AIDS RESEARCH AND HUMAN RETROVIRUSESVolume 12, Number 12, 1996Mary Ann Liebert, Inc.

HIV-Specific Cellular and Humoral Immune Responses inPrimary HIV Infection

ELIZABETH CONNICK,1 DAVID G. MARR,1 XING-QUAN ZHANG,1 STEPHEN J. CLARK,2MICHAEL S. SAAG,2 ROBERT T. SCHOOLEY,1 and TYLER J. CURIEL1

ABSTRACT

Primary human immunodeficiency virus (HIV) infection is characterized by a high-titer viremia that declinesprecipitously within weeks, most likely as a result of host immune responses. Peripheral blood mononuclearcells (PBMCs) and plasma of four recently HIV-infected individuals were examined to assess the humoral andcellular immune responses potentially involved in early suppression of viral replication. Neutralizing anti-bodies against autologous viral isolates were low or undetectable in three subjects studied. Cellular cytotoxi-city was assayed using Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (B-LCLs) infectedwith recombinant vaccinia that express HIV-1 proteins. HIV envelope-specific cytotoxicity, which was not me-

diated by CD8+ cells nor human leukocyte antigen (HLA) class I restricted, developed in PBMCs of all foursubjects early after primary infection, but was not correlated with declines in viremia. Gag-specific cytotoxicT lymphocyte (CTL) activity was observed in freshly isolated PBMCs of two subjects, and HIV-specific CTLcell lines were cultured from PBMCs of three subjects shortly after HIV infection. Antibody-dependent cel-lular cytotoxicity (ADCC) developed early in all four subjects, and was temporally correlated with declinesin viremia in two subjects in whom viral load was well characterized. These data suggest that both CTL re-

sponses and ADCC may be critical to control of viral replication in acute HIV infection.

INTRODUCTION causes of the failure of immune surveillance that presages theemergence of AIDS and the terminal phase of HIV infection.

Primary HIV-1 infection is associated in 30-70% of cases A number of virus-specific immune responses have beenwith a symptomatic mononucleosis-like illness that pre- demonstrated in chronic HIV infection, including neutralizing

cedes seroconversion and is characterized by high titers of antibodies,17-24 HIV-specific cytotoxic T lymphocytesplasma and cell-associated viremia.1-16 In concert with sero- (CTLs),25-35 and antibody-dependent cellular cytotoxicityconversion, initial symptoms resolve and titers of viremia by (ADCC).36-49 A few studies that examined the immune re-

all measures are seen to decline from 10- to 1000-fold.12-16 sponse in acute HIV infection have reported a temporal asso-

Clinical latency usually follows, and is characterized by the ab- dation between the appearance of CTL precursors and declinessence of symptoms and more restricted viral replication. The in plasma viremia during acute HIV infection,50-53 as well as

resolution of symptoms and the marked decrease in viremia that weak or absent neutralizing antibody responses during this crit-occur in conjunction with seroconversion strongly suggest that ical period.54-59 Little is known about ADCC during acute HIVimmune mechanisms are critical in limiting HIV infection to a infection, however, as there is only one report of ADCC beingchronic persistent disease as opposed to an acutely lethal one. present in an individual who seroconverted as a result of a lab-A better understanding of immune responses in early HIV in- oratory accident.60 We examined humoral and cellular immunefection could provide insights into the most significant, and po- responses in four recently HIV-infected individuals to deter-tentially protective, immune responses to this disease, mine the relationship between control of plasma viremia dur-Moreover, understanding the role of the immune response in ing acute HIV infection and neutralizing antibodies, CTL ac-

the initial control of viremia might ultimately shed light on the tivity, and ADCC.

'Division of Infectious Diseases, Department of Medicine, University of Colorado Health Sciences Center and Veterans Affairs Medical Center,Denver, Colorado 80220.

infectious Disease Division, Department of Medicine, University of Alabama, Birmingham, Alabama 35294.

1129

1130 CONNICK ET AL.

MATERIALS AND METHODS

SubjectsSubject 1 (HOBR) (previously reported in Refs. 15, 16, 53,

and 70). A 28-year-old homosexual man was hospitalized withfever and persistent vomiting. One month earlier he had en-

gaged in anal-receptive intercourse with his HIV-infected part-ner. Two weeks before admission, he had developed fever, sore

throat, nausea, and diarrhea. On hospital admission he was

febrile to 38.9°C, and had an erythematous, macular rash on hisface and trunk. His absolute CD4+ lymphocyte count was

794/jixl. His anti-HIV-1 enzyme-linked immunosorbent assay(ELISA) was negative, but serum from an emergency room

specimen drawn 3 days earlier was positive for HIV p24 anti-gen. HIV-1 was cultured from his plasma and peripheral bloodmononuclear cells (PBMCs) at this time. His symptoms re-

solved over 3 to 4 days. A repeat HIV-1 antibody test 2 weekslater showed a positive ELISA and Western blot. Subsequently,he remained asymptomatic with CD4+ lymphocyte counts rang-ing from 600 to 1500//xl. He was lost to follow-up 1 year afterhis seroconversion.

Subject 2 (WHRI). A 28-year-old homosexual male presentedwith a recent diagnosis of HIV-1 antibody seroconversion. Hehad had multiple sexual partners over the preceding months andhad tested negative for HIV-1 antibody 6 weeks earlier. Fourweeks before presentation, he developed a febrile illness char-acterized by sore throat, lymphadenopathy, and headache,which resolved within 5 days. Two weeks prior to presentation,he sought repeat HIV antibody testing, and was found to haveseroconverted. At the time of presentation he had no culturableplasma virus or detectable p24 antigenemia. HIV-1, however,was cultured from his PBMCs. His initial CD4+ lymphocytecount was 5%6/pl, but it dropped to 430 cells//xl 4 months af-ter seroconversion, and he was started on zidovudine. Eightmonths after seroconversion he was lost to follow-up.

