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HIV
By Alick Mwambungu Dublin Institute of Technology Republic of Ireland
Contents
Introduction – History and Overview of HIV
Pathogenesis – Structure and Lifecycle of HIV
Effect On The Immune System – Destruction of Cells and Resulting Immunodeficiencies
Diagnosis and Treatment
What is HIV?
Genus Retroviridae
Lentivirus, which literally means slow virus - it takes such a long time to develop adverse effects in the body.
This virus attacks the immune system
There are two strains – HIV 1 & HIV 2
It belongs to a group of retroviruses.
These contain RNA, the genetic material of HIV
The outer layer of the HIV virus cell is covered in coat proteins, which can bind to certain WBCs. This allows the virus to enter the cell, where it alters the DNA.
The virus infects and destroys the CD4 lymphocytes which are critical to the body’s immune response.
History of HIV
The HIV virus first came to light during the early 1980’s.
A number of healthy gay men in New York began to develop rare opportunistic infections & cancers, that were resistant to treatment.
One such viral opportunistic infection is cytomegalovirus that causes blindness & inflammation of the colon
Transmission HIV is transmitted from person to person in several ways:
Through unprotected sex with an infected person
Exposure to infected blood
By the use of contaminated equipment for injections (by drug users or medical treatment in developing countries)
From an infected mother to her baby before or during birth, or by breast feeding
HIV Origins
Research teams in the U.S.A & France made independent research discoveries of the virus.
French researchers discovered a virus linked to AIDS in 1983, they called it Lymphadenopathy-Associated Virus (LAV)
In 1984, American researchers isolated a virus that caused AIDS, calling it Human Tlymphotropic Virus type 111 (HTLV-111)
These two viruses were later found to be the same virus - HIV
HIV Origin
The emergence of HIV & AIDS has resulted in countless debates as to where it originated from
It’s suspected that it originated from S.I.V (Simian Immunodeficiency Virus)
SIV affects Monkeys
HIV Origins
Certain strains of SIV closely resemble the two types of HIV
HIV 1 – was difficult to link with SIV
In 1999 SIVcpz closely related to HIV 1
Originated from chimpanzees but it has significant differences from HIV-1
HIV 2 closely related to SIVsm
Originated from the green monkey
Conspiracy Theories
Two different SIV infections combined to form a new third virus, leading to zoonosis
Several theories regarding zoonosis
Hunter Colonialism – Transferred by hunting & killing chimpanzees - Colonial rule in Africa, forced labour - Eating SIV infected chimpanzees
Conspiracy Theories -
Oral Polio Vaccine Grown on kidney cells from chimpanzees Infected with SIVcm2 leading to HIV 1
Contaminated needles - Not sterilising needles between patients - Sharing of needles
Manmade
Linked to the CIA
Early known cases of HIV
Plasma sample taken from male adult in the Congo in 1959
A 1998 analysis of the plasma sample from 1959 has suggested that HIV-1 was introduced into humans around the 1940s or the early 1950s
Tissue sample taken from a teenager, St. Louis in 1969 – died
Tissue sample from Norwegian sailor- died 1976
Another sample was dated back to the end of the 19th Century
Incidence
~ 42 million people have been infected with HIV to date.
AIDS has caused millions of deaths
Only conditions typical of old age, such as heart disease & stroke cause more fatalities worldwide
In Africa, HIV has reached epidemic proportions, responsible for 1 in 5 deaths in 1998
In the UK 37,900 HIV + in 1999
Global Incidence
Global Incidence Trends
More than 25 million people have died of AIDS since 1981
Africa has 12 million AIDS orphans
At the end of 2006, women accounted for 48% of all adults living with HIV worldwide, and for 59% in subSaharan Africa
Young people (under 25 years old) account for half of all new HIV infections worldwide - around 6,000 become infected with HIV every day
In developing and transitional countries, 6.8 million people are in immediate need of life-saving AIDS drugs; of these, only 1.65 million are receiving the drugs
Global Incidence Trends
During 2006 around four million adults and children became infected with HIV (Human Immunodeficiency Virus), the virus that causes AIDS
By the end of the year, an estimated 39.5 million people worldwide were living with HIV/AIDS
The year also saw around three million deaths from AIDS, despite recent improvements in access to antiretroviral treatment.
World Statistical Regions of HIV
Structure of HIV
Genome and Proteins of HIV
• The genome of HIV is encoded on two identical strands of RNA when the virus is in the free form. • It has nine open reading frames (leading to nine primary translation products) but 15 proteins are actually made in all as a result of cleavage of three of the primary products. • The GAG gene and the GAG and POL genes together are translated into polyproteins which are then cleaved by a protease.
