1. VECTORS IN VACCINE DELIVERY To transfer the desired gene into a target cell, a carrier is required. Such vehicles of gene delivery are known as vectors. 2 main classes – Viral vectors – Non viral vectors

4. Cancer Vaccines CD4 T Cell TCR Class II MHC Class I MHC Activated Dendritic Cell Tumor Antigen Cytokines = HELP CD8 T Cell Activated CD8 T Cells Traffic to Tumor and Lysis Cells Burch et al, 2000; Small et al 2000; Fong et al, 1997.

5. PSA LFA-3ICAM-1B7-1 Co-Stimulatory Molecules Target Antigen Plasmid DNA Vaccinia Virus Fowlpox Virus rV-PSA-TRICOM rF-PSA-TRICOM Packaging Cell Line Vaccine Viral Vaccines – Same Idea: But Starting At A Different Step PSA= prostate-specific antigen. Madan et al, 2009; Sonpavde et al, 2011; Drake, 2010. ProstVac VF

6. CD4 T Cell TCR CD8 T Cell TCR Class II MHC Class I MHC Epithelial Cells ACTIVATED CD8 T Cell Madan et al, 2009; Sonpavde et al, 2011.

7. The only cell-based therapy currently approved for the treatment of prostate cancer. 1.he antigen prostatic acid phosphatase (PAP), which is present in 95% of prostate cancer cells, and 2.an immune signaling factor granulocyte- macrophage colony stimulating factor (GM-CSF) that helps the APCs to mature.

9. Basic research Gene therapy vaccines APPLICATIONS

12. MOST COMMON VIRAL VECTORS Retroviruses Adenoviruses Adeno-associated viruses Herpes simplex viruses can create double-stranded DNA copies of their RNA genomes. Can integrate into genome. HIV, MuLV, Rous sarcoma virus dsDNA viruses that cause respiratory, intestinal, and eye infections in humans. Virus for common cold ssDNA viruses that can insert their genetic material at a specific site on chromosome 19 dsDNA viruses that infect a neurons. Cold sores virus

14. 1.their basic biology has been studied extensively 2.they have previously been shown to be relatively safe for use in humans. Coxsackievirus and adenovirus receptor (CAR)

16. ADENOVIRUSES: vaccines Adenovectors are also being evaluated as vaccines for animal diseases such as rabies, and foot and mouth disease The first gene therapy licensed product, Gendicine, is based upon an adenovector encoding tumor suppressor p53 and was approved in 2003 in China for use in the treatment of squamous cell head and neck cancer adenovectors are being developed in efforts to make vaccines for infectious diseases, including HIV malaria,38 SARS,39,40 and Ebola. Further, a nasal application of an adenovirus expressing an influenza gene has demonstrated safety and immunogenicity in humans are used in not only single-modality vaccine regimens but also mixed-modality prime-boost strategies, for instance, following a prime with plasmid DNA vaccines.

18. ADENO-ASSOCIATED VIRUSES 1.the virus is dependent on the co-infection and helper functions of other viruses such as adenovirus and herpesvirus for efficient replication. 2. No human disease has been associated with AAV; instead, it was shown to have beneficial effects for the host as it inhibits the activity of oncogenic viruses such as papilloma virus and adenovirus. 3.AAV exists in several serotypes; type 2 has been most explored as a vector for delivering foreign genetic material.

19. Viral Entry. The cell tropism of AAV varies between the different serotypes, thus making a specific serotype more suited for a particular application depending on the target tissue. AAV-2 is able to infect a variety of cells, including liver, lung, muscle, and central nervous system, and is believed to enter cells primarily by binding to heparin sulfate proteoglycans, expressed throughout the body. Upon binding to the target cell receptor, the AAV is internalized by endocytosis, is released in the cytosol upon acidification of the late endosome, and subsequently travels to the nucleus

20. Poxviruses The poxviruses comprise a family of complex double-stranded DNA viruses. They belong to the largest family of virus infectious for humans. Canarypox and fowlpox have been utilized, because they infect but do not replicate in human cells. There is thus no concern about preexisting immune responses to these vectors. Variants of avipox members have been used in several preclinical and clinical studies Orthopox: smallpox virus(variola), v acciniavirus, cowpox virus, monkeyp ox virussmallpoxv acciniacowpoxmonkeyp ox

21. HERPEX SIMPLEX VIRUS VECTOR

22. VIRAL VECTORS 1) RETROVIRUS VECTOR SYSTEM The recombinant retroviruses have the ability to integrate into the host genome in a stable fashion. Can carry a DNA of size – less than 3.4kb Target cell - dividing

23. Retro virus:

28. IDEAL FEATURES OF A VECTOR: ☻High concentration of virus allowing many cells to be infected or transduced or transduced ☻Convenience and reproducibility of production ☻Ability to transduce dividing and non-dividing cells ☻Ability to integrate into a site-specific location in the host chromosome, or to be successfully maintained as stable episomes. ☻A transcriptional unit that can respond to manipulation of its regulatory elements ☻Ability to target the desired type of cell ☻No components that elicit an immune response

29. Group 1 (fowlpox group) = primed with an adenovirus-derived vector (expressing SIV genes) boost with a fowlpox virus-derived vector. Group 2 (Adeno group) = primed with fowlpox virus-derived vector boost with adenovirus-derived vector Control = “immunized” with empty vectors. Two groups of 9 animals each (3 controls and 6 vaccinees) Experimental Plan/ Time line of blood withdrawal for quantification of specified genes PI= preimmunization, FP= final priming, DOI= day of infection/challenge 29 Ad5-SIV comprised: Gag/ Pol- VACCINE TRIALS USING VIRAL VECTORS: AN EXAMPLE

30. Experimental Plan/ Time line of blood (PBMCs) withdrawal for quantification of specified genes Wpi= weeks post infection Pre inf= pre infection ex vivo evaluation of FAM26F expression upon SIV-infection Exp-1 included 24 animals Exp-2 included 18 animals Challenged with repeatedly with low dose of SIVmac251 Pre inf1wpi24wpi Pre inf24wpi 48wpi 12wpi 2wpi 12wpi Exp-1 Exp-2 30