Anti-HCV core antibody: A potential new marker of occult and otherwise serologically silent HCV infection
Authors: Tomasz I. Michalak, Tram N.Q. Pham
Journal: Journal of Hepatology (2008)

Hepatitis C virus (HCV) frequently causes chronic hepatitis C (CHC), which with time may advance to liver fibrosis, cirrhosis, and hepatocellular carcinoma. In the natural course of HCV infection, HCV RNA can be found in serum 1–2 weeks after viral encounter, while HCV-specific antibodies (to core, envelope and/or nonstructural [NS] proteins) are usually not detectable until 8–9 weeks [[1]Strader D.B. Wright T. Thomas D.L. Seeff L.B. Diagnosis, management, and treatment of hepatitis C.Hepatology. 2004; 39: 1147-1171Crossref PubMed Scopus (1574) Google Scholar]. Nevertheless, in clinical practice, screening for anti-HCV antibodies remains the first step towards identifying HCV-infected individuals, although a positive finding must be accompanied by HCV RNA detection before a diagnosis of active infection can be made [[2]Alter M.J. Kuhnert W.L. Finelli L. Guidelines for laboratory testing and result reporting of antibody to hepatitis C virus. Centers for Disease Control and Prevention.MMWR Recomm Rep. 2003; 52: 1-13PubMed Google Scholar]. There are several immunoassays (EIA) currently available for the screening of anti-HCV antibodies. These include second-generation Abbott HCV EIA 2.0 (Abbott Diagnostics, IL, USA), and third-generation Ortho HCV Version 3.0 ELISA (Ortho-Clinical Diagnostics, NY, USA) [[2]Alter M.J. Kuhnert W.L. Finelli L. Guidelines for laboratory testing and result reporting of antibody to hepatitis C virus. Centers for Disease Control and Prevention.MMWR Recomm Rep. 2003; 52: 1-13PubMed Google Scholar]. The fourth-generation EIA Innotest-HCV-AbIV (Innogenetics, Belgium) is thought to be superior for sensitivity and specificity over that of third generation assays [[3]Innotest HCV Ab IV: the first fourth generation assay with unique antigens from different genotypes. Microgen Bioproducts Newsl 2008;28:1–4.Google Scholar]. Although all of these tests employ a cocktail of recombinant HCV antigens which may include core, envelope, and/or NS, they differ with respect to the actual sequence of the peptides used for antibody capture. In the past few years, data from several research groups have revealed the presence of occult HCV infection (OCI), as evidenced by small amounts of HCV RNA in serum and a low-level virus replication in peripheral blood mononuclear cells (PBMC) and/or liver, in persons with spontaneous or therapeutically-induced resolution of hepatitis C [4Pham T.N.Q. MacParland S.A. Mulrooney P.M. Cooksley H. Naoumov N.V. Michalak T.I. Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C.J Virol. 2004; 78: 5867-5874Crossref PubMed Scopus (283) Google Scholar, 5Radkowski M. Gallegos-Orozco J.F. Jablonska J. Colby T.V. Walewska-Zielecka B. Kubicka J. et al.Persistence of hepatitis C virus in patients successfully treated for chronic hepatitis C.Hepatology. 2005; 41: 106-114Crossref PubMed Scopus (272) Google Scholar, 6Castillo I. Rodriguez-Inigo E. Lopez-Alcorocho J.M. Pardo M. Bartolome J. Carreno V. Hepatitis C virus replicates in the liver of patients who have a sustained response to antiviral treatment.Clin Infect Dis. 2006; 43: 1277-1283Crossref PubMed Scopus (93) Google Scholar, 7Di Liberto G. Roque-Afonso A.M. Kara R. Ducoulombier D. Fallot G. Samuel D. et al.Clinical and therapeutic implications of hepatitis C virus compartmentalization.Gastroenterology. 2006; 131: 76-84Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 8Ciancio A. Smedile A. Giordanino C. Colletta C. Croce G. Pozzi M. et al.Long-term follow-up of previous hepatitis C virus positive nonresponders to interferon monotherapy successfully retreated with combination therapy: are they really cured?.Am J Gastroenterol. 