•
•
•
GAG polyprotein is cleaved to into four proteins: matrix protein (p17) which anchors the gp160 receptor capsid protein (p24) which forms a capsule around the RNA genome the nucleocapsid protein which binds to the HIV packaging signal on viral RNA p6 which plays a role in incorporating proteins into new virons. ENV gene is translated to gp160 which is then cleaved by a host cell protease found in the Golgi body to: Gp41 Gp120 Together these form the HIV receptor
• POL polyprotein is cleaved to: Protease, an enzyme required for maturation and functioning of other viral proteins Reverse transcriptase which is responsible for incorporation into the host genome. Integrase which cuts the host DNA and attaches the proviral genome to the cut ends. • Six other proteins are made by HIV • Three of these are incorporated into the virus: Tat and Rev are regulatory proteins Vpu (HIV-1) indirectly assists in assembly • The others are not found in the mature virus:
The HIV receptor
•
Gp160 is composed of gp41 and gp120 and forms the receptor for binding to the host cell (CD4 positive cells).
•
The gp41 portion is half embedded in the membrane envelope and interacts with gp120 portion on the exterior side of the membrane.
•
Each receptor is composed of 3 subunits of gp41 and 3 subunits of gp120.
The HIV Receptor
Lifecycle of HIV
HIV particles enter the body in a fluid as it can not survive without a support medium.
The virus targets any cell expressing CD4, including T helper cells, macrophages, dendritic cells and monocytes.
• Receptor Interactions •
The first stage of infection involves the binding of the gp120 portion of the receptor on the HIV to the CD4 receptor on the host cell.
•
This binding also requires the interaction of the virus with a chemokine co-receptor
•
Of the chemokine receptor family the two most important co-receptors for HIV are CCR5 on macrophages and CXCR4 on T-cells.
•
Attachment of HIV to a co-receptor first allows for a more stable and intimate association with the CD4 receptor.
•
This interaction with the CD4 receptor causes a conformational change in the gp120 portion, which in turn causes a change in the gp41 portion, initiating the membrane fusion step of the infection.
2. Cell Membrane Fusion and Integration •
Virus particles enter the CD4 positive host cell.
•
HIV is transported in as a pre-integration complex and p17 carries nuclear localisation signals which are a sequence of proteins recognised by the host cell as belonging in the nucleus
•
Intergrase protein cleaves the host genome and reverse transcriptase reads the sequence of viral RNA and transcribes it into a complementary DNA sequence of the host.
•
HIV is now a genetic disease, once the viral genome is integrated it becomes a permanent component of the host cell.
3. Viral Replication •
As with all viruses HIV is an obligate intracellular parasite – it can only replicate when incorporated with the host’s genome.
•
Once integrated the virus can remain dormant in the host cells, or can begin the production of new RNA.
•
Non-genomic RNA is translated into large polyproteins. This is then cleaved into structural proteins which are assembled around genomic RNA.
•
Protease completes cleavage of polypeptides into fully functional proteins resulting in a multiple mature viral particles being released from the cell.
•
This causes cell lysis which is one of the most destructive mechanisms in the depletion of CD4 T-cell populations.
Mature Viral Particles Escaping From A T-Helper Cell
Infection of the Lymphocytes • HIV can exist in two forms, the M-trophic strain which is associated with infection of macrophages and the T-trophic which infects T-helper cells. •
Due to the high error frequency of HIV polymerase one strain can readily mutate with the other strain.
• The M-trophic strain has an important role in the initial infection as the primary site of infection for HIV is the macrophage. • The infected macrophage then acts as a “trojanhorse” in bringing the virus to the lymphatic system since it’s job is antigen presentation.
•
After infection of the macrophage the M-trophic genome undergoes mutation and T-trophic strains are produced.
•
The generation of the T-trophic strain results in the infection of the T-helper cell populations within the lymph nodes and lymphatic tissue.
•
T-cells are attracted to the lymph nodes and lymphatic system by two chemokines produce by the infected macrophages - MIP-1alpha and MIP-1beta
•
Migration and proliferation of T-helper cells in response to the infection means more and more cells will be continuously infected.
•
HIV is also thought to have the ability to infect Tmemory cells. This is an important factor in persistence of the disease in the lymphatic system over decades. Eventually there is loss of lymph node architecture.
Effects of HIV on the immune system 3 areas: 1. Destruction of CD4+ T cells population 2. Immune effects due to HIV infection 3. Progression of HIV infection to AIDS
Destruction of CD4+ T cells
Direct virological mechanisms Host’s immune responses
1.Direct virological mechanisms
HIV destroys individual infected cells through cell lysis or the formation of syncytia when large numbers of uninfected cells fuse with the infected cell.
This results in the deaths of potentially hundreds of uninfected cells.
The half-life of an actively infected CD4+ T cell is less than 1.5 days.
2.Host’s immune responses
Both humoral and cell-mediated immune responses partially control the viral production but in this process they destroy the infected CD4+T cells, leading to a gradual decline of CD4+ T cells
HIV-specific CTLs kill infected CD4+ T cells
Antibodies that recognize a variety of HIV antigens are produced - Antibody dependent cell-mediated cytotoxicity
Apoptosis of infected cells
Immune responses fail to eradicate all viruses. Viral load is maintained at low level Continuous decline of CD4+ T cells
Role of CD4+ T cells
Role: secrete cytokines that enable activation of B cells, Tc cells and macrophages
CD4+ T cells - TH1 cells and TH2 cells
TH1 cells – secrete IFN-γ => phagocyte-mediated immunity.
