Therapeutic vaccination against chronic viral infections

Review

Therapeutic vaccination

Therapeutic vaccination against chronic viral infections Pierre Vandepapelière Chronic viral infections such as those caused by hepatitis B virus, human papilloma virus, herpes simplex virus, and HIV, in theory, present logical targets of active specific immunotherapy. Indeed, immunological mechanisms are involved in several aspects of their pathogenesis and natural course, such as virus persistence, destruction of infected cells and control of viral replication. Therapeutic vaccination could therefore be an adequate replacement for, or adjunct to, existing therapies. Almost all approaches to therapeutic vaccination have been evaluated in those four disease areas. Despite encouraging results in animals none of these attempts has, so far, been completely successful in the human setting. Immune HBV However, with a better understanding immunity tolerance of the immunological mechanisms HBeAg + involved in the control of disease successful therapeutic vaccines, used alone or in combination with   HBsAg other therapies, are an achievable   HBeAg goal.   Anti-HBe

decades, the prevalence of genital herpes has increased worldwide. In the USA, the proportion of HSV2 seropositive adults increased from 16·4% for the period 1976–1980 to 21·9% for 1988–1994,4 which represents a 30% increase during a period of high awareness regarding the importance of safe sexual practices. HSV not only causes painful recurrent lesions, but also has important psycho-social consequences, and genital HSV infection increases the risk of acquisition of HIV infection. Since the HIV epidemic began, more than 60 million people have been infected with the virus, 20 million have died as a result, and currently 40 million adults and children are living with HIV.5

  

Immune clearance HBeAg+/ anti-HBe+

Anti-HBe+ immunity

        

        

Reactivation pre core mutation

        

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Introduction

HBV-DNA

In this review, four viruses will be ALT considered: hepatitis B virus (HBV), HIV, herpes simplex virus (HSV), and Tolerant +++ Good response “Healthy Pre core mutant CHB human papilloma virus (HPV). Poor response to therapy Aggressive disease asymptomatic Immunotherapeutic approaches can to therapy Active disease carriers” Liver failure/cirrhosis be divided into four categories No active disease Liver failure/ High risk HCC Not treated Few complications cirrhosis depending on whether they are active No active disease High risk HCC High risk HCC or passive, specific or non-specific. This review will focus only on active, specific immunotherapy, or Figure 1. Serological, virological, and clinical patterns of CHB: evolution over time. HCC=hepatocellular carcinoma. therapeutic vaccination. The selection of viruses in this Although mass vaccination has drastically decreased review is dictated by the fact that research and development on therapeutic vaccines is most active in those four areas. All the prevalence of hepatitis B infection in a few countries, four viruses cause chronic or latent infections, with clear, the pool of carriers worldwide remains essentially although different, immunological implications. Each virus unchanged. Currently available therapies, mainly causes a major public health problem worldwide, despite the interferon
and lamivudine, are not fully satisfactory in availability of preventive vaccines for HBV and antiviral terms of safety or efficacy profiles. Numerous therapies are drugs for HPV, HSV, and HIV, and they are among the most common chronic viral infections in humans. Over 350 PV is Director, clinical R&D HIV vaccines and anti-infective million individuals are chronic carriers of HBV1 and the therapeutic vaccines, GlaxoSmithKline Biologicals, Rue de l’Institut virus causes up to 1 million deaths annually. HPV infects 89, B-1330, Rixensart, Belgium. 20–80% of sexually active adults,2 and is recognised as the Correspondence: Dr Pierre Vandepapelière. Tel +32 2 656 8733; major causative agent of cervical cancer.3 Over the past two fax +32 2 656 9072; email [email protected]

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Therapeutic vaccination

Mode of transmission and pathogenesis

Virus

Lysis (%)

SI

To support the strategies behind the research into therapeutic vaccination of HBV, HPV, HSV, and HIV infections, some understanding of the characteristics of the transmission, pathogenesis Lysis of Professional APC infected and immune mechanisms involved in persistence Liver hepatocytes cell and control of each of these viruses is required. The main mode of transmission is through the B cell sexual route for all four infections. However, HBV Helper effect and HIV are also largely transmitted through the Helper effect parenteral route and from mother to child; HSV CD8+ CD4+ and HPV are almost exclusively transmitted T cell CTL through the sexual mucosae. Rarely, HSV and HPV can be transmitted from the mother to the neonate Cytokines leading occasionally to neonatal herpes disease or to asymptomatic HPV carriage, or recurrent respiratory papillomatosis, respectively. The fact Down-regulation that HBV and HIV can be transmitted through the of viral gene expression blood is linked with the permanent presence of the viruses in blood. Correlated with the viraemia are Neutralisation of circulating the systemic disease manifestations, liver disease for virus HBV and infection of CD4+ lymphocytes for HIV. Chronic hepatitis B (CHB) is an evolving Figure 2. Immune responses to HBV infection. Redrawn from an original supplied by infection, changing over time in clinical, C Ferrari. immunological, and virological profile according to the balance between the immune response and viral available for the treatment of HPV-induced genital warts. activity (figure 1). The immune response to HBV infection is However, the rate of recurrence and of reinfection is multifactorial, involving several compartments of the still very high with currently no effective treatment to immune response (figure 2). Predominant is the cytotoxic eradicate the subsequent latent infection and thereby T lymphocyte (CTL) response resulting in at least partial effect a cure. There is also no approved therapy for chronic viral clearance through lysis of infected cells.6 Inflammatory HPV cervical infection. Despite the ability to detect cytokines, such as interferon  and tumor necrosis factor and cure most cases of local disease, 35% of patients (TNF)
, also aid intracellular inactivation of the virus.7 A strong CD4+ response especially against the core develop persistent or metastatic disease for which there is no consistently effective therapy. In the case of antigen during acute hepatitis B seems to be essential to HIV infection, although highly active antiretroviral prevent chronicity in addition to initiating viral clearance8 therapy (HAART) is very effective, having greatly (figure 3). However, evidence points to a combined humoral decreased the morbidity and mortality associated with and cellular immune response for the induction and HIV infection in the USA, the epidemic is worsening throughout most of the world. Indeed, more than 90% of Acute hepatitis Chronic hepatitis 50 HIV-infected individuals do not have HBV core 40 access to HAART. There is therefore a clear medical 30 need for new therapies. Active, 20 specific immunotherapy or therapeutic 10 vaccination is in theory a logical strategy, in view of the numerous 0 immunological mechanisms involved HBV core 18–27 80 in the pathogenesis of these four chronic 60 infections. The objective of this review is to 40 summarise previous approaches for 20 the development of therapeutic vaccination, for HBV, HPV, HSV and 0 Patients HIV infections, to review the current research and development in this field, and to identify the targets and challenges Figure 3. HLA class II and class I restricted T cell responses to HBV during acute and chronic ahead. hepatitis B. Adapted from reference 8.