Subject 3 (BAKE). A 21-year-old homosexual man presentedwith a 5-day history of vomiting, pharyngitis, dysphagia, oralulcérations, rash, and fever, and was hospitalized for dehydra-tion. Twenty-four days prior to admission he had engaged inunprotected anal receptive intercourse with his partner, who hadAIDS. At the time of admission, his HIV-1 antibody ELISAtest was negative. He had high levels of serum p24 antigen,however, and HIV-1 was cultured from both his plasma and hisPBMCs. His illness resolved over 1 week. Six weeks later hetested positive for HIV-1 antibody by both ELISA and Westernblot. His CD4+ lymphocyte count was 425 cells/^,1 at admis-sion, but dropped to 281 cells//xl within 42 days of his sero-

conversion illness. He was started on zidovudine, and subse-quently entered a randomized, double-blinded study ofnevirapine in combination with zidovudine or didanosine. Heremains asymptomatic 2.5 years after infection, with CD4+lymphocyte counts from 200 to 350//J.1.

Subject 4 (MNX). A 28-year-old woman, whose boyfriendwas an intravenous drug user, was referred for evaluation of a

recent diagnosis of primary HIV-1 infection. One month ear-

lier she had presented to another hospital with fever, pharyngi-tis, rash, adenopathy, photophobia, and headache. Her serum

p24 antigen was positive, and her Western blot for HIV was

initially indeterminate, but subsequently became positive. Her

acute illness resolved over a few weeks, except for headaches.Her initial CD4+ lymphocyte count was 409 cells//il. Shestarted zidovudine 2 months after her seroconversion, butswitched to didanosine, and than zalcitabine because of side ef-fects. She remains asymptomatic 2 years after her seroconver-

sion, with stable CD4+ lymphocyte counts between 350 and450 cells//xl.

Clinical specimensSubjects 1, 2, and 3 presented to hospitals affiliated with the

University of Alabama at Birmingham (Birmingham, AL).Subject 4 presented to a hospital affiliated with the Universityof Colorado Health Sciences Center (Denver, CO). The sub-jects were studied prospectively, with written consent, accord-ing to protocols approved by the institutional review boards ateach university. Peripheral blood was obtained by venipunctureinto tubes containing acid citrate dextrose or sterile syringescontaining preservative-free heparin. Samples from theUniversity of Alabama were sent by overnight courier to theUniversity of Colorado Health Sciences Center, where cyto-toxicity assays and lymphocyte cultures were performed within24 hr of collection.

MediaR-10 consists of RPMI 1640 (GIBCO, Grand Island, NY),

10% heat-inactivated fetal bovine serum (FBS; HyClone, SaltLake City, UT), 4 mM L-glutamine (GIBCO), penicillin (100U/ml) and streptomycin (100 ¿ig/ml) (GIBCO), and 10 mM N-2-hydroxyethylpiperazine-A/'-2-ethanesulfonic acid (HEPES;Sigma, St. Louis, MO). R-15 and R-20 are made in the same

way except that the concentration of FBS is 15 and 20%, re-

spectively. R-10-50 and R-10-5 consist of R-10 with 50 or 5U/ml, respectively, of recombinant human interleukin 2 (rhlL-2) (a gift of M. Gately, Hoffman-La Roche, Nutley, NJ).

Preparation of lymphocytes and plasmaThe PBMCs were isolated from whole blood by Ficoll-

Hypaque (Histopaque; Sigma) density gradient centrifugation,washed three times with RPMI 1640, suspended in R-10, andcounted on a hemocytometer. Plasma was stored at

—

70°C.

HTV-1 quantitative viral cultures, p24 antigen testing,and quantitative competitive-polymerase chain reaction

HIV-1 quantitative viral cultures and p24 antigen testingwere performed at the University of Alabama, Birmingham as

previously described.15 Quantitative competitive polymerasechain reaction (QC-PCR) on subject 1 (HOBR) was performedat Genelabs Technologies (Redwood City, CA) by M. Piatakand J. Lifson, as previously described.15,16

Isolation of HTV-1HIV was isolated from either frozen cultured PBMCs from

a primary culture or uncultured PBMCs according to theNational Institute of Health (NIH) AIDS Clinical Trials Group(ACTG) consensus protocol.61 Virus from subject 1 was pas-saged three times before an acceptable titer was achieved. Virusfrom subject 2 was passaged twice before recovery, and virus

IMMUNE RESPONSE TO HIV INFECTION 1131

from subject 3 was passaged once. The viral stock was titratedusing the NIH ACTG consensus protocol,62 and the tissue cul-ture infective dose at which 50% of cultures would be infected(TCID50) was calculated by the Spearman-Karber method.63

Neutralizing antibody assayPlasma was heat inactivated at 56°C for 1 hr, serially diluted

fivefold from a dilution of 1:5 to 1:3125, and 0.1 ml was in-cubated with 0.1 ml of phosphate-buffered saline (PBS;GIBCO) containing 100 TCID50 of autologous virus for 1 hr.Plasma and virus were cultured overnight with 2 X 106 phyto-hemagglutinin (PHA) blasts in 2 ml of R-10-5, then centrifugedat 300 X g, washed two times in PBS, and resuspended in 2 mlof R-10-5 in 24-well plates. Each plasma dilution was per-formed in triplicate. Plasma from an HIV-uninfected donor was

used as a negative control, and HIV immune globulin (HIVIG)(contributed by A. Prince through the AIDS Research andReference Reagent Program, Rockville, MD), known to neu-

tralize some HIV strains, was also tested in parallel. Cultureswere fed on day 4 by partial medium exchange, and super-natants were assayed for p24 antigen on day 7. The reciprocalneutralizing titer was defined as the reciprocal of the highestdilution at which there was a 50% or greater reduction in p24antigen production compared to the seronegative control.

