Human Immunodeficiency Virus (HIV)

1. Human Immunodeficiency Virus (HIV) GayaneArutunyan Monica Brown David Yang

2. Patient • 28 year old male • Bad case of thrush (oral candidiasis) • Low grade fever • Lost 20 pounds in the last year without dieting • Serious bouts of diarrhea • Stool Sample tested positive for Giardia lamblia. • Difficulty breathing • Radiographic examination showed bilateral infiltrate (characteristic of Pneumocystosis carinii pneumonia)

3. Radiographic Results (A.D.A.M., 2006)

4. Patient • 28 year old male • Bad case of thrush (oral candidiasis) • Low grade fever • Lost 20 pounds in the last year without dieting • Serious bouts of diarrhea • Stool Sample tested positive for Giardia lamblia. • Difficulty breathing • Radiographic examination showed bilateral infiltrate (characteristic of Pneumocystosis carinii pneumonia) • Heroin addict and admits to sharing needles at a “shooting gallery”

5. Human Immunodeficiency Virus (HIV) Icosahedral or Wedge-shaped Nucleocapsid gp120 gp160 gp41 Reverse Transcriptase single stranded (+) sense RNA Lipid Membrane (NIH, 2005)

6. HIV genome Two copies (+) ss RNA (Dimmock, et. al, 2007)

7. Types of HIV • Two species of HIV infect humans: • HIV-1 • More virulent, relatively easy to transmit • Majority of HIV infections globally • 3 types of HIV-1: (based on alterations in env gene) • Clades M, N, and O • HIV-2 • Less transmittable • Largely confined to West Africa (Gao, et. al; 1999) (Keele, et. al; 2006) (Reeves, et. al; 2002) (Thompson, et. al; 2002)

9. Laboratory Tests • Enzyme Linked Immunosorbent Assay (ELISA) • Direct : Tests for the virus • Indirect : Tests for antibodies to the virus • Enzyme Linked Immunosorbent Assay (ELISA) • Direct : Tests for the virus • Indirect : Tests for antibodies to the virus

10. Indirect ELISA Rinse Rinse HIV Antigen Patient’s serum Rinse No Color Change Color Change Enzyme-labeled antihuman IgG

11. Laboratory Tests • Enzyme Linked Immunosorbent Assay (ELISA) • Direct : Tests for the virus • Indirect : Tests for antibodies to the virus • Western Blot Analysis • Direct : analyze samples for specific protein(s) • Indirect : analyze samples for antibodies against a specific protein(s) • Enzyme Linked Immunosorbent Assay (ELISA) • Direct : Tests for the virus • Indirect : Tests for antibodies to the virus • Western Blot Analysis • Direct : analyze samples for specific protein(s) • Indirect : analyze samples for antibodies against a specific protein(s)

12. Indirect Western Blot Analysis

13. HIV – Western Blot Results gp160 = viral envelope precursor (env) gp120 = viral envelope protein binds to CD4 p31 = reverse transcriptase (pol) p24 = viral core protein (gag) HIV (+) serum HIV (-) serum (Univ. of Arizona, 1996)

14. Patient Risk Assessment • High Risk Behaviors: • Sharing of infected drug injection needles/syringes • Accidental needle stick (healthcare professionals) • Unprotected sex with infected individual • Blood transfusions/Organ transplants • Rare, only occurs in underdeveloped countries with inadequate blood donor testing programs • Transmission from infected mother-to-fetus during pregnancy or delivery • Transmission from breast milk of infected mother to her baby

16. Prevention of HIV Infection • Known Methods of Preventing HIV Infection: • Avoid sexual contactwith infected individuals • Avoid sharing needles/syringesthat could be contaminated with HIV • Avoid any type of contact with the bodily fluidof an infected individual

17. Prevention of HIV Infection • New Methods for Prevention of HIV: • Intravaginal lime or lemon juice to kill the HIV virus before entry • Intravaginal oestrogen: Karotinizing the vagina with estrogen • Male circumcision: Removal of the inner foreskin removes the main site of HIV entry into the penis, resulting in a sevenfold reduction in susceptibility to infection • Post-coital penile hygiene: Wiping the penis immediately after intercourse with lime or lemon juice or vinegar should kill the virus before it has had a chance to infect.

