Virusassociated Malignancies

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Epstein-Barr Virus

EBV, also designated human herpesvirus 4, is associated with malignancies of B-cells, epithelial cells, T-cells, natural killer cells, and muscle (56,57). The development of EBV-positive tumors is associated with the latent life cycle of the virus during which it expresses up to nine viral proteins that provide targets for CTLs.

Adoptive Immunotherapy

LPDs of stem cell transplant recipients have provided an excellent system for testing the biologic efficacy of ex vivo expanded, adoptively transferred antigen-specific CTL lines. LPD represents the most immunogenic tumor (type 3 latency) among EBV-related malignancies, as all EBV latency-associated proteins are expressed by the virally transformed lymphocytes. Virus-infected B-cells expressing type 3 latency do not evade the immune response and are seen only in severely immunocompromised individuals. For example, EBV-LPD occurs in up to 20% of recipients of T-cell-depleted stem cells from HLA-mismatched or unrelated donors. No antiviral agents are reproducibly effective against EBV-LPD. Immunotherapy with unmanipulated donor leukocytes, although effective in some cases, is associated with a high incidence of disease progression, and survivors have a high incidence of graft-versus-host disease (GvHD) (58). We have shown that LPD can be prevented or eradicated and GvHD avoided by using selectively expanded EBV-specific CTLs either prophylactically or as treatment for overt disease (59). EBV-transformed B-cell lines are relatively easy to

Epstein Barr Virus Infection Timeline

Fig. 3. Timeline for generation of Epstein-Barr virus-specific cytotoxic T-lymphocytes (EBV-CTL). To ensure that CTL lines are available by the time patients are at high risk for EBV-lymphoproliferative disease (EBV-LDP) (1-2 months after stem cell transplantation), we initiate the lines as soon as the donor is identified. The first step is the generation of the lym-phoblastoid cell lines (LCLs), which takes about 4-6 weeks. Activation, expansion, and safety testing of the CTL line takes an additional 4-6 weeks. The first 26 patients received CTLs that had been genetically marked with a retrovirus vector carrying the neomycin resistance gene. Marking efficiencies of 0.5-10% allowed us to track the in vivo persistence of the CTLs and to determine their involvement in any toxicity. An initial dose escalation study revealed that low numbers of CTLs were biologically effective, and so all patients currently receive one dose of 107 CTL per m2. The target date of infusion is day 45, at which time graft-versus-host disease (GvHD), if it is to occur, should be apparent. Endogenous EBV-specific CTLs return at about 8-9 months post transplant. Virus load monitoring is also useful for timely intervention.

Fig. 3. Timeline for generation of Epstein-Barr virus-specific cytotoxic T-lymphocytes (EBV-CTL). To ensure that CTL lines are available by the time patients are at high risk for EBV-lymphoproliferative disease (EBV-LDP) (1-2 months after stem cell transplantation), we initiate the lines as soon as the donor is identified. The first step is the generation of the lym-phoblastoid cell lines (LCLs), which takes about 4-6 weeks. Activation, expansion, and safety testing of the CTL line takes an additional 4-6 weeks. The first 26 patients received CTLs that had been genetically marked with a retrovirus vector carrying the neomycin resistance gene. Marking efficiencies of 0.5-10% allowed us to track the in vivo persistence of the CTLs and to determine their involvement in any toxicity. An initial dose escalation study revealed that low numbers of CTLs were biologically effective, and so all patients currently receive one dose of 107 CTL per m2. The target date of infusion is day 45, at which time graft-versus-host disease (GvHD), if it is to occur, should be apparent. Endogenous EBV-specific CTLs return at about 8-9 months post transplant. Virus load monitoring is also useful for timely intervention.

establish and provide a continuous source of APCs to activate EBV-specific CTL lines from seropositive donors (Fig. 3). Since 1993, we have infused donor-derived, EBV-specific CTL lines into over 60 recipients of stem cell transplants (60,61).

The most important result of the study was that none of the patients who received prophylactic CTLs developed EBV-LPD, in contrast to about 12% of controls (61). Gene-marking studies showed that the infused CTL lines could expand in vivo in response to EBV reactivation and then persist for as long as 5 years. Infusion of CTLs into patients with a high virus load resulted in a dramatic drop in the virus load to low or undetectable levels. Further evidence for antitumor effects came from four patients who developed frank lymphoma before they were treated with CTLs. Subsequent infusions of the activated T-lymphocytes produced complete remissions in three of the four cases (61). Marking studies showed that the EBV-specific CTLs home to tumor sites, where they accumulate or expand and ultimately cause lymphoma regression.

