40 million (est.) 200140 million (est.) 2001

3 million(est.) 20013 million(est.) 2001

5 million (est.) 20015 million (est.) 2001

Time Time course course of HIV of HIV infection infection and and AIDSAIDS

HIV HIV infection:infection:

Time Time course of course of CD4+ CD4+ counts & counts & opportunisticopportunistic infectionsinfections

Dr. Luc Montagnier Dr. Luc Montagnier co-discoverer of HIVco-discoverer of HIV

Dr. Robert GalloDr. Robert Gallo

co-discoverer of co-discoverer of HIVHIV

HIV PropertiesHIV Properties

-Human T-cell lymphotropic virus-Human T-cell lymphotropic virus

-Lentivirus subgroup of retroviruses-Lentivirus subgroup of retroviruses

-D-type nucleocapsid-D-type nucleocapsid

-Surrounded by a lipid envelope-Surrounded by a lipid envelope

HIV Replication in Host CellsHIV Replication in Host Cells

-Virus Entry Into Cells--Virus Entry Into Cells-

1. The HIV envelope protein gp120 binds to the 1. The HIV envelope protein gp120 binds to the CD4 cellular receptor and chemokine CD4 cellular receptor and chemokine coreceptor on the surface of host cells.coreceptor on the surface of host cells.

2. The binding of gp120 unlatches the gp41 2. The binding of gp120 unlatches the gp41 protein, causing it to penetrate the host cell protein, causing it to penetrate the host cell membrane.membrane.

3. The virus lipid envelope becomes fused with 3. The virus lipid envelope becomes fused with the host cell membrane resulting in virus entry the host cell membrane resulting in virus entry into the cell.into the cell.

HIV gp120 and gp41HIV gp120 and gp41

-The viral proteins gp120 and -The viral proteins gp120 and gp41are embedded in the lipid gp41are embedded in the lipid envelope.envelope.

-gp120 & gp41 mediate virus entry -gp120 & gp41 mediate virus entry into the host cell.into the host cell.

-There is enormous antigenic -There is enormous antigenic variation in the gp120 protein.variation in the gp120 protein.

gp120 and gp41: roles in virus entry into hostgp120 and gp41: roles in virus entry into host

HIV Reverse TranscriptaseHIV Reverse Transcriptase

-The reverse transcriptase(RT) is -The reverse transcriptase(RT) is carried by the virus in the core carried by the virus in the core complexed to the RNA genome.complexed to the RNA genome.

-The RT is used to copy the dsRNA -The RT is used to copy the dsRNA genome of the virus into a dsDNA genome of the virus into a dsDNA copy.copy.

-Drugs that inhibit the RT are used in -Drugs that inhibit the RT are used in HIV therapy. E.g. AZT, DDI.HIV therapy. E.g. AZT, DDI.

HIV reverse transcriptaseHIV reverse transcriptase

HIV reverse transcriptase with RNA substrateHIV reverse transcriptase with RNA substrate

Reverse transcriptase active site inhibitorReverse transcriptase active site inhibitor

HIV ProteaseHIV Protease

-The primary product of viral protein -The primary product of viral protein synthesis is a long continuous synthesis is a long continuous polyproteinpolyprotein

-The polyprotein consists of all of the -The polyprotein consists of all of the viral proteins covalently attached.viral proteins covalently attached.

-One end of the polyprotein folds into -One end of the polyprotein folds into the the virus proteasevirus protease which cuts the rest which cuts the rest of the polyprotein into pieces.of the polyprotein into pieces.

HIV Protease: active site with inhibitorHIV Protease: active site with inhibitor

HIV IntegraseHIV Integrase

-The integrase is brought into the cell -The integrase is brought into the cell in the core of the virus.in the core of the virus.

-The integrase catalyzes the covalent -The integrase catalyzes the covalent integration of the double stranded integration of the double stranded DNA copy of the virus into the host DNA copy of the virus into the host genome.genome.

Viral integrase Viral integrase inserts virus DNA inserts virus DNA into human DNAinto human DNA

HIV integraseHIV integrase

HIV Replication in Host CellsHIV Replication in Host Cells

-Virus Entry Into Cells--Virus Entry Into Cells-

- Chemokine coreceptor polymorphisms - Chemokine coreceptor polymorphisms explain the partial resistance to HIV infection explain the partial resistance to HIV infection and longer progression to AIDS seen in some and longer progression to AIDS seen in some people.people.

