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British Medical Bulletin 2004 70(1):29-49; doi:10.1093/bmb/ldh024
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Published online 31 August 2004

British Medical Bulletin, Vol. 70 © The British Council 2004; all rights reserved

Hepatitis vaccines

Peter Karayiannis, Janice Main and Howard C. Thomas

Department of Medicine A, Faculty of Medicine, St Mary’s Campus, Imperial College, London, UK

Correspondence to: P. Karayiannis, Department of Medicine A, Faculty of Medicine, Division of Medicine, St Mary’s Campus, Imperial College, London W2 1NY, UK. E-mail: p.karayiannis{at}imperial.ac.uk


   Introduction
Top
 Introduction
Hepatitis A vaccines
 Hepatitis B vaccines
 Hepatitis C vaccines
 Hepatitis D vaccines
 Hepatitis E vaccines
 Conclusions
 References
 
The field of hepatitis virus vaccination continues to undergo constant change. Safe and effective vaccines are now available for the prevention of hepatitis A virus (HAV) and hepatitis B virus (HBV) infections. However, controversies regarding their use in health programmes continue, and the emergence of HBV vaccine escape mutants continues to provide challenges for vaccine manufacturers. The development of a hepatitis C virus (HCV) vaccine remains a formidable challenge, but there is a more optimistic outlook for the development of a hepatitis E virus (HEV) vaccine.


   Hepatitis A vaccines
Top
 Introduction
 Hepatitis A vaccines
Hepatitis B vaccines
 Hepatitis C vaccines
 Hepatitis D vaccines
 Hepatitis E vaccines
 Conclusions
 References
 
HAV is an enterally transmitted member of the Picornaviridae, and the only member of the hepatovirus genus. The RNA genome of the virus is single stranded, positive sense and about 7.5 kb long. It contains a single open reading frame (ORF) that encodes the viral polyprotein, which is post-translationally cleaved by a viral protease to yield the components of the capsid, and a range of non-structural proteins including the viral RNA polymerase and the protease.

Until the beginning of the 1990s the only prophylaxis available against HAV was human normal immune globulin (HNIG), also known as gamma globulin, derived from pooled blood-donor plasma. This has been used for passive immunization since the 1940s,1 and has been administered to travellers visiting areas of high HAV endemicity and to close contacts in HAV outbreaks.2,3 Its efficacy depends on the prevalence of protective antibodies against HAV (anti-HAV) in the blood-donor population, which has been declining in Western countries in the past 20 years or so. There have been reports of failed prophylaxis, but nevertheless, in the setting of an HAV outbreak, HNIG appears to reduce the clinical severity of an attack even if given too late to prevent infection. The injection can be painful. Administration within 2 weeks of exposure appears to prevent infection and administration after that time can modify the disease severity. HNIG within the United Kingdom is derived from imported plasma in view of the theoretical risk of new variant Creutzfeldt–Jakob disease (vCJD) in UK blood donors. HNIG normally affords 3 months of protection against HAV. Its use is now much more limited because of the development of an effective HAV vaccine, safety concerns and reduced efficacy.

The development of a safe and effective vaccine for HAV, which became available in 1992, has been a major advance.4,5 HAV is grown in human diploid cells and inactivated by treatment with formaldehyde (Havrix and VAQTA). The vaccine in alum is administered intramuscularly in the deltoid region. The initial three-dose immunization schedule given at 0, 1 and 6 months has been more recently replaced by a two-dose regimen, given at 0 and 6–12 months. Studies have shown that the second dose can in fact be given at any time between 24 and 72 months without affecting the immune response.6–9 Development of anti-HAV 1 month after the final dose normally occurs in 100% of vaccinees, whether these are infants (1–2 years),10–12 children and adolescents (2–18 years),13,14 or adults (≥18 years).8,15–19 Geometric mean titres of anti-HAV 1 month after the last dose are high (3802–4133 mIU/ml),8,13,14 but there is a gradual decline over subsequent years to, for example, 661 mIU/ml at 5 years14 and 242 mIU/ml at 12 years.15,20–22 Mathematical modelling studies suggest that antibody may persist for 20–30 years, irrespective of vaccinee group.13,14,21,23 Infants under the age of 1 year with maternal anti-HAV achieve slightly lower seroconversion rates (93–100%) and lower geometric mean titres.24–28 Nevertheless, studies in such infants, as well as adults with booster doses of vaccine 6–12 years after primary vaccination, report a substantial and rapid immune response to HAV antigen, suggesting that protection may be long lasting even when anti-HAV is no longer detectable.7,8,24,29,30 This suggests the existence of a robust recall response and long-lasting immune memory that make booster immunization of immunocompetent individuals unnecessary.31

