Introduction
Viruses linked to cancers in humans are the Epstein-Barr virus (EBV), associated with lymphomas and nasopharyngeal cancer, hepatitis B virus (HBV) and hepatitis C virus (HCV), both associated with cancer of the liver, human papillomaviruses (HPV), associated with cancer of the cervix, human T lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2), associated with adult T-cell leukemia and with hairy-cell leukemia, respectively, and human herpesvirus 8 (HHV-8), associated with Kaposi sarcoma. No vaccine exists against these viruses except HBV. This chapter will describe vaccines under development against EBV, HCV and HPV.
Epstein-Barr virus
In the 1950s, Denis Burkitt described the existence of B-cell lymphomas in 2–14 year-old African children from malaria endemic areas. In 1964, continuous B-lymphocyte cell lines derived from these tumors were found by Epstein and Barr to spontaneously release a herpesvirus. It was Gertrud and Werner Henle who demonstrated that the Epstein-Barr virus (EBV) is ubiquitous in the human population where it is usually the cause of infections that are not apparent though it may cause infectious mononucleosis. The more severe, albeit rare, result of EBV infection is malignant transformation and cancer development in various forms, including Burkitt’s lymphoma and nasopharyngeal carcinoma, one of the most common cancers in China.
The primary site of Epstein-Barr virus (EBV) infection is the oropharyngeal cavity. Children and teenagers are commonly afflicted usually after oral contact, hence the name “kissing disease”. Based on serology, about 95% of the world adult population has been infected with EBV and, following primary infection, remains lifelong carriers of the virus. In developed countries, exposure to EBV occurs relatively late: only 50–70% of adolescents and young adults are EBV seropositive. About 30% of the seronegative group will develop infectious mononucleosis as a result of primary EBV infection. The disease is characterized by fever, sore throat, generalized lymphadenopathy, splenomegaly, intense asthenia, hyper-lymphocytosis (>50%) with atypical lymphocytes and elevated transaminase levels. In developing countries, EBV antibodies are acquired early in life and the disease is mostly asymptomatic.
EBV is associated with Burkitt’s B-cell lymphoma and nasopharyngeal carcinoma. Burkitt's lymphoma (BL) is a malignant form of tumor associated with EBV that is endemic to central parts of Africa and New Guinea with an annual incidence of 6–7 cases per 100 000 and a peak incidence at 6 or 7 years of age. The epidemiological involvement of EBV in Burkitt's lymphoma is based on the recognition of the EBV viral genome in tumor cells, associated with an elevated antibody titre against EBV viral capsid antigen (VCA). The highest prevalence of BL is found in the "lymphoma belt," a region that extends from West to East Africa between the 10th degree north and 10th degree south of the equator and continues south down the Eastern coast of Africa. This area is characterized by high temperature and humidity, which is probably the reason why an association of malaria with BL was suspected at one time. In African countries such as Uganda, in the lymphoma belt, the association of BL with EBV is very strong (97%), whereas it is weaker elsewhere (85% in Algeria; only 10–15% in France and the USA).
Nasopharyngeal cancer (NPC) incidence rates are less than 1 per 100 000 in most populations, except in populations in southern China, where an annual incidence of more than 20 cases per 100 000 is reported. Isolated northern populations such as Eskimos and Greenlanders also show high incidence. There is a moderate incidence in North Africa, Israel, Kuwait, the Sudan and parts of Kenya and Uganda. Men are twice as likely to develop NPC as women. The rate of incidence generally increases from ages 20 to around 50. In the USA, Chinese-Americans comprise the majority of NPC patients, together with workers exposed to fumes, smoke and chemicals, implying a role for chemical carcinogenesis. Studies related to nutrition and diet have shown an association between eating highly salted foods and NPC. Vitamin C deficiency at a young age also may be a contributing factor. Finally, a study of HLA haplotypes revealed a genetically distinct subpopulation in southern China, with an increased frequency of haplotype A-2/B-Sin-2 which may account for the higher disease incidence in the area.
