- Email: [email protected]
Antibody pattern to hepatitis C virus antigens in patients with acute and chronic liver diseases
Antibody pattern to hepatitis C virus antigens in patients with acute and chronic liver diseases G J Padron’, E Arus*, L Rivera3, Z Rodriguez4, B Gra6, L Blitz-Dorfman’
A Fernandez5,
‘Center for Genetic Engineering and Biotechnology, PO Box 6162, Havana; 2Hospital Luis Diaz Soto, H del Este, Havana; 3Hospital Carlos J Finlay, Marianao, Havana; 4Docent Blood Bank, Marianao, Havana; 5Hospital Julio Trigo, A Naranjo, Havana; ‘jlnstitute of Gastroenterology, Vedado, Havana, Cuba; ‘Regional Viral Reference Laboratory, University of Zulia, Maracaibo, Venezuela
Summary The present paper describes the antibody pattern in the early stages of hepatitis C virus (HCV) infection and compares it with that obtained in chronic carriers. Samples were tested for anti-HCV from 470 consecutive patients admitted to gastroenterology services at three Havana hospitals, with serological and biochemical signs of non-A, non-B hepatitis, including those referred from three Havana blood banks because of a positive result in anti-HCV in pre-donation screening. Four anti-HCV enzyme immunoassay (EIA) systems and two supplementary assays were used for antibody detection. These results show that antibodies to viral core antigens are the first and commonest among Cuban patients infected with HCV, and generally, they can reveal the infection before the supplementary tests do. LiaTek HCV has proved to be a good tool for the confirmation of enzyme-linked immunosorbent assay (ELISA) positive samples. Key words: Non-A, non-B Serodiagn.
Immunother.
hepatitis,
HCV, antibodies
Infect. Disease 1995, Vol. 7, 23-26,
Introduction The present availability of different antigenic proteins and peptides derived from the putative nucleotide sequence of the hepatitis C virus (HCV) has considerably improved the screening and diagnosis of this infection. Nevertheless, little is known about the biological role of the antibodies elicited to different viral proteins and their significance in the natural history of the disease. In chronic infection, as well as in acute cases, different antibody patterns have been observed, but knowledge about the kinetics of the appearance of different antibodies to HCV antigens is still poor. In the present paper we describe the antibody pattern in the early Received: 11 June 1994 Accepred: IO August 1994 Correspondence and reprint requests to: GJ Padron, Center for Genetic Engineering and Biotechnology. PO Box 6162, Havana, Cuba 0 1995 Elsevier Science B.V. All rights reserved 0888.0786/95/$09,50
March
stages of HCV infection and obtained in chronic carriers.
compare
it with
that
Materials and methods We studied 470 consecutive patients admitted to gastroenterology services of three Havana hospitals from September 1990 to April 1993, with serological and biochemical signs of non-A, non-B hepatitis (typical illness with abnormal values of alanine transaminase (ALT) and no serological markers of other hepatotropic virus), including those referred from three Havana blood banks because of a positive result in anti-HCV pre-donation screening. Serum samples were obtained and frozen at -20” C until assay, with no more than two freezing before antiHCV testing using two commercially available enzyme immunoassay (EIA) systems: Ortho HCV enzymelinked immunosorbent assay (ELISA) 2nd generation (Ortho Diagnostic Systems, Beerse, Belgium), UBI HCV EIA (Organon Teknika BV, Boxtel,
24
Table
Serodiagn.
1.
Immunother,
Characteristics
Infect.