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Table 1. Mechanisms associated with HBV persistence Factors associated with induction of persistence Neonatal induction ● Transplacental passage of HbeAg in utero ● Immaturity of the immune system ● MHC genetic characteristics Adult infection ● Immunodepression at the time of infection ● MHC genetic characteristics Factors associated with maintenance of persistence HBV viral protein in excess in serum ● HBeAg ● HBsAg

Virus mutants and variants Modulation of immune system by viral proteins HBV infection of extra-hepatic sites

maintenance of complete control of infection.9 The maintenance or clearance of CHB infections is also dependent upon the balance between Th1 and Th2 cells: Th1 cells are predominant during the clearance phase with Th2 dominant during the tolerant phase.10 Numerous factors have been associated with the induction and maintenance of persistence of which viral factors such as circulating HBeAg, HbsAg, and viral induced immune disturbances are predominant6 (table 1). Over 100 types of papillomaviruses have been described in animals and in people. In human beings HPV are subdivided into low risk, benign HPV types, and high risk HPV types, which are associated with the development of cancers mainly in the genital area; classification is also according to the infected tissue—ie, cutaneous or mucosal (table 2). Maintenance of HPV chronicity is likely to be multifactorial, resulting from a combination of viral location, suppressed cellular immune responses, and genetic host factors. HPV infections are usually confined to the epithelium displaying no evidence of a viraemic phase and limited exposure to the systemic immune system. The inadequate immune response probably results from the infection being retained in their host cells in addition to some viral effects on local Langerhans cells and on antigen presentation. Host factors (eg, HLA class II genes, MHC class II phenotypes) also seem to be involved in the course of HPV infection.11,12 Both HSV1 and HSV2 cause genital herpes upon infection of mucocutaneous surfaces; once inside the epithelial cell, the virus replicates ultimately causing cell death (figure 4). Concurrently, virus is transported to neuronal cell nuclei in the ganglia innervating the mucocutaneous zone of infection where the virus may replicate and establish a clinically silent latent infection. This latent infection can reactivate spontaneously or following various stimuli, resulting in the transfer of virus to epithelial cells adjacent to the nerve endings, causing recurrent infections that result either in herpetic skin lesions or in asymptomatic shedding of the virus. Although not fully elucidated, the mechanisms of establishment, maintenance, and reactivation of latent virus are known to involve both viral and host factors. The viraemic phase of HSV infection however has no role in the pathogenesis of either initial or recurrent genital herpes.

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The humoral response in the prevention of infection of mucocutaneous cells is predominantly due to the production of antibodies primarily directed against various envelope glycoproteins.13 With respect to the cellular immune response, both CD4+ and CD8+ T-cell responses are key to the control of infection.14 An association between the level of circulating interferon
15 or of interleukin 216 and the time to the next herpetic recurrence in human beings has been demonstrated. These results, together with the observation that a worsening of the disease occurs during immunosuppression, supports the concept that an adequate cellular response is key for the control of recurrent HSV infections. HIV is a member of the lentivirus group of retroviruses. Retroviruses have the unique ability to transcribe their RNA to DNA, and are capable of integrating their genome into the host cell DNA early in infection (figure 5). Analysis of nucleic acid from HIV-1 have revealed large variations both within and between patients; genomic recombination between two different HIV-1 populations frequently occurs in vivo resulting in genetic shifts. Subclassifications of HIV are defined mainly by biological, genetic, or antigenic phenotypes. A further classification in “clades” is based on phylogenetic analysis of env and gag sequences with different clades showing diverse geographical distribution. Ten clades have been identified to date, designated A–H, J, and NT. The pathogenesis of HIV disease is multifactorial and multiphasic, a consequence of complex interactions between the virus and a variety of host factors with overlapping phases of persistent viral replication, inappropriate immune cell activation, and immunodeficiency. The primary HIV-1 infection is a transient condition accompanied by an initial rapid rise in plasma viraemia, a decrease in blood CD4+ T-cell count, and a large increase in blood CD8+ T-lymphocyte count, which generally correlates inversely with plasma viral load. While the viral load is controlled, CTL levels remain high, only falling late in disease when the viral load increases (see figure 6). The majority of patients will progress inexorably towards HIV disease (AIDS), a process accompanied by a progressive decline in CTL activity; chronic infection is characterised by a lack of fully functional CD4 and CD8 HIV-1-specific T-cells with proliferative and cytolitic capacity, respectively. Table 2. Most common HPV types and associated diseases Group

Virus types Major

Associated diseases

Minor

Low risk Cutaneous HPV1 HPV2 HPV3

Verruca plantaris Verruca vulgaris Verruca plana

Mucosal

Genital warts Laryngeal papillomatosis Tumor of Buschke-Loewenstein

HPV6 HPV11

High risk Cutaneous HPV8 Muscosal

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Epidermodysplasia verruciformis (leading to carcinoma)

HPV16–18 31-33-45- Flat warts 35-39-51- Cervical dysplasia and carcinoma 52-56-68 Carcinoma of penis, vulva, vagina, anus

