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Vaccines for the 21st Century: A Tool for Decisionmaking APPENDIX 28 Summary of Workshops     Herpes Simplex   326     Epstein-Barr Virus   329     Hepatitis C Virus   334     Human Papillomavirus   338     Dengue Hemorrhagic Fever   342     Chlamydia trachomatis   347     Tuberculosis   351     Histoplasmosis and Coccidioidomycosis   355     Group A Streptococci   359     Helicobacter pylori   363     Neisseria gonorrhoea   367     Adjuvants   372     Antigen Delivery Systems   377     DNA Vaccines   381     Preventive Vaccine for Diabetes   386     Cytokine Modification of Autoreactivity   389     T-Cell Subset Choice on the Outcome of Autoimmune Disease   392     Antigen-Induced Programmed T-Cell Death as a New Approach to Immune Therapy   395     Induction, Propagation, and Immunoregulation of Autoimmune Diseases of the Central Nervous System   399     Peptide-Mediated Regulation of Autoimmunity   403     Stimulation and Costimulation   407     Viral Therapeutic Vaccines—Hepatitis B   412     CD8 CTL to Mutated Oncoproteins and Fusion Proteins   415     CTL Screening for Tumor Antigens   420     Immunity to Oncogenic Self-Proteins   425     Cytokines and Their Local Environments   429

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Vaccines for the 21st Century: A Tool for Decisionmaking HERPES SIMPLEX1 Pathobiology. Primary herpes simplex virus (HSV) infection occurs through mucosal surfaces, followed by Kaposi’s varicelliform eruption. Immune host responses control this primary infection within 14 to 21 days. What is unique is the retrograde neuronal transport of the virus to the dorsal root ganglia, where a cycle of replication occurs and the virus becomes latent until a provocative stimulus leads to reactivation. At that time, anterior grade transport returns the virus to the mucosal surfaces, where replication ensues. Incidence and Burden. By adulthood, about 80 percent of the U.S. population has been exposed to nongenital HSV type 1 and is at risk for reactivation, leading to herpes simplex labialis. There are about 750,000 new cases of genital infection per year in the United States, and overall seroprevalence of HSV-2 is about 60 million individuals. Genital herpes generates about 600,000 physician visits and 20,000 unnecessary caesarian sections annually. Two more serious infections are herpes simplex encephalitis and neonatal herpes, each with about 1,500 cases per year. The cost of neonatal herpes is at least $750 million per year, primarily in indirect costs for the long-term management of neurological sequelae from which these babies suffer. Seroprevalence of HSV type 2 is increasing rapidly. In 1992, seroprevalence for the U.S. population at large was about 31 percent; for persons of color, between 50 and 55 percent. Acquisition is a function of the number of sexual partners to whom an individual is exposed: for heterosexual men with greater than 50 partners, the probability of acquiring HSV-2 is 80 percent or higher; for heterosexual women with more than 50 partners, over 90 percent. Women are more likely to acquire HSV-2 than men. As with other genital ulcerative disease, there is also a four- or fivefold increase in the risk of acquiring HIV infection. Rationale and Goals of Vaccine. Studies of couples with discordant serostatus suggest that prior HSV-1 infection confers some degree of protection from acquiring HSV-2 infection. When the male is positive for HSV-2 and the female is seronegative, her probability of acquiring HSV-2 is 20 percent during the calendar year. This becomes particularly significant if she becomes pregnant during that year. If the female is seropositive for HSV-1, however, the probability drops to 10 percent. (The reverse also holds: if the male is positive for HSV-1 rather than seronegative, his probability of acquiring HSV-2 drops from 10 percent to 5 percent.) These findings suggest that a vaccine that could seroconvert the mother from at-risk to lower-risk could lower the probability of transmission to the newborn. The goal of this vaccine would be to prevent or at least reduce the severity of primary infection, and possibly to reduce the frequency and clinical symptoms of reoccurrences. We know that exogenous reinfection is extremely 1   Based on a presentation by Richard J.Whitley, M.D.

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Vaccines for the 21st Century: A Tool for Decisionmaking uncommon in the immune-competent host, and that HSV-1 antibodies reduce both the probability and the clinical symptoms of primary HSV-2 infection. Similarly, fetal transmission is 10 times less likely when there are preexisting HSV-2 antibodies, compared with primary infection. And the duration of primary HSV-2 infections are reduced in seropositive individuals from 21–28 days to 10–14 days. Approaches to Vaccine Development. Four approaches have been attempted in developing such a vaccine. Live virus by autoinoculation has been defined as a failure in studies over the past 100 years. HSV has 13 glycoproteins in its envelope, two of which (B and D) are required for infectivity and potent inducers of neutralizing antibodies. Subunit vaccines for glycoprotein B, glycoprotein D, and a combination of B and D are under investigation. Vector glycoprotein genes using either canary pox or a vaccinia vector have been. Engineered herpes simplex virus is a potential future candidate for clinical trials. Subunit Vaccines. Clinical trials are currently evaluating monoclonal antibody therapy using glycoprotein B and D combinations to treat neonatal HSV infection and HSV encephalitis. Other subunit trials underway include one therapeutic trial of approximately 800 volunteers and 2 primary prevention trials of approximately 500 couples each. In the latter prophylactic trial, one partner in each monogamous relationship has recurrent HSV-2 and the other is HSV-2-seronegative. The former, therapeutic trial is based on the results of a preliminary trial of 100 volunteers between the ages 18 and 55, who averaged 4 to 14 outbreaks per year and had not received acyclovir for 3 months prior to therapy. Half received 100 micrograms of glycoprotein D-2 with alum adjuvant; half received placebo; and there was a booster at 2 months. In those who received the vaccine, the average number of recurrences dropped from 0.5 to 0.42 per month, and the median from 6 to 4 per year. Genetically Engineered HSV Virus. Genetic stability is a concern in all genetically engineered vaccines; reversions to or recombinations with wild-type viruses, when they do occur, should be less virulent than the current strain. In addition, live attenuated viruses abdicated the ability to become latent and therefore, under some circumstances, might be reactivated. This is why deletions in the gamma 134.5 gene are particularly promising: they debilitate the virus’ ability to become latent and subsequently reactivate (see below). The herpes simplex virus has a genome that is only 150,000 base pairs (150 kB) long, in an architecture consisting of a unique long segment and a unique short segment. There are internal repeats bonding the unique long and short segments so that the virus could invert upon itself, leaving four equimotor isomers in any population. There is a 70-percent homology between HSV-1 and HSV-2. The starting point for an engineered vaccine is an intratypic hybrid identified as R-70–20 and was developed about 10 years ago. It consists of the unique long segment of HSV-1, a segment of the unique long segment of HSV-