- ~1% of Caucasians are very resistant to HIV - ~1% of Caucasians are very resistant to HIV infection and posses a 32 base pair deletion infection and posses a 32 base pair deletion variant of the CCR5 coreceptor.variant of the CCR5 coreceptor.

-Coreceptor antagonists may find use in HIV -Coreceptor antagonists may find use in HIV therapytherapy

1. HIV Reproduction In the Body1. HIV Reproduction In the Body

-During Primary infection the virus -During Primary infection the virus infects Langerhans cells (dendritic) cell infects Langerhans cells (dendritic) cell in the mucosa-for sexually acquired in the mucosa-for sexually acquired HIV.HIV.

-The Langerhans cells spread and -The Langerhans cells spread and establish the infection in lymphatic establish the infection in lymphatic tissues including the lymph nodes.tissues including the lymph nodes.

2. HIV Reproduction In the Body2. HIV Reproduction In the Body

-Infection of the lymph nodes is -Infection of the lymph nodes is followed by massive viremiafollowed by massive viremia

- This leads to wide dissemination of - This leads to wide dissemination of HIV in lymphoid organs and the central HIV in lymphoid organs and the central nervous system.nervous system.

3. HIV Reproduction In the Body3. HIV Reproduction In the Body

-The virus continues to replicate in -The virus continues to replicate in the lymph nodes but its replication is the lymph nodes but its replication is controlled by specific immune controlled by specific immune response including CD8+ cells response including CD8+ cells (cytotoxic T cells)(cytotoxic T cells)

Anthony Fauci, MDAnthony Fauci, MD

Director,Director,

National Institute of National Institute of Allergy and Infectious Allergy and Infectious Diseases, NIHDiseases, NIH

HIV Replication in Germinal Centers of the Lymph HIV Replication in Germinal Centers of the Lymph Nodes (indicated by the presence of HIV-RNA)Nodes (indicated by the presence of HIV-RNA)

HIV Replication in Germinal Centers of the Lymph Nodes HIV Replication in Germinal Centers of the Lymph Nodes

HIV-specific CTL responses in a rapid HIV-specific CTL responses in a rapid progressor and long-term nonprogressor. progressor and long-term nonprogressor.

1.1. The rapid progressor exhibited a The rapid progressor exhibited a rapid CD4+ count decline and developed rapid CD4+ count decline and developed AIDS at 13 months with a consistently AIDS at 13 months with a consistently high plasma viral load (>300,000 high plasma viral load (>300,000 copies/mL of HIV RNA).copies/mL of HIV RNA). 2.2. The nonprogressor remains well at The nonprogressor remains well at 19 years with a CD4+ cell count greater 19 years with a CD4+ cell count greater than 1000/µL and viral load less than 400 than 1000/µL and viral load less than 400 HIV RNA copies/mL.HIV RNA copies/mL.

HAART 101HAART 101

HAART 101HAART 101

HHighly ighly AActive ctive AAntintirretroviral etroviral TTherapyherapy

A combination of anti-HIV drugs that A combination of anti-HIV drugs that can reduce viral replication and viral can reduce viral replication and viral load to low or undetectable load to low or undetectable levelslevels(<50copies viral RNA/ml serum).(<50copies viral RNA/ml serum).

The decrease in viral load can allow for The decrease in viral load can allow for the restoration of CD4+ T-lymphocyte the restoration of CD4+ T-lymphocyte numbersnumbers(>200CD4+ cells/mm(>200CD4+ cells/mm33)) and and reconstitution of the immune system.reconstitution of the immune system.

HAART 101HAART 101

Immune reconstitution results in the Immune reconstitution results in the reduction in severity or the elimination reduction in severity or the elimination of opportunistic infections.of opportunistic infections.

This is accompanied by a reversal of This is accompanied by a reversal of wasting and an increase in lean body wasting and an increase in lean body mass.mass.

HAART 101HAART 101

Combination antiretroviral therapy Combination antiretroviral therapy can include:can include:a Nucleoside Analoguea Nucleoside Analogue

a Non-nucleoside Analoguea Non-nucleoside Analogue

a Protease Inhibitora Protease Inhibitor

3, 4 or 5 anti-HIV drugs may be 3, 4 or 5 anti-HIV drugs may be combined.combined.