TH2 cells – secrete IL-4 => Ig synthesis.
Some studies demonstrate that the proportion of TH1 T cells decreases in HIV-infected patients but TH2 T cells increases. - disrupt the normal balance of cytokines.
Immune defects due to HIV infection Almost all aspects of immunity are affected as the disease progresses. Impaired cell mediated responses CD4+ T cell Rapid loss of memory helper T cells and the inability to replace these cells leads to increasing immunodeficiency.
CD4+ T cells that have bound gp120 may not be available to interact with class II MHC molecules on APC => T cell responses are inhibited.
Autoimmune response can occur as the HIV receptor shares homology with the MHC II molecules.
HIV becomes a component of the CD4 T-cell genome and as a result everytime the cell is activated there is further viral replication. Cytokines produced by innate immunity activate infected CD4+ T cells.
Immune defects due to HIV infection CD8+ T cell
Full differentiation of CD8+CTLs requires cytokines that are produced by CD4+ T cells Progressive decline of CD4+ T cells over time, is associated with decreased activation and survival of CD8+ cytotoxic T cells resulting in a decreased ability to destroy virally infected cells. HIV Nef protein inhibits expression of Class I MHC molecules. High mutation rates of HIV also allow the virus to escape adaptive immune responses.
Immune defects due to HIV infection B cells – impaired humoral response B-cell hyperreactivity Polyclonal hypergammaglobulinemia due to enhanced nonspecific IgG and IgA production. Impaired Ab-isotype switching and inability to respond to specific antigen. High incidence of B-cell lymphomas
Lymph nodes HIV kills cells in the lymph nodes Early HIV infection: destruction of dendritic cells Late stage: extensive damage, tissue necrosis, a loss of follicular dendritic cells and germinal centres. An inability to trap Ag or support activation of T+B cells
Immune defects due to HIV infection
Impaired Innate Immunity Macrophages
macrophage is infected directly by HIV or phagocytosis of other infected cells or Fc receptor-mediated endocytosis.
•Act as a reservoir for the virus • Normal functions are impaired which include •Phagocytosis + killing of organisms •Ag presentation •Chemotaxis •Cytokine secretion exhibit defective phagocytic function and impaired chemotaxis due to imbalance of cytokines production. Neutrophils -
Immunodeficiency
Immunodeficiency - due to defects in one or more components of the immune system
Main causes for immunodeficiency in HIV infection - destruction of lymphoid tissue and depletion of CD4+ T cells.
The CD4+ T cell count gives an indication of the level of damage that has already occurred to the immune system as a result of HIV infection.
Progression of HIV infection
After initial infection with HIV, there is usually an acute flu-like illness. This illness may include Fever Headache Tiredness Enlarged lymph nodes But after this most individuals are clinically asymptomatic for years. This is called the clinical latency period.
Progression of HIV infection Exposure to HIV normal Acute HIV disease
Immune competence
Slightly reduced
Abnormal
Clinical latency period -declining CD4+ T cell amount
AIDS
Opportunistic infections
Severely impaired
Time
Progression to AIDS
During the latency period, lymph nodes and the spleen are sites of continuous HIV replication and cell destruction.
The immune system remains competent at handling most infections with opportunistic microbes but the number of CD4+ T cells steadily declines.
Symptoms often experienced months to years before the onset of AIDS. Lack of energy Weight loss Frequent fevers and sweats Persistent or frequent yeast infections Persistent skin rashes Dysfunction of CNS
Progression to AIDS
Final stage of HIV infection - AIDS
Occurs when the destruction of peripheral lymphoid tissue is complete and the blood CD4+ T cell count drops below 200 cells/mm3. (Healthy adults usually have CD4+ T-cell counts of 1,000 or more).
AIDS – acquired immunodeficiency syndrome – is marked by development of various opportunistic infections and malignancies.
The level of virus in the blood and CD4+ T cell count can predict the risk of developing AIDS. Viral titres often accelerate as the patient progresses towards AIDS.
Without treatment, at least 50% of people infected with HIV will develop AIDS within ten years.
Opportunistic Infections
Opportunistic infections can be caused by bacteria/viruses/fungi/protozoa that are able to invade the body only when the immune system is weakened.
Common HIV-related opportunistic infections and diseases:
Bacterial diseases: tuberculosis, bacterial pneumonia, septicaemia
Protozoal diseases: PCP(pneumocystis carinii pneumonia), targets the lungs
Fungal diseases: candidiasis, cryptococcosis (most often appears as meningitis)
Opportunistic Infections
Loss of cellular immunity - associated with increased susceptibility to intracellular pathogens such as viruses.
Cytomegalovirus, herpes simplex
HIV-associated malignancies: Kaposi's sarcoma, lymphoma, squamous cell carcinoma and cervical cancer.
