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Viral hepatitis: diagnosis, treatment and prevention
Immunology Today, vol. 5, No. 7, 1984
most other soft tissues and usually presents between 40 and 60 years with an equal sex distribution. Before the advent of chemotherapy it was fatal within a year in 80 % of patients. Kornblut postulated that it was caused by a tissue hypersensitivity reaction which followed an upper respiratory tract infection. There is no diagnostic test. Survival has improved dramatically with the use of
185 cyclophosphamide, azathioprine and adjuvant steroids. N. Rasmussen (Copenhagen) proposed that Wegner's should be classified as an autoimmune disease. Certain HLA antigens, B7, B8, DR2 and DR3, were abnormally frequent among patients. Over half the patients have one or more autoantibodies, most frequently to smooth muscle and rheumatoid
Viral hepatitis: diagnosis, treatment and prevention from C. R. Howard With over 200 million people chronically infected, hepatitis B remains the most serious persistent virus infection of man. The recent availability of a vaccine offers an opportunity to control, and eventually reduce these numbers. Although the prime consideration is the protection of those at risk of acquiring infection from such individuals, a reduction in the numbers of carriers will ultimately reduce the large numbers of people in areas of high endemicity who are predisposed to developing primary liver cancer later in life as a result of previous exposure to the virus. By contrast, hepatitis A is never associated with chronic liver infection but its ease of transmission by the oral route presents a major health problem in endemic areas and to those travelling to regions of the world with poor public sanitation. Hitherto unrecognized agents, collectively termed 'non-A, non-B viruses', now account for the majority of posttransfusion hepatitis cases since the introduction of routine tests for the screening of blood for markers of hepatitis B. M a n y of these cases become chronic infections with severe consequences. Additional epidemic forms of non-A, non-B together with the hepatitis B-associated delta agent complete the list of viruses discussed during the symposium*.
Hepatitis A virus Viral hepatitis type A appears to be declining in Europe and North America, most cases being limited to travellers returning from endemic areas or individuals exposed to contaminated shellfish or inadequate sanitation. Notwithstanding, interest continues to abound in the properties of this virus. R. H. Purcell (Bethesda) reviewed the present state of knowlege, reiterating *The 1984 International Symposium on Viral Hepatitis, organized by the University of California at San Francisco, 8-10 M a r c h 1984.
that, unlike hepatitis B, the viraemia is brief and at a low level. In contrast, the titre excreted in the stools early during the acute phase may be as high as 109 infectious particles per ml of extract. There also appears to be a direct relationship between incubation period of the disease and the titre of virus in the inoculum. Although structurally resembling the picornaviruses, the growth properties of the virus in tissue culture differs markedly from, say, those observed with poliovirus. In particular, the replicative cycle of hepatitis A virus is long in duration, extending over several weeks with fresh isolates. In addition, mature virus particles remain largely cell-associated, thus requiring physical methods for release and further passage in fresh cells. Attempts to attenuate the virus sufficiently for use as a h u m a n vaccine were summarized (R. Daemer, Bethesda; P. Provost, West Point). The group at the National Institute of Health have followed the interesting possibility of attenuation by continually passaging early harvest material, thereby decreasing the time required for a full cycle of replication. Some evidence of attenuation for susceptible chimpanzees was found after 20 passages.
Hepatitis B virus The hepatitis B virus genome consists of a partial double-stranded DNA structure with four open reading frames of any significant size. Two of these correspond to the genes coding for the major surface (S) polypeptide of mol. wt 25 000 and the internal core component of tool. wt 19 000 respectively. The remaining genes are believed to code for the virus-specific DNA polymerase and a fourth protein designated ' X ' . Evidence was presented that the latter peptide may be present in monkey kidney cells either transfected with recombinant viral DNA or in extracts of cultured hepatoma cells (A. Moriaty, La Jolla). Transcription occurs primarily from two promoters but
factor. Analysing the serological reactions Rasmussen noted that 9 of 12 patients had antibodies to normal granulocytes and monocytes but not lymphocytes. This was not found in 22 000 other sera. The antibodies disappeared during remission. IT] Adrian Drake-Lee is in the Royal National Throat, Nose and Ear Hospital, London.
initiation of R N A polymerase activity may occur at up to eight sites (W. J. Rutter, University of California, San Francisco). Analysis of the region coding for the surface polypeptide which contains various antigenic reactivities (HBsAg) showed that transcription may begin upstream at two separate sites to give larger proteins of tool. wts 71 000 and 45 000 respectively. Direct evidence for the presence of these larger forms in 22 n m HBsAg particles purified from serum was shown by W. H. Gerlich (University of Goettingen) and both may contain the additional amino acid sequence representing a receptor site for polymerized albumin on the virus particle, suggesting that a possible 'carrier' protein may be responsible for mediating the attachment of virus to new host cells. Indeed the relative amounts of these minor components appeared enhanced in sera containing large amounts of hepatitis B virus. Comparison of individual DNA clones suggests that there is extensive variation in predicted amino acid sequence for this peptide region, possibly indicative of multiple infection in the individual from whom the virus was originally isolated. However, A. R. Neurath (New York Blood Center) pointed out that selected domains of the major 25 000 tool. wt HBsAg peptide appear to be distantly related to amino acid sequences found in native human albumin, offering a possible explanation of the close association between this host protein and virus-specific molecules. Expression of the surface gene is enhanced up to 100-fold by a sequence between the S and X genes (W. J. Rutter, University of California, San Francisco). Although expression of various hepatitis B gene products may be achieved by transfeetion of cell lines with cloned viral DNA, efforts to demonstrate directly in vitro the presence of virus in serum have been notoriously unsuccessful. However, the co-cultivation of adult hepatocytes with a rat epithelial liver cell line may now provide a suitable tissue culture system for studying all stages of virus replication and provide suitable targets for cellular immunity studies. N. Theze and colleagues (Htpital Pontchaillou,