Experience with CTL treatment of advanced EBV-LPD has illustrated two common pitfalls of such therapy. First, if the tumor occurs in a sensitive anatomic location, the inflammatory response can be damaging. One of our patients with bulky disease in the nasopharynx showed increased swelling after the CTL infusion, requiring intubation and tracheotomy (61). Second, mutation of important CTL epitopes becomes increasingly likely with tumor progression. Analysis of tumor tissue from a patient whose disease resisted CTL therapy revealed a deletion in viral DNA that removed two immunodominant epitopes against which the CTLs were specific (59,62).

Recipients of solid organ transplants may be at particularly high risk for EBV-LPD if they are seronegative prior to transplantation or if they receive organs, such as gut, that carry a high B-cell load, or if they receive prolonged, intensive immunosuppressive therapy for repeat episodes of graft rejection. Surprisingly, it has been possible to generate EBV-specific CTL lines from organ recipients even after they have developed lymphoproliferative disease. This suggests that EBV-specific CTL precursors are present in the circulation but are unable to expand in vivo during treatment with immunosuppressive drugs. If such cells are moved to a supportive culture environment, they can respond to activation and proliferation signals. In the case of seronegative recipients, it may be possible to activate CTLs by using APCs such as DCs, since they are able to activate CTLs from naive precursors in vitro (47). Prophylaxis with CTLs is probably not an option in these patients, since with time, in the absence of viral antigen and the presence of immunosuppressive drugs, the infused CTL may be lost. However, because regular monitoring of the virus load can permit early intervention, CTLs should be prepared in advance for patients with a high-risk status and then infused when virus DNA appears or they seroconvert.

Patients with immunodeficiency disorders are also at increased risk for EBV-LPD. As with solid organ recipients, it has been possible to generate EBV-specific CTLs from some of these patients and to use them as therapeutic agents (63).

EBV-positive Hodgkin's disease is another candidate for treatment with EBV-specific CTLs. Five patients who received autologous EBV-specific CTLs in a phase I study had temporary clinical improvements, including increases in EBV-specific CTL precursor frequency, reductions in high virus loads, resolution of type B symptoms, and stabilization of disease (64). Current improvements include the generation of CTL lines that are specific for the limited range of viral antigens expressed in Reed-Sternberg cells.

Vaccination

The high incidence of nasopharyngeal carcinoma (NPC) in southern China (1-2% of the general population) has been associated with early infection by EBV (65). A vaccine that could prevent or delay primary infection with EBV by establishing mucosal immunity might decrease the incidence of EBV-associated NPC (66). The gp340 envelope glycoprotein of EBV, the principal target for neutralizing antibodies, binds to the cellular receptor for the C3d component of complement, enabling virus to enter the B-cells. A vaccine based on purified gp340 has been shown to reduce the virus load and protect against EBV-associated LPD in cotton-top tamarins (2). When tested in Chinese children, live recombinant vaccinia virus engineered to express gp340 induced EBV-specific immune responses with protection against and/or delay of EBV infection (67). However, the outcome of this study may not be evident for 40 years.

Because the CTL-mediated immune response is necessary for control of EBV infection, the possibility of priming T-cells by peptide immunization, before primary infection, has been raised. One advantage of accelerating the CTL response to primary infection is that one may be able to limit subsequent colonization of the B-lymphocyte pool (2). This approach would require peptides that are customized to the patients' HLA phenotype (17) or perhaps a polytope vaccine, as described previously (45). An effective vaccine against EBV would be especially useful for seronegative recipients of solid organs from seropositive donors. Such patients are generally at greater risk of developing EBV-LPD (68).

Human Papillomavirus

Infection with HPV is widespread throughout population. Over 80 strains of this virus cause a spectrum of tumors of skin and mucous membranes. HPV-1 and HPV-6 appear to be largely responsible for benign skin warts and genital warts, respectively, whereas 90% of cervical carcinomas are associated with HPV-16 and HPV-18 (69). Despite the wide involvement of HPV in sexually transmitted disease and its association with malignancy, specific therapy for HPV is still not available. Surgical or chemical removal of the wart leaves the viral episome in the basal epithelium, and host cell transformation can result from random integration of oncogenic HPV DNA into the genome (70).

Prophylaxis with vaccines for genital papillomaviruses might prevent infection by eliciting neutralizing antibodies (70). To be effective, such vaccines must establish an immunologic barrier at the anogenital epithelium by selective stimulation of a secretory IgA-mediated anti-HPV virion response in the genital mucosa (70). A phase I trial of L1 (the major capsid protein) particles to vaccinate HPV-11-naive adults is imminent. Detection of type-specific HPV DNA in the genital tract or the development of HPV-induced anogenital lesions will serve as end points of this proposed vaccination trial (71).