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Vaccines for the 21st Century: A Tool for Decisionmaking 2, and the unique short segment of HSV-2. A 12-kB deletion of internal repeats from the HSV-2 short domain, mapping for a series of glycoproteins (including G, J, GD, GI, and GE), was inserted. This vaccine considerably reduces virulence in rodent models, is stable upon serial passage in mouse brains, and is safe and efficacious in aotis monkeys at concentrations up to 107 PFU administered by any route, including intracerebrally. It was safe in humans at up to 104.5 PFU (higher doses were not studied). This vaccine required two doses, implying that it was overly attenuated for administration to humans. HSV can also be attenuated through deletions and stop codes. The gamma 134.5 gene resides in the inverted repeats in two copies on the unique long segment of the HSV-2 genome; when 134.5 is deleted, it leaves a virus with an LD-50 upon inoculation directly into the central nervous system of a mouse of 106 PFU, compared with 102 PFU for the restored wild-type virus. A stop-code on the carboxy terminus of the genome can similarly attenuate the virus. Using the latter technique with HSV-2, a recombinant vaccine was generated that also deleted the structural components of UL-55 and UL-56. This vaccine has significantly reduced neurovirulence in mice—LD-50 is 5.6 X 105 PFU, compared with less than 50 PFU for the wild type in that circumstance—and is safe in the aotis monkey at up to 106 PFU. In terms of efficacy, the vaccine appears to protect guinea pigs from disease at dosages in the range of 104 to 105 PFU. Immunized aotis monkeys survive challenges with wild-type virus at up to 105 PFU when given intravaginally. These results suggests that this is, at least potentially, a genetically engineered vaccine that will provide a broader immune response than is encountered with subunit vaccines. Both the method of attenuation and the route of administration seem to influence the efficacy of these candidate vaccines. When mice were inoculated intranasally and then challenged intranasally with either wild virus or HSV-2, there was a reduction of mortality of 20 percent for those inoculated with 134.5-deletion mutants, and 13 percent when a stop code is place at the carboxy terminus. When mice are immunized intranasally and challenged intravaginally, however, the effect is not as great. Researchers are currently looking for IgA and IgG2a in the vaginal secretions of these mice to learn more about these immune responses. Animal Models. Two new animal models have emerged from this work. The first is a test of the virulence of the virus; the aotis monkey is exquisitely sensitive for the evaluation of genetically engineered viruses, which are inoculated directly into the eye. The second is a rodent model to establish the genetic stability of attenuated vaccines. It involves inoculating virus into the mouse brain, harvesting tissue, raising the virus again, and reinoculating virus into mouse brains on subsequent occasions; nine passages will usually select unstable variants or a reversion to wild type. Inoculation into mouse brain can also be used to evaluate the virulence of attenuated virus. In response to questions from the audience, Dr. Whitley added the following:

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Vaccines for the 21st Century: A Tool for Decisionmaking Researchers do not at present have a marker of host immune response that is predictive of vaccine efficacy. Both neutralizing antibodies and cell-mediated immune responses have been investigated and eliminated. Gamma-134.5-deletion vaccines will eventually be tested in immunocompromised models. Subunit vaccines currently require three doses to provide persistent antibody levels, although the duration and intensity of immune response may increase with a new adjuvant. R-70–20 acted more like a dead vaccine, requiring two doses. There are hopes that replication-competent viruses like the 134.5-deletion mutants will provide enhanced immunogenic effects with one shot rather than two. There has been little work on cellular responses to these vaccines or indeed to primary and recurrent herpes at all. Some researchers are doing CTLs on mice, and others are looking at T-cell responses in humans who are seropositive but have no clinical recurrences. T-cell responses following primary infection are robust, but no one has done comprehensive CTLs or tried to dissect out specific T-cell response in all of these human populations. The target population for HSV-2 immunization is adolescents as they are about to begin sexual activity, perhaps 10 or 12 years old, and the vaccine should provide at least 10 years of resistance to the wild-type virus. It will probably be impractical to test for serologic status for HSV-1 or HSV-2 prior to immunization. EPSTEIN-BARR VIRUS2 Epstein-Barr virus (EBV) is another extremely common virus, but it is linked to a growing list of pathologies. Unlike HSV, there have been few vaccine trials, but a great deal is known about the disease processes. This knowledge could be used to generate partial or complete immunity to block these diseases. Incidence and Burden. Worldwide, about 95 percent of the adult population is infected with EBV. If the primary infection comes in late adolescence, about 50 percent of seronegatives will develop the clinical manifestation called infectious mononucleosis (IM). IM is a significant disease, with at least 125,000 new cases recorded each year in the United States. About 70 percent of patient will resolve their symptoms in 2 to 4 weeks and is the major cause of lost time for new Army recruits. However, 30 percent develop more extensive impairment that may not resolve for 3 to 4 months. About 1 percent of cases develop complications, including neurological, bone marrow, liver involvement, and 2   Based on a presentation by Nancy Raab-Traub, M.D.