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With respect to HSV, the establishment of viral latency in the sensory neurons protects the virus from Virus transmission to mucosal surface or abraded epithelium any kind of immune attack; neuronal cells do not display MHC class I or II Viral replication antigens that are needed for stimulation Release from of CD4+ or CD8+ T cells. Only the Uptake by sensory nerves nerve ending genetic information of the latent virus is present in the neuronal cells where no Retrograde transport Anteregrade transport to sensory ganglia virion or capsid can be detected. to mucosal and cutaneous sites Furthermore, cell lysis due to immune Establishment of attack is undesirable since the neuronal latent infection cells will not be replaced and could result in neurosensorial defects. It is therefore probable that the immune response could only reach the virus after reactivation. Therapeutic vaccines will need to act on the reactivated virus both during clinically symptomatic and asymptomatic reactivation. Ideally Productive replication in sensory neurons the objectives for immunotherapy should include the reduction in Figure 4. Schematic representation of the pathogenesis of HSV infection. Redrawn from an original symptomatic recurrent episodes, the courtesy of L Stanberry. reduction of virus transmission during clinical episodes, the reduction of This lack of responsiveness is due to an inability to properly asymptomatic shedding to reduce viral transmission outside respond to HIV-1 antigens as opposed to a loss of CD4 the clinical recurrences, and potentially the reduction of viral T-cell numbers. Of crucial importance is the presence of reactivation. fully functional antigen presenting cells (APCs), such as Although chemotherapeutic treatment of HIV is capable dendritic cells, to provide a bridge between the helper T-cell of controlling viraemia, drug-resistant virus can potentially and the CTL. emerge, and the requirements of the patient to adhere to aggressive regimens, with high toxicity, can be unacceptable for possibly lifelong treatment. HAART was originally Rationale for therapeutic vaccines believed to have the potential to eradicate the HIV-1 virus.17 Objectives of immunotherapy Theoretically, viral eradication is feasible in HPV infections However, subsequent data revealed that replicationthanks to its localisation limited to mucocutaneous cells, competent HIV-1 remains even in patients in whom infection whereas, realistically, only control of the infection is a more has been suppressed for a long period.18 Additionally, HAART not only fails to eradicate the virus it also fails to enable total achievable objective for HBV, HSV, and HIV. For low-risk HPV infection, available therapies are restoration of immune responses specific for HIV.19 Since eradication of HIV-1 may be an unachievable goal, reasonably efficacious in the clearance of warts. Therefore, the objective of a therapeutic vaccine for patients with low-risk the current objectives of a therapeutic vaccine must be the HPV must be to reduce the high recurrence rate observed, adequate control of the virus to prevent clinical disease and either as monotherapy or in combination with ablative or virus transmission. With respect to chronic progressive infection, the aim must be to mimic the immune system of the topical therapies. The objectives for a therapeutic vaccine against high-risk “long-term non-progressor” patient,20 whereby viraemia is HPV are complicated by the diversity of diseases caused by contained, in the absence of any immunological damage, HPV, the diversity of HPV types causing the same disease, and without progression towards disease. Practically, an effective the spontaneous regression observed with some infections therapeutic vaccine could either delay initiation of HAART, (figure 7). A therapeutic vaccine, therefore, should be allow prolonged HAART therapeutic interruption, or composed of the most prevalent HPV subtypes, or be cross- improve the immune status or the control of viral load in type efficacious, with the aim to prevent reinfection by the conjunction with HAART. In addition, the virus load same subtype as well as by other subtypes for at least several should be maintained at sufficiently low levels to prevent years. If clinical lesions are present, the vaccine should also transmission. induce their regression together with total viral clearance. In the case of HBV, an effective vaccine would need to Supportive evidence of efficacy/rationale induce an adequate immune response resulting in a reversion Although to date no candidate therapeutic vaccine has of the tolerant state or so called healthy-asymptomatic carrier shown efficacy in human beings, the supportive evidence for state combined with clearance of the infected hepatocytes and the concept of therapeutic vaccines for HBV, HPV, HSV, and HIV is extensive. control of viral replication. Viral shedding Lesion formation

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Therapeutic vaccination

reported from the 1930s to the 1990s, albeit in inconclusive, uncontrolled studies in which the placebo effect was Neutralising Infectious HIV not fully determined. Although some CD8 T cells antibodies virion efficacy has been reported with two inactivated vaccines, “Lupidon” and the CD4 coMHC I + receptors peptide Skinner vaccine, in controlled studies,26,27 the data have yet to be substantiated due to limitations in the design of the studies. Reverse ds RNA transcriptase Results from studies in animal models Antigen RNA have provided strong evidence in support Assembly processing DNA of the concept, especially in female guineapigs where administration of HSV DNA Integrase glycoproteins with potent adjuvants Structural reduced recurrent genital HSV disease protein dsDNA Transcription Regulatory and asymptomatic viral shedding.28 In protein the same model, replication-impaired Viral RNA transcript and DNA vaccines have also shown some effect on recurrent herpes.29,30 Genomic Translation viral RNA In the case of HIV infection, it is Viral mRNA believed that the failure of humoral and cell-mediated immunity to HIV underlies the increasing viral burden and the decline in CD4 cell count observed Figure 5. HIV cycle of cellular infection. during the onset of severe clinical In the case of CHB, it has been established that the disease. Indeed a rapid decrease in acute viraemia in patients destruction of hepatocytes observed during chronic was observed following induction of a strong CTL response infection is not caused by a direct cytopathic viral effect but and high levels of CTLs have been associated with a by the host immune response. Furthermore, the persistence reduction in disease progression and an improvement in phase is due to virus and host interactions governed survival rate.20,31 The role of cell-mediated immunity in HIV vaccines is predominantly by the immune response to viral antigens during chronicity6 The correlation of spontaneous supported by extensive pre-clinical and clinical data. Passive seroclearance of the disease with acute exacerbations, immunoprophylaxis has resulted in the control of viral load resulting in effective control by the immune system and and disease, thereby conferring complete protection, against attenuation of the disease, provides the strongest argument simian immunodeficiency virus (SIV) in macaques.32 In in favour of active immunotherapy. Further evidence HIV-infected individuals, the beneficial effect of both HIVincludes the ability to effect clearance of HBsAg by passive specific CD4+ and CD8+ T-cell responses in seropositive transfer of bone marrow from a naturally HBV immune individuals has been demonstrated in a number of studies.33 donor.21 Induction of HBe seroconversion has additionally CD8+ CTL responses following primary infection are been demonstrated following treatment with associated with the control of viral replication during acute immunomodulatory agents (eg, interferon
, thymosin
), infection, in addition, strong CTL responses are observed in and also some candidate therapeutic vaccines.22 long-term non-progressors31 and in highly-exposed, Supportive evidence for the rationale of a therapeutic seronegative African sex workers.34 In chronically infected vaccination in the treatment of HPV-induced lesions and for individuals, a significant inverse correlation was established prevention of their recurrences is extensive and includes: between HIV-specific CTL frequency and plasma RNA documented spontaneous regression of lesions with level.35 The protective role of CTL was further substantiated concomitant vigorous humoral and lymphoproliferative by the characterisation of virus mutants escaping CTL immune responses; massive T cell invasion and restoration recognition.36 of the T-helper response; induction of regression by early proteins and structural proteins in animals; and an HIV RNA increase in the prevalence and severity CD4+ of HPV infections, in addition to count the risk of carcinogenesis in highHIV-1 specific risk HPV-induced lesions, in CTL 2–4 weeks 3–10 years 2–3 years immunosuppressed individuals.23—25 Encouraging data regarding the Infection Clinical latency Overt AIDS potential of therapeutic vaccination in the treatment of HSV infections were Figure 6. Kinetics of viral load and immune response during the phase of HIV infection.