Formalin-inactivated HAV has also been coupled to immunopotentiating reconstituted influenza virosomes (IRIVs) and evaluated as a potential vaccine. The latter were prepared by combining phosphatidylcholine, phosphatidylethanolamine, phospholipids originating from the influenza virus envelope, influenza virus haemagglutinin and neuraminidase.32 Immunization with this aluminium-free vaccine resulted in 100% seroconversion in infants (6–7 months) and children (5–7 years) who received two doses of the vaccine at 0 and 12 months.33 The geometric mean titres of anti-HAV following immunization with this vaccine appear to be comparable to those obtained with the alum-based vaccine,33 as does the projected duration of protective immunity (>20 years).34

The hepatitis A vaccine is recommended for travellers to areas of high HAV endemicity, and should be considered in food handlers, homosexual men and sewage workers. In addition, individuals with chronic liver disease and those who are immunocompromised should be vaccinated, as the chance of severe disease in these patients following exposure to HAV is increased. In some countries, the vaccine has been incorporated in childhood vaccine programmes. Different formulations have been developed including combination vaccines with HBV (discussed later) or typhoid.35 The vaccine is generally well tolerated, with occasional reports of mild local reactions or, more rarely, fever and malaise.

Data regarding the use of HAV vaccine in outbreaks are limited.36 Public health and hygiene measures are of vital importance in controlling an outbreak. HNIG offers some degree of immediate protection for selected individuals. HAV vaccines take several days to establish immunity, which is then long term. It has been suggested that HAV vaccination can prevent infection if given within a week of exposure, but it is recognized that there are often delays in diagnosis of the primary case. There are also concerns that those who have been successfully vaccinated may nevertheless become infected and although they may demonstrate no symptoms or signs of infection, they may still excrete infectious virus.37


   Hepatitis B vaccines
Top
 Introduction
 Hepatitis A vaccines
 Hepatitis B vaccines
Hepatitis C vaccines
 Hepatitis D vaccines
 Hepatitis E vaccines
 Conclusions
 References
 
HBV is a member of the Hepadnaviridae and is the smallest DNA virus to infect humans. Its compact genome is 3.2 kb long, partially double stranded and circular. There are four partially or totally overlapping ORFs. The surface antigen (HBsAg) ORF encodes for three envelope proteins, which are co-terminal and bear the ‘a’ antigenic determinant, antibodies against which are virus neutralizing (anti-HBs). The precore/core ORF encodes two proteins: the precore/core protein, which is the precursor for the soluble ‘e’ antigen (HBeAg), and the nucleocapsid antigen (HBcAg) or core protein. The other two ORFs encode for the x protein (HBxAg) and the DNA polymerase/reverse transcriptase. As a humoral immune response to HBsAg confers protection against infection, HBsAg has been used as an immunogen in vaccine development.

HBV is transmitted parenterally and through sexual contact, causing both acute and chronic hepatitis. Infants born to HBV carrier mothers are at increased risk of becoming infected perinatally, as are siblings and peers of carrier children through horizontal transmission. The risk of chronicity varies according to the age at the time of infection, with rates of 90–95% in those infected perinatally38 and 5% in adults. Host genetic factors appear important in determining outcome following infection.39 Higher rates of chronicity are seen in those with immunosuppression, including HIV infection. Acute hepatitis B can range from a mild or subclinical infection to a life-threatening disease with fulminant hepatitis. Chronic infection can lead eventually to cirrhosis with risks of liver failure or the development of hepatocellular carcinoma (HCC) in up to 30% of cases. Prophylactic vaccination offers the means of interrupting the transmission of the virus, reducing the pool of infected individuals and preventing the long-term sequelae of chronic liver disease. Although therapeutic vaccination of chronic carriers has also been attempted, this has been largely unsuccessful so far and will not be reviewed here.

