What are the surface proteins on HIV and what are their functions? What envelops and protects the genome?
How many copies of the genome are contained within in virus? What is used to transcribe the genome?
HIV is an enveloped virus with a lipid bilayer acquired as it buds from infected cells. The two virus-surface proteins are gp120 (binding to CD4 on target cell) and gp41 (fusion of virus and cell membranes). Inside are two copies of the RNA virus genome-about 10kb in size and associated with the RNA are copies of the reverse transcriptase enzyme. The RNA is protected by a capsid (also called nucleocapsid) structure made of subunits called p24.
Explain the stages of infection of HIV in cells.
Stages in infection of cells are:
1) Virus binding which consists of binding of gp120 to CD4;
2) Virus entry which consists of subsequent binding to gp120 to a chemokine receptor (also called a co-
receptor) triggering a change in the gp120 and gp41 to cause fusion of membranes. At this time the capsid enters the cytoplasm;
3) reverse transcription-using nucleotides in the cytoplasm, reverse transcriptase begins to make DNA copies of the RNA genome;
4) Integration of HIV DNA-the HIV DNA escapes from the capsid, enters the nucleus and becomes integrated into the host cell genome using the virus-encoded integrase enzyme. At this stage the DNA is called provirus.
5) Expression of virus genes;
6) Budding of new virions.
What two chemokine receptors does HIV bind to during infection?
On what cells are these chemokine receptors contained? What strains bind to which receptors?
How does the virus protect itself from antibodies?
Most resting T cells express the chemokine receptor CXCR4 and when activated T cells express CCR5. Macrophages express both. Initial infection during sexual transmission nearly always transfers virus strains that use CCR5-called R5 strains. Later during infection, more pathogenic strains of virus can arise that infect cells through CXCR4-called X4 strains. The virus uses sequential binding of CD4, then chemokine receptor, then fusion events to infect the cell. This protects the virus from antibodies since antibody epitopes are only exposed to antibodies after fusion cascade is triggered. CD4 inserts into a deep cleft in gp120-hence additional protection from antibody (since gp120 is not exposed/minimally exposed to the environment).
When a patient is infected with HIV through sexual transmission, what are the likely cells that are infected first?
Where do infected cells go?
What site within the body is thought to be a massive site of viral infection and T-cell depletion? What cells become depleted?
When a patient is infected through sexual transmission, the first cells infected are macrophages, T cells, or dendritic cells-all of these express CD4 and can be infected by HIV. Infected cells travel to local lymph nodes and spread infection to other immune cells. Recent evidence indicates lymphoid tissue within the GI tract then becomes a site of massive virus infection and T cell depletion. Memory cells become depleted (have lots of CCR5). While naïve cells are also depleted, they are replenished by new cells from the thymus.
How long is the acute infection phase? How long after HIV exposure does it occur?
What are the symptoms during this phase?
Describe CD4 and viurs levels during this phase.
When are CD8 and antibody responses detected?
What is the set point? Why is it important?
Can viral antigens be detected during the acute phase of infection?
Acute Infection; 2-4 weeks after exposure. Lasts on average about 10 days. Mild to Severe flu-
like symptoms (fever, myalgias, fatigue). At this time the virus titer increases logarithmically to a peak. CD4 cell numbers decrease significantly. Little or no virus immunity is detected but then finally increases and begins to control the virus. Both CD8 responses and antibody responses can be detected towards the end of this period. The virus titer begins to decrease and reaches a point called the virus set point which varies between individuals but is predictive of speed of progression. CD4 numbers can increase partially. Virus antigens can be detected in blood during acute infection.
When is risk of transmission the highest?
When viral levels are the highest-acute infection, AIDS
Approximatley how long is the asymptomatic/chronic/latent infection phase of HIV?
What has changed the average length of this phase?
What are virus levels like at this stage? What are CD4 levels like?
Are virus antigens detectable during this phase?
Asymptomatic, also called chronic or latent Infection; before HAART, this averaged around 10 years in the United States. With HAART the average is much longer. Virus stays low at set point, largely under the control of host immunity, but there is a consistent slow reduction in CD4 cell number due to low-level virus replication. Virus antigens are mostly undetectable at this stage.
HAART: Highly active anti-retroviral therapy. Since the introduction of HAART in developed countries, fewer patients progress to AIDS.
What occurs during AIDS? Why does this happen? What are CD4 levels like in this phase?
Are virus antigens detectable during this phase?
AIDS; opportunistic infections as a result of low CD4. CD4 numbers become very low- generally, less than 200/mm3 - increased risk of being symptomatic. Virus antigens can become detectable again.
What four tests are used to diagnose HIV? (just list)
Rapid test (in office)
test for HIV antigens
Explain how ELISA works in diagnosis of HIV.