© 1984, ElsevierSciencePubfishemB.V., Amsterdam 0167 4919/84/$02.00
186 Paris) reported that full expression of viral DNA and antigen may be obtained using hepatocytes obtained by liver perfusion of kidney donors and that such cells maintained at least some differentiated functions up to six weeks after culture. Viral HBsAg and complete virions were seen and genome copies detected by hybridization from 5 to 16 days after infection. If confirmed, this would represent a significant breakthrough in our understanding of the virology of hepatitis B. There is now a clear relationship between hepatocelhilar carcinoma and hepatitis B infection. In Taiwan, for example, it is estimated that 40% of deaths in hepatitis B carriers are a direct result of hepatoma (R. P. Beasley, University of Washington, Taipei). A similar relationship exists between primary liver cancer of woodchucks and a virus related to hepatitis B, the woodchuck hepatitis virus. It has been well documented in this animal model system that viral DNA can be found integrated into host cell DNA, a finding similar to that obtained with cultured human hepatoma cell lines probed with hepatitis B cDNA. A unique and distinct pattern of viral DNA sequences has been found in various human tumours of either monoclonal or polyclonal origin, characterized as multiple rearrangements, duplications, inversions and deletions of specific sequences. In contrast, chronic infection in the absence of tumours seems to be associated with colinear sequences of viral genes with perhaps a few deletions (D. A. Shafritz, Albert Einstein College of Medicine, New York). Hybridization probes may also be Used as an index of active replication in carriers of hepatitis B. In collaboration with Professor S. J. Hadziyannis (Hippokration General Hospital, Athens) the New York group reported that up to 50% of patients with antibody to the e antigen, a marker normally associated with low or negligible viraemia, could be found to contain viral DNA in the serum and this was associated with active expression of the internal core antigen (HBcAg) in the liver of these individuals. The remaining patients showed an absence of DNA in serum and no HBcAg expression in hepatocytes but clustering of cells in the liver positive for HBsAg. The hypothesis was presented that these two groups represented two ends of a spectrum of viral disease in chronically infected individuals: some patients actively express all antigens associated With ongoing virus replication accompanied by active cellular immune responses directed against infected hepatocytes. The other extreme is a non-active form with integration of viral DNA into high
Immunology Today, vol. 5, No. 7, 1984
molecular weight cellular sequences which may subsequently become rearranged over a number of years together with host sequences under the influence of other mutagenic factors prior to cellular transformation. Selection of clones containing integration at specific sites then occurs to produce clonal growth and neoplasia which escape normal surveillance mechanisms. The increasing availability of monoclonal antibodies to HBsAg has permitted several studies designed to detect antigen in immune complexes (J. R. Wands, Massachussetts General Hospital, Boston; S. E. Brown, London School of Hygiene and Tropical Medicine, London). This has led to a re-evaluation of the possible role of hepatitis B virus in primary liver cancer patients negative for HBsAg but positive for antibody. In a study of such cases in West Africa where hepatitis B infection commonly occurs in childhood, the majority of anti-HBs sera contain antigen detectable by monoclonal antibody in immune complexes preferentially bound by Clq (S. E. Brown, London School of Hygiene and Tropical Medicine, London). This provides further evidence of the link between hepatitis B and hepatoma, and suggests very low expression of viral gene products in the presence of antibody, presumably over a period of many years subsequent to the initial hepatitis B infection. It is widely accepted that liver damage accompanying hepatitis virus infection is immunologicaUy mediated, although the role of specific effector cells, e.g. cytotoxic T-cells, is still very unclear. Evidence involving cellular cytotoxicity is fragmentary, largely due to the problems inherent in producing suitable target cells in vitro bearing both viral and the appropriate M H C antigens. A theoretical model to overcome these limitations would be to take peripheral B-cells and transfect with hepatitis B-specific gene sequences in order to obtain sufficient numbers of target cells expressing both viral determinants and class I molecules at the plasma membrane. Syngeneic T-cells could then be prepared either from the peripheral population or by liver biopsy and expanded using antibodies to T3 in the presence of IL2 (J. D. Stobo, University of California, San Francisco). To date, no data has yet been obtained from the application of these techniques although several aspects of antigen expression on the surface of infected hepatocytes have been examined. For example, it is known that the general density of class I M H C antigen increases during interferon treatment of chronic hepatitis although experiments using hepatoma
cell lines and monoclonal antibodies do not show co-migration of host and HBsAg at the cell surface (H. C. Thomas, Royal Free Hospital, London). One important point that is likely to be the subject of further study is the possible expression of core (HBcAg) antigen at the surface of hepatocytes infected with hepatitis B. Thomas noted that IgM antibodies to HBcAg often appeared at the time of increasing liver cell damage and the duration of the response generally correlated with the clinical course of illness. This aspect is likely to receive further attention as work not covered by this symposium is beginning to show that specific recognition of HBcAg at the cell surface by immune T cells may occur (A. W. L. F. Eddleston, King's College Hospital, London) and that core antibodies may even be protective (E. Tabor, Food and Drug Administration, Bethesda).