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Therapeutic vaccination

rationale was to try to break the immunological tolerance by exposing the tolerant host to antigens that differed slightly from the tolerated antigen. Unfortunately the results Latent Clinical disease Subclinical obtained were too limited to allow firm infection infection conclusions on the efficacy of this vaccine. Low risk High risk Due to the potential oncogenic CIN I/II Genital warts properties of HSV2, several inactivated Potentiating purified vaccines have been developed, factors: having undergone fractionation to CIN I/II •MHC Regression Progression remove viral DNA, with possible Clearance Growth •Immunosuppression further fractionation to obtain mainly Multiplication •Co-carcinogens Recurrences the envelope glycoproteins. However, Carcinoma •Repeated/persistent infections in situ a number of shortcomings are associated with the inactivated subunit vaccines, which include consistency in Invasive the concentrations of immunogens, cancer complete inactivation of virus, total elimination of potentially oncogenic Figure 7. Disease cycle of genital HPV. viral DNA, and cost of production. Results have been disappointing with the early Development of therapeutic vaccines glycoprotein vaccines in the treatment of recurrent genital Historical The concept of therapeutic vaccination pre-dates the herpes. Two have shown some efficacy. The Skinner availability of antibiotics when they were unsuccessfully used vaccine, a purified envelope vaccine comprising a mixture to treat invasive bacterial infections.37 Therapeutic of HSV1 glycoproteins, showed some positive clinical immunisation against HPV started in the 1920s with effects.27,45 The only marketed therapeutic HSV vaccine in subsequent investigations involving vaccination with wart some European countries is “Lupidon”, which has been extracts reported over the following 60 years. However, only shown to be efficacious in genital or orolabial herpes and recently has consolidated research been conducted with ocular herpes.26 Interestingly, the dosing regimen in these therapeutic vaccines for HPV lesions as opposed to the early studies involved numerous regular subcutaneous injections studies with autogenous vaccines. From the 1920s to 1960s, over a prolonged treatment duration, which contrasts with HSV vaccines were prepared using non-attenuated or partly most other studies. This observation, together with the attenuated virus from the patient’s own lesions, from temporary efficacy following vaccination reported with heterologous lesions, or from infected animal tissue. A wide other vaccines, indicates that vaccination for herpes may variety of vaccine types have been investigated for HSV, with need sustained administration akin to suppressive antiviral drug treatment. no or very limited success. One of the earliest immunotherapeutic agents developed Research into the use of therapeutic vaccination against HBV has been more restricted, due to the relatively recent for HIV is “Remune”, a whole killed gp120-depleted HIV-1 discovery of the virus being the cause of the disease and the vaccine in incomplete Freund’s adjuvant (table 4). Remune development of effective preventive vaccines in the 1980s. significantly increased lymphocyte proliferative responses to Recent investigations have evaluated these prophylactic p24 antigen in HIV-1 seropositive patients82 and enhanced HIV-1 antigen-specific T-helper-cell function.83 Results vaccines as such or improved for the treatment of CHB. Vaccines were initially investigated as a potential therapy from further studies have indicated a beneficial effect of for HIV disease in 1985.38 Early candidate vaccines were based Remune on the control of HIV infection. In humans, on envelope glycoproteins (gp120 and gp160). Since then Remune was shown to have an effect on the major comore than 6000 healthy volunteers have participated in 60 receptors for HIV-1 and their ligands.124 different phase I/II trials of 30 different candidate vaccines. An overview of the types of vaccines evaluated for Recombinant subunit vaccines therapeutic use in the treatment of HBV, HPV, HSV and The availability of genetic engineering in the 1980s resulted HIV infections is provided below. For more complete in the research and development of recombinant subunit descriptions, readers should refer to the tables 3, 4, 5, and 6 vaccines. These are generally regarded as safer than inactivated vaccines because there is no possibility of the and to other reviews.22,39—41 vaccine containing contaminated DNA or infectious virions, although they contain fewer antigens and are less Inactivated vaccines Active immunotherapy for treatment of CHB was first immunogenic than inactivated whole virion or envelope attempted in 1982 with a prophylactic plasma-derived HBsAg vaccines. However, immunogenicity can be enhanced by the vaccine (“Heptavax”-B, Merck, Sharp & Dohme).68 The use of adjuvants. Sexual contact with HPV-infected partner

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Results from phase II studies with recombinant protein- both humoral and cellular immune responses. Additionally, based prophylactic vaccines in patients with CHB have been chimeric L1 VLPs can be combined with L2 alone or disappointing, including “Genhavac” B (Aventis Pasteur), together with early proteins, thereby having the potential to which contains HBsAg and preS2 antigen,69,70 and act as an immunostimulant and as a carrier of other “Hepagene” (Medeva Ltd), comprising HBsAg, part of antigens, plasmid DNA, costimulatory proteins and foreign preS1, and all of preS2.74,75 antigen such as HIV1 gp41.128 In the mice tumor regression Investigations with specific, potent, Th1-oriented model, chimeric VLPs have been tested therapeutically with adjuvant systems, rather aluminium, are currently underway. very positive results.129 Immunogenicity results using L1 Vaccines containing HBV envelope proteins together with VLPs in healthy volunteers and patients with genital warts the adjuvant system AS2, containing 3D-monophosphoryl are promising.130,131 Attention has focused on the HSV glycoproteins B and D lipid A (MPL) and QS21 (Quillaria Saponaria) and an oil in water emulsion, induced high levels of anti-HBs antibodies, a (gB, gD) as subunit purified vaccines because they are targets strong lymphoproliferative response, high interferon , for both protective humoral and cell-mediated immunity. secretion and a potent CTL stimulation in healthy Vaccines containing gD, or a combination of gB and gD, volunteers.76—78 Encouraging preliminary efficacy results have have been shown to be immunogenic in animals and people. been obtained in CHB patients.113 Data from animal models indicated the importance of With respect to high-risk HPVs, development of adjuvants.48,132,133 Initial results with a vaccine developed by protein-based vaccines has focused on the early proteins, Chiron Biocine (gD+Alum; gBgD+MF-59) were promising especially E6 and E7, which are constantly expressed by the with the demonstration of significant decreases in the tumor cells, in combination with adjuvant and/or vehicles to Table 3. Therapeutic HSV vaccines enhance the cellular immune Vaccine or company Vaccine description References response. Inactivated whole-cell virus vaccine A HPV6 L2E7 fusion protein Eli Lilly Formalin-inactivated vaccine; rabbit kidney cells 42 Heat-killed virus 26 vaccine (Cantab Pharmaceuticals) "Lupidon" H: HSV1, G: HSV2 for the treatment of genital Dundarov vaccine Formalin-inactivated vaccine 43 warts has shown a trend towards Rabbit kidney culture fewer recurrences, albeit in Five strains of HSV1 and/or HSV2 an uncontrolled study. Both Inactivated purified envelope vaccines proliferative responses to L2E7 Kutinova vaccine HSV1 lectin purified glycoproteins on Al(OH)3; 44 and clearance of warts were formalin inactivated 114 HSV1 mixed glycoproteins observed. Although addition of Ac NFU1 (S) MRC5 Formalin-inactivated, detergent extraction 27, 45 MPL as an adjuvant enhanced (Skinner vaccine) Cappel HSV2 glycoproteins 46 125 immunogenic responses, Lederle gD1 purified by immunoaffinity chromatography+alum 47 further phase II efficacy studies Recombinant subunit vaccines revealed no difference from GD 100 µg+alum 48–51 placebo and the development was Chiron-Biocine GBgD+MTP+PE+MF-59 terminated (unpublished results). GBgD+MF-59; expressed in CHO cells Positive high-risk HPV-DNA GlaxoSmithKline Biologicals Truncated gD2 (20 g) produced on CHO+alum+MPL 52–54 Truncated gD1 produced on CHO cells 55 sequences are associated with Genentech HSV2 glycoprotein expressed on baculovirus+Al (PO4) 56 virtually all cervical cancers, with Lederle Praxis Biologicals gD related peptides HPV 16 and 18 being the most Takeda Recombinant+tgD1-IL2 57 prevalent types.126 Regression of Genetically engineered HSV mutants: replication defective mutants tumour lesions in the murine DISC vaccine Deletion of non structural protein ICP 4-8-27 58 model have been demonstrated, 5 Black Z virus Recombination of a mutant HSV-1 ICP8 gene 59 together with both humoral and into a HSV-2 wild type strain cellular immune responses, Genetically engineered HSV mutants: single-cycle mutants following vaccination with R7017–R7020 Deletion of sequences in HSV1 to reduce 60 recombinant HPV16 E7 protein virulence and neutrotropin Addition of gG and gD from HSV2 plus MPL and QS21 as ICP34.5 deleted HSV Deletion of neurovirulence gene ICP34.5 61 adjuvants.127 gH deleted mutant (SC16AgH) HSV-1 mutant lacking glycoprotein H, necessary 62 Capsid proteins (L1 and L2), (DISC vaccine) for virion entry into cells which have mainly been RAV9395 Deletion of UL55, UL56 and 1 34.5 genes 63 Functions as a live attenuated HSV-2 vaccine developed for prophylactic use, have the ability to self assemble Other vaccines into virus like particles (VLPs) Viral vectors Vaccinia: VgD52 (HSV1) 64 VP176 (gD1), VP221 (Gd2) 65 when expressed recombinantly in Adenovirus: gB1 (AdgB8) 66 various systems. L1 VLPs are Plasmids encoding for gC, gD, gE from HSV1 (50 g) 67 highly immunogenic, producing DNA vaccine