The first HBV vaccine, manufactured from HBsAg particles derived from the plasma of chronic HBV carriers, became available in the early 1980s. Subsequent recombinant vaccines were expressed in yeasts such as Saccharomyces cerevisiae (Engerix-B, Recombivax-HB) and, more recently, in Chinese hamster ovary (CHO) cells (Hepacare). In adults, the vaccine is administered intramuscularly into the deltoid at 0, 1 and 6 months. The anterior thigh is recommended for babies. Minor skin reactions have been recorded and there are rarer accounts of systemic upset. Large studies have confirmed that there is no association between the vaccine and the onset or relapse of multiple sclerosis.40,41

The targeting of HBV vaccines within any population varies from country to country. In countries with high seroprevalence universal vaccination programmes have been instituted, and HBV vaccination within 12 h of birth, given together with hepatitis B immune globulin (HBIg), is recommended for infants born to carrier mothers.42 Similar strategies have been developed in some countries where the seroprevalence rates are lower, such as the United States and much of Western Europe, but in the UK and Scandinavia HBV vaccine is restricted to those with a high-risk lifestyle (e.g. homosexual men or intravenous drug users), certain occupational groups, such as health care workers, and patients on dialysis programmes.

The first plasma-derived vaccine was trialled in homosexual men from New York and shown to be highly immunogenic and safe and to have very good protective efficacy.43,44 There has been similar experience with the recombinant vaccines in numerous other studies carried out throughout the world and targeting different groups at risk. For example, more recent data from the United States show that seroprotection levels were achieved in 83–100% and 69–99% of those immunized with Engerix or Recombivax, respectively, depending on the vaccinee group.45 Studies comparing recombinant vaccines suggest that Engerix, which contains more antigen, induces higher antibody levels and response rates than Recombivax.46 The response to the vaccine is determined by measuring anti-HBs levels 1–4 months after the last dose of the vaccine, and the minimum protection level is set at 10 mIU/ml.47 It is suggested that one should aim for levels above 100 mIU/ml when vaccinating health care workers. A number of factors that determine non-response to the vaccine have been identified, including smoking, obesity, male sex and increasing age.46,48 In addition, lower response rates have been noted in renal dialysis and immunosuppressed patients. Certain MHC haplotypes appear to be important in antigen presentation and achieving an immune response to vaccine.49

Initial uncertainty about the duration of vaccine-related immunity is dissipating as long-term follow-up studies are published. A number of such studies, where monitoring continued up to 12 years after vaccination, showed that anti-HBs levels declined over time and that half of the vaccinees had levels below 10 mIU/ml.47 However, it was noted that there were no symptomatic HBV cases (HBsAg negative) among these groups of vaccinees, mainly from endemic areas, although detection of anti-HBc in some indicated infection and increases in anti-HBs indicated an anamnestic response. In a recent study from Hong Kong, protective levels of antibody were still present in 60.4%, 81.4% and 79%, respectively, of three groups of children vaccinated with a two- or three-dose recombinant vaccine, or a three-dose plasma-derived vaccine, when tested 12 years later.50 Experience has been similar in other studies in children51–53 and health care personnel.54 However, a strong anamnestic response is also apparent following booster immunization of individuals with waning or suboptimal antibody levels47,55,56 accompanied by sharp rises in anti-HBs. It is likely that a similar response would occur if the vaccinees were exposed to infectious virus. As a consequence, boosters are not recommended in the United States and their use is being reconsidered in the UK. However, a Gambian study of HBV vaccination given in infancy and early childhood showed that, nevertheless, there were risks of HBV infection in the teenage years particularly if the initial antibody response had been low.57 Therefore there may be an argument for boosters where the risk of exposure is relatively high. Patients with immunodeficiency, such as those with advanced HIV infection,58 those taking immunosuppressants or those on dialysis programmes,59 have a reduced response rate and boosters may also be important in these groups.

