Lecture 9-10 AIDS

Why is AIDS unique?

1. New disease to our species and society

2. Originate from the mutation of a virus common in nonhuman primates

3. Changed the tropisms of several pathogens common to humans (including imparting neurologic tropisms)

4. Allowed the reemergence in human of pathogens that were nearly eradicated [tuberculosis]

5. Continues to demonstrate the role of cellular immunity in the evolution of several human diseases

Treatment for AIDS

no treatment exists for eradication of iAIDS for nfected person with HIV

AIDS vaccine

NONE

beginning of AIDS

5 cases of rare pneumonia called Pneumocystis carinii → Kaposi’s sarcoma → chronic generalized lymphadenopathy

Common to all patients was a severe depression of cellular immunity

Initial name of AIDS

GRID [Gay-related immunodeficiency] 

AIDS possibilities

A new disease within the gay community
   >Use of recreational drugs used by gay men to heighten the sexual experience (e.g., nitrates)?
  >The practice of anal sex?
  >A combination of two or more factors associated with the gay lifestyle?

•A new strain of cytomegalovirus

•A fungus that produced cyclosporin A

Puzzle Pieces

•Hemophilacs who were not gay but who received blood transfusions also developed AIDS

•Implied that AIDS was caused by a blood-borne infectious agent similar to hepatitis B

•Cats suffer from a leukemia (T-cell proliferation) caused by a feline retrovirus

•Feline leukemia virus can also cause a depletion of T cells that leads to an immune suppression similar to AIDS in humans 

Adult T-cell leukemia

•In 1977, Gallo’s laboratory isolated two retroviruses from patients with adult T-cell leukemia
    > Human T-cell lymphotropic (leukemia) virus type 1 (HTLV-1)
    > Human T-cell lymphotropic (leukemia) virus type 2 (HTLV-2)

•Both were found to be
-transmitted by blood transfusion and sexual routes
-HIV/infected CD4+ T lymphocytes

•Primary HTLV-1 infection found to be asymptomatic, but progresses to adult T-cell leukemia over 15 - 20 years (long incubation period)

HIV/AIDS previous names

•Lymphadenopathy-associated virus (LAV) 

•AIDS-related virus (ARV)

•Human T-cell lymphotropic virus type 3 (HTLV-3) [same as LAV]

The etiologic agent of AIDS is…

HIV

Animal Studies

•Similar retroviruses that cause immunodeficiency have been recognized in animals
  >Feline immunodeficiency virus
  >Simian immunodeficiency virus

Epidemiologic studies

•AIDS appears as a clinical entity only after appearance of HIV infection

•HIV can be detected and/or isolated from 100% of patients with AIDS

•HIV can not be detected and/or isolated in patients immuno-suppressed by drugs, X-irradiation, or cancers

Blood-transmission studies

•Transfusions that occurred during 1982 - 1983 showed direct correlation between HIV infection of the donor and development of AIDS in the recipient

There has been a dramatic decrease in transfusion-associated AIDS now that our blood supply is screened for HIV infection

In vitro (test tube) studies

•HIV infects and kills CD4+ T lymphocytes in culture, the same cell population affected in the body during evolution of AIDS

HIV

pathogenic human retrovirus [from reverse transcription]

Retroviruses

EVERY virion has 2 copies of the RNA genome

Genome RNA converted to DNA by reverse transcriptase

A DNA copy of the genome (provirus) is integrated into host cell DNA

Among retroviruses, human immunodeficiency virus is classified as a

lentivirus

Lentiviruses

Human immunodeficiency virus type 1
Simian immunodeficiency virus
Feline immunodeficiency virus

HIV Types

HIV type 1 (HIV-1)
  •Prevalent throughout the world
  •Slow but progressive deterioration of the immune system (5-8 years)

HIV type 2 (HIV-2)
  •Found primarily in West Africa
  • Slow but progressive deterioration of the immune system (11-19 years)

HIV-1 Subtypes/Clades

•A subtype nomenclature for classification of HIV-1 isolates, Determined by genetic diversity → cluster in specific geographical areas

•The major (M) group contains 9 subtypes (A-D, F-H, J, K)

•The B subtype is predominant in United States & Europe

What subtype of HIV-1 is most prevalent in USA/Europe?

