Course Contents An introduction to Vaccinology Vaccine history

Course Contents • An introduction to Vaccinology • Vaccine history and types • Epidemiologic basis of Vaccinology • Role of combination vs. single vaccines • The immune responses to the pathogen & vaccine development • Immunology of Vaccines • Antigen and Vaccine Delivery Strategies • Determinants of Vaccine Availability • Vaccine development strategies: New approaches • Live versus attenuated vaccines • Role of multinational companies in vaccines production • DNA as vaccine • Peptide & Subunits vaccine • Adjuvants in vaccines • Population Genetic analysis: immunity to vaccine • Recombinant vaccine • Animal models of vaccine testing • Vaccine Delivery Systems • Practical issues in relation to trials • Ethical issues related to clinical evaluation of vaccines • Vaccine safety • Review of vaccines in current use • Vaccine-economics • Future of Vaccines/Vaccination

Course Contents PART 1 : INTRODUCTION • Definition • History • Types • Combination vs single vaccine Part 2: Principles of Vaccine Design Immunologic Memory: T and B Cells memory • Antigen Processing and Presentation by MHC Class I, II, and Nonclassical Molecules • Understanding the Mucosal Immune System for Better Mucosal Vaccine Design Part 3: ANIMAL MODELS FOR VACCNE TESTING • Utility of Mouse Models in Vaccine Design and Development, • Utility of Nonhuman Primate Models for Vaccines,

Part 4: Delivery Systems • Transcutaneous Immunization via Vaccine Patch Delivery System • Needle-free Jet Injection for Vaccine Administration • Oral Vaccines: An Old Need and Some New Possibilities Adjuvants: Part 6: Regulatory Considerations • Regulatory Issues • Role of international companies • Vaccine economics PART 7: Population genetics and vaccines • Ethical issues • Vaccine safety Part 5: Evaluating Vaccine Efficacy • Trials in human and practical issues

VACCINE DELIVERY SYSTEMS

VACCINE DELIVERY SYSTEMS 1. VIRAL VECTORS BASED 2. NON VIRAL

VACCINE DELIVERY SYSTEMS • • MUCOSAL DELIVERY OF VACCINES LIPOSOMAL DELIVERY SYSTEMS VIROSOMES DELIVERY SYSTEMS POLIMERIC NANOPARTICLE DELIVERY SYSTEMS DENDRIMER-BASED DELIVERY SYSTEMS NEEDLE-FREE DELIVERY EDIBLE VACCINES DNA VACCINES:

Nanopatch A stamp-size patch similar to an adhesive bandage contains about 20, 000 microscopic projections per square inch. When worn on the skin, it will deliver vaccine directly to the skin, which has a higher concentration of immune cells than the muscle tissue does, which is one of the tissues where injections commonly deliver vaccines.

1. Dermal administration by nanopatches thus increases the effectiveness of vaccination, 2. requiring less vaccine than injection.

LIPOSOMAL DELIVERY SYSTEMS

• Liposomes and their derivatives “lipoplexes” (liposome/DNA complexes) are hollow spherical constructs of phospholipid bilayers capable of entrapping hydrophilic moieties in the aqueous compartment and hydrophobic moieties in the lipid bilayers with cholesterol imparting rigidity to the bilayer. • Viruses, proteins, glycoproteins, nucleic acids, carbohydrates, and lipids can be entrapped and targeted at cellular and subcellular level for evoking immune responses • Diphtheria liposomal vaccine shows good immunogenicity and tolerance in humans

VIROSOMES DELIVERY SYSTEMS Virosomes represent vesicular systems into which antigens can be loaded into virosomes or adsorbed onto the virosomal surface through hydrophobic interactions

VIROSOMES DELIVERY SYSTEMS • Virosomes are small spherical unilamellar lipid membranes of nucleocapsid including vesicles (150 nm) embedded with viral membrane proteins such as hemagglutin and neuraminidase of influenza virus but devoid the genetic material of the source virus. • Once they have delivered the antigens, the virosomes are completely degraded within the cells

DENDRIMER-BASED DELIVERYSYSTEMS • Dendrimers are branched, synthetic polymers with layered Architectures. to bind the DNA and get it into the cell.

