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Serologic confirmation of human T-lymphotropic virus type I infection in

healthy blood and plasma donors

Article  in  Blood · December 1989

DOI: 10.1182/blood.V74.7.2585.bloodjournal7472585 · Source: PubMed

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 BJ Poiesz and LT PierikDW Anderson, JS Epstein, TH Lee, MD Lairmore, C Saxinger, VS Kalyanaraman, D Slamon, W Parks, healthy blood and plasma donorsSerological confirmation of human T-lymphotropic virus type I infection in

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Blood, Vol 74. No 7 (November 15), 1989: pp 2585-259 1 2585

Serological Confirmation of Human T-Lymphotropic Virus Type I Infection inHealthy Blood and Plasma Donors

By David W. Anderson, Jay S. Epstein, Tun-Hou Lee, Michael D. Lairmore, Carl Saxinger, V.S. Kalyanaraman,

Dennis Slamon, Wade Parks, Bernard J. Poiesz, Lauren T. Pierik, Helen Lee, Richard Montagna,

Patrick A. Roche, Alan Williams, and William Blattner

We wished to develop criteria for serological confirmation

of human T-lymphotropic virus type I (HTLV-l) infection in

healthy donors. Selected serum or plasma samples reac-

tive by HTLV-l enzyme immunosorbent assay or gel-

agglutination assays with at least one viral-specific band on

Western immunoblot (WIB) were tested in six laboratories

by four WIBs and four radioimmunoprecipitation assays

(RIPAs) for antibodies to HTLV-l proteins encoded by gag

(p19 and p24). env (gp46 and/or gp6l). and tax (p41?)

genes. One hundred forty-two donor sera were obtained

from 38 Japanese. 69 American, and 35 Caribbean blood or

plasma donors. Among these samples. WIB assays

appeared more sensitive to p24 antibodies. whereas RIPAs

were significantly more sensitive to gp6l antibodies. All

sera (1 37) with gp6l antibodies had p24 antibodies. Of the

H UMAN T-CELL lymphotropic virus type I (HTLV-I),

the first known human retrovirus, is associated with an

aggressive lymphoma, adult T-cell leukemia/lymphoma

(ATL), and a degenerative demyelinating neurological dis-

ease called tropical spastic paraparesis (TSP) in the Carib-

bean and HTLV-I-associated myelopathy (HAM) in

Japan.”2 Development of AlL may require years to decades

after infection with HTLV-I.3’4 Preliminary data from Japa-

nese patients with HAM and a history of blood transfusion

suggest a mean interval of 4 years between transfusion and

development of neurologic symptoms (Osame M, unpub-

lished observations, November, 1988). HTLV-I endemic

areas have been identified in southern Japan, the Caribbean,

and parts of Africa.5 HTLV-lI is a closely related retrovirus

which shares considerable genomic homology with HTLV-I

and is believed to be indistinguishable from HTLV-I with

current serologic techniques. Little is known about the

epidemiology and clinical outcomes of HTLV-II infections

except for their apparent high frequency among drug abusers

and occasional isolation from patients with T-hairy cell

leukemia and some B-cell lymphoid malignancies. In the

absence of virus isolation or characterization of gene

sequences by polymerase chain reaction (PCR), the virus

type of seropositives in the United States is best classified as

HTLV-l/ll. Antibodies to HTLV-I/II were recently

detected in 10 (0.025%) of 39,898 random blood donors in

eight US cities,6 and HTLV-I/lI seroconversion in multiply

transfused US hospital patients has been documented.7

On November 29, 1988, enzyme immunoassay (EIA)