Hepatitis B vaccines Currently licensed vaccines consist of purified HBsAg particles and have been shown to be both safe and efficacious. Perhaps the most exhaustive study of vaccinated individuals at high risk of acquiring hepatitis B has been conducted by the New York Blood Center among the homosexual population of New York City. The latest results presented by Cladd Stevens showed that among the 92% of men who seroconverted to the vaccine less than 9% had a reduced level of antibody approaching the margin required for complete protection some four to five years after immunization. Natural exposure to the virus in such vaccines has occasionally been manifested by an increase in both protective antibody to HBsAg and antibody to the inner core (HBcAg) component of the virus although there have been neither cases of overt clinical illness nor the appearance of circulating HBsAg. One problem in the use of the vaccine, however, is in immunocompromised patients, e.g. those on maintenance haemodialysis, where seroconversion rates are relatively poor, often below 70 %. Interestingly, much better results can be obtained in this group of patients using relatively impure vaccine preparations (J. Desmyter, Rega Institute, Leuven). Perhaps the greatest challenge is a reduction in the number of chronic carriers by the immunization of children, especially those born to hepatitis B carrier mothers in highly endemic regions, e.g. tropical parts of Africa, Asia and the Far East. The problem is particularly acute in Asia where a high proportion of such mothers have very high levels of virus replication and
Immunology Today, vol. 5, No. 7, 1984
transmission to the infant may occur during or shortly after birth. The use of immunoglobulin containing high levels of antibody to HBsAg merely delays the onset of disease in this group of individuals who usually stand a better than 80% chance of acquiring chronic hepatitis B. However, the combined use ofimmunoglobulin and vaccine offers an opportunity to develop the infant's own immunity whilst passively protected. A muhicentre study conducted among Asian immigrants in the USA showed that there may have been failure of the vaccine (subtype ad) to protect newborn infants against exposure to virus coated with HBsAg of the heterologous subtype ay (C. Stevens, New York Blood Center, New York). If confirmed, this is contrary to previously reported studies in vaccinated adults in whom good protection against a heterologous subtype of hepatitis B virus has been observed. A dearer understanding of human antibody responses to HBsAg particle vaccines is dearly needed in order to identify the epitopes responsible for the induction of a protective antibody response. A new approach to this was presented by C. R. Howard and colleagues (London School of Hygiene and Tropical Medicine, London) who found a synthetic peptide representing a nine amino acid length of the naturally occurring primary gene product of the surface gene bound a major proportion of human antibodies specific for HBsAg in high titre pooled immunoglobulin. Additionally, all seroconversions in vaceinees were associated with reactivity to this same peptide, although measurements of antibody affinity showed that cydization of this peptide was a prerequisite for maximum binding, indicating the importance of protein conformation in this antigen-antibody system. This type of analysis offers an opportunity to ask questions in more general terms as to the possible role of antibody affinity to selected epitopes in determining the outcome of hepatitis B infection. Of interest was the preliminary findings that affinity values of antibody in the sera of antibody-positive HBsAg negative chronic liver disease patients were significantly lower than those observed in sera from other individuals. An alternative approach to understanding immune responses to hepatitis B vaccines is the use of antiidiotype antibodies that recognize a common idiotype present on human antibodies to HBsAg present in the sera of both immunized individuals and others naturally exposed to the virus (R. Kennedy, Baylor College of Medicine, Houston). The idiotype-anti-idiotype reaction was inhibited by a synthetic
187 peptide minimizing part of the HBsAg major polypeptide, and the rabbit idiotype was capable of priming the immune response in mice both to this peptide and the naturally occurring antigen. This may prove useful for examining in detail why certain individuals, notably the immunosuppressed, respond poorly or not at all to the conventional hepatitis B vaccine. It is widely recognized that there is an urgent requirement to develop further so-called 'second generation' vaccine materials that would avoid both the problem of an adequate supply of plasma from suitable hepatitis B carriers and also the attendent problem of biological safety inherent in the use of plasma from individuals at high risk of exposure to other unrelated or hitherto unrecognized agents. Perhaps the most immediate new source of vaccine material is the expression of HBsAg in S. cerevisiae transfected with suitable plasmids carrying the surface gene sequence. The yields of yeast-derived antigen have been improved over recent years, and the meetingheard the first results of a limited trial in volunteers who received the new vaccine (F. Deinhardt, Max v Pettenkofer Institute, Munich). At the present time, there appears to be no evidence of undesirable side reactions directed against contaminating antigen of yeast origin, although further trials will be required to exhaustively eliminate this possibility. This yeast-derived vaccine will offer the first real possibility to seriously contemplate vaccination on a much wider scale, particularly in areas endemic for hepatitis B, as the use of currently available vaccines is essentially restricted to those at high risk of infection.
Hepatitis B-associated delta agent The delta agent, first described by M. Rizetto and colleagues (Molinette Hospital, Turin), is a distinct agent from hepatitis B but requires the presence of the hepatitis B virus for its replication, acquiring the outer HBsAg-reactive coat on release from infected hepatocytes. This requirement for a helper function is not restricted to hepatitis B, as the delta agent can replicate in woodchucks simultaneously infected by the related hepatitis virus of this rodent (J. L. Gerin, Georgetown University, Rockville and R. H. Purcell, NIAID, Bethesda). Delta infection in man results in liver cell damage distinct from hepatitis B, although simultaneous infection with both agents results in a hepatitis limited in duration to the normal course of acute hepatitis B infection. Occasionally, however, serum or fulminant hepatitis results with release of delta antigen into
the circulation. In contrast, superinfection of individuals already persistently infected with hepatitis B commonly results in chronic hepatitis and they may also become carriers of the second agent. This exacerbation of clinical disease has been recently seen in several outbreaks of severe liver disease among isolated populations in both Veneuzuela (S. C. Hadler, Centers for Disease Control, Atlanta) and Columbia (K. Ljunggren, University Hospital, Lund). There is good reason to believe that the use of a cheap and effective hepatitis B vaccine would also protect against delta infection. There is evidence that batches of normal immunoglobulin prepared in the USA prior to the routine screening of blood donors for markers of hepatitis B in 1973 contained antibody to delta and the titre closely correlated with the titre of antibody to the hepatitis B core component (A. Ponzetto, Molinette Hospital, Turin). This indicates that deha infections are not necessarily a recent introductions into the hepatitis B carrier population of North America. At the present time, it is estimated that less than 4% of hepatitis B carriers in the USA have delta antibody, although this rises to over 50% in carriers with a history of drug abuse (N. Nath, American Red Cross, Bethesda and I. K. Mushahwar, Abbott Laboratories, North Chicago). Although little is known of the nature of the dependence on hepatitis B replication, hepadnavirus DNA synthesis and expression was reported to be depressed in patients with delta-positive chronic active hepatitis (Hadziyannis, Hippokration General Hospital, Athens and D. A. Shafritz, Albert Einstein College of Medicine, New York). Attempts to clone the small 550 000 mol wt. R N A molecule found associated with the delta agent were reported at this meeting (A. Smedile, Molinette Hospital, Turin), and eDNA probes should prove useful in elucidating the nature of the delta antigen. The presence of delta agent in the serum of patients with chronic liver disease was successfully demonstrated using blot hybridization techniques at an enhanced level of sensitivity compared with serological methods.
