Terry Kotrla, MS, MT(ASCP)BB

Unit 4Part 6 Human Immunodeficiency

Virus

IntroductionEtiologic agent of Acquired

Immunodeficiency Syndrome (AIDS).Discovered independently by Luc

Montagnier of France and Robert Gallo of the US in 1983-84.

Former names of the virus include:Human T cell lymphotrophic virus (HTLV-III)Lymphadenopathy associated virus (LAV)AIDS associated retrovirus (ARV)

IntroductionHIV-2 discovered in 1986, antigenically distinct

virus endemic in West Africa.One million people infected in US, 30 million

worldwide are infected.Leading cause of death of men aged 25-44 and 4th

leading cause of death of women in this age group in the US.

Reduced mortality resulting from the use of highly active antiretroviral therapies is a major factor contributing to the number of persons in the United States living with HIV disease.

Additionally, more than 56,000 new HIV infections are estimated to occur annually

HIV in the US

Characteristics of the virusIcosahedral (20 sided), enveloped virus

of the lentivirus subfamily of retroviruses.

Retroviruses transcribe RNA to DNA.Two viral strands of RNA found in core

surrounded by protein outer coat.Outer envelope contains a lipid matrix within

which specific viral glycoproteins are imbedded.

These knob-like structures responsible for binding to target cell.

Characteristics of the virus

HIV The outer shell of the virus

is known as the Viral enevlope.

Embedded in the viral envelope is a complex protein known as env which consists of an outer protruding cap glycoprotein (gp) 120, and a stem gp14.

Within the viral envelope is an HIV protein called p17(matrix), and within this is the viral core or capsid, which is made of another viral protein p24(core antigen).

Structural GenesThree main structural genes:

Group Specific Antigen (Gag)Envelope (Env)Polymerase (Pol)

Group Specific Antigen (Gag)Located in nucelocapsid of virus.Icosahedryl capsid surrounds the internal

nucleic acids made up of p24 andp15.p17 lies between protein core and envelope

and is embedded in the internal portion of the envelope.

Two additional p55 products, p7 and p9, are nucleic acid binding proteins closely associated with the RNA.

Envelope (Env)Envelope (Env) gene codes for envelope

proteins gp160, gp120 and gp41.These polyproteins will eventually be cleaved

by proteases to become HIV envelope glycoproteins gp120 and gp41.

gp160 cleaved to form gp120 and gp41.gp120 forms the 72 knobs which protrude from

outer envelope.gp41 is a transmembrane glycoprotein antigen

that spans the inner and outer membranes and attaches to gp120.

gp120 and gp41 both involved with fusion and attachment of HIV to CD4 antigen on host cells.

Polymerase (Pol)Polymerase (Pol) codes for p66 and p51

subunits of reverse transcriptase and p31 an endonuclease.Located in the core, close to nucleic acids.Responsible for conversion of viral RNA into

DNA, integration of DNA into host cell DNA and cleavage of protein precursors.

Viral Replication http://tinyurl.com/3425m69

First step, HIV attaches to susceptible host cell.Site of attachment is the CD4 antigen found

on a variety of cellshelper T cellsmacrophagesmonocytesB cellsmicroglial brain cellsintestinal cells

T cells infected later on.

Early Phase HIV InfectionIn early phase HIV

infection, initial viruses are M-tropic. Their envelope glycoprotein gp120 is able to bind to CD4 molecules and chemokine receptors called CCR5 found on macrophages

Viral Replication

In late phase HIV infection, most of the viruses are T-tropic, having gp120 capable of binding to CD4 and CXCR4 found on T4-lymphocytes.

Viral ReplicationThe gp120 protein on virus binds

specifically to CD4 receptor on host cell with high affinity.

Gp41 causes fusion of the virus to the cell membrane.After fusion virus particle enters cell.Viral genome exposed by uncoating particle.

Viral ReplicationReverse transcriptase produces viral DNA

from RNA.Becomes a provirus which integrates into

host DNA.Period of latency occurs.

Viral ReplicationAfter a period of latency lasting up to 10

years viral replication is triggered and occurs at high rate.

CD4 cell may be destroyed in the process, body attempts to replace lost CD4 cells, but over the course of many years body is unable to keep the count at a safe level.