Depletion of CD8+ cells

Lymphocytes of the CD8+ surface phenotype were depletedfrom freshly isolated PBMCs by MicroCELLector T-25 cellculture flasks for selection of CD8+ cells (Applied ImmuneSciences, Menlo Park, CA) according to the specifications ofthe manufacturer, with the exception that aggregated humangammaglobulin (lot 001666; Calbiochem, La Jolla, CA) (100pg/ml) was used instead of gammaglobulin. Analysis of cellsurface markers by flow cytometry of both the unseparated andCD8+ cell-depleted PBMCs showed depletion of CD8+ cellsto fewer than 5% of lymphocytes. Mean viability of recoveredcells was >98% as determined by trypan blue dye exclusion.

Target cell lines

Epstein-Barr virus (EBV)-transformed B lymphoblastoidcell lines (B-LCLs) were established on study subjects as de-scribed.25 The cell line K562 (CCL 243; American TypeCulture Collection [ATCC], Rockville, MD) is susceptible to

lysis by natural killer cells, and the Daudi cell line (providedby G. Harrison, University of Colorado Health Sciences Center)is susceptible to lymphokine-activated killer cells. Cell line 131(provided by B. Walker, Massachusetts General Hospital,Boston, MA) and cell line X005 were EBV-transformed B-LCLs derived from chronically HIV-infected subjects. Cell lineEC is a B-LCL derived from an HIV-uninfected individual.

Recombinant vaccinia vectors

Recombinant vaccinia vectors that express Env (vPE16[HTLV IIIB]), as well as a control recombinant vaccinia ex-

pressing Escherichia coli /3-galactosidase (vSC8), were pro-vided by B. Moss (NIAID, NIH). Vaccinia recombinant vDKl,

which expresses HIV-1 Gag, was provided by D. Kuritzkes(University of Colorado Health Sciences Center).

Preparation of target cells

B-LCLs suspended at 1 X 106/ml were infected with re-

combinant vaccinia viruses and labeled with Na2[5lCr]04 (DuPont, Boston, MA) as described.26

Cytotoxicity assay51Cr-labeled target cells were aliquoted into round-bottomed

wells of a 96-well microtiter plate at 1 X 104 cells/well.Subjects' unstimulated PBMCs were added to wells to achieveeffectontarget (E:T) ratios of 100:1, 50:1, or 25:1 in a final vol-ume of 200 pi. Data points were determined in triplicate. Plateswere incubated at 37°C for 6 hr, centrifuged for 5 min at 300 X

g at 4°C, and 51Cr release determined by measuring counts perminute (cpm) on a y counter (model 5500; BeckmanInstruments, Fullerton, CA) of 100 pi of supernatant sampledfrom each well. When lymphocyte cell lines were used as ef-fector cells, the 51Cr release assays were performed at E:T ra-

tios of 10:1 with 5 X 103 target cells/well and 4-hr incubations.Maximum 51Cr release was determined by detergent lysis oftarget cells with 5% Triton X-100, and spontaneous release was

determined by incubating target cells in the absence of effec-tor cells. Spontaneous release was less than 30% of maximumrelease in all reported assays unless otherwise noted. Percentspecific cytotoxicity was calculated as previously described.25An assay was judged to demonstrate HIV-specific cytotoxicityif the percent specific cytotoxicity against a target cell ex-

pressing a specific HIV protein was 10 or greater than that forthe target cell expressing the control protein (ß-galactosidase).

Cytotoxic T lymphocyte cell linesHIV-1-specific CTL cell lines were derived using a method

modified from Walker et al.28 Freshly isolated PBMCs were

seeded into flat-bottomed 96-well microtiter plates at 50 or 25cells/well in R-10-50. Irradiated (3000 rad) allogeneic PBMCsfrom HIV-seronegative donors (2 X 105 cells/well) and theanti-CD3 monoclonal antibody 12F6 (0.05 pg/ml; provided byJ. Wong, Massachusetts General Hospital, Boston, MA),64 were

added as a stimulus to T cell proliferation. The final volume ofeach well was 200 pi. Plates were incubated at 37°C with 5%C02 and fed one or two times weekly with partial medium ex-

changes. Cell lines were restimulated with additional 12F6, ir-radiated allogeneic feeder cells, and rhIL-2 every 10 to 14 days.Cells from wells exhibiting proliferation were assayed for cy-tolysis of autologous target cells expressing HIV-1 antigens.Cell lines able to lyse autologous target cells were expandedfor further studies.

Phenotypic analysis of cell surface markersThe expression of cell surface antigens was analyzed by flow

cytometry on a fluorescence-activated cell sorter (EPICS III;Coulter Electronics, Hialeah, FL) using fluorescein- or phyco-erythrin-conjugated monoclonal antibodies specific for the Tcell surface molecules CD3, CD4, and CD8, or the B cell sur-

face molecule CD 19. Appropriate murine isotypic negative con-

trols were included in all analyses.

1132

Human Leukocyte Antigen typingHLA typing was performed on PBMCs at the University of

Alabama or B-LCLs or PBMCs at the University of ColoradoHealth Sciences Center, using standard sérologie techniques.