B subtype

Origin of AIDS

•Sequence homology studies provide evidence that the AIDS virus originated through mutation of simian immunodeficiency virus (SIV)
   >SIV of chimpanzee ---> HIV-1
  >SIV of sooty mangebey monkey ---> HIV-2

•HIV-1 thought to have appeared in humans between 1884 and 1924, more focused estimate 1908
   >Fragments of HIV-1 genome recovered from blood sample collected from a man in Africa in 1959 (Kinshasa, the Congo)
  >Fragments of HIV-1 genome recovered from blood sample collected from a woman in Africa in 1960 (Kinshasa, the Congo

HIV-1 Genome

•gag (group-specific antigen) [capsid, matric and nucleocapsid protein]

•pol (DNA polymerase) [reverse transcriptase, protease, integrase] 

•env (envelope proteins) [gp120, gp41, gp160 →precursor protein, cleaved to make them]

•several regulatory genes …

  > vpr (structural regulatory protein) found in virion of HIV [opens up nuclear pore that allows nucleocapsid to get into nucleus]

HIV-1 Major Receptors/Tropisms

1.T-cell-tropic strains of HIV-1
>CD4 molecule
  >CXCR4 chemokine receptor

2. Macrophage-tropic strains of HIV-1

  >CD4 molecule
>CCR5 chemokine receptor

3 activities of Reverse Transcriptase (3 types of enzymes)

•RNA-dependent DNA polymerase activity of reverse transcriptase copies the entire single-stranded RNA genome to make RNA-DNA molecule
[Uses single-stranded RNA genome as template to make a single-stranded DNA copy of genome]

•RNase H activity of reverse transcriptase removes the template genome RNA probably as genome RNA is copied into single-stranded complementary DNA strand
[Selectively dissolves the RNA part of the RNA-DNA hybrid]

DNA-dependent DNA polymerase activity of reverse transcriptase uses single-stranded DNA as a template to produce a linear double-stranded DNA copy (proviral DNA) of original single-stranded RNA
[Uses single-stranded DNA copy of genome to make a double-stranded DNA copy of genome (provirus or proviral DNA)]

Proviral DNA Integration

•Integrase binds to two ends of the linear proviral DNA, brings them together and in close proximity to cellular DNA

•Integrase removes two 3’ terminal nucleotides of each strand of the proviral DNA

•Proviral DNA is inserted into host DNA by concerted cleavage and ligation reaction

•Host enzymes carry out repair synthesis of gap

Maturation

•Full-length transcript from proviral DNA can serve as genome RNA

•env protein (gp160) travels to lumen of the ER for glycosylation→ cleaved into gp120 and gp41 by protease, → trans-located to the plasma membrane where they are inserted

•gag and pol travel to the inner surface of plasma membrane →bind to newly synthesized genome RNA

•Assembly around genome RNA causes curvature of plasma membrane as the virion grows → roughly spherical particle is formed as the envelopment process proceeds → enveloped particles released → the protease cleaves the gag and pol polyproteins to yield individual structural + enzymatic proteins

•Structural proteins rearrange to form the viral core of mature virions that are now infectious

Who is susceptible to HIV-1?

EVERYONE, but not everyone is susceptible to development of AIDS after infection

Transmission of HIV-1

direct contact with infected body fluids (Not transmitted via aerosols)
-Blood & blood product, Semen & cervicovaginal secretions, Amniotic fluid, Breast milk, Saliva, Tears, Urine, Cerebrospinal fluid NOT IN SWEAT

NOT transmitted through Handshaking, Hugging, Kissing on the cheek, Deep kissing (?), Sharing of household items, Toilet seats, doorknobs, hot tubs

Insect-borne transmission of HIV-1

NOT POSSIBLE

AIDS epidemic thought to be from mosquito, but from drug abuse and prostitution 

•Age-specific rates of HIV-1 infection and disease do not support insect transmission

•Insects (or cultured insect cells) do not support the replication of HIV-1

Routes of HIV-1 Transmission

•Sexual route

•Blood route

•Mother to child

Sexual Route of HIV-1

Anal intercourse → vaginal intercourse → oral sex 

Cofactors contributing ot HIV-1 infection

•Genital lesions produced by other sexually transmitted diseases
(gonorrhea is possible exception, since not direct lesion)

•Lack of circumcision among males
  >Increased susceptibility to genital herpes, warts
  >No increased susceptibility to syphilis
•Alcohol consumption[get drunk, don’t use condom]

Blood Route of HIV-1

  •Transfusion of blood or blood products

  •Sharing of contaminated needles (not abuse of drugs)

  • Transplantation of contaminated tissues / organs

What about healthcare providers and needle sticks?
>0.1 - 0.3% if blood known to be HIV-1 (+)
>If not known, depends on prevalence of HIV-1 in community
[If prevalence is 1:10,000, probability is 1:10 million]
[If prevalence is 1:100, probability is 1:100,000]

