Epstein-Barr virus is the pathogenic agent for several diseases, both infectious and cancerous. It is the source of infectious mononucleosis, and the cause of many cases of Burkitt's lymphoma, B-cell lymphomas, some T-cell lymphomas, some gastric carcinomas, Hodgkin's disease, X-linked lymphoproliferative syndrome, nasopharyngeal carcinoma, gastric carcinoma and oral hairy leukoplakia. Many of these diseases are fatal and are caused by reactivation of the latent Epstein-Barr virus. Since the discovery of EBV in association with Burkitt's Lymphoma in the 1970s, research has been underway to develop a vaccine. The considerations and advances in this work are examined below. As mentioned in the immunology section, cell-mediated immunity is thought to play an important role in creating an immune response to EBV. Cytotoxic T cells target all the EBV latent gene protein products except for EBNA1. This makes synthetic peptides a realistic possibility for a vaccine (1). A Whole Virus Vaccine One of the earliest and most successful methods of creating vaccines has been the use of live, attenuated forms of the pathogen. This approach evokes both humoral and cellular immunity because the full repertoire of viral proteins are presented in the same quantity and context as in the virulent form. This method has proven sucessful in developing the chickenpox vaccine, currently the only herpesvirus vaccine liscenced by the FDA. However, in the case of EBV the viral DNA itself may be oncogenic. Another obstacle towards developing such a vaccine is the absence of an efficient in vitro culture system for the production of live EBV (1,2). Inactivated, killed forms of the virus would probably be ineffective because cell-mediated immunity is necessary for the recognition and elimination of intracellular pathogens. [back to top] Synthetic Peptide Vaccines A more detailed knowledge of the immune response is necessary for the development of a peptide vaccine than for any of the other methods. There is a need to know the specific B and T cell epitopes and their interactions with agretopes to form MHC complexes. Synthetic peptide vaccines are generally poorly immunogenic. Lipid carriers known as ISCOMS (immunostimulating complexes) and other delivery systems such as miscles, liposomes, and solid matrix-antibody antigen (SMAA) help to promote the uptake by macrophages or target cells by increasing immunogenicity and delivering proteins via the cytostolic pathway (3). The EBV virus membrane antigen, (MA) has been the target of most subunit vaccine development. This antigen can be detected on both the envelopes of virions and on the plasma membrane of virus producer cell lines. Three particular glycoproteins with similar antigenic determinants have been found-- gp340 (of molecular weight 340,000-350,000 daltons), gp220 (220,000-270,000 daltons), and gp85 (85,000 daltons). The first two proteins are derived by splicing a single gene and do not change the reading frame. Neutralizing antibodies that recognize MA will also neutralize the virus. For this reason, it is believed that gp340/220 would be a good candidate for an EBV vaccine (1). The first task in producing a subunit vaccine is the production and purification of the proteins in question. EBV cell lines established to date have given quite low protein yields; which would be even more problematic should a vaccine need to be produced. The production of recombinant gp340 has been attempted in e-coli, yeast, and plasmids but in all cases there has been reduced or lost expression of the protein or anomalous carbohydrate modification (1). Another key requirement for vaccine development is an animal model. EBV was found to cause tumors in two new world species primates, the owl monkey and the cottontop tamarin. A breeding colony of the endangered species, the cottontop tamarin was chosen and challenged with a large dose of EBV and a number of immunizations. Neutralizing serum antibodies were produced against EBV by the cottontop tamarins: "This animal model reproduced key aspects of human EBV infection, including oral transmission, atypical lymphocytosis, lymphadenopathy, activation of CD23+ peripheral blood B cells, sustained serologic responses to lytic and latent EBV antigens, latent infection in the peripheral blood, and virus persistence in oropharyngeal secretions. This system may be useful for studying the pathogenesis, prevention, and treatment of EBV infection and associated oncogenesis"(10). The final delivery requirement is the addition of an immunostimulatory adjuvant. ISCOMS and muramyl dipeptide (MDP) pluronic polymer formulation have been under development for several years but are still not licensed for humans (6). A gp340/ISCOMs vaccine which uses gp340 from EBV membranes in vitro and was very effective at stimulating protective immunity in cottontop tamarins. Protection was achieved by three, 2 microgram doses (4). Although a less effective adjuvant, alum is currently the only FDA liscensed adjuvant available for human use. In a study by Finerty et al., animal models were used to test a gp340/alum vaccine in cottontop tamarins and in rabbits. The adjuvant elicited protective immunity against EBV-induced lymphoma in three out of five cottontop tamarins, suggesting that alum should be used as part of a human trial of a gp340-based subunit vaccine (5). In addition, rabbits immunized with gp340/alum showed the same diversity of antibody responses as rabbits immunized with gp340/SAF-1, an experimental adjuvant claimed to be more effective than alum (5). TRIALS A phase I clinical trial conducted by Aviron (1999) demonstrated safety and immunogenicity for a subunit vaccine containing the gp220/350 surface glycoprotein. The proprietary adjuvant