HIV HIV

By Alick MwambunguBy Alick Mwambungu

Dublin Institute of TechnologyDublin Institute of Technology

Republic of IrelandRepublic of Ireland

ContentsContents

Introduction – History and Overview of HIVIntroduction – History and Overview of HIV

Pathogenesis – Structure and Lifecycle of HIVPathogenesis – Structure and Lifecycle of HIV

Effect On The Immune System – Destruction of Effect On The Immune System – Destruction of Cells and Resulting ImmunodeficienciesCells and Resulting Immunodeficiencies

Diagnosis and TreatmentDiagnosis and Treatment

What is HIV?What is HIV?

Genus RetroviridaeGenus Retroviridae

Lentivirus, which literally means slow virus Lentivirus, which literally means slow virus - it takes such a long time to develop - it takes such a long time to develop adverse effects in the body. adverse effects in the body.

This virus attacks the immune systemThis virus attacks the immune system

There are two strains – HIV 1 & HIV 2There are two strains – HIV 1 & HIV 2

It belongs to a group of retroviruses.It belongs to a group of retroviruses.

These contain RNA, the genetic material of These contain RNA, the genetic material of HIVHIV

The outer layer of the HIV virus cell is The outer layer of the HIV virus cell is covered in coat proteins, which can bind to covered in coat proteins, which can bind to certain WBCs. This allows the virus to certain WBCs. This allows the virus to enter the cell, where it alters the DNA.enter the cell, where it alters the DNA.

The virus infects and destroys the CD4 The virus infects and destroys the CD4 lymphocytes which are critical to the lymphocytes which are critical to the body’s immune response.body’s immune response.

History of HIVHistory of HIV

The HIV virus first came to light during the The HIV virus first came to light during the early 1980’s. early 1980’s.

A number of healthy gay men in New York A number of healthy gay men in New York began to develop rare opportunistic began to develop rare opportunistic infections & cancers, that were resistant to infections & cancers, that were resistant to treatment.treatment.

One such viral opportunistic infection is One such viral opportunistic infection is cytomegalovirus that causes blindness & cytomegalovirus that causes blindness & inflammation of the coloninflammation of the colon

TransmissionTransmission

HIV is transmitted from person to person in several HIV is transmitted from person to person in several ways:ways:

Through unprotected sex with an infected personThrough unprotected sex with an infected person

Exposure to infected bloodExposure to infected blood

By the use of contaminated equipment for By the use of contaminated equipment for injections (by drug users or medical treatment in injections (by drug users or medical treatment in developing countries)developing countries)

From an infected mother to her baby before or From an infected mother to her baby before or during birth, or by breast feedingduring birth, or by breast feeding

HIV OriginsHIV Origins Research teams in the U.S.A & France Research teams in the U.S.A & France

made independent research discoveries of made independent research discoveries of the virus.the virus.

French researchers discovered a virus French researchers discovered a virus linked to AIDS in 1983, they called it linked to AIDS in 1983, they called it Lymphadenopathy-Associated Virus (LAV)Lymphadenopathy-Associated Virus (LAV)

In 1984, American researchers isolated a In 1984, American researchers isolated a virus that caused AIDS, calling it Human T-virus that caused AIDS, calling it Human T-lymphotropic Virus type 111 (HTLV-111)lymphotropic Virus type 111 (HTLV-111)

These two viruses were later found to be These two viruses were later found to be the same virus - HIVthe same virus - HIV

HIV OriginHIV Origin

The emergence of HIV & AIDS has resulted The emergence of HIV & AIDS has resulted in countless debates as to where it in countless debates as to where it originated fromoriginated from

It’s suspected that it originated from S.I.V It’s suspected that it originated from S.I.V (Simian Immunodeficiency Virus)(Simian Immunodeficiency Virus)

SIV affects MonkeysSIV affects Monkeys

HIV OriginsHIV Origins

Certain strains of SIV closely resemble the two Certain strains of SIV closely resemble the two types of HIVtypes of HIV

HIV 1 – was difficult to link with SIVHIV 1 – was difficult to link with SIV

In 1999 SIVcpz closely related to HIV 1In 1999 SIVcpz closely related to HIV 1

Originated from chimpanzees but it has Originated from chimpanzees but it has significant differences from HIV-1significant differences from HIV-1

HIV 2 closely related to SIVsmHIV 2 closely related to SIVsm

Originated from the green monkeyOriginated from the green monkey

Conspiracy TheoriesConspiracy Theories

Two different SIV infections combined to Two different SIV infections combined to form a new third virus, leading to zoonosisform a new third virus, leading to zoonosis