Antibody-dependent cellular cytotoxicity assayPBMCs isolated from an HIV-uninfected healthy donor were

used as effector cells. CEM-NKr cells (contributed by P.Cresswell through the AIDS Research and Reference ReagentProgram, Rockville, MD) were used as target cells. Two sepa-rate aliquots of 2 X 106 target cells were incubated with 100pCi of Na2[51Cr]04 (Du Pont) for 1.5 hr at room temperaturewhile constantly being rotated. One of the aliquots of targetcells was simultaneously incubated with 3.9 pg of HIV-1Sf2gpl20 (contributed by K. Steimer, Chiron Corporation, throughthe AIDS Research and Reference Reagent Program, Rockville,MD). Target cells were washed three times with PBS, and 100-pl aliquots containing 2 X 103 cells were placed into 96-wellround-bottom plates. Subject plasma was heat inactivated at56°C for 1 hr and diluted in RIO to give final plasma dilutionsof 1:80, 1:400,1:2,000,1:10,000, and 1:50,000 in the microtiterwells. Every assay included heat-inactivated plasma from an

HIV-seronegative individual as a negative control, and HIVIGas a positive control. The plasma (50 /¿l/well) was incubatedfor 30 min with target cells, and then 50 pi of effector cellscontaining 8 X 104 cells was added. Effectors and targets were

also incubated with 50 pi of RIO to determine the backgroundcytotoxicity in the absence of plasma. Plates were incubated for6 hr at 37°C, supernatants were harvested, and determinationsof spontaneous lysis, maximum lysis, and percent cytotoxicitywere made as described above for the cytotoxicity assays.Spontaneous lysis was less than 14% in all assays. Each datapoint was determined in triplicate. The percent specific ADCCwas determined by subtracting the percent cytotoxicity againstuncoated target cells from the percent cytotoxicity against tar-get cells coated with gpl20. In all assays the HIVIG mediatedspecific ADCC greater than 40%, and the seronegative controldemonstrated less than 6% specific ADCC.

RESULTS

Assays of plasma viremia

Longitudinal assays of plasma virus were performed in sub-jects 1 and 3 (Fig. 1), both of whom were studied from the timeof their symptomatic primary infection. Subject 1 had the high-est levels of p24 antigenemia, culturable virus, and viral RNAcopy number 21 days after the onset of his illness. Viral RNAcopy number declined 1.2 login by day 29 and remained stableuntil his last visit, 10 months after his initial illness. He was

asymptomatic at this time and subsequently lost to follow-up.Subject 3 had a more rapid decline in plasma viremia than sub-ject 1. His peak levels of culturable plasma virus were observedon day 6 after the onset of his acute retroviral illness, and no

culturable virus was detected in his plasma by day 12. His lev-els of culturable plasma virus remained low or undetectable upto 10 months after his primary infection. Plasma viremia was

not assayed in a longitudinal fashion in either subject 2 or 4,

CONNICK ET AL.

Days from onset of acute retroviral illness

FIG. 1. Longitudinal determinations of plasma HIV-1 RNAcopy number by QC-PCR, p24 antigen levels, and culturablevirus. (A) subject 1; (B) subject 3. (A) is modified from Ref.16 and is reprinted here with the permission of the authors andthe publisher, Current Science Publication.

because both entered the study several weeks after their acuteretroviral illnesses. Subject 2, however, did not have p24 anti-genemia or culturable plasma virus at day 31, when he enteredthe study.

Neutralizing antibody assaysVirus was isolated for autologous neutralization studies on

day 17, day 31, and day 6 after the onset of illness in subjects1, 2, and 3, respectively. Virus was not isolated from PBMCsof subject 4 at an early time point. Subjects' plasma from vary-ing time points as well as pooled HIVIG were assayed for neu-

tralization of these early viral isolates.Plasma from two of three subjects demonstrated low titers

of neutralizing antibodies (Table 1). Plasma from subject 1 hada reciprocal neutralizing titer of 10 at the time of his high titerviremia (day 17), and his plasma neutralizing antibody titer con-

tinued to be low or undetectable on days 57 and 218, when ma-

jor decreases in plasma viremia had already occurred. Plasmafrom subject 2 had a reciprocal neutralizing titer of 50 on day

IMMUNE RESPONSE TO HIV INFECTION 1133

Table 1. Reciprocal Neutralizing Titers3 in Assays of Subject Plasma with Autologous Virus

Subject I Subject 2 Subject 3

Virus isolate HTVIG Day 17 Day 57 Day 218 Day 31 Day 255 Day 6 Day 97

Subject 1Subject 2Subject 3

505010

10 <10 1050 50

<10 <10

"Reciprocal neutralizing titer is defined as the reciprocal of the highest plasma dilution at which there was a50% or greater reduction in p24 antigen production compared to a seronegative control.

31 after the onset of his acute retroviral illness, at a time whenhe had no detectable p24 antigenemia or culturable plasmavirus. His titer remained at this level 255 days later. Subject 3had no detectable neutralizing antibodies either at day 6, when

plasma viremia was high, or on day 97 when culturable viruswas no longer detectable in his plasma. HIVIG demonstratedlow titers of neutralizing antibodies against all three viral iso-lates.

o

U

u

17 57 218Time after onset of acute retroviral illness, days

8 21 42 57 97 181Time af1er onset of acute retroviral illness, days

oo

Uoao8.