The beneficial effects of HBV vaccination are becoming increasingly apparent, particularly in reducing new infections. For example, in Taiwan, with a chronic carrier rate of 15–20%,60 the proportion of babies who become carriers has decreased from 86–96% to 12–14% in those born to highly infectious HBeAg positive carrier mothers, and from 10–12% to 3–4% in those born to anti-HBe positive mothers who are less infectious.61 Fifteen years after the start of the vaccination programme, the prevalence of HBsAg in children aged <15 years had decreased from 9.8% in 1984 to 0.9% in 1999, and in adolescents aged 15–20 years it was 7%.62,63 By 2001, the prevalence in adolescents had decreased even further to 4.7% in males and 3.4% in females.64 The incidence of HCC has also declined as a result of HBV vaccination65,66 from 0.7 per 100 000 children in the period 1981–1986 to 0.57 in the period 1986–1990 and 0.36 in the period 1990–1994. Fulminant hepatic failure in infants has also decreased dramatically in the period since the start of vaccination.61 A recent study reported similar results for children ≥1 year, but fulminant hepatic failure remained a significant problem in infants aged <1 year.67 These infants were more likely to have been born to anti-HBe positive mothers and not to have received HBIg according to the vaccination schedule. This should be borne in mind in view of recent reports that suggest that the omission of HBIg from vaccination schedules does not compromise the immune response.68–70 It seems highly likely that these infants may have been infected with the precore variant of the virus, the transmission of which can result in fulminant liver failure.71,72

New vaccines which incorporate pre-S gene products have been developed and have led to reduced non-response rates.56,73 A triple recombinant vaccine containing pre-S1, pre-S2 and S components has been shown to be immunogenic not only in naive individuals, but also in previous non-responders.74 Attempts to overcome non-response have also included DNA immunization75 and combined vaccine and granulocyte–macrophage colony-stimulating factor.76 Enhanced vaccine immunogenicity has been obtained with other adjuvants such as MF59,77,78 AS0479 and CpG.80

Hepatitis A and B combination vaccines have also been developed (Twinrix, AmBirix, Comvax), and such vaccines could easily be incorporated into vaccination programmes that already use the monovalent HBV vaccine. Existing data suggest that the combined vaccines probably result in immune responses equivalent to those induced by either monovalent vaccine.81–86 However, in a recent study, although all vaccinees were still positive for anti-HAV 24 months post-vaccination, the figures in the same vaccinees for anti-HBs seroprotection levels (>10 mIU/ml) declined from 97.9% and 100% a month after the last vaccine dose to 93.3% and 96.2% 24 months later in the two- and three-dose schedules, respectively.87 Information on the duration of antibody responses following vaccination with a combined vaccine, and whether booster immunization may be necessary, is not comprehensive at the moment.

A vaccine escape mutant (G145R) was detected in 1989 in a child born to an HBV-infected mother.88 The child had become infected despite a satisfactory antibody response. Subsequent studies have detected this and other variants in vaccinees, in patients treated with monoclonal or polyclonal antibodies during liver homografting for end-stage HBV-induced liver disease and also in chronic HBV carriers. Although only small numbers have become infected with these viruses, it is thought that emergence of HBsAg mutants will become more frequent as vaccination programmes are extended in areas where neonatal exposure is common. Indeed, a recent study from Taiwan has shown an increase in the prevalence of HBs variants in children from 7.8% in 1984, before universal vaccination, to 28.1% in 1994, after universal vaccination.89 Therefore there is a need for careful epidemiological surveillance, and vaccine manufacturers are already evaluating future strategies, such as inclusion of the pre-S region or the vaccine escape mutant sequences into the existing HBsAg vaccines.

The use of HBV vaccines has limited the need for HBIg in the routine clinical setting. HBIg is generally administered to babies of HBV carriers (with HBV vaccine in the contralateral side), as already discussed. It is also administered in non-immune individuals who have been exposed to a risk of HBV infection, such as a needlestick injury, where the needle was contaminated with blood from an HBV-infected subject. HBIg, combined with lamivudine, is also administered to HBV-infected patients undergoing liver transplantation for HBV-induced cirrhosis or HCC to limit re-infection of the graft. HBIg is expensive and treatment is required over long periods in many of these patients.