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Vaccines for the 21st Century: A Tool for Decisionmaking even fatal IM. This extensive morbidity might be decreased by appropriate immunotherapy, but there is at present no effective antiviral therapy. In most cases EBV establishes latency in the B-lymphocytes. However, EBV is associated with a variety of aplastic diseases, including 80 percent of AIDS-related lymphomas. Among post-transplant proliferative disorders, anywhere from 25 percent to 100 percent (depending on organ) occur in patients who are having a primary EBV infection. Similarly, there are many similarities between IM and Hodgkin’s disease—patients with a history of IM are 2 to 3 times more likely to develop Hodgkin’s lymphoma, and 70 percent of Hodgkin’s-like lymphocytes are EBV-positive. EBV is also associated with Burkitt’s lymphoma, which is relatively rare in the United States but is endemic in Africa. EBV is associated with the vast majority of nasopharyngeal carcinoma, which occurs when the virus is reactivated in mucosal lymphocytes and infects epithelial cells, which develop abnormalities and rapidly becomes dysplasia or carcinoma in situ. EBV is now being described in an increasing number of T-cell lymphomas, a significant proportion of Hodgkin’s disease, and in discrete substantive gastric carcinomas. Other related diseases include hairy leukoplakia, which was originally described in AIDS patients. Pathobiology. EBV can establish both permissive infection in epithelial cells and lymphocytes and latent, nonpermissive transforming infections in lymphocytes. Pathogenesis involves introduction of the virus into the oral epithelial cells, which are usually permissive to viral infection, and virus is then secreted into the saliva. Secondarily to this epithelial infection, virus enters the B-lymphocyte, where it circularizes, forming an extra chromosomal episome in the nucleus. One week after the initial infection, as many as 5 percent to 20 percent of peripheral blood lymphocytes are infected with EBV, including a wide variety of cell types: B-lymphocytes, Reed-Sternberg-like cells, plasmacytoid cells, and even a small percentage of T-lymphocytes. This is basically a latent infection: most of these cells do not make viruses but instead begin to express viral gene products that are associated with the process of cellular transformation. Many of these gene products are very potent targets of cytotoxic T-lymphocytes (CTLs), which must be generated to control the infection and the transformed lymphocytes. In the absence of T-cell response, these lymphocytes grow uncontrolled and eventually develop into a lymphoma. Viral Genome. Analysis of the viral genome can distinguish latent from replicative infection and is informative in other ways. Like HSV, the EBV genome is a double-stranded DNA molecule with variants, but with a simpler structure. It is about 190 kB long, but instead of inverted repeats, it has multiple copies of a 500-base-pair direct repeat at each end of the genome. Variants are highly heterogenous, with anywhere from 1 to 20 copies of the terminal fragments at either end of the genome. This assay was able to distinguish between the variant form from the larger episomal form, but it also revealed that (within each cell) each of the up to 100 copies of the EBV episome was identical with regard to the number of terminal repeats. This also suggested that every cell within a tumor would be identical, from which researchers deduced that the

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Vaccines for the 21st Century: A Tool for Decisionmaking epithelial tumors associated with EBV (e.g., nasopharyngeal and salivary gland carcinomas) were also clonal. This was confirmed by comparing the clonality predicted by EBV terminal studies and the clonality found in immunoglobulin rearrangements. This indicated that most of the tumors associated with EBV were clonal proliferations and, more importantly, that these proliferations had developed from a single EBV-infected progenitor cell. This has been confirmed in post-transplant proliferative disorders, Hodgkin’s lymphoma, Burkitt’s lymphoma, gastrocarcinomas, cancers of the salivary gland, and the new T-cell lymphomas that are being reported in Taiwan and Japan. In nasopharyngeal carcinoma, patients have extremely high titers to replicative antigens and, while the Southern blot test does show a single clonal EBV fragment, there is also a faint ladder array indicating that at least a few cells go into permissive infection and make additional virus. The principal exception is hairy leukoplakia, for which the tests show no evidence of a fused band representing an episomal compartment, but rather an abundant ladder array indicating that this is a permissive infection. In the majority of cases, however, the diseases associated with EBV are associated with latent infection. EBV Gene Functions. As noted, EBV gene products are expressed by infected cells, and researchers have identified the function of many of these products. One of the most important is Epstein-Barr nuclear antigen type one (EBNA-1), which binds to a specific DNA sequence in the EBV genome (the origin of plasma replication) and allows replication by DNA prolimerase. As a result, these infections are not susceptible to any kind of antiviral therapy directed toward the prolimerase. EBNA-1 also has the ability to induce the expression of the rad genes, which may be critical in some of the genomic rearrangements that have been described in Burkitt’s lymphoma. No CTLs have been identified that are specific for EBNA-1, apparently due to a unique sequence in the EBNA-1 gene that mimics a cellular gene and prevents it from being recognized by class one antigens. Five additional nuclear antigens (EBNA-2 through 6) have viral regulatory functions. Three of them are essential for transformation of B-lymphocytes. EBNA-2 transactivates these other genes, and three of them regulate the expression of latent membrane protein one (LMP-1) in the transformed lymphocytes. Almost all of the CTL targets are in these genes, which are expressed uniquely in B-lymphocytes. LMP-1 is considered to be the viral oncogene: it promotes transformation and cellular division; it is the only gene that transforms rodent cells and cultures; and it is essential to the activation of a B-lymphocyte. As a result, it is able to inhibit apoptosis both in B-lymphocytes (by inducing expression of BCL-2) and in epithelial cells (through a p53-dependent mechanism that occurs through the induction of the A-20 gene). The LMP-1 gene is expressed in post-transplant lymphomas and other malignancies associated with EBV; it appears to be a

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Vaccines for the 21st Century: A Tool for Decisionmaking critical gene for neoplastic growth. LMP-2 associates with LMP-1 in transformed cells and appears to block activation of the B-lymphocyte by interfering with the signals that would induce antibody synthesis. In doing so, LMP-2 helps to maintain the latent infection. A last set of EBV genes expressed in infected cells are called Epstein-Barr encoded RNAs (EBERs). They are RNA polymerase-3 transfers, but their function is not yet known. They are not essential for transformation, but they are extremely abundant (50,000 to 1 million copies per infected cell) and very stable. EBERs can be very useful for identifying infected tissues, and because they are not expressed in hairy leukoplakia (a permissive infection) they may be useful in identifying repositories of latent infection. Levels of Latency. Research on the actual behavior of EBV in each of the associated pathologies has shown that there are actually three levels of latency. Type one latency is a minimal level of expression, characteristic of Burkitt’s lymphoma. Only EBNA-1 and the EBERs are expressed, so the infected cells do not become targets for cytotoxic T-cells. The peripheral blood lymphocytes of normal individuals who have been exposed to EBV also express EBNA-1 and EBERs, and they are also believed to express LMP-2, helping to maintain them in a state of latent infection. In type two latency, additional genes are expressed. Specifically, LMP-1 and LMP-2, two genes that are regulated by the EBNAs, are expressed in the absence of the EBNAs. In other words, the critical transforming genes are being expressed but not the key CTL targets, which may be important in the development of disease for which this is the characteristic state of latency, including nasopharyngeal carcinoma, Hodgkin’s disease, and T-cell lymphoma. In type three latency, the entire array of EBV latent genes are expressed, including not only the oncogenes but also EBNAs 2 through 6, which are the key CTL targets, as well as high levels of EBERs whose function is as yet unknown. This type of infection, which is typical of peripheral blood lymphocytes in infectious mononucleosis and most of the cells in post-transplant proliferative disease, should be very amenable to control by the immune system. In replicative infection, one vital gene called ZEBRA is the replication activator that turns on the expression of the viral replicative antigens (including polymerase and thymidine kinase) and the structural proteins (including the viral caps antigen, or VCA, and glycoproteins 350/220, 110, 85, 42, and 27). The glycoproteins of EBV are much less complex than those of HSV, and one in particular—gp350/220—is the main target of neutralizing antibodies and hence the principal focus of research attention. Approaches for Vaccine Development. One approach that is being pursued is recombinant gp350, which does induce antibody-dependent cytotoxicity. In cotton-top tamarinds, this vaccine protects against lymphoma after a parenteral challenge. This approach would probably protect humans in pathologies that are dependent on replication, such as IM and nasopharyngeal carcinoma. In a very limited trial in China, 10 children were infected with gp350 in vaccinia and 10 got placebo; after a year, all 10 on placebo became infected with EBV,