CO

o

[ PBMC vs auto vac/env

I I PBMC vs auto vac/lac

Q CD8+ depleted PBMC vs

ES CD8+ depleted PBMC vs

53 PBMC vs alio vac/env

EO PBMC vs alio vac/lac

auto vac/env

auto vac/lac

74 108 255Time after onset of acute retroviral illness, days

Time after onset of acute retroviral illness, days

FIG. 2. Envelope-specific cytolysis by PBMCs developed in all four subjects, was unaffected by depletion of CD8+ cells, andwas directed against allogeneic as well as autologous envelope-expressing cells. (A) Subject 1; (B) subject 2; (C) subject 3; (D)subject 4. auto, Autologous B-LCL target cells; alio, allogeneic B-LCL target cells. Methods: Autologous and allogeneic B-LCLtarget cells were infected with recombinant vaccinia expressing either Illb envelope (vac/env) or control protein (vac/lac). A por-tion of freshly isolated PBMCs was depleted of CD8+ cells, using antibody-coated flasks. Cytotoxicity was assayed in 6-hr 51Crrelease assays by incubating unfractionated PBMCs or CD8+-depleted PBMCs with target cells. The E:T ratio was 50:1 in allinstances except subject 2 on day 255, when it was 100:1. Spontaneous 51Cr release for subject 1 was 50% in the assay on day57, and 31% in the assay on day 218. Allogeneic cell lines used were unmatched at class I HLA loci except for a BW6 matchfor subject 3. Class I and class II HLA types of the subjects and allogeneic target cell lines were as follows: subject 1: A3, A26,B7, B35, Cw7, Cw7/DR10,17, DRw52, DQw5; allogeneic cell line used in assay for subject 1 was cell line X005: A2, A29/30,B44, Bw62, Bw4-6, Cw3/class II HLA unknown, subject 3: A2, A3, B7, B8, Bw6, Cw7, Cw8/DR17(3), DR14(6), DR52,DQ2-5(1). The allogeneic cell line used in the assay for subject 3 on days 8 and 20 was EC: A31-, B14, Bw60, Bw6, Cw3,DR17(3)-4, DRw52-53, DQ2-7(3). The allogeneic cell line used in assays on days 42 and 57 was cell line 131: Al, A33, B51-,Bw4/DR2,5, DRw52, DQw2.

1134 CONNICK ET AL.

Cytotoxicity assays

Because transformation and expansion of autologous B-LCLs usually requires a minimum of 6 weeks, assays for en-

velope and Gag-specific cytotoxicity using autologous B-LCLswere not performed until days 57, 74, 97, and 92 in subjects 1,2, 3, and 4, respectively. Assays using allogeneic target celllines were performed as early as day 17 for subject 1 and day8 for subject 3.

Envelope-specific cytotoxicity was detected in PBMCs fromsubjects 1, 2, and 4 at the first time point tested, which was day17, day 74, and day 92, respectively, after the onset of their pri-mary illnesses (Fig. 2A, B, and D). PBMCs from subject 3,however, demonstrated very little envelope-specific cytotoxic-ity until 6 weeks after the onset of his symptoms (Fig. 2C). Inall four subjects, envelope-specific cytotoxicity was demon-strable despite depletion of CD8+ cells, and in subjects 1 and3, envelope-specific cytotoxicity was demonstrated against al-logeneic target cells unmatched at HLA class I loci. Some al-logeneic target cells shared HLA class II loci with the subjects.

The appearance of envelope-specific cytotoxicity did notseem to be associated with changes in plasma viremia in thetwo subjects in whom viremia was assayed. Although envelope-specific cytotoxicity was present in subject 1 at day 17, his levelof plasma viremia was rising at this time and did not declinesubstantially until 1 week later. On the other hand, plasmaviremia in subject 3 declined markedly by day 12 after the on-

set of his acute retroviral illness, whereas envelope-specific cy-totoxicity did not appear in his PBMCs until 6 weeks after theonset of his illness.

Gag-specific cytotoxicity was found in PBMCs from two ofthe four subjects. Subjects 1 and 2 had Gag-specific cytotoxi-city at the first time point assayed, which was day 57 and day74, respectively, following their primary illnesses (Fig. 3).Unlike the case with envelope-specific cytotoxicity, in neitherinstance was Gag-specific cytotoxicity demonstrable against al-logeneic target cells. This type of Gag-specific cytotoxicity hasbeen described by other investigators in patients with long-standing HIV infection, and when the effector population hasbeen identified it has almost always been HLA class I-restricted

o'S22>^U

o

o

clCO

8

H PBMC vs auto vac/gagI"! PBMC vs auto vac/lac

E3 PBMCvs alio vac/gagE3 PBMC vs alio vac/lac

0 CD8+ depleted PBMC vs auto vac/gagS CD8+ depleted PBMC vs auto vac/lac

Day 74 Day 108 Day 255

Days from onset of acute retroviral illnes

FIG. 3. PBMCs from subjects 1 (A) and 2 (B) demonstrated Gag-specific cytotoxicity against autologous, but not allogeneicantigen-expressing target cells at the first times tested. In subject 1, depletion of CD8+ cells from PBMCs reduced Gag-specificcytolysis. Methods: Autologous or allogeneic B-LCL target cells were infected with recombinant vaccinia viruses expressingHIV-1 Gag (vac/gag) or control protein (vac/lac). Unfractionated PBMCs or CD8+-depeleted PBMCs were incubated with tar-get cells in a 6-hr 51Cr release assay. The E:T ratio was 50:1 in the case of subject 1, and 100:1 in the case of subject 2. Spontaneousrelease was 50% in the assay shown for subject 1 on day 57. Spontaneous release was 33% for the allogeneic target cell line forsubject 2 on day 108. The allogeneic target cell line used in the assay for subject 1 on day 113 was X005, which is completelyunmatched from subject 1 at class I loci (see caption to Fig. 2). The class I HLA type of subject 2 was: A2, A24, B8, B44, Bw4,6.The allogeneic cell target cell line used in the assay for subject 2 on day 255 was EC, which is class I matched only at Bw6 (seecaption to Fig. 2). auto, autologous B-LCL target cells; alio, allogeneic B-LCL target cells.