Recent studies have shown that EBV also is associated with B-cell malignancies such as Hodgkin’s lymphoma (HL) and lymphoproliferative disease in immunosuppressed patients, as well as with some T-cell lymphomas and other epithelial tumors such as gastric cancers. These tumors are characterized by the presence of multiple extrachromosomal copies of the viral genome in tumor cells and the expression of part of the EBV genome.
EBV, together with HHV-8 (Kaposi sarcoma-associated virus), belongs to the genus Lymphocryptovirus, in the subfamily Gammaherpesvirinae, family Herpesviridae. These are complex enveloped DNA viruses, which multiply in the nucleus of the host cell (see 5.3). EBV infects resting human B-lymphocytes and epithelial cells, multiplies in the latter and establishes latent infection in memory B-lymphocytes. Thus, infected individuals may produce virions, carry virus-specific CTLs, produce EBV-specific antibody, and yet harbor latently infected memory B-cells. These maintain the latent EBV genome as an episome that expresses only part of its genetic information, including EBV nuclear antigens EBNA-1 (a latent DNA replication factor), EBNA-2 (a transcriptional activator) and EBNA-3A and -3C (involved in the establishment of latency), together with integral membrane proteins LMP-1 and LMP-2 which play major roles in maintenance of latency and escape from the immune response of the host. Latently infected cells do not produce the B7 coactivator receptor and, therefore, are not killed by CTLs. When peripheral blood from an infected individual is cultured, latently infected B-cells begin to replicate and yield immortalized progeny lymphoblasts that can be indefinitely propagated in the laboratory.
The major EBV external surface glycoprotein is a 350 kD antigen, gp350/220, which binds the CD21 receptor on B-cells. Another envelope glycoprotein, gp42, is responsible for the fusion between the virus envelope and the host cell membrane. The EBV genome, a 172 kbp linear double-stranded DNA molecule, becomes circular for replication and latency. Viral capsid antigens (VCA) are late gene products.
The development of an EBV vaccine could protect individuals against primary infection and hence presumably reduce the burden of EBV-associated cancers.
The principal target of EBV neutralizing antibodies is the major virus surface glycoprotein gp350/220. Several vaccine candidates based on gp350/220 have been developed. Live recombinant vaccinia virus vectors have been used to express the gp350/220 antigen and were found to confer protection in primates and elicit antibodies in EBV-negative Chinese infants.
Soluble recombinant gp350/220 produced in CHO cells was found to be safe in humans but needed strong adjuvants to elicit acceptable immunogenicity (co-development by MedImmune, GSK and Henogen). Phase II clinical trials of this candidate vaccine are under way.
Clinical trials of an EBNA-3A peptide are being conducted in Australia.
Hepatitis C Virus
Updated 8 February2010 by Ellie Barnes UK
The need for an HCV vaccine
Hepatitis C virus (HCV) in a virus that infects the liver. Most people that are infected develop persistent infection. A proportion of people (20-50%) develop progressive liver disease leading ultimately to liver cirrhosis, liver failure and hepatocellular carcinoma
[1].
HCV is globally distributed and it is estimated that up to 170 million people (3% of the worlds population) are infected world wide
[2].
Hepatitis C is transmitted via infected blood. In parts of Asia and Africa 2-10% of people are infected and infection is frequently due to the use of contaminated blood products, medical instruments and tattooing. In Western Europe and North America less than 1% of the population are infected and infection is largely confined to at risk populations including those that received blood transfusions before the screening of infected blood products and intra-venous drug users. Sexual transmission and peri-natal transmission is unusual occurring in approximately 5% of cases. In 20% of people no cause of infection can be established.
A vaccine that prevents and treats HCV infection is urgently required. The target population would be at risk groups in developed countries and the entire population in many developing countries. No such vaccine currently exists but a number of approaches are currently in development.