EIA (ELISA) is performed on serum or plasma. If positive, retest to confirm. Recently, rapid tests were approved for use. Positive ELISA are confirmed by western blot. ELISA for HIV antibody is performed by 1) coating a surface with HIV antigens. 2) add patient serum and allow antibodies to bind to antigens 3) add a secondary antibody that is labeled with enzyme (e.g. goat anti-human horseradish peroxidase). Add enzyme substrate and see color change.
Explain how Western Blot is used in the diagnosis of HIV.
What is an indeterminant blot? What is the patient advised to do in this case?
Western blot for HIV antibody is performed by electrophoresis of HIV virus to separate proteins of different sizes (i.e. gp120 is 120 kilodaltons in molecular weight, gp41 is 41 kd, p24 is 24 kd). Separated proteins are transferred (blotted) to nitrocellulose. The nitrocellulose is exposed to patient serum followed by labeled second antibody as in ELISA. Develop and get color change to see if there are antibodies to specific HIV proteins in the serum.
Sometimes a western blot will be indeterminant (one band matching HIV virus proteins). In this case, patient is advised to return in one month for retesting.
What are the pros and cons to rapid testing?
What antigen is commonly tested for in the diagnosis of HIV? Why?
Rapid testing is faster and can be performed conveniently in the office. Also, more HIV positive people receive their results. However, because the test is not as sensitive some people receive false positives.
Testing of patients also includes a test for HIV antigens (usually p24) since this can be in blood during acute infection before antibody is present.
What are the issues with testing methods for HIV?
Problems in HIV testing: During acute infection-antibody has not yet been expressed at high levels, but patient is HIV infected and therefore a patient may show up as negative for antibodies to HIV by the ELISA/EIA test. Western blot is less sensitive than screening tests-therefore in early infection will get a positive in ELISA and negative in WB. Delays in reporting are also an issue. During neonatal testing-all infants born to infected mothers will have antibody to HIV whether infants are infected or not due to trans-placental transfer of IgG. (less than 5% of infants born to HIV+ mothers become infected in U.S.). Vaccinated persons may be positive for antibody but not infected. Cross reactivity with antigens from other viruses-most notable HTLV I and II (human T cell leukemia viruses-retroviruses).
What are the virus and immune mediated mechansims of CD4 depeltion in HIV?
HIV infects CD4 cells and CD4 depletion leading to immunodeficiency is the hallmark of the disease. What are the mechanisms of CD4 depletion? There is a consensus that generalized immune activation contributes to depletion of the CD4+ cell reservoir. Depletion rates may be higher in the gut.
Virus Mediated effects on CD4 cells:
- Virus infection is directly cytopathic for cells
- Binding of virus or free gp120 to uninfected cells induces aberrant signaling/dysfunction/apoptosis.
Immune Mediated effects on CD4 cells
- Binding of virus or free gp120 to uninfected cells leads to immune recognition-antibody, opsonization, ADCC
- Infected cells are destroyed by CTL activity.
What effect do HAART drugs have on viral levels?
What is contained within HAART drugs to combat the virus? Why is immunity not completely restored with use of HAART?
What effect do HAART drugs have on CD4 levels and function?
• Highly active anti-retroviral treatment (HAART)- combination of drugs-suppresses virus substantially in nearly all patients
– virus becomes undetectable in most after wks
• HAART cocktail of RT (reverse transcriptase) and protease inhibitors
• This restores immunity to some degree but not completely-probably since memory cells were depleted in early infection
• During suppression of virus at acute stage, CD4 cell counts in most cases partially rebound towards normal
• CD4 cell function in most cases partially rebounds towards normal
What are the options for vaccination against HIV? How are they proposed to work?
Preventive vaccines. While several vaccine strategies have been successful in preventing infection in animal models of HIV infection, until recently there were a number of notable failures in vaccine trials in humans. However, in a trial in Thailand (reported in 2009) a canarypox vector vaccine (ALVAC-HIV) boosted with a recombinant glycoprotein vaccine (AIDSVAX B/E) led to a 31% reduction of HIV incidence in vaccine recipients. The immune responses that enabled protection are a focus of intensive post-trial studies.
Therapeutic vaccines. Another emerging concept is that a vaccine will eventually be available, but instead of preventing infection altogether (sterilizing immunity), infection will still occur, but the damage to the immune system will be blunted since the immune system will keep virus replication at lower levels (acute and virus set point will be lower).
By what mechanism has HIV thought to possibly cure patients?
Approaches to "cure" infected patients: Renewed interest in curing HIV was partly stimulated by a report of a bone-marrow transplant of CCR5-deleted stem cells to an HIV-positive patient, who seemed to eliminate detectable HIV after engraftment of this tissue. Perhaps gene therapy could modify this receptor to eliminate HIV in infected patients.