AMERICAN JOURNAL OF CLINICAL PATHOLOGY Editorial
Hepatitis C Virus, Antibodies, and Infectivity Paradox, Pragmatism, and Policy
Within the last few years, heroic studies have culminated in the expression of segments of the genome of hepatitis C virus (HCV), the causative agent of most cases of post-transfusion non-A, non-B hepatitis.12 The expressed protein (c 100-3) is thought to represent a nonstructural component of the virus. Nevertheless, it reacts with immunoglobulin molecules from individuals infected with HCV and it has been used as the capture reagent in immunoassays that are now used routinely to screen donations of blood.3,4 It is possible that the use of this test has reduced post-transfusion HCV infection by 50 to 85% or more. In fact, direct assessment of the incidence of infection among transfused cardiac surgery patients shows that on implementation of the test, the frequency of infectivity decreased by 83%, from 0.18 to 0.03%.5 These data are, of course, extremely encouraging, but they also confirm that there are situations in which HCV infection (and infectivity) occur in the absence of detectable levels of antibodies to c 100-3. At this time, it is not known whether there is any relationship between antibodies to c 100-3 and neutralizing antibodies to HCV or indeed, whether such antibodies exist at all. It can be seen that, even in an individual, the relationship between antibodies to the c 100-3 peptide of HCV and the presence of HCV virions, or of neutralizing an-
It is not surprising that most immunoglobulin preparations have detectable levels of antibodies to HCV. All of the materials discussed by Dodd and colleagues6 were prepared from plasma collected before implementation of any testing for anti-HCV and indeed, until now, source plasma has not been tested for anti-HCV. Clinical trials of tests for antibodies to HCV suggest that the prevalence of anti-HCV among commercial donors of source plasma is 6 to 10%.7,8 Why, then did three immunoglobulin preparations give equivocal or nonconfirmable results? The authors suggest that the Baxter product was made from plasma that had been screened to eliminate units with high ALT levels. In fact, all source and voluntary plasma is routinely tested for ALT levels in the United States. Some products, however, are manufactured entirely from plasma recovered from voluntary donations to community blood centers, where the prevalence rates for antiHCV reactivity are on the order of 0.5 to 0.7% (CT Fang, personal communication, 1991). These products are the ones identified as Baxter Immunoglobulin IV (properly known as Immune Globulin Intravenous [Human], Polygam), Sandoglobulin, and Varicella-Zoster Ig, the very same preparations that showed variable and/or non-
4
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
tibodies to HCV, is unclear. Surely, such relationships must be much more obscure when the questions are asked in the context of products derived from pools of thousands of individual plasma donations. In this issue of the American Journal of Clinical Pathology, Dodd (no relation) and others6 provide pause for further reflection. They tested a number of commercially available immunoglobulin preparations for anti-HCV, using enzyme immunoassay tests based on the c 100-3 antigen. The reader should be aware that these tests are designed for use with serum or plasma and that results obtained with other samples may not be reliable. In this study, however, the use of a blocking test to assess specificity, plus the finding that at least some results were nonreactive, lend credence to the data. Unfortunately, the experiments with fractionated animal plasma provide only limited information about nonspecificity because the probe reagents for the enzyme immunoassay tests are designed to detect human, not animal immunoglobulins.
Many viruses have evolved mechanisms that not only permit them to evade the host immune response but also to establish a state of persistent infection. Viruses are complex and infection often results in the elaboration of antibodies directed against a variety of antigenic sites; however, not all such antibodies are necessarily effective in eliminating the virus. As a result, infectious virus and antibodies to that virus often co-exist in the same individual. In some circumstances, the virus may even be present in the circulation. Consequently, testing for antibodies can be used to identify blood donations that may be infectious for certain viruses. This approach has proved remarkably effective in the case of the human immunodeficiency virus: careful selection and anti-human immunodeficiency virus testing of all donors, both voluntary and paid, have profoundly reduced the risk of transfusiontransmitted acquired immune deficiency syndrome.
