James L Gulley MD PhD Philip M Arlen MD and Jeffrey Schlom PhD

The Revised Authoritative Guide To Vaccine Legal Exemptions

Vaccines Have Serious Side Effects

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Contents

Introduction

Properties of Pox Viruses

Prime and Boost With Pox Viruses

T-Cell Costimulatory Molecules

Dendritic Cell Vaccines With Pox Vectors

Cytokine Expression Driven by Pox Vectors

Future Development of Recombinant Pox Virus Vaccines

References

1. introduction

Tumor-associated antigens (TAAs) are by definition either weakly immunogenic or functionally nonimmunogenic. Vaccine strategies must be developed in which the presentation of these TAAs to the immune system results in far greater activation of T cells than is being achieved naturally in the host. One approach to create an inflammatory milieu designed to trigger a robust immune response to TAAs involves the use of recombinant viral vectors to deliver the appropriate genetic material. Though several viral vector platforms are currently being evaluated, the pox virus family of vectors possesses several properties that make these vectors extremely attractive for use in anticancer vaccines. These include: a) the capacity to integrate the entire tumor antigen gene, parts of that gene, and/or multiple genes (including genes for costimulatory molecules and cytokines), b) the ability to infect antigen-presenting cells (APCs) allowing them to process the antigens, and c) the relative low cost. The ability to incorporate multiple transgenes within one vector is unique to these large virions, and allows for potential synergy of the incorporated gene products. This chapter will provide some insight into the strengths and weaknesses of pox viral vaccines, as well as the preclinical studies and early clinical trials of pox viral vaccine strategies for cancer therapy.

From: Handbook of Cancer Vaccines Edited by: M. A. Morse, T. M. Clay, and H. K. Lyerly © Humana Press Inc., Totowa, NJ

Life Cycle Pox Viridae

(Replication del'cctivc)

Fig. 1. The pox virus family contains two groups that have been studied as vectors for cancer vaccines. The orthopox group contains vaccinia, which replicates in mammalian tissue. MVA (modified vaccinia Ankara) is derived from vaccinia; however, it is missing the genetic material required for replication. The avipox vectors are replication defective in mammalian tissue.

(Replication del'cctivc)

Fig. 1. The pox virus family contains two groups that have been studied as vectors for cancer vaccines. The orthopox group contains vaccinia, which replicates in mammalian tissue. MVA (modified vaccinia Ankara) is derived from vaccinia; however, it is missing the genetic material required for replication. The avipox vectors are replication defective in mammalian tissue.

Table 1 Properties of Pox Viruses

Can insert a large amount of foreign DNA

-Multiple transgenes (up to seven) Will infect a large range of cells Recombinant genes remain stably expressed Replication accurate Efficient post-translational processing Recombinant proteins are highly immunogenic

2. properties of pox viruses

Members of the Poxviridae family can be divided into those that are capable of replicating in mammalian species (e.g., Orthopoxvirus group) and those that can infect mammalian cells but cannot complete the replication process (e.g., Avipoxvirus group; see Fig. 1). There are advantages and disadvantages to the use of each as vectors. Some of the viruses that are replication competent have been extensively studied and have well-defined safety profiles (1). These vectors can continue to infect additional cells producing more TAA until they are eradicated by the immune system. Often vaccines associated with these replication-competent vectors are more immunogenic than those using replication-defective vectors. The replication-defective vectors, however, are theoretically safer because they can infect mammalian cells only once. Several properties of pox viruses have led to their extensive use as expression vectors, including their capacity for carrying large amounts of transgene DNA and their wide range of hosts (2,3). These viruses have several advantages, including the ability to make stable recombinant vectors with accurate replication and efficient post-translational processing of the transgene, and the fact that they do not integrate into eukaryotic DNA (see Table 1).