screening tests designed to detect antibodies to HTLV-l were

licensed by the US Food and Drug Administration (FDA).8

Implementation of HTLV-I EIA screening ofdonated whole

blood and cellular components is currently underway in

many blood establishments nationwide. An estimated I 3,600

US blood donors may test positive in the first year of

HTLV-I EIA screening. (The number of blood donors test-

ing positive [repeatably reactive] in the first year of HTLV-I

EIA screening was estimated as the product of 8 x 106 blood

donors times 0.17%6 repeatably reactive rate). Although

1 37 sera positive for p24 and gp6l antibodies. p19

antibodies were detected in 1 29 sera, and p41? antibodies

were detected in 1 08. In sera with p1 9 antibodies and

antibodies to env- or tax-encoded proteins, p24 antibodies

were always present. Antibodies to p4O� were not found in

the absence of gp6l antibodies. Virological evidence of

infection was found in seven American donors by lympho-

cyte coculture (one HTLV-l. one HTLV-ll) or by polymerase

chain reaction (three HTLV-l, two HTLV-ll). Sera from all

seven donors showed p24 and gp46 and/or gp6l antibod-

ies. We suggest that seroreactivity to both p24 and gp46

and/or gp6l by WIB or RIPA or both are suitable criteria to

confirm but not to distinguish HTLV-l and HTLV-ll infec-

tions.

S 1989 by Grune & Stratton, Inc.

blood from these donors will not be used for transfusion, less

than one of six is likely to be confirmed positive with

additional, more specific serological tests.6 Thus, criteria

used to confirm HTLV-I/II seropositivity will affect notifi-

cation and counseling strategies used for thousands of

healthy donors.

As with screening tests for human immunodeficiency

virus-I (HIV-l), a distantly related but not serologically

cross-reactive retrovirus, current approaches to confirmation

of l-ITLV-I/Il seropositivity rely on additional, more specific

tests such as Western immunoblot (WIB) and radioimmuno-

precipitation assay (RIPA).9”#{176} Both WIB and RIPA enable

separate demonstration of antibodies to HTLV-I proteins

encoded by viral genes. However, there are important differ-

ences between WIB and RIPA in terms of viral antigen

From the Center for Biologics Evaluation and Review, FDA.

Bethesda, MD; Department of Cancer Biology. Harvard School of

Public Health, Boston, MA; Laboratory of Tumor Cell Biology and

the Viral Epidemiology Section. National Cancer Institute. Bet hes-

da, MD; Retrovirus Diseases Branch, Center for Disease Control,

Atlanta, GA; Bionetics Research, Rockville, MD; Division of

Hematology/Oncology, UCLA School of Medicine, Los Angeles,

CA; Department of Pediatrics/Immunology. University of Miami.

Miami, FL; Division of Hematology/Oncology. Department of

Medicine, SUNY at Syracuse, Syracuse. NY; Abbott Laboratories,

North Chicago, IL; Cellular Products, Buffalo, NY; E. I. Dupont

De Nemours, Wilmington. DE; and American Red Cross Jerome H.

Holland Laboratory, Rockville. MD.

Submitted March 20, 1989; accepted July i I, /989.

Address reprint requests to Jay S. Epstein. MD. Chief, Labo-

ratory of Retrovirology, Division of Blood and Blood Products,

Center for Biologics Evaluation and Research, Food and Drug

Administration, 8800 Rockville Pike, Bethesda, MD 20892.

The publication costs ofthis article were defrayed in part by page

charge payment. This article must therefore be hereby marked

“advertisement” in accordance with 18 U.S.C. section 1 734 solely to

indicate thisfact.

� I 989 by Grune & Stratton, Inc.