In people with AIDS, these infections are often severe and sometimes fatal.
Diagnosis of HIV
Initial test for HIV is an indirect ELISA test Economic, rapid, performed easily, high sensitivity and specificity Detects anti-HIV antibodies in patient serum Antibodies are generally detectable within 3 months of infection Antibodies are typically directed at the envelope glycoproteins (gp120 and gp41) Absence of antibody, as in ‘window period’ does not exclude the presence of the virus which can be detected by PCR amplification approx ten days after infection ‘Window period’ – time between infection and detection of serological viral marker Direct ELISA for p24 antigen can also be used although the false negative rate is higher
Diagnosis of HIV
Although very sensitive, ELISA may yield nonspecific reactions resulting in false positive results
Positive or indeterminate ELISA tests for anti-HIV antibodies are confirmed by immunoblotting (Western Blotting) which identifies specific HIV virus proteins
PCR can also be used
Detects pro-viral DNA or viral RNA
It is highly sensitive and specific but is more costly than ELISA
Can be used to test infants born to HIV-infected mothers
Principle of ELISA Test
Recombinant HIV antigens, corresponding to viral proteins of HIV-1 and HIV-2 are coated on a solid phase Patient serum is added and if any antibodies are present, an antibody-antigen complex is formed Unbound material is removed by washing The Ab-Ag complex is detected by adding an enzymeconjugated secondary antibody that binds to the primary antibody. Washing step A substrate for the enzyme is added Coloured reaction product formed and absorbance is measured Intensity of the colour of the solution is proportional to the amount of antibody present
Indirect ELISA test
Western Blotting
Confirms HIV infection Proteins are separated by electrophoresis and transferred to a nitrocellulose membrane by the passage of an electric current The proteins are treated with antibodies Similar to ELISA technique, addition of secondary antibodies with an enzyme attached allows the use of colour to detect a particular protein A discrete protein band represents the specific antigen that the antibody recognizes The bands from a positive Western blot are from antibodies binding to specific proteins and glycoproteins from the HIV virus The CDC recommends that the blot should be positive for two of the p24, gp41 and gp120/160 markers (gp160 is the precursor form of gp41 and gp120, the envelope protein)
Western blot
HIV Western blot
Treatment of HIV
Eradication of HIV infection not possible with currently available drugs Viral replication can not be completely suppressed Latently infected CD4+ T cells established at early stage Goals of antiretroviral therapy are to: - Suppress viral replication - Restore and/or preserve immune function - Improve quality of life - Reduce HIV-associated morbidity and mortality Combinations of antiretroviral drugs are used Referred to as HAART (highly active antiretroviral therapy) Suppress levels of plasma viraemia for long periods Plasma viraemia is a strong prognostic factor in HIV infection
Antiretroviral Drugs Significant declines in AIDS related morbidity and mortality are seen as a result of HAART Several strategies for development of effective antiviral drugs Potential therapies based on knowledge of the way in which HIV gains access into the cells and its method of replication Targets for therapeutic anti-retroviral drugs: - Inhibiting reverse transcription - Inhibiting proteases - Inhibiting integrase – interferes with integration of viral DNA into host genome - Inhibiting fusion – prevents virus from fusing with host cell
Inhibition of Viral Replication
Inhibition of activity of reverse transcriptase (RT), an enzyme responsible for viral RNA being reverse transcribed to cDNA Example: Zidovudine or AZT (azidothymidine) – first type of agent to be developed for treatment of HIV infection AZT is an analogue of thymidine that competes with natural nucleotides at the RT active site for incorporation into DNA Introduction of AZT into a growing cDNA chain of the retrovirus causes termination of the chain, yielding inactive proviral DNA A different approach - drugs such as nevirapine and delaviridine (non-nucleoside analogues) which inhibit the action of the reverse transcriptase enzyme
Action of AZT
Disadvantages:
The administered AZT is used not only by the HIV-1 RT but also by human DNA polymerase Incorporation of AZT into the DNA of the host cells kills them Precursors of red blood cells especially sensitive to AZT, results in anaemia and other side effects RT inhibitors have several disadvantages: Toxic Select for resistant viral variants quickly when used alone Lack of effect of cells already effected with HIV, which no longer require RT to complete the viral replication cycle
Protease Inhibitors
Prevent the assembly of new infectious viruses
Inhibits activity of viral protease enzyme necessary to cleave viral precursor proteins (gag proteins) into the p24 and p17 virion components, required for viral activity
Disadvantages:
Resistance appears after only a few days, single mutation required In contrast, resistance to AZT takes months to develop as it requires three or four mutations in the viral reverse transcriptase
Therapeutic Options
Combination of RT inhibitors protease inhibitors results in potent anti-viral activity
In most cases, two nucleoside analogues and one protease inhibitor are taken together
HAART lowers plasma viral loads in many cases to levels not detectable by current methods
Has improved the health of AIDS patients to the point that they can function at a normal level
Effect of Treatment on Viral Load
By Alick Mwambungu Dublin Institute of Technology Republic of Ireland
Contents
Introduction – History and Overview of HIV
Pathogenesis – Structure and Lifecycle of HIV
Effect On The Immune System – Destruction of Cells and Resulting Immunodeficiencies
Diagnosis and Treatment
What is HIV?