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frequency of recurrences in patients with genital herpes,49 but no efficacy was shown in a subsequent study.50 A second subunit therapeutic vaccine developed by GlaxoSmithKline Biologicals consists of a recombinant HSV2 gD2t in combination with a potent adjuvant, MPL. This formulation was shown to induce a strong humoral and cellular immunity, predominantly of the Th1 type, both in HSV seropositive and HSV seronegative subjects.52,53 The importance of the Th1 type immunity and of the adjuvants has been clearly demonstrated when this vaccine was shown to have clear prophylactic efficacy in HSV-negative women.54 The Chiron vaccine which contained gD+gB+MF-59, a Th2 biased adjuvant, had previously failed to demonstrate efficacy in the same setting.134 Isolation of HIV closely followed the successful development of a recombinant protein vaccine for HBV; it was hoped that a similar approach could be effective in the treatment of HIV. The earliest generations of HIV-1 vaccines were initially limited to monomeric gp120 or gp160 proteins. First generation envelope antigens induced in vitro neutralising antibodies; however, these antibodies were mainly directed against homologous laboratory-adapted viral strains, being ineffective to neutralise clinical isolates of HIV-1.135 Vaxgen subsequently developed “AIDSVAX”, a second generation clade B bivalent gp120 based on one laboratory and one primary isolate of HIV, which is currently being evaluated in phase III trials as a prophylactic vaccine.88 The potential of gp160 has mostly been studied with “VaxSyn”, a recombinant HIV envelope subunit gp160 vaccine. Although early phase I studies showed VaxSyn to be immunogenic and safe,136 subsequent trials in HIV-1infected individuals have failed to show any efficacy.137 p24 VLP has been developed as a candidate vaccine against HIV infections. Results have been disappointing with only a marginal effect on immunogenicity in HIV-positive individuals.90 Promising results have been obtained in rhesus monkeys immunised with a combination of gp120 and nef/tat regulatory proteins in the AS02 adjuvant and subsequently challenged intravenously with a highly pathogenic, partially Table 4. Therapeutic HBV vaccines Vaccine/company Vaccine description Inactivated purified envelope vaccines

References

MSD

68

HBsAg+alum (“Heptavax”) S-pre S2+alum (“Genhevac”) S-pre S1-pre S2 (complete L)+alum

69, 70 71

S-pre S2+adjuvant (MF-59) 72, 73 S + pre S1+pre S2+Alum (“Hepagene”) 74, 75 S + AS2 adjuvant (MPL-QS21— 76–78 oil/water emulsion)

Other vaccines Cytel Corporation Core lipopeptide Powderject HBs-DNA vaccine Shanghai Institute HBsAg-anti-HBs Ab complex of Biological Products

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Genetically engineered HSV mutant vaccines

Several groups have focused on genetically engineered, replication impaired mutants of HSV for therapeutic vaccination. The potential advantages of such as approach are two-fold: endogenous expression of viral proteins resulting in a broad, multiclonal immune stimulation, and a safety profile akin to that of inactivated virus vaccines. Through genetic manipulation, potentially harmful sequences can be deleted including those needed for replication, whilst genes for promoting immunogenicity can be added. There are, however, potential safety issues regarding the use of mutants in respect of their potential to establish latency, to reactivate, to recombine with virulent wild-type virus, and to induce oncogenicity.29 Efficacy has been demonstrated with these vaccines used prophylactically in various animal models, one of which, a HSV-2 replication-impaired vaccine (Cantab Pharmaceuticals, DISC/TA-HSV vaccine), was found to be immunogenic in HSV seropositive and seronegative volunteers,52,53 but in patients showed limited efficacy139 and the company has since announced the end of the program. Other genetically modified vaccines have also reached early clinical development but were not pursued. Antigen-antibody complexes

One group in Shanghai, China, has combined HBsAg and anti-HBs antibodies, thus forming immune complexes to induce humoral and cytotoxic T-cell immune response. Immunogenic effects and efficacy using the solid matrix antigen-antibody complex (SMAA) have been demonstrated in HBV-infected ducks.140 A complex formed from plasmaderived HBsAg hepatitis vaccine and anti-HBs human hepatitis B immunoglobulins showed potentially efficacious effects in HBeAg-positive CHB patients.81 However, this approach has the disadvantage of using human plasmaderived components. HBV core antigen as epitope carrier

Recombinant protein based vaccines Aventis Pasteur Biotechnology General Ltd Chiron Corporation Medeva Ltd GlaxoSmithKline Biologicals

heterologous SHIV; CD4 cell counts were maintained, viral load was strongly reduced, with no fatalities more than 24 months after virus challenge.89 This vaccine has entered clinical studies in both the prophylactic and therapeutic settings. Since the same adjuvant has promoted protection in man against malaria challenge,138 this approach is promising.