   Hepatitis C vaccines
Top
 Introduction
 Hepatitis A vaccines
 Hepatitis B vaccines
 Hepatitis C vaccines
Hepatitis D vaccines
 Hepatitis E vaccines
 Conclusions
 References
 
The hepatitis C virus (HCV) is an enveloped RNA-containing particle about 60 nm in diameter. The virus has been classified within the Flaviviridae and assigned to its own hepacivirus genus. The positive-sense RNA genome of the virus contains a single ORF that encodes the viral polyprotein. This is post-translationally cleaved to yield the structural and non-structural proteins of the virus. The structural proteins, which include the core protein and the two envelope glycoproteins E1 and E2, are released from the amino-terminal third of the polyprotein by host signal peptidases. However, the non-structural (NS) proteins are processed by virus-encoded proteases.90,91

The structural proteins, particularly the envelope glycoproteins, have been targeted as potential candidates for vaccine development. However, there are concerns associated with the genetic variability of the virus. There are six genotypes of the virus, each subdivided into several subtypes.92 The RNA polymerase of the virus lacks proof-reading capacity so that misincorporation of nucleotides occurs during RNA replication. Therefore, at any one time, the virus circulates in the serum as a population of closely related variants known as quasi-species.93 The amino-terminal end of E2 exhibits marked variation and is known as hypervariable region 1 (HVR1).94 It appears to come under significant humoral immune selection pressure.95 This region contains epitopes which induce virus-neutralizing antibodies that are, however, isolate specific. Such antibodies are capable of neutralizing some of the quasi-species but not all, hence the persistence of viral infection. Moreover, as described below, such antibodies emerge late in the infection, do not reach high titres and wane with time.96

The absence of a tissue culture system has hampered development further. In addition, until recently the chimpanzee was the only animal model of HCV infection. The recent development of human liver xenografts, capable of being infected with HCV in SCID mice, has somewhat alleviated the situation.

Protein vaccines

Early immunization experiments in chimpanzees using the E1 and E2 glycoproteins expressed in cell culture by recombinant vaccinia viruses led to production of antibodies against both E1 and E2. Following challenge with a homologous inoculum, five of the seven immunized animals were protected from infection. The two remaining animals with the lowest antibody titres developed hepatitis which, however, did not progress to chronicity. In contrast, all non-immunized animals developed chronic infection.97 Subsequent neutralization experiments in chimpanzees showed that, whereas sera obtained 2 years after acute infection were capable of neutralizing HCV in acute phase samples, serum taken 11 years later was not capable of doing so. Therefore such antibodies were isolate specific and thus incapable of neutralizing diverse quasi-species populations, as normally found in serum.98

Other approaches to recombinant protein based vaccination include the use of a core-ISCOM formulation tested in rhesus macaques and shown to have primed strong CD4+ and CD8+ T-cell responses, and a Th0-type response based on cytokine profiles.99 In addition, HBsAg virus-like particles (VLPs) have been used as vehicles for the display of HVR1 epitopes within the a determinant. Injection of mice with combinations of VLPs expressing different HVR1 epitopes from 1a or 1b strains induced antibodies of higher titre than individual VLPs, thus demonstrating synergism.100

DNA vaccines

Recent findings suggest that recovery from HCV infection is usually associated with a strong and multispecific T-cell response.101 Therefore the induction of CD4+ proliferative and CD8+ cytotoxic T-cell responses may be a more desirable attribute for an effective HCV vaccine than induction of the humoral responses normally associated with adjuvanted vaccines. In this respect, DNA immunization may offer an alternative strategy. Early experiments in mice using HCV core protein indicated that this approach results in high anti-HCV titres, causes T-cell proliferation and induces cytotoxic T-lymphocyte (CTL) responses.102,103 Such immune responses have also been obtained with plasmid constructs encoding E2,104,105 NS3106,107 and NS5A,108 and with polycistronic constructs encoding both structural and non-structural proteins.109,110 Similar results have been obtained in rabbits and macaques immunized with a plasmid construct encoding all HCV structural proteins.111 However, it has been observed that in mice there was a hierarchy in CTL responses to HCV structural proteins (E2 > core > E1) when these were expressed as a polyprotein.112 In addition, the breadth of CTL responses generated by NS3 DNA was restricted to a single major epitope.106 These findings imply that the target protein and potential epitopes that may be recognized by the immune system must be very carefully selected and evaluated.