Genus Retroviridae
Lentivirus, which literally means slow virus - it takes such a long time to develop adverse effects in the body.
This virus attacks the immune system
There are two strains – HIV 1 & HIV 2
It belongs to a group of retroviruses.
These contain RNA, the genetic material of HIV
The outer layer of the HIV virus cell is covered in coat proteins, which can bind to certain WBCs. This allows the virus to enter the cell, where it alters the DNA.
The virus infects and destroys the CD4 lymphocytes which are critical to the body’s immune response.
History of HIV
The HIV virus first came to light during the early 1980’s.
A number of healthy gay men in New York began to develop rare opportunistic infections & cancers, that were resistant to treatment.
One such viral opportunistic infection is cytomegalovirus that causes blindness & inflammation of the colon
Transmission HIV is transmitted from person to person in several ways:
Through unprotected sex with an infected person
Exposure to infected blood
By the use of contaminated equipment for injections (by drug users or medical treatment in developing countries)
From an infected mother to her baby before or during birth, or by breast feeding
HIV Origins
Research teams in the U.S.A & France made independent research discoveries of the virus.
French researchers discovered a virus linked to AIDS in 1983, they called it Lymphadenopathy-Associated Virus (LAV)
In 1984, American researchers isolated a virus that caused AIDS, calling it Human Tlymphotropic Virus type 111 (HTLV-111)
These two viruses were later found to be the same virus - HIV
HIV Origin
The emergence of HIV & AIDS has resulted in countless debates as to where it originated from
It’s suspected that it originated from S.I.V (Simian Immunodeficiency Virus)
SIV affects Monkeys
HIV Origins
Certain strains of SIV closely resemble the two types of HIV
HIV 1 – was difficult to link with SIV
In 1999 SIVcpz closely related to HIV 1
Originated from chimpanzees but it has significant differences from HIV-1
HIV 2 closely related to SIVsm
Originated from the green monkey
Conspiracy Theories
Two different SIV infections combined to form a new third virus, leading to zoonosis
Several theories regarding zoonosis
Hunter Colonialism – Transferred by hunting & killing chimpanzees - Colonial rule in Africa, forced labour - Eating SIV infected chimpanzees
Conspiracy Theories -
Oral Polio Vaccine Grown on kidney cells from chimpanzees Infected with SIVcm2 leading to HIV 1
Contaminated needles - Not sterilising needles between patients - Sharing of needles
Manmade
Linked to the CIA
Early known cases of HIV
Plasma sample taken from male adult in the Congo in 1959
A 1998 analysis of the plasma sample from 1959 has suggested that HIV-1 was introduced into humans around the 1940s or the early 1950s
Tissue sample taken from a teenager, St. Louis in 1969 – died
Tissue sample from Norwegian sailor- died 1976
Another sample was dated back to the end of the 19th Century
Incidence
~ 42 million people have been infected with HIV to date.
AIDS has caused millions of deaths
Only conditions typical of old age, such as heart disease & stroke cause more fatalities worldwide
In Africa, HIV has reached epidemic proportions, responsible for 1 in 5 deaths in 1998
In the UK 37,900 HIV + in 1999
Global Incidence
Global Incidence Trends
More than 25 million people have died of AIDS since 1981
Africa has 12 million AIDS orphans
At the end of 2006, women accounted for 48% of all adults living with HIV worldwide, and for 59% in subSaharan Africa
Young people (under 25 years old) account for half of all new HIV infections worldwide - around 6,000 become infected with HIV every day
In developing and transitional countries, 6.8 million people are in immediate need of life-saving AIDS drugs; of these, only 1.65 million are receiving the drugs
Global Incidence Trends
During 2006 around four million adults and children became infected with HIV (Human Immunodeficiency Virus), the virus that causes AIDS
By the end of the year, an estimated 39.5 million people worldwide were living with HIV/AIDS
The year also saw around three million deaths from AIDS, despite recent improvements in access to antiretroviral treatment.
World Statistical Regions of HIV
Structure of HIV
Genome and Proteins of HIV
• The genome of HIV is encoded on two identical strands of RNA when the virus is in the free form. • It has nine open reading frames (leading to nine primary translation products) but 15 proteins are actually made in all as a result of cleavage of three of the primary products. • The GAG gene and the GAG and POL genes together are translated into polyproteins which are then cleaved by a protease.