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Vaccines for the 21st Century: A Tool for Decisionmaking but only 2 of the 10 who got gp350 were infected. Much has been done in terms of designing gp350 to be produced in the cells and working on a strategy for clarification in high quantities. Abnormal viral response in the absence of antibodies to fusion protein is a concern. T-cell epitope vaccines are also being investigated, because researchers have identified so many of the CTL epitopes that are critical for EBV infections. Experiments with SCID mice indicate that it is possible to transfer CTLs and thereby control viral proliferations. Such a vaccine might provide prophylactic protection for transplant recipients and (in combination with gp350) in nasopharyngeal carcinoma. Tumors may not express key antigens, or viral functions may impair peptide presentation. Ultimately, however, the goal would be to make a genetically altered EBV vaccine. This would be essential to protect against EBV-related malignancies such as Burkitt’s lymphoma and nasopharyngeal carcinoma, where the infection would have to be eliminated to prevent the disease. As detailed above, many of the necessary steps have been taken in this direction. Many people are infected with both type 1 and type 2 EBV, suggesting that wild-type infection is not protective. Animal Models. Animal models are somewhat limited. Cotton-top tamarinds develop lymphomas when EBV is injected parenterally, and they can be protected with gp350 vaccine. SCID-human chimeric mice also develop EBV lymphoproliferative diseases, and can be protected with transferred CTLs; but they may not be very useful for evaluating vaccines because of transient T-cell function and the absence of appropriate lymphokine synthesis. A new and promising prototype is a mouse EBV homologue, although it might be more similar to herpes saimiri. You can induce infection by internasal inoculation, and the mice develop lymphoid proliferations that have some similarities to EBV infection. However, the most encouraging animal model is a rhesus EBV that is highly homologous to EBV, and has identical patterns of mucosal infection and disease. Also, SIV-infected animals develop lymphomas. This may be the ideal system to test a genetically altered EBV virus. In response to questions from the audience, Dr. Raab-Traub added the following: No research is being done to analyze the mucosal infection or to induce mucosal immune response. There have been anecdotal reports of defective viral genomes in some seronegative subjects. These genomes, which lack the transforming EBV genes that generated immunoglobulin-A response in saliva, might offer some protection. 100 percent of infected B-lymphocytes become immortalized. Recombinant gp350 vaccine should be ready for testing in a small seronegative population soon. It would be effective against both type 1 and type 2 EBV.

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Vaccines for the 21st Century: A Tool for Decisionmaking HEPATITIS C VIRUS3 Hepatitis C virus (HCV) is a recent field of research but a major cause of disease. What used to be known as “non-A and non-B hepatitis” was recognized in the mid-1970s with the development of serological tests for the hepatitis A and B viruses, and the cause was identified and named in 1988. Researchers are only beginning to understand the interaction of HCV with the host and the ensuing immune responses. With the technology now available in the areas of recombinant antigen production, adjuvants, and genetic immunization, researchers hope to have at least some impact on transmission and disease development at some point in the future. Burden and Epidemiology. HCV is a truly global problem and is the major infectious disease problem in Japan. In the United States, there have been 150,000 new cases of HCV infection per year for the past decade. From 50 percent to 60 percent of these infections progress to persistent viremia and chronic persistent hepatitis. Of patients with chronic hepatitis, about 20 percent will progress to cirrhosis, and about 20 percent of cirrhotics with HCV will undergo liver failure. As a result, HCV is most common cause for liver transplantation in the United States. In addition, it is now clear that hepatocellular carcinoma is associated with HCV, with cirrhosis as a precondition, and that about 10 percent of cirrhotics with HCV will develop cancer over a period of time. Research in Japan indicates that mean time from infection to cirrhosis is 20 years, and to cancer 30 years, so this is a very indolent type of disease. At the same time, research at NIH indicates that in a minority of patients cirrhosis can develop in a few years. Conversely, around 15 percent of post-transfusion HCV infections become negative over the long term, so there are some spontaneous resolvers. The biggest risk factor for contracting hepatitis C is intravenous drug use, constituting about half of U.S. infections. About 30 percent to 40 percent of cases have no known risk factor. Transfusion is now a negligible risk, with the introduction of a blood screening test. There is a measurable incidence in health care workers who are exposed to infected blood, and data from Japan indicated that mothers can transmit the virus to their babies. The issue of sexual transmission is controversial, but studies show that multiple heterosexual partners are a risk factor, while the incidence in homosexual men is extremely low; apparently HCV can be transmitted sexually, but it is inefficiently transmitted that way. Viral Genome. HCV belongs to the Flaviviridae family, along with flavivirus and pestiviruses. It is an RNA virus that does not integrate into cellular DNA, and instead replicates only through RNA replication intermediates. The virus is highly heterogenous: it can change rapidly in the infected host, and at 3   Based on a presentation by Michael Houghton, M.D.