The responses described above can be potentiated by varying the route of plasmid administration (intradermal delivery by a gene gun), by the inclusion of other potent helper T-cell epitopes in the constructs or by co-injection of plasmids encoding cytokines such as interleukins 2 and 12 (IL-2 and IL-12).113 More recent attempts at enhancing both humoral and cellular immune responses after DNA immunization include increases in immunostimulatory CpG motifs in plasmid construct backbones,114,115 deglycosylation116 or truncation of envelope glycoproteins117 and cationic liposome gene delivery.118 Significantly enhanced CD8+ T-cell responses were also obtained against HCV E2 encoding DNA after co-immunization with an IL-12 mutant construct. The latter had an N-glycosylation site mutation (N220L) of the p40 subunit which forms heterodimers with p35 and p19 to yield IL-12 and IL-23, respectively. This mutation led to reduced secretion of p40; it did not affect heterodimer formation but, in contrast, augmented antigen-specific T-cell immunity compared with the wild-type constructs.119,120 Finally, constructs expressing NS3 together with its co-factor NS4A resulted in enhanced protein expression and were more immunogenic than NS3 alone.121

Experiments in non-human primates have shown that DNA immunization with a plasmid encoding E2 is capable of inducing antibodies that block E2 uptake by cells in vitro.122 Similar experiments in chimpanzees followed by HCV challenge did not lead to protection from infection. However, unlike the control animal that became a chronic carrier following challenge, the immunized animals recovered from the infection.123 Therefore there was modification of disease outcome but no prevention of infection, as was the experience with protein-based vaccines. Modification of disease was also the outcome in two other chimpanzees that were primed with a multicomponent DNA construct encoding C/E1/E2, as well as NS3, followed by protein boosting.124

Other approaches

Viral vectors constitute another efficient vehicle for delivering heterologous genes to infected cells. Expression of the recombinant proteins in such cells mimics natural infection and induces both humoral and cellular immune responses. Immunization of mice with such vectors expressing either structural or non-structural HCV proteins, followed by challenge with recombinant vaccinia viruses expressing the relevant HCV protein, have prevented infection or reduced vaccinia replication drastically. Viral vectors used so far to achieve this outcome include recombinant adenoviruses,125–127 semliki forest virus,128 vesicular stomatitis virus129 and poxviruses such as canarypox. Recombinant canarypox viruses,109,130 as well as recombinant adenoviruses,131 have been used to boost immune responses primed by DNA immunization. Protection against recombinant vaccinia expressing HCV proteins has also been demonstrated following immunization with bacterial vectors such as the Calmette–Guerin bacillus encoding HCV NS5a132 and an attenuated Salmonella–HCV NS3 recombinant.133 Finally, other than recombinant vaccinia, Listeria monocytogenes has also been used as the challenge inoculum.134

It was recently demonstrated that mimotopes (HVR1 peptide mimics) engineered at the N-terminus of E2 induce antibodies cross-reacting with many natural HVR1 variants following DNA immunization,135 thus increasing the chances of neutralization of a broader quasi-species range. In another approach, expression of structural proteins in insect cells by recombinant baculoviruses resulted in the formation of HCV-like particles capable of inducing high titres of anti-E2 antibodies and virus-specific cellular immune responses with gamma interferon production. These responses appeared to be dependent on the maintenance of particle integrity, since such responses diminished when immunizing with heat-denatured particles.136 More recent work has demonstrated protection from challenge with recombinant vaccinia expressing the HCV structural proteins following immunization of mice with these VLPs. Moreover, adoptive transfer of lymphocytes from immunized animals afforded protection from challenge, which was abrogated by either CD4 or CD8 depletion.137 Immune responses after VLP immunization were boosted further by use of adjuvants such as AS01B and CpG 10105.138