0006-4971/89/7407-0007$3.00/0

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2586 ANDERSON ET AL

preparation and the physicochemical milieu of antigen-

antibody interaction. Viral antigen commonly used in WIB

formats is derived from density-gradient purified virions

rather than extracts of infected cells. Viral proteins in WIB

formats therefore consist predominantly of virus particle-

associated structural proteins (gag-encoded proteins p19,

p24, and their precursor p55 and env-encoded proteins

gp2lE, gp46, and their precursor gp6I/68). After boiling,

sodium dodecyl sulfate (SDS) treatment, reduction and

electrophoretic transfer, all of which may modify the anti-

gens, the antigen-antibody interaction occurs on a two-

dimensional nitrocellulose membrane. In contrast, viral anti-

gens commonly used in RIPA formats are extracts from

chronically-infected cells and include structural as well as

nonstructural (tax-encoded p4(Y) regulatory proteins at van-

ous stages of posttranslational modification. The antigen-

antibody interaction in a RIPA occurs in a solution (three-

dimensional) of whole cellular lysate under nondenatuning

conditions.Investigators have used various confirmatory criteria and

differing combinations of WIB and RIPA to identify asymp-

tomatic HTLV-I/II seropositive persons.9”#{176} Indeed, valida-

tion of serological confirmatory criteria in this setting is

complicated by the inability of current assays to distinguish

antibodies to HTLV-I from antibodies to HTLV-II, a lack of

common positive reference sera, and incomplete knowledge

ofvariability in the pattern of HTLV-I-specific antibodies or

HTLV-Il-specific antibodies among persons after infection.

An additional complication is insufficient data regarding the

comparative performance of WIB and RIPA in detecting

variations in the pattern of antibodies to specific HTL V-I/I!

antigens, particularly when different cell lines are used for

antigen production.

Using a variety of assays in six different laboratories, we

undertook to characterize the pattern of HTLV-I/II-

specific antibodies in healthy blood and plasma donors. Our

investigation represents a consensus approach to confirma-

tion of HTLV-l/II seropositivity and serves as the basis for

current Public Health Service and blood bank guidelines for

confirming positive results of antibody screening. The results

obtained provide a rationale for use of antibodies to certain

gag and env gene products in confirmatory criteria as well as

for assessing the suitability of WIB and RIPA for use in

confirmatory strategies.

MATERIALS AND METHODS

Positive and negative reference sera. The Center for BiologicsEvaluation and Research (CBER), US FDA distributed to each ofsix collaborating laboratories and one in-house laboratory (Table 1)

eight uncoded sera (positive reference sera) from US patientsclinically diagnosed as having ATL (three cases) or TSP (five cases)(B.J. Poiesz, unpublished observations, December, 1987). All caseshad evidence of HTLV-I-specific sequences in peripheral lympho-cyte DNA extracts amplified by PCR,” including six patients withHTLV-l-specific sequences found by Southern blotting of unampli-

fled lymphocyte DNA extracts. Each laboratory chose to studyantibodies to one or several of the gag-encoded core proteins p19,p24, and precursor p55, the env-encoded glycoproteins gp46 or gp6l,gp63, gp65, or gp68 (hereafter called gp6l) or the tax-encodedtranscriptional activator p4#{216}X(Table 1 ). These particular viralproteins were selected for study because, with the exception of gp68,

each represents the product of only one HTLV-I gene.22’23 Uncodedsera from 20 normal US blood donors (negative reference sera) were

also provided. Normal donors included 18 males, mean age 21 ± 4

years (SD) and two females (aged 20 and 34 years) who donatedblood in Maryland and were found nonreactive for HIV-1 antibodies(HIV-1 EIA, Abbott Laboratories, N. Chicago, IL), hepatitis B coreantibodies (Corzyme, Abbott) and hepatitis B surface antigen

(Auszyme, Abbott). For all nine positive reference sera, eachlaboratory found strong seroreactivity to all viral proteins listed for

its assays in Table 1 . No detectable seroreactivity was found by anylaboratory for any of the 20 negative reference sera. To investigatenonspecific interference, 37 sera representing a variety of serologicalconditions that might interfere with detection of HTLV-I/II-

specific antibodies were distributed under code. These sera wereobtained from Boston Biomedica, Medford, MA, and included twosera from multiply transfused hospital patients, one grossly lipemicserum, one serum with antiHLA DR antibodies, two sera with

antibodies to HIV, three sera reactive for rheumatoid factor, andsera with immunoreactivity to each of the following infectious agents

