JOURNAI OF CI INICAI MICROBIOI 0c,. JUIV 1989. P. 1577-15810095-11 37/89/071577-05$02.00/0Copyright V 1989. American Society for Microbiology

Antibody Response to Human Immunodeficiency Virus Type 1Protease according to Risk Group and Disease Stage

CHARLES A. B. BOUCHER.' MICHAFL H. DE JAGER.' CHRISTINE DEBOUCK,- LEON G. EPSTEIN,'3FRANK DE WOLF,.'4 TOM F. W. WOLFS,' AND JAAP GOUDSMIT'*

HIunan Retrorirzîs Laborator+, A((demi(4Me(fical Center,1 a"(d Departtment of Inféctious Diseases, Municipal HealthService,4 Anstezerdain, The Netherlands: Deparutnent ofMolecular Genetics, Sinith KMine & Frech Lahoratories, King of

Prussia, Penn.svlhania 19479 ; and lniher.itv of Me(icine and Dentistrv-New' Jersev Medical School and Children 'sHospital AIDS Program, Newark, New Jersey 07103-2757-

Received 17 November 1988/Accepted 20 March 1989

Three groups with different routes of human immunodeficiency virus type 1 (HIV-1) transmission(homosexual men, hemophiliacs, and children) were studied for serum antibodies to a recombinant form of theHIV-1 protease using an enzyme-linked immunoassay. At 1 year after seroconversion, defined as the momentantibodies to HIV-1 proteins were first detected, 56% (34/61) of the homosexual men had antibodies toprotease, and 2 years after seroconversion this percentage was 63% (24/38). Within this 2-year period theseantibodies were no longer detected in 16% (9/56). A similar pattern was observed in 20 hemophiliacs whoseroconverted after exposure to HIV-1-contaminated blood products. We found that 63% (160/255) ofhomosexual men in Centers for Disease Control stage II or III, 60% (9/15) of patients with acquiredimmunodeficiency syndrome (AIDS)-related complex, and 36% (14/39) of patients with AIDS had antibodies toprotease. In 255 homosexual men in Centers for Disease Control stage II or III, antibodies to protease weresignificantly more frequently found in samples lacking HIV-1 antigen (P < 0.001) and possessing antibodies toHIV-1 core proteins (P < 0.001). Twenty-four persons who developed AIDS were studied longitudinally: 58%(14/24) had antibodies to protease 1 year before developing symptoms; 29% (7/24) showed a decline and 29%(7/24) showed a loss of antibodies to protease at the onset of symptoms. Within a group of 47 HIV-1-infectedchildren, 90% (18/20) with a stable disease course were persistently protease antibody positive, versus 4 of 27children (15%) with an unstable disease course (P = 0.0001). These data indicate that HIV-1 protease isexpressed and antigenic in most HIV-1-infected individuals and that a decline or absence of antibodies toprotease is strongly associated with unstable disease in children and AIDS in adults.

The human immunodeficiency virus type 1 (HIV-1) is thecause of the acquired immunodeficiency syndrome (AIDS;4, 11, 20). The humoral antibody response after HIV-1infection is broad, showing antibodies to structural proteins(HIV-1 Ab) (envelope [3]. core [18], and polymerase [21]) aswell as to several regulatory proteins, including the transac-tivator protein (Tat) (1), negative factor (Nef) (2). virioninfectivity factor (Vif) (2), regulator of expression of virionproteins (Rev) (16), and two proteins without a knownfunction, Vpr and Vpu (22. 25). Retroviral protease is anenzyme that cleaves Gag and Gag-polymerase precursorpolyproteins into functional proteins of mature virus parti-cles, and this processing is necessary for the infectivity ofvirus particles. In vitro mutagenesis of the protease-codingregion of murine leukemia virus and HIV-1 resulted inprotease-defective virus lacking infectivity (15, 17). Thetranslation product of the HIV-1 protease-coding regionexhibits autoprocessing, resulting in a functional Il-kilo-dalton protein (8). To what extent and at what time duringnatural infection this 11-kilodalton protease is expressed are

not known.Studies of HIV-1-infected individuals have shown a de-

cline in antibodies to pl7f"Z-' and p24'"' core proteins (HIV-1cAb) prior to development of symptoms of immunodefi-ciency (18, 19). This decline in HIV-1 cAb is followed by theappearance of HIV-1 core antigens (HIV-1 cAg) in the serum