Non-A, non-B hepatitis Little progress has been made in defining the agents of so-called non-A, non-B hepatitis, that is cases of acute or chronic infection that are serologically negative for markers of hepatitis A and B. This group of infections now accounts for the majority of post- transfusion hepatitis, and it has been estimated that 1-2 % of the population in the USA have one of these agents over half with no clinical
188
Immunology Today, vol. 5, No. 7, 1984
symptoms (J. H. Hoofnagle, NIADDK, Bethesda). Of considerable concern is the high frequency of persistence of liver disease in patients with chronic non-A, non-B hepatitis. In one stud),, 68 % of cases showed continuing signs of liver damage one year after onset of disease many with chronic active hepatitis progressing to cirrhosis (H. J. Alter, Clinical Center, Bethesda). All attempts to define a serological marker for non-A, non-B hepatitis have proved unsuccessful; despite over 30 reports in the literature identifying specific antigens, multicentre studies co-ordinated by H. Alter of the Clinical Center, National Institute of Health have shown conclusivety that none of these are specific. A
collaborative study among various groups at N I H have found that a rheumatoid fhctor-like IgM is frequently present in acute phase, chronic non-A, non-B sera which reacts with antibody in various categories of"multiply transfused individuals. This IgM molecule is not demonstrated by" conventional rheumatoid factor tests but is adsorbed by aggregated IgG and is destroyed on reduction (S. M. Feinstone, NIAID, Bethesda), and this may account for the claims from various laboratories that they have developed a test for a specific antigen. Certainly one of the major difficulties in the study of non-A, non-B hepatitis is the apparently low titre of infectivity in most units of contaminated blood. Although
The T-cell antigen receptor
Paradigm regained from Jonathan Howard About 18 months ago, Jensenius and Williams reviewed the state of knowledge about the T-cell receptor, and summarized their views as follows: 'It has been dogma that the antigen receptors on T lymphocytes must have recognition sites that are essentially the same as those of antibodies and that the receptors would be found if structures related to immunoglobulin (Ig) V-domains could be identified on T lymphocytes. In a critical assessment we conclude that there is no sound basis for these views.'1. The extraordinary torrent of information that has been pouring out of North America since then has shown how very completely wrong Jensenius and Williams were. It is true that the methods used to solve the problem were novel. The solution turns out to be so Ig-like that some of my colleagues have declared themselves to be disappointed. The culmination of this process of restoring the immunoglobulin paradigm to its place comes with the recent publication of the complete primary sequence of both the alpha and beta chains of the putative receptor of a mouse cytotoxic T-cell clone 2. Before discussing the implications of this latest advance, let us recapitulate briefly the chronology of this amazing story. At the end of 1982James Allison and colleagues at the University of Texas, Smithville, prepared a monoclonal antibody against a mouse T ceil lymphoma membrane marker. The antigenic determinant was 'idiotypic' in the sense that it was borne only by the immunizing lymphoma and not by other superficially similar cell lines: a tumour-specific antigen, in other words. The molecule identified on the © 1984, Elsevier Science Publishers B.V,, Amsterdam
cell surface was a disulphide-bridged heterodimer, the two chains both having apparent relati;ce molecular weights on SDS gels in the region of 40 000. Because of the disulphide bridge, the molecule ran off the diagonal on two-dimensional SDS gels in which the first dimension was unreduced, and the second reduced. Allison and his colleagues were able to show that a molecule with this characteristic property was also carried on normal T cells, and serological crossreactivity betwen the lymphoma molecule and the normal counterpart was demonstrated by means of a rabbit antiserum raised against immunoprecipitated lymphoma material a. The possibility that this molecule might be related to the T cell receptor was explicit in the first paper from this group 4. Two other groups were also making antiidiotypic antibodies against T cells, but now against either antigen-specific mouse T cell hybrids or against antigenspecific human T cell clones. Monoctonal antibodies were developed which interfered with specific T cell function, and showed idiotypic specificity. In both cases the molecules identified on the cell surface were also disulphide-bridged heterodimers with two chains of around 40 000 relative molecular weight 5'6. The intimate association between this molecule and specific antigen recognition by T cells was shown by the important discovery that the idiotypic specificity could predict the fine specificity of both antigen and M H C recognition 7. Work at the protein level was rapidly foUowed by eDNA cloning. Mark Davis and colleagues s'9 looked for T cell
0167 - 4919/841rd)2,00
chimpanzee transmission studies clearly show that there are at least two distinct agents involved, no definite isolation of virus has been reported. At least one form may be an enveloped virus judging by its sensitivity to lipid solvents with a diameter less than 80 nm (D. A. Bradley, Center for Disease Control, Atlanta), although studies of faecal material collected from a recent epidemic non-A, non-B in Nepal suggests that a hepatitis A virus-like particle 27 nm in diameter may also be implicated (M. A. Kane, Centers for Disease Control, Atlanta).