Disease
1995; 7: No 1
of the studied population
(n
= 470)
Antibody
pattern in acute infection after
RIBA-
Character
HCV negative HCV indeterminate HCV positive Total biopsied Normal liver Non-specific changes Acute hepatitis Chronic persistent hepatitis Chronic active hepatitis Chronic active hepatitis + cirrhosis
No. of patients
%
244 13 213 92 14 14 19 15 27 3
51.9 2.8 45.3 43.2 15.2 15.2 20.7 16.3 29.3 3.3
Netherlands) and an EIA test developed in our laboratory based on core antigens’. All negative samples were tested using Innotest HCV Ab (Innogenetics, Antwerp, Belgium) as a fourth EIA system. Samples from acute patients, as well as those from chronic patients, were randomly chosen during the whole study period, and tested by two supplementary systems: Chiron RIBA HCV test system 2nd generation (Chiron Corporation, Emeryville, CA, USA) and LiaTek HCV (Organon). All procedures were performed following the manufacturers’ instructions. Results From 470 studied patients 213 (45.3%) were positive to HCV by EIA. Thirteen (2.8%) yielded indeterminate results by both supplementary tests. Liver biopsy on 92 of the positive cases showed that 14 of them had no evidence of liver damage, 14 non-specific damage, 19 acute hepatitis and 45 chronic hepatitis (Table 1). According to RIBA-2, antibodies to core protein were the first detected and the strongest serological reaction in nine out of 12 acute cases (75%). They were present in all of the 41 chronic patients studied. Antibodies to non-structural protein C33c (NS3) were the second most frequently observed, both in acute and chronic cases (66.7% and 90.2%, respectively) with a less important role for the two NS4 antigens. Fifteen samples yielded indeterminate results; eight of them among chronic and seven among acute patients (Table 2). A quantitative analysis of the RIBAresults, taking into account the magnitude of the reactions (e.g. considering positive those samples with one band
Table
2. RIBA positivity pattern among Cuban patients with HCV infection Patients
Total
RIBA antigens 5-l- 1 c- 100-3
Chronic Acute Total
Table 3.
29 4 33
26 5 31
c33c
c22
37 8 45
41 9 50
41 12 53
Indererminare
8 7 15
Sample
1 2 3 4 5 6 7 8-9 IO 11 12
RIBA antigens
Result
5-l- 1
c- 100-3
c33c
c22
_ _
-
2+ -
I+ 2+ -
I+ I+ I+ -
3+ 4+ I+ _
_
2+ 2+
4+ 4+ 3+ 3+
+I3+ 4+ 4+ 4+ 3+ 2+ 4+ 4+
4+ 3+
Indeterminate Indeterminate Positive Positive Positive Indeterminate Indeterminate Indeterminate Indeterminate Positive Indeterminate Positive
reacting over 2+) could considerably reduce the number of indeterminate results, especially in acute cases, where the beginning of the immunological response is characterized by the presence of antibodies to individual antigens (Table 3). LiaTek HCV yielded less indeterminate results both in acute and chronic patients (26.3% and 8.9%, respectively) and the antibody pattern was also most frequently represented by different core antigens, followed by the NS4 band. In fact, no sample was reactive only to NS5, which explains the failure to detect a sample reactive only by Innotest Ab EIA. Five out of seven RIBA indeterminate acute patients were positive by LiaTek and one RIBApositive case remained indeterminate by LiaTek HCV. The follow-up demonstrated seropositivity in all of these patients, despite four RIBA indeterminate results. Acute hepatitis was later confirmed by liver biopsy. The use of a different criterion for positivity and multiple core antigens are factors influencing the better performance of LiaTek HCV in both acute and chronic patients (Tables 4 and 5). Five out of 19 acute patients were followed monthly before infection. In all cases infection was initially evident through rises in ALT of more than 2.5 the upper limit of normal during 1 and 8 months (average 2.5 months) before the appearance of ELISA detectable antibody. In eight chronic ELISA HCV positive patients, who were indeterminate by RIBA-2, seven were positive after LiaTek. In all seven, antibodies to core protein were the only antibodies detected and five of them showed reactivity of 2+ or greater. Discussion Antibodies to viral core antigen are the most frequently found among Cuban patients infected with HCV. Individuals infected with HCV, especially in the early phase of infection may yield indeterminate results with RIBAin spite of polymerase chain
Padron et al.: Antibody
Table 4.
LiaTek HCV positivity
Table 5.