2006; 101: 1811-1816Crossref PubMed Scopus (23) Google Scholar, 9Lee W.M. Polson J.E. Carney D.S. Sahin B. Gale M. Reemergence of hepatitis C virus after 8.5 years in a patient with hypogammaglobulinemia: evidence for an occult viral reservoir.J Infect Dis. 2005; 192: 1088-1092Crossref PubMed Scopus (60) Google Scholar]. Parallel to this revelation, in a series of studies conducted by Dr. Carreno and colleagues [10Castillo I. Pardo M. Bartolome J. Ortiz-Movilla N. Rodriguez-Inigo E. de Lucas S. et al.Occult hepatitis C virus infection in patients in whom the etiology of persistently abnormal results of liver-function tests is unknown.J Infect Dis. 2004; 189: 7-14Crossref PubMed Scopus (225) Google Scholar, 11Castillo I. Rodriguez-Inigo E. Bartolome J. de Lucas S. Ortiz-Movilla N. Lopez-Alcorocho J.M. et al.Hepatitis C virus replicates in peripheral blood mononuclear cells of patients with occult hepatitis C virus infection.Gut. 2005; 54: 682-685Crossref PubMed Scopus (120) Google Scholar, 12Bartolome J. Lopez-Alcorocho J.M. Castillo I. Rodriguez-Inigo E. Quiroga J.A. Palacios R. et al.Ultracentrifugation of serum samples allows detection of hepatitis C virus RNA in patients with occult hepatitis C.J Virol. 2007; 81: 7710-7715Crossref PubMed Scopus (82) Google Scholar], OCI was also identified in individuals who were apparently non-reactive for anti-HCV antibodies by standard clinical assays but had persistently elevated liver enzyme levels in sera. However, unlike OCI which continues after termination of symptomatic hepatitis C mentioned above, the etiology of this form of low-level HCV carriage is unknown. For clarity, these two potentially distinctive forms of OCI are referred to here as secondary and crytogenic, respectively. It is necessary to emphasize that their identification was possible only after introduction of highly sensitive nucleic acid amplification assays capable of detecting HCV RNA at levels below the cut-offs of standard clinical tests, i.e., <2.5 virus genome equivalents (vge)/reaction. In this issue of the Journal, Quiroga and colleagues [[13]Quiroga J. Castillo I. Llorente S. Bartolomé J. Barril G. Carreño V. Identification of serologically silent occult hepatitis C virus infection by detecting immunoglobulin G antibody to a dominant HCV core peptide epitope.J Hepatol. 2009; 50: 256-263Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar] report findings of whether patients with cryptogenic OCI carry antibodies of IgG class directed against an immunodominant epitope of HCV core protein, and whether the aforementioned antibodies may be relevant to the identification of this otherwise serologically silent form of OCI. In a cohort of over 140 patients examined, 40% were found positive for the IgG anti-HCV core antibody, including 10% of individuals who were antibody non-reactive at the time of the first sample testing. This finding is quite intriguing given that the assay only evaluated antibody against a single N-terminal epitope (amino acids 5–19) of the core protein. Nevertheless, one may argue that the data are not entirely unexpected for a couple of reasons. First, the core polypeptide, with several conserved epitope clusters, is thought to be the most immunogenic among the HCV proteins [[14]Chien D.Y. McFarland J. Tabrizi A. Kuo C. Houghton M. Kuo G. et al.Distinct subtypes of hepatitis C virus defined by antibodies directed to the putative core, NS4, and NS5 region polypeptides.in: Nishioka N. Suzuki S. Mishiro S. Oda T. Viral hepatitis and liver disease. Springer-Verlag, Tokyo1983: 320-324Google Scholar], and is able to elicit a more pronounced humoral response than the other HCV antigens in acute, chronic or resolved hepatitis C [15Chen M. Sallberg M. Sonnerborg A. Weiland O. Mattsson L. Jin L. et al.Limited humoral immunity in hepatitis C virus infection.Gastroenterology. 1999; 116: 135-143Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 16Lechmann M. Ihlenfeldt H.G. Braunschweiger I. Giers G. Jung G. Matz B. et al.T- and B-cell responses to different hepatitis C virus antigens in patients with chronic hepatitis C infection and in healthy anti-hepatitis C virus-positive blood donors without viremia.Hepatology. 1996; 24: 790-795PubMed Google Scholar, 17Nikolaeva L.I. Blokhina N.P. Tsurikova N.N. Voronkova N.V. Miminoshvili M.I. Braginsky D.M. et al.Virus-specific antibody titres in different phases of hepatitis C virus infection.J Viral Hepat. 2002; 9: 429-437Crossref PubMed Scopus (35) Google Scholar]. Second, in patients with resolved or progressing CHC, the most frequently identifiable antibodies are those directed against epitopes located towards the N-terminal half of the core protein [[16]Lechmann M. Ihlenfeldt H.G. Braunschweiger I. Giers G. Jung G. Matz B. et al.T- and B-cell responses to different hepatitis C virus antigens in patients with chronic hepatitis C infection and in healthy anti-hepatitis C virus-positive blood donors without viremia.Hepatology. 