The use of the HBc particle as an epitope carrier moiety is supported by a number of properties of the HBc particle, which include an increased immunogenicity at B-cell, T-cell, and CTL levels to its own epitopes in addition to a high acceptance of insertions of foreign epitopes with concomitant increase in immunogenicity. Promising results with constructions of preS1 and preS2 epitopes fused to various sites in the core antigen have been obtained, with induction of a strong cellular and humoral immune response, without the need for additional adjuvantation.141 Chimeric HPV 16 E4-HBc recombinant particles have also been shown to exhibit HPV-16-E4 reactivity.142

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Table 5. Therapeutic HIV vaccines Vaccine/company Vaccine description Inactivated whole cell virus vaccine

References

Remune/Immune Corporation

82–84

Whole killed HIV-1, depleted gp120, clade A envelope, clade G gag; incomplete Freund's adjuvant +HAART +prior ODN immunostimulatory DNA

85, 86 87

Recombinant gp160 Bivalent gp120 Recombinant gp120, nef/tat genes; AS2 adjuvant

32 88 89

Envelope vaccines VaxSyn AIDSVAX/Vaxgen GlaxoSmithKline Biologicals

Core proteins/gene products vaccines p24 VLP/Chiron Pharmaceuticals p24 and p17 core proteins p24 VLP +/-zidovudine

90 91

Viral vectors ALVAC1452 (vCP1452) ALVAC-HIV (vCP205) Aventis Pasteur NYVAC-SIV Eurovax Coxackie-HIV Fowlpox (rFPV) Modified vaccinia virus Ankara (MVA) Merck Research Laboratories

Recombinant canarypox carrier: gag, pol, env, nef genes +/-p24E-V3 MN +gp120 +prior HAART Vaccinia virus+SIV genes/peptides +prior HAART Recombinant viral vector +HIV-1 gag/pol-ITN DNA prime/MVA boost Recombinant adenovirus 5

DNA vaccine HIV-1 gene constructs Merck Research Laboratories

nef, rev, tat genes +/-HAART Cytokine-augmented DNA Recombinant adenovirus/DNA MVA/DNA

Other vaccines Tat protein

Tat toxoid

Peptide vaccines

92 93 94 95, 96 97 98 99 100, 101 102, 103 102, 103

a lipidated E7 peptide, although no clinical response was detected.119,148 In early stage clinical disease, vaccination with an E7 peptide vaccine elicited CTL and interferon  responses with partial or complete regression in 50% of patients.149 Immunogenic enhancement of the potency of HPV-16 E7 peptide-based vaccines using adjuvants, including Montanide ISA 51,120 immune stimulatory complexes (ISCOMS),150 and immunostimulatory carriers (ISCAR)121 has been variable and may reflect the importance of choice of adjuvant, together with route of administration, in the determination of immunogenic effects. DNA vaccines

The use of naked DNA for vaccine immunotherapy is currently the 104–106 subject of intensive investigation 107, 108 (for review see151). The basis for 109 this vaccination is injection of a 102, 103 DNA plasmid containing a specific nucleic acid sequence that leads to 110 in vivo expression of the encoded gene products and subsequent development of specific cellular and humoral immune responses directed against these antigens. As for subunit vaccines, nucleic acid-based vaccines can express few viral proteins making their immunogenicity narrow; unlike subunit vaccines, they induce cell-mediated responses without needing adjuvants at least in small animals. Their immunogenicity in non-human primates and in human beings is much weaker when they are used alone. Safety concerns using this concept

MHC class I CTLs are crucial for the prevention, control, and cure of infectious diseases in general and of HBV infection in particular. The administration of peptides associated with MHC class I molecules will, in theory, induce specific CTL activity. A lipoprotein-based therapeutic vaccine (Cytel Coorporation) was found to elicit CTL production in healthy volunteers143 and to a lesser extent CHB patients, although no Table 6. Therapeutic HPV vaccines clinical efficacy was demonstrated.144 Vaccine/company Vaccine description References A number of phase I and II Live vector vaccines studies have been conducted, Xenova Recombinant vaccinia; modified E6 and E7 111 following encouraging preclinical from HPV16 and 18 Recombinant vaccinia 112 data, using peptides derived from the Sig/E7/LAMP-1 HPV E6 and E7 protein combined Protein-based vaccines L2E7 fusion protein, HPV6 113, 114 with the human HLA-A*0201 MHC Xenova/GlaxoSmithKline class I allele.145 In terminally ill Biologicals HPV11 L1 VLP on Al(OH)3 115 cervical cancer patients, stabilisation Med Immune CLS, Australia HPV16 E6–E7 protein with chelating "Iscomatrix" 116 of disease and tumor regression were Stress Gene Biotechnologies HPV16 E7+BCG HS/65 117 observed in some patients, with Chiron Pharmaceuticals HPV16 E2 protein with MF59 adjuvant 118 limited T-cell immune response Other vaccines detected.146,147 E6 and E7-specific Peptide HLA H2 restricted HPV16 E7 peptide+PADRE 119 cytotoxic responses have been elicited HPV16 E7 peptide+PADRE+Montanide ISA 51 adjuvant 120 HPV16 E7 peptide conjugated with ISCAR 121 in patients with advanced cervical DNA plasmids expressing early/structural proteins 122 cancer following treatment with E6 or DNA DNA plasmid+gold microparticles injected subcutaneously 123 E7 HLA-A*0201-binding peptides or