NS4
NS5
Core- 1
Core-2
57 16 73
36 13 49
52 15 67
56 15 71
Antibody
1
2 3 4 5 6 7 8 9 10 11 12
Total Core-4
Core-3 44 11 55
49 13 62
68 19 87
I+
3+ 2+ 2+ I+ +I+I3+ +I4+
6 5 11
Result
LiaTek antigens
I+ -
Indeterminate
pattern in acute infection after LiaTek HCV
Sample NS4
25
pattern among Cuban patients with HCV infection LiaTek antigens
Patients
Chronic Acute Total
pattern to HCV antigens
NS5
Core- 1
Core-2
Core-3
-
+I+II+ +II+ +l+I-
+I-
-
2+ 2+ 2+ I+ I+ 2+ -
2+ 3+ 4+ 3+ I+ 2+ 2+ -
-
4+
2+
+I-
reaction (PCR) positive results*, particularly in those with anti-core positivity”m5. There are also qualitative differences between NS4 antigens used in both supplementary assays, as can be seen from Tables 3 and 4, where patients 1, 6 and 7 are positive to antiNS4 antibodies by LiaTek and remain negative by RIBA-2. These results show that anti-HCV positive patients may exhibit different antibody responses to specific viral antigens. Discrepant results found between RIBAand LiaTek supplementary tests in patients in the earliest stage of the acute disease may be explained by the heterogeneity among antibodies elicited in different individuals after infection. These findings may be due to differences, both at the viral nucleotide and aminoacid levels, between HCV strain+. If during the course of the disease a heterogeneous viral population is formed, one should expect a wider spectrum of antigen recognition. Another explanation could be based on individual differences in the immunological response to different viral antigens. Further studies are necessary to elucidate this finding. In spite of the many cases that still remain indeterminate by RIBA-2, especially in the acute phase of the disease, patients reactive for only one antigen by RIBAmay still be infectious’. On the other hand, in the light of the present results, the manufacturer’s criteria of indeterminate RIBA results are obviously inadequate. If samples showing a strong reactivity (2+ or higher) for just one antigen are to be considered positive, all false indeterminate results would turn
Core-4
+I-
I+
I+
2+ +l-
3+ I+
+I-
+II+
-
_
_ -
4+
2+
2+
Indeterminate Positive Positive Positive Positive Positive Positive Positive Indeterminate Positive Indeterminate Positive
positive (Table 3). ELISA is more sensitive than RIBAfor the detection of anti-HCV antibodies, as it is unable to detect all positive seraX especially during the acute phase of the disease. No significant difference was found regarding the severity of the clinical illness among HCV positive and indeterminate patients (data not shown). Present results show that antibodies to core protein are the first elicited and the most frequent sign of HCV infection among Cuban patients with acute as well as chronic HCV infection and, in some cases, they can show the infection before supplementary tests. In our experience, LiaTek HCV has proved to be a better tool for confirming ELISA positive samples.
Acknowledgements The authors thank Organon Teknika, Ortho Diagnostic Systems and Innogenetics BV for providing the diagnostic systems used in this research.
References Padrdn G, Lemos G, Sanchez G et al. Evaluacibn de un sistema para la detecibn de anticuerpos contra un antigen0 sintetico del core del virus de hepatitis C y su prevalencia en donantes de sangre. Biotecnologia Aplicada
1994;
11:165-70
Van der Poel CL, Brester D, Reesnik HW. Plaisier AA, Schaasberg W, Leentvaar-Kuypers A et al. Early antihepatitis C virus response with second generation C22iC22 ELISA. VOX Sanpinis 1992; 62: 208-12
26
3
4
5 6
Serodiagn.
Immunother.