1996; 24: 790-795PubMed Google Scholar], the same region Quiroga et al. chose to study. Despite the apparent success in specificity, the sensitivity of the IgG anti-HCV core detection in cryptogenic OCI was low, as over 60% of the patients remained nonreactive. On the one hand, it could be that the antibody levels fluctuate or are present at levels below the detection limit of the assay, which is not surprising since HCV-specific antibodies identifiable by current assays can be narrowly focussed, low in titers and delayed in appearance [[15]Chen M. Sallberg M. Sonnerborg A. Weiland O. Mattsson L. Jin L. et al.Limited humoral immunity in hepatitis C virus infection.Gastroenterology. 1999; 116: 135-143Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar]. Since the HCV core protein contains multiple immunodominant B cell epitopes outside the amino acid 5–19 sequence [[16]Lechmann M. Ihlenfeldt H.G. Braunschweiger I. Giers G. Jung G. Matz B. et al.T- and B-cell responses to different hepatitis C virus antigens in patients with chronic hepatitis C infection and in healthy anti-hepatitis C virus-positive blood donors without viremia.Hepatology. 1996; 24: 790-795PubMed Google Scholar], it is conceivable that seemingly seronegative patients with cryptogenic OCI may have anti-HCV antibodies which are directed against other epitopes of the protein. In this regard, detection of seronegative OCI may be improved through the establishment of an assay which could simultaneously screen for antibodies specific for different epitopes of the core protein. On the other hand, one has to also look at the level of viral antigens available to trigger humoral immunity. HCV replication in patients with OCI is generally very low. The difficulty of even detecting HCV RNA in serum [[12]Bartolome J. Lopez-Alcorocho J.M. Castillo I. Rodriguez-Inigo E. Quiroga J.A. Palacios R. et al.Ultracentrifugation of serum samples allows detection of hepatitis C virus RNA in patients with occult hepatitis C.J Virol. 2007; 81: 7710-7715Crossref PubMed Scopus (82) Google Scholar] in individuals with cryptogenic OCI is suggestive of low, if any, circulating viral antigens. As such, this level of antigenic stimulation may simply be below the threshold required to induce and/or sustain a detectable humoral immune response. In their latest work, Quiroga et al. [[13]Quiroga J. Castillo I. Llorente S. Bartolomé J. Barril G. Carreño V. Identification of serologically silent occult hepatitis C virus infection by detecting immunoglobulin G antibody to a dominant HCV core peptide epitope.J Hepatol. 2009; 50: 256-263Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar] stated that the number of HCV-infected hepatocytes detected by in situ hybridization was seemingly higher in patients who tested positive for the anti-HCV core antibody than those who did not. However, unless one could actually quantify the level of HCV RNA and/or viral protein in these infected hepatocytes, it would be very difficult to equate a higher number of infected cells with IgG anti-HCV core antibody positivity. Along this line, despite that signs of necroinflammation were thought to be more frequently observed in individuals who were anti-HCV core antibody reactive, the data available do not allow for ascribing any direct pathogenic relevance of the OCI to the persistently elevated liver enzymes found in these individuals. Although PBMC from patients with cryptogenic OCI have been shown to be a reservoir for HCV replication [10Castillo I. Pardo M. Bartolome J. Ortiz-Movilla N. Rodriguez-Inigo E. de Lucas S. et al.Occult hepatitis C virus infection in patients in whom the etiology of persistently abnormal results of liver-function tests is unknown.J Infect Dis. 2004; 189: 7-14Crossref PubMed Scopus (225) Google Scholar, 11Castillo I. Rodriguez-Inigo E. Bartolome J. de Lucas S. Ortiz-Movilla N. Lopez-Alcorocho J.M. et al.Hepatitis C virus replicates in peripheral blood mononuclear cells of patients with occult hepatitis C virus infection.Gut. 2005; 54: 682-685Crossref PubMed Scopus (120) Google Scholar], it was not established if PBMC, especially B and T cells, from this patient cohort were infected with HCV, and if so, whether virus expression in immune cells in patients tested positive for IgG anti-HCV core antibody would be different from those who did not. Along