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relate to the oncogenic potential following possible integration into the host genome, but this has never been demonstrated despite extensive evaluation. DNA vaccines with plasmids carrying genes encoding various HBV envelope proteins have induced high antibody titres and a strong cellular response after a single injection, both in mice and chimpanzees.152 A plasmid encoding HBsAg coated on gold particles using a transdermal gene delivery system (Powderject XR1) administered to healthy volunteers elicited a low but protective antibody response.80 Investigations are ongoing with other specific genetic constructions. Of potential significance with respect to HBV therapeutic vaccination is the co-delivery of DNA vaccines with cytokines, which have been shown to modulate the T-helper response towards predominantly Th1 or Th2, depending upon the type of cytokines.153 In the treatment of HPV infections, DNA plasmids constructed expressing various altered early proteins and/or structural proteins are under investigation.122,154 The development of HSV DNA vaccines has been relatively limited. Plasmid encoding for envelope glycoproteins (eg, gC, gD and gE) have been shown to induce both humoral and cellular immune responses in animals and to provide protection following HSV challenge.155,156 DNA vaccines have currently only been evaluated for prophylactic usage. Antigen-specific immune responses against HIV-1 have been induced following DNA vaccination of primates.157 Encouraging results have also been obtained using DNA vaccine therapeutically in chimpanzees.158 Among the first human trials of DNA-based vaccines, asymptomatic HIVinfected individuals immunised with DNA constructs containing the nef, rev, or tat genes resulted in detectable memory cells and specific cytotoxicity.104 Following the subsequent administration of HAART, induction of new HIVspecific responses were additionally observed.105 The immune response as well as the protection against viral challenge in rhesus monkeys of a DNA vaccine expressing env and gag antigen were significantly increased by coadministration of a plasmid encoding for interleukin-2 and the Fc portion of IgG.107 This vaccine did not protect against infection but showed a good effect against the development of disease.108 There was a significant correlation between the level of CTL response and the control of viraemia. However, a single nucleotide mutation within an immunodominant gag CTL epitope in an animal with undetectable plasma viral RNA resulted in viral escape from CTLs and clinical disease progression.159 This observation outlines the potential limitations of vaccine strategies based exclusively on the induction of a CD8 response, both for prophylactic and therapeutic vaccination. Viral vector vaccines

Viral vectors offer the potential for effective gene transfer to mammalian cells thereby introducing immunogenic proteins to the immune system. On injection into the host, replication occurs with expression of the foreign gene product, thereby inducing humoral and cellular immune responses to both vector and foreign gene product.

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Although both antibody and CTL responses against HBV have been elicited in mice and primates with a variety of vectors,160 limited investigations have been conducted to date. A recombinant vaccinia vaccine expressing modified HPV E6 and E7 induced HPV-specific CTL responses in patients with pre-invasive and invasive cervical cancer.111 Another recombinant vaccinia vaccine, Sig/E7/LAMP-1, has also shown a significant antitumour effect and specific CTL cell responses which were enhanced following combined therapy with Sig/E7/LAMP-1 DNA vaccine.112 Several vaccines have been investigated for the treatment of HSV with a variety of viral vectors such as adenovirus, adeno-associated virus, canarypox, varicella-zoster virus, Salmonella typhimurium and vaccinia. They generally are immunogenic and protective against challenge in animal models,39 but have not been evaluated therapeutically in animals or people. A number of organisms are being evaluated as potential vectors for HIV vaccines;161 the most commonly tested has been the poxvirus. VCP1452 (ALVAC1452) is a recombinant canarypox virus vaccine carrying the core gag, pol, env, and nef sequences of HIV-1 and has been evaluated alone or in combination with gp120 protein. In early investigations in uninfected volunteers, clade B-based canarypox vaccines were shown to elicit broad CTL reactivities capable of recognising viruses belonging to genetically diverse HIV-1 clades.92 Further studies in HIVnegative volunteers showed the potential of ALVAC as a means of inducing a cellular immune response, and also as a possible priming agent for other subunit antigens such as envelope glycoproteins, pseudoparticles, or new peptides.93,94 The “prime-boost” combined approach is increasingly becoming the focus of development strategies in this area. Several recombinant attenuated vaccinia vectors, adenovirus vectors, and recombinant canarypox vectors have been evaluated in phase I trials alone and in combination with a recombinant protein envelope or DNA boost. All recombinant viral vectors have been safe and immunogenic to date and have been shown to effectively prime the immune response to an envelope or DNA boost. Preclinical studies with NYVAC, a highly attenuated vaccinia virus that can be combined with SIV or HIV genes or peptides, have shown promising efficacy results. Early SIV challenge studies in monkeys vaccinated with NYVAC-SIV demonstrated control over viral load with delayed disease progression.97,162 A recombinant modified vaccinia Ankara (MVA) vaccine has been extensively evaluated alone or in combination with DNA vaccine.102,103 In a challenge study in rhesus monkeys comparing several vaccine vector delivery systems—plasmid DNA vectors, MVA, and a replication incompetent adenovirus type 5 (Ad5) vector—expressing the SIV gag protein,109 the Ad5 vaccine elicited the most effective cellular immune response and the best protection against disease after viral challenge. All those vaccine formulations are currently in phase I clinical trials both in HIV-uninfected and HIV-infected human volunteers.

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Combination of antiviral drugs and therapeutic vaccination strategies

The potential for a synergistic reaction on combining immunomodulatory therapy with antiviral drugs in the treatment of HIV and HBV is a very appealing concept. In principle, an antiviral could reduce the viral load, in addition to any viral-induced immunotolerance, thereby allowing an enhanced response by the immunotherapeutic compound. In CHB patients, lamivudine therapy has induced restoration of the T-cell responsiveness,163 while HAART also induces an immune reconstitution in HIV infected patients.164 Promising results were obtained in HBV-infected woodchucks (WHBV model) using a new antiviral (L-FMAU, Triangle Pharmaceuticals) and WHsAg vaccine in alum.165 Phase II studies are underway with HBsAg together with the adjuvant system AS2, containing MPL and QS21, (GlaxoSmithKline Biologicals) and with a HBV vaccine comprising preS2 and S antigens in MF59 adjuvant (Chiron Corporation), both in combination with lamivudine. Strong proliferative responses have been demonstrated to different whole-killed HIV-1 and core proteins from different clades after treatment with Remune in HIV-1 infected subjects receiving antiviral therapy.85 In a recent study in SIV-infected macaques undergoing treatment with HAART, cellular immune responses were demonstrated following treatment with a recombinant attenuated poxvirus vaccine (NYVAC), but only in those animals whose viral load had been markedly reduced by HAART; when HAART was stopped, the rebound in viral load was significantly greater in those animals receiving placebo than those receiving the vaccine.98 A strategy in the treatment of HIV that is gaining considerable interest is the use of vaccines in association with a procedure named structured therapeutic interruption (STI) to enhance induction or restoration of immune responsiveness in HAART-treated patients (for review see166); theoretically the preserved immunity in these patients could be boosted by exposing temporarily and repetitively the immune system to the natural virus by altering periods with and without antiretroviral therapy. STI in the treatment of HIV-1 shortly after infection has been shown to induce a better immune control of viral replication than during chronic infection.166 Improved cellular immune responses induced by a recombinant canarypox vaccine expressing env, gag, pol, and nef genes used together with recombinant gp160 (ALVAC 1452/gp160) in HAART-treated primary infection patients were recently shown to temporally suppress HIV-1 rebound kinetics after drug treatment interruption and enhanced cellular immune responses.95 Promising results from a further STI study have demonstrated significant increases in the frequency of CTL responses and number of epitopes recognised post-STI.167 Larger studies have been initiated to substantiate these results and include the QUEST trial, which aims to assess the efficacy of ALVAC with or without Remune vaccine in addition to HAART in primary infection patients to delay the viral load rebound after stopping both HAART and vaccination.168 Therapeutic interruption

