These antibodies will be valuable in designing future HIV vaccines
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Mapping the innate immune response initiated locally in the vagina upon exposure to the virus:
Haase et al. (Nature 2009) observed growing clusters of infected immune cells in the vaginas of monkeys exposed to simian immunodeficiency virus (SIV), the monkey version of HIV.
What was observed?
What was a strategy developed from the findings?
* First, plasmacytoid dendritic cells rush to the scene; these spur chemokine and cytokine response.
* In turn brings CD4+ T cells. Become infected by the virus, fuelling its spread throughout the body.
**New strategy**
* Glycerol monolaurate (GML) blocks the growth of microorganisms such as Staphylococcus and Chlamydia.
* In cultured human vaginal epithelial cells exposed to HIV, GML blocks production of molecules that appear during inflammation and that are thought to increase susceptibility to HIV infection.
* Applied GML gel to five rhesus macaques that had been vaginally exposed to SIV - blocked acute infection in all five of the monkeys.
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**Summary**
* **AIDS as a disease was first identified in 1981 in groups of high-risk individuals: homosexual men, intravenous drug users and haemophiliacs.**
* **Currently estimated that 30 million people are infected with the AIDS virus.**
* **Virus causing AIDS was isolated in 1983 and called the human immunodeficiency virus (HIV). A second strain, called HIV-2, was identified in 1986.**
* **HIV is a retrovirus, i.e. its genetic material consists of RNA. It infects CD4+ cells, mainly CD4 T cells but also monocytes and dendritic cells.**
* **Clinically HIV infection is characterised by the development of opportunistic infections, especially with the yeast Pneumocystis carinii, and the development of a rare tumour, Kaposi's sarcoma.**
* **Development of AIDS can take over 15 years from infection. Following infection and seroconversion there is a latent period that is largely a clinical.**
* **Although the latent period is aclinical there is an extensive amount of viral destruction and replication; and also destruction and replacement of CD4 T cells. However, there is a gradual net loss of CD4 T cells, leading eventually to immunosuppression and the development of opportunistic infections, which are fatal without treatment.**
* **HIV infection induces cellular depletion and early abnormalities of CD4+ T cells; decreases CD8+ T-cell counts and function (cellular immunity); causes deterioration of specific antigen responses (humoral immunity); and leads to alteration of innate immunity through impairment of cytolytic activity and cytokine production by natural killer cells.**
* **Most successful HIV chemotherapy is triple therapy with a combination of reverse transcriptase inhibitors and viral protease inhibitors. However, this therapy has significant side-effects, is difficult to comply with and very expensive. Suppresses but does not clear HIV due to Vif blocking Inteferon path**
* **HIV vaccines are faced with the problem of the high mutation rate of the virus and logistical problems of testing the efficacy of a vaccine against a chronic infection.**
* **Microbiocides or inhibit Pyroptosis might be a way forward? Mutation rate effects?**