(three sera per agent): Toxoplasma gondii. cytomegalovirus

(CMV), rubella, Herpes simplex, and Epstein-Barr virus (EBV)

early antigen. In addition, three sera were positive for hepatitis Bsurface antigen, one serum was positive for hepatitis B core antibod-

ies, three sera were reactive by the heterophile test, three sera were

reactive with antinuclear antibodies, and three sera were reactive bythe rapid plasma reagin test. No seroreactivity was found by any

laboratory (laboratory 3 performed WIB but not RIPA on thesesamples) in any of the 37 samples. Thus, the performance of each

assay was similar among seven laboratories, and common positiveand negative reference sera were available as standards for assessingreactivities of unknown test sera.

Donor samples. Serum or plasma samples from 153 HTLV-Iseroreactive blood and plasma donors from Japan, the United States,and the Caribbean basin were obtained by CBER from bloodestablishments and manufacturers (Table 2). Samples were

accepted for study if they met the following criteria: (a) volume aSmL, (b) repeatably reactive by HTLV-l EIA or gel-agglutinationscreening tests, (c) seroreactive to at least one HTLV-I viral protein

on WIB or reactive on HTLV-I indirect immunofluorescent assay

(I FA), and (d) remained frozen at less than - 20#{176}Csince blooddrawing. Of I 53 samples received in an 8-month period, I 42 (donorsamples) met the above criteria for study and represented similarly

significant numbers ofJapanese (38), American (69) and Caribbean(35) donors. Of 142 donor samples, 37 were serum and the remain-

der were plasma. Identical aliquots of each donor sample weresimultaneously distributed under code to seven collaborating labora-

tories for investigation by serological assay methods as listed inTable I. All donor samples were found nonreactive by HIV-l EIA(Organon-Teknika, Durham, NC) except two plasma samples from

Martinique, both of which showed no HIV-specific bands by HIV-lWIB (Biotech/DuPont HIV Western Blot Kit, DuPont, Wilming-

ton, DE).Statistical methods. Associations between discrete variables

were evaluated for significance by Fisher’s exact test (corrected for atwo-tailed distribution). Differences in the performance of WIB andRIPA for detection of p24 and gp6l antibodies were evaluated byMcNemar’s paired chi-square test.

RESULTS

Comparison of WIB and RIPA for detection of p24

antibodies. Seroreactivity to the viral core p24 antigen was

assessed by four WIB and four RIPAs performed in different

laboratories (Table 3). If two or more WIB assays were

positive, the sample was designated consensus positive (ie

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CONFIRMATION OF HTLV-l/Il SEROPOSITIVITY 2587

Table 1 . Serologic Methods of Collabor ating Laboratories

Method SeroreactivitiesLaboratory Assay Type Antigen Source (Reference No.) Evaluatedt

1 . Retrovirus Diseases Branch WIB MT-2 cells 12 gag-p 1 9, p24, p55

Centers for Disease Con- env-gp46 and gp6 1/68trol RIPA MT-2 cells gag-p24, p55

Atlanta, GA env-gp6 1/68

2. Laboratory of Tumor Cell Bi- Competitive EIA HUT 102 cells 13, 14 Positive/indeterminate/negative

ology WIB HUT 102 cells 15 gag-p19, p24, p55National Cancer Institute env-gp46

Bethesda, MD

3. Department of Cancer Biol- WIB MJ cells 16 gag-p 19,p24,p55ogy RIPA MT-2 cells 1 7 env-gp63/65

Harvard School of Public env-gp6 1/68

Health

Boston, MA

4. Bionetics Research, Inc. WIB HUT 102 cells 18 gag-p19 and p24

Rockville, MD p24 RIPA HUT 102 cells gag-p24

5. Division of Hematology! RIPA SLB-1 cells 6 gag-p24

Oncology env- gp6 1

UCLA School of Medicine tax-p40’