•
•
•
GAG polyprotein is cleaved to into four proteins: matrix protein (p17) which anchors the gp160 receptor capsid protein (p24) which forms a capsule around the RNA genome the nucleocapsid protein which binds to the HIV packaging signal on viral RNA p6 which plays a role in incorporating proteins into new virons. ENV gene is translated to gp160 which is then cleaved by a host cell protease found in the Golgi body to: Gp41 Gp120 Together these form the HIV receptor
• POL polyprotein is cleaved to: Protease, an enzyme required for maturation and functioning of other viral proteins Reverse transcriptase which is responsible for incorporation into the host genome. Integrase which cuts the host DNA and attaches the proviral genome to the cut ends. • Six other proteins are made by HIV • Three of these are incorporated into the virus: Tat and Rev are regulatory proteins Vpu (HIV-1) indirectly assists in assembly • The others are not found in the mature virus:
The HIV receptor
•
Gp160 is composed of gp41 and gp120 and forms the receptor for binding to the host cell (CD4 positive cells).
•
The gp41 portion is half embedded in the membrane envelope and interacts with gp120 portion on the exterior side of the membrane.
•
Each receptor is composed of 3 subunits of gp41 and 3 subunits of gp120.
The HIV Receptor
Lifecycle of HIV
HIV particles enter the body in a fluid as it can not survive without a support medium.
The virus targets any cell expressing CD4, including T helper cells, macrophages, dendritic cells and monocytes.
• Receptor Interactions •
The first stage of infection involves the binding of the gp120 portion of the receptor on the HIV to the CD4 receptor on the host cell.
•
This binding also requires the interaction of the virus with a chemokine co-receptor
•
Of the chemokine receptor family the two most important co-receptors for HIV are CCR5 on macrophages and CXCR4 on T-cells.
•
Attachment of HIV to a co-receptor first allows for a more stable and intimate association with the CD4 receptor.
•
This interaction with the CD4 receptor causes a conformational change in the gp120 portion, which in turn causes a change in the gp41 portion, initiating the membrane fusion step of the infection.
2. Cell Membrane Fusion and Integration •
Virus particles enter the CD4 positive host cell.
•
HIV is transported in as a pre-integration complex and p17 carries nuclear localisation signals which are a sequence of proteins recognised by the host cell as belonging in the nucleus
•
Intergrase protein cleaves the host genome and reverse transcriptase reads the sequence of viral RNA and transcribes it into a complementary DNA sequence of the host.
•
HIV is now a genetic disease, once the viral genome is integrated it becomes a permanent component of the host cell.
3. Viral Replication •
As with all viruses HIV is an obligate intracellular parasite – it can only replicate when incorporated with the host’s genome.
•
Once integrated the virus can remain dormant in the host cells, or can begin the production of new RNA.
•
Non-genomic RNA is translated into large polyproteins. This is then cleaved into structural proteins which are assembled around genomic RNA.
•
Protease completes cleavage of polypeptides into fully functional proteins resulting in a multiple mature viral particles being released from the cell.
•
This causes cell lysis which is one of the most destructive mechanisms in the depletion of CD4 T-cell populations.
Mature Viral Particles Escaping From A T-Helper Cell
Infection of the Lymphocytes • HIV can exist in two forms, the M-trophic strain which is associated with infection of macrophages and the T-trophic which infects T-helper cells. •
Due to the high error frequency of HIV polymerase one strain can readily mutate with the other strain.
• The M-trophic strain has an important role in the initial infection as the primary site of infection for HIV is the macrophage. • The infected macrophage then acts as a “trojanhorse” in bringing the virus to the lymphatic system since it’s job is antigen presentation.
•
After infection of the macrophage the M-trophic genome undergoes mutation and T-trophic strains are produced.
•
The generation of the T-trophic strain results in the infection of the T-helper cell populations within the lymph nodes and lymphatic tissue.
•
T-cells are attracted to the lymph nodes and lymphatic system by two chemokines produce by the infected macrophages - MIP-1alpha and MIP-1beta
•
Migration and proliferation of T-helper cells in response to the infection means more and more cells will be continuously infected.
•
HIV is also thought to have the ability to infect Tmemory cells. This is an important factor in persistence of the disease in the lymphatic system over decades. Eventually there is loss of lymph node architecture.
Effects of HIV on the immune system 3 areas: 1. Destruction of CD4+ T cells population 2. Immune effects due to HIV infection 3. Progression of HIV infection to AIDS
Destruction of CD4+ T cells
Direct virological mechanisms Host’s immune responses
1.Direct virological mechanisms
HIV destroys individual infected cells through cell lysis or the formation of syncytia when large numbers of uninfected cells fuse with the infected cell.
This results in the deaths of potentially hundreds of uninfected cells.
The half-life of an actively infected CD4+ T cell is less than 1.5 days.
2.Host’s immune responses
Both humoral and cell-mediated immune responses partially control the viral production but in this process they destroy the infected CD4+T cells, leading to a gradual decline of CD4+ T cells
HIV-specific CTLs kill infected CD4+ T cells
Antibodies that recognize a variety of HIV antigens are produced - Antibody dependent cell-mediated cytotoxicity
Apoptosis of infected cells
Immune responses fail to eradicate all viruses. Viral load is maintained at low level Continuous decline of CD4+ T cells
Role of CD4+ T cells
Role: secrete cytokines that enable activation of B cells, Tc cells and macrophages
CD4+ T cells - TH1 cells and TH2 cells
TH1 cells – secrete IFN-γ => phagocyte-mediated immunity.