Several theories regarding zoonosisSeveral theories regarding zoonosis

Hunter ColonialismHunter Colonialism – – Transferred by hunting & killing Transferred by hunting & killing

chimpanzeeschimpanzees- Colonial rule in Africa, forced labourColonial rule in Africa, forced labour- Eating SIV infected chimpanzeesEating SIV infected chimpanzees

Conspiracy TheoriesConspiracy Theories

Oral Polio VaccineOral Polio Vaccine- Grown on kidney cells from chimpanzeesGrown on kidney cells from chimpanzees- Infected with SIVcm2 leading to HIV 1Infected with SIVcm2 leading to HIV 1

Contaminated needlesContaminated needles- Not sterilising needles between patients- Not sterilising needles between patients- Sharing of needles- Sharing of needles

ManmadeManmade

Linked to the CIALinked to the CIA

Early known cases of HIVEarly known cases of HIV

Plasma sample taken from male adult in the Plasma sample taken from male adult in the Congo in 1959 Congo in 1959

A 1998 analysis of the plasma sample from 1959 A 1998 analysis of the plasma sample from 1959 has suggested that HIV-1 was introduced into has suggested that HIV-1 was introduced into humans around the 1940s or the early 1950s humans around the 1940s or the early 1950s

Tissue sample taken from a teenager, St. Louis in Tissue sample taken from a teenager, St. Louis in 1969 – died1969 – died

Tissue sample from Norwegian sailor- died 1976Tissue sample from Norwegian sailor- died 1976

Another sample was dated back to the end of the Another sample was dated back to the end of the 1919thth Century Century

IncidenceIncidence ~ 42 million people have been infected ~ 42 million people have been infected

with HIV to date.with HIV to date.

AIDS has caused millions of deathsAIDS has caused millions of deaths

Only conditions typical of old age, such as Only conditions typical of old age, such as heart disease & stroke cause more heart disease & stroke cause more fatalities worldwidefatalities worldwide

In Africa, HIV has reached epidemic In Africa, HIV has reached epidemic proportions, responsible for 1 in 5 deaths proportions, responsible for 1 in 5 deaths in 1998in 1998

In the UK 37,900 HIV + in 1999In the UK 37,900 HIV + in 1999

Global IncidenceGlobal Incidence

Global Incidence TrendsGlobal Incidence Trends More than 25 million people have died of AIDS More than 25 million people have died of AIDS

since 1981since 1981

Africa has 12 million AIDS orphansAfrica has 12 million AIDS orphans

At the end of 2006, women accounted for 48% of At the end of 2006, women accounted for 48% of all adults living with HIV worldwide, and for 59% all adults living with HIV worldwide, and for 59% in sub-Saharan Africain sub-Saharan Africa

Young people (under 25 years old) account for Young people (under 25 years old) account for half of all new HIV infections worldwide - around half of all new HIV infections worldwide - around 6,000 become infected with HIV every day6,000 become infected with HIV every day

In developing and transitional countries, 6.8 In developing and transitional countries, 6.8 million people are in immediate need of life-million people are in immediate need of life-saving AIDS drugs; of these, only 1.65 million are saving AIDS drugs; of these, only 1.65 million are receiving the drugsreceiving the drugs

Global Incidence TrendsGlobal Incidence Trends

During 2006 around four million adults and During 2006 around four million adults and children became infected with HIV (Human children became infected with HIV (Human Immunodeficiency Virus), the virus that causes Immunodeficiency Virus), the virus that causes AIDSAIDS

By the end of the year, an estimated 39.5 By the end of the year, an estimated 39.5 million people worldwide were living with million people worldwide were living with HIV/AIDSHIV/AIDS

The year also saw around three million deaths The year also saw around three million deaths from AIDS, despite recent improvements in from AIDS, despite recent improvements in access to antiretroviral treatment.access to antiretroviral treatment.

World Statistical Regions of HIVWorld Statistical Regions of HIV

Structure of HIVStructure of HIV

Genome and Proteins of HIVGenome 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 GAG polyprotein is cleaved to into four proteins: proteins: 

matrix protein (p17) which anchors the gp160 matrix protein (p17) which anchors the gp160 receptorreceptor

capsid protein (p24) which forms a capsule capsid protein (p24) which forms a capsule around the RNA genomearound the RNA genome

the nucleocapsid protein which binds to the the nucleocapsid protein which binds to the HIV packaging signal on viral RNA HIV packaging signal on viral RNA

p6 which plays a role in incorporating proteins p6 which plays a role in incorporating proteins into new virons. into new virons.