18. Immunological Basis for Opportunistic Infection • HIV targets cells that express CD4 and can infect macrophages, dendritic cells, and activated T cells • During the non-symptomatic phase of HIV infection, HIV has a low affinity towards T cells and higher affinity towards macrophages → slow death rate of T cells • Once the virus becomes lymphotropic, it begins to infect activated T cells far more efficiently due to increased affinity for CD4 • Functional T cell levels begin to decrease, eventually to a point where the T cell population is too small to recognize the full range of Ags that could potentially be detected • The lack of full Ag recognition → core symptoms of acquired immune deficiency syndrome (AIDS). CD4 T cell depletion during AIDS allows various pathogens to escape T cell recognition, thus allowing opportunistic infections that would normally elicit a helper T cell response to bypass the immune system

19. Immunological Basis for Opportunistic Infection • Nef protein – advances the endocytosis and degradation of cell surface proteins, including CD4 and MHC proteins • Downregulates expression of host CD4 • Downregulates expression of host MHC class I molecules • MHC class I molecules function to present peptide fragments to cytotoxic T cells to destroy virally infected cells. • Induces phosphorylation of MA proteins to increase viral infectivity (increased viral replication) • MA and Vpr proteins aid in facilitating the transport of HIV pre-integration complex across nuclear membrane. • Alter T cell signaling to promote viral replication.

20. Opportunistic Infections CD4 Count Diseases 200-500/μL Pneumonia (usually caused by bacteria) Tuberculosis in the lungs Oral or vaginal yeast infections Shingles (viral skin infection) Oral hairy leukoplakia Kaposi’s sarcoma 100-200/μL All of the above plus: Pneumoniadue to Pneumocystis carinii (PCP) Chronic diarrhea 50-100/ μL All of the above plus: Encephalitis (usually due to toxoplasmosis) Esophagitis due to yeast or viruses Meningitis (usually due to cryptococcus)

21. Opportunistic Infections CD4 Count Diseases (50-100/µL Tuberculosis outside the lungs Continued) Chronic herpes simples virus (HSV infection) Primary brain lymphoma <50 /µL All of the above, plus: Widespread infection due to Mycobacterium avium complex Retinitis, diarrhea, encephalitis due to cytomegalovirus

22. Epidemiology • The virus was first successfully identified and classified in 1981. • The appearance of rare diseases amongst HIV infected persons first alerted scientists to the then unknown virus. • At end of 2003 in the U.S. 1,039,000 - 1,185,000 persons infected with HIV/AIDS • Global (2006 statistics): 39.5 million infected with HIV/AIDS • 4.3 million new infections in 2006; 2.8 million (65%) of these were in sub-Saharan Africa • Increases in Eastern Europe and Central Asia, where there may have been a more than 50% rise in infection rates since 2004 • In 2006, 2.9 million deaths due to AIDS-related illnesses

23. 2003

24. Human Immunodeficiency Virus (HIV) has an incubation period of about 10 years and eventually leads to Acquired Immunodeficiency Syndrome (AIDS), resulting in the impairment of the immune system. This can lead to death from infections, secondary diseases from opportunistic bacteria and/or viruses that are usually harmless to people, or many different types of cancers. Common diseases associated with HIV infection: Kaposi's sarcoma (KS) Pneumocystis carinii pneumonia (PCP) Mycobacterium avium complex (MAC) HIV and Disease • Early Symptoms: • Most don’t exhibit symptoms when first infected • However, may have flu-like symptoms (fever, headache, tired, enlarged lymph nodes) 1-2 months after exposure • Very infectious during this period

25. HIV and Disease Later Symptoms: • More sever symptoms may not appear until after 10yrs, however this varies with each individual • Decline in number of CD4 + T cells • The most advanced stage of AIDS is classified as having < 200 CD4+ T cells/cubic millimeter of blood (in healthy adults CD4+ T-cell counts = 1,000+) • Onset of AIDS is characterized by: • weight loss, • fevers/sweats, • fatigue • rashes/flaky skin, • persistent yeast infections, • Pelvic inflammatory disease in women will not respond to treatments, • short-term memory loss, • frequent and severe herpes infections, • shingles • coma • deaths