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Vaccines for the 21st Century: A Tool for Decisionmaking least 6 major types and 40 subtypes have been identified around the world. The 1A and 1B subtypes account for most infections in the United States. The genome consists of about 10,000 nucleotides, with a 5' terminal iris and an internal ribosome entry site. It makes a large polyprotein that is cleaved post-translationally and co-translationally by a combination of host enzymes and viral proteases. It seems that the host signal peptidase is primarily involved in processing the nuclear capsid and the two envelope glycoproteins (gpE1 and gpE2). The presumed nonstructural proteins (NS) appear to be processed by the action of two viral proteases, one in the NS3 domain that is a trypsin-like protease, and another that spans the NS2 and NS3 genes that appears to be a metallic protease. Immunology. Preliminary work on the correlates of immunity have shown that peripheral CD4-positive T-cells respond to HCV nonstructural protein 3 (NS3) very early in the infection, and that this response persists following recovery. In chronic patients, however, there is very little T-cell response. There is evidence that the protective immune response is short-lived and weak, at best. For example, a study of polytransfused thalassemic children found that patients who normalized after an initial episode of acute hepatitis nevertheless developed a second infection that progressed to chronic persistent hepatitis. Both infections involved the HCV-1-B subtype. Viral Persistence. HCV is remarkably adept at persisting in the host in the face of an apparently substantial immune response. In the livers of about 50 percent of patients with chronic hepatitis, researchers are able to identify CTLs of varying specificity to either structural protein or to nonstructural proteins in the polyprotein precursor. CTLs also infiltrate the livers of chimpanzees with chronic infections. It is not known how the virus persists in the face of such a response. One theory is that HCV has evolved a mechanism for abrogating a lymphokine action; another is that the virus inhibits CTL induction in vivo. It is possible that there may be immune-privilege sites in the host that have not been identified. Another theory is based on recent work suggesting that there are CTL escape mutants in chronically infected chimpanzees. An otherwise conserved epitope in nonstructural protein 3 (NS3), which is the target for a strong CTL response, mutated over time, leading researchers to speculate that escape variants might emerge. Immune Escape. Virtually every patient with chronic non-A and non-B hepatitis, whether from transfusion or IV drug use, has circulating levels of antibodies to the envelope glycoproteins gpE1 and gpE2. HCV can be difficult to grow in vitro, but work done in Japan suggests that the virus mutates over time to evade this humeral immune response. Using an RNA binding assay, researchers were able to show that serum taken from 1978 to 1982 contained antibodies that would neutralize HCV taken in 1977. In the same patient, however, the antibodies that neutralized the 1977 virus did not neutralize virus

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Vaccines for the 21st Century: A Tool for Decisionmaking theoretically be eligible for immunotherapy. In practice, because some tumors express different tumor antigens, about 60 percent of melanoma patients could be eligible, and lower percentages of patients with other tumor types. In a preliminary study, patients received three different dosages (30, 100, or 300 micrograms) of MAGE-1 peptide, without adjuvant, subcutaneously, at monthly intervals. After three injections, there was little or no toxicity, no tumor response, and no CTL response. In a second study, patients received either 100 or 300 micrograms of MAGE-3 peptide, again subcutaneously and at monthly intervals. There was no toxicity, but significant tumor response and at least one possible case of CTL response. One melanoma patient showed considerable response by the day of the third injection, but unfortunately died a month later of a brain metastasis. A second patient with 100 metastases around a skin graft showed significant regression after three injections and one year later is tumorfree. A third patient, designated AVL3, had primary melanomas removed in 1990, but in April 1995 had several metastases in the lung; by October 1995 the patient was disease-free, although the tumor subsequently relapsed. In response to questions from the audience, Dr. van der Bruggen added the following: Human patients show tumor response but no CTL response, either spleen or peripheral blood; experimental mice show CTL responses but no regression of tumors. Researchers have not had the opportunity to look for tumor-infiltrating lymphocytes, nor have they looked for antibodies against MAGE-1 and MAGE3, although this is planned. Researchers cannot explain why regressions are observed only after the third injection. One member of the audience speculated that CTLs have fairly short memory, and since the tumor itself may not be a good source of antigen, multiple injections are needed to maintain a high level of CTLs. It may be possible to elicit a CD4 restricted response (e.g., to tumor lysate) as a supplement to the initial CD8 response.

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Vaccines for the 21st Century: A Tool for Decisionmaking IMMUNITY TO ONCOGENIC SELF-PROTEINS25 Immunity to Mutated Ras. Researchers asked whether or not oncogenic proteins can be targeted for vaccine and T-cell therapy. They first examine mutated ras as a prototype. Ras is activated by point mutations that are common in diverse tumors. Ras is present in about 15 percent of all human tumors, including 50 percent of colon cancer and 95 percent of pancreas cancer. Animal experiments demonstrated that ras can function as a tumor-specific antigen, eliciting both helper T-cell and CTL responses that can decrease the growth of tumor in vitro and in vivo. Researchers have also found existent immune responses in a few patients with pancreatic and colon cancers. Most are antibody responses to normal ras, but a small number have a very restricted antibody response to the mutated protein. Other patients have a very specific T-cell response to the mutated segment of ras, but there is a problem in trying to focus immune attack against a single epitope. More importantly, oncogenic proteins that are activated by mutation have increased function, and proteins that have increased function don’t have to be present in abundant amounts. Immunity to HER-2/neu. Rather than looking at immune responses to proteins that weren’t abundant, researchers asked whether or not it was possible to focus an immune attack against an oncogenic protein that is present in large amounts. Their prototype was HER-2/neu, a very large nonmutated protein that is expressed in very low amounts in some normal tissues, but is amplified in about 25 percent of breast cancer patients. When amplified, it is overexpressed and substantially more abundant. The structure of HER-2/neu includes a very large extracellular domain, so large that there are potentially epitopes for every individual. Initial experiments revealed that 15 percent or 16 percent of breast cancer patients have existent antibody responses to HER-2/neu, including 42 percent of patients with documented overexpression of the protein. In the latter case, the immune response is assumed to be elicited by virtue of the fact that the protein is overexpressed. Unfortunately, some normal patients also have a response, between 2 percent and 5 percent based on screening of blood donors. Experience has shown that by setting the cutoff level high enough—in this case, a titer of less than 1:500—it is possible to exclude virtually all responses in normal individuals while retaining a much more specific response for breast cancer patients. In many cases, the responses are extremely low, between 1:100 and 1:500, but five patients out of 96 had very substantial antibody responses, with titers of greater than 1:12,000. As it happened, these same five patients also had stage I or stage II breast cancer, as opposed to more advanced disease. In general, 25   Based on a presentation by Martin Cheever, M.D.