• ENVENV gene is translated to gp160 which is then gene is translated to gp160 which is then

cleaved by a host cell protease found in the cleaved by a host cell protease found in the Golgi body to: Golgi body to:

Gp41 Gp41 Gp120 Gp120 • Together these form the HIV receptor 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 proteinsVpu (HIV-1) indirectly assists in assembly

• The others are not found in the mature virus:

The HIV receptor

• Gp160Gp160 is composed of is composed of gp41gp41 and and gp120gp120 and and forms the receptor for binding to the host cell forms the receptor for binding to the host cell (CD4 positive cells). (CD4 positive cells).

• The gp41 portion is half embedded in the The gp41 portion is half embedded in the membrane envelope and interacts with membrane envelope and interacts with gp120 portion on the exterior side of the gp120 portion on the exterior side of the membrane.membrane.

• Each receptor is composed of 3 subunits of Each receptor is composed of 3 subunits of gp41 and 3 subunits of gp120.gp41 and 3 subunits of gp120.

The HIV Receptor

Lifecycle of HIV

HIV particles enter the body in a fluid as it can not HIV particles enter the body in a fluid as it can not survive without a support medium.survive without a support medium.

The virus targets any cell expressing CD4, The virus targets any cell expressing CD4, including T helper cells, macrophages, dendritic including T helper cells, macrophages, dendritic cells and monocytes.cells and monocytes.

• The first stage of infection involves the binding of The first stage of infection involves the binding of the gp120 portion of the receptor on the HIV to the the gp120 portion of the receptor on the HIV to the CD4 receptor on the host cell.CD4 receptor on the host cell.

• This binding also requires the interaction of the This binding also requires the interaction of the virus with a chemokine co-receptorvirus with a chemokine co-receptor

• Of the chemokine receptor family the two most Of the chemokine receptor family the two most important co-receptors for HIV are CCR5 on important co-receptors for HIV are CCR5 on macrophages and CXCR4 on T-cells. macrophages and CXCR4 on T-cells.

• Attachment of HIV to a co-receptor first allows for a Attachment of HIV to a co-receptor first allows for a more stable and intimate association with the CD4 more stable and intimate association with the CD4 receptor. receptor.

• This interaction with the CD4 receptor causes a This interaction with the CD4 receptor causes a conformational change in the gp120 portion, which conformational change in the gp120 portion, which in turn causes a change in the gp41 portion, in turn causes a change in the gp41 portion, initiating the membrane fusion step of the infection.initiating the membrane fusion step of the infection.

1.Receptor Interactions

2. Cell Membrane Fusion and Integration

• Virus particles enter the CD4 positive host cell. Virus particles enter the CD4 positive host cell.

• HIV is transported in as a pre-integration complex HIV is transported in as a pre-integration complex and p17 carries nuclear localisation signals which and p17 carries nuclear localisation signals which are a sequence of proteins recognised by the host are a sequence of proteins recognised by the host cell as belonging in the nucleus cell as belonging in the nucleus

• Intergrase protein cleaves the host genome and Intergrase protein cleaves the host genome and reverse transcriptase reads the sequence of viral reverse transcriptase reads the sequence of viral RNA and transcribes it into a complementary DNA RNA and transcribes it into a complementary DNA sequence of the host. sequence of the host.

• HIV is now a genetic disease, once the viral genome HIV is now a genetic disease, once the viral genome is integrated it becomes a permanent component of is integrated it becomes a permanent component of the host cell.the host cell.

3. Viral Replication3. Viral Replication• As with all viruses HIV is an obligate intracellular As with all viruses HIV is an obligate intracellular

parasite – it can only replicate when incorporated parasite – it can only replicate when incorporated with the host’s genome. with the host’s genome.

• Once integrated the virus can remain dormant in the Once integrated the virus can remain dormant in the host cells, or can begin the production of new RNA.host cells, or can begin the production of new RNA.

• Non-genomic RNA is translated into large Non-genomic RNA is translated into large polyproteins. This is then cleaved into structural polyproteins. This is then cleaved into structural proteins which are assembled around genomic RNA. proteins which are assembled around genomic RNA.

• Protease completes cleavage of polypeptides into Protease completes cleavage of polypeptides into fully functional proteins resulting in a multiple fully functional proteins resulting in a multiple mature viral particles being released from the cell.mature viral particles being released from the cell.

• This causes cell lysis which is one of the most This causes cell lysis which is one of the most destructive mechanisms in the depletion of CD4 T-destructive mechanisms in the depletion of CD4 T-cell populations. cell populations.