A therapeutic vaccine would also be an invaluable adjunct to current treatment options for HCV. Today's gold-standard treatment for HCV consists of pegylated-interferon and ribavirin
[3]. This has been a remarkable advance is the treatment of HCV and may result in the permanent eradication of the virus in infected people. Nevertheless, it is currently prohibitively expensive, has an extensive side effect profile and is often ineffective. The current cure rates using this treatment is 45% for genotype-1 and -4 infection, 70% for genotype-3 and 80% for genotype-2, Once cirrhosis is established, a cure is less likely still and liver transplantation may be the only option.
There is currently intense research activity to establish new antiviral agents that target the HCV polymerase and protease, similar to the drugs currently used for HIV infection. Some of these are now in an advanced stage of development and may become available for clinical use in 2011
[4]
[5]. However, these new therapies are only effective for genotype-1 infection where they are expected to increase the cure rate to 70%, will be given in addition to interferon and ribavirin, and are likely to add to the cost of a treatment that is already unaffordable to most countries.
The challenges facing an HCV vaccine
One of the major challenges facing the development of a vaccine for HCV is the high degree of genetic diversity that is exhibited by the virus, estimated to be 10 fold higher than that seen in HIV. This is because the viral polymerase lacks proof reading capacity. The envelope protein, which is the major target for HCV antibodies, is particularly diverse. Based on the degree of genetic similarity, HCV has been classified into six major genotypes, that may be further subdivided into subtypes
[6]
[7]. Sequence homology between genotypes is approximately 80%. Specific genotypes are in general located in distinct geographical locations, whilst a small number of subtypes (1a, 1b, 2a and 3a) have recently become more widely distributed associated with modern practices such as medical injections, blood products and intravenous drug use. Other factors that have hindered vaccine development for HCV include the lack of an accessible animal model and the fact that the virus cannot be easily grown in the laboratory. It may not be possible to develop a vaccine that targets all HCV genotypes, but genotype specific vaccines that are administered in regions where specific genotypes dominate is a realistic goal.
Following primary (acute) infection with HCV a significant proportion of people will spontaneously eradicate the infection
[1].
This is quite different from HIV where viral persistence is inevitable following infection. In this setting, the induction of an effective immune response against HCV is thought to play a crucial role in eradicating the virus. Once chronic infection is established the antibody response persists, but the T cell response is weak or undetectable. It is not clear if the loss of T cell responsiveness is the cause or the consequence of chronic infection.
The exacts correlates of a successful immune response are not yet fully defined and probably involve interplay between multiple components of the immune system. This would include cytokines -chemicals such as interferons that inhibit virus production in infected cells, antibodies that bind to the virus and block viral entry, and T cells that target and kill infected cells.
To date, all successful vaccines against other infections work by inducing a protective antibody response. However, in HCV the predominant target for HCV antibodies is the highly variable envelope region and whilst it has been shown that these antibodies may prevent infection in chimpanzee models, these are only effective against a limited number of HCV strains
[8].
There is now strong evidence to show that a major component of a protective immune reaction includes the generation of robust CD8+ and CD4+ T cell responses that target parts of the HCV virus that is presented to T cells in association with self (HLA class I and II) molecules
[9]
[10]
[11]
[12]
[13]
. To some extent T cells must also deal with the problem of variability, but these may also target the more conserved "internal" proteins of the virus. Broadly reactive T cells that target multiple parts of the virus, a strong T cell response, and a "functional" T cell response that produces the right kind of cytokines and proliferate well will be required.
A number of groups are currently working to develop both T cell and antibody based vaccines to prevent and also to treat HCV infection. As a treatment it is thought that a vaccine may be more effective in the presence of a lower viral load. For this reason, the concept of vaccination as an adjunct to current therapy in the presence of a lowered viral load has emerged.
Many different approaches have been tried in HCV vaccine development. However, only a small fraction of animal and primate studies have progressed to human studies.