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following vaccination of patients while still on HAART is a design currently broadly used for the evaluation of therapeutic vaccines and numerous clinical studies are planned or ongoing. Prolonged antiviral therapy is effective against frequently recurring genital herpes, but the disease often returns on discontinuation of treatment. On combining the immune modulator imiquimod and an HSV2 glycoprotein vaccine, both the duration and extent of protection from recurrences was enhanced compared with imiquimod alone.169

Conclusions Immunotherapy and especially active, specific immunotherapy or therapeutic vaccination is in theory a very promising approach for the treatment of chronic viral infections. Deficient immunological mechanisms are involved in the establishment of chronic infection, in its persistence, and/or in the pathogenesis of clinical disease. In some cases, successful spontaneous control of the infection is associated with profound modifications in the systemic or local immune patterns with intense activation of specific immune mechanisms. Usually, those immune responses are T-cell associated, especially T-helper 1 and cytotoxic T lymphocyte responses. Also, numerous studies in various animal models have delivered positive results with candidate therapeutic vaccines, mainly in HPV and HSV infections. The concept of therapeutic vaccination is almost one century old, and virtually all possible vaccine designs have been or are being evaluated for the treatment of HBV, HIV, HSV, and HPV. Unfortunately, until now, none has clearly proven efficacy. The most ancient and the largest experience lies in the HSV field. Despite very promising results in guineapigs and some anecdotal positive studies in man, recent large, well-controlled studies have been disappointing. An inactivated whole-cell vaccine is claimed to have efficacy and is marketed in some European countries. It is the only vaccine that has been administered regularly over a long period. This, together with the short-lasting efficacy observed early after the stopping of classic vaccination schedules with other HSV vaccines, could indicate that immune control of chronic viral infections such as HSV or HIV requires long-term, regular vaccine administration. Therapeutic vaccines to HBV and HPV are, in theory, the most feasible approaches to control of chronic infections in view of the immune-correlated control of infection observed naturally. All attempts at vaccination in CHB have failed so far, probably because they have used formulations only derived from preventive vaccines—ie, based on surface antigens. This approach contradicts the observed predominant CD4 and CD8 responses to the core antigen during HBeAg seroconversion and the suspected tolerogenic role of HBsAg. In addition, it is likely that the immune approach will have to be adapted to the virological, serological, and clinical status of the infection. The selection of the right antigens will also be critical in the development of HPV therapeutic vaccines. Although several candidate vaccines are promising, the pathogenesis of the infection and disease is so complex that clinical demonstration of efficacy will be very long and difficult.

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Search strategy and selection criteria Data for this review were identified by searches of Medline, Current Contents, and references from relevant articles. Numerous articles were identified through searches of the extensive files of the authors. Search terms were “therapeutic vaccine”, “immunotherapy”, “HIV”, “HBV”, “HSV”, and “HPV”. English language papers were reviewed.

HIV chronic infection will probably be one of the most challenging targets for the development of therapeutic vaccination. Evidence supporting the approach is limited to interpretation of SHIV monkey results in the prophylactic setting and to rare cases of natural control of infection in anecdotal long-term non-progressors. In addition, the outcome of HIV infection is precisely a progressive destruction of the immune cells that could have a key role in viral control. Most of the current strategies for an HIV vaccine concentrate on the induction of a CD8+ T-cell response. However, it has been shown recently that a single point mutation can allow a viral escape and development of disease. Therefore, a broader immune response, including a CD4+ T cell and

References 1 2 3

4 5 6 7

8

9 10 11 12 13

15 14 16

17

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a strong antibody response is likely to be needed, both for prophylactic and therapeutic vaccination. Future therapeutic HIV vaccine approaches will likely combine different vaccine strategies in prime-boost regimens to broaden and increase the immune responses. In addition, therapeutic vaccination is currently combined with antiretroviral therapy and the objective of its development is limited to temporary cessation of HAART. In conclusion, there is a strong rationale for therapeutic vaccination as an alternative approach to the treatment of chronic viral diseases. However, the development of therapeutic vaccines has proven to be more complex than initially expected. Future strategies should pay attention to features such as patient characteristics, mode and duration of administration, careful selection of antigens, and adequate immune stimulations. Acknowledgements

I thank Sarah Benns for her contribution to this manuscript in terms of literature research and editorial assistance. Conflicts of interest

I have no conflicts of interest to declare.

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Clinical picture Silent cardiac echinococcosis An 11-year-old boy was admitted because of sudden left hemiparesis. Chest radiographs, electrocardiogram, and fundoscopic examination were normal. Blood count showed only microcytic anaemia. A magnetic-resonance imaging (MRI) brain scan disclosed multiple brain cysts (0·8–4cm) mainly in the posterior parietal regions (figure, top). Liver function tests were normal. ELISA titres of echinococcal antibodies were positive (1/16384). Three superficial brain cysts were excised and histological examination confirmed the diagnosis. Extensive imaging with ultrasound and computed tomography (CT) did not disclose involvement of any other abdominal or retroperitoneal organ. Albendazole for primary brain echinococcosis was started at 400mg/day. Improvement in motor function followed and the child was discharged. In the first follow-up, a grade-two systolic heart murmur at the apex was noticed. An echocardiogram showed an intracavitary mass in the left ventricle (figure, bottom). The mass was attached to the cordae tendini of the posterior mitral leaflet and was in contact with the posterior wall of the left ventricle. There was moderate mitral regurgitation. A cardiac MRI disclosed a 22·5cm mass without typical cystic components. The mass was excised and all but one of the cordae were preserved. Histological examination of the specimens showed necrotic larvae and the echinococcal capsule attached on the cordae. Repeat echocardiograms at 1 and 3 months showed moderate mitral regurgitation and no evidence of new cyst formation. A new CT scan showed a decrease in size of the six remaining brain cysts. The antibody titre was 1/512 at that time. Periklis A Davlouros, Ignatios Ikonomidis, Konstantinos Frimas, and Antonis S Manolis Top. T1 weighted brain MRI scan showing multiple cystic lesions and cerebral oedema. Bottom. Long axis parasternal two-dimensional echocardiographic view. An echogenic mass (arrow) attached on the posterior left ventricular wall and mitral valve cordae. RV=right ventricle, LV=left ventricle, LA=left atrium, Ao=aortic root, MV=mitral valve.

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Departments of Cardiology and Paediatrics, Patras Medical School General University Hospital, Rio, Patras, Greece. Correspondence: Antonis S Manolis, Professor of Cardiology, 41 Kourempana Street, Agios Dimitrios, Athens 173 43, Greece. Email [email protected]

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