Nucleoside Reverse Transcriptase Nucleoside Reverse Transcriptase Inhibitors: Inhibitors: NRTINRTIss

AZTAZT DDIDDI 3TC3TC

NRTIs are phosphorylated to the NTP NRTIs are phosphorylated to the NTP level and then inhibit the RT by causing level and then inhibit the RT by causing premature chain terminationpremature chain termination

Non Nucleoside Reverse Transcriptase Non Nucleoside Reverse Transcriptase Inhibitors: Inhibitors: nNRTIsnNRTIs

DelavirdineDelavirdine EfavirenzEfavirenz

Competitive inhibitors of the RT.Competitive inhibitors of the RT.

HAART 101HAART 101

The average HIV infected individual The average HIV infected individual makes between 10makes between 1066 and 10 and 1077 virus virus particles per day.particles per day.

Virus load in untreated individuals is Virus load in untreated individuals is often in excess of 10,000 copies of often in excess of 10,000 copies of virus RNA/ml of serum.virus RNA/ml of serum.

HAART 101HAART 101

Treatment success is measured by a Treatment success is measured by a decrease in viral load of at least 1 log decrease in viral load of at least 1 log (10 fold).(10 fold).

Ideally the viral load should fall Ideally the viral load should fall below the level of detection: <50 below the level of detection: <50 copies virus RNA/ml serum.copies virus RNA/ml serum.

In addition, CD4+ lymphocyte counts In addition, CD4+ lymphocyte counts should rise above 200 cells/mmshould rise above 200 cells/mm33 serum.serum.

HAART 101HAART 101

Treatment failure is similarly Treatment failure is similarly indicated by an increase in viral load.indicated by an increase in viral load.

Treatment failure may be due to:Treatment failure may be due to:

Drug toxicityDrug toxicity

Drug Holidays/Non-complianceDrug Holidays/Non-compliance

Development of drug resistant Development of drug resistant virusvirus

Establishment of Viral Establishment of Viral Reservoirs in HIV InfectionReservoirs in HIV Infection

<Primary Infection<Primary Infection

<Establishment of infection <Establishment of infection in Lymphoid Tissuein Lymphoid Tissue

<Establishment of Viral <Establishment of Viral ReservoirReservoir

HAART 101HAART 101

Is It Possible To “Cure” HIV InfectionIs It Possible To “Cure” HIV Infection

The largest reservoir of virus is in The largest reservoir of virus is in the lymph nodes in quiescent CD4+ the lymph nodes in quiescent CD4+ cells and dendritic cells.cells and dendritic cells.

HIV infected cells in the nervous HIV infected cells in the nervous system, including microglia, may also system, including microglia, may also function as latent reservoirs of HIV.function as latent reservoirs of HIV.

Reservoirs of HIVReservoirs of HIV

Reservoirs of HIV are Reservoirs of HIV are established at the time of primary established at the time of primary infection.infection.

These reservoirs persist despite These reservoirs persist despite HAART that effectively controls HAART that effectively controls plasma viremia. Neither HAART plasma viremia. Neither HAART nor an HIV-specific immune nor an HIV-specific immune response eradicates HIV response eradicates HIV reservoirs.reservoirs.

HAART 101HAART 101

Quiescent cells are refractile to Quiescent cells are refractile to treatment since antiretroviral therapy treatment since antiretroviral therapy only eliminates HIV infected cells when only eliminates HIV infected cells when the virus is actively replicating.the virus is actively replicating.

Quiescent cells form a “reservoir” of Quiescent cells form a “reservoir” of latent HIV that can emerge when latent HIV that can emerge when therapy fails(resistance) or is therapy fails(resistance) or is discontinued(noncompliance/drug discontinued(noncompliance/drug holiday).holiday).

HAART 101HAART 101

Strategies for eliminating the latent Strategies for eliminating the latent reservoir of HIV are aimed at “curing” reservoir of HIV are aimed at “curing” HIV disease.HIV disease.

An approach to promoting virus An approach to promoting virus replication in latently infected immune replication in latently infected immune cells is to specifically or non-specifically cells is to specifically or non-specifically stimulate them by immunization or stimulate them by immunization or cytokines.cytokines.