Finally, immunization of mice with immature dendritic cells (DCs) infected with a recombinant adenovirus encoding HCV C/E1 proteins has been shown to induce lower CD4+ and CD8+ T-cell responses compared with animals immunized with a recombinant encoding NS3 protein only. No such differences in immune responses were seen in animals immunized with mature DCs infected with the same recombinants. It was also observed that HCV/E1 expression inhibited DC maturation mediated by tumour necrosis factor {alpha} (TNF-{alpha}) and CD40L.139 These findings have implications for future development of therapeutic vaccines against HCV. Racanelli and colleagues140 used DCs transfected with a cytopathic pestivirus RNA replicon encoding HCV NS3 to immunize HLA-A2 transgenic mice. Time-delayed DC apoptosis and antigen presentation in vivo induced protection from challenge with recombinant vaccinia. Therefore antigen expressed in DCs that apoptosed cross-primed CD8+ T cells.

Summary

In summary, in order to be effective an HCV vaccine must induce a high-titre, long-lasting and broadly cross-reactive anti-E2 response, and at the same time induce a strong multispecific cell-mediated immune response to structural and/or non-structural proteins of the virus. No such vaccine is yet available.


   Hepatitis D vaccines
Top
 Introduction
 Hepatitis A vaccines
 Hepatitis B vaccines
 Hepatitis C vaccines
 Hepatitis D vaccines
Hepatitis E vaccines
 Conclusions
 References
 
The hepatitis D virus (HDV) is a small spherical particle measuring 36 nm in diameter. HDV is defective in that it requires HBV for the provision of its outer coat, which consists of HBsAg. The delta antigen (HDAg) forms the nucleocapsid of the virus and this in turn encloses its circular single-stranded RNA genome (1700 nucleotides). There are at least three distinct viral genotypes.141,142

Protection against infection with the helper virus (HBV) by immunization with the hepatitis B vaccine will also prevent infection with HDV. Protection of individuals who are already chronic HBV carriers from superinfection with HDV is more difficult. Since HDAg is the only protein that the virus encodes, this has been tried as a potential vaccine, with the aim of inducing a cellular immune response that would lead to accelerated lysis of infected cells and thus reduce spread of the virus through the liver. The woodchuck (groundhog) carries the woodchuck hepatitis virus, a hepadnavirus closely related to HBV, and can be superinfected with HDV. Procedures inducing a predominantly humoral response to HDAg have increased the period of viraemia and hepatitis,143 while immunization precedures stimulating the cellular response (recombinant vaccinia) have shortened the period of viraemia.144

DNA immunization has also been employed to induce anti-HD specific immunity. Plasmid constructs encoding either large or small HDAg induced humoral immune responses, which were weak with the large antigen but stronger with the small antigen. Both antigens were equally good at eliciting a Th1-biased T-cell response in immunized mice.145,146 However, similar experiments in woodchucks followed by HDV challenge did not prevent infection, but led instead to modification of the course of the ensuing disease.147


   Hepatitis E vaccines
Top
 Introduction
 Hepatitis A vaccines
 Hepatitis B vaccines
 Hepatitis C vaccines
 Hepatitis D vaccines
 Hepatitis E vaccines
Conclusions
 References
 
The enterically transmitted non-A non-B hepatitis virus, now known as HEV, is a spherical non-enveloped particle, measuring 32–34 nm in diameter. The single-stranded positive-sense RNA genome of the virus is polyadenylated and approximately 7.5 kb long. It contains three ORFs (1–3). ORF 1 encodes the non-structural proteins of the virus including the RNA-dependent RNA-polymerase, whilst ORF 2 encodes the nucleocapsid protein (length, 660 amino acids) and ORF 3 encodes a protein (length, 122 amino acids) of unknown function.148,149 The virus is distantly related to the caliciviruses, but should probably be placed in its own new family group.

Epidemics of HEV infection have been associated with poor hygiene and sanitary conditions resulting in contamination of drinking water with sewage. Outbreaks have occurred throughout Asia, Africa, the Middle East and Central America, and infection is particularly severe in pregnancy. Although sporadic cases have been reported in the West, these have usually been linked to travel in endemic areas. Occasionally, sporadic cases occur in the absence of such travel. The recent detection of the virus in rats in Western countries suggests that these and other animal reservoirs, such as swine, sheep, cattle and goats, with high prevalence rates of antibody to HEV (anti-HEV)150 may be the source of such infections in humans.