Infect. Disease 1995; 7: No 1
Chan S-W, Simmonds P, McOmish F, Yap PL, Mitchell R, Dow B, Follet E. Serological response to infection with three different types of hepatitis C virus. Lancet 1991; 338: 1391 Puoti M, Zonaro A, Ravaggi A, Marin MC, Castelnuovo F, Cariani E, Hepatitis C virus RNA and antibody response in the clinical course of acute hepatitis C virus infection. Hepatology 1992; 16: 877-81 Tong CYW, Codd AA, RIBAband intensity and PCR in HCV infection. Lancet 1992; 340: 117 Transfusion Safety Study Group. Possible HCV reinfection of hemophiliacs with antecedent anti-c22-3 and
7
8
anti-c33c but no anti-clOO-3. (abstr). Vth International Symposium on Viral Hepatitis, Madrid, Spain, 30 January-l February 1992 Halfon S, Rousseau S, Tamalet C, Antoni M, Gerolami V, Levy M et al. Indeterminate second-generation hepatitis C recombinant immunoblot test: detection of hepatitis C virus infection by polymerase chain reaction J Infect Disease 1992; 166: 449 Lazizi Y, Elfassi E, Pillot J. Detection of hepatitis C virus sequences in sera with controversial serology by nested polymerase chain reaction. J Clin Microbial 1992; 30: 931-4
A Fernandez5,
‘Center for Genetic Engineering and Biotechnology, PO Box 6162, Havana; 2Hospital Luis Diaz Soto, H del Este, Havana; 3Hospital Carlos J Finlay, Marianao, Havana; 4Docent Blood Bank, Marianao, Havana; 5Hospital Julio Trigo, A Naranjo, Havana; ‘jlnstitute of Gastroenterology, Vedado, Havana, Cuba; ‘Regional Viral Reference Laboratory, University of Zulia, Maracaibo, Venezuela
Summary The present paper describes the antibody pattern in the early stages of hepatitis C virus (HCV) infection and compares it with that obtained in chronic carriers. Samples were tested for anti-HCV from 470 consecutive patients admitted to gastroenterology services at three Havana hospitals, with serological and biochemical signs of non-A, non-B hepatitis, including those referred from three Havana blood banks because of a positive result in anti-HCV in pre-donation screening. Four anti-HCV enzyme immunoassay (EIA) systems and two supplementary assays were used for antibody detection. These results show that antibodies to viral core antigens are the first and commonest among Cuban patients infected with HCV, and generally, they can reveal the infection before the supplementary tests do. LiaTek HCV has proved to be a good tool for the confirmation of enzyme-linked immunosorbent assay (ELISA) positive samples. Key words: Non-A, non-B Serodiagn.
Immunother.
hepatitis,
HCV, antibodies
Infect. Disease 1995, Vol. 7, 23-26,
Introduction The present availability of different antigenic proteins and peptides derived from the putative nucleotide sequence of the hepatitis C virus (HCV) has considerably improved the screening and diagnosis of this infection. Nevertheless, little is known about the biological role of the antibodies elicited to different viral proteins and their significance in the natural history of the disease. In chronic infection, as well as in acute cases, different antibody patterns have been observed, but knowledge about the kinetics of the appearance of different antibodies to HCV antigens is still poor. In the present paper we describe the antibody pattern in the early Received: 11 June 1994 Accepred: IO August 1994 Correspondence and reprint requests to: GJ Padron, Center for Genetic Engineering and Biotechnology. PO Box 6162, Havana, Cuba 0 1995 Elsevier Science B.V. All rights reserved 0888.0786/95/$09,50
March
stages of HCV infection and obtained in chronic carriers.
compare
it with
that
Materials and methods We studied 470 consecutive patients admitted to gastroenterology services of three Havana hospitals from September 1990 to April 1993, with serological and biochemical signs of non-A, non-B hepatitis (typical illness with abnormal values of alanine transaminase (ALT) and no serological markers of other hepatotropic virus), including those referred from three Havana blood banks because of a positive result in anti-HCV pre-donation screening. Serum samples were obtained and frozen at -20” C until assay, with no more than two freezing before antiHCV testing using two commercially available enzyme immunoassay (EIA) systems: Ortho HCV enzymelinked immunosorbent assay (ELISA) 2nd generation (Ortho Diagnostic Systems, Beerse, Belgium), UBI HCV EIA (Organon Teknika BV, Boxtel,
24
Table
Serodiagn.
1.
Immunother,
Characteristics
Infect.
