Original Paper

Vox Sang 1992;62:208-212

C. L. Van der Poela9 D. Bresters" H. W. Reesinka3b9 A.A. D. PlaisieF W. Schaasbergb A. Leentvaar- Kuypers Q.-L. Chood, S. Quand, A. Politod M . Houghtond, G. Kuod P. N . Lelie b, H. 'I: M . Cuypers

a Red Cross Blood Bank Amsterdam, " Central Laboratory of the Netherlands

Red Cross Blood Transfusion Service, Department of Infectious Diseases, Municipal Health Service, Amsterdam, The Netherlands Chiron Corp., Emeryville, Calif., USA

Early Antihepatitis C Virus Response with Second-

................................................................................................. Abstract Detection of early antibody to hepatitis C virus (HCV) by a new second- generation C200/C22 anti-HCV enzyme-linked immunosorbent assay (EL- ISA) and a four-antigen recombinant immunoblot assay (4-RIBA) was com- pared with the first-generation anti-HCV ClOO ELISA using sequential serum samples of 9 recipients who were infected with HCV, as detected by polym- erase chain reaction after transfusion of blood products. Within 26 weeks after transfusion, 9/9 (100%) recipients seroconverted with C200/22 ELISA, and 6/9 (67%) seroconverted with ClOO ELISA. Compared with ClOO ELISA, C200/ C22 ELISA seroconversion occurred simultaneously in 3 cases, 5-6 weeks earlier in 3 other cases, and 20 weeks earlier in 1 case. Seven of 9 (78%) recipients became positive, and 2/9 (22%) became indeterminate with 4-RIBA. In 8 cases with clinical posttransfusion hepatitis non-A, non-B (PTH- NANB), anti-HCV C200/C22 ELISA seroconversion occurred 2-17 (mean 6) weeks after the onset of hepatitis. In 6 cases of PTH-NANB, anti-HCV ClOO ELISA seroconversion occurred 2-26 (mean 9) weeks after the onset of hepa- titis. It is concluded that the second-generation C200/C22 ELISA is more sensitive than the ClOO ELISA for the detection of antibody during early HCV infection. Indeterminate 4-RIBA results are found in the early phase of HCV infection. .....................

Introduction

Anti-HCV antibody testing with the first-generation ClOO assays was found to be specific, but not very sensitive during the early phase of infection with hepatitis C virus (HCV) [l]. In the acute phase of hepatitis non-A, non-B (NANB) the test was found reactive in only 2040% of the cases. Furtheimore, anti-HCV antibody seroconversion with ClOO enzyme-linked immunosorbent assay (ELISA)

in recipients of blood with posttransfusion hepatitis non-A, non-B (PTH-NANB) was shown to occur as late as 52 weeks after infection, or not at all [l-51. Recently, two additional recombinant HCV proteins, encoded by nonstructural and structural regions of the HCV genome, were developed by Chiron (Emmeryville, Calif.) [2]. A recombinant antigen of the nonstructural NS3 region named C33 and a recombinant antigen of the nucleocap- sid named C22c [6] were added to the previously used 5-1-1

Received: August 9. 1Y91 Revised manuscript Red Cross Blood Bank Amsterdam 0042-wO7/92/0h24-0208 received: Dec. 13,1991 PO Box 9 137 $2.7S/0 Accepted: Dec. 17. 1991

C . L. van der Poel. MD. PhD 0 1992 S . Karger AG. Basel

NL-1006 AC Amsterdam (The Netherlands)

and ClOO antigens [7] in the format of a second-generation four-antigen recombinant immunoblot assay (4-RIBA), which was shown to be a very specific confirmatory assay for anti-HCV ClOO ELISA-positive blood donor samples [2]. Also, the addition of the new antigens in the 4-RIBA allowed more sensitive HCV antibody testing in recipients of blood wih PTH-NANB than with the first-generation anti-C100 ELISA [2,8].

In a new second-generation anti-HCV ELISA (C200/ C22 ELISA), the C33 (expressed as one polypeptide with C100, named C200) and C22c antigens were added on the solid phase to the ClOO antigen of the first-generation anti-HCV ELISA. We have compared the sensitivity of this second-generation C200/C22 ELISA with the ClOO ELISA and 4-RIBA, for detection of anti-HCV antibody during the early phase of HCV infection in 9 recipients with transfusion-acquired HCV infection, which was con- firmed by polymerase chain reaction (PCR).