Early sero-epidemiological studies using antibody assays based on recombinant proteins expressed in Escherichia coli or insect cells, or using synthetic peptides, have yielded results of varying sensitivity and specificity depending on whether full-length, truncated or modified proteins were used in the diagnostic enzyme-linked immunosorbent assays (ELISAs). HEV strain differences may also contribute to this problem. Such studies have established the presence of antigenic epitopes at the carboxyl ends of both ORF 2 and ORF 3 proteins.148,149 Moreover, IgG HEV antibody to ORF 3 waned during convalescence, whilst that to the ORF 2 gene product persisted for several years in both convalescent human sera and sera from experimentally infected primates. Thus the carboxyl end of the ORF 2 protein (amino acids 394–607) is thought to contain a major conformational epitope which evokes virus-neutralizing antibodies. More recently, terminally truncated proteins expressed in E.coli or insect cells have been shown to form LVPs which present additional conformational epitopes.

Therefore the ORF 2 gene product is the main candidate for a prophylactic vaccine against HEV. A fusion protein of trpE to the carboxy-terminal two-thirds of the capsid protein (ORF2) was the first to be used in immunization studies in cynomolgus macaques and shown to afford protection from the development of hepatitis following challenge with live HEV.151 Animals were also protected from HEV hepatitis following passive immunization with anti-ORF 2 specific antibodies.152,153 More recently, immunization with SVPs produced in a baculovirus or prokaryotic system conferred protection against disease, and more importantly protected against challenge with both homologous and heterologous HEV inocula.154–156 Further work with recombinant HEV capsid protein expressed in insect cells revealed two major proteolytically processed products of molecular weight 56 and 53 kDa. The latter is truncated at its carboxyl end and lacks the neutralization epitope. Whereas the p56 afforded protection from challenge with live HEV, rhesus macaques immunized with p53 were not protected.157 A preclinical immunogenicity and efficacy trial of a recombinant HEV vaccine also in rhesus macaques has shown that two doses of the vaccine were essential for optimum efficacy.158 This same HEV candidate vaccine (produced by Novavax Inc. under sponsorship from GlaxoSmithKline) has already been used in human volunteers, all of whom seroconverted following either a two- or three-dose schedule (Dr R.Purcell, Viral Hepatitis Meeting, Atlanta, GA, 2000). A phase II–III trial is presently under way in Nepal.

DNA vaccination, using recombinant plasmids containing sequences from ORF 2, has been employed in immunization studies in mice and rats.159–161 Such approaches have also employed chimaeric plasmids encoding immune enhancement proteins such as cytotoxic T-lymphocyte antigen 4,162 IL-2 and granulocyte–macrophage colony-stimulating-factor.163 Anti-HEV induced by nucleic acid immunization was of high titre and was long lasting. Moreover, these antibodies competed effectively with convalescent sera and recognized LVPs. Finally, DNA immunization by gene gun of cynomolgus macaques with a plasmid containing the entire HEV ORF2 sequence induced anti-HEV production which protected the animals against challenge with a heterologous strain.164


   Conclusions
Top
 Introduction
 Hepatitis A vaccines
 Hepatitis B vaccines
 Hepatitis C vaccines
 Hepatitis D vaccines
 Hepatitis E vaccines
 Conclusions
References
 
Protection against HAV, HBV and HEV can be achieved by the use of formaldehyde-inactivated virus or recombinant subunit vaccines. Protective immunity against HBV also affords protection against HDV infection. Clinically adequate vaccines against HCV are not yet available, but protection against homologous strains of the virus has been achieved in animal studies. The existence of multiple viral genotypes of HCV and of many quasi-species within each viral isolate presents a substantial unmet challenge.


   References
Top
 Introduction
 Hepatitis A vaccines
 Hepatitis B vaccines
 Hepatitis C vaccines
 Hepatitis D vaccines
 Hepatitis E vaccines
 Conclusions
 References
 

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