HAART 101HAART 101

Toxicity Associated with HAARTToxicity Associated with HAART

LipodystrophyLipodystrophy

>Abnormal fat metabolism and >Abnormal fat metabolism and distribution.distribution.

HAART 101HAART 101

LipodystrophyLipodystrophy

Loss of fat in the arms, legs, Loss of fat in the arms, legs, buttocks or face.buttocks or face.

Buildup of fat around the:Buildup of fat around the:

Gut (“Crix Belly”/”Protease Gut (“Crix Belly”/”Protease Paunch”),Paunch”),

Back of the neck and Back of the neck and shoulders shoulders (“Buffalo Hump”)(“Buffalo Hump”)

Breasts.Breasts.

HAART 101HAART 101

LipodystrophyLipodystrophy

Hyperlipidemias: elevated blood Hyperlipidemias: elevated blood cholesterol and triglycerides.cholesterol and triglycerides.

Hyperglycemias: elevated blood Hyperglycemias: elevated blood glucose.glucose.

Both of the above are frequent Both of the above are frequent enough and serious enough to warrant enough and serious enough to warrant monitoring and intervention.monitoring and intervention.

HAART 101HAART 101

Examples of Other Side EffectsExamples of Other Side Effects::

Straightening of hair and Straightening of hair and darkening of nails (AZT).darkening of nails (AZT).

Stevens-Johnson syndrome, a Stevens-Johnson syndrome, a potentially fatal skin rash potentially fatal skin rash (nevirapine).(nevirapine).

Diarrhea and facial pain Diarrhea and facial pain (ritonavir)(ritonavir)

Drug Resistant HIVDrug Resistant HIV

incidence of resistant virus frequently incidence of resistant virus frequently within months of initiation of therapy.within months of initiation of therapy.

Monotherapy results in a very high Monotherapy results in a very high The mutation rate for HIV is determined The mutation rate for HIV is determined by the lack of error correction during by the lack of error correction during nucleic acid replication, e.g. a sloppy nucleic acid replication, e.g. a sloppy reverse transcriptase.reverse transcriptase.

Drug Resistant HIVDrug Resistant HIV

It is estimated that every possible It is estimated that every possible base substitution in the HIV genome is base substitution in the HIV genome is produced each day.produced each day.

A rationale for combination therapy is A rationale for combination therapy is that it decreases the probability of that it decreases the probability of selecting multiple resistant mutations selecting multiple resistant mutations in a single virus particle.in a single virus particle.

This has been borne out in human This has been borne out in human studies.studies.

Guidelines for Antiretroviral Therapy:

Updated August 13, 2001.

Department of Health and Human Services and H. J. Kaiser Foundation.

A. Fauci and J. Bartlett, Co-chairs of Panel.

www.hivatis.org

1-800-448-0440

Patients who are Candidates for HAARTPatients who are Candidates for HAART

Acute HIV syndromeAcute HIV syndrome

Within 6 months of seroconversionWithin 6 months of seroconversion

<350 CD4+ T cells/ul<350 CD4+ T cells/ul

>30,000 copies/ml HIV RNA (bDNA >30,000 copies/ml HIV RNA (bDNA assay)assay)

Treatment GoalsTreatment Goals

Maximal and durable suppression of Maximal and durable suppression of viral load.viral load.

A one-logA one-log1010 decrease at 8 weeks and decrease at 8 weeks and no detectable virus (<50 copies/ml) at no detectable virus (<50 copies/ml) at 4-6 months.4-6 months.

Restoration/preservation of immune Restoration/preservation of immune function measured by CD4+ T cells function measured by CD4+ T cells and clinical picture.and clinical picture.

Treatment Success is Monitored byTreatment Success is Monitored by

Plasma HIV RNA levels (viral load)Plasma HIV RNA levels (viral load)

CD4+ T cell countCD4+ T cell count

Antiretroviral drug resistanceAntiretroviral drug resistance

Drug complianceDrug compliance

Drug toxicityDrug toxicity

Rationale for Viral Load TestingRationale for Viral Load Testing

A dose response association A dose response association between decrease in plasma viremia between decrease in plasma viremia and improved clinical outcome and improved clinical outcome (survival and AIDS defining diagnoses) (survival and AIDS defining diagnoses) has been demonstrated in 18 studies has been demonstrated in 18 studies with over 5,000 patients.with over 5,000 patients.