Destruction of large numbers of CD4 cause symptoms of HIV to appear with increased susceptibility to opportunistic infections, disease and malignancy.

HIV (arrows) Infecting a T-lymphocyte

Viral ReplicationMethods of transmission:

Sexual transmission, presence of STD increases likelihood of transmission.

Exposure to infected blood or blood products.Use of contaminated clotting factors by

hemophiliacs.Sharing contaminated needles (IV drug users).Transplantation of infected tissues or organs.Mother to fetus, perinatal transmission

variable, dependent on viral load and mother’s CD 4 count.

Transmission

Primary HIV SyndromeMononucleosis-like, cold or flu-like

symptoms may occur 6 to 12 weeks after infection.lymphadenopathyfeverrashheadacheFatiguediarrheasore throatneurologic manifestations.no symptoms may be present

Primary HIV SyndromeSymptoms are relatively nonspecific.HIV antibody test often negative but

becomes positive within 3 to 6 months, this process is known as seroconversion.

Large amount of HIV in the peripheral blood.

Primary HIV can be diagnosed using viral load titer assay or other tests.

Primary HIV syndrome resolves itself and HIV infected person remains asymptomatic for a prolonged period of time, often years.

Clinical Latency PeriodHIV continues to reproduce, CD4 count

gradually declines from its normal value of 500-1200.

Once CD4 count drops below 500, HIV infected person at risk for opportunistic infections.

The following diseases are predictive of the progression to AIDS:persistent herpes-zoster infection (shingles)oral candidiasis (thrush)oral hairy leukoplakiaKaposi’s sarcoma (KS)

Oral Candidiasis (thrush)

Oral Hairy Leukoplakia

Being that HIV reduces immunologic activity, the intraoral environment is a prime target for chronic secondary infections and inflammatory processes, including OHL, which is due to the Epstein-Barr virus under immunosuppressed conditions

Kaposi’s sarcoma (KS)Kaposi’s sarcoma

(shown) is a rare cancer of the blood vessels that is associated with HIV. It manifests as bluish-red oval-shaped patches that may eventually become thickened. Lesions may appear singly or in clusters.

AIDSCD4 count drops below 200 person is

considered to have advanced HIV diseaseIf preventative medications not started the HIV

infected person is now at risk for:Pneumocystis carinii pneumonia (PCP)cryptococcal meningitistoxoplasmosis

If CD4 count drops below 50:Mycobacterium aviumCytomegalovirus infectionslymphomadementiaMost deaths occur with CD4 counts below 50.

Other Opportunistic Infections Respiratory system

Pneumocystis Carinii Pneumonia (PCP) Tuberculosis (TB) Kaposi's Sarcoma (KS)

Gastro-intestinal system Cryptosporidiosis Candida Cytomegolavirus (CMV) Isosporiasis Kaposi's Sarcoma

Central/peripheral Nervous system Cytomegolavirus Toxoplasmosis Cryptococcosis Non Hodgkin's lymphoma Varicella Zoster Herpes simplex

Skin Herpes simple Kaposi's sarcoma Varicella Zoster

Infants with HIVFailure to thrivePersistent oral candidiasisHepatosplenomegalyLymphadenopathyRecurrent diarrheaRecurrent bacterial infectionsAbnormal neurologic findings.

Immunologic ManifestationsEarly stage slight depression of CD4 count,

few symptoms, temporary.Window of up to 6 weeks before antibody is

detected, by 6 months 95% positive.During window p24 antigen present, acute

viremia and antigenemia.

Immunologic ManifestationsAntibodies produced to all major antigens.

First antibodies detected produced against gag proteins p24 and p55.

Followed by antibody to p51, p120 and gp41As disease progresses antibody levels

decrease.

Immunologic ManifestationsImmune abnormalities associated with

increased viral replication.Decrease in CD4 cells due to virus budding from

cells, fusion of uninfected cells with virally infected cells and apoptosis.

B cells have decreased response to antigens possibly due to blockage of T cell/B cell interaction by binding of viral proteins to CD4 site.

CD8 cells initially increase and may remain elevated.

As HIV infection progresses, CD4 T cells drop resulting in immunosuppression and susceptibility of patient to opportunistic infections.