(13). A third but later determinant of disease progression is a

decline in the amount of CD4t lymphocytes (9). By using the

Corresponding author.

disappearance of HIV-1 cAb and persistent HIV-1 antigen-emia, it was possible, in a prospective study, to predict thedevelopment of 80% of al AIDS cases (9). However, evenwhen a decline in the number of CD4+ lymphocytes was alsotaken into account, a small group with a grave short-termprognosis remained unidentified. To study the expression ofHIV-1 protease in natural infection and in a search foradditional prognostic markers, the antigenicity of HIV-1protease was examined. A bacterially expressed proteasewas used as the antigen in an enzyme-linked immunoaissay(EIA). Using this assay, we analyzed sera collected overtime from several cohorts of HIV-1-infected individuals forantibodies to protease in relation to disease progression.

MATERIALS AND METHODS

Study population. Sera to be tested were obtained fromfour cohorts. The first group included participants in aprospective study on the prevalence and incidence of HIVinfection and risk factors for AIDS. This study was started inOctober 1984, and until May 1986, 961 asymptomatic menliving in The Netherlands who had at least two homosexualcontacts in the preceding 6 months were enrolled. Clinicaldata and blood samples were collected every 3 months. Ofthe 961 men, 238 persons were HIV-1 Ab-positive in the firstsample taken. Between October 1984 and October 1987, 74of the remaining 723 men seroconverted for HIV-1 Ab.Owing to different dates of HIV-1 Ab seroconversion, theduration of the follow-up period, after seroconversion, var-ied from 3 months to over 2 years. Of these 312 infectedmen, 255 remained asymptomatic (Centers for Disease Con-

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1578 BOUCHER ET AL.

trol [CDC] stage Il) or had persistent generalized lymphad-enopathy only (CDC stage III), 17 developed AIDS-relatedcomplex (ARC; CDC stage IV A), and 40 developed AIDS(CDC stage IV B-E) (5). The samples used to detect anti-bodies to protease were the latest drawn samples for persons

in CDC stage Il or III, and for patients in CDC stage IV, thesamples were drawn at the time ARC or AIDS was diag-nosed. The second group consisted of 143 patients withAIDS diagnosed at the AIDS Unit, Academic MedicalCenter, Amsterdam, The Netherlands. Serum specimenswere obtained within 3 months of the AIDS diagnosis. Thethird group comprised hemophiliacs who were participantsin a prospective study which began in 1983 in The Nether-lands (24). Of 157 patients negative at entry, clinical data andblood samples were collected every 6 months. Of these, 20developed HIV-1 Ab during follow-up; their mean age was

26 years; 6 of them were under 18. Ail 20 remained asymp-

tomatic or developed persistent generalized lymphadenopa-thy only.The fourth group consisted of 47 children with HIV-1

infection. These children were monitored at the New JerseyChildren's Hospital AIDS Program, Newark. By using thenew CDC classification system for children under 13 years ofage (6), all of these children were classified as P2 (sympto-matic). The clinical condition of the children was rated stableor unstable by determining their pulmonary status, theirneurological status, and the presence of opportunistic or

major bacterial infections (10).None of the patients was receiving treatment with either

zidovudine or interferon at the time serum samples were

drawn.Detection of HIV-1 Ab, HIV-1 cAg, and HIV-1 cAb. HIV-1

Ab were detected with two commercially available enzyme-linked immunosorbent assays. Initially purified human T-celllymphotropic virus type IIIB (HTLV-IIIB) was used as theantigen (Vironostika; Organon Teknika, Oss, The Nether-lands). Later, bacterially produced recombinant HIV-1 Agconsisting of a mixture of viral core and envelope proteinswere used (Abbott recombinant HIV-1 EIA; Abbott Labo-ratories, Chicago, Ill.) (7). The moment of seroconversionwas standardized by retrospectively testing all samples fromseroconverters using the Abbott recombinant HIV-1 EIA.Seropositivity was confirmed by either immunoblotting or a