Los Angeles, CA

6. Department of Pediatrics! p24 RIPA HUT 102 cells 19, 20 gag-p24

Immunology

University of Miami

Miami, FL

7 . Laboratory of Retrovirology IFA MT-2 cells 2 1 Positive, indeterminate, negative

CBER, FDA

Bethesda, MD

Laboratories 4 and 6 performed this assay by measuring counts from immunoprecipitable ‘25l-p24 in a y-counter, whereas laboratories 1 , 2, and 5subjected precipitable proteins to gel electrophoresis and autoradiography.

tEach laboratory determined which seroreactivities to report based on previous experience or validation experiments with monoclonal antibodies orboth.

�:50% concordance) by WIB. Likewise, if two or more

RIPAs showed evidence of p24 antibodies, the sample was

consensus positive (ie, a50% concordance) by RIPA. Of 142

donor samples tested, I 39 were consensus positive for p24

antibodies by WIB and included all 1 34 that were consensus

positive by RIPA. In terms of its performance as a group of

assays, WIB appeared to be more sensitive for p24 antibodies

than RIPA, but this difference did not reach statistical

significance (P = 0.074). We considered all 139 donor sam-

ples consensus positive by WIB to have antibodies to p24.

Table 2. Donor Sera Characteristics

Donation Donor n/5ex/Mean Age Initial 5creeningNationality Site Type N hiT) ± SD Test (Manufacturer)

Japanese Japan B 29 21/M/38 ± 1 1

1/F/44 -

HTLV-l gel-agglutination

(Fujirebio�)Japanese Okinawa B 9 5/M/48 ± 1 1

4/F/46 ± 13

HTLV-l EIA (DuPont)

Japanese-American Hawaii B 34 28/M/49 ± 1 1

6/F/50 ± 14

HTLV-l EIA (DuPont)

American United States B 22 14/M/40 ± 15

8/F/40 ± 8HTLV-l EIA (Abbott, 4;

DuPont, 15; CPI. 3)American United States P 13 9/M/39 ± 10

4/F/36 ± 4

HTLV-l EIA (Abbott, 13)

Jamaican Jamaica B 1 1 8/M/3 1 ± 43/F/45 ± 12

HTLV-l EIA (DuPont)

Martinique Martinique B 24 1 2/M/49 ± 8

12/F/53 ± 7

HTLV-l EIA (Abbott)

Abbreviations: B, blood donor; P. plasma donor.

#{149}Ageand sex data could not be obtained for seven Japanese blood donors.

tFuiirebio is located in Tokyo, Japan.

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Seroreactivity to p 19 and p24 was determined by consensus among

four independent WIBS. Seroreactivity to gp 6 1 was determined by

consensus among three independent RIPAS.

2588 ANDERSON ET AL

Table 3. Numbers of Donor Samples With Seroreactivity to p24

by WIB and RIPA

No. Positive Testsfor p24 Antibodies RIPA5 a 2 RIPAS < 2

WlBassays�2 134 5

WIB assays <2 0 3

Each sample was tested by four WIBS and four RIPAS. The number of

donor samples is given for each of four groups of assay results. Each

group is arbitrarily defined according to whether a majority (two or more)

of WIBS or RIPAS were positive for p24 antibodies. P = .074 for

detection of p24 antibodies by WIB in more donor samples (ie, 139

samples) than by RIPA (ie. 134 samples).