Mature Viral Particles Escaping From A T-Helper Mature Viral Particles Escaping From A T-Helper CellCell

• 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 “trojan-horse” in bringing the virus to the lymphatic system since it’s job is antigen presentation.

Infection of the Lymphocytes

• After infection of the macrophage the M-trophic After infection of the macrophage the M-trophic genome undergoes mutation and T-trophic strains genome undergoes mutation and T-trophic strains are produced. are produced.

• The generation of the T-trophic strain results in the The generation of the T-trophic strain results in the

infection of the T-helper cell populations within the infection of the T-helper cell populations within the lymph nodes and lymphatic tissue. lymph nodes and lymphatic tissue.

• T-cells are attracted to the lymph nodes and T-cells are attracted to the lymph nodes and lymphatic system by two chemokines produce by the lymphatic system by two chemokines produce by the infected macrophages - MIP-1alpha and MIP-1beta infected macrophages - MIP-1alpha and MIP-1beta

• Migration and proliferation of T-helper cells in Migration and proliferation of T-helper cells in response to the infection means more and more cells response to the infection means more and more cells will be continuously infected.will be continuously infected.

• HIV is also thought to have the ability to infect T-HIV is also thought to have the ability to infect T-memory cells. This is an important factor in memory cells. This is an important factor in persistence of the disease in the lymphatic system persistence of the disease in the lymphatic system over decades. Eventually there is loss of lymph node over decades. Eventually there is loss of lymph node architecture.architecture.

Effects of HIV on the immune systemEffects of HIV on the immune system

3 areas:3 areas:

1. Destruction of CD4+ T cells population1. Destruction of CD4+ T cells population

2. Immune effects due to HIV infection2. Immune effects due to HIV infection

3. Progression of HIV infection to AIDS3. Progression of HIV infection to AIDS

Destruction of CD4+ T cellsDestruction of CD4+ T cells Direct virological mechanismsDirect virological mechanisms Host’s immune responsesHost’s immune responses

1.Direct virological mechanisms1.Direct virological mechanisms

HIV destroys individual infected cells through cell HIV destroys individual infected cells through cell lysis or the formation of syncytia when large numbers lysis or the formation of syncytia when large numbers of uninfected cells fuse with the infected cell. of uninfected cells fuse with the infected cell.

This results in the deaths of potentially hundreds of This results in the deaths of potentially hundreds of uninfected cells.uninfected cells.

The half-life of an actively infected CD4+ T cell is less The half-life of an actively infected CD4+ T cell is less than 1.5 days.than 1.5 days.

2.Host’s immune responses2.Host’s immune responses

Both humoral and cell-mediated immune Both humoral and cell-mediated immune responses partially control the viral production responses partially control the viral production but in this process they destroy the infected but in this process they destroy the infected CD4+T cells, leading to a gradual decline of CD4+T cells, leading to a gradual decline of CD4+ T cellsCD4+ T cells

HIV-specific CTLs kill infected CD4+ T cellsHIV-specific CTLs kill infected CD4+ T cells

Antibodies that recognize a variety of HIV Antibodies that recognize a variety of HIV antigens are produced - Antibody dependent cell-antigens are produced - Antibody dependent cell-mediated cytotoxicitymediated cytotoxicity

Apoptosis of infected cellsApoptosis of infected cells

Immune responses fail to eradicate all viruses. Immune responses fail to eradicate all viruses. Viral load is maintained at low levelViral load is maintained at low level Continuous decline of CD4+ T cellsContinuous decline of CD4+ T cells

Role of CD4+ T Role of CD4+ T cellscells

Role: Role: secrete cytokines that secrete cytokines that enable activation of B cells, Tc enable activation of B cells, Tc cells and macrophagescells and macrophages

CD4+ T cells - TCD4+ T cells - THH1 cells and 1 cells and TTHH2 cells2 cells

TTHH1 cells – secrete IFN-1 cells – secrete IFN-γγ => => phagocyte-mediated phagocyte-mediated immunity.immunity.

TTHH2 cells – secrete IL-4 => Ig 2 cells – secrete IL-4 => Ig synthesis.synthesis.

Some studies demonstrate Some studies demonstrate that the proportion of Tthat the proportion of THH1 T 1 T cells decreases in HIV-infected cells decreases in HIV-infected patients but Tpatients but THH2 T cells 2 T cells increases. - increases. - disrupt the normal disrupt the normal balance of cytokines.balance of cytokines.

Almost all aspects of immunity are affected as the disease Almost all aspects of immunity are affected as the disease progresses.progresses.