Death comes due to immuno-incompetence.

‘typical’ primary HIV-1 infection

symptoms

HIV-1 p24 antigen

0 1 2 3 4 5 6 / 2 4 6 8 10

weeks years

HIV antibodies

Time following infection

HIV viral load

HIV proviral DNA

symptoms

‘window’period

1° infection

Laboratory Diagnosis of HIV InfectionMethods utilized to detect:

AntibodyAntigenViral nucleic acidVirus in culture

ELISA TestingFirst serological test developed to detect

HIV infection.Easy to perform.Easily adapted to batch testing.Highly sensitive and specific.

Antibodies detected in ELISA include those directed against: p24, gp120, gp160 and gp41, detected first in infection and appear in most individuals

ELISA TestingELISA tests useful for:

Screening blood products.Diagnosing and monitoring patients.Determining prevalence of infection.Research investigations.

ELISA TestingDifferent types of ELISA techniques used:

indirectcompetitivesandwich

ELISAs are for screening only, false positives do occur and may be due to AI disease, alcoholism, syphilis, and immunoproliferative diseases.

ELISA Sandwich

Other Screening TestsAgglutination tests using latex particles,

gelatin particles or microbeads are coated with HIV antigen and will agglutinate in the presence of antibody.

Dot-Blot Testing utilizes paper or nitrocellulose impregnated with antigen, patient serum is filtered through, and anti-antibody is added with enzyme label, color change is positive.A rapid, cost-effective and may become an

alternative to standard ELISA and Western blot testing.

Western BlotMost popular confirmatory test.

Utilizes a lysate prepared from HIV virus.The lysate is electrophoresed to separate out

the HIV proteins (antigens).The paper is cut into strips and reacted with

test sera.After incubation and washing anti-antibody

tagged with radioisotope or enzyme is added.Specific bands form where antibody has

reacted with different antigens.Most critical reagent of test is purest quality

HIV antigen.The following antigens must be present: p17,

p24, p31, gp41, p51, p55, p66, gp120 and gp160.

Western BlotAntibodies to p24 and p55 appear earliest

but decrease or become undetectable.Antibodies to gp31, gp41, gp 120, and

gp160 appear later but are present throughout all stages of the disease.

Western BlotInterpretation of results.

No bands, negative.In order to be interpreted as positive a

minimum of 3 bands directed against the following antigens must be present: p24, p31, gp41 or gp120/160.

CDC criteria require 2 bands of the following: p24, gp41 or gp120/160.

DNA PCRDNA PCRRNA PCRRNA PCR

p24 Agp24 Ag3rd gen ELISA1st gen ELISA

Detuned ELISA1wk 2wk 3wk 2mo 6mo 1yr 2yr 3yr +8yr

gp160gp120

p68p55p53

gp41-45

p40

p34

p24

p18

p12

gp160gp120

p68p55p53

gp41-45

p40

p34

p24

p18

p12

gp160gp120

p68p55p53

gp41-45

p40

p34

p24

p18

p12

early recent / established advanced

Spectrum Spectrum of anti-HIV of anti-HIV

testing testing

Western BlotExpensive – $ 80 - 100technically more

difficultvisual interpretationlack standardisation

- performance- interpretation- indeterminate reactions

– resolution of ??‘Gold Standard’ for

confirmation

Western BlotIndeterminate results are those samples that

produce bands but not enough to be positive, may be due to the following:prior blood transfusions, even with non-HIV-1 infected

bloodprior or current infection with syphilisprior or current infection with malariaautoimmune diseases (e.g., diabetes, Grave’s disease,

etc)infection with other human retrovirusessecond or subsequent pregnancies in women.run an alternate HIV confirmatory assay.

Quality control of Western Blot is critical and requires testing with strongly positive, weakly positive and negative controls.

Indirect immunofluorescenceCan be used to detect both virus and

antibody to it.Antibody detected by testing patient serum

against antigen applied to a slide, incubated, washed and a fluorescent antibody added.

Virus is detected by fixing patient cells to slide, incubating with antibody.

Detection of p24 HIV antigenThe p24-antigen screening assay is an EIA

performed on serum or plasma. P24 antigen only present for short time,

disappears when antibody to p24 appears.