TH2 cells – secrete IL-4 => Ig synthesis.
Some studies demonstrate that the proportion of TH1 T cells decreases in HIV-infected patients but TH2 T cells increases. - disrupt the normal balance of cytokines.
Immune defects due to HIV infection Almost all aspects of immunity are affected as the disease progresses. Impaired cell mediated responses CD4+ T cell Rapid loss of memory helper T cells and the inability to replace these cells leads to increasing immunodeficiency.
CD4+ T cells that have bound gp120 may not be available to interact with class II MHC molecules on APC => T cell responses are inhibited.
Autoimmune response can occur as the HIV receptor shares homology with the MHC II molecules.
HIV becomes a component of the CD4 T-cell genome and as a result everytime the cell is activated there is further viral replication. Cytokines produced by innate immunity activate infected CD4+ T cells.
Immune defects due to HIV infection CD8+ T cell
Full differentiation of CD8+CTLs requires cytokines that are produced by CD4+ T cells Progressive decline of CD4+ T cells over time, is associated with decreased activation and survival of CD8+ cytotoxic T cells resulting in a decreased ability to destroy virally infected cells. HIV Nef protein inhibits expression of Class I MHC molecules. High mutation rates of HIV also allow the virus to escape adaptive immune responses.
Immune defects due to HIV infection B cells – impaired humoral response B-cell hyperreactivity Polyclonal hypergammaglobulinemia due to enhanced nonspecific IgG and IgA production. Impaired Ab-isotype switching and inability to respond to specific antigen. High incidence of B-cell lymphomas
Lymph nodes HIV kills cells in the lymph nodes Early HIV infection: destruction of dendritic cells Late stage: extensive damage, tissue necrosis, a loss of follicular dendritic cells and germinal centres. An inability to trap Ag or support activation of T+B cells
Immune defects due to HIV infection
Impaired Innate Immunity Macrophages
macrophage is infected directly by HIV or phagocytosis of other infected cells or Fc receptor-mediated endocytosis.
•Act as a reservoir for the virus • Normal functions are impaired which include •Phagocytosis + killing of organisms •Ag presentation •Chemotaxis •Cytokine secretion exhibit defective phagocytic function and impaired chemotaxis due to imbalance of cytokines production. Neutrophils -
Immunodeficiency
Immunodeficiency - due to defects in one or more components of the immune system
Main causes for immunodeficiency in HIV infection - destruction of lymphoid tissue and depletion of CD4+ T cells.
The CD4+ T cell count gives an indication of the level of damage that has already occurred to the immune system as a result of HIV infection.
Progression of HIV infection
After initial infection with HIV, there is usually an acute flu-like illness. This illness may include Fever Headache Tiredness Enlarged lymph nodes But after this most individuals are clinically asymptomatic for years. This is called the clinical latency period.
Progression of HIV infection Exposure to HIV normal Acute HIV disease
Immune competence
Slightly reduced
Abnormal
Clinical latency period -declining CD4+ T cell amount
AIDS
Opportunistic infections
Severely impaired
Time
Progression to AIDS
During the latency period, lymph nodes and the spleen are sites of continuous HIV replication and cell destruction.
The immune system remains competent at handling most infections with opportunistic microbes but the number of CD4+ T cells steadily declines.
Symptoms often experienced months to years before the onset of AIDS. Lack of energy Weight loss Frequent fevers and sweats Persistent or frequent yeast infections Persistent skin rashes Dysfunction of CNS
Progression to AIDS
Final stage of HIV infection - AIDS
Occurs when the destruction of peripheral lymphoid tissue is complete and the blood CD4+ T cell count drops below 200 cells/mm3. (Healthy adults usually have CD4+ T-cell counts of 1,000 or more).
AIDS – acquired immunodeficiency syndrome – is marked by development of various opportunistic infections and malignancies.
The level of virus in the blood and CD4+ T cell count can predict the risk of developing AIDS. Viral titres often accelerate as the patient progresses towards AIDS.
Without treatment, at least 50% of people infected with HIV will develop AIDS within ten years.
Opportunistic Infections
Opportunistic infections can be caused by bacteria/viruses/fungi/protozoa that are able to invade the body only when the immune system is weakened.
Common HIV-related opportunistic infections and diseases:
Bacterial diseases: tuberculosis, bacterial pneumonia, septicaemia
Protozoal diseases: PCP(pneumocystis carinii pneumonia), targets the lungs
Fungal diseases: candidiasis, cryptococcosis (most often appears as meningitis)
Opportunistic Infections
Loss of cellular immunity - associated with increased susceptibility to intracellular pathogens such as viruses.
Cytomegalovirus, herpes simplex
HIV-associated malignancies: Kaposi's sarcoma, lymphoma, squamous cell carcinoma and cervical cancer.