Impaired cell mediated responsesImpaired cell mediated responsesCD4+ T cellCD4+ T cell Rapid loss of memory helper T cells and the inability to Rapid loss of memory helper T cells and the inability to

replace these cells leads to increasing immunodeficiency. replace these cells leads to increasing immunodeficiency.

CD4+ T cells that have bound gp120 may not be available CD4+ T cells that have bound gp120 may not be available to interact with class II MHC molecules on APC => T cell to interact with class II MHC molecules on APC => T cell responses are inhibited. responses are inhibited.

Autoimmune response can occur as the HIV receptor shares Autoimmune response can occur as the HIV receptor shares homology with the MHC II molecules.homology with the MHC II molecules.

HIV becomes a component of the CD4 T-cell genome and as HIV becomes a component of the CD4 T-cell genome and as a result everytime the cell is activated there is further viral a result everytime the cell is activated there is further viral replication.replication.

Cytokines produced by innate immunity activate infected Cytokines produced by innate immunity activate infected CD4+ T cells.CD4+ T cells.

Immune defects due to HIV Immune defects due to HIV infectioninfection

Immune defects due to HIV infectionImmune defects due to HIV infection CD8+ T cellCD8+ T cell

FFull differentiation of CD8+CTLs requires cytokines ull differentiation of CD8+CTLs requires cytokines that are produced by CD4+ T cellsthat are produced by CD4+ T cells

Progressive decline of CD4+ T cells over time, is Progressive decline of CD4+ T cells over time, is associated with decreased activation and survival of associated with decreased activation and survival of CD8+ cytotoxic T cells resulting in a decreased ability CD8+ cytotoxic T cells resulting in a decreased ability to destroy virally infected cells. to destroy virally infected cells.

HIV Nef protein inhibits expression of Class I MHC HIV Nef protein inhibits expression of Class I MHC molecules.molecules.

High mutation rates of HIV also allow the virus to High mutation rates of HIV also allow the virus to escape adaptive immune responses. escape adaptive immune responses.

Immune defects due to HIV Immune defects due to HIV

infectioninfection B cells – impaired humoral responseB cells – impaired humoral response

B-cell hyperreactivity B-cell hyperreactivity Polyclonal hypergammaglobulinemia due to enhanced Polyclonal hypergammaglobulinemia due to enhanced

nonspecific IgG and IgA production.nonspecific IgG and IgA production. Impaired Ab-isotype switching and inability to respond to Impaired Ab-isotype switching and inability to respond to

specific antigen.specific antigen. High incidence of B-cell lymphomas High incidence of B-cell lymphomas

Lymph nodesLymph nodes

HIV kills cells in the lymph nodesHIV kills cells in the lymph nodes Early HIV infection: destruction of dendritic cellsEarly HIV infection: destruction of dendritic cells Late stage: extensive damage, tissue necrosis, a loss of Late stage: extensive damage, tissue necrosis, a loss of

follicular dendritic cells and germinal centres.follicular dendritic cells and germinal centres. An inability to trap Ag or support activation of T+B cellsAn inability to trap Ag or support activation of T+B cells

Impaired Innate ImmunityImpaired Innate Immunity

MacrophagesMacrophages 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

Neutrophils Neutrophils - exhibit defective phagocytic function and impaired chemotaxis due to imbalance of cytokines production.

Immune defects due to HIV Immune defects due to HIV

infectioninfection

ImmunodeficiencyImmunodeficiency

Immunodeficiency - due to Immunodeficiency - due to defects in one or more defects in one or more components of the immune components of the immune systemsystem

Main causes for Main causes for immunodeficiency in HIV immunodeficiency in HIV infection - infection - destruction of destruction of lymphoid tissue and lymphoid tissue and depletion of CD4+ T depletion of CD4+ T cells.cells.

The CD4+ T cell count The CD4+ T cell count gives an indication of the gives an indication of the level of damage that has level of damage that has already occurred to the already occurred to the immune system as a result immune system as a result of HIV infection.of HIV infection.

normal

Severely impaired

Abnormal

Slightly reduced

Time

Acute HIV disease

Exposure to HIV

Clinical latency period-declining CD4+ T cell amount

AIDS

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Opportunistic infections

Progression of HIV infection

Progression of HIV infectionProgression of HIV infection

After initial infection with After initial infection with HIV, there is usually an HIV, there is usually an acute flu-like illness.acute flu-like illness.

This illness may includeThis illness may include Fever Fever Headache Headache Tiredness Tiredness Enlarged lymph nodesEnlarged lymph nodes

But after this most But after this most individuals are clinically individuals are clinically asymptomatic for years. asymptomatic for years. This is called the clinical This is called the clinical latency period.latency period.