Viral load testing should be done at Viral load testing should be done at time of diagnosis and every 3-4 time of diagnosis and every 3-4 months after virus load <50 HIV RNA months after virus load <50 HIV RNA copies/ml.copies/ml.

Resistance to Antiretroviral Drugs Resistance to Antiretroviral Drugs During HAART.During HAART.

A high incidence of drug A high incidence of drug resistance occurs with resistance occurs with monotherapy: e.g. AZT.monotherapy: e.g. AZT.

Resistance to Antiretroviral Drugs Resistance to Antiretroviral Drugs During HAARTDuring HAART..

While drug resistance is less While drug resistance is less frequent during combination frequent during combination therapy, treatment failure can be therapy, treatment failure can be attributed to the selection of attributed to the selection of singly or multiply drug resistant singly or multiply drug resistant HIV variants.HIV variants.

Monitoring Drug Resistant HIVMonitoring Drug Resistant HIV

The emergence of drug resistant The emergence of drug resistant HIV in patients on HAART is HIV in patients on HAART is monitored using genotyping or monitored using genotyping or phenotypic testing of viral phenotypic testing of viral isolates.isolates.

This diagram outlines the RT-PCR and This diagram outlines the RT-PCR and sequencing strategy utilized in the sequencing strategy utilized in the ViroSeq™ HIV-1 Genotyping SystemViroSeq™ HIV-1 Genotyping System

ViroSeq™ HIV-1 ViroSeq™ HIV-1 Genotyping Genotyping System ReportSystem Report

Phenotyping Assays:Phenotyping Assays:

measure the ability of HIV measure the ability of HIV isolated from patients to grow isolated from patients to grow in the presence of various in the presence of various concentrations of HIV drugs.concentrations of HIV drugs.

The VIRADAPT and GART studies The VIRADAPT and GART studies indicated that the short-term indicated that the short-term virologic response to therapy was virologic response to therapy was significantly greater when the significantly greater when the choice of drugs was guided by choice of drugs was guided by resistance testing.resistance testing.

HIV Reservoir

Effective “autovaccination” that results in long-term immune control of HIV following interruptions of therapy does not occur in the vast majority of chronically infected individuals.

HIV Reservoir

Repeated cycles of interrupted HAART (2 months on/1 month off) result in consistent control of plasma viremia after the “on-HAART” period; however, virus rebounds during the “off HAART” periods.

HIV Reservoir

Repeated cycles of interruptions of HAART (7 days on/7 days off) for up to 22 cycles result in:

A. persistent suppression of plasma viremia.

B. No deleterious effects on CD4+ cells.

C. No expansion of HIV reservoirs

D. No detectable emergence of phenotypic or genotypic resistance.

HIV Reservoir

The long-term clinical effects (beneficial and deleterious) of structured interruptions of HAART remain to be determined by expanded clinical trials.

This is being actively investigated by Fauci et al.

Promising New Therapies Promising New Therapies in Advanced Stages of in Advanced Stages of Clinical TestingClinical Testing

Drugs that Modulate Immune Responses to HIV Infection in Clinical Trials:

Interleukin-2

Drugs that Block HIV Entry into Cells in clinical trials.

HIV Cell Fusion andHIV Cell Fusion andthe Infection Processthe Infection Process

Attachment

AttachmentT cell via CD4 receptors (dk. blue) on cell membrane surface

Co-Receptor Binding

HIV gp 120 protein binds to co-receptors on CD4+ T cell

HIV Insertion

HIV gp 41 protein, composed of HR1 and HR2 subunits, “harpoons” CD4+ T cell

Insertion of the GP41 into the Host Cell

Fusion

HIV gp 41 protein folds on itself bringing HIV membrane and CD4+ T cell membrane togetherFusion of the HIV Envelope with the

Host Cell Membrane

Successful HIV Cell Infection

HIV genetic material transfers to CD4+ T cell

Successful HIV Infection

Fusion Inhibition

T-20 (yellow) Binds HIV gp 41

HIV Cell Fusion and Infection Inhibited

HIV binding to CD4+ cells: Interaction of gp120 with CD4 and chemokine coreceptors.