DENDRIMER-BASED DELIVERYSYSTEMS • Dendrimers, available under the trademark name of “Starburst” serve as nonviral gene transfer agents, enhancing the transfection of DNA by endocytosis and, ultimately, into the cell nucleus. • A novel approach for the treatment of renal cell carcinomas uses a chimeric molecule comprising a granulocyte macrophage colony stimulating factor (GM-CSF) attached to a G 250 kidney cancer specific antigen which is transfected in to the cancerous cell by the use of dendrimer

POLIMERIC NANOPARTICLE DELIVERY SYSTEMS Polymeric nanoparticles because of their size are preferentially taken up by the mucosa associated lymphoid tissue. They are extensively reviewed for nasal and oral delivery of vaccines

EDIBLE VACCINES

CONCLUSION • Vaccine drug delivery systems are gaining popularity these days due to the benefits they offer. • As they avoid the need to administer booster doses and provide a long term therapy in small dose. • Needle free technologies, Edible vaccines on the other hand open an attractive avenue for the oral delivery of vaccines.

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

Cancer Vaccines CD 4 T Cell Activated Dendritic Cell TCR Class II MHC TCR Tumor Antigen Cytokines = HELP Class I MHC CD 8 T Cell Activated CD 8 T Cells Traffic to Tumor and Lysis Cells Burch et al, 2000; Small et al 2000; Fong et al, 1997.

Viral Vaccines – Same Idea: But Starting At A Different Step Co-Stimulatory Molecules PSA Prost. Vac VF LFA-3 ICAM-1 B 7 -1 Target Antigen Vaccinia Virus Fowlpox Virus Plasmid DNA Packaging Cell Line r. V-PSA-TRICOM r. F-PSA-TRICOM PSA= prostate-specific antigen. Madan et al, 2009; Sonpavde et al, 2011; Drake, 2010. Vaccine

Prost. Vac VF CD 4 T Cell TCR Class II MHC TCR Class I MHC Epithelial Cells CD 8 T Cell ACTIVATED CD 8 T Cell Madan et al, 2009; Sonpavde et al, 2011.

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

APPLICATIONS Basic research Gene therapy vaccines

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

diseases ranging from subclinical infections, in which the person does not even notice the infection, to upper respiratory infections, to diseases of many organ systems As opposed to Lentiviruses, adenoviral DNA does not integrate into the genome and is not replicated during cell division.

Viral Entry. The breadth of diseases caused by adenoviruses reflects the extensive tropism of the virus. This ability to infect a number of different types of cells is one of the reasons that adenoviruses have been developed as vectors for delivering genes for both vaccine and gene therapy applications

Adenoviruses are generally quite species-specific, complicated the preclinical studies of adenovectors. PRE-EXISTING IMMUNITY • Exploring types other than 5 • virus with altered surface proteins or chimeric virus carrying the surface proteins of another serotype An adenovector can package 105% of its genome size into its rigid capsid, and thus, a transgene of 3 -4 kb can be included for replication-competent vectors. Replicationincompetent vectors can house transgenes ranging from 7 -8 to up to 10 kb depending upon how many original early regions are deleted

• • • their basic biology has been studied extensively the viral genome can accommodate large heterologous transgene insertions, they readily infect quiescent and dividing cells, they can be amplified to high titers and they have previously been shown to be relatively safe for use in humans.

ADENOVIRUSES: vaccines The first gene therapy licensed product, Gendicine, is based upon an adenovector encoding tumor suppressor p 53 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 Adenovectors are also being evaluated as vaccines for animal diseases such as rabies, and foot and mouth disease • 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.