Disease
1995; 7: No 1
of the studied population
(n
= 470)
Antibody
pattern in acute infection after
RIBA-
Character
HCV negative HCV indeterminate HCV positive Total biopsied Normal liver Non-specific changes Acute hepatitis Chronic persistent hepatitis Chronic active hepatitis Chronic active hepatitis + cirrhosis
No. of patients
%
244 13 213 92 14 14 19 15 27 3
51.9 2.8 45.3 43.2 15.2 15.2 20.7 16.3 29.3 3.3
Netherlands) and an EIA test developed in our laboratory based on core antigens’. All negative samples were tested using Innotest HCV Ab (Innogenetics, Antwerp, Belgium) as a fourth EIA system. Samples from acute patients, as well as those from chronic patients, were randomly chosen during the whole study period, and tested by two supplementary systems: Chiron RIBA HCV test system 2nd generation (Chiron Corporation, Emeryville, CA, USA) and LiaTek HCV (Organon). All procedures were performed following the manufacturers’ instructions. Results From 470 studied patients 213 (45.3%) were positive to HCV by EIA. Thirteen (2.8%) yielded indeterminate results by both supplementary tests. Liver biopsy on 92 of the positive cases showed that 14 of them had no evidence of liver damage, 14 non-specific damage, 19 acute hepatitis and 45 chronic hepatitis (Table 1). According to RIBA-2, antibodies to core protein were the first detected and the strongest serological reaction in nine out of 12 acute cases (75%). They were present in all of the 41 chronic patients studied. Antibodies to non-structural protein C33c (NS3) were the second most frequently observed, both in acute and chronic cases (66.7% and 90.2%, respectively) with a less important role for the two NS4 antigens. Fifteen samples yielded indeterminate results; eight of them among chronic and seven among acute patients (Table 2). A quantitative analysis of the RIBAresults, taking into account the magnitude of the reactions (e.g. considering positive those samples with one band
Table
2. RIBA positivity pattern among Cuban patients with HCV infection Patients
Total
RIBA antigens 5-l- 1 c- 100-3
Chronic Acute Total
Table 3.
29 4 33
26 5 31
c33c
c22
37 8 45
41 9 50
41 12 53
Indererminare
8 7 15
Sample
1 2 3 4 5 6 7 8-9 IO 11 12
RIBA antigens
Result
5-l- 1
c- 100-3
c33c
c22
_ _
-
2+ -
I+ 2+ -
I+ I+ I+ -
3+ 4+ I+ _
_
2+ 2+
4+ 4+ 3+ 3+
+I3+ 4+ 4+ 4+ 3+ 2+ 4+ 4+
4+ 3+
Indeterminate Indeterminate Positive Positive Positive Indeterminate Indeterminate Indeterminate Indeterminate Positive Indeterminate Positive
reacting over 2+) could considerably reduce the number of indeterminate results, especially in acute cases, where the beginning of the immunological response is characterized by the presence of antibodies to individual antigens (Table 3). LiaTek HCV yielded less indeterminate results both in acute and chronic patients (26.3% and 8.9%, respectively) and the antibody pattern was also most frequently represented by different core antigens, followed by the NS4 band. In fact, no sample was reactive only to NS5, which explains the failure to detect a sample reactive only by Innotest Ab EIA. Five out of seven RIBA indeterminate acute patients were positive by LiaTek and one RIBApositive case remained indeterminate by LiaTek HCV. The follow-up demonstrated seropositivity in all of these patients, despite four RIBA indeterminate results. Acute hepatitis was later confirmed by liver biopsy. The use of a different criterion for positivity and multiple core antigens are factors influencing the better performance of LiaTek HCV in both acute and chronic patients (Tables 4 and 5). Five out of 19 acute patients were followed monthly before infection. In all cases infection was initially evident through rises in ALT of more than 2.5 the upper limit of normal during 1 and 8 months (average 2.5 months) before the appearance of ELISA detectable antibody. In eight chronic ELISA HCV positive patients, who were indeterminate by RIBA-2, seven were positive after LiaTek. In all seven, antibodies to core protein were the only antibodies detected and five of them showed reactivity of 2+ or greater. Discussion Antibodies to viral core antigen are the most frequently found among Cuban patients infected with HCV. Individuals infected with HCV, especially in the early phase of infection may yield indeterminate results with RIBAin spite of polymerase chain
Padron et al.: Antibody
Table 4.
LiaTek HCV positivity
Table 5.