•Likelihood of transmission also depends on needle bore, depth of needlestick, volume injected, and amount of virus in inoculum 

Mom → Baby Route of HIV-1

Rate of transmission is 12% - 50% (average = 25%)

Intrauterine transmission occurs by passage of virus from mother to fetus across the placenta

Transmission occurs at time of birth by exposure to infected blood during vaginal delivery

Transmission occurs at time of breast-feeding via infected breast milk
~12% risk for continuous breast-feeding

Hep B vs HIV needle stick

Risk of infection (one needlestick)

HIV is <0.5% (if known to be HIV+)
HBV is ~18% (if known to be HBV+)

Universal Precautions for AIDS

Handle all blood, body fluids, unfixed tissues, and potential fomites as though they are infected with a dangerous pathogen

Pathogenesis (target cells, receptors, tropisms)

•T-lymphocytes (T-cells) & macrophages are primary targets for HIV-1 infection

•CD4 molecule is expressed on surface of T cells & macrophages
>CD4 molecule serves as primary receptor for HIV-1 and binds to distinct domains on envelope gp120 molecule of HIV-1

•Set of co-receptors (chemokine receptors) expressed on T cells & macrophages
>Co-receptors bind to distinct domains on envelope gp120 molecule of HIV-1 (CXCR4 for T cells; CCR5 for macrophages)
>Determines tropism of HIV-1 strains
>Uncovers gp41 that fuses envelope and plasma membrane

Dendritic cells represent an important target for HIV-1 infection during primary infection

•Dendritic cells=  APCS found in blood + skin/genital tract (Langerhans cells)
>Because of location in genital tracts, dendritic cells are primary targets for HIV-1 infection during sexual transmission

•Dendritic cells express CD4 molecule, when immature they initially express CCR5 co-receptor, but can express CXCR4 co-receptor upon maturation

Organization of HIV

Types →Subtypes → Strains

HIV-1 Strains & Tropisms

All use CD4 molecule as primary receptor

R5 Strains
>Use CCR5 chemokine receptor
>Tropism for macrophages / (immature) dendritic cells

X4 Strains
>Use CXCR4 chemokine receptor
>Tropism for CD4+ T cells / (mature) dendritic cells

R5X4 Strains
>Use either CCR5 or CXCR4 chemokine receptors
>Tropism for macrophages / dendritic cells and CD4+ T cells

Stages of HIV-1 Infection

•Primary infection

•Asymptomatic infection (“latent period”)

•Symptomatic disease progression (AIDS)

Primary HIV-1 Infection

•HIV-1 (R5 strains) infects dendritic cells + macrophages at the mucosal epithelium (CCR5 co-receptor) → HIV-1 travels to lymph nodes → enters the blood within 2 days of infection, cause viremia → Explosive virus replication associated with viremia (10⁷ [million] HIV-1 / ml)

•~80% of persons develop flu-like symptoms (fever, headache, lymphadenopathy, night sweats) associated with skin rash on the trunk that abates within 2 weeks of onset, but persistent lymph-adenopathy and headache (meningitis) can continue for months

•Marked reduction of viremia due to HIV-1-specific immunity

•HIV-1-infected macrophages persist as reservoir (provirus)

Asymptomatic HIV-1 Infection

•A period of variable duration measured in years with few clinical manifestations

•Low levels of HIV-1 detected within peripheral blood

•Extensive HIV-1 replication within lymph nodes that is associated with progressive deterioration of immune functions (immune dysregulation) → Destruction of the lymph node tissue

•High mutation + antigenic modulation → generation of numerous HIV-1 strains

•Virus replication controlled by cellular immunity via CD8+ T-cell responses, which enhanced by a dominant Th1 CD4+ T-cell cytokine profile

•Many peripheral blood resting CD4+ T cells harbor virus in a latent state

•Asymptomatic period is usually 5-8 years, person remains infectious and can transmit virus

•Begin to see gradual loss of CD4+ T-cell function and number

Symptomatic disease progression (AIDS)

•HIV-1 levels ↑ in peripheral blood with concomitant increase in the diversity of HIV-1 strains

•X4 strains of HIV-1 predominate with tropism for CD4+ T cells (CD4+ T helper cells)

•Hypergammaglobulinemia appears due to chronic activation of B cells (polyclonal B cell activation)

•Type 1 cytokine production shifts to Type 2 cytokine production (Th1/Th2 shift)