DODD
All of these factors have resulted in a perplexing controversy. Should plasma destined for the preparation of intravenous immunoglobulins be tested for anti-HCV and, if reactive, eliminated from the pools? On the one hand, it has been suggested that because the majority of lots of current products are safe, it would be unwise to change current practice without understanding the consequences of such an action, which might have adverse effects on the viral load." On the other hand, because most anti-HCV-positive units clearly harbor infectious virus, it would be illogical to continue to include these units and their bioburden in the pools.9 The question is more than academic because the United States Food and
Drug Administration (FDA) has specifically recommended against such testing,4 yet products manufactured in the United States are routinely exported to Europe, where a requirement for testing recently was published. Furthermore, a significant amount of plasma collected from voluntary donations is also fractionated in the United States, where voluntary donors are tested for antiHCV and, if reactive, are deferred from future donation; 4 until this time, however, plasma from such individuals has been included in plasma pools. Fortunately, resolution of this dilemma is at hand. The USFDA performed studies to define whether intravenous immunoglobulin preparations derived from anti-HCV (c 100-3) nonreactive plasma are infectious for HCV. Screened pools were prepared and, on injection into two chimpanzees, were found to be infectious for HCV. This was not unexpected because the current c 100-3 test is acknowledged to have less than 100% sensitivity. The plasma pools were routinely fractionated by seven FDAlicensed manufacturers using their standard procedures. The resulting immunoglobulin preparations were administered to three chimpanzees, each of which received the same dose of each of the seven preparations. After almost 9 months of observation, none of the chimpanzees exhibited any evidence of infection with HCV.12 These data strongly suggest that removal of anti-c 100-3 reactive units from plasma pools will not result in an immunoglobulin product that is uniformly infectious for HCV. In September 1991, these data were presented to the FDA Blood Products Advisory Committee. This committee recommended that plasma destined for fractionation should be tested for anti-HCV and that seropositive units should not be included in the pools. In October 1991, the FDA accepted and acted on these recommendations. In addition, the FDA recommended that urgent attention be given to the development of procedures that can be used to achieve viral inactivation in immunoglobulin preparations. Although we still do not fully understand the relationships between HCV antibody, viremia, and immunity, rational policy has been developed from a paradoxical situation by the pragmatic application of laboratory methods. ROGER Y. DODD, Ph.D.
Head, Transmissible Diseases Laboratory American Red Cross Jerome H. Holland Laboratories Rockville, Maryland REFERENCES 1. Choo Q-L, Kuo G, Weiner AJ, et al. Isolation of a cDNA clone derived from a blood borne non-A, non-B viral hepatitis genome. Science 1989;244:359-362.
Vol. 97 • N o . I
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
confirmable anti-HCV reactivities. In fact, it is likely that these products had low or undetectable levels of anti-HCV and that the results for these particular products are most appropriately interpreted as nonspecific findings. In this context, it should be noted that methods used for the manufacture of intravenous immunoglobulins differ significantly from those used to prepare conventional immune globulin and, furthermore, that procedures differ widely among manufacturers. Much more intriguing are the issues of HCV infectivity and viral neutralizing capacity of immunoglobulin preparations. As Dodd and colleagues6 note, there have been a number of instances in which intravenous immunoglobulin preparations have been implicated in the transmission of non-A, non-B hepatitis, generally attributable to HCV. These events have not been explained adequately, but their rarity suggests that they may be a result of some failure or error in manufacturing process control. 910 No intravenous immunoglobulin preparation licensed in the United States and produced in manufacturing (as opposed to pilot) facilities has been associated with the transmission of non-A, non-B hepatitis. The implication is that the procedures used for the preparation of immunoglobulins may partition HCV away from the final product and/or that there are steps that normally inactivate the virus during preparation. Finlayson and Tankersley" have suggested that neutralizing or precipitating antibodies to HCV normally could contribute to this absence of infectivity. However, as yet there is no convincing evidence that neutralizing antibodies to HCV may be found in immunoglobulin preparations. In part, this lack of evidence may be a result of the absence of any laboratory system for the examination of HCV infectivity. In addition, there have been many controlled clinical studies in which immunoglobulin preparations were evaluated for post-exposure prophylaxis against parenteral hepatitis transmission. Although efficacy against hepatitis B infection generally was demonstrable, these studies yielded conflicting evidence for protection against non-A, non-B hepatitis.
