Alison Fitzgerald, MD

PGY-3

November 25, 2013

CLASSICS

CLINICAL SCENARIO – NOVEMBER 2000• 34 year old M presents to PCP for annual exam and blood work

• PMHx: None

• PSHx: None

• Social Hx:

• Worked as a research associate in a prestigious lab; currently pursing a master’s degree in political science in Washington, DC

• Grew up in MA and CA; attended private liberal arts college in CA

• homosexual male; 2 male partners in the past while in monogamous relationships; occasionally engaged in unprotected sex; several previous sexual relationships with women, also occasionally engaging in unprotected sex

• No tobacco, occasional EtOH

• Medications: None

• Allergies: None

BLOOD WORK FROM THIS VISIT…

• HIV positive

PHONE CALL FROM FRIEND…

• He was appropriately devastated by news

• He does not know what to do

• How did this happen to him???

THIS BRINGS US BACK TO THE EARLY 80’S• (December 1970: Robert Gallo first reports finding reverse transcriptase in human leukemia

cells)

• December 1980: Bernard Poiesz and Frank Ruscetti report isolation of the first human cancer virus, HTLV

• June 1981: UCLA’s Michael Gottlieb reports first six cases of what will later be called AIDS

• June 1981: CDC publish a Morbidity and Mortality Weekly Report describing cases of a rare lung infection, Pneumocystis carinii pneumonia in 5 young, previously healthy, gay men; 2 had died by the time the report was published; 270 cases reported by year-end and 121 had died

• July 1981: Cases of Kaposi’s Sarcoma reported affecting 41 gay men

• January 1982: first American AIDS clinic is established in San Francisco

• I was in middle school!

• Ronald Reagan was elected president in 1980

ALSO IN THE EARLY 80’S

MORE FROM THE EARLY 80’S• November 1982: Gallo reports the isolation of a substrain of his human retrovirus, called

HTLV-II, from a leukemic human T-cell line

• December 1982: CDC reports a case of AIDS in an infant who received blood transfusions

• January 1983: CDC reports cases of AIDS in female sexual partners of males with AIDS

• March 1983: Gallo sends Science a paper reporting the detection of HTLV in 2 East Coast homosexuals with AIDS

• March 1983: Harvard’s Max Essex submits a paper to Science reporting that 25-40% of AIDS patients have antibodies to a mysterious protein on the surface of HTLV-infected cells

• May 20, 1983: Professor Luc Montagnier of the Pasteur Institute in France reports the discovery of a retrovirus named Lymphadenopathy Associated Virus (LAV) that could be the cause of AIDS

ISOLATION OF A T-LYMPHOTROPIC RETROVIRUS FROM A PATIENT AT RISK FOR ACQUIRED IMMUNE DEFICIENCY SYNDROME (AIDS)

F BARRE-SINOUSSI, JC CHERMANN, F REY, MT NUGEYRE, S CHAMARET, J GRUEST, C DAUGUET, C AXLER-BLIN, F VEZINET-BRUN, C ROUZIOUX, W ROZENBAUM, AND L MONTAGNIER

SCIENCE 20 MAY 1983: 868-871

ACQUIRED IMMUNE DEFICIENCY SYNDROME (AIDS) – WHAT WAS KNOWN• Recently been recognized in several countries

• Disease had been reported mainly in homosexual males with multiple partners

• Epidemiological studies suggested horizontal transmission by sexual routes, as well as by IV drug administration and blood transfusion

• The pronounced depression of cellular immunity and the quantitative modifications of subpopulations of T lymphocytes suggest that T cells or a subset of T cells might be a preferential target for the putative infectious agent

• The depressed cellular immunity may result in serious opportunistic infections in AIDS patients, many of whom develop Kaposi’s sarcoma

• A picture of persistent multiple lymphadenopathies has also been described in homosexual males and infants who may or may not develop AIDS; the histological aspect of such lymph nodes is that of reactive hyperplasia; such cases may correspond to an early or a milder form of the disease

FINDING THE VIRUS… WHAT THEY REPORTED

• The isolation of a novel retrovirus from a lymph node of a homosexual patient with multiple lymphadenopathies. The virus appears to be a member of the human T-cell leukemia virus (HTLV) family

FINDING THE VIRUS… HOW THEY DID IT• The retrovirus was propagated in culture of T lymphocytes from a healthy donor and from

umbilical cord blood of newborn humans

• Viral core proteins were not immunologically related to the p24 and p19 proteins of subgroup I of HTLV. However, serum of the patient reacted strongly with surface antigen(s) present on HTLV-1-infected cells. AND the ionic requirements of the viral reverse transcriptase were close to that of HTLV.