commercially available recombinant EIA (Envacor; Ab-bott). Quantification of antibodies to core proteins in Enva-cor was achieved by comparing the optical density of a

sample with the optical density of a serial twofold dilution ofa reference serum. A sample showing fewer antibodies than

the 1-to-16 dilution of the reference serum was considerednegative. Serum samples were assayed for unbound HIV-1cAg by using a sandwich-type EIA (Abbott) (12). The assay

is most sensitive for core protein p24 of HIV-1. By using a

titration curve of the positive control, it is possible toquantitate the amount of antigen to a cutoff value of 50pg/ml.

Detection of T-lymphocyte subsets. Peripheral blood mono-nuclear cells were isolated from heparinized venous bloodby density gradient centrifugation on Ficoll-Hypaque. CD4`and CD8 lymphocytes were enumerated by an indirectimmunofluorescence technique using monoclonal antibodies(Central Laboratory of The Netherlands Red Cross BloodTransfusion Service, Amsterdam) and a flow cytometrysystem (Epics-C; Coulter Electronics Ltd., Luton, UnitedKingdom). A low CD4' lymphocyte count was defined as

CD4 lymphocyte numbers below 0.5 x 109/liter.Detection of antibodies to HIV-1 protease. The protease

was produced in Eschelirhia coli as a transnational fusion tothe first 56 amino acids of E. coli galactokinase. The expres-sion plasmid, designated PRO1 (8), consisted of a 272-base-pair DdcI restriction fragment (protease residues 1 to92) from the BH10 clone of HTLV-IIIB inserted in thepOTSKF33 fusion vector (12) downstream and in-frame withthe amino terminus of galactokinase. The recombinant fu-sion protein was produced and purified as previously de-scribed (1).The presence of antibodies to protease was determined by

using a direct enzyme-linked noncompetitive solid-phaseimmunoassay. Microdilution plates were coated with therecombinant protein using 1 pxg per well in 100 pl ofphosphate-buffered saline (PBS) for 16 h at room tempera-ture. After being coated, wells were washed six times withPBS-Tween 20 (0.1%, [vol/vol]). Nonspecific binding siteswere blocked by incubation for 1 h at 37°C with 4% normalgoat serum in PBS-Tween 20, followed by another wash withPBS-Tween 20. Sera to be tested were diluted 1:100 inPBS-Tween 20 with 4% normal goat serum and incubated for1 h at 37°C. After another washing step, horseradish perox-idase-conjugated goat anti-human immunoglobulin G (KPL,Gaithersburg, Md.) was added, diluted 1:500 in PBS-Tween20 with 4% normal goat serum, and the wells were incubatedfor 1 h at 37°C. Bound antibodies were visualized usingortho-phenylenediamine, and the reaction was stopped with1 N H2S04 after a coloring time of 10 min. Optical densitywas read at 450 nm (reference filter, 630 nm). Interassayvariation was controlled by the inclusion on each plate of a

protease-positive human sample in a serial dilution.

TABLE 1. Development of antibodies to HIV-1 protease in homosexual men after HIV-1 Ab seroconversion

No. with HIV-1 Ab at time (mo) after seroconversion'Homosexual men

0-3 3-6 6-9 9-12 12-15 15-18 18-21 21-24 >24

No. of persons" 74 70 65 61 54 48 45 38 27HIV-1 protease antibody:

Positive 1 20 30 31 29 26 24 22 17Newly positive '2 15 6 3 2 2 1 2 2Becoming negative 0 2 1 3 1 1 1 0 0

% Positive 31 47 54 51 56 56 53 63 70Cumulative % positive" 31 50 55 56 57 58 56 63 70

The moments HIV-1 Ab was first detected were aligned and designated time zero." The diffreent points of time of HIV-1 Ah seroconversion account ftr the gradual decline in number of persons (e.g.. 61 persons seroconverted for HIV-1 Ab

1 year ago. and 27 persons seroconverted more than 2 years ago).Cumulative percent positive reflects Hall persons who showed antibodies to protease ait any time during the follow-up and is not corrected for decline of

antibodies in some individuals.