Comparison of WIB and RIPA for detection of env-

encoded proteins gp46 and/or gp6I. Seroreactivity to env-

encoded proteins was assessed in different laboratories by

three WIB assays for envelope gp46 and/or precursor gp6l

and by three RIPAs for gp6l (Table 4). Iftwo or more WIB

assays were positive (ie, greater than 50% concordance), the

sample was designated consensus positive by WIB. Likewise,

if two or more RIPAs showed evidence of gp6l antibodies

(ie, greater than 50% concordance), the sample was consen-

sus positive by RIPA. Of 142 donor samples tested, 137 were

consensus positive for gp6I antibodies by RIPA and included

all I 25 samples that were consensus positive by WIB for

either gp46 or gp6l. As a group, RIPAs detected antibodies

to env-encoded proteins in significantly more donor samples

than did WIB (P= .0015). We considered all 137 donor

samples consensus positive by RIPA to have antibodies to

gp6 I.

Correlation ofp24 and gp6l antibodies. It is important

that the 137 donor samples consensus positive by RIPA for

gp6l antibodies were all consensus positive by WIB for p24

antibodies. These I 37 samples were also repeatably reactive

when blindly tested by each of the FDA-licensed l-ITLV-I

EIA tests (Abbott, Dupont, and Cellular Products, Buffalo,

NY). When tested by HTLV-l IFA (Table I), 131 ofthe 137

samples were positive and six were indeterminate owing to

weak reactivity.

Five samples, all from US donors, were not seroreactive by

consensus to gp6I (Table 5) and showed predominantly p19

seroreactivity. Antibodies to p19 and p24 were compared on

four WIB in different laboratories (Table 6). One hundred

thirty-one donor samples were seroreactive by consensus (ie,

two or more WIB assays positive for each p19 and p2’!) to

both p1 9 and p24; I 29 of these were also seroreactive by

consensus to gp6 I . Among the remaining I 1 donor samples,

Table 4. Numbers of Donor Samples With Seroreactivity

togp6l by WIB and RIPA

No. Positive Tests for gp46and/or gp6 1 Antibodies RIPAS a 2 RIPAS < 2

WlBassays�2 125 0

WlBassays<2 12 5

Each sample was tested by three WIBS and three RIPAS. The number

of donor samples is given for each of four groups of assay results. Each

group is arbitrarily defined according to whether a majority (two or more)

of WIBS or RIPAS were positive for gp46 and/or gp6 1 antibodies. P

.00 1 5 for detection of gp6 1 antibodies by RIPA in more donor samples

(ie, 1 37 samples) than by WIB (ie 1 25 samples).

Table 5. HTLV-l/lI-Speciflc Antibodies Detected by WIB and

RIPA in Blood Donor Samples Lacking Consensus

Positivity for Anti-GP61

Antibodies Detected Antibodiesby WIB Detected by RIPA

Viral proteins p 1 9 p24 gp46 gp6 1 p24 p40’ gp6 1

Total number of assays used 4 4 2 2 4 1 3

Sample

1 3 1 0 0 0 0 0

2 3 1 0 0 1 0 0

3 3 1 0 0 1 0 0

4 2 2 0 0 1 0 0

5 4 2 1 0 1 0 0

Number of assays positive.

eight were seroreactive by consensus to p24 and not to p19;

there was a significant association (P = .007) between the

presence of antibodies to p24 and antibodies to gp6I among

these I I samples. In contrast, none of three donor samples

with p19 antibodies in the absence of p24 antibodies was

seroreactive to env-encoded proteins. Antibodies to the tax-

encoded protein p40’ were detected by RIPA in 108 donor

samples, of which 108 were seroreactive to both p24 and

gp6 I.

Virological studies of peripheral blood lympho-

cytes. Follow-up studies of peripheral blood lymphocytes

(PBLs) were performed for seven US blood donors who were

positive for HTLV-I/II antibodies by EIA screening tests

and who were available for further studies (Table 7). Anti-

bodies to gag and env were identified by consensus in all

seven cases. Lymphocyte cocultures in two donors were

positive by endonuclease restriction mapping for HTLV-I in

one case and human T-lymphotropic virus type II (HTLV-

II) in one case. Lymphocyte DNA extracts from five other

donors were positive for HTLV-l-specific sequences but

negative for HTLV-II-specific sequences by PCR in three

cases and positive for HTLV-II-specific sequences but nega-

tive for HTLV-I-specific sequences by PCR in two cases.

