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Commentary:
On DNA vaccines and prolonged expression of immunogens Tomsˇ Hanke Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom Persistent hepatitis B virus (HBV) infection represents a major public health concern because of its association with chronic liver disease and the propensity of the disease to progress to cirrhosis and hepatocellular carcinoma. Despite the availability of a prophylactic vaccine effective in a majority of the population, alternative vaccination strategies are being sought to induce protective responses in healthy non-responders and to boost and broaden T cell responses in chronically infected patients, which may lead to a better control of the virus and/or its eventual complete clearance. In this issue of the European Journal of Immunology immunization of BALB/c mice intramuscularly with a DNA vaccine encoding the hepatitis virus B surface antigen (HBsAg) was shown to result in prolonged secretion of HBsAg into the serum and the elicitation of HBsAgspecific antibodies. In fact, the vaccine was so efficient that the antibodies and HBsAg formed circulating immune complexes and induced kidney and liver lesions similar to those observed in chronically infected patients. This commentary discusses these results in terms of the safety of plasmid DNA-vectored genetic vaccines in general, the use of DNA vaccines expressing HBsAg for the treatment of chronic hepatitis and the consequences of prolonged immunogen expression for the development of protective immune responses.
Received 14/2/06 Accepted 1/3/06 [DOI 10.1002/eji.200635986]
Key words: Antibody DNA vaccines Hepatitis B virus Persistence Safety
See accompanying article http://dx.doi.org/10.1002/eji.200535468
According to the World Health Organization, an estimated 350 million people worldwide are chronically infected with hepatitis B virus (HBV). Following acute infection, approximately 10% of adults and as many as
Correspondence: Tomsˇ Hanke, MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, The John Radcliffe, Oxford OX3 9DS, United Kingdom Fax: +44-1865-222502 e-mail:
[email protected] Abbreviations: HBV: hepatitis B virus HBsAg: hepatitis virus B surface antigen CIC: circulating immune complexes f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
90% of infants infected perinatally, the most common route of transmission, develop chronic infection. Although many of these individuals eventually clear the virus or achieve a state of non-replicative infection, prolonged chronic hepatitis leads to the development of cirrhosis, liver failure, or hepatocellular carcinoma in up to 40% of patients causing over 1 million deaths annually [1]. The outcome of HBV infection is determined by the vigor and quality of the orchestrated CD4+ T, CD8+ T and B cell-mediated immune responses mounted by each individual [1]. In general, the T cell response in www.eji.de
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acute hepatitis B is strong and multispecific in patients who ultimately clear the virus, while it is weak and narrow in those who go on to develop chronic hepatitis. There is a highly successful prophylactic HBV vaccine formulated as a yeast-produced recombinant hepatitis virus B surface antigen (HBsAg) protein, which induces a vigorous immune response with protective immunity as defined by the anti-HBsAg antibody titer [2]; however, results with the same vaccine in the therapeutic setting have been disappointing, possibly due to a less efficient induction of cell-mediated immune responses. Thus, various multivalent vaccines and combinations of vaccines with drugs and cytokines are being explored both to overcome the non-responsiveness to the prophylactic HBsAg vaccine and to boost and broaden weak HBV-specific T-cell responses in patients with chronic hepatitis B. In particular, genetic vaccines vectored by plasmid DNA are thought to be well suited for this purpose as they elicit both CD8+ T cells and antibodies to HBV proteins [3, 4]. Although the T cell immunogenicity of DNA vaccines alone in the first clinical trials has been unimpressive, it is still early days. There is a great potential to improve DNA vaccines through more efficient delivery, adjuvantation and/or the use of heterologous prime-boost regimens. In the study in this issue of the European Journal of Immunology, Zi et al. [5] describe the immunization of BALB/c mice with a DNA vaccine encoding HBsAg. Not only did their vaccine achieve persistent levels of HBsAg and anti-HBsAg antibody in the sera of immunized mice, levels which were sustained for several months, the HBsAg and antibody also formed circulating immune complexes (CIC), which induced and were found in multiple liver and kidney lesions. Using the same DNA vector expressing enhanced green fluorescent protein, the authors were able to demonstrate sustained enhanced green fluorescent protein expression in the injected muscle cells for at least 6 months post delivery, but this time in the absence of lesions in the liver or kidneys. To confirm that the presence of the HBsAg CIC are the pathological basis for the observed damage, Zi et al. [5] induced similar lesions in normal mice by passive transfer of the immunized mice sera taken at the peak of HBsAg CIC levels, and in HBsAg-transgenic mice injected with the anti-HBsAg antibody alone. The authors concluded that the lesions caused by the CIC cast doubts on the safety of therapeutic DNA-mediated immunization for chronic hepatitis B infection. Whether or not this is the case, Zi et al. certainly highlighted some interesting issues. First, it should be stated that CIC are normally formed in patients who clear the virus and recover from acute infection [1, 6]. Abnormal CIC levels or composition are found both in the serum and pathologic lesions of most chronically infected patients and have been f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Highlights implicated in the pathology of mainly extrahepatic disease. Their role in liver damage has been less certain because CIC formation correlates both with disease activity and HBV replication. Thus, Zi and colleagues[5] provided a clear demonstration that the HBsAg CIC alone are sufficient to cause liver and kidney lesions similar to those observed in chronically