IMMUNE RESPONSE TO HIV INFECTION 1135

Auto vac/gag

j Auto vac/lac

alio vac/gag

Cell line B Cell line K

u

o

ex

-fv^Al

[_J Auto vac/env

[ Auto vac/gag

] Auto vac/lac

E3 K562

a.Cell line 50.24

Ixj Auto vac/cnv

I 1 Auto vac/lac

5a Alio vac/cnv

_3 Daudi

Cell line 25.6

FIG. 4. HIV-specific CTL cell lines were isolated fromPBMCs of subjects 1, 3, and 4, shortly after their primary ill-nesses. (A) Gag-specific cytotoxic cell lines cultured fromPBMCs taken from subject 1 on day 57 after the onset of hisacute retroviral illness. (B) An envelope-specific cell line cul-tured from PBMCs taken from subject 3 on day 8 after the on-

set of his acute retroviral illness. (C) An envelope-specific cy-totoxic cell line cultured from PBMCs taken from subject 4 on

day 36 after the onset of her acute retroviral illness. Methods:Autologous B-LCL target cells were infected with recombinantvaccinia expressing Illb env (vac/env), gag (vac/gag), or thecontrol protein (vac/lac). Cell lines were incubated with B-LCLor the K562 or Daudi cell lines in a 4-hr 51Cr release assay, atan E:T ratio of 10:1. The allogeneic target cell line used in theassay for subject 1 was EC, which is unmatched to subject 1 atHLA class I loci (see caption to Fig. 2). The class I HLA typeof subject 4 was as follows: A30-, B18-, Cw2, Cw5. The al-logeneic B-LCL used in the assay for subject 4 was from sub-ject 3 (see caption to Fig. 2), which is also unmatched at classI HLA loci to subject 4. Auto, autologous B-LCL target cells;alio, allogeneic B-LCL target cells.

CD8+ CTLs.25,29,30 In subject 1, Gag-specific cytotoxicity per-sisted on repeat testing 6 months later, and was found to be me-

diated primarily by CD8+ cells, as demonstrated by depletionstudies. Gag-specific cytotoxicity was present in the PBMCs ofsubject 2 at days 74 and 108, but was undetectable 255 daysafter the onset of his acute retroviral illness. Depletion studieswere not performed on this subject. PBMCs from subject 3 andsubject 4 did not demonstrate Gag-specific cytotoxicity againsteither allogeneic or autologous B-LCLs (data not shown).

HTV-1-specific cytotoxic T lymphocyte lines

HIV-specific CTL cell lines were isolated from all four sub-jects. From subjects 1,3, and 4, CTLs were isolated at the firsttime point tested, which was day 57, day 8, and day 36, re-

spectively, after the onset of their acute retroviral illnesses (Fig.4). Two Gag-specific cell lines were isolated from subject 1(Fig. 4A). Phenotypic analysis of cell surface markers showedthat both cell lines were mixed populations of CD4+ and CD8+T lymphocytes. One envelope-specific CTL line, 50.24, was

isolated from PBMCs taken from subject 3 (Fig. 4B). Analysisof cell surface phenotypic markers showed that over 80% ofthe cells were T lymphocytes, and that the major portion ofthese T cells were CD8+ cells, with <4% CD4+ lymphocytes.One envelope-specific CTL cell line was isolated from subject4 (Fig. 4C). Analysis of cell surface markers showed that over

90% of the cells in this cell line were of the CD3+CD8+ phe-notype.

Although preliminary assays suggested that cell lines withcytolytic activity against envelope- and Gag-expressing targetcells were present in stimulated PBMCs from subject 2 (datanot shown), none of these cell lines survived long enough toallow their definitive identification as CTLs. Six months afterhis primary illness, CD8+ envelope-specific CTL cell lines were

isolated from the blood of subject 2 (data not shown).

Antibody-dependent cellular cytotoxicity assaysPlasma from all four subjects mediated ADCC (Fig. 5).

Subjects 2 and 4 both had high titers of antibodies capable ofmediating ADCC at the first time tested, day 31 and day 36,respectively, following the onset of their acute retroviral ill-nesses (Fig. 5B and D). Plasma from subject 1 did not mediateADCC at day 17 or day 22 following the onset of his illness,but suddenly developed the ability to mediate ADCC betweenday 22 and day 29 (Fig. 5A). The timing of the developmentof his ADCC coincides almost exactly with a 1.2 logio decreasein his plasma viremia. Subject 3, on the other hand, already hadlow titers of antibodies that mediated ADCC on day 8 of hisseroconversion illness and the capacity of his plasma to medi-ate ADCC increased over time (Fig. 5C). The presence of an-

tibodies capable of mediating ADCC quite closely preceded thedecline in his plasma viremia evident on day 12.

DISCUSSION

The first interaction between HIV and the immune system,which occurs during primary infection, reveals strikingly ef-fective clearance of infectious virus from the plasma and re-

duction in viral replication.12-16 The PBMCs and plasma of four

1136 CONNICK ET AL.

2,000 10,000 50,000 No plasma

Reciprocal Plasma Dilution

UuQ<

a.

80 400 2,000 10,000 50,000 No plasma

Reciprocal Plasma Dilution

UUQ<

a.cri<->

cuu

2,000 10,000 50,000 No plasma

Reciprocal Plasma Dilution

UUQ<

D.

Eil

80 400 2,000 10,000 50,000 No plasma

Reciprocal Plasma Dilution

FIG. 5. Plasma from all subjects shortly after their acute retroviral illnesses mediated ADCC against CEM-NKr cells incubatedwith gpl20. (A) Subject 1; (B) subject 2; (C) subject 3; (D) subject 4. "Day" refers to the time after the onset of the subject'sacute retroviral illness, with day 1 being the first day of illness. Methods: Various dilutions of subject plasma were incubated for30 min with CEM-NKr cells previously labeled with 51Cr and preincubated with or without gpl20. PBMCs from a single HIV-seronegative donor were then added at an effector-to-target cell ratio of 40:1. Percent cytotoxicity was determined in a 6-hr 51Crrelease assay. Percent specific ADCC was determined by subtracting the cytotoxicity against CEM-NKr cells alone from cyto-toxicity against CEM-NKr cells labeled with gpl20. Although not shown here, every assay included plasma from an HIV-seroneg-ative individual that never demonstrated specific ADCC greater than 5%, and HIVIG, which always mediated specific ADCCgreater than 40%.

recently HIV-infected individuals were examined to assess thehumoral and cellular HIV-specific immune responses poten-tially involved in early suppression of viral replication.