Progression to AIDSProgression to AIDS During the latency period, lymph nodes and the spleen During the latency period, lymph nodes and the spleen

are sites of continuous HIV replication and cell are sites of continuous HIV replication and cell destruction. destruction.

The immune system remains competent at handling most The immune system remains competent at handling most infections with opportunistic microbes but the number of infections with opportunistic microbes but the number of CD4+ T cells steadily declines.CD4+ T cells steadily declines.

Symptoms often experienced months to years before the Symptoms often experienced months to years before the onset of AIDS.onset of AIDS.

Lack of energy Lack of energy Weight loss Weight loss Frequent fevers and sweats Frequent fevers and sweats Persistent or frequent yeast infections Persistent or frequent yeast infections Persistent skin rashes Persistent skin rashes Dysfunction of CNSDysfunction of CNS

Final Final stage of HIV infection - AIDS stage of HIV infection - AIDS

Occurs when the destruction of peripheral lymphoid tissue Occurs when the destruction of peripheral lymphoid tissue is complete and the blood CD4+ T cell count drops below is complete and the blood CD4+ T cell count drops below 200 cells/mm200 cells/mm33. (Healthy adults usually have CD4+ T-cell . (Healthy adults usually have CD4+ T-cell counts of 1,000 or more). counts of 1,000 or more).

AIDS – acquired immunodeficiency syndrome – is marked by AIDS – acquired immunodeficiency syndrome – is marked by development of various opportunistic infections and development of various opportunistic infections and malignancies.malignancies.

The level of virus in the blood and CD4+ T cell count can The level of virus in the blood and CD4+ T cell count can predict the risk of developing AIDS. Vpredict the risk of developing AIDS. Viral titres often iral titres often accelerate as the patient progresses towards AIDS.accelerate as the patient progresses towards AIDS.

Without treatment, at least 50% of people infected with HIV Without treatment, at least 50% of people infected with HIV will develop AIDS within ten years.will develop AIDS within ten years.

Progression to AIDSProgression to AIDS

Opportunistic InfectionsOpportunistic Infections

Opportunistic infections can be caused by Opportunistic infections can be caused by bacteria/viruses/fungi/protozoa that are able to invade the bacteria/viruses/fungi/protozoa that are able to invade the body only when the immune system is weakened. body only when the immune system is weakened. 

Common HIV-related opportunistic infections and diseases:Common HIV-related opportunistic infections and diseases:

Bacterial diseases: tuberculosis, bacterial pneumonia, Bacterial diseases: tuberculosis, bacterial pneumonia, septicaemia septicaemia

Protozoal diseases: PCP(pneumocystis carinii pneumonia), Protozoal diseases: PCP(pneumocystis carinii pneumonia), - - targets the lungs targets the lungs

Fungal diseases: candidiasis, cryptococcosis (most often Fungal diseases: candidiasis, cryptococcosis (most often appears as meningitis) appears as meningitis)

Loss of cellular immunity - associated with Loss of cellular immunity - associated with increased susceptibility to intracellular increased susceptibility to intracellular pathogens such as viruses.pathogens such as viruses.

Cytomegalovirus, herpes simplexCytomegalovirus, herpes simplex

HIV-associated malignancies: Kaposi's HIV-associated malignancies: Kaposi's sarcoma, lymphoma, squamous cell sarcoma, lymphoma, squamous cell carcinoma and cervical cancer.carcinoma and cervical cancer.

In people with AIDS, these infections are often In people with AIDS, these infections are often severe and sometimes fatal.severe and sometimes fatal.

Opportunistic InfectionsOpportunistic Infections

Diagnosis of HIVDiagnosis of HIV Initial test for HIV is an indirect ELISA test Initial test for HIV is an indirect ELISA test Economic, rapid, performed easily, high sensitivity and Economic, rapid, performed easily, high sensitivity and

specificityspecificity Detects anti-HIV antibodies in patient serumDetects anti-HIV antibodies in patient serum Antibodies are generally detectable within 3 months of Antibodies are generally detectable within 3 months of

infectioninfection Antibodies are typically directed at the envelope Antibodies are typically directed at the envelope

glycoproteins (gp120 and gp41)glycoproteins (gp120 and gp41) Absence of antibody, as in ‘window period’ does not Absence of antibody, as in ‘window period’ does not

exclude the presence of the virus which can be exclude the presence of the virus which can be detected by PCR amplification approx ten days after detected by PCR amplification approx ten days after infectioninfection