• HTLV-II virus (type-C retrovirus) also identified in T cells from a patient with hairy cell leukemia; highly related to HTLV-I

• The new retrovirus appears to be different from HTLV-I and –II but, along with the previous HTLV isolates, it was tentatively concluded that it belongs to a family of T-lymphotropic retroviruses that are horizontally transmitted in humans and may be involved in several pathological syndromes including AIDS

PATIENT 1• 33 year-old homosexual male who sought medical consultation in December 1982 for cervical

lymphadenopathy

• Examination showed axillary and inguinal lymphadenopathies

• No fever or weight loss

• H/o several episodes of gonorrhea; treated for syphilis in September 1982

• Had more than 50 sexual partners per year

• Traveled to many countries including North Africa, Greece and India

• Last trip to NYC was in 1979

LYMPH NODE FROM PATIENT 1

• Lymph node bx revealed numerous T lymphocytes (OKT3+): 62% were T lymphocytes, 44% were T-helper cells and 16% were suppressor cells

• Cells were put into culture medium and after 15 days, a reverse transcriptase activity was detected in the culture supernatant by using the ionic conditions described for HTLV-I

• Peripheral blood lymphocytes were cultured in the same way and were consistently negative for reverse transcriptase

VIRUS TRANSMISSION• Using a culture of T lymphocytes from an adult healthy donor

• On day 3, half the culture was cocultivated with the lymphocytes from the biopsy from patient 1

• Reverse transcriptase activity was detected on day 15 of the coculture, and it had the same characteristics as that released by patient 1’s cells

• Cells from the uninfected culture of lymphocytes from the healthy donor did not release reverse transcriptase activity

• The cell-free supernatant of the infected coculture was used to infect 3-day old cultures of T lymphocytes from two umbilical cords, LC1 and LC5; after a lag period of 7 days, reverse transcriptase activity was detected in both of the cord lymphocyte cultures (identical cultures which had not been infected remained negative)

• Implications: the virus could be propagated on normal lymphocytes from either newborns or adults

PROVING THAT IT WAS A RETROVIRUS

• Its use of reverse transcriptase

• Its density in a sucrose gradient (1.16)

• By its labeling with [3H]uridine

• Electron microscopy of the infected umbilical cord lymphocytes showed characteristic immature particles with dense crescent (C-type) budding at the plasma membrane

ELECTRON MICROSCOPY OF THE INFECTED UMBILICALCORD LYMPHOCYTES SHOWED CHARACTERISTICIMMATURE PARTICLES WITH DENSECRESCENT (C-TYPE) BUDDING AT THE PLASMAMEMBRANE

NEW ISOLATE INDISTINGUISHABLE FROM HTLV-I ISOLATES?

• Antibodies used were specific for the p19 and p24 core proteins of HTLV-I

• Monoclonal ab to p19 and a polyclonal goat ab to p24 were used in an indirect fluorescent assay against infected cells from the biopsy of patient 1 and lymphocytes obtained from a healthy donor and infected with the same virus

• The virus-producing cells did not react with either type of antibody, whereas two lines of cord lymphocytes chronically infected with HTLV-I and used as controls showed strong surface fluorescence

• When serum from patient 1 was tested against infected lymphocytes from the biopsy, the surface fluorescence was as intense as that of the control HTLV-producing lines. This suggests that the serum of the patient contains antibodies that recognize a common antigen present on HTLV-I-producing cells AND on the patient’s lymphocytes

• Similarly, cord lymphocytes infected with the virus from patient 1 DID NOT react with antibodies to p19 or p24