5
6
AMERICAN JOURNAL OF CLINICAL PATHOLOGY Editorial
2. Alter HJ, Purcell RH, Shih JW, et al. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N Engl J Med 1989;321: 1494-1500. 3. Kuo G, Choo Q-L, Alter HJ, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989;244:362-364. 4. CDC. Public Health Service inter-agency guidelines for screening donors of blood, plasma, organs, tissues, and semen for evidence of Hepatitis B and Hepatitis C. MMWR 1991;40(RR-4):1-17. 5. Nelson K, Donahue J, Munoz A, et al. Risk of hepatitis C virus (HCV) infection in cardiac surgery patients and effectiveness of screening. Third International Symposium on HCV 1991; 118 (abstr). 6. Dodd LG, McBride JH, Gitnick GL, Howanitz PJ, Rodgerson DO. Prevalence of non-A, non-B hepatitis (NANB)/hepatitis C virus
7. 8. 9. 10. 11. 12.
(HCV) antibody in human immune globulins. Am J Clin Pathol 1992;97:108-113. Abbott Laboratories. Hepatitis C virus encoded antigen (recombinant c 100-3) HCV EIA. Manufacturer's Product Insert, 1990. Ortho Diagnostics Division. Hepatitis C virus encoded antigen (recombinant cl00-3) Ortho HCV ELISA test system. Manufacturer's Product Insert, 1990. Habibi B, Garretta M. Screening for hepatitis C virus antibody in plasma for fractionation. Lancet 1990;335:855-856. Rousell RH, Budinger MD, Pirofsky B, SchiffRI. Prospective study on the hepatitis safety of intravenous immunoglobulin, pH 4.25. Vox Sang 1991;60:65-68. Finlayson JS, Tankersley DL. Anti-HCV screening and plasma fractionation: The case against. Lancet 1990;335:1274-1275. Biswas R, Mitchell F, Wilson L, et al. Hepatitis C and therapeutic immunoglobulin product safety: A chimpanzee study. Third International Symposium on HCV 1991;115 (Abstr).
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
A.J.C.P.-January 1992
Hepatitis C Virus, Antibodies, and Infectivity Paradox, Pragmatism, and Policy
Within the last few years, heroic studies have culminated in the expression of segments of the genome of hepatitis C virus (HCV), the causative agent of most cases of post-transfusion non-A, non-B hepatitis.12 The expressed protein (c 100-3) is thought to represent a nonstructural component of the virus. Nevertheless, it reacts with immunoglobulin molecules from individuals infected with HCV and it has been used as the capture reagent in immunoassays that are now used routinely to screen donations of blood.3,4 It is possible that the use of this test has reduced post-transfusion HCV infection by 50 to 85% or more. In fact, direct assessment of the incidence of infection among transfused cardiac surgery patients shows that on implementation of the test, the frequency of infectivity decreased by 83%, from 0.18 to 0.03%.5 These data are, of course, extremely encouraging, but they also confirm that there are situations in which HCV infection (and infectivity) occur in the absence of detectable levels of antibodies to c 100-3. At this time, it is not known whether there is any relationship between antibodies to c 100-3 and neutralizing antibodies to HCV or indeed, whether such antibodies exist at all. It can be seen that, even in an individual, the relationship between antibodies to the c 100-3 peptide of HCV and the presence of HCV virions, or of neutralizing an-
It is not surprising that most immunoglobulin preparations have detectable levels of antibodies to HCV. All of the materials discussed by Dodd and colleagues6 were prepared from plasma collected before implementation of any testing for anti-HCV and indeed, until now, source plasma has not been tested for anti-HCV. Clinical trials of tests for antibodies to HCV suggest that the prevalence of anti-HCV among commercial donors of source plasma is 6 to 10%.7,8 Why, then did three immunoglobulin preparations give equivocal or nonconfirmable results? The authors suggest that the Baxter product was made from plasma that had been screened to eliminate units with high ALT levels. In fact, all source and voluntary plasma is routinely tested for ALT levels in the United States. Some products, however, are manufactured entirely from plasma recovered from voluntary donations to community blood centers, where the prevalence rates for antiHCV reactivity are on the order of 0.5 to 0.7% (CT Fang, personal communication, 1991). These products are the ones identified as Baxter Immunoglobulin IV (properly known as Immune Globulin Intravenous [Human], Polygam), Sandoglobulin, and Varicella-Zoster Ig, the very same preparations that showed variable and/or non-
4
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
tibodies to HCV, is unclear. Surely, such relationships must be much more obscure when the questions are asked in the context of products derived from pools of thousands of individual plasma donations. In this issue of the American Journal of Clinical Pathology, Dodd (no relation) and others6 provide pause for further reflection. They tested a number of commercially available immunoglobulin preparations for anti-HCV, using enzyme immunoassay tests based on the c 100-3 antigen. The reader should be aware that these tests are designed for use with serum or plasma and that results obtained with other samples may not be reliable. In this study, however, the use of a blocking test to assess specificity, plus the finding that at least some results were nonreactive, lend credence to the data. Unfortunately, the experiments with fractionated animal plasma provide only limited information about nonspecificity because the probe reagents for the enzyme immunoassay tests are designed to detect human, not animal immunoglobulins.