•Absolute numbers of CD4+ T cells decline (immune deficiency) + Delayed-type hypersensitivity (DTH) T-cell functions decline [patient become anergic] + Protective CD8+ T-cell functions decline = Appearance of numerous opportunistic infections

Antigenic modulation

•HIV-1 exhibits a high mutation rate
>5X greater than influenza virus, 10,000-100,000 mutations per day

•High mutation rate + antigenic modulation generates numerous antigenically distinct strains of HIV-1, which may show spectrum of virulence

•Appearance of X4 strains that show tropism for CD4+ T cells (latent HIV-1 infection in resting CD4+ T cells)

Th1 vs Th2 CD4+ T Cells

•Th1 CD4+ T cells → Stimulates cellular immunity

•Th2 CD4+ T cells → Stimulates antibody production

CD4+ T-cell numbers

•Persons without HIV-1 infection have 700-1000 CD4+ T cells/microliter of peripheral blood

•HIV-1-infected persons are considered to have “normal” CD4+ T-cell counts if the number is x>500 CD4+ T cells/µL of peripheral blood

•A person is classified as having AIDS if CD4+ T-cell count drops to x< 200 CD4+ T cells/µl of peripheral blood (CDC classification of AIDS)

Outcomes

•Typical progressors
>Develop AIDS 8-10 years after primary infection
>75 - 80% of HIV-1-infected persons

•Rapid progressors
>Develop AIDS 2-3 years after primary infection
>~10% of HIV-1-infected persons

•Nonprogressors (long-term nonprogressors)
>Healthy 15-30 years after HIV-1 infection
>10-17% of HIV-1-infected persons (1:500)

Nonprogressors/long-term survivors

•Virus load in blood is far less than in progressors due to strong immune response
>Broadly reactive neutralizing antibodies
>Potent antiviral CD8+ T-cell activity 

•Lymph nodes show normal architecture, but should be destroyed

•HIV-1 isolates replicate poorly in culture (attenuated strains?)

Exhibit mutations in gene(s) for CCR5 (highly resistant to R5 strains

Clinical progression of HIV-1

•Appearance of thrush, chronic herpes zoster, & chronic genital herpes are early signs of clinical progression (CD4+ T cell counts are X>200 cells/µL of peripheral blood)

•P carinii pneumonia, Toxoplasma encephalitis, & cryptococcal meningitis occur when CD4+ T cell counts are x<200 cells/µL of peripheral blood (CDC definition of AIDS)

•Mycobacterium avium complex, CMV disease, PML, and lymphoma occur when CD4+ T cells are x<50 cells/µL of peripheral blood

HIV - Associated Dementia (HAD)

~20% of AIDS patients develop dementia before death

•Clinical course is usually abrupt over weeks / months, not insidious over years
>Initial presentation includes forgetfulness and loss of concentration   followed by behavioral changes (apathy, social withdrawal, irritability)
>Disease progresses to loss of fine motor control, unsteadiness of gait, and tremors
>Most deteriorate into global cognitive dysfunction, mutism, incontinence, and generalized spasticity although patient is awake
>Death occurs 3-6 months after onset of dementia

Brain shrinks in size due to tissue wasting 

•HIV-1-associated dementia can occur despite use of antiviral drugs

When in the course of HIV-1 infection does the brain become infected with HIV-1?

HIV-1 recovered from cerebrospinal fluid within 2 weeks of primary infection
>Primary infection associated with flu-like symptoms and headache, photophobia, nuchal rigidity (meningitis)

Antiretrovirus Drugs

GOALS

1. Slow/prevent HIV-1 replication→ slow HIV-1-induced immunosuppression → stop the manifestation of a number of clinical diseases caused by opportunistic infections and malignancies

2. Slow/ prevent clinical manifestations caused by direct HIV-1 infection (wasting disease, retinal disease, HIV-associated dementia)

Management of HIV-1

Antiretrovirus Drugs

Antiviral Drugs

Reverse Transcriptase Inhibitors

Protease Inhibitors

Combination Antiretroviral Therapy (ART)

Integrase Inhibitors

Prophylaxis/PrEP

Antiviral drugs

•Advantages to very early or very late steps
>Attachment, adsorption (entry), release
>Inhibitors do not need to enter cell to be active

•Advantages to steps that involve enzymes
>Steps in genome replication, assembly, maturation
>Enzymes make good targets for antiviral drugs

They are at low concentrations within the cell, they a well-understood mechanistically, and pharmaceutical companies have experience in developing enzyme inhibitors as drugs

Most antiviral drugs used clinically are enzyme inhibitors that target genome replication, assembly, maturation