The HTLV-I/lI-specific antibody pattern of the four

HTLV-I infected donors was not distinguishable from that of

the three HTLV-II infected donors by WIB or RIPA.

DISCUSSION

Serological diagnosis of asymptomatic HTLV-I/II infec-

tion among persons living in the United States presents

several challenges. The epidemiology of HTLV-I and

HTLV-ll in the general US population has not been evalu-

ated systematically. Retroviral infection involves proviral

Table 6. Numb

C

ers of Donor Samples With Seroreactivity by

onsensus to p1 9. p24. and gp6l

Seroreactive

toSeroreactive Not Seroreactivetogp6l togp6l

p19+p24

p24 only

pl9only

129 2

8 0

0 3

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CONFIRMATION OF HTLV-I/II SEROPOSITIVITY 2589

Table 7. HTLV-I/Il-Specific Antibodies Detected by WIB and RIPA in Blood Donor Samples

With Virological Evidence of Retroviral Infection

Antibodies DetectedbyWIB

Antibodies Detected

byRIPA

Viral proteins p 1 9 p24 gp46 gp6 1 p24 p40’ gp6 1

Total number of assays used 4 4 2 2 4 1 3Virus identified (technique used)

HTLV-l (Lymphocyte coculturet) 4

HTLV-I (PCR*) 4

4 1

4 2

2

2

2

4

0

1

3

3

HTLV-l (PCR*) 1 4 1 1 3 1 2

HTLV-l (PCR�) 1

HTLV-ll (Lymphocyte coculturet) 1

4 0

4 20

13

41

12

3HTLV-II (PCR#) 1 4 0 0 2 1 3

HTLV-ll (PCR#) 4 4 2 2 4 1 3

Number of assays positive.

tCocultivated lymphocytes were extracted, and whole DNA was digested with the following restriction endonucleases: Xbal , EcoR 1 . and BamHI.Southern blots of digested DNA were probed with an AccI to HincIl fragment from the 3’ end of the HTLV-l genome (bases 7346-8534) and a BamHI toBamHI fragment from the 5’ end of the HTLV-Il genorne (bases 36 1 -5090) (Slamon D, unpublished observations. February, 1989).

*Whole DNA extracts from PBLs were subjected to PCR with the following primer pairs: SK43, 44, a tax gene sequence common to HTLV-I andHTLV-ll; SK54, 55. a pot gene sequence specific for HTLV-l; and SK58, 59, a polgene sequence specific for HTLV-lI.’#{176}

#Foung S, personal communication, January, 1989.

integration; therefore, diagnostic virological methods such as

lymphocyte coculture or PCR should allow direct identifica-

tion of viral sequences in cellular DNA of a person infected

many years before. However, these methods are not yet

available for widespread routine use. Thus, serological tests

will be used both to screen for and confirm most cases of

HTLV-l/ll infection in the immediate future. To examine

serological confirmatory strategies we studied a cross-section

of serum and plasma samples from two foreign HTLV-I

endemic areas and the United States that were repeatably

reactive on HTLV-l screening tests and had evidence of

viral-specific antibodies on WIB or IFA. The presence of

antibodies to specific viral proteins was defined by a consen-

sus ofseroreactivities in both WIB and RIPA formats.

Our data confirm the observations of other investigators6’9

that the presence of antibodies to p24 and env-encoded

proteins are highly correlated in sera from healthy donors.

Antibodies to p19 usually accompanied p24 and gp6l anti-

bodies. Antibodies to gp46 and/or gp6I were detected in 9%

fewer donor samples by WIB assays than by RIPAs

(P = .001 5). The capability of RIPAs to detect antibodies to

env-encoded proteins not apparent on WIB has been

observed by other investigators6’9 for small numbers of sam-

pIes; the physicochemical basis for this remains unclear.