Many viruses have evolved mechanisms that not only permit them to evade the host immune response but also to establish a state of persistent infection. Viruses are complex and infection often results in the elaboration of antibodies directed against a variety of antigenic sites; however, not all such antibodies are necessarily effective in eliminating the virus. As a result, infectious virus and antibodies to that virus often co-exist in the same individual. In some circumstances, the virus may even be present in the circulation. Consequently, testing for antibodies can be used to identify blood donations that may be infectious for certain viruses. This approach has proved remarkably effective in the case of the human immunodeficiency virus: careful selection and anti-human immunodeficiency virus testing of all donors, both voluntary and paid, have profoundly reduced the risk of transfusiontransmitted acquired immune deficiency syndrome.
DODD
All of these factors have resulted in a perplexing controversy. Should plasma destined for the preparation of intravenous immunoglobulins be tested for anti-HCV and, if reactive, eliminated from the pools? On the one hand, it has been suggested that because the majority of lots of current products are safe, it would be unwise to change current practice without understanding the consequences of such an action, which might have adverse effects on the viral load." On the other hand, because most anti-HCV-positive units clearly harbor infectious virus, it would be illogical to continue to include these units and their bioburden in the pools.9 The question is more than academic because the United States Food and
Drug Administration (FDA) has specifically recommended against such testing,4 yet products manufactured in the United States are routinely exported to Europe, where a requirement for testing recently was published. Furthermore, a significant amount of plasma collected from voluntary donations is also fractionated in the United States, where voluntary donors are tested for antiHCV and, if reactive, are deferred from future donation; 4 until this time, however, plasma from such individuals has been included in plasma pools. Fortunately, resolution of this dilemma is at hand. The USFDA performed studies to define whether intravenous immunoglobulin preparations derived from anti-HCV (c 100-3) nonreactive plasma are infectious for HCV. Screened pools were prepared and, on injection into two chimpanzees, were found to be infectious for HCV. This was not unexpected because the current c 100-3 test is acknowledged to have less than 100% sensitivity. The plasma pools were routinely fractionated by seven FDAlicensed manufacturers using their standard procedures. The resulting immunoglobulin preparations were administered to three chimpanzees, each of which received the same dose of each of the seven preparations. After almost 9 months of observation, none of the chimpanzees exhibited any evidence of infection with HCV.12 These data strongly suggest that removal of anti-c 100-3 reactive units from plasma pools will not result in an immunoglobulin product that is uniformly infectious for HCV. In September 1991, these data were presented to the FDA Blood Products Advisory Committee. This committee recommended that plasma destined for fractionation should be tested for anti-HCV and that seropositive units should not be included in the pools. In October 1991, the FDA accepted and acted on these recommendations. In addition, the FDA recommended that urgent attention be given to the development of procedures that can be used to achieve viral inactivation in immunoglobulin preparations. Although we still do not fully understand the relationships between HCV antibody, viremia, and immunity, rational policy has been developed from a paradoxical situation by the pragmatic application of laboratory methods. ROGER Y. DODD, Ph.D.