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ANTIBODY RESPONSE TO HIV-1 PROTEASE 1579

TABLE 2. Development of antibodies to HIV-1 protease inhemophiliacs before and after HIV-1 Ab seroconversion

No. with HIV-1 Ab at time (mo) afterHemophiliacs seroconversion

-3 0-6 6-12 12-18 18-24 >24

No. of persons"' 20 20 20 19 14 ilHIV-1 protease antibody 1 10 4 0 1 Onewly positive

" The moments HIV-1 Ab \as first detected were aligned and designatedtime zero.

b The different points of time of HIV-1 Ab seroconversion accouunt for thegradual decline in number of persons (c.g.. 20 persons seroconverted forHIV-1 Ab 1 year ago. and Il persons seroconverted more than 2 years ago).

Statistics. Statistics used were the chi-square test, includ-ing the Yates correction, and Fisher exact test (23).

RESULTS

Antigenicity of protease. To obtain a cutoff value, serumsamples of 80 HIV-1 Ab-seronegative homosexual men weretested in the protease EIA. By using the mean absorbanceplus four times the standard deviation as the cutoff value.only one HIV-Ab-negative sample showed a value above thecutoff, indicating a specificity of approximately 99%. Withina group of 74 seroconverted homosexual men who werestudied longitudinally, a variable interval was observedbetween the moment of HIV-1 Ab (recombinant core andenvelope proteins) seroconversion and the moment antibod-ies to recombinant protease were first detected (Table 1).One person had antibodies to protease in a sample drawn 3months before seroconversion for HIV-1 Ab; 22 developedprotease antibodies at the same time as the HIV-1 Ab. At 1year after HIV-1 Ab seroconversion, 56% (34/61) of theindividuals had antibodies to protease. Another year later,this percentage was 63%c (24/38). In hemophiliacs, the pat-tern of protease antibody development was similar to thatobserved in homosexual men (Table 2). Of the 56 serocon-verters in the group of homosexual men who developedantibodies to protease, 9 individuals (16%) lost detectableprotease antibodies during follow-up.

Presence of antibodies to HIV-1 protease in relation toclinical status and prognostic markers. To determine therelationship between the presence of protease antibody andthe stage of infection, we determined antibodies to HIV-1protease retrospectively in 255 individuals classified as CDCstage Il or III, in 15 of 17 individuals who progressed toARC, and in 39 of 40 individuals who developed AIDS whileparticipating in our cohort study. In the population of 143hospitalized AIDS patients, protease antibody could bedetermined in 134 patients. Of 255 individuals in CDC stageIl or III, 63% (160/255) had antibodies to protease. Withinthe ARC group, 60% (9/15) were protease antibody positive,

in contrast to 36% (14/39) of the AIDS cohort group and 271%(36/134) of the hospitalized AIDS group (9 patients withAIDS were not tested) (Table 3).Next we analyzed the relationship in CDC stage Il or III

between antibody to protease and the prognostic markers:HIV-1 cAg, HIV-1 cAb, and CD4' lymphocyte counts(Table 4). In the protease antibody-positive group, 73%(116/160) also showed the presence of HIV-1 cAb, versus

46% (44/95) of protease antibody-negative persons (P <0.001). Only 11%W (17/158) of the protease antibody-positivepersons were positive for HIV-1 cAg, versus 35%C (33/95) ofthe protease antibody-negative persons (P < 0.001; in twopersons, HIV-1 Ag was not determined). The number ofCD4' lymphocytes was determined in 213 men. Of 133protease antibody-positive men, 62%, (83/133) showed CD4'numbers above 0.5 x 109/liter, versus 56%c (45/80) in theprotease antibody-negative cohort (P = 0.5).