infected patients. So what are the implications for the safety of HBsAgexpressing DNA vaccines? To date, DNA vaccines in preclinical and clinical studies have been invariably very safe and well tolerated. The vaccine DNA molecules alone have not caused any adverse effects on blood biochemical and haematological values, caused no detectable organ pathology or systemic toxicity, and there has been no evidence of autoimmunity, appearance of anti-nuclear antibodies or development of antibodies to double stranded DNA [7–11]. As for genetic toxicity, chromosomal integration is a highly unlikely event, which nevertheless remains an important theoretical risk that has to be considered in pre-clinical studies. Integration may result in an insertional mutagenesis, which may cause activation of oncogenes, inactivation of tumour-suppressor genes or chromosomal instability resulting in chromosomal breakages and/ or rearrangements. It has been shown that upon injection, most plasmid DNA is rapidly degraded [12]; for a short period of time some vaccine DNA could be detected sporadically at various sites around the body, but by 6–7 weeks after vaccination the plasmids were only detected at the injection site [13–15]. Particularly in the injected muscle cells, vaccine DNA may persist for several months as extrachromosomal DNA [13] and, when re-isolated, displays bacterial methylation. In the study on HBsAg DNA vaccine mentioned above, Zi et al. [5] note that their vector plasmid was derived from pcDNA3, which contains a mammalian origin of replication and as such has never been intended for clinical use because of safety concerns relating to the possible amplification of the vaccine DNA. Thus, while this study has been useful to exemplify some of the principles of DNA vaccination in an animal model, plasmids used to vector genetic vaccines currently in clinical trials do not typically include mammalian origin of replication and as such are designed for a short burst of immunogen expression without persistence of the gene or the corresponding protein. Although persistence of administered vaccine sequences at the injection site has not been considered to pose a significant safety concern in terms of genetic toxicity [13], persistent expression of the passenger gene products will require a careful case-by-case evaluation. As for the safety of prolonged HBsAg expression, it should be noted that Zi et al. [5] vaccinated nave mice, which is in effect a prophylactic setting. In chronic HBV www.eji.de
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infection, immune complexes are most likely already circulating in the patients' sera [1] and it is not at all clear whether the DNA vaccine contribution will exacerbate the problem of abnormal CIC and help to reach the pathological threshold. Overall, the vaccine stimulation of T cells might still be beneficial and the decisive consideration, although safer vaccines can be designed which focus on induction of T cells and minimize or avoid the induction of antibodies. Furthermore, to make the DNA vaccines less persistent and therefore safer, allowing them to deliver a boost to T cell responses while decreasing the longevity of HBsAg shedding into the circulation, an intradermal route might be used instead of an intramuscular one. Intradermal application of DNA vaccines has been shown to be superior for T cell induction to direct intramuscular injection and skin growth ensures removal of the plasmid-transformed HBsAg-producing cells. Vigorous cytotoxic T lymphocyte responses may also contribute to the eventual eradication of these persistent HBsAg factories [16, 17] and it is of note that the vaccine of Zi et al. [5] did not induce strong HBsAg CTL. At first, it might be intuitive that long-lasting immunogen expression is a desirable vaccine feature for induction of long-lasting protection, however, as discussed this might not be the case. Undoubtedly, the main goal of prophylactic (but not necessarily therapeutic) vaccination is induction of a long-lasting protective immune response. What constitutes a protective response remains debatable and depends upon the biology of the pathogen in question and its interaction with the host. Thus, protection may be best conferred by, and therefore the vaccine should induce, activated effector responses at the port of entry, which are ready to attack the incoming virus instantly; however, maintenance of effector T cells requires continuous or frequent restimulation e.g. by a persisting genetic vaccine, because effector T cells are short-lived and capable of only limited or no proliferation. Thus for this scenario, persisting immunogen expression is desirable [18], although care has to be taken to avoid induction of tolerance, anergy or exhaustion of T cells, as well as the avoidance of triggering autoimmunity. Alternatively, a vigorous and appropriate T cell priming with an optional strong boost using truly non-persisting vaccines should generate a high frequency of 'good quality' central memory T cells capable of rapid expansion and expression of effector functions upon antigenic re-exposure [19]. Compared to effectors, the memory T cell response to incoming virus is delayed, although some effector functions such as cytokine production can be activated within a few hours. Memory T cells should be long lasting in the absence of a persistent antigenic stimulation. Here, persistent antif 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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gen expression may have a negative effect by interfering with the memory development [20]. With some data emerging that, at least for T cell responses and for some particular infections, protection correlates with the presence of central memory T cells, the idea of persisting vaccines is becoming less and less appealing. In conclusion, a prudent approach to the long-term safety of DNA and for that matter any genetic vaccines would be to follow volunteers participating in these early trials for life to firmly establish the long-term effects, or their absence, of these modern biologicals. However, there are substantial ethical, financial, legal and practical implications associated with such longterm 'flagging' of trial participants.
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