6.1 INTRODUCTION
Antibodies to the second envelope protein may appear as the first serological markers in acute HCV infection and appear to persist in chronic infection. However, detection of anti-E2 has been hampered by the lack o f suitable antigens. The E2 glycoprotein has recently been expressed in Chinese hamster ovary (CHO) cells, purified and used to develop an anti-E2 enzyme immunoassay (Lesniewski et al. 1995).
Anti-E2 has been found in over 95% o f HCV RNA positive patients. It appears that quantitative levels of this antibody correlate with the level of viraemia (Yuki et al. 1996; Leon et al. 1996). Serial analysis of sera taken from patients infected with HCV who have undergone successful anti-viral treatment with interferon alpha indicates that anti-E2 may disappear following therapy (Yokosuka etal. 1993; Saracco et al. 1994).
The aims of this study were to determine the prevalence o f anti-E2 in stored sera taken from a cohort of anti-HCV positive Irish women who were all infected by HCV genotype lb in 1977. In this study I evaluated whether the absence o f anti-E2 distinguished patients who had spontaneous resolution of HCV infection from those with persistent disease.
6.2 PATIENT DETAILS
Evaluation of the anti-E2 assay was carried out on stored sera from 87 anti- HCV positive Irish women who were infected by HCV genotype lb from contaminated anti-D immunoglobulin given in 1977 (Power et al. 1995). All women were positive for anti-HCV antibodies by ELISA-3. HCV RNA was estimated using nested PCR with primers directed towards the 5’ non-coding region of HCV (Okamoto et al. 1990). Sixteen sera were from women who were RIBA-2 and HCV RNA positive (group 1). Fifty sera were from women who were who were reactive on RIBA testing but repeatedly negative for HCV RNA (group 2). Twenty one sera were from women who had normal liver function tests, indeterminate RIBA and who were repeatedly negative on testing for HCV RNA (group 3) (table 6.1).
Number of sera tested RIBA HCV 2.0 (Chiron)
HCV RNA
Group 1 16 positive positive
Group 2 50 positive negative
Group 3 2 1 indeterminate negative
6.3 ANTI-E2 ASSAY
Purified HCV E2 antigen produced from CHO cells was supplied coated onto polystyrene beads. Human serum or plasma was diluted into specimen diluent and incubated with the bead. Antibody to E2 bound to the bead and unbound materials were removed with washing. Bound immunoglobulins (mainly IgG) were then detected by subsequent incubation with horseradish peroxidase labelled goat antibody to human IgG and development with o- phenylenediamine-2HCl (OPD). The oxidised OPD generated an orange- yellow colour proportional to the amount of anti-E2 bound. After stopping the reaction with IN H2SO4, the colour intensity was read
spectrophotometrically at 492nm (Quantum II). Samples were assayed either neat or at various dilutions (1:20, 1:400) and anti-E2 concentrations were extrapolated from a standard curve generated from a set o f five provided calibrated samples.
6.4 RESULTS
All 16 sera (100%) from patients in group 1 had high titres of anti-E2 antibodies (median value 5.61 x 10^ arbitrary anti-E2 units/ml). In group 2, 31/50 sera (62%) were above the cut-off for anti-E2 antibodies (median value 4.26 X 10^ units/ml). However in group 3, only 3/21 sera (14%) were positive
for anti-E2 antibodies (median value 31 units/ml). The concentration of anti- E2 was significantly lower (p<0.0001) in groups 2 and 3 when each was compared to group 1 (one-way analysis o f variance, Kruskal-Wallis test) (figure 6.1).
6.5 CONCLUSIONS
A proportion o f patients tested for HCV antibodies will have an indeterminate reaction on a confirmatory RIBA test despite testing positive by ELISA. In such patients who are persistently HCV RNA negative, and who lack other supportive evidence of hepatic dysfunction, the lack of confirmation may indicate a false-positive ELISA (Tobler et al. 1994). Demonstration of additional specific antibodies to HCV in this situation should reveal those patients who have persistent infection with HCV.
The recombinant anti-E2 used in our study is derived from a type la genotype of HCV, and the patients tested from the Irish cohort are known to have been infected with type lb HCV from contaminated anti-D immunoglobulin. The presence o f high levels of HCV anti-E2 antibodies in our viraemic patients therefore confirms that there is no loss of activity of the anti-E2 assay across subtypes la and lb. This suggests that there are likely to be conserved E2 epitopes among type la and type lb viruses as previously described (Lesniewski et al. 1995).
All the Irish women tested for HCV antibodies and HCV RNA in the above study have a documented exposure to HCV, yet a proportion of these individuals appear to have cleared the virus and have no histological evidence o f liver damage. In this group we have shown that a high proportion of anti- HCV positive women with past exposure to HCV, but indeterminate RIBA who are persistently HCV RNA negative are also anti-E2 negative.
Lack of anti-E2 in patients who have cleared HCV poses questions regarding the role of neutralizing antibodies directed towards the envelope
region of HCV. It is possible that successful antibody binding to the underlying viral protein may occur in some patients infected with HCV leading to viral clearance. The presence of neutralizing epitopes within E2 has been suggested in part by the use o f bindings assays that measure the attachment of recombinant envelope proteins to mammalian cells. (Rosa et al. 1996).
Our study indicates that in those individuals who clear HCV, anti-E2 eventually disappears from the serum after a period of time. This is in marked contrast to a related flavivirus, HGV (or GBV-C) where absence of anti-E2 correlates with viraemia. Furthermore the presence of anti-E2 antibodies is associated with loss of detectable HGV RNA and recovery from HGV infection (Tacke et al. 1997). The fundamental differences in the role o f anti-E2 in HCV and HGV infection may relate to the different degrees of glycosylation of the envelope protein of each virus (Jarvis et al. 1996).
In summary loss or absence of anti-E2 may be useful in confirming clearance of HCV and a state of ‘immunity’ in patients who have been previously exposed to HCV. Proof of loss of infectivity in the Irish women will need to be ascertained by ongoing look-back studies o f transmission of HCV by blood transfusion.
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