Other Fusion Inhibitors:

Agents that bind to the cellular chemokine coreceptor: CXCR4

The agents bind to CXCR4 preventing the virus gp120 from binding to it. This blocks virus fusion and entry into the cell.

JM-3100 appears to bind to CXCR4 (fusin) and block the interaction between CXCR4 and the V3 loop of gp120.

ALX40-4C appears to bind to CXCR4 (fusin) and block the interaction between CXCR4 and the V3 loop of gp120.

Integrase Inhibitors:

Inhibit the HIV integrase and prevent the virus dsDNA from entering the host genome.

Integration of the virus DNA into the host genome is an obligate step in HIV replication.

The HIV integrase

(-)-Arctigenin is a potent inhibitor of HIV-1 integrase

1-MO-3,5-DCQA, a dicaffeoylquinic acid, was isolated from an aqueous extract of Achyrocline satureiodes. It is a potent inhibitor of the HIV-1 integrase.

HIV-1 integrase has been shown to be a potential target of cobalamins.

Hydroxyurea is an inhibitor of the cellular enzyme Ribonucleotide Reductase that perturbs dNTP and ddNTP pool sizes. Hydroxyurea has been shown to have a potentiating effect when given in combination with nucleoside analogues such as ddI.

An extensive listing of Approved and Experimental Drugs can be Found at the NIAID/NIH website:

http://www.niaid.nih.gov/daids/dtpdb/clasdrug.HTM

IDEAL CHARACTERISTICS OF AN AIDS VACCINE

1.1. Efficacy in preventing transmission by mucosal Efficacy in preventing transmission by mucosal and parenteral routesand parenteral routes

2.2. Excellent safety profile, with minimal risk of Excellent safety profile, with minimal risk of adverse reactions even in unscreened "real world" adverse reactions even in unscreened "real world" populationspopulations

3.3. Single dose administrationSingle dose administration

4.4. Long-lived effect resulting in protection many years Long-lived effect resulting in protection many years after vaccinationafter vaccination

Status of HIV Vaccine Candidates 1Status of HIV Vaccine Candidates 1

HIV peptides (protein fragments) Status: In phase I trials Advantages: Simple & cheap to make.

Safety. Disadvantages: only a limited number of peptides can be included.

Combination of Elements: e.g. pure gp120 protein + virus vector. Status: In phase II trials. Advantages: Should stimulate both cell & humoral immunity. Disadvantages: Weak immunogens/narrow specificity

Whole, killed HIV Status: Not under study in Humans. Advantages: Might present HIV surface proteins in a natural conformation Disadvantages: May include active virus.

Status of HIV Vaccine Candidates 2Status of HIV Vaccine Candidates 2

Live, attenuated HIV: Status: No human studies only primates. Advantages: Most similar to HIV; gives good protection in animal models. Disadvantages: May cause disease.

Naked DNA: containing 1 or more HIV genes. Status: In phase 1 trials. Advantages: Simple and inexpensive. Disadvantages: Possible integration of HIV genes into human cells. Modest immunogens.

Viral Surface Proteins: such as gp120. Status: In phase II and III trials. Advantages: Safe & easy to make.Disadvantages: Vaccine-elicited antibodies have failed to recognize HIV from patients.

Status of HIV Vaccine Candidates 3Status of HIV Vaccine Candidates 3

Status of HIV Vaccine Candidates 4Status of HIV Vaccine Candidates 4

Live bacterial vectors: bacteria engineered

to carry genes encoding HIV proteins.

Status: In phase I trialsAdvantages: Simple & inexpensive.safe Disadvantages: limited # of HIV genes; unknown stability of vectors.

Live viral vectors: non-HIV viruses which carry genes for HIV proteins.Status: In phase II trials.Advantages: Control over amount and types of HIV proteins made.Disadvantages: Difficult to make; weakly immunogenic.

Pseudovirions: non-replicating HIV-like particles.Status: Close to phase I trials.Advantages: present HIV surface proteins in a natural conformation.Disadvantages: hard to make

Replicons: non-HIV viruses with HIV genes; incomplete replicationStatus: Close to phase I trials Advantages: can carry many HIV genes. Disadvantages: Difficult to make.

Status of HIV Vaccine Candidates 5Status of HIV Vaccine Candidates 5