‘‘Window period’ – time between infection and Window period’ – time between infection and detection of serological viral markerdetection of serological viral marker

Direct ELISA for p24 antigen can also be used although Direct ELISA for p24 antigen can also be used although the false negative rate is higherthe false negative rate is higher

Diagnosis of HIVDiagnosis of HIV Although very sensitive, ELISA may yield non-Although very sensitive, ELISA may yield non-

specific reactions resulting in false positive specific reactions resulting in false positive resultsresults

Positive or indeterminate ELISA tests for anti-HIV Positive or indeterminate ELISA tests for anti-HIV antibodies are confirmed by immunoblotting antibodies are confirmed by immunoblotting (Western Blotting) which identifies specific HIV (Western Blotting) which identifies specific HIV virus proteinsvirus proteins

PCR can also be usedPCR can also be used

Detects pro-viral DNA or viral RNADetects pro-viral DNA or viral RNA

It is highly sensitive and specific but is more It is highly sensitive and specific but is more costly than ELISAcostly than ELISA

Can be used to test infants born to HIV-infected Can be used to test infants born to HIV-infected mothersmothers

Principle of ELISA TestPrinciple of ELISA Test Recombinant HIV antigens, corresponding to viral Recombinant HIV antigens, corresponding to viral

proteins of HIV-1 and HIV-2 are coated on a solid phaseproteins of HIV-1 and HIV-2 are coated on a solid phase Patient serum is added and if any antibodies are Patient serum is added and if any antibodies are

present, an antibody-antigen complex is formedpresent, an antibody-antigen complex is formed Unbound material is removed by washingUnbound material is removed by washing The Ab-Ag complex is detected by adding an enzyme-The Ab-Ag complex is detected by adding an enzyme-

conjugated secondary antibody that binds to the conjugated secondary antibody that binds to the primary antibody. primary antibody.

Washing stepWashing step A substrate for the enzyme is addedA substrate for the enzyme is added Coloured reaction product formed and absorbance is Coloured reaction product formed and absorbance is

measuredmeasured Intensity of the colour of the solution is proportional to Intensity of the colour of the solution is proportional to

the amount of antibody presentthe amount of antibody present

Indirect ELISA testIndirect ELISA test

Western BlottingWestern Blotting

Confirms HIV infectionConfirms HIV infection Proteins are separated by electrophoresis and Proteins are separated by electrophoresis and

transferred to a nitrocellulose membrane by the transferred to a nitrocellulose membrane by the passage of an electric currentpassage of an electric current

The proteins are treated with antibodiesThe proteins are treated with antibodies Similar to ELISA technique, addition of secondary Similar to ELISA technique, addition of secondary

antibodies with an enzyme attached allows the use of antibodies with an enzyme attached allows the use of colour to detect a particular proteincolour to detect a particular protein

A discrete protein band represents the specific antigen A discrete protein band represents the specific antigen that the antibody recognizesthat the antibody recognizes

The bands from a positive Western blot are from The bands from a positive Western blot are from antibodies binding to specific proteins and glycoproteins antibodies binding to specific proteins and glycoproteins from the HIV virusfrom the HIV virus

The CDC recommends that the blot should be positive The CDC recommends that the blot should be positive for two of the p24, gp41 and gp120/160 markers for two of the p24, gp41 and gp120/160 markers (gp160 is the precursor form of gp41 and gp120, the (gp160 is the precursor form of gp41 and gp120, the envelope protein)envelope protein)

Western blotWestern blot

HIV Western blotHIV Western blot

Treatment of HIVTreatment of HIV Eradication of HIV infection not possible with currently Eradication of HIV infection not possible with currently

available drugsavailable drugs Viral replication can not be completely suppressedViral replication can not be completely suppressed Latently infected CD4+ T cells established at early Latently infected CD4+ T cells established at early

stagestage Goals of antiretroviral therapy are to:Goals of antiretroviral therapy are to:

- Suppress viral replication- Suppress viral replication- Restore and/or preserve immune function- Restore and/or preserve immune function- Improve quality of life - Improve quality of life - Reduce HIV-associated morbidity and mortality- Reduce HIV-associated morbidity and mortality

Combinations of antiretroviral drugs are usedCombinations of antiretroviral drugs are used Referred to as HAART (highly active antiretroviral Referred to as HAART (highly active antiretroviral

therapy)therapy) Suppress levels of plasma viraemia for long periodsSuppress levels of plasma viraemia for long periods Plasma viraemia is a strong prognostic factor in HIV Plasma viraemia is a strong prognostic factor in HIV

infectioninfection

Antiretroviral DrugsAntiretroviral Drugs

Significant declines in AIDS related morbidity and Significant declines in AIDS related morbidity and mortality are seen as a result of HAARTmortality are seen as a result of HAART