Materials and Methods

Historical Data From recipients of blood products who participated in a prospec-

tive study on PTH-NANB in 1984-1986, stored serum samples ob- tained sequentially before and 2, 4, 6, 8, 10, 12, 16, 20, 26, and 232 weeks after transfusion were available for anti-HCV C2001C22 ELISA testing [5, 91. Historical data from previously performed studies on these recipients were used for comparison: methods of alanine aminotransferase (ALT) testing and the definition of PTH- NANB [9],first-generationanti-HCVClOOELISA testing [5],confir- matory testing with 4-RIBA [2], PCR testing with 36' (NS3) primers on plasma samples, freshly obtained from the recipients at follow-up in 1990 [2]. Onset of hepatitis was defined as the sampling date of the first sample with ALT 22.5 times the upper limit of normal [9].

Selection of Patients We previously described 1 recipient without PTH-NANB who

seroconverted with anti-HCV ClOO ELISA and 4-RIBA in 1984-86 and became PCR-positive in 1990 [2]. We also described 9 recipients with PTH-NANB (in 1984-86) of whom 5 were positive and 3 were negative with PCR with NS3 primers [lo] in 1990, and 1 was not tested in PCR with NS3 primers because a fresh blood sample in 1990 could not be obtained [2]. Of these latter 4 recipients, we retested with a more sensitive PCR, using primers of the highly conserved 5' untrans- IatedregionoftheHCVgenome [11,12], the available plasmasamples obtained in 1990and stored serum samples taken during and immedi- ately after the ALTpeak in 1984-86. With 5' untranslated PCR, HCV infection was thus established in 3 additional recipients: one (con- tinuously 4-RIBA-indeterminate) recipient was PCR-positive in 1984-86 as well as in 1990, one (4-RIBA-positive in 1984-86 and 4-RIBA-negative in 1990) was PCR-positive in 1984-86 and negative in 1990, and one (CRIBA-positive) recipient who refused follow-up in 1990 was PCR-positive in 1984-86.

In the present study, from a total of 9 HCV-infected recipients, representing 8 recipients with clinical PTH-NANB and 1 without PTH-NANB, the sequential serum samples of the 1984-86 PTH- NANB study were tested with anti-HCV C200K22 ELISA.

Laboratory Tests Second-generation anti-HCV ELISA testing was performed with

C200/C22 anti-HCV ELISA (Ortho, Raritan, N.J.) according to the manufacturer's instructions. Briefly: 20 pl of serum specimen at U10 dilution in diluent were pipetted into microwell strip plates, coated with the recombinant HCVC100, C200/C22 and C22c antigens. After incubation at 37°C for 60 min and 5 washings with phosphate-buf- fered salinelpolysorbate, 200 pl of murine monoclonal anti-IgG- horseradish peroxidase conjugate was added. After incubation for 60 min at 37"C, plates were washed 5 times and incubated with 200 p1 of o-phenylene diamine substrate at room temperature for 30 min, after which the reaction was stopped with 50 pl of 4 N H2S04, and absorbance was read at 492 and 620 nm. ELISA optical density (OD) reading results were considered reactive when equal to or higher than the cutoff. Cutoff values were defined as 0.600 + mean absorbance of 3 negative controls.

To discriminate between passive and active antibodies, all 9 series of serum samples were additionally tested at a 1:20 dilution in the ClOOELISA,andatl:20,1:200andl: 2,000dilutionsintheC2OO/C22 ELISA; serum dilutions were made in anti-HCV-negative normal donor AB serum. Passive antibody transmission was diagnosed when an ELISA test with undiluted serum was negative before transfusion and became positive in the first sample (obtained at 2 weeks) thereaf- ter. The moment of anti-HCV ELISA seroconversion was designated (a) when, in case no passive antibody occurred, a recipient became anti-HCVELISA-positive after transfusion, (b) when, incase passive antibody was found, either a more than twofold increase of ELISA OD value or a rise in ELISA titer occurred >4 weeks after trans- fusion.

Statistics Statistical methods included Wilcoxon's matched pairs signed-

ranks tests, performed with SPSS PC+@ software.

Results

All 9 recipients were anti-HCV-negative before trans- fusion. Within 26 weeks after transfusion, all 9 HCV-in- fected recipients seroconverted with C200/C22 ELISA as compared to 6/9 (66.7%) when ClOO ELISA was used. One recipient seroconverted with ClOO ELISA after 32 weeks, and 2 did not seroconvert with ClOO ELISA. With 4-RIBA, 7/9 (78%) recipients became positive with- in 26 weeks after transfusion, and 2 (22%) remained in- determinate (1 with isolated anti-C22c and 1 with isolated anti-C33). In the 7 recipients with both ClOO and C200/ C22 ELISA seroconversion, C200/C22 ELISA serocon- version occurred simultaneously with ClOO ELISA sero- conversion in 3/7 cases, 5-6 weeks earlier in 3 other cases,