Head, Transmissible Diseases Laboratory American Red Cross Jerome H. Holland Laboratories Rockville, Maryland REFERENCES 1. Choo Q-L, Kuo G, Weiner AJ, et al. Isolation of a cDNA clone derived from a blood borne non-A, non-B viral hepatitis genome. Science 1989;244:359-362.
Vol. 97 • N o . I
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
confirmable anti-HCV reactivities. In fact, it is likely that these products had low or undetectable levels of anti-HCV and that the results for these particular products are most appropriately interpreted as nonspecific findings. In this context, it should be noted that methods used for the manufacture of intravenous immunoglobulins differ significantly from those used to prepare conventional immune globulin and, furthermore, that procedures differ widely among manufacturers. Much more intriguing are the issues of HCV infectivity and viral neutralizing capacity of immunoglobulin preparations. As Dodd and colleagues6 note, there have been a number of instances in which intravenous immunoglobulin preparations have been implicated in the transmission of non-A, non-B hepatitis, generally attributable to HCV. These events have not been explained adequately, but their rarity suggests that they may be a result of some failure or error in manufacturing process control. 910 No intravenous immunoglobulin preparation licensed in the United States and produced in manufacturing (as opposed to pilot) facilities has been associated with the transmission of non-A, non-B hepatitis. The implication is that the procedures used for the preparation of immunoglobulins may partition HCV away from the final product and/or that there are steps that normally inactivate the virus during preparation. Finlayson and Tankersley" have suggested that neutralizing or precipitating antibodies to HCV normally could contribute to this absence of infectivity. However, as yet there is no convincing evidence that neutralizing antibodies to HCV may be found in immunoglobulin preparations. In part, this lack of evidence may be a result of the absence of any laboratory system for the examination of HCV infectivity. In addition, there have been many controlled clinical studies in which immunoglobulin preparations were evaluated for post-exposure prophylaxis against parenteral hepatitis transmission. Although efficacy against hepatitis B infection generally was demonstrable, these studies yielded conflicting evidence for protection against non-A, non-B hepatitis.
5
6
AMERICAN JOURNAL OF CLINICAL PATHOLOGY Editorial
2. Alter HJ, Purcell RH, Shih JW, et al. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N Engl J Med 1989;321: 1494-1500. 3. Kuo G, Choo Q-L, Alter HJ, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989;244:362-364. 4. CDC. Public Health Service inter-agency guidelines for screening donors of blood, plasma, organs, tissues, and semen for evidence of Hepatitis B and Hepatitis C. MMWR 1991;40(RR-4):1-17. 5. Nelson K, Donahue J, Munoz A, et al. Risk of hepatitis C virus (HCV) infection in cardiac surgery patients and effectiveness of screening. Third International Symposium on HCV 1991; 118 (abstr). 6. Dodd LG, McBride JH, Gitnick GL, Howanitz PJ, Rodgerson DO. Prevalence of non-A, non-B hepatitis (NANB)/hepatitis C virus
7. 8. 9. 10. 11. 12.
(HCV) antibody in human immune globulins. Am J Clin Pathol 1992;97:108-113. Abbott Laboratories. Hepatitis C virus encoded antigen (recombinant c 100-3) HCV EIA. Manufacturer's Product Insert, 1990. Ortho Diagnostics Division. Hepatitis C virus encoded antigen (recombinant cl00-3) Ortho HCV ELISA test system. Manufacturer's Product Insert, 1990. Habibi B, Garretta M. Screening for hepatitis C virus antibody in plasma for fractionation. Lancet 1990;335:855-856. Rousell RH, Budinger MD, Pirofsky B, SchiffRI. Prospective study on the hepatitis safety of intravenous immunoglobulin, pH 4.25. Vox Sang 1991;60:65-68. Finlayson JS, Tankersley DL. Anti-HCV screening and plasma fractionation: The case against. Lancet 1990;335:1274-1275. Biswas R, Mitchell F, Wilson L, et al. Hepatitis C and therapeutic immunoglobulin product safety: A chimpanzee study. Third International Symposium on HCV 1991;115 (Abstr).
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
A.J.C.P.-January 1992