Four distinct targets during HIV-1 replication are susceptible to antiviral therapy

•Provirus DNA formation (reverse transcriptase)

•Nucleocapsid formation (assembly)

•Integration of provirus DNA into host DNA (integrase)

•Maturation of virion (protease)

Reverse Transcriptase Inhibitors

Azidothymidine (AZT) was first antiretroviral drug, proof of concept

>Transiently increased CD4+ T-cell counts
>Reduced opportunistic infections
>Increased survival time

Well tolerated in pregnant women {no malformation, premature birth, fetal distress] and reduces transmission form 25% to 8%

Toxicities
Toxic to bone marrow (severe anemia / neutropenia)
Painful peripheral neuropathy and numbness
Pancreatitis
Abnormalities of the liver (hepatitis)
Generalized clinical symptoms of fever, rash, and/or diarrhea

AZT Mech. of Action

•Like acyclovir, azidothymidine is a nucleoside analogue with an altered structure

•A thymidine base is attached to a ribose in which the normal 3’ OH is converted to an azido (N₃) group

•Like acyclovir, azidothymidine leads to premature chain termination when incorporated into growing DNA molecule AND/OR Preferentially blocks HIV reverse transcriptase

Protease inhibitors

Ritonavir

•A peptidomimetic drug now used as a protease inhibitor to manage HIV-1 infection
>Developed by determining crystal structure of HIV-1 protease
>Binds to HIV-1 protease as substrate, but not cleaved, remains tightly bound to HIV-1 protease

•Two possible mechanisms of action …
>gag polyprotein not cleaved, so resulting particles are immature and not infectious (primary action)
>pol polyprotein not cleaved, so reverse transcriptase is not available for genome replication (secondary action)

Toxicities
-Lipodystrophy, a redistribution of fat from the face and limbs to the gut
-Concern that long-term use will lead to coronary disease / stroke due to adverse effect on lipid metabolism

Protease mechanism of actions

>gag polyprotein not cleaved, so resulting particles are immature and not infectious (primary action)
>pol polyprotein not cleaved, so reverse transcriptase is not available for genome replication (secondary action)

Combination Antiretroviral Therapy (ART)

•Combination therapy using protease inhibitor in combination with two reverse transcriptase inhibitors (or other antiretrovirus drugs)

•Clinical trials have demonstrated additive / synergistic therapeutic efficacy (HIV-1-infected persons & AIDS patients)
>Decreases HIV-1 load within the peripheral blood
>Improves immune functions (CD4+ T cells)
>Prevents onset of many opportunistic infections and   malignancies

•A dramatic decrease in AIDS-related mortality and morbidity

•Transformed AIDS into a chronic disease of HIV-1 infection

•Has not reduced new cases of HIV-1 infection

integrase inhibitors

•~12 years were needed to develop clinically usable inhibitors of integrase due to difficulty in crystallization of molecule

•Four integrase inhibitors approved/licensed, Provide “effective virus suppression”

•May be taken in combination with other HIV-1 drugs to minimize adaptation of the virus

•May offer hope to HIV-1-infected persons who have developed resistance to HAART / ART

Prophylaxis

Combination of antiretroviral drugs to prevent HIV infection

•PrEP reduces the risk of HIV infection from sex by ~99%

•Several PrEP available that consist of two antiretroviral drugs (reverse transcriptase inhibitors without or with integrase inhibitor)
>Truvada
>Descovy
>Dovato
>Biktarvy

Lenacapavir
•Binds directly to the interface between HIV-1 virus capsid protein (p24) subunits thereby interfering with progeny virus assembly

•Administered by injection twice a year

•99.9% of Phase 3 clinical trial administered drug did not acquire HIV-1 infection (only 2 cases out of 2,180 patients became HIV-1 infected)

Premature (Accelerated) Aging

Persons with chronic HIV-1 infection are living longer, but as a group are now showing development of age-related disorders

-Cardiovascular disease
-Cognitive impairment
=Type II diabetes
-Osteoporosis
-Age-related macular degeneration

Estimated that persons with chronic HIV-1 infection are showing accelerated aging by as much as 15 years

Vaccination

A vaccine has not been developed yet

•HIV-1 directly attacks and destroys components of the immune system, can establish a latent / persistent infection via provirus DNA, can cross an intact blood-brain barrier and infect the brain, ans exhibits high mutation that generates numerous antigenic variants

>Which strain(s) of HIV-1 should be included in a vaccine?
>Which subtype(s) of HIV-1 should be included in a vaccine?