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Vaccines for the 21st Century: A Tool for Decisionmaking antibody titer tended to be highest in patients with early breast cancer, and to decrease with more advanced disease. However, HER-2/neu was overexpressed in about 25 percent of patients with advanced breast cancer, where it was associated with more aggressive disease. Researchers speculate that the existent immunity occurs early on in the course of disease and prevents the progression of some patients. Researchers have shown that the antibody response is to the whole protein and to both the intracellular and extracellular domains. This appears to be due to cell breakdown, which releases previously sequestered segments of the protein. The extracellular domain functions as a growth factor receptor, so that when it is amplified and overexpressed, the signalling through this protein is part and parcel of the aggressiveness of the disease. Some monoclonal antibodies to HER-2/neu have agonistic effects, some have antagonistic effects; hence, antibodies to the extracellular domain might either inhibit or stimulate the growth of breast cancer cells. Researchers studied the patients with the highest antibody titer in greater detail. In several of these patients, the IgG antibody binds to the same tumor cells that overexpress HER-2/neu. Using epitope mapping, they have discovered a segment of the extracellular domain that is rich in cystine and thus a potential binding site. The researchers are now investigating whether this is a functional antibody. Some patients with an IgG antibody response also showed a proliferative T-cell response to HER-2/neu. The patient with the highest antibody response also had the greatest T-cell response, to both whole protein and to peptides from the intra- and extracellular domains. Other patients showed proliferative response to peptides but not whole protein. Researchers have not yet mapped all of the epitopes involved. Animal Models. To learn how to immunize with the HER-2/neu protein, researchers needed to develop an animal model. In the mouse, the neu sequence is not evident and has not been closed. Rat neu protein is quite homologous to human HER-2/neu, but when researchers immunized rats with purified neu protein in complete Freund’s adjuvant, they could find neither proliferative T-cell response nor antibody response. Earlier researchers had developed a vaccinia virus vector that expressed the extracellular domain of rat neu protein. While this vector was immunogenic in mice, however, it too failed to elicit a response in rats. Those researchers had concluded that the failure to elicit a response was due to tolerance to self. However, subsequent screening has identified patients with existent immune response, proving that is possible to overcome tolerance to HER-2/neu. Researchers therefore began to focus on immunizing to fragments of the protein. They found that immunizing rats with groups of intracellular domain neu peptides resulted in peptide-specific T-cell and antibody responses, and that the T-cells that responded to peptide also responded to whole protein. They were also able to immunize to peptides from the extracellular domain, but the response was much weaker. This may be due to a biological principle—i.e.,

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Vaccines for the 21st Century: A Tool for Decisionmaking tolerance of extracellular domain is more stringent because it is a shed protein that is more available to the thymus for induction of tolerance—or it may be because researchers merely picked the wrong peptides to work with. Sequencing shows that the peptides to which rats were immunized are identical in the human protein (rat neu and human HER-2/neu are about 89 percent homologous at the amino acid level). Hence, the antibodies that will immunoprecipitate the rat protein will also immunoprecipitate the human protein. These are also peptides to which at least some human patients have responded. Researchers now plan to go forward with a vaccine trial in which humans patients will be immunized with peptides identified as immunogenic in rats. The adjuvant used in the rat studies was complete Freund’s, which is too toxic for standard use in humans, so they plan to use GM-CSF as an adjuvant. Instead of growing dendritic cells in vitro with GM-CSF, the cytokine is injected intradermally with the peptide. Animal tests have shown that it is possible to generate immune response to intra- and extracellular domain peptides with GM-CSF as the adjuvant, and indeed that the DTH assay is much stronger that when using complete Freund’s adjuvant. Researchers have submitted to an IRB a protocol to immunize patients with breast and ovarian cancers with peptides. FDA has signed off, and the protocol should begin within months. Data should be available before 2000 on whether these peptides are toxic and/or efficacious in vivo. Autoimmune Cancer Therapy. Generating an immune response to self-protein does not resolve the issue of whether it is possible to induce an aggressive autoimmune response as a form of cancer therapy. To answer this question, researchers focused on the prostate—once the prostate becomes malignant, it is often removed, at which point any prostate tissue left in the body is by definition malignant. If it were possible to induce a rapidly destructive, aggressive autoimmune prostatitis, it would have therapeutic benefit. There are several problems in this use of autoimmune prostatitis; not the least is immunological tolerance to self-proteins. There is no information available on which prostate-specific proteins are immunogenic; nor have experiments confirmed that autoimmunity can destroy normal prostate tissue. In addition, most autoimmune disease is relapsing and often resolves spontaneously, while this strategy requires a rapid and aggressive autoimmunity that can eradicate the organ. (Parenthetically, there is a general lack of attention to CTLs in the field of autoimmunity, possibly because autoimmunity isn’t mediated by CTLs, or possibly because CTLs are too difficult to deal with.) Researchers first tried to immunize to prostatic acid phosphatase (PAP), a common and well-characterized marker for human prostate tumor for which the sequence of a rodent model is also known. PAP is a glycoprotein secreted exclusively by prostate epithelial cells. It is expressed by all normal prostate tissue and by most prostate cancers. And while portions of the molecule are similar to

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Vaccines for the 21st Century: A Tool for Decisionmaking acid phosphatases from other tissues, other portions are prostate-specific. All of these factors made it an appealing target. As with HER-2/neu, purified rat PAP with complete Freund’s adjuvant induced neither helper T-cell nor antibody response. Peptides induced helper T-cell responses in female rats, which had little or no protein in their systems, and in two cases the response was peptide-specific. But rat peptides failed to induce any response in male rats. Trying another approach, researchers first immunized male rats to homologous human PAP peptides, then immunized with rat peptides, and this sequential immunization did induce helper T-cell and antibody responses to rat PAP. Researchers believe that their ability to immunize females but not males is related to the peptides they used, which are far less abundant in females than in males; they are currently investigating this supposition. Even when they succeeded in immunizing male rats, however, researchers found that there was none of the inflammation to the prostate that would have been expected. This suggests that tumor immunotherapy vaccination is very ineffective for inducing any antitumor response. Researchers suspect that it will be necessary to use T-cell therapy to get the precursor frequency high enough to support an immune response. The researchers have not yet tested or tried to elicit CTLs. Researchers are currently trying to determine which proteins are most immunogenic in autoimmune prostatitis, and will focus in the future on those among them that are expressed by prostate cancer. Relatively little is known about autoimmunity and prostate in either humans or rats—only that prostate inflammation can be induced in rats immunized to prostate homogenate. Previous studies used multiple injections of homogenate with repeated use of complete Freund’s, which is no longer allowed. This results in antibody response to a variety of different proteins, which are now being identified, but immunization with whole prostate doesn’t get a response to PAP. By selecting out the fractions of protein that induce the greatest antibody response, and then immunizing to these fractions alone, researchers were able to generate a very substantial, rapidly progressive, destructive autoimmune prostatitis. The researchers are now concentrating their efforts on (1) identifying the exact targets of this response and (2) learning how to induce this response prospectively and at will. Considerable additional research will be needed before these results can be used for therapeutic benefits in human prostate cancer. Autoimmune prostatitis is an ill-defined syndrome, and none of the relevant antigens have been identified. Animal experiments may reveal how to maximize the destructive response, but it will still be necessary to identify the human proteins that are homologous to rat proteins, and verify that the homologous antigen is immunogenic in humans, before instituting human vaccine and T-cell therapy trials for prostate cancer. In response to questions from the audience, Dr. Cheever added the following:

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Vaccines for the 21st Century: A Tool for Decisionmaking Several strains of rats are used in these studies. Whole prostate homogenate appears to contain fractions that somehow suppress or block the immune response, as well as fractions that induce a response. Researchers use the Western blot test to determine which induce an antibody response, and then select for them. Ras and other oncoproteins still represent a valid approach, but it will apply only to a small subset of patients. The advantage of working on a protein like HER-2/neu is the greater likelihood of getting a response in every individual who has that very common protein. CYTOKINES AND THEIR LOCAL ENVIRONMENTS26 Potential Energy Model. A certain threshold level of response against a particular tumor antigen is required for rejection of that tumor. The endogenous level of immunity against the antigen is below this threshold; vaccination must enhance existent immunity sufficiently to raise the response above this threshold level. This has implications for the choice of antigen and vaccine strategy: Even if the endogenous level of immunity to antigen A is close to the thre-shold, if the vaccine approach is weak, the response will not reach the threshold, and the vaccine fails. On the other hand, if the endogenous level of immunity to antigen B is much lower, even a strong vaccine may not be able to raise the response to the threshold, and the vaccine still fails. An extreme example of the latter case is an antigen expressed in the thymus, with tolerance generated by clonal deletion; the endogenous immunity is negative, and no vaccine approach could hope to produce a therapeutic response. Consequently, the ideal strategy is to identify a good antigen whose endogenous level of immunity is relative close to the threshold, along with a strong vaccine approach. This involves issues of endogenous self-tolerance, repertoire, and vaccine approaches. At the present time, however, there is almost an embarrassment of riches in terms of different approaches to cancer vaccines—peptides, recombinant adenovirus, pox virus, Listeria, BCG, etc. It may well be impossible to test each of them reasonably in patients unless there is first a concerted effort to compare them rigorously, in a head-to-head fashion, in the appropriate animal models. Role of Cytokines. For many cancers these target antigens still aren’t known, although it is assumed that tumor cells themselves are important—the 26   Based on a presentation by Drew Pardoll, M.D.

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Vaccines for the 21st Century: A Tool for Decisionmaking antigens relevant to immune response are in there, somewhere. However, another avenue of research has concentrated on the lack of signal 2 as the missing ingredient in immune response against tumors. There was evidence from earlier studies that one way to activate T-cells against tumor antigens was to provide signal 1 and signal 2 on the same cell, and that an important signal 2 for CTLs was IL-2 (or lymphokines made by helper T-cells). Based on this paradigm, researchers began introducing cytokine genes into tumor cells, beginning with IL-2, in order to produce a whole-cell vaccine that contained all of the antigens and presented peptide as MHC signal 1 and signal 2 on the same cell. Aided by the use of defective retroviral vectors, they inserted a wide range of cytokine genes into a weakly immunogenic tumor, vaccinated animals, and compared the resulting protection against challenge with wild-type tumor cells. The most effective vaccine involved tumor cells transduced with a GM-CSF gene. This was a surprise at the time, but other groups soon reported that GM-CSF has the unique and interesting function of inducing hematopoietic progenitors to differentiate not only into granulocytes and macrophages, but also into dendritic cells. It may be that these high-potency APCs, differentiating locally in the presence of GM-CSF, have something to do with the enhanced systemic immune response to GM-CSF-transduced tumor cells. Subsequent research has tried to explain how this process works. Paracrine Cytokine Adjuvant. What turned out to be important physiologically, in addition to the particular cytokine, is the elaboration of that cytokine at the site of the antigen. In a sense, the GM-CSF-transduced tumor cell actually represents a timed-release depot for two sets of molecules: its own antigens, and GM-CSF. Importantly, it also replaces the complex “black boxes” of conventional adjuvants (BCG, C.parvum, mycobacterium, TB) with a single molecule, and in doing so generated a systemic antitumor immune response that was more potent than mixing irradiated tumor cells with conventional adjuvants. Researchers have learned that APCs that differentiated at the site of the vaccine, under control of GM-CSF, actually ingest antigens from tumor cells and process them into both the Class I and Class II pathways, an example of crosspriming. As the APCs are ingesting and processing antigens, they are travelling to the draining lymph node, where one can first identify activated Class I- and Class II-restricted CTLs and helper cells. Once activated, these cells leave the draining lymph node and circulate systemically. An important implication is that effector-phase CD4 is very important, in addition to CD8-positive cells. Consequently, the best vaccines will involve epitopes that actually represent tumor antigens. For this reason, there should be concern over the use of “universal helper epitopes,” which are not expressed by the tumor cell. Clinical Trials. These results led researchers to initiate a Phase I trial in patients with metastatic renal cancer who had undergone nephrectomy to remove the primary tumor. They used a retroviral vector to transduce human GM-CSF gene into tumor cells, expanded these cells, irradiated them at doses that