Within the group of 173 patients with AIDS, the relation-ship between the prognostic markers and the antibodies toprotease was also studied. HIV-1 cAg was more oftenpresent in protease antibody-negative patients (69% [81/1181)as opposed to protease antibody-positive patients (53%r26/49]; P = 0.08; in 6 patients, HIV-1 cAg was not deter-mined). Furthermore, 17% (18/108) of protease antibody-negative AIDS patients were HIV-1 cAb positive, versus

33% (16/48) of protease antibody-positive patients (P = 0.03;in 17 patients, HIV-1 cAb was not determined).

Next, the value of the combination of HIV-1 cAg andeither absence of HIV-1 cAb or absence of antibodies toHIV-1 protease was determined. Of 166 AIDS patients(cohort and hospital population) at diagnosis, 33 (20%) were

both HIV-1 cAg negative and HIV-1 cAb positive. Thus, in80% of the patients, HIV-1 cAb was not detectable and/orHIV-1 cAg was present (in 17 patients, the combination was

not determined). Of 171 AIDS patients, 150 (88%/) were

protease antibody negative and/or antigenemic (in 12 pa-

tients, the combination was not determined).To study the development of antibodies to protease over

time, serum samples from 47 American HIV-1-infectedsymptomatic children were tested. Within the group of 27children with unstable disease course, several patterns were

observed. Six children developed antibodies to proteaseduring follow-up, four were persistently positive, seven lostantibodies. and ten were persistently negative. Of the group

of 20 children with stable disease, 18 were persistentlypositive and 2 were persistently negative. Thus, 90%c (18/20)of children with stable disease were persistently proteaseantibody positive, versus only 15%c (4/27) of children withunstable disease (P = 0.0001). Of all 47 children, 12 diedduring follow-up; 9 of these 12 (75%) either lost antibodies or

were persistently negative (3 were persistently positive).Within the group of 35 children still alive, 10 (28%) either lostantibodies or were persistently negative (P < 0.006).

TABLE 3. Presence of antibodies to protease and three prognostic markers in different clinical stages of HIV-1 infection

C1p- Protease antibody positive 74 HIV-1 cAb positive Û/ HIV-1 cAg positive CD4+ lymphocytes(no. positive/total) (no. positive/total) (no. positive/total) <0.5 x 10"/liter

CDC Il or III 63 (160/255) 64 (162/254) 20 (50/253) 40 (85/213)ARC 60 (9/15) 44 (7/16) 41 (7/17) 50 (8/16)AIDS cohort 36 (14/39) 25 (9/36) 66 (25/38) 74 (14/19)AIDS hospital 27 (36/134) 23 (29/125) 60 (83/139) ND'AIDS combined 29 (50/173) 24 (38/161) 61 (108/177) ND

" ND. Not determined.

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TABLE 4. Presence of three prognostic markers in relation toantibodies to HIV-1 protease

% Persons with or without antibodies to

Group protease (no./total)With Without

CDC Il or III 63 (160/255) 37 (95/255)HIV-1 Ag positive Il (17/158) 35 (33/95)HIV-1 Ab positive 73 (116/160) 46 (44/95)CD4+ lymphocytes 62 (83/133) 56 (45/80)

>0.5 x 109/liter

CDC IV B-E 29 (50/173) 71 (123/173)HIV-1 Ag positive 53 (26/49) 69 (81/118)HIV-1 Ab positive 33 (16/48) 17 (18/108)

In 24 adult AIDS patients, a serum sample drawn a mean

of 1 year before diagnosis was compared for proteaseantibody with a serum sample drawn at the time of diagnosis.At 1 year before diagnosis, 58% (14/24) had protease anti-bodies. At the moment of AIDS diagnosis, 42% (10/24) werestill negative, 29% (7/24) showed a decline in proteaseantibodies (determined by comparing optical densities), and29% (7/24) had lost protease antibodies.The results of this longitudinal study are comparable to the

results of the transectional study on the larger groups of thecohort.