Recombinant protein vaccines
Recombinant HCV envelope (E1/E2) vaccines
The first candidate therapeutic vaccine was administered to humans in 2003, and consisted of recombinant HCV-E1 protein in alum adjuvant (InnoVAC-C developed by Innogenetics, Ghent, Belgium). The rationale for this was that pre-existing antibodies to HCV envelope proteins have been associated with a better response to interferon therapy. Following multiple injections, this vaccine induced both antibody and T cell responses in healthy and treatment naïve HCV infected patients
[14]
[15]
. Twenty-four patients received two courses of 6 injections and liver histology was assessed before and after vaccination. Plasma virus levels did not change but liver biopsy showed histological improvement in 9 patients. The observed increase in HCV-E1 antibody levels correlated with a decline in alanine transaminase levels (a measure of liver inflammation) and new E1 specific T cell responses were generated in 21 patients. Further studies on this vaccine have not been published and the company stopped its HCV vaccine programme in 2008.
A phase I clinical trials in healthy subjects of an E1/E2 heterodimer was conducted by Chiron/Novartis (Emeryville, California), following successful challenge experiments in chimpanzees (http://clinicaltrials.gov/ct2/show/study/NCT00500747). This study is currently unpublished. The same company is also developing a T cell vaccine using HCV core protein produced in yeast adsorbed onto immunostimulating complex matrix (ISCOM), following studies in macaques showing induction of both CD8+ and CD4+ T cell responses
[16]
.
Peptide Vaccines
A candidate therapeutic vaccine, IC41, was developed by Intercell AG (Vienna, Austria), based on 5 synthetic peptides containing 5 HCV HLA A2 restricted CD8+T cell epitopes, and 3 CD4+ T cell epitopes, in addition to a T cell adjuvant poly-L-arginine. This vaccine has been shown to generate proliferative HCV specific T cell responses and also IFN-gamma specific responses in ELISpot assays in both healthy and chronically infected HCV infected patients in phase I studies
[17]
. A larger phase II study included 60 HLA -A2 patients with genotype 1 infection, who had previously failed to respond to interferon based therapy
[18]
. Of these, 36 patients received 6 vaccinations with IC41 whilst the remainder received peptides or adjuvant alone. T cell proliferative responses and IFN-gamma ELISpot responses were observed in 67% and 42% of the IC41 vaccinees respectively. In 3 patients a decline of > 1 log viraemia was observed. The observed T cell responses were generally weak. More recently the same vaccine was administered to patients during combination pegylated-interferon and ribavirin therapy. Weak T cell responses were observed in vaccinated patients. However, no control arm of unvaccinated patients was included for comparison
[19]
.
A further study using a "personalised" approach has assessed the immunogenicity of four CD8+ A24 peptides in Freund's adjuvant administered to 12 patients with HCV, that had previously failed to respond to interferon therapy
[20]
. Antibody and CD8+ T cell responses to the peptides were assessed pre-vaccination in each individual and only those peptides that induced an immune response were then used for a further 14 vaccinations given 2 weeks apart. Augmentation of peptide specific T cell responses were reported in the majority of patients following 7 vaccinations, the first time point at which responses were assessed.
Finally, a T cell vaccine using HCV core protein produced in yeast adsorbed onto immunostimulating complex matrix (ISCOM) is under development by Novartis, following studies in macaques showing induction of both CD8+ and CD4+ T cell responses
[16].
Yeast based protein vaccine
Heat-killed recombinant Saccharomyces cerevisiae expressing a core-NS3 fusion protein (GI-5005) is developed as a candidate HCV vaccine by Globeimmune (Louisville, Colorado). A phase II clinical trial including 140 treatment naïve and prior non-responders to interferon recently reported an increase in rapid virological response and end of treatment response when vaccination is administered with interferon and ribavirin compared to a control group of standard therapy alone. No immunological data to support these findings has been published
(http://www.globeimmune.com)
.