27. Precautions • Pre-Exposure • Assume any patient’s blood and other bodily fluids are potentially infectious • Protective barriers • Hands and all other skin surfaces must be immediately washed after any contact with blood or body fluids • Extra precaution must be taken in the handling and proper disposal of any sharp instruments used on a patient Post-Exposure • In post-exposure cases, the Center for Disease Control and Prevention (CDC) recommends the post exposure prophylaxis (PEP) • Antiretroviral medications (Tenofovir disoproxil fumarate and emtricitabine) administered to HIV-infected women during labor and delivery has shown ~50% reduction in the risk of mother-to-child transmission • Antiretroviral regimens have been shown to be associated with an 80% reduction in risk of HIV infection among healthcare personnel following needle sticks and other accidental exposures, when treatment is initiated promptly

28. Treatments • Reverse Transcriptase (RT) Inhibitors – interrupt early stage of viral replication to slow spread of HIV in body and delay start of opportunistic infections • Nucleoside/nucleotide RT inhibitors • Insert faulty DNA building blocks into HIV genome • Prevents completion of DNA chain → no replication • Non-nucleoside RT inhibitors • Bind to reverse transcriptase and prevents reverse transcription • May slow the spread of HIV in the body and delay the start of opportunistic infections. Abacavir Combivir Lamivudine Trizivir Zalcitabine Stavudine Zidovudine Didanosine Delavirdine Efavirenz Nevirapine

29. Treatments Kaletra Saquinavir Ritonavir Amprenavir Fortovase Nelfinavir • Protease Inhibitors – interrupt late stage of viral replication in the HIV life cycle • Side Effects: nausea, diarrhea, gastrointestinal symptoms, serious effects from interaction with other drugs • Fusion Inhibitors – new class of drugs • Fuzeon (enfuvirtide or T-20) interferes with HIV-1’s ability to enter the cell by blocking fusion of the virus to the host cell membrane • HIV cannot enter and infect immune cells • Designed to be used in combination with other anti-HIV treatments • reduces the level of HIV infection in blood • may be effective against HIV resistant to current antiviral treatments • Side Effects: pneumonia, trouble breathing, low bp, chills/fever, skin rash, blood in urine, vomiting Indinavir

30. Treatments • HAART • Treatment has significantly reduced number of deaths from AIDS in the U.S. • Reduces the amount of virus circulating in blood to nearly undetectable levels, although HIV is still present by hiding places such as the brain, lymph nodes, testes, & retina of the eye • Side Effects: severe; decrease in RBCs or WBCs, inflammation of pancreas, painful nerve damage, even death in some cases

31. Vaccines Currently, no vaccines approved for use by the FDA Two types in development: Therapeutic Vaccine - intended to boost the immune systems of those already infected Preventive Vaccine – intended to generate an immune response in an uninfected person to prevent future infection

32. Possible Targets of a Theoretical HIV Vaccine: • gp120, gp160 and gp41 viral surface proteins • to prevent it from binding to the host cell receptor CD4. • Cyclophilin A • At the cell surface – to block from binding to the heparin sulfate receptors • Within the cytoplasm – to keep from expanding the viral core. • Without expansion of the viral core, reverse transcription cannot occur. • Reverse transcriptase • Unable to synthesize new copies of the viral genome. • CCR5 (chemokine) receptor • Cleavage – to inactivate the receptor. • Approximately 13% of people of northern European descent have a naturally occurring deletion of 32 base pairs in the CCR5 gene results. • This mutant CCR5 receptor never reaches the surface of their cells. • Homozygotes for this mutation (1-2% Caucasians) have a resistance to HIV infection.

33. Preventive HIV Vaccines Under Development: • Subunit vaccines – "component" or "protein" vaccines that contain an individual viral components rather than the whole virus • The subunits are made through genetic engineering and administered to induce an anti-HIV immune response • The weak response however may not prevent future infections • Recombinant vector vaccines– use of non-HIV viruses that do not cause disease in humans or have been rendered unable to cause disease (attenuated) • viruses are used as vectors carry copies of HIV genes into human cells, in which HIV proteins will be produced • HIV proteins can stimulate an anti-HIV immune response. The response may be stronger since the recombinant vector vaccines delivers several HIV genes • Vectors being studied for use in HIV vaccines are ALVAC (canarypox virus), MVA (type of cowpox virus), VEE (normally a horse virus), and adenovirus-5 (human virus; usually doesn’t cause serious disease)