209

Table 1. Onset of hepatitis and anti-HCV seroconversion with first- and second-generation HCVantibody assays in 9 recipientswith PCR-confirmed HCV infection

~

Recipient Weeks after transfusion

onset of anti-HCV antibody serownversion

ELISAs 4-RIBA hepatitis

(ALT22.5N) ClOO C2OO/C22" indeter- positive minate or positive

R1 R2 R3 R4 R5 R6 R7 R8 R9

5 7 6 3 6 8 7

10 no hepatitis

17 11 13 8 32 12

-h 20 12 12 23 23 9 9 -b 12

26 20

11 8

16 20 12 23 9

12 20

17 13 26

17 26

9

20

-

-

'' Anti-HCV C2001C22 ELISA seroconversion, compared to ClOO ELISA seroconversion, p = 0.0679 (NS) Wiloxon signed-ranks test.

Also anti-HCV ClOO ELISA-negative at follow-up in 1990 [2].

Table 2. C100, C200/C22 ELISA, and 4-RIBA results in 53 sero- conversion samples of 9 recipients with PCR-confirmed HCV in- fection

Recipient Number of Serum samples reactive for serum samples anti-HCV antibody total, %

ELISAs 4-RIBA after exclusion of Passive ClOO C200/C22a indeter- positive ELISA minate or antibody positive

R1 R2 R3 R4 R5 R6 RI R8 R9

6 7 5 2

10 8 6 6 3

4 6 7 7 2 5 0 2 5 5 2 2 6 6 0 5 2 3

The serum samples were obtained 2-32 weeks after transfusion, and those withpossiblepassiveantibodyreactivitywereexcluded(see text).

Sensitivity of C2001C22 ELISA as comprared to ClOO ELISA: p = 0.0431 by Wilcoxon signed-ranks test.

and 20 weeks earlier in 1 case. In these 7 cases, serocon- version occurred at a mean of 14 weeks after transfusion (range 8-23) with C200/C22 ELISA, versus a mean of 19 weeks (range 9-32) with ClOO ELISA (nonsignificant by Wilcoxon signed-ranks test).

C200/C22 ELISA seroconversion occurred significant- ly earlier than 4-RIBA positivity (p = 0.0431, Wilcoxon signed-ranks test), but not significantly earlier than reac- tivity with one or more antigens (indeterminate or posi- tive) in 4-RIBA (nonsignificant by Wilcoxon signed-ranks test).

In 8 cases of clinical PTH-NANB, anti-HCV C200/C22 ELISA seroconversion occurred 1.6-17.1 (mean 5.7) weeks after onset of hepatitis. Two recipients with PTH- NANB did not seroconvert in anti-HCV ClOO ELISA. In 6 cases of PTH-NANB, anti-HCV ClOO ELISA serocon- version occurred 2-26 (mean 9) weeks after the onset of hepatitis. The onset of hepatitis and of ClOO ELISA, C200/C22 ELISA and 4-RIBA seroconversion in the 9 HCV-infected recipients is summarized in table 1.

Passive antibody was found in U9 recipients with ClOO ELISA and in 7/9 with C200/C22 ELISA; all samples with passive antibody were negative in both ELISAs at 1:20 serum dilution. In 6/7 cases the passive antibody reactivity in the C200/C22 ELISA was accompanied by reactivity in 4-RIBA: isolated anti-C22c was present in 5 cases and anti-C100 + C33 + C22c in 1 case.

When the serum samples were excluded, in which pas- sive antibody caused the ELISA or 4-RIBA reactivity, a subset of 53 serum samples, taken 2-32 weeks after trans- fusion, was obtained (table 2). In this subset, 28/53 (53%) serum samples were positive with ClOO ELISA, 41/53 (77%) were positive with C200/C22 ELISA, 25/53 (47%) were reactive with two or more HCV antigens (positive) in the 4-RIBA and 40/53 (76%) were reactive with one or more HCV antigens (indeterminate or positive). C200/ C22 ELISA was significantly more sensitive than ClOO ELISA and 4-RIBA positivity (two or more bands), but similar to reactivity with one or more bands (indetermi- nate or positive) in 4-RIBA (p=O.O431, p=0.0180 and p = 0.3173, respectively, by Wilcoxon signed-ranks tests).