DNA Vaccination:
ChronVac-C is a DNA based vaccine using plasmids expressing HCV proteins NS3 and 4a made by Tripep AB (Stockholm, Sweden). This is co-administered intra-muscularly with electroporation (EP) as a delivery system. EP consists of a number of short electrical pulses, which are said to be painful but shortlived. EP has been shown to enhance cellular responses probably through creation of pores in the cell membranes of target cells that enhance vaccine delivery. Additionally the damage to the cell membranes is thought to enhance a local inflammatory response at the site of vaccination
[21]
[22]
[23]
. EP, at least in mice is said to enhance the immunogenic response induced by DNA vaccination 10 fold
[24]
. A phase I clinical trial in 12 treatment naïve, genotype-1 HCV infected patients, with low viral load (< 800,000 IU/mL) is currently underway. Interim results suggest that 4/6 showed a decline in viral load of >0.5 logs with a concomitant increase in T cell reactivity in 3 of these patients
[25]
.
DNA vaccination using plasmids encoding structural proteins, has been combined with core protein (CIGB-230, Centro de Ingenieria Genetica y Biotecnologia, Havana, Cuba) in a recent trial of 6 intramuscular injections, 4 weeks apart in 15 patients with genotype-1 infection, who were non-responders to previous interferon therapy
[26]
. Weak anti-HCV proliferative responses were generated in some patients. However, whilst some patients showed a small increase in IFN-gamma ELISpot responses, others showed a reduction in responses following the last vaccination.
Virally vectored vaccines:
Transgene S.A. (Strasbourg, France) are developing a candidate therapeutic vaccine (TG4040) based on modified vaccinia Ankara (MVA) encoding the HCV NS3/4/5B proteins. Initial chimpanzee studies used a heterologous prime boost regimen of DNA encoding core-E2 and NS3 that showed reduced viremia upon viral challenge, although 3/4 animals developed persistent infection
[27]
. Phase I studies of 15 HCV infected human subjects are currently underway, in a dose escalation study involving 3 subcutaneous injections given weekly of MVA encoding NS3-5, followed by a boost with the same vector a month later in a sub-group of patients
[28]
. Early results show that three patients developed HCV specific T cell responses in response to vaccination.
Replicative defective adenoviral vectors that are genetically engineered to encode the non-structural proteins (NS3-NS5B) of a genotype-1b HCV strain have been developed by Okairos (Rome, Italy), and are currently in phase I clinical trials (Oxford, UK). This follows earlier studies in chimpanzees using a heterologous prime boost regimen of adenoviral/DNA (electroporated plasmid) vectors encoding the HCV NS proteins. This strategy induced broad CD8+ and CD4+ HCV specific T cell responses in 4/5 animals that were protected from heterologous viral challenge
[29]
. Since anti-adenoviral antibodies can limit the efficacy of these vectors, a number of adenoviral vectors derived from rare human adenoviral serotypes and also from chimpanzee adenovirus, to which humans have been rarely exposed, have been developed. A Phase I study of healthy volunteers, using a double prime/heterologous boost with two different adenoviral vectors has recently been shown to be highly immunogenic. A therapeutic vaccine approach using the same vectors in combination with interferon and ribavirin is currently underway in Oxford, UK.