Neutralizing antibodies to autologous virus were low or un-

detectable in the three subjects in which they were studied.These findings are consistent with those of other investigators,who have found low or absent levels of neutralizing antibodiesearly in HIV infection, without apparent correlation to declinesin viremia.50,54-59

Envelope-specific cytotoxicity, which was not mediated byCD8+ cells or HLA class I restricted, was identified in all foursubjects early after infection, but did not appear to be corre-

lated with declines in viremia in either subject 1 or 3. The pre-cise nature of the effector population that mediated envelopecytotoxicity was not determined. One peculiarity of the vac-

cinia recombinant vPE16 that was used in these assays is thatit causes HIV envelope to be expressed not only as peptide inassociation with HLA molecules, but also as full-length gpl20on the surface of the cell. Other investigators have reported en-

velope-specific cytotoxicity in the PBMCs of HIV-infected in-

dividuals directed against similar target cells and when the ef-fector mechanism was characterized it appeared to be ADCCmediated by natural killer (NK) cells that had previously boundcytophilic antibody.41,44,46 Nevertheless, the ability of plasmato mediate ADCC in subjects 1 and 3 did not correlate with en-

velope cytotoxicity. In subject 1 envelope-specific cytotoxicitypreceded ADCC by 1 week, and in subject 3 envelope-specificcytotoxicity was not detectable as early as was ADCC. CD4+CTLs have been reported in HIV disease,65,66 and could po-tentially have been the effectors of envelope-specific cytotoxi-city since the allogeneic target cells shared some HLA class IIalíeles with the subjects. In addition, Heinkelein et al.61 haveobserved that CD4+ cells from both HIV-seropositive andseronegative individuals can lyse B-LCLs that express HIV en-

velope on their surface, and this killing is not HLA restricted,but appears to result after binding of gpl20 by CD4. Full-lengthenvelope on the surface of target cells may also have served as

a target for NK cells. Thus, there are multiple possible candi-dates for the effectors of the envelope-specific cytotoxicity ob-served.

IMMUNE RESPONSE TO HIV INFECTION 1137

HIV-specific CTLs derived from in vitro stimulation ofPBMCs were obtained from three of four subjects at the earli-est time point attempted. In the case of subject 3, CTLs were

derived from PBMCs at day 8 of his acute retroviral illness,immediately prior to a profound decrease in his viremia. Theone subject from whom CTLs were not initially isolated most

likely had them as well, since preliminary assays were sugges-tive of the presence of CTLs, and Gag-specific CTL activitywas detected in his unstimulated PBMCs subsequently. Thesedata are consistent with the findings of two other groups,50-53who have detected CTL precursors early in HIV infectionthrough in vitro stimulation of PBMCs, and who have definedmore precisely than this study the close temporal relationshipbetween the appearance of HIV-specific CTL activity and de-clines in viral load. In addition, Mackewicz et al.59 have re-

ported noncytolytic viral suppressive activity mediated byCD8+ cells early after seroconversion. Similar evidence of thecorrelation of the appearance of CD8+ CTLs and their precur-sors with decreasing viremia has also been reported in rhesusmacaques acutely infected with SIV.68,69

Gag-specific cytotoxicity, which probably reflected CD8+CTL activity, was detected in fresh PBMCs from subjects 1 and2 on day 57 and day 74, respectively, after the onset of theiracute retroviral illnesses. Although activated Gag-specificCTLs may have been present in the PBMCs of these subjectsat earlier time points, owing to technical limitations of the as-

say this was the first time at which these assays could be per-formed. The Gag-specific cytotoxicity of subject 1 was de-tectable up to 5 months after infection. This subject was recentlyreported by others to have no detectable HIV-specific CTLs infrozen PMBCs taken from this same time period, and subse-quently thawed and stimulated in vitro with anti-CD3 mono-

clonal antibody.53 This discrepancy in findings likely reflectseither poor functional survival of CTLs after being frozen andthawed, or another technical problem, since cytotoxicity againstthe negative control was unusually high in their assay. It is un-

likely that CTL activity would be observed in unstimulatedPBMCs without the existence of CTL precursors.

Cytotoxic T lymphocyte activity in unstimulated PBMCs hasbeen well documented in chronic HIV infection,25,32,34,35 butthis is the earliest report of its occurrence following acute in-fection. The phenomenon of detectable CTL activity in un-

stimulated PBMCs is unique to chronic HIV infection, and thisdistinctively heightened response is presumed to be secondaryto ongoing in vivo priming of the immune response from chronicviremia. The Gag-specific CTL activity that we detected maybe the functional correlate of the oligoclonal expansion ofCD8+ T cells following acute HIV infection described byPantaleo et al.10 in several seroconverters. Indeed, subject 1 was

also included in their study (subject 6) and found to have an

expanded variable domain /3-chain (V/3) subset (V/37) at day35, and other expanded V/3 subsets at later time points. Theevanescent nature of subject 2's Gag-specific CTL activity, on

the other hand, may parallel the transient oligoclonal expan-sions of CD8+ T cells seen in some of their other seroconvert-ers.