DISCUSSION

The antigenicity of protease in natural HIV-1 infectionwas studied, using a bacterially expressed HIV-1 proteasegene product as the antigen in an EIA. A majority (70%) ofHIV-1-infected individuals monitored over a period longerthan 2 years after infection was found to have antibodies toHIV-1 protease. Some individuals failed to develop anantibody response to protease; no significant differences in

optical densities of samples drawn before or after HIV-1 Abseroconversion were found in these individuals (data notshown). Since viral protease is expressed and cleaved froma Gag-polymerase precursor (14) and some of the proteaseantibody-negative individuals showed the presence of coreproteins (HIV-1 Ag) in their serum, one can assume thatprotease itself is exposed to the immune system.

Protease antibody negativity could be explained by varia-tion in antigenic epitopes of protease among various HIV-1strains, resulting in restricted reactivity, although this is notlikely given the relatively low variation between sequenced

luu -

80 -

60 Antigen >50 pg/ml

40-~40- anti-protease positive_ ~sCD4ecells >0,5.109/L

20anti-core positive

O-CDC Il/lit ARC (CDC tV-A) AIOS (CDC IV B-E)

FIG. 1. Presence of four serological markers in progressingdisease stages.

HIV-1 protease genes. Indeed, comparing the amino acidsequences from the pol gene of three HIV-1 strains (HTLV-IIIB, LAV-1, and SF-2), a maximal difference of 4% wasobserved versus 15%c for the envelope gene (26). Variation inthe individual response to the epitopes present on HIV-1protease is another explanation for the failure to developantibodies to protease.Three prognostic markers-HIV-1 cAg, HIV-1 cAb, and

CD4+ lymphocyte numbers in a transectional cohort ofinfected persons in CDC stage Il or 1I1-were compared forthe presence of antibodies to viral protease. In persons withasymptomatic infection, the presence of antibodies to pro-tease showed a relationship with both the presence of HIV-1Ab and the absence of HIV-1 Ag.

Antibodies to protease were found less frequently inpatients with AIDS, suggesting that these antibodies are lostin the later stages of disease. Since this study was notperformed longitudinally, another explanation might be thatpersons who never develop an antibody response to proteaseare at increased risk of progression to AIDS. However, asmall subgroup of 14 initially protease antibody-positiveinfected homosexual men studied longitudinally showed adecline or loss of antibodies to protease at the moment AIDSwas diagnosed. Within a group of HIV-1-infected children,the majority of those with a stable disease course werepersistently positive in the protease antibody assay. Follow-up of this group is indicated to determine whether thetransition to unstable disease is paralleled by a decline ordisappearance of antibodies to protease.At present it is not clear what causes the decline in

protease antibodies. The disappearance of detectable anti-bodies in serum might be due to the formation of immunecomplexes with viral protease. Alternatively, the decline inprotease antibodies may result from B-cell dysfunction in thelater stages of disease. In co[nparing CDC stage Il or III withstage IV A, no difference was found regarding the percent-age of protease antibody-positive individuals. This suggeststhat the decline in antibodies to protease is a late effectrather than an early marker (Fig. 1), although the size of thegroup of patients with ARC is too small to draw definiteconclusions. More longitudinal studies are needed, however,to determine the time span between the decline of proteaseantibodies and the development of disease. Considering thatthe decline in protease antibodies seems to be a late effectand that protease is a scarce antigen, antibodies to proteaseare less likely to be important in preventing disease.

In conclusion, during natural infection with HIV-1, mostpeople develop an antibody response to protease, showingthat the viral protease is expressed during infection and quiteantigenic. The differences in protease reactivity amonggroups in different stages of disease indicate that the amountof detectable antibodies to protease declines with diseaseprogression.

ACKNOWLEDGMENTS

We thank Erna Albers for her excellent help in preparing themanuscript and José Houweling for helping with statistical analysis.

This study was partly supported by The Netherlands Foundationfor Preventive Medicine (grants PF 28-1026 and PF 28-1028) andPublic Health Ser-vice grant NS 25032 (to L. G. Epstein) from theNational Institutes of Health.

LITERATURE CITED1. Aldovini, A., C. Debouck, M. B. Feinberg, M. Rosenberg, S. K.