C. R. Itoward is in the School of Hygiene and Tropical Medicine, London W1, UK.
specific messages whose genomic sequences were rearranged in mature T cells. For this elegant approach to work it was necessary both that the T-cell receptor message should not hybridize significantly with Ig message, and that the T-cell receptor genes should rearrange during T-cell ontogeny in a manner for which only immunoglobulin genes provide a precedent. In other words, this experiment assumed the falsity of the immunoglobulin paradigm for the purpose of sequence, while taking the same paradigm for granted for the purpose of rearrangement. In the event this schizophrenic approach was rewarded and the first sequence of a mouse T cell receptor beta chain was obtained. It was immediately apparent how very' immunoglobulin-like this sequence was in overall organization 9. Not only was the molecule built from two disulphide-bridged domains of exactly the same size and general structm-e as Ig domains, but there was strong amino acid sequence homology at several critical sites adjacent to the intra chain bridges. It was also clear that the N-terminal domain was variable, and the C-terminal constant; there was evidence for independent J and more recenfly D segments between the two domains. To an extraordinary degree the beta chain of the mouse T cell receptor resembled the first two domains of an immunoglobulin chain, with the addition of a hydrophobic transmembrane segment and a short intracytoplasmic tail. The m R N A for the/3chain of a human T-cell receptor was isolated from a T-cell line and identified by sequence homology with Ig1°. More recendy the disposition of V, J and C genes of the mouse and human T cell receptor beta chain have been investigated in the germ-line configuration. An upstream V family is followed at an unknown distance by a tandem pair of
most other soft tissues and usually presents between 40 and 60 years with an equal sex distribution. Before the advent of chemotherapy it was fatal within a year in 80 % of patients. Kornblut postulated that it was caused by a tissue hypersensitivity reaction which followed an upper respiratory tract infection. There is no diagnostic test. Survival has improved dramatically with the use of
185 cyclophosphamide, azathioprine and adjuvant steroids. N. Rasmussen (Copenhagen) proposed that Wegner's should be classified as an autoimmune disease. Certain HLA antigens, B7, B8, DR2 and DR3, were abnormally frequent among patients. Over half the patients have one or more autoantibodies, most frequently to smooth muscle and rheumatoid
Viral hepatitis: diagnosis, treatment and prevention from C. R. Howard With over 200 million people chronically infected, hepatitis B remains the most serious persistent virus infection of man. The recent availability of a vaccine offers an opportunity to control, and eventually reduce these numbers. Although the prime consideration is the protection of those at risk of acquiring infection from such individuals, a reduction in the numbers of carriers will ultimately reduce the large numbers of people in areas of high endemicity who are predisposed to developing primary liver cancer later in life as a result of previous exposure to the virus. By contrast, hepatitis A is never associated with chronic liver infection but its ease of transmission by the oral route presents a major health problem in endemic areas and to those travelling to regions of the world with poor public sanitation. Hitherto unrecognized agents, collectively termed 'non-A, non-B viruses', now account for the majority of posttransfusion hepatitis cases since the introduction of routine tests for the screening of blood for markers of hepatitis B. M a n y of these cases become chronic infections with severe consequences. Additional epidemic forms of non-A, non-B together with the hepatitis B-associated delta agent complete the list of viruses discussed during the symposium*.
Hepatitis A virus Viral hepatitis type A appears to be declining in Europe and North America, most cases being limited to travellers returning from endemic areas or individuals exposed to contaminated shellfish or inadequate sanitation. Notwithstanding, interest continues to abound in the properties of this virus. R. H. Purcell (Bethesda) reviewed the present state of knowlege, reiterating *The 1984 International Symposium on Viral Hepatitis, organized by the University of California at San Francisco, 8-10 M a r c h 1984.
that, unlike hepatitis B, the viraemia is brief and at a low level. In contrast, the titre excreted in the stools early during the acute phase may be as high as 109 infectious particles per ml of extract. There also appears to be a direct relationship between incubation period of the disease and the titre of virus in the inoculum. Although structurally resembling the picornaviruses, the growth properties of the virus in tissue culture differs markedly from, say, those observed with poliovirus. In particular, the replicative cycle of hepatitis A virus is long in duration, extending over several weeks with fresh isolates. In addition, mature virus particles remain largely cell-associated, thus requiring physical methods for release and further passage in fresh cells. Attempts to attenuate the virus sufficiently for use as a h u m a n vaccine were summarized (R. Daemer, Bethesda; P. Provost, West Point). The group at the National Institute of Health have followed the interesting possibility of attenuation by continually passaging early harvest material, thereby decreasing the time required for a full cycle of replication. Some evidence of attenuation for susceptible chimpanzees was found after 20 passages.
Hepatitis B virus The hepatitis B virus genome consists of a partial double-stranded DNA structure with four open reading frames of any significant size. Two of these correspond to the genes coding for the major surface (S) polypeptide of mol. wt 25 000 and the internal core component of tool. wt 19 000 respectively. The remaining genes are believed to code for the virus-specific DNA polymerase and a fourth protein designated ' X ' . Evidence was presented that the latter peptide may be present in monkey kidney cells either transfected with recombinant viral DNA or in extracts of cultured hepatoma cells (A. Moriaty, La Jolla). Transcription occurs primarily from two promoters but
factor. Analysing the serological reactions Rasmussen noted that 9 of 12 patients had antibodies to normal granulocytes and monocytes but not lymphocytes. This was not found in 22 000 other sera. The antibodies disappeared during remission. IT] Adrian Drake-Lee is in the Royal National Throat, Nose and Ear Hospital, London.