Anti-HIV-1 bound to membrane, incubated with patient serum, second anti-HIV-1 antibody attached to enzyme label is added (sandwich technique), color change occurs.

Optical density measured, standard curve prepared to quantitate results.

Detection of p24 HIV antigenPositive confirmed by neutralizing reaction,

preincubate patient sample with anti- HIV, retest, if p24 present immune complexes form preventing binding to HIV antibody on membrane when added.

Test not recommended for routine screening as appearance and rate of rise are unpredictable.

Sensitivity lower than ELISA.

Detection of p24 HIV antigenMost useful for the following:

early infection suspected in seronegative patient

newbornsCSFmonitoring disease progress

Polymerase Chain Reaction (PCR)Looks for HIV DNA in the WBCs of a person.PCR amplifies tiny quantities of the HIV DNA

present, each cycle of PCR results in doubling of the DNA sequences present.

The DNA is detected by using radioactive or biotinylated probes.

Once DNA is amplified it is placed on nitrocellulose paper and allowed to react with a radiolabeled probe, a single stranded DNA fragment unique to HIV, which will hybridize with the patient’s HIV DNA if present.

Radioactivity is determined.

Virus isolationVirus isolation can be used to definitively

diagnose HIV.Best sample is peripheral blood, but can

use CSF, saliva, cervical secretions, semen, tears or material from organ biopsy.

Cell growth in culture is stimulated, amplifies number of cells releasing virus.

Cultures incubated one month, infection confirmed by detecting reverse transcriptase or p24 antigen in supernatant.

Viral Load TestsViral load or viral burden is the quantity of

HIV-RNA that is in the blood.RNA is the genetic material of HIV that

contains information to make more virus.

Viral Load TestsViral load tests measure the amount of

HIV-RNA in one milliliter of blood.Take 2 measurements 2-3 weeks apart

to determine baseline.Repeat every 3-6 months in conjunction

with CD4 counts to monitor viral load ant T-cell count.

Repeat 4-6 weeks after starting or changing antiretroviral therapy to determine effect on viral load.

Testing of NeonatesDifficult due to presence of maternal IgG

antibodies.Use tests to detect IgM or IgA antibodies,

IgM lacks sensitivity, IgA more promising.Measurement of p24 antigen.PCR testing may be helpful but still not

detecting antigen soon enough: 38 days to 6 months to be positive.

Treatment - The Move Toward Lower Pill Burdens

Dosing Daily pill burdenRegimen

1996

Zerit/Epivir/Crixivan 10 pills, Q8H

20023 pills, BIDCombivir (AZT/3TC)/EFV

1998Retrovir/Epivir/Sustiva 5 pills, BID

20033 pills, QDViread/ Emtriva/Sustiva

20042 pills, QDTruvada/Sustiva

Sustiva + Truvada TreatmentSustiva + Truvada (FTC + tenofovor) is one of

the most popular and effective starting HIV regimens.

Many patients will have dream,sleep, central nervous system effects particularly in the first month (due to the Sustiva).

Upset stomach, bloating, gas, loose stools is also fairly common during the first month and for most patients is fairly mild.

HIV levels in the blood will often drop by > 99% in the first month and the CD4 count (marker of immune system function) will often increase providing protection against AIDS related diseases within weeks or months of starting the medication.

TruvadaTruvada is made up of HIV drugs from a class

called nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), also known as “nukes.”

NRTIs block reverse transcriptase, a protein that HIV needs to make more copies of itself.

Prevents HIV from altering the genetic material of healthy CD4 cells.

Prevents the cells from producing new virus and decreases the amount of virus in the body.

May slow down HIV disease

References http://www.cat.cc.md.us/courses/bio141/lecguide/unit2/viruses/

hivlc.html#translat

http://pathmicro.med.sc.edu/lecture/HIV3.htm

http://www.avert.org/hivstages.htm http://www.aidsinfo.nih.gov/guidelines/ http://www.hopkins-aids.edu/publications/pocketguide/

pocketgd0105.pdf http://www.modares.ac.ir/sci/saman_h/Pages/applications.htm http://hivinsite.ucsf.edu/InSite?page=kb-02&doc=kb-02-02-

02-02 http://www.hivandhepatitis.com/recent/test/realtime/

061604_f.html