NS4
NS5
Core- 1
Core-2
57 16 73
36 13 49
52 15 67
56 15 71
Antibody
1
2 3 4 5 6 7 8 9 10 11 12
Total Core-4
Core-3 44 11 55
49 13 62
68 19 87
I+
3+ 2+ 2+ I+ +I+I3+ +I4+
6 5 11
Result
LiaTek antigens
I+ -
Indeterminate
pattern in acute infection after LiaTek HCV
Sample NS4
25
pattern among Cuban patients with HCV infection LiaTek antigens
Patients
Chronic Acute Total
pattern to HCV antigens
NS5
Core- 1
Core-2
Core-3
-
+I+II+ +II+ +l+I-
+I-
-
2+ 2+ 2+ I+ I+ 2+ -
2+ 3+ 4+ 3+ I+ 2+ 2+ -
-
4+
2+
+I-
reaction (PCR) positive results*, particularly in those with anti-core positivity”m5. There are also qualitative differences between NS4 antigens used in both supplementary assays, as can be seen from Tables 3 and 4, where patients 1, 6 and 7 are positive to antiNS4 antibodies by LiaTek and remain negative by RIBA-2. These results show that anti-HCV positive patients may exhibit different antibody responses to specific viral antigens. Discrepant results found between RIBAand LiaTek supplementary tests in patients in the earliest stage of the acute disease may be explained by the heterogeneity among antibodies elicited in different individuals after infection. These findings may be due to differences, both at the viral nucleotide and aminoacid levels, between HCV strain+. If during the course of the disease a heterogeneous viral population is formed, one should expect a wider spectrum of antigen recognition. Another explanation could be based on individual differences in the immunological response to different viral antigens. Further studies are necessary to elucidate this finding. In spite of the many cases that still remain indeterminate by RIBA-2, especially in the acute phase of the disease, patients reactive for only one antigen by RIBAmay still be infectious’. On the other hand, in the light of the present results, the manufacturer’s criteria of indeterminate RIBA results are obviously inadequate. If samples showing a strong reactivity (2+ or higher) for just one antigen are to be considered positive, all false indeterminate results would turn
Core-4
+I-
I+
I+
2+ +l-
3+ I+
+I-
+II+
-
_
_ -
4+
2+
2+
Indeterminate Positive Positive Positive Positive Positive Positive Positive Indeterminate Positive Indeterminate Positive
positive (Table 3). ELISA is more sensitive than RIBAfor the detection of anti-HCV antibodies, as it is unable to detect all positive seraX especially during the acute phase of the disease. No significant difference was found regarding the severity of the clinical illness among HCV positive and indeterminate patients (data not shown). Present results show that antibodies to core protein are the first elicited and the most frequent sign of HCV infection among Cuban patients with acute as well as chronic HCV infection and, in some cases, they can show the infection before supplementary tests. In our experience, LiaTek HCV has proved to be a better tool for confirming ELISA positive samples.
Acknowledgements The authors thank Organon Teknika, Ortho Diagnostic Systems and Innogenetics BV for providing the diagnostic systems used in this research.
References Padrdn G, Lemos G, Sanchez G et al. Evaluacibn de un sistema para la detecibn de anticuerpos contra un antigen0 sintetico del core del virus de hepatitis C y su prevalencia en donantes de sangre. Biotecnologia Aplicada
1994;
11:165-70
Van der Poel CL, Brester D, Reesnik HW. Plaisier AA, Schaasberg W, Leentvaar-Kuypers A et al. Early antihepatitis C virus response with second generation C22iC22 ELISA. VOX Sanpinis 1992; 62: 208-12
26
3
4
5 6
Serodiagn.
Immunother.
Infect. Disease 1995; 7: No 1
Chan S-W, Simmonds P, McOmish F, Yap PL, Mitchell R, Dow B, Follet E. Serological response to infection with three different types of hepatitis C virus. Lancet 1991; 338: 1391 Puoti M, Zonaro A, Ravaggi A, Marin MC, Castelnuovo F, Cariani E, Hepatitis C virus RNA and antibody response in the clinical course of acute hepatitis C virus infection. Hepatology 1992; 16: 877-81 Tong CYW, Codd AA, RIBAband intensity and PCR in HCV infection. Lancet 1992; 340: 117 Transfusion Safety Study Group. Possible HCV reinfection of hemophiliacs with antecedent anti-c22-3 and
7
8
anti-c33c but no anti-clOO-3. (abstr). Vth International Symposium on Viral Hepatitis, Madrid, Spain, 30 January-l February 1992 Halfon S, Rousseau S, Tamalet C, Antoni M, Gerolami V, Levy M et al. Indeterminate second-generation hepatitis C recombinant immunoblot test: detection of hepatitis C virus infection by polymerase chain reaction J Infect Disease 1992; 166: 449 Lazizi Y, Elfassi E, Pillot J. Detection of hepatitis C virus sequences in sera with controversial serology by nested polymerase chain reaction. J Clin Microbial 1992; 30: 931-4