Optimal antibody binding to major epitopes of gp6l may

require the conformational presentation of antigens as in the

milieu ofsolubilized cellular lysate used in a RIPA. Alterna-

tively, ultracentrifugal purification and other preparative

processes commonly used in WIB assays may adversely

affect gp6l epitope presentation. In contrast, WIB appears

to be the more sensitive format for p24 antibody detection,

although this trend did not reach statistical significance

(P = .074).

Several serological confirmatory criteria for HTLV-l were

proposed recently. Agius et al’#{176}required antibodies to both

p19 and p24 by WIB whereas Fang et al9 required antibodies

to two or more gene products by a combination of WIB and

RIPA. We showed that seroreactivity to gp6I could be

detected by RIPA in 129 of 131 (98.5%) of donor samples

with p19 and p24 antibodies by WIB. In this regard, the two

sets of confirmatory criteria are similar. However, as shown

in Table 6, two donor samples without gp6I antibodies

showed p19 and p24 antibodies by consensus and in three

other samples p24 antibodies were detected by one of four

WIB assays. Confirmatory criteria that require antibodies to

at least two independent gene products would not be satisfied

for these five samples and are thus more conservative than a

requirement ofantibodies only to p19 and p24.

We have assumed that HTLV-I/Il-specific antibodies to

two independent gene products are prudent confirmatory

criteria. On the basis of the present data, we suggest that

antibodies to p24 and to the env-encoded proteins gp46

and/or gp6l be demonstrated by one or a combination of

serologic tests. This strategy would maintain a requirement

for antibodies to two independent gene products while

excluding the presence of antibodies to p1 9 or p40’ as criteria

to be used in confirmation; ie, we found no samples with p19

antibodies but without p24 antibodies that contained anti-

bodies to gp6I at routine sample dilutions. Furthermore,

antibodies to p405 were present only in a subset (79%) of

samples with p24 and gp6I antibodies. Conversely, we have

no evidence that persons lacking p24 and gp46 and/or gp6l

antibodies are infected with HTLV-I or HTLV-II. All seven

samples for which the donor’s lymphocytes yielded virologi-

cal evidence of HTLV-I or HTLV-II infection (Table 7)

showed seroreactivity to p24 and gp#{243}l.It is of interest that

p19 antibodies were detected by two or more WIBs in only

three of these seven samples whereas antibodies to p40’ were

found in six of seven samples. In the absence of more

virologic data, samples which show antibodies to at least one

suspected HTLV-I gene product (such as p19 only, p19 and

p28, p40’ only) should be regarded as indeterminate to leave

open the question of infection in these cases.

We recommend examination of samples by WIB for p24

and gp46 and/or gp6l antibodies. If antibodies to p24 are

present but antibodies to gp46 and/or gp6I are lacking, a

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REFERENCES

2590 ANDERSON ET AL

RIPA for gp6I antibodies is recommended. Application of

this algorithm to our samples with p24 antibodies by WIB

would result in the need for a RIPA to evaluate further 7 of

32 (22%) US donor samples and 6 of 34 (18%) Japanese-

American (Hawaii) donor samples for gp6l antibodies. In

contrast, only I of 29 (3%) Japanese, 0 of I I Jamaican, and 0

of 24 Martinique donor samples with p24 antibodies by WIB

would have required further evaluation by RIPA. These

differences among the populations may relate to higher

measurable antibody titers among persons living in areas of

greater prevalence of HTLV-I infection, as in the general

populations of Japan and Caribbean countries.8 The lower

concordance rate in US samples may reflect differences in

virus type, HTLV-l versus HTLV-II, or differences in

expression of virus in vivo resulting in titer differences to

various antigens, or both. Further studies carefully charac-

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epitope mapping of various reactivities should help clarify

these alternatives.

The confirmatory criteria we developed have obvious

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infection with HTLV-I can not be distinguished from

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