The immunogenicity of cervical cancer is supported by the observation that it progresses rapidly in immunosuppressed patients, and spontaneous remissions are associated with lymphocyte infiltration (73). Furthermore, the detection of CTL activity against HPV-16-E7-encoded immunogenic peptides in some patients with cervical intraepithelial neoplasia or cervical cancer suggested that natural immunity might be strengthened with immunotherapeutic approaches (72). There is considerable interest in using HPV vaccines to eliminate residual cancer, precancerous lesions, or warts. About 60% of cervical carcinomas express the transforming proteins of HPV-16 (E6 and E7) (49). Hence, these proteins are obvious targets for any immunotherapeutic approaches that successfully exploit tumor rejection antigens in murine models (74). Immunization with an immunodominant peptide of HPV-16-E7 protected animals against lethal challenge with a tumor expressing this epitope, whereas immunization with peptide-pulsed DCs could eradicate established tumor (12).

Vaccination with peptides has been applied in clinical trials for cervical cancer. A pep-tide vaccine consisting of two HPV-16-E7 HLA-A*0201-restricted CTL peptides and a helper peptide has been tested in women with end-stage cervical cancer (73,75), but no correlation between vaccine dose and clinical outcome was observed. Steller et al. (76) also tested the effectiveness of a lipidated form of HPV-16-E7 covalently linked to a helper peptide. Activation of CTL responses, detected by IFN-7 release assay, were demonstrated in two of three evaluable patients who had been vaccinated with this peptide.

Genetic immunization with a recombinant vaccinia virus expressing the E6 and E7 epitopes of HPV-16 and HPV-18 (mutated to abrogate the transforming capacity of the virus, while retaining the predicted epitopes) was tested in eight patients with late-stage cervical cancer (77). Vaccinia antibody responses were noted in all patients, with three demonstrating HPV-specific antibody responses. HPV-specific CTLs could be detected in one of three patients. Such an approach might be more effective in patients with less advanced cancers or preinvasive lesions, or perhaps even in HPV-16 or -18 carriers without detectable lesions.

To eliminate basal cells infected with HPV, some investigators have proposed E1 and E2 proteins of HPV as targets for immunotherapy, as they are required for maintenance of the episomal state (70). Papilloma virus-like particles (VLPs), formed by the assembly of the major capsid protein of papillomaviruses in the absence of other viral proteins, can be engineered to carry other proteins (78,79), whereas a VLP bearing an HPV-16-E7 epitope protected mice from challenge with E7-transformed tumor cells (79).

Hepatitis B Virus

HBV replicates in the liver and causes hepatic dysfunction (80). Most HBV infections are self-limiting and are effectively controlled by the immune system. A small fraction of HBV infections can become persistent or chronic if the immune system fails to resolve the infection completely. Persons with chronic HBV infection are at substantially increased risk of developing cirrhosis and primary hepatocellular carcinoma. Although no specific HBV sequence has been implicated in the development of hepatocellular carcinoma, the virus appears to act as a mutagenic agent, and viral gene expression may not be required for tumor growth. IFN-a treatment is helpful in some cases, but a satisfactory medical treatment for chronic HBV infection is still unavailable. Immunologic intervention with the conventional hepatitis B vaccine relies on primary prevention. The vaccine is composed of a highly purified preparation of hepatitis B surface antigen (HbsAg). Virtually 100% of persons who develop HBsAg antibody titers of > 10 mIU/mL after primary vaccination are protected against primary infection. However, the vaccine is of no benefit to individuals already infected or who have chronic disease. The ultimate goal of hepatitis B vaccination is to decrease the incidence of HBV-related chronic liver disease and hepatocellular carcinoma. Recent studies in Taiwan have demonstrated a reduction in the incidence of primary liver cancer in children born after the implementation of routine hepatitis B vaccination programs (80).

From 2.5 to 5% of immunized adults with HbsAg do not develop an antibody response. This rate increases to 40% in high-risk patients, such as those on hemodialysis (81). Chimpanzees that received intramuscular vaccination with plasmid DNA encoding the major and middle HBV envelope proteins developed strong group-, subtype-, and preS2-specific antibodies compared with those achieved by traditional antigen-based vaccination (31). Thus, genetic immunization may lead to a lower rate of unresponsiveness.

Patients who successfully clear HBV develop a strong HLA class I-restricted CTL response, whereas in those with chronic hepatitis B, the response is weak or unde-tectable. Hence, a vaccine capable of inducing a CTL response to HBV may be capable of eradicating chronic infection and thereby eliminating the risk of developing cirrhosis or hepatocellular carcinoma. Twenty-six normal volunteers were immunized with an HLA-A*0201-restricted CTL epitope from the HBV core antigen linked to a tetanus toxoid-derived helper epitope with palmitic acid residues used as adjuvants (82,83). This vaccination proved to be safe and generated primary HBV-specific CTL responses. This was the first demonstration that a CTL peptide can induce a primary CTL response in humans and provides the rationale for a larger clinical trial in patients chronically infected with HBV (83).

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