Discussion

Our study shows that all 9 recipients with PCR-con- firmed HCV infection seroconverted within 26 weeks af- ter transfusion, when second-generation anti-HCV C200/ C22 ELISA was used. Only 6 seroconversions were de- tected in the same period with first-generation ClOO EL-

2 10 Van der Poel/Bresters/ReesinWPlaisier/Schaasberg/ Early Anti-HCV Detection Leentvaar-Kuypers/Choo/Quan/Poli to/Houghton/Kuo/Lelie/ Cuypers

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0 60 XHI 160 200 260

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I -

I - ..

c22 - c930 - cmo -

I- 6-1-1 __ E l l 2 - ElA 1 ~

I- ..

---,

6-1-1

EIA a ElA I

PCR + 1 / ALT \ 0 60 Iw 160 200

Y L

ALT UIL

100

1000

100

1090

1966 day8 almr lrmsfusion 1990

donor n o r y . n l R 1

6-1-1 6-1-1

E l l 2 E l l 1 200 E l l 1 200

ALT 100 100

PCRnl P C R + o w iw 160 200 ma so0 o 60 mo iw 200 aw 300 560

1084 days s l l u lranaluelon 19w 1966 day8 after l r m s f u s b n 1 0 0 0

0 W 100 160 200 ZW 500

1984 day. allsr lransluslon

100

1000

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1986 day8 aller Iransfu4on

Fig. 1. Anti-HCV recognition patterns in 9 HCV-infected recip- ients of blood. All recipients were PCR-positive either with NS3 primers or with 5' untranslated primers (see text). Recipient R7 was PCR-positive in 1986 but PCR-negative in 1990. The antibody reac-

tivity is depicted in horizontal bars above the graph: - = negative test result; 0 = passive antibody; I = active antibody. The antibody reactivity of the implicated blood donors is depicted likewise at the ultimate left side of the graphs.

ISA. Thus, the addition of two immunogenic structural and nonstructural recombinant proteins in the assay sig- nificantly increases the sensitivity of anti-HCV ELISA testing in the early stage of infection. Nevertheless, the latency phase between the onset of hepatitis and detection of anti-HCV with C200 ELISA ranged from 2 to 17 weeks (mean 6 weeks).

The antibody recognition patterns to the different HCV proteins among the respective infected recipients is quite unpredictable (fig. 1). We have also described [2] that 3 recipients of 3 different units of HCV-infected fresh frozen plasma from 1 donor resulted in 3 patterns of anti- body response and also in 3 patterns of HCV disease (re- cipients R1, R8 and R9, fig. 1). Therefore it appears diffi- cult to predict the course of infection from the specific antibody recognition pattern. This inconsistent antibody response stresses the need for direct virus detection for diagnostic purposes. Because of low HCV titers of an estimated 102-10" viral particledm1 [lo] this can only be

performed by amplification of HCV genomic sequences, preferably with PCR of the highly conserved 5' untrans- lated region [ll, 121. But HCVPCR testingis as yet limited to specialized laboratories and the procedures are not yet standardized.

As shown in this study, 4-RIBA-indeterminate reac- tivities may represent early HCV infection. By current criteria, 4-RIBA positivity [2] is not sensitive enough to confirm all C200/C22 ELISA-positive samples, especially in early HCV infection. Apparently, in early seroconver- sion an individual may be C200/C22 ELISA-positive but 4-RIBA-negative during a short period. It remains to be established if this would be of importance in the setting of blood donor screening, since under such circumstances more often chronic carriers rather than acute seroconvert- ing individuals are found. The prevalence of PCR-/4-RI- BA-confirmed HCV infection among our blood donors is low: 0.04% [13]. Since HCV infection is chronic in 80% of the cases [2], and since blood donors are selected for the

211

absence of recent exposure to parenterally transmitted viruses, the incidence of new HCV infections in this pop- ulation is probably lower. Since the C200/C22 ELISA allowed antibody detection in 2 chronically infected recip- ients who stayed ClOO ELISA-negative for more than 4-6 years, it can be expected that the C200/C22 ELISA will increase the sensitivity of anti-HCV blood donor screening.

We conclude that the second-generation C200/C22 ELISA is more sensitive than the ClOO ELISA for detec- tion of antibody during early HCV infection. Indetermi-

nate 4-RIBA results may represent individuals infected with HCV, especially in the early phase of infection.

Acknowledgements

We thank Ortho Diagnostics for HCV tests; Mrs. S . Koenis-de Koning, Mr. H. Van Drimmelen, Mrs. D. Mulder-Folkerts, Mrs. I . N. Winkel, Mrs. P. Exel-Oehlers, and Mr. E. Bakker for technical assistance; Prof. Dr. W. G. Van Aken and Prof. Dr. R. A. Coutinho for comments on the manuscript, and Mrs. A. Roukens and Mr. E Feijen for preparation of the typescript.

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