initiation of R N A polymerase activity may occur at up to eight sites (W. J. Rutter, University of California, San Francisco). Analysis of the region coding for the surface polypeptide which contains various antigenic reactivities (HBsAg) showed that transcription may begin upstream at two separate sites to give larger proteins of tool. wts 71 000 and 45 000 respectively. Direct evidence for the presence of these larger forms in 22 n m HBsAg particles purified from serum was shown by W. H. Gerlich (University of Goettingen) and both may contain the additional amino acid sequence representing a receptor site for polymerized albumin on the virus particle, suggesting that a possible 'carrier' protein may be responsible for mediating the attachment of virus to new host cells. Indeed the relative amounts of these minor components appeared enhanced in sera containing large amounts of hepatitis B virus. Comparison of individual DNA clones suggests that there is extensive variation in predicted amino acid sequence for this peptide region, possibly indicative of multiple infection in the individual from whom the virus was originally isolated. However, A. R. Neurath (New York Blood Center) pointed out that selected domains of the major 25 000 tool. wt HBsAg peptide appear to be distantly related to amino acid sequences found in native human albumin, offering a possible explanation of the close association between this host protein and virus-specific molecules. Expression of the surface gene is enhanced up to 100-fold by a sequence between the S and X genes (W. J. Rutter, University of California, San Francisco). Although expression of various hepatitis B gene products may be achieved by transfeetion of cell lines with cloned viral DNA, efforts to demonstrate directly in vitro the presence of virus in serum have been notoriously unsuccessful. However, the co-cultivation of adult hepatocytes with a rat epithelial liver cell line may now provide a suitable tissue culture system for studying all stages of virus replication and provide suitable targets for cellular immunity studies. N. Theze and colleagues (Htpital Pontchaillou,
© 1984, ElsevierSciencePubfishemB.V., Amsterdam 0167 4919/84/$02.00
186 Paris) reported that full expression of viral DNA and antigen may be obtained using hepatocytes obtained by liver perfusion of kidney donors and that such cells maintained at least some differentiated functions up to six weeks after culture. Viral HBsAg and complete virions were seen and genome copies detected by hybridization from 5 to 16 days after infection. If confirmed, this would represent a significant breakthrough in our understanding of the virology of hepatitis B. There is now a clear relationship between hepatocelhilar carcinoma and hepatitis B infection. In Taiwan, for example, it is estimated that 40% of deaths in hepatitis B carriers are a direct result of hepatoma (R. P. Beasley, University of Washington, Taipei). A similar relationship exists between primary liver cancer of woodchucks and a virus related to hepatitis B, the woodchuck hepatitis virus. It has been well documented in this animal model system that viral DNA can be found integrated into host cell DNA, a finding similar to that obtained with cultured human hepatoma cell lines probed with hepatitis B cDNA. A unique and distinct pattern of viral DNA sequences has been found in various human tumours of either monoclonal or polyclonal origin, characterized as multiple rearrangements, duplications, inversions and deletions of specific sequences. In contrast, chronic infection in the absence of tumours seems to be associated with colinear sequences of viral genes with perhaps a few deletions (D. A. Shafritz, Albert Einstein College of Medicine, New York). Hybridization probes may also be Used as an index of active replication in carriers of hepatitis B. In collaboration with Professor S. J. Hadziyannis (Hippokration General Hospital, Athens) the New York group reported that up to 50% of patients with antibody to the e antigen, a marker normally associated with low or negligible viraemia, could be found to contain viral DNA in the serum and this was associated with active expression of the internal core antigen (HBcAg) in the liver of these individuals. The remaining patients showed an absence of DNA in serum and no HBcAg expression in hepatocytes but clustering of cells in the liver positive for HBsAg. The hypothesis was presented that these two groups represented two ends of a spectrum of viral disease in chronically infected individuals: some patients actively express all antigens associated With ongoing virus replication accompanied by active cellular immune responses directed against infected hepatocytes. The other extreme is a non-active form with integration of viral DNA into high
Immunology Today, vol. 5, No. 7, 1984
molecular weight cellular sequences which may subsequently become rearranged over a number of years together with host sequences under the influence of other mutagenic factors prior to cellular transformation. Selection of clones containing integration at specific sites then occurs to produce clonal growth and neoplasia which escape normal surveillance mechanisms. The increasing availability of monoclonal antibodies to HBsAg has permitted several studies designed to detect antigen in immune complexes (J. R. Wands, Massachussetts General Hospital, Boston; S. E. Brown, London School of Hygiene and Tropical Medicine, London). This has led to a re-evaluation of the possible role of hepatitis B virus in primary liver cancer patients negative for HBsAg but positive for antibody. In a study of such cases in West Africa where hepatitis B infection commonly occurs in childhood, the majority of anti-HBs sera contain antigen detectable by monoclonal antibody in immune complexes preferentially bound by Clq (S. E. Brown, London School of Hygiene and Tropical Medicine, London). This provides further evidence of the link between hepatitis B and hepatoma, and suggests very low expression of viral gene products in the presence of antibody, presumably over a period of many years subsequent to the initial hepatitis B infection. It is widely accepted that liver damage accompanying hepatitis virus infection is immunologicaUy mediated, although the role of specific effector cells, e.g. cytotoxic T-cells, is still very unclear. Evidence involving cellular cytotoxicity is fragmentary, largely due to the problems inherent in producing suitable target cells in vitro bearing both viral and the appropriate M H C antigens. A theoretical model to overcome these limitations would be to take peripheral B-cells and transfect with hepatitis B-specific gene sequences in order to obtain sufficient numbers of target cells expressing both viral determinants and class I molecules at the plasma membrane. Syngeneic T-cells could then be prepared either from the peripheral population or by liver biopsy and expanded using antibodies to T3 in the presence of IL2 (J. D. Stobo, University of California, San Francisco). To date, no data has yet been obtained from the application of these techniques although several aspects of antigen expression on the surface of infected hepatocytes have been examined. For example, it is known that the general density of class I M H C antigen increases during interferon treatment of chronic hepatitis although experiments using hepatoma
cell lines and monoclonal antibodies do not show co-migration of host and HBsAg at the cell surface (H. C. Thomas, Royal Free Hospital, London). One important point that is likely to be the subject of further study is the possible expression of core (HBcAg) antigen at the surface of hepatocytes infected with hepatitis B. Thomas noted that IgM antibodies to HBcAg often appeared at the time of increasing liver cell damage and the duration of the response generally correlated with the clinical course of illness. This aspect is likely to receive further attention as work not covered by this symposium is beginning to show that specific recognition of HBcAg at the cell surface by immune T cells may occur (A. W. L. F. Eddleston, King's College Hospital, London) and that core antibodies may even be protective (E. Tabor, Food and Drug Administration, Bethesda).