Arya, and F. Wong-Staal. 1986. Synthesis of the completetrans-activation gene product of human T-lymphotropic virus

J CLIN MlT ROBIOL.

a)

Q)

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ANTIBODY RESPONSE TO HIV-1 PROTEASE 1581

type 111 in Escherichia coli: demonstration of immunogenicity invivo and expression in vitro. Proc. Nati. Acad. Sci. USA83:6672-6676.

2. Arya, S. K., and R. C. Gallo. 1986. Three novel genes of humanT-lymphotropic virus type 111; immune reactivity of their prod-ucts with sera from acquired immune deficiency syndromepatients. Proc. Natl. Acad. Sci. USA 83:2209-2213.

3. Barin, F., M. F. McLane, J. S. Allan, T. H. Lee, J. E.Groopman, and M. Essex. 1985. Virus envelope protein ofHTLV 1II represents major target antigen for antibodies inAIDS patients. Science 228:1094-1096.

4. Barre-Sinoussi, F., J. C. Chermann, F. Rey, M. T. Nugyere, S.Chamaret, J. Gruest, C. Dauguet, C. Axier-Blin, F. Brun-Vezinet, C. Rouzioux, W. Rozenbaum, and L. Montagnier. 1983.Isolation of a T-lymphotropic retrovirus from a patient at riskfor acquired immune deficiency syndrome (AIDS). Science220:868-871.

5. Centers for Disease Control. 1986. Classification system forhuman T-lymphotropic virus type III/lymphadenopathy associ-ated virus infections. Morbid. Mortal. Weekly Rep. 35:334-339.

6. Centers for Disease Control. 1987. Classification system forhuman immunodeficiency virus (HIV) infection in childrenunder 13 years of age. Morbid. Mortal. Weekly Rep. 36:'25-236.

7. Dawson, G. J., J. S. Heller, C. A. Wood, R. A. Guttierez, J. S.Webber, J. C. Hunt, S. A. Hojvat, D. Senn, S. G. Devare, andR. H. Decker. 1988. Reliable detection of individuals seroposi-tive for the human immunodeficiency virus (HIV) by competi-tive immunoassays using Escherichia coli-expressed HIV struc-tural proteins. J. Infect. Dis. 157:149-155.

8. Debouck, C., J. G. Gorniak, J. E. Strickler, T. D. Meek, B. W.Metcalf, and M. Rosenberg. 1987. Human immunodeficiencyvirus protease expressed in Escherichia coli exhibits autopro-cessing and specific maturation of the gag precursor. Proc. Natl.Acad. Sci. USA 84:8903-8906.

9. de Wolf, F., J. M. A. Lange, J. T. M. Houweling, R. A.Coutinho, P. T. Schellekens, J. van der Noordaa, and J. Goud-smit. 1988. Numbers of CD4+ cells and the levels of coreantigens of and antibodies to the human immunodeficiency virusas predictors of AIDS among seropositive homosexual men. J.Infect. Dis. 158:615-622.

10. Epstein, L. G., C. A. B. Boucher, S. H. Morrison, E. M. Connor,J. M. Oleske, J. M. A. Lange, J. van der Noordaa, M. Bakker, J.Dekker, H. Scherpbier, H. van den Berg, K. Boer, and J.Goudsmit. 1988. Persistent HIV-l antigenemia in children cor-relates with disease progression. Pediatrics 82:919-924.

11. Gallo, R. C., S. Z. Salahuddin, M. Popovic, G. M. Shearer, M.Kaplan, B. F. Haynes, T. J. Palker, R. Redfield, J. Oleske, B.Safai, G. White, P. Foster, and P. D. Markham. 1984. Frequentdetection and isolation of cytopathic retroviruses (HTLV-1I)from patients with AIDS and at risk for AIDS. Science 224:500-503.

12. Goudsmit, J., C. Debouck, R. H. Meloen, L. Smit, M. Bakker,D. M. Asher, A. V. Wolff, C. J. Gibbs, Jr., and D. C. Gajdusek.1988. Human immunodeficiency virus type 1 neutralizationepitope with conserved architecture elicits early type-specificantibodies in experimentally infected chimpanzees. Proc. Natl.Acad. Sci. USA 85:4478-4482.