Several strategies for development of effective Several strategies for development of effective antiviral drugsantiviral drugs

Potential therapies based on knowledge of the way in Potential therapies based on knowledge of the way in which HIV gains access into the cells and its method of which HIV gains access into the cells and its method of replication replication

Targets for therapeutic anti-retroviral drugs:Targets for therapeutic anti-retroviral drugs:

- Inhibiting reverse transcription- Inhibiting reverse transcription

- Inhibiting proteases- Inhibiting proteases

- Inhibiting integrase – interferes with integration of viral - Inhibiting integrase – interferes with integration of viral DNA into host genome DNA into host genome

- Inhibiting fusion – prevents virus from fusing with host - Inhibiting fusion – prevents virus from fusing with host cellcell

Inhibition of Viral ReplicationInhibition of Viral Replication

Inhibition of activity of reverse transcriptase (RT), an Inhibition of activity of reverse transcriptase (RT), an enzyme responsible for viral RNA being reverse enzyme responsible for viral RNA being reverse transcribed to cDNAtranscribed to cDNA

Example: Zidovudine or AZT (azidothymidine) – first Example: Zidovudine or AZT (azidothymidine) – first type of agent to be developed for treatment of HIV type of agent to be developed for treatment of HIV infectioninfection

AZT is an analogue of thymidine that competes with AZT is an analogue of thymidine that competes with natural nucleotides at the RT active site for natural nucleotides at the RT active site for incorporation into DNAincorporation into DNA

Introduction of AZT into a growing cDNA chain of the Introduction of AZT into a growing cDNA chain of the retrovirus causes termination of the chain, yielding retrovirus causes termination of the chain, yielding inactive proviral DNAinactive proviral DNA

A different approach - drugs such as nevirapine and A different approach - drugs such as nevirapine and delaviridine (non-nucleoside analogues) which inhibit delaviridine (non-nucleoside analogues) which inhibit the action of the reverse transcriptase enzymethe action of the reverse transcriptase enzyme

Action of AZTAction of AZT

Disadvantages:Disadvantages:

The administered AZT is used not only by the HIV-1 The administered AZT is used not only by the HIV-1 RT but also by human DNA polymeraseRT but also by human DNA polymerase

Incorporation of AZT into the DNA of the host cells Incorporation of AZT into the DNA of the host cells kills themkills them

Precursors of red blood cells especially sensitive to Precursors of red blood cells especially sensitive to AZT, results in anaemia and other side effectsAZT, results in anaemia and other side effects

RT inhibitors have several disadvantages:RT inhibitors have several disadvantages: ToxicToxic Select for resistant viral variants quickly when used Select for resistant viral variants quickly when used

alonealone Lack of effect of cells already effected with HIV, which Lack of effect of cells already effected with HIV, which

no longer require RT to complete the viral replication no longer require RT to complete the viral replication cyclecycle

Protease InhibitorsProtease Inhibitors Prevent the assembly of new infectious virusesPrevent the assembly of new infectious viruses

Inhibits activity of viral protease enzyme necessary to Inhibits activity of viral protease enzyme necessary to cleave viral precursor proteins (gag proteins) into the cleave viral precursor proteins (gag proteins) into the p24 and p17 virion components, required for viral p24 and p17 virion components, required for viral activityactivity

Disadvantages:Disadvantages:

Resistance appears after only a few days, single Resistance appears after only a few days, single mutation requiredmutation required

In contrast, resistance to AZT takes months to develop In contrast, resistance to AZT takes months to develop as it requires three or four mutations in the viral as it requires three or four mutations in the viral reverse transcriptase reverse transcriptase

Therapeutic OptionsTherapeutic Options

Combination of RT inhibitors protease inhibitors Combination of RT inhibitors protease inhibitors results in potent anti-viral activityresults in potent anti-viral activity

In most cases, two nucleoside analogues and one In most cases, two nucleoside analogues and one protease inhibitor are taken togetherprotease inhibitor are taken together

HAART lowers plasma viral loads in many cases to HAART lowers plasma viral loads in many cases to levels not detectable by current methods levels not detectable by current methods

Has improved the health of AIDS patients to the Has improved the health of AIDS patients to the point that they can function at a normal levelpoint that they can function at a normal level

Effect of Treatment on Viral LoadEffect of Treatment on Viral Load