Human papillomavirus
Human papillomavirus (HPV) causes cervical cancer, the second biggest cause of female cancer mortality worldwide. Estimates of the number of cervical cancer deaths are around 250,000 per year. The prevalence of genital HPV infection in the world is around 440 million. There are over 100 genotypes of HPV, 40 of which infect human mucosal areas of the upper digestive tract and the ano-genital tract. These are grouped into "high-risk" and "low-risk" types according to the degree of risk of development of cancer after infection with each genotype. Genital HPV infection is extremely common and most often causes no symptoms. A proportion of individuals infected with low-risk HPV types such as HPV-6 or HPV-11 will develop genital warts, whereas a subset of women with high-risk HPVs such as HPV-16 or HPV-18 will develop preneoplastic lesions of cervical intraepithelial neoplasia (CIN). Low-grade cervical dysplasias are common and most regress spontaneously. In contrast, the minority of lesions that progress to high-grade dysplasias tend to persist and/or progress to carcinomas in situ before becoming invasive cancers. The majority of adenocarcinomas of the cervix and of squamous cell cancers (SCC) of the vulva, vagina, penis and anus are caused by HPV-16 and HPV-18 (together accounting for about 70% of cases globally), the remaining 30% being due to other high-risk HPV types (such as HPV-31, -33, -35, -39, -45,-51, -66). The relative importance of different high-risk types varies between countries and regions, but type 16 has the greatest contribution to cervical cancer in all regions. HPV is also associated with other cancers of the anus, head and neck, and rarely, recurrent respiratory papillomatosis in children.
About 500 000 cases of cervical cancer are estimated to occur each year, over 80% of which occur in developing countries, where neither population-based routine screening (eg Papanicolau smear test) nor optimal treatment is available. The highest estimated incidence rates of cervical cancer occur in Africa, Central and South America and Asia.
Epidemiological studies in the USA have reported that 75% of the 15–50 year-old population is infected with genital HPV over their lifetime, 60% with transient infection, 10% with persistent infection (confirmed by detection of HPV DNA in genital samples), 4% with mild cytological signs, and 1% with clinical lesions.
HPV belongs to the family Papovaviridae. These are small nonenveloped icosahedral viruses with an 8 kbp-long double-stranded circular DNA genome. The papillomavirus genome comprises early and late genes that encode early proteins E1–E7 and late proteins L1–L2. The early proteins are nonstructural proteins involved in replication and transcription of the genome (E1–E5) or in host cell tumoral transformation (E6 and E7), whereas L1 and L2 are the structural capsid proteins of the virion. The low-grade cervical dysplasias correspond to productively infected cells that actively shed virus, whereas high-grade dysplasias and cancers do not produce virions: viral gene expression in these cells is limited to the E6 and E7 oncogenes that are transcribed from randomly integrated viral DNA. The E7 protein is thought to induce cell proliferation and disrupt the cell cycle regulation by inactivation of the Rb family proteins, whereas E6 blocks cell apoptosis by directing the p53 tumor suppressor protein to the proteasome.
Prophylactic HPV vaccine candidates are based on recombinant capsid protein L1 and aim to elicit neutralizing antiviral antibodies to protect against infection, while therapeutic vaccine candidates are based on viral oncogenic proteins E6 and E7, with or without L1, and aim to induce cell-mediated immune responses to eliminate the transformed tumor cells.
The most advanced and promising approach for a prophylactic vaccine involves the use of noninfectious recombinant virus-like particles (VLPs) which self-assemble spontaneously from pentamers of the L1 capsid protein. These VLPs can be produced in baculovirus-infected insect cells, in yeast, or in other cell substrates. They induce high titres of virus-neutralizing antibodies even in the absence of an adjuvant. In preclinical studies, vaccination of animals resulted in excellent protection from homologous virus challenge, and passive transfer of antibodies from the vaccinated animals also conferred protection,confirming the importance of neutralizing antibodies.
Two prophylactic vaccine candidates are at the level of Phase III clinical evaluation and the companies have filed for licensure. GSK is focusing on a bivalent HPV-16,-18 VLP vaccine candidate and Merck is developing a tetravalent vaccine based on VLPs from HPV-6, -11, -16, and -18. Both showed very high efficacy in proof-of-principle studies and the manufacturer have announced results showing almost 100% protection against high-grade cervical cancer precursors caused by HPV types 16 and 18 in women aged 16-25 years. Other candidate prophylactic HPV vaccines are in development and entering clinical trials.
A number of therapeutic vaccine candidates also have been developed, several of which have undergone Phase I/II clinical evaluation.