this line, it would be of interest to determine whether HCV infection of lymphoid cells in general, and B cells in particular, would lead to an alteration in the ability to make antibody by B cells. In addition to humoral immunity, exposure to HCV also mounts a cellular immune response, although its strength varies depending on a number of host and virus-related factors. To date, there have been many reports which documented the presence of HCV-specific T cell responses in individuals who had no detectable anti-HCV antibody, as determined by standard clinical assays [18Kamal S.M. Amin A. Madwar M. Graham C.S. He Q. Al Tawil A. et al.Cellular immune responses in seronegative sexual contacts of acute hepatitis C patients.J Virol. 2004; 78: 12252-12258Crossref PubMed Scopus (73) Google Scholar, 19Zeremski M, Shu MA, Brown Q, Wu Y, Des J, Busch MP, et al. Hepatitis C virus-specific T-cell immune responses in seronegative injection drug users. J Viral Hepat 2008 [Epub ahead of print].Google Scholar, 20Quiroga J.A. Llorente S. Castillo I. Rodriguez-Inigo E. Pardo M. Carreno V. Cellular immune responses associated with occult hepatitis C virus infection of the liver.J Virol. 2006; 80: 10972-10979Crossref PubMed Scopus (44) Google Scholar]. For example, sexual contacts of patients with acute hepatitis C were found to exhibit both proliferative and cytotoxic T cell responses to recombinant HCV antigens without developing HCV-specific antibodies [[18]Kamal S.M. Amin A. Madwar M. Graham C.S. He Q. Al Tawil A. et al.Cellular immune responses in seronegative sexual contacts of acute hepatitis C patients.J Virol. 2004; 78: 12252-12258Crossref PubMed Scopus (73) Google Scholar]. Similarly, T cells from injection drug users who were persistently negative for anti-HCV antibodies could make high levels of gamma-interferon following ex vivo stimulation with HCV peptides [[19]Zeremski M, Shu MA, Brown Q, Wu Y, Des J, Busch MP, et al. Hepatitis C virus-specific T-cell immune responses in seronegative injection drug users. J Viral Hepat 2008 [Epub ahead of print].Google Scholar]. In an earlier work [[20]Quiroga J.A. Llorente S. Castillo I. Rodriguez-Inigo E. Pardo M. Carreno V. Cellular immune responses associated with occult hepatitis C virus infection of the liver.J Virol. 2006; 80: 10972-10979Crossref PubMed Scopus (44) Google Scholar], Quiroga et al. documented the presence of HCV-specific T cell responses, albeit intermittently, in more than 50% of patients with cryptogenic OCI. Taken together, the data argue that testing of appropriately tailored HCV-specific humoral and cellular immune responses may provide a surrogate marker to the diagnosis of past exposure to low-dose HCV leading to primary OCI, to the confirmation of the existence of secondary OCI, and to the determination of the kinetics and potential reactivation of OCI. In summary, the data by Quiroga et al. [[13]Quiroga J. Castillo I. Llorente S. Bartolomé J. Barril G. Carreño V. Identification of serologically silent occult hepatitis C virus infection by detecting immunoglobulin G antibody to a dominant HCV core peptide epitope.J Hepatol. 2009; 50: 256-263Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar] offer novel insights which could potentially pave the way towards better diagnosis of OCI in patients with abnormally high liver enzymes of currently unknown etiology. The finding that a number of patients with cryptogenic OCI who were initially nonreactive for IgG anti-HCV core antibodies became positive upon subsequent testing underscores the necessity of screening serial samples, as has been recognized in previous works [5Radkowski M. Gallegos-Orozco J.F. Jablonska J. Colby T.V. Walewska-Zielecka B. Kubicka J. et al.Persistence of hepatitis C virus in patients successfully treated for chronic hepatitis C.Hepatology. 2005; 41: 106-114Crossref PubMed Scopus (272) Google Scholar, 21Pham T.N.Q. Mulrooney-Cousins P.M. Mercer S.E. MacParland S.A. Inglot M. Zalewska M. et al.Antagonistic expression of hepatitis C virus and alpha interferon in lymphoid cells during persistent occult infection.J Viral Hepat. 2007; 14: 537-548Crossref PubMed Scopus (26) Google Scholar]. The study also highlights the need for the development of clinically applicable assays detecting anti-HCV core antibodies at a high sensitivity, an effort which is actively pursued by some laboratories in the industry.