was supplied by SmithKline Beecham. The trial was a randomized, double-blind study, containing 67 healthy adult participants. The study indicated that the vaccine was tolerated and safe. Evidence of an immune response was shown by laboratory tests from participant sera (2). Clinical trials of a peptide vaccine with an EBNA-3A epitope are currently being conducted in Australia. (2) The Queenland Institute of Medical Research has conducted a phase I trial designed to test the safety and immunogenicity of an Epstein-Barr virus cytotoxic T cell epitope vaccine. The synthetic peptide, FLRGRAYGL (an HLA B8 restricted epitope from EBNA-3A), is in an emulsion of tetanus toxoid, water, and oil adjuvant Montanide ISA 720. Healthy volunteers who are EBV negative and who are HLA B8 positive have shown no adverse reactions to date (7). [back to top] Recombinant Vector Vaccines Recombinant vector vaccines have the advantage of being able to enter target cells, through the use of attenuated viruses or bacteria, and deliver genes encoding for major antigens of the pathogen. Adequate quantities of viral proteins are then manufactured and processed within the host cell, with or without viral vector replication. Poxvirus shells expressing EBV have demonstrated immunogenicity in experimental animals but have produced relatively small immune responses in human trials. Vaccina virus recombinants have been used to produce gp340 (1). A vaccina virus recombinant expressing gp340, vMA1 has been tested in both cotton-top tamarins and common marmosets. The M81 strain of EBV demonstrated decreased replication the group of marmosets innoculated with the vaccine (11). In China, a live recombinant virus vaccine based on the liscensed vaccina strain Tien Tan, has been designed and is being used in human vaccine trials. The vaccina promotor expresses the EBV membrane antigen BNLF-1 MA which is gp220/340. It has been shown that the protection against and/or retardation of EBV infection is possible in humans through the use of live, safe vaccina vectors (8). Recombinant vaccina viruses have several advantages. They are inexpensive and simple to produce, thereby making them a viable possibility for mass vaccinations in developing countries. They also offer the excellent possibility of multiple vaccinations through a single vector since poxviruses have a large capacity for transporting foreign DNA A disadvantage is that any live vaccine is a potential problem with the current AIDS epidemic (1). Another potential vaccine vector is the Oka vaccine strain of varicella zoster virus (VZV). Appropriate expression of gp340/220 was observed in the recombinant. The expression of EBV proteins in the VZV vaccine could provide an convenient method of multivalent vaccination (9). Adenoviruses are becoming increasingly popular as carriers of foreign genes. The construction of an adenovirus type 5 recombinant which combines a non-replicating virus with the EBV gp340/220 genes has been tested in animal models. A serum response to gp340 was detected in vivo, but there was no antibody response to in vitro EBV. The benefit of using adenoviruses is their ability to infect the tonsils and salivary glands, producing oral secretory antibodies in the same target areas as natural EBV infection. However, EBV-specific IgA may enhance EBV infection of epithelial cells, thereby ameliorating viral infection. (1) TRIALS A phase I clinical trial of the recombinant vaccina virus mentioned above has been successful in China. The trial was performed in three distinct human populations: EBV-positive adults exposed to the vaccina virus, EBV-positive children not exposed to the vaccina virus, and EBV-negative infants not exposed to the vaccina virus. No significant antibody variations were observed in the adults but EBV-neutralizing titers increased in the vaccinated children, while antibodies to the viral capsid antigen remained the same. All vaccinated infants developed MA antibodies with neutralizing properties in vitro (8). [back to top] Future considerations and challenges The developments of a vaccine are still in the preliminary stages. It is not known whether gp220/350 will prove to be an effective vaccine in humans. The correlates of immunology are not well understood. The study of EBV in animal models needs to be continued to better understand the molecular biology of the virus. The gp220/350 may be adequate to protect against primary infection and even more importantly, one would hope that a protective response against EBV-associated tumors would be illicited. Yet in the oncogenic forms, the gene products are expressed differently. Little about the CTL specifities for EBV antigen are known. Many of the other EBV antigens remain to be evaluated in potential combination with or superiority over gp340 and gp220. The next stages in vaccine development should result in a phase II study by SmithKline Beecham Biologicals and data collected from the Australian phase I study. Increased studies of immunotherapy should prove successful at preventing some cases of PTLD. The success of this anti-cancer therapy has great potential in many opportunistic malignancies. There is just cause for the continued development of a vaccine against Epstein-Barr virus despite the fact that it will probably not target Burkitt's lymphoma or Hodgkin's disease patients. Infectious mononucleosis is a prevalent disease in the United States and other developed nations. The cost of a vaccine would probably result in long-term benefits from decreased health care costs and fewer days of loss of productivity. A vaccine against mononucleosis may contribute to delayed onset of EBV, decreasing the incidence of diseases such as Burkitt's lymphoma in young African children.