In people with AIDS, these infections are often severe and sometimes fatal.
Diagnosis of HIV
Initial test for HIV is an indirect ELISA test Economic, rapid, performed easily, high sensitivity and specificity Detects anti-HIV antibodies in patient serum Antibodies are generally detectable within 3 months of infection Antibodies are typically directed at the envelope glycoproteins (gp120 and gp41) Absence of antibody, as in ‘window period’ does not exclude the presence of the virus which can be detected by PCR amplification approx ten days after infection ‘Window period’ – time between infection and detection of serological viral marker Direct ELISA for p24 antigen can also be used although the false negative rate is higher
Diagnosis of HIV
Although very sensitive, ELISA may yield nonspecific reactions resulting in false positive results
Positive or indeterminate ELISA tests for anti-HIV antibodies are confirmed by immunoblotting (Western Blotting) which identifies specific HIV virus proteins
PCR can also be used
Detects pro-viral DNA or viral RNA
It is highly sensitive and specific but is more costly than ELISA
Can be used to test infants born to HIV-infected mothers
Principle of ELISA Test
Recombinant HIV antigens, corresponding to viral proteins of HIV-1 and HIV-2 are coated on a solid phase Patient serum is added and if any antibodies are present, an antibody-antigen complex is formed Unbound material is removed by washing The Ab-Ag complex is detected by adding an enzymeconjugated secondary antibody that binds to the primary antibody. Washing step A substrate for the enzyme is added Coloured reaction product formed and absorbance is measured Intensity of the colour of the solution is proportional to the amount of antibody present
Indirect ELISA test
Western Blotting
Confirms HIV infection Proteins are separated by electrophoresis and transferred to a nitrocellulose membrane by the passage of an electric current The proteins are treated with antibodies Similar to ELISA technique, addition of secondary antibodies with an enzyme attached allows the use of colour to detect a particular protein A discrete protein band represents the specific antigen that the antibody recognizes The bands from a positive Western blot are from antibodies binding to specific proteins and glycoproteins from the HIV virus The CDC recommends that the blot should be positive for two of the p24, gp41 and gp120/160 markers (gp160 is the precursor form of gp41 and gp120, the envelope protein)
Western blot
HIV Western blot
Treatment of HIV
Eradication of HIV infection not possible with currently available drugs Viral replication can not be completely suppressed Latently infected CD4+ T cells established at early stage Goals of antiretroviral therapy are to: - Suppress viral replication - Restore and/or preserve immune function - Improve quality of life - Reduce HIV-associated morbidity and mortality Combinations of antiretroviral drugs are used Referred to as HAART (highly active antiretroviral therapy) Suppress levels of plasma viraemia for long periods Plasma viraemia is a strong prognostic factor in HIV infection
Antiretroviral Drugs Significant declines in AIDS related morbidity and mortality are seen as a result of HAART Several strategies for development of effective antiviral drugs Potential therapies based on knowledge of the way in which HIV gains access into the cells and its method of replication Targets for therapeutic anti-retroviral drugs: - Inhibiting reverse transcription - Inhibiting proteases - Inhibiting integrase – interferes with integration of viral DNA into host genome - Inhibiting fusion – prevents virus from fusing with host cell
Inhibition of Viral Replication
Inhibition of activity of reverse transcriptase (RT), an enzyme responsible for viral RNA being reverse transcribed to cDNA Example: Zidovudine or AZT (azidothymidine) – first type of agent to be developed for treatment of HIV infection AZT is an analogue of thymidine that competes with natural nucleotides at the RT active site for incorporation into DNA Introduction of AZT into a growing cDNA chain of the retrovirus causes termination of the chain, yielding inactive proviral DNA A different approach - drugs such as nevirapine and delaviridine (non-nucleoside analogues) which inhibit the action of the reverse transcriptase enzyme
Action of AZT
Disadvantages:
The administered AZT is used not only by the HIV-1 RT but also by human DNA polymerase Incorporation of AZT into the DNA of the host cells kills them Precursors of red blood cells especially sensitive to AZT, results in anaemia and other side effects RT inhibitors have several disadvantages: Toxic Select for resistant viral variants quickly when used alone Lack of effect of cells already effected with HIV, which no longer require RT to complete the viral replication cycle
Protease Inhibitors
Prevent the assembly of new infectious viruses
Inhibits activity of viral protease enzyme necessary to cleave viral precursor proteins (gag proteins) into the p24 and p17 virion components, required for viral activity
Disadvantages:
Resistance appears after only a few days, single mutation required In contrast, resistance to AZT takes months to develop as it requires three or four mutations in the viral reverse transcriptase
Therapeutic Options
Combination of RT inhibitors protease inhibitors results in potent anti-viral activity
In most cases, two nucleoside analogues and one protease inhibitor are taken together
HAART lowers plasma viral loads in many cases to levels not detectable by current methods
Has improved the health of AIDS patients to the point that they can function at a normal level
Effect of Treatment on Viral Load
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