TABLE 1. INDIRECT IMMUNOFLUORESCENCE ASSAY

Cell type Immunofluorescence (percent positive)Antibody to p19 Antibody to p24 Serum from patient 1

Normal blood lymphocytes

N 10916 - - -

LC1 - - -

HTLV-producing cells

C91/PL + (90 to 100) + (90 to 100) + (90 to 100)

C10/MJ2 + (90 to 100) + (90 to 100) + (90 to 100)

Virus-producing cells from

Patient 1 + (90 to 100)

LC1/patient 1 +/- (0.5 to 2)

Patient 2 + (90 to 100)

PATIENT 2• T lymphocytes were also cultured from another patient who presented with multiple

lymphadenopathies and who had been in close contact with an AIDS case

• These lymphocytes DID NOT produce viral reverse transcriptase, however, they reacted in the immunofluorescence assay with serum from patient 1

• AND serum from patient 2 reacted strongly with control HTLV-producing lines

• So now it was important to determine which viral antigen was recognized by antibodies present in the two patients’ sera using polyacrylamide gel electrophoresis

P25 PROTEIN PRESENT IN VIRUS-INFECTED CELLS FROM PATIENT 1 AND IN LC1 CELLS INFECTED WITH THIS VIRUS, WAS SPECIFICALLY RECOGNIZED BY SERUM FROM PATIENTS 1 AND 2 BUT NOT BY ANTISERUM TO HTLV-1 P24 OR SERUM OF NORMAL DONORS

AND CONVERSELY…

• The p24 present in control HTLV-infected cell extracts was recognized by antibodies to HTLV but NOT by serum from patient 1

• A weak band (lane 2, Fig. 3B) could hardly be seen with serum from patient 2, suggesting some similarities of the p25 protein from this patient’s cells with HTLV-I p24

• When purified, labeled virus from patient 1 was analyzed and 3 major proteins could be seen: the p25 protein and proteins with MW of 80K and 45K

CONCLUSIONS OF THIS PAPER

• The retrovirus from patient 1 contains a major p25 protein, similar in size to that of HTLV-1 but different immunologically

• The role of this virus in the etiology of AIDS remains to be determined

• The virus-producing lymphocytes seemed to have no increased growth potential in vitro compared to the uninfected cells. Therefore, the multiple lymphadenopathies may represent a host reaction against the persistent viral infection rather than hyperproliferation of virus-infected lymphocytes

• Other factors, such as repeated infection by the same virus or other bacterial and viral agents may, in some patients, overload this early defense mechanism and bring about an irreversible depletion of T cells involved in cellular immunity

THE TRIO• Luc Antoine Montagnier (born 18 August 1932) is a French virologist and joint recipient with

Françoise Barré-Sinoussi and Harald zur Hausen of the 2008 Nobel Prize in Physiology or Medicine for his discovery of the human immunodeficiency virus (HIV).

SCIENCE, 4 MAY 1984 VOL 224, ISSUE 4648, PAGES 446-526

CONTRIBUTIONS BY RC GALLO• Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from

patients with AIDS and pre-AIDS, M Popovic, MG Sarngadharan, E Read, and RC Gallo, Science 4 May 1984: 497-500

• Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS, RC Gallo, SZ Salahuddin, M Popovic, GM Shearer, M Kaplan, BF Haynes, TJ Palker, R Redfield, J Oleske, B Safai, and al. et, Science 4 May 1984: 500-503

• Serological analysis of a subgroup of human T-lymphotropic retroviruses (HTLV-III) associated with AIDS, J Schupbach, M Popovic, RV Gilden, MA Gonda, MG Sarngadharan, and RC Gallo, Science 4 May 1984: 503-505

• Antibodies reactive with human T-lymphotropic retroviruses (HTLV-III) in the serum of patients with AIDS, MG Sarngadharan, M Popovic, L Bruch, J Schupbach, and RC Gallo, Science 4 May 1984: 506-508

POLITICS SURROUNDING AIDS• December 6, 1983: congressional subcommittee releases The Federal Response to AIDS, a

report criticizing the U.S. Government for failure to invest sufficient funding in AIDS surveillance and research