13. Goudsmit, J., F. de Wolf, D. A. Paul, L. G. Epstein, J. M. A.Lange, W. J. A. Krone, H. Speelman, E. C. Wolters, J. van derNoordaa, J. M. Oleske, H. J. van der Helm, and R. A. Coutinho.1986. Expression of human immunodeficiency virus antigen(HIV-Ag) in serum and cerebrospinal fluid during acute andchronic infection. Lancet i:177-180.

14. Jacks, T., M. D. Power, F. R. Masiarz, P. A. Luciw, P. J. Barr,and H. E. Varmus. 1988. Characterization of ribosomal frame-shifting in HIV-1 gag-pol expression. Nature (London) 231:280-283.

15. Katoh, I., Y. Yoshinaka, A. Rein, M. Shibuya, T. Odaka, and S.Oroszlan. 1985. Murine leukemia virus maturation: proteaseregion required for conversion from "immature" to "mature"core form and virus infectivity. Virology 145:280-292.

16. Knight, D. M., F. A. Flomerfelt, and J. Ghrayeb. 1987. Expres-sion of the art/trs protein of H1V and study of its role in viralenvelope synthesis. Science 236:837-840.

17. Kohl, N. E., E. A. Emini, W. A. Schleif, L. J. Davis, J. C.Heimbach, R. A. F. Dixon, E. M. Scolnick, and I. S. Sigal. 1988.Active human immunodeficiency virus protease is required forviral infectivity. Proc. Natl. Acad. Sci. USA 85:4686-4690.

18. Lange, J. M. A., R. A. Coutinho, W. J. A. Krone, L. F.Verdonck, S. A. Danner, J. van der Noordaa, and J. Goudsmit.1986. Distinct IgG recognition patterns during progression ofsubclinical and clinical infection with Iymphadenopathy associ-ated virus/human T lymphotropic virus. Br. Med. J. 292:228-230.

19. Lange, J. M. A., F. de Wolf, W. J. A. Krone, S. A. Danner,R. A. Coutinho, and J. Goudsmit. 1987. Decline of antibodyreactivity to outer viral core protein pl7 is an earlier serologicalmarker of disease progression in human immunodeficiency virusinfection than anti-p24 decline. AIDS 1:155-159.

20. Levy, J. A., A. D. Hoffman, S. M. Kramer, J. A. Landis, J. M.Shimabukuro, and L. S. Oshiro. 1984. Isolation of lymphotropicretroviruses from San Francisco patients with AIDS. Science225:840-842.

21. Steimer, K. S., K. W. Higgins, M. A. Powers, J. C. Stephans, A.Gyenes, C. George-Nascimento, P. A. Luciw, P. J. Barr, R. A.Hallewell, and R. Sanchez-Pescador. 1986. Recombinant poly-peptide from the endonuclease region of the acquired immunedeficiency syndrome retrovirus polymerase (pol) gene detectsserum antibodies in most infected individuals. J. Virol. 58:9-16.

22. Strebel, K., T. Klimkait, and M. A. Martin. 1988. A novel geneof HIV-1. vpu, and its 16-kilodalton product. Science 241:1221-1223.

23. Svinscow, T. D. V. 1987. Statistics at square one. p. 46-48.British Medical Association. London.

24. Wolfs, T. F. W., C. Breederveld, W. J. A. Krone, L. van derHoek, M. Bakker, J. Goudsmit, and the Dutch HaemophiliaGroup. 1988. HIV-antibody seroconversions in Dutch haemo-philiacs using heat-treated and non heat-treated coagulationfactor concentrates. Thromb. Haemostasis 59:396-399.

25. Wong-Staal, F., P. K. Chanda, and J. Ghrayeb. 1987. Humanimmunodeficiency virus: the eighth gene. AIDS Res. Hum.Retroviruses 3:33-39.

26. Yasanuga, T., N. Sagata, and Y. Ikawa. 1986. Protease genestructure and env gene variability of the AIDS virus. FEBSLett. 199:145-150.

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