Pox viral vectors are unique in that their viral DNA replication and RNA transcription take place exclusively in the cytoplasm and can be demonstrated in enucleate cells. Because the vector's RNA is transcribed in the cytoplasm, the expression of any inserted transgenes requires pox virus promoters. Ideally, an open reading frame of the TAA is

Vaccinia Vector

Fig. 2. A diagram of a vaccinia-infected cell is shown with an exaggerated view of the endoplasmic reticulum (ER), cis, medial, trans Golgi, and the trans-Golgi network (C, M, T, and TGN, respectively). The major stages of the virus life cycle are listed. Following late gene expression, previrion forms assemble to form intracellular mature virus (IMV). The IMV is targeted to the TGN and following envelopment, intracellular enveloped virus (IEV) is formed. IEV are propelled to the cell surface by the polymerization of actin filaments. Once released, the virus may remain attached to the membrane as cell-associated enveloped virus (CEV) or be released into the medium as extracellular enveloped virus (EEV). Reproduced with permission from reference 10.

Fig. 2. A diagram of a vaccinia-infected cell is shown with an exaggerated view of the endoplasmic reticulum (ER), cis, medial, trans Golgi, and the trans-Golgi network (C, M, T, and TGN, respectively). The major stages of the virus life cycle are listed. Following late gene expression, previrion forms assemble to form intracellular mature virus (IMV). The IMV is targeted to the TGN and following envelopment, intracellular enveloped virus (IEV) is formed. IEV are propelled to the cell surface by the polymerization of actin filaments. Once released, the virus may remain attached to the membrane as cell-associated enveloped virus (CEV) or be released into the medium as extracellular enveloped virus (EEV). Reproduced with permission from reference 10.

juxtaposed to a viral promoter resulting in gene expression levels typical for that promoter. This cytoplasmic replication abolishes many safety concerns inherent in other recombinant viral vaccines that replicate in the nuclear compartment in close proximity to cellular genes.

When infecting a cell, a pox virus first undergoes endocytosis and the viral core is released into the cytoplasm (see Fig. 2). The viral transcriptase is then activated and viral mRNA is produced inside the cytoplasm within minutes after infection. This process also shuts down normal cellular transcription inside the nucleus. Among the first genes transcribed are ones encoding for proteins that complete the uncoating of the viral core and other "early" genes that are transcribed prior to reproduction of viral DNA. There are about 100 of these early genes, many with unique promoters, that can be replaced with transgenes encoding TAAs. With vaccinia infection in humans, viral DNA replication occurs within 1.5-6 h following infection. The process of virion assembly and mature particle budding from the membrane completes the life cycle. Multiple virions budding simultaneously from the membrane causes cellular lysis.

With pox vectors that are replication defective in mammalian species (e.g., fowlpox), the early genes are transcribed; however, viral DNA cannot be replicated nor can the late viral gene products that aid in assembly be made. Thus transgenes on early promoters are made within 30 min after infection with peak expression of proteins occurring in about

Table 2

Serious Complications of Vaccinia in Patients at Least 20 Yr of Age on Revaccination in a 10-State Survey (1968)^

auto-inoculation 25.0/106 vaccinations generalized vaccinia 9.1/106 vaccinations eczema vaccinatum 4.5/106 vaccinations post-vaccinia encephalomyelitis 4.5/106 vaccinations vaccinia gangrenosum 6.8/10 vaccinations

Note. It should be noted that these patients were given the vaccine by scarification without occlusive dressings and that patients with immunodeficiency states or skin conditions were not excluded, as is done in current clinical trials. 'c a Lane JM, Ruben FL, Neff JM, Millar JD. Complications of smallpox vaccination, 1968: results of ten statewide surveys. J Infect Dis 1970; 122:303-9.

Vaccinia (Smallpox) Vaccine Recommendations of the Immunization Practices Advisory Committee (ACIP) MMWR 40(RR14): 1-10. 12/13/1991

http://www.cdc.gov/mmwr/preview/mmwrhtml/00042032.htm

cVaccinia (Smallpox) Vaccine Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2001 MMWR 50(RR10): 1-25 06/22/2001

http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5010a1.htm http://www.cdc.gov/mmwr/PDF/RR/RR5010.pdf

6 h. During this process few viral proteins are translated, decreasing the potential for a host immune response to the vector. Cells infected with these virions also shut down cellular transcriptions and the cells slowly die over 14-21 d, generally by apoptosis. These cells do not release infectious virions.

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