34. Preventive HIV Vaccines Under Development: • DNA vaccines–introduce HIV genes into the body, but don’t rely on a virus vector • “Naked" DNA containing HIV genes injected directly into body so that the cells will take it up and produce HIV proteins • HIV proteins will induce immune response against HIV • Prime-Boost Vaccination Strategy • Use one type of vaccine, then administer a second type later to stimulate different parts of the immune system to boost up the overall defense of the body

35. Who Can Benefit from HIV Vaccines? • Healthcare professionals • Blood/organ recipients (especially in underdeveloped countries) • Countries where a significant number of the population has people infected with HIV and has poor health care • Individuals in high risk groups

36. References Dimmock, N.J., Easton, A.J., and K.N. Leppard. Introduction to Modern Virology, 6th Edition. Blackwell publishing Ltd. 2007. Gao, F., Bailes, E., Robertson, D. L., Chen, Y., Rodenburg, C. M., Michael, S. F., Cummins, L. B., Arthur, L. O., Peeters, M., Shaw, G. M., Sharp, P. M., and Hahn, B. H. (1999). "Origin of HIV-1 in the Chimpanzee Pan troglodytes troglodytes". Nature397 (6718): 436-441. doi:10.1038/17130. PMID 9989410. Keele, B. F., van Heuverswyn, F., Li, Y. Y., Bailes, E., Takehisa, J., Santiago, M. L., Bibollet-Ruche, F., Chen, Y., Wain, L. V., Liegois, F., Loul, S., Mpoudi Ngole, E., Bienvenue, Y., Delaporte, E., Brookfield, J. F. Y., Sharp, P. M., Shaw, G. M., Peeters, M., and Hahn, B. H. (2006). "Chimpanzee Reservoirs of Pandemic and Nonpandemic HIV-1". ScienceOnline 2006-05-25. doi:10.1126/science.1126531 McQueen, Nancy. Microbiology 401 lectures material. California State University, Los Angeles. 2007 MedLinePlus. "HIV ELISA/western blot." U.S. National Library of Medicine. Last accessed April 16, 2007. http://www.nlm.nih.gov/medlineplus/ency/article/003538.htm Reeves, J. D. and Doms, R. W (2002). "Human Immunodeficiency Virus Type 2". J. Gen. Virol.83 (Pt 6): 1253-1265. PMID 12029140 Thomson, M. M., Perez-Alvarez, L. and Najera, R. (2002). "Molecular epidemiology of HIV-1 genetic forms and its significance for vaccine development and therapy". Lancet Infect. Dis.2 (8): 461-471. PMID 12150845 http://www.niaid.nih.gov/factsheets/hivinf.htm http://hivinsite.ucsf.edu/InSite?page=kb-02&doc=kb-02-01-01 http://www.cdc.gov/hiv http://aidsinfo.nih.gov/ContentFiles/HIVPreventionVaccines_FS_en.pdf http://www.cdc.gov/hiv/resources/factsheets/At-A-Glance.htm http://www.globalrph.com/hivupdate.htm http://www.tpan.com/publications/pa/jan_feb_06/fuzeon.shtml http://data.unaids.org/pub/GlobalReport/2006/2006GR-PrevalenceMap_en.pdf http://www.who.int/hiv/topics/me/en/index.html

37. Picture references • Dimmock, N.J., Easton, A.J., and K.N. Leppard. Introduction to Modern Virology, 6th Edition. Blackwell publishing Ltd. 2007. • http://wildiris3.securesites.net/cms_prod/files/course/192/WAHIV4_2007_fig1.jpg • http://www.eduwhere.com/images/lab_goggles.jpg • http://www.osha.gov/needlesticks/biohazard-sample2.jpg • http://www1.istockphoto.com/file_thumbview_approve/2209865/2/istockphoto_2209865_hiv_negative.jpg • http://www.inmarkinc.com/images/hinew02.jpg • http://www.ipngos.org/images/vaccine.jpg • http://www.who.int/hiv/facts/en/hiv_global2003.jpg • http://www.stanford.edu/group/virus/retro/2005gongishmail/hiv2.jpg • http://pathmicro.med.sc.edu/images/global.gif • http://www.metrokc.gov/HEALTH/apu/healthed/background/global.gif • http://www.niaid.nih.gov/factsheets/howhiv.htm • http://www.biology.arizona.edu/immunology/activities/western_blot/west2.html

38. Questions?