Hepatitis B vaccines Currently licensed vaccines consist of purified HBsAg particles and have been shown to be both safe and efficacious. Perhaps the most exhaustive study of vaccinated individuals at high risk of acquiring hepatitis B has been conducted by the New York Blood Center among the homosexual population of New York City. The latest results presented by Cladd Stevens showed that among the 92% of men who seroconverted to the vaccine less than 9% had a reduced level of antibody approaching the margin required for complete protection some four to five years after immunization. Natural exposure to the virus in such vaccines has occasionally been manifested by an increase in both protective antibody to HBsAg and antibody to the inner core (HBcAg) component of the virus although there have been neither cases of overt clinical illness nor the appearance of circulating HBsAg. One problem in the use of the vaccine, however, is in immunocompromised patients, e.g. those on maintenance haemodialysis, where seroconversion rates are relatively poor, often below 70 %. Interestingly, much better results can be obtained in this group of patients using relatively impure vaccine preparations (J. Desmyter, Rega Institute, Leuven). Perhaps the greatest challenge is a reduction in the number of chronic carriers by the immunization of children, especially those born to hepatitis B carrier mothers in highly endemic regions, e.g. tropical parts of Africa, Asia and the Far East. The problem is particularly acute in Asia where a high proportion of such mothers have very high levels of virus replication and
Immunology Today, vol. 5, No. 7, 1984
transmission to the infant may occur during or shortly after birth. The use of immunoglobulin containing high levels of antibody to HBsAg merely delays the onset of disease in this group of individuals who usually stand a better than 80% chance of acquiring chronic hepatitis B. However, the combined use ofimmunoglobulin and vaccine offers an opportunity to develop the infant's own immunity whilst passively protected. A muhicentre study conducted among Asian immigrants in the USA showed that there may have been failure of the vaccine (subtype ad) to protect newborn infants against exposure to virus coated with HBsAg of the heterologous subtype ay (C. Stevens, New York Blood Center, New York). If confirmed, this is contrary to previously reported studies in vaccinated adults in whom good protection against a heterologous subtype of hepatitis B virus has been observed. A dearer understanding of human antibody responses to HBsAg particle vaccines is dearly needed in order to identify the epitopes responsible for the induction of a protective antibody response. A new approach to this was presented by C. R. Howard and colleagues (London School of Hygiene and Tropical Medicine, London) who found a synthetic peptide representing a nine amino acid length of the naturally occurring primary gene product of the surface gene bound a major proportion of human antibodies specific for HBsAg in high titre pooled immunoglobulin. Additionally, all seroconversions in vaceinees were associated with reactivity to this same peptide, although measurements of antibody affinity showed that cydization of this peptide was a prerequisite for maximum binding, indicating the importance of protein conformation in this antigen-antibody system. This type of analysis offers an opportunity to ask questions in more general terms as to the possible role of antibody affinity to selected epitopes in determining the outcome of hepatitis B infection. Of interest was the preliminary findings that affinity values of antibody in the sera of antibody-positive HBsAg negative chronic liver disease patients were significantly lower than those observed in sera from other individuals. An alternative approach to understanding immune responses to hepatitis B vaccines is the use of antiidiotype antibodies that recognize a common idiotype present on human antibodies to HBsAg present in the sera of both immunized individuals and others naturally exposed to the virus (R. Kennedy, Baylor College of Medicine, Houston). The idiotype-anti-idiotype reaction was inhibited by a synthetic
187 peptide minimizing part of the HBsAg major polypeptide, and the rabbit idiotype was capable of priming the immune response in mice both to this peptide and the naturally occurring antigen. This may prove useful for examining in detail why certain individuals, notably the immunosuppressed, respond poorly or not at all to the conventional hepatitis B vaccine. It is widely recognized that there is an urgent requirement to develop further so-called 'second generation' vaccine materials that would avoid both the problem of an adequate supply of plasma from suitable hepatitis B carriers and also the attendent problem of biological safety inherent in the use of plasma from individuals at high risk of exposure to other unrelated or hitherto unrecognized agents. Perhaps the most immediate new source of vaccine material is the expression of HBsAg in S. cerevisiae transfected with suitable plasmids carrying the surface gene sequence. The yields of yeast-derived antigen have been improved over recent years, and the meetingheard the first results of a limited trial in volunteers who received the new vaccine (F. Deinhardt, Max v Pettenkofer Institute, Munich). At the present time, there appears to be no evidence of undesirable side reactions directed against contaminating antigen of yeast origin, although further trials will be required to exhaustively eliminate this possibility. This yeast-derived vaccine will offer the first real possibility to seriously contemplate vaccination on a much wider scale, particularly in areas endemic for hepatitis B, as the use of currently available vaccines is essentially restricted to those at high risk of infection.
Hepatitis B-associated delta agent The delta agent, first described by M. Rizetto and colleagues (Molinette Hospital, Turin), is a distinct agent from hepatitis B but requires the presence of the hepatitis B virus for its replication, acquiring the outer HBsAg-reactive coat on release from infected hepatocytes. This requirement for a helper function is not restricted to hepatitis B, as the delta agent can replicate in woodchucks simultaneously infected by the related hepatitis virus of this rodent (J. L. Gerin, Georgetown University, Rockville and R. H. Purcell, NIAID, Bethesda). Delta infection in man results in liver cell damage distinct from hepatitis B, although simultaneous infection with both agents results in a hepatitis limited in duration to the normal course of acute hepatitis B infection. Occasionally, however, serum or fulminant hepatitis results with release of delta antigen into
the circulation. In contrast, superinfection of individuals already persistently infected with hepatitis B commonly results in chronic hepatitis and they may also become carriers of the second agent. This exacerbation of clinical disease has been recently seen in several outbreaks of severe liver disease among isolated populations in both Veneuzuela (S. C. Hadler, Centers for Disease Control, Atlanta) and Columbia (K. Ljunggren, University Hospital, Lund). There is good reason to believe that the use of a cheap and effective hepatitis B vaccine would also protect against delta infection. There is evidence that batches of normal immunoglobulin prepared in the USA prior to the routine screening of blood donors for markers of hepatitis B in 1973 contained antibody to delta and the titre closely correlated with the titre of antibody to the hepatitis B core component (A. Ponzetto, Molinette Hospital, Turin). This indicates that deha infections are not necessarily a recent introductions into the hepatitis B carrier population of North America. At the present time, it is estimated that less than 4% of hepatitis B carriers in the USA have delta antibody, although this rises to over 50% in carriers with a history of drug abuse (N. Nath, American Red Cross, Bethesda and I. K. Mushahwar, Abbott Laboratories, North Chicago). Although little is known of the nature of the dependence on hepatitis B replication, hepadnavirus DNA synthesis and expression was reported to be depressed in patients with delta-positive chronic active hepatitis (Hadziyannis, Hippokration General Hospital, Athens and D. A. Shafritz, Albert Einstein College of Medicine, New York). Attempts to clone the small 550 000 mol wt. R N A molecule found associated with the delta agent were reported at this meeting (A. Smedile, Molinette Hospital, Turin), and eDNA probes should prove useful in elucidating the nature of the delta antigen. The presence of delta agent in the serum of patients with chronic liver disease was successfully demonstrated using blot hybridization techniques at an enhanced level of sensitivity compared with serological methods.