• 1983: After a NY physician is threatened with eviction from his building for treating AIDS patients, Gay Men’s Health Crisis and Lambda Legal file the first AIDS discrimination lawsuit

• 1984: Community-based AIDS service organizations join together to form AIDS Action, a national organization in Washington, D.C., to advocate on behalf of people and communities affected by the epidemic, to educated the Federal Gov’t, and to help shape AIDS-related policy and legislation

• October 1984: SF officials order bathhouses closed due to high-risk sexual activity occurring in these venues; NY and LA follow suit within the year

MORE POLITICS…• 1985: Ryan White, an Indiana teenager with hemophilia who contracted AIDS though a blood

transfusion, is refused entry to his middle school. He goes on to speak publicly against AIDS stigma and discrimination

• October 2, 1985: Rock Hudson dies of AIDS-related illness; he leaves $250K to help set up the American Foundation for AIDS research (amfAR); Elizabeth Taylor serves as the National Chairman

• 1985 - Blood tests to detect HIV are developed

• September 17, 1986: Reagan mentions AIDS publicly for the first time, vowing in a letter to Congress to make AIDS a priority

• March 19, 1987: FDA approves the first antiretroviral drug, zidovudine (AZT); Congress approves $30 million in emergency funding to states for AZT

• July 1987: Congress adopts the Helms Amendment, which banned the use of Federal funds for AIDS education materials that “promote or encourage, directly or indirectly, homosexual activities.”

MEDIA• 1987: Randy Shilt’s book about the early years of the HIV/AIDS epidemic, And the Band

Played On: Politics, People and the AIDS epidemic, is published

• And the Band Played On was used as the basis for a 1993 Emmy-winning HBO movie of the same name. It was produced by Aaron Spelling, directed by Roger Spottiswoode, and starred Matthew Modine as epidemiologist Don Francis and Richard Masur as Dr. William Darrow at the Centers for Disease Control. Alan Alda portrayed controversial viral researcher Robert Gallo

AIDS IN 2000…

• UN Security Council meets to discuss the impact of AIDS on peace and security in Africa

• In his State of the Union address on January 27, Clinton announces the launch of the Millennium Vaccine Initiative to create incentives for developing and distributing vaccines against HIV, TB and malaria

• April 30: Clinton declares that HIV/AIDS is a threat to US national security

• July 23: G8 Summit release a statement acknowledging the need for additional HIV/AIDS resources

AIDS TODAY• 35.3 million - Number of people living with AIDS/HIV worldwide in 2012; 2.3 million - New

infections worldwide in 2012; 260,000 - New infections in children worldwide in 2012; 1.6 million - AIDS-related deaths worldwide in 2012; 70.8 percent of the world's people living with HIV/AIDS live in Sub-Saharan Africa

• July 24, 2012 - Doctors announce during the 19th International AIDS Conference that Timothy Ray Brown, the Berlin patient, has been "cured" of HIV. Brown, diagnosed with leukemia, underwent a bone marrow transplant in 2007 using marrow from a donor with an HIV-resistant mutation. He no longer has detectable HIV

• March 3, 2013 - Researchers announce that a baby born infected with HIV has been "functionally cured." The child, born in Mississippi, was given high doses of antiretroviral drugs within 30 hours of being born. Two years later, doctors were unable to detect evidence of HIV in the child's blood

• 2013 - President Obama's budget request for 2014 fiscal year includes $6 billion for global aids prevention, $1.65 billion for the Global Fund to Fight HIV/AIDS, Tuberculosis and Malaria

REFERENCES• F. Barre-Sinoussi et al., Science 220, 868 (1983)

• M Popovic, MG Sarngadharan, E Read, RC Gallo, Science 224, 497 (1984)

• RC Gallo et al., Science 224, 500 (1984)

• J Schupbach et al., Science 224, 503 (1984)

• MG Sarngadharan, M Popovic, L Bruch, J Schupbach, RC Gallo, Science 224, 506 (1984)

• 30 Years of HIV/AIDS Timeline, AIDS.GOV

• A Historical Look at AIDS Research from 1970-1987, Chicago Tribune, November 1989

• www.Wikipedia.org

• www.CNN.com

• www.CDC.gov

THANK YOU