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Vaccines for the 21st Century: A Tool for Decisionmaking inhibited replication but not immunogenicity, and then vaccinated patients with three monthly injections at two different doses, half the dose intradermal and half subcutaneous. Researchers didn’t expect and didn’t find any toxicity; the important results were immunological. In order to determine whether paracrine elaboration of GM-CSF generated human immune responses, patients were randomized in a double-blind fashion to receive either irradiated tumor cells or irradiated tumor cells transduced with the GM-CSF gene. DTH was chosen as the simplest and most reproducible assay for in vivo immune response. A total of 30 days after vaccination, 1 million nontransduced cells were injected at a distant site, and the diameter of induration, edema, and erythema was measured to determine DTH response. When the blind was broken, the results indicated that the lower vaccine dose (4 million cells) did not induce significant immune responses. However, the higher dose (40 million cells) did produce some fairly impressive DTH responses in patients who had received the GM-CSF-transduced vaccine. Even more encouraging were the results of the biopsy analysis of infiltrates at the DTH site. In addition to the quantitative difference, roughly 50 percent of the infiltrating cells in GM-CSF-transduced patients were eosinophils, whereas no eosinophils were found infiltrating the DTH sites of nontransduced patients. This was the same result observed in animal tests. Anecdotally, one of the three patients that received the higher dose of transduced vaccine showed a fairly significant clinical response. This patient had multiple pulmonary metastases from his renal cancer that had progressed very rapidly over the 2 months between surgery and initial vaccination. After 1 vaccination, there was evidence of regression, and after the third vaccination there was a 95-percent reduction in the volume of metastatic tumor. While anecdotal, this suggests that there may be a therapeutic correlate to the observed immune response. Allogeneic Vaccines. In these renal cancer patients, as in the melanoma patients discussed above, the vaccine did not induce a response against normal tissue; patients showed no impairment or autoimmunity of their remaining kidney, just as melanoma patients did not develop vitiligo. This suggests that it should be possible to generate responses against them without generating a clinically prohibitive autoimmune disease. At the same time, there is mounting evidence that the immunorelevant antigens in tumors are shared, as was the case in the MAGE proteins (see above). This may provide a rationale for using a genetically modified allogeneic vaccine. Tumor antigens are presented to T-cells not by the tumor but by APCs derived from host bone marrow. This implies that it may not be necessary to match HLA between the vaccine and the patient. Certainly, generic vaccines would be far less labor-intensive and less expensive than individualized vaccines, and—since quantities would no longer be limited by the growth potential

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Vaccines for the 21st Century: A Tool for Decisionmaking of the individual’s tumor—it would also be possible to vaccinate with higher doses. Antigen-Specific Tumor Vaccines. Researchers also hope to use activated T-cells to identify the relevant antigens for antigen-specific vaccines. One group has been pursuing this concept in the mouse model using CT26, an NMU-induced colon cancer, in order to identify the repertoire of antigens that are being recognized by the CD8 arm of the immune response following vaccination with GM-CSF-transduced tumor cells. Their technique involves taking bulk T-cells from the draining lymph nodes (rather than tumor-specific CTLs), eluting peptides from Class I molecules, fractionating them with reverse-phase HPLC, and using surrogate targets to assay peptide fractions for bioactivity. The results indicated that there was only one bioactive fraction, suggesting that the majority of CD8-positive immune response was focused on a single peptide among the many presented by the CT26 tumor. These results have been repeated in 40 separate experiments. This peptide, called AH1, had a molecular weight of 1,128 and was doubly charged. It sensitized surrogate target cells down to a concentration of 5 X 10-12 molar. Upon sequencing, it proved to be a peptide derived from an endogenous murine MuLV gene that is normally completely silent in the BALB/c genome but is reactivated by altered methylation, much like MAGE-1 (see above). (The latter finding has led to new interest in endogenous human retroviruses, which also seem to be reactivated in human tumors.) AH1 is not expressed in normal tissues from BALB/c mice, including normal colon and small intestine epithelium, but it is turned on in a number of different tumors. Interestingly, if T-cells from vaccinated animals are stimulated for two rounds with AH1 plus IL-2 and then adoptively transferred back into animals with CT26 tumors, most of the animals are cured. However, there is no significant response when tumor-bearing animals are vaccinated with AH1-pulsed dendritic cells or other approaches. Researchers believe that they should examine several other approaches for introducing these gene products in vivo, such as viruses that target them to both the Class I and Class III MHC pathways. In addition, there are some interesting recombinant viral and bacterial approaches that should be compared head-to-head with endogenous tumor antigen models before deciding which approaches will be taken to clinical trials, with their tremendous investment of time, effort, and expense. In response to questions from the audience, Dr. Pardoll added the following: Although GM-CSF up-regulates B7 in macrophages, it does not do so in tumor cells and, hence, this cannot explain their increased immunogenicity. In fact, the major effect of transducing B7 into tumors is to provide a target molecule for NK cells to more actively lyse the tumor cells. A B7-transduced tumor cell isn’t nearly as good an APC as a bone marrow progenitor that differentiates into a dendritic cell in the presence of GM-CSF.

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Vaccines for the 21st Century: A Tool for Decisionmaking Researchers have compared paracrine GM-CSF (either by transduction or by time-release microspheres) with BCG and C. parvum in seven or eight different tumor models. The resulting systemic immune response generated by GM-CSF is between 1.5 and 4.0 logs more potent than either adjuvant. While researchers have learned a tremendous amount from their experiments, they are also certain that transducing autologous tumor explants is not feasible for large-scale application in the general patient population. Microsphere approaches obviate the need for GM-CSF transduction, and the question is moot if immunodominant tumor antigens are in fact shared. However, it may be 15 to 30 years before investigators identify the relevant antigens for all of the important tumors. GM-CSF significantly up-regulates both Th-1 and Th-2 lymphokines. A single vaccination with B7-transduced tumor cells does not evoke a measurable response that maps to the tumor’s MHC type. There is a small response to a second vaccination. Tumor antigens will ultimately prove to be very important, but at present the best strategy for identifying immunorelevant antigens is to use whole-cell vaccines and let the immune system indicate which of the 50,000 or 100,000 antigens in that tumor it is capable of responding to. There is no data to support the assertion that viral sequences bind to MHC better than self-sequences. However, high-affinity binding generates a much more profound tolerance. A total of 10 percent or 15 percent of tumors turn off MHC Class I, in which case CTL response is irrelevant. In such cases, NK cells can be brought into the response to replace CD8 responses. Once the relevant antigens are defined, melanoma and cervical cancer are both logical targets for cancer vaccines. So, too, are cancers of dispensable tissues (e.g., ovary, prostate, breast). Eventually, tumor vaccines might be used prophylactically, to prevent tumors caused by viruses before they occur (e.g., human papilloma virus, hepatitis C virus).

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