Non-A, non-B hepatitis Little progress has been made in defining the agents of so-called non-A, non-B hepatitis, that is cases of acute or chronic infection that are serologically negative for markers of hepatitis A and B. This group of infections now accounts for the majority of post- transfusion hepatitis, and it has been estimated that 1-2 % of the population in the USA have one of these agents over half with no clinical
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symptoms (J. H. Hoofnagle, NIADDK, Bethesda). Of considerable concern is the high frequency of persistence of liver disease in patients with chronic non-A, non-B hepatitis. In one stud),, 68 % of cases showed continuing signs of liver damage one year after onset of disease many with chronic active hepatitis progressing to cirrhosis (H. J. Alter, Clinical Center, Bethesda). All attempts to define a serological marker for non-A, non-B hepatitis have proved unsuccessful; despite over 30 reports in the literature identifying specific antigens, multicentre studies co-ordinated by H. Alter of the Clinical Center, National Institute of Health have shown conclusivety that none of these are specific. A
collaborative study among various groups at N I H have found that a rheumatoid fhctor-like IgM is frequently present in acute phase, chronic non-A, non-B sera which reacts with antibody in various categories of"multiply transfused individuals. This IgM molecule is not demonstrated by" conventional rheumatoid factor tests but is adsorbed by aggregated IgG and is destroyed on reduction (S. M. Feinstone, NIAID, Bethesda), and this may account for the claims from various laboratories that they have developed a test for a specific antigen. Certainly one of the major difficulties in the study of non-A, non-B hepatitis is the apparently low titre of infectivity in most units of contaminated blood. Although
The T-cell antigen receptor
Paradigm regained from Jonathan Howard About 18 months ago, Jensenius and Williams reviewed the state of knowledge about the T-cell receptor, and summarized their views as follows: 'It has been dogma that the antigen receptors on T lymphocytes must have recognition sites that are essentially the same as those of antibodies and that the receptors would be found if structures related to immunoglobulin (Ig) V-domains could be identified on T lymphocytes. In a critical assessment we conclude that there is no sound basis for these views.'1. The extraordinary torrent of information that has been pouring out of North America since then has shown how very completely wrong Jensenius and Williams were. It is true that the methods used to solve the problem were novel. The solution turns out to be so Ig-like that some of my colleagues have declared themselves to be disappointed. The culmination of this process of restoring the immunoglobulin paradigm to its place comes with the recent publication of the complete primary sequence of both the alpha and beta chains of the putative receptor of a mouse cytotoxic T-cell clone 2. Before discussing the implications of this latest advance, let us recapitulate briefly the chronology of this amazing story. At the end of 1982James Allison and colleagues at the University of Texas, Smithville, prepared a monoclonal antibody against a mouse T ceil lymphoma membrane marker. The antigenic determinant was 'idiotypic' in the sense that it was borne only by the immunizing lymphoma and not by other superficially similar cell lines: a tumour-specific antigen, in other words. The molecule identified on the © 1984, Elsevier Science Publishers B.V,, Amsterdam
cell surface was a disulphide-bridged heterodimer, the two chains both having apparent relati;ce molecular weights on SDS gels in the region of 40 000. Because of the disulphide bridge, the molecule ran off the diagonal on two-dimensional SDS gels in which the first dimension was unreduced, and the second reduced. Allison and his colleagues were able to show that a molecule with this characteristic property was also carried on normal T cells, and serological crossreactivity betwen the lymphoma molecule and the normal counterpart was demonstrated by means of a rabbit antiserum raised against immunoprecipitated lymphoma material a. The possibility that this molecule might be related to the T cell receptor was explicit in the first paper from this group 4. Two other groups were also making antiidiotypic antibodies against T cells, but now against either antigen-specific mouse T cell hybrids or against antigenspecific human T cell clones. Monoctonal antibodies were developed which interfered with specific T cell function, and showed idiotypic specificity. In both cases the molecules identified on the cell surface were also disulphide-bridged heterodimers with two chains of around 40 000 relative molecular weight 5'6. The intimate association between this molecule and specific antigen recognition by T cells was shown by the important discovery that the idiotypic specificity could predict the fine specificity of both antigen and M H C recognition 7. Work at the protein level was rapidly foUowed by eDNA cloning. Mark Davis and colleagues s'9 looked for T cell
0167 - 4919/841rd)2,00
chimpanzee transmission studies clearly show that there are at least two distinct agents involved, no definite isolation of virus has been reported. At least one form may be an enveloped virus judging by its sensitivity to lipid solvents with a diameter less than 80 nm (D. A. Bradley, Center for Disease Control, Atlanta), although studies of faecal material collected from a recent epidemic non-A, non-B in Nepal suggests that a hepatitis A virus-like particle 27 nm in diameter may also be implicated (M. A. Kane, Centers for Disease Control, Atlanta).
C. R. Itoward is in the School of Hygiene and Tropical Medicine, London W1, UK.
specific messages whose genomic sequences were rearranged in mature T cells. For this elegant approach to work it was necessary both that the T-cell receptor message should not hybridize significantly with Ig message, and that the T-cell receptor genes should rearrange during T-cell ontogeny in a manner for which only immunoglobulin genes provide a precedent. In other words, this experiment assumed the falsity of the immunoglobulin paradigm for the purpose of sequence, while taking the same paradigm for granted for the purpose of rearrangement. In the event this schizophrenic approach was rewarded and the first sequence of a mouse T cell receptor beta chain was obtained. It was immediately apparent how very' immunoglobulin-like this sequence was in overall organization 9. Not only was the molecule built from two disulphide-bridged domains of exactly the same size and general structm-e as Ig domains, but there was strong amino acid sequence homology at several critical sites adjacent to the intra chain bridges. It was also clear that the N-terminal domain was variable, and the C-terminal constant; there was evidence for independent J and more recenfly D segments between the two domains. To an extraordinary degree the beta chain of the mouse T cell receptor resembled the first two domains of an immunoglobulin chain, with the addition of a hydrophobic transmembrane segment and a short intracytoplasmic tail. The m R N A for the/3chain of a human T-cell receptor was isolated from a T-cell line and identified by sequence homology with Ig1°. More recendy the disposition of V, J and C genes of the mouse and human T cell receptor beta chain have been investigated in the germ-line configuration. An upstream V family is followed at an unknown distance by a tandem pair of