Whether detectable CTL activity in unstimulated PBMCs isbeneficial in HIV infection remains to be determined. On theone hand, it could play a direct role in the initial control of vi-ral replication. On the other hand, CTL activity could simply

be an epiphenomenon stimulated by poorly controlled viremia,or even an HIV-specific immune response that contributes tothe pathogenesis of the disease. Although not seen in all HIV-infected individuals, CTL activity in PBMCs is more readilydemonstrated in patients with stable HIV disease, and less fre-quently detected in subjects with advanced HIV disease.32,71Harrer etal.35 have reported vigorous CTL activity in long-termnonprogressors, suggesting that HIV-specific CTL activity doesnot contribute to disease progression. One study found Gag-specific CTL activity was associated with long-term survival,72although others have found that Gag-specific CTL precursorsdo not protect against disease progression.33 Indeed, Pantaleoet al.10 suggested that the most dramatic oligoclonal expansionsof CD8+ cells were associated with more rapid progression toAIDS. Similarly, Ferbas et al.34 found that the most vigorousCTL activity in long-term nonprogressors was found in thosewith the highest viral load, suggesting that CTL activity maybe driven by viral replication rather than vice versa. Two of our

subjects did not demonstrate Gag-specific CTLs in unstimu-lated PBMCs, possibly because they did not mount substantialGag-specific CTL responses, or their Gag-specific CTLs mayhave recognized epitopes not encoded by the gag sequence con-

tained by the vaccinia recombinant used in our assays. It is dif-ficult to generalize from a small number of cases, but there were

no obvious differences in the clinical course of HIV disease be-tween those subjects in our study who did and those subjectswho did not have detectable Gag-specific cytotoxicity.

All four subjects were found to have plasma capable of me-

diating ADCC, and in subjects 1 and 3 ADCC appeared to betemporally correlated with declines in plasma viremia. Prior tothis study, the presence of ADCC in primary HIV infection hasbeen reported only once,60 and this was in an individual whohad already seroconverted. Thus, the ADCC seen in subjects 1and 3 represents the earliest reports of ADCC in primary HIVinfection. ADCC has been documented at all stages of HIV dis-ease, and it has always been found to be mediated by antibod-ies to HIV envelope.36-49 Our findings contrast with those ofMoore et a/.,58 who in a detailed study of the development ofanti-gpl20 antibody in three seroconverters found that serum

gpl20 antibodies did not develop until after major declines inviremia had already occurred. They did not assay for ADCC,however, and it is possible that their assays for gpl20 were notsensitive enough to detect small amounts of antibody capableof mediating ADCC. Baum et al.49 reported that low or de-creasing ADCC titers following seroconversion are highly pre-dictive of disease progression. These data in conjunction withour findings suggest that ADCC may be an important effectormechanism in clearing the viremia of primary infection and con-

trolling subsequent viral replication in HIV disease.One limitation of this study is that it examined immune re-

sponses only in peripheral blood. There is increasing evidencethat a significant degree of the host immune response to HIVinfection occurs in lymph nodes, and that immune responses inperipheral blood may not mirror responses in lymph nodes.73,74Examination of early immune responses in lymphoid organs,although technically difficult, could provide important insightsinto the initial control of HIV replication that are not possiblethrough study of PBMCs.

A second limitation is that our study was biased toward sub-jects with symptomatic primary HIV infection. All four of our-

1138 CONNICK ET AL.

subjects had symptomatic primary HIV infection, and three ofthe four required hospitalization for their acute retroviral ill-nesses. Evidence from several studies suggests that patientswith symptomatic acute retroviral infection, or those withmore prolonged symptoms, progress more rapidly toAIDS.6,7,9-11,75,76 Indeed, three of the four subjects in our studyhad CD4+ lymphocyte counts below 500 cells/ju.1 within 1 yearof infection, consistent with accelerated courses of HIV dis-ease. It is possible that individuals with symptomatic primaryinfection are relatively deficient in the immune responses mostcritical to containing HIV infection. In addition, it has been sug-gested that viral factors, such as a syncytium- or non-syncytium-inducing phenotype, may play an important role in the immuneresponse and clinical course of HIV infection.77-79 A study ofboth symptomatic and asymptomatic individuals with primaryHIV infection would be useful in identifying the viral pheno-types and immune responses to primary infection in a widerspectrum of cases.

In summary, our study suggests that CTLs and ADCC mayboth play important roles in controlling initial viral replicationin acute HIV infection. Further delineation of the CTL andADCC responses both in acute and chronic HIV infection isneeded to ascertain which types of responses are beneficial. Inaddition, studies of immune responses in a wider population ofseroconverters are warranted, since the symptomatic serocon-

verters usually studied tend to have much more acceleratedcourses of HIV disease than average. A better understanding ofthe immune response in early HIV infection could provide im-portant insights into the development of vaccines or other im-mune based therapies for HIV disease. In addition, a better un-

derstanding of these immune responses may be useful inshedding some light on the enigma of persistent viral replica-tion and the inability of the host to eradicate HIV disease de-spite vigorous virus-specific immune responses.

ACKNOWLEDGMENTS

We thank the four subjects who participated in these stud-ies. We also thank David Skolnick, Rick Schlichtemeier,Patricia Uherova, and Russell K. Young for technical assis-tance, Peggy Bakker for assistance in obtaining clinical spec-imens, Rita Sangaline for performing the flow cytometry stud-ies, Mary Ann Leukel and Dr. Henry Claman for HLA typingperformed at the University of Colorado Health SciencesCenter, Dr. Jeffrey Lifson and Dr. Michael Piatak for per-mission to use their QC-PCR data from subject 1, Dr. LindaBaum for advice concerning the methodology of ADCC as-

says, and Dr. Elizabeth McFarland for thoughtful commentson the manuscript. Financial support for this research was pro-vided by Public Health Service Grants R01-CA37461 (R.T.S.,E.C.) and 1T32 AI07447-01. Previously, this work was pre-sented in part at the VIII International Conference on AIDS/HISTD World Congress, Amsterdam, The Netherlands, July19-24, 1992 (abstract PoA2172); Keystone Symposia on

Molecular and Cellular Biology, Frontiers in HIVPathogenesis, Alburquerque, New Mexico, March 29-April 4,1993 (abstract Q107); and the IX International Conference on

AIDS/IV STD World Congress, Berlin, Germany, June 7-11,1993 (abstract PO-A22-0488).

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