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. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response. AAV can infect both dividing and non -dividing cells and may incorporate its genome into that of the host cell. These features make AAV a very attractive candidate for creating viral vectors for gene therapy

The virion can contain up to 5 kb of inserted foreign genetic material flanked by the AAV inverted terminal repeats

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

• AAV vectors have been shown to induce antiviral responses upon transduction of cells. However, these responses seem to be of relatively low level and need molecular adjuvants capable of further activating the innate and specific immune response. 1. The r. AAV has been explored as a vaccine vector in several preclinical animal models. In one study, macaques displayed robust immune responses and partial viral control after SIV challenge following a single immunization using r. AAV-carrying SIV genes. 2. it is currently being evaluated in a phase II trial as a vaccine vector for delivering HIV genes. 3. In addition to several ongoing and completed clinical trials using recombinant AAV-2 for gene therapy,

Poxviruses Orthopox: smallpox virus(variola), vacciniavirus, cowpox virus, monkeypox virus The poxviruses comprise a family of complex double-stranded DNA viruses. They belong to the largest family of virus infectious for humans with genomes of 150 -300 kb. Smallpox, which is the most virulent one, has been successfully eradicated by vaccination. 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

Both enveloped extracellular vaccinia (EEV) and intracellular mature virion (IMV) forms are infectious. IMVs enter by fusing to cell membranes, while EEV entry involves endocytosis in an acidic Milieu Via Heparin sulfate present on many cells. Poxviruses have ideal properties as vectors foreign genes for two reasons. 1. Their genome is large and sufficiently stable to carry large amounts of foreign materials and still retain transcriptional and translational capacity, 2. poxviruses perform their whole life cycle in the cytoplasm of somatic human cells. They carry the early genes and enzymes necessary for their own transcription and translation and therefore do not enter the nucleus, a property that is valuable in vaccine constructions.

Current Vaccine Strategies. aimed at developing vaccines against a variety of infectious agents such as malaria, tuberculosis, and HIV Attenuated vaccinia recombinants based on NYVAC 71 and modified vaccinia Ankara (MVA) have entered several clinical trials. HIV VACCINE TRIALS: 1. loss of several vaccinia virulence factor genes by deletion. 2. Foreign genes such as HIV env or gag-pol have been introduced by homologous recombination into these deletions. • The use of a DNA vaccine prime followed by modified vaccinia Ankara carrying multiple HIV genes gave very high response rates in a recent phase 1 clinical trial. • early neonatal immunization with these vectors

MALARIA : There has been substantial clinical progress in the use of pox vectors for malaria, with a phase IIb trial in children in Africa. A NYVAC construct was made to contain eight different stage-related genes from Plasmodium falciparum malaria. This construct induced immune responses to all except one of the encoded malaria antigens. Moreover, MVA and the fowl pox strain F 9 have been used for human trials against malaria

RABBIES: A vaccinia recombinant rabies vaccine for use in animals (Raboral VR-G) was successfully made and licensed. Wild animals are immunized orally by vaccine placed in edible bait, and this vaccine has been distributed worldwide NDV: the efficacy of a recombinantfowlpox virus harboring genes from Newcastle disease virus (NDV) significantly protected chickens from developing NDV disease.

virus belonging to the family Rhabdoviridae, a family also containing rabies viruses. VSV exists in several serotypes. In humans, the virus causes primarily mild and self-limiting flu-like symptoms, although more serious conditions such as encephalitis have been reported Viral Entry and Replication. Spikes composed of trimers of the viral glycoprotein cover the enveloped and bullet shaped Virion. The cellular receptors for VSV are unknown, although evidence indicates that phosphatidyl serine, a component ubiquitously present in plasma membranes, is involved. 83, 84 The viral glycoprotein attaches to target cells, resulting in the virus being internalized by endocytosis and released into the cytoplasm upon acidification. The viral membrane subsequently fuses to a cellular vesicle. Replication takes place in the cytoplasm and starts when the viral RNA-dependent RNA polymerase transcribes five subgenomic m. RNA which are translated into five viral proteins: nucleocapsid, phosphoprotein, matrix protein, polymerase, and glycoprotein. The virus assembles in two steps and subsequently buds through the host plasma membrane, causing cell death and lysis. 80

Preexisting Immunity. With the exception of a few areas in the world, the seroprevalence in humans of VSV antibodies is believed to be very low. 80 However, the envelope glycoprotein of VSV efficiently induces neutralizing antibodies 85 capable of preventing reinfection as well as hampering the effect of a subsequent boost using VSV as a vaccine vector. 86 For homologous prime-boost regimes, it has been possible to circumvent this effect by boosting with a second recombinant VSV where the glycoprotein is exchanged for another VSV serotype. 87

Expression of Foreign Proteins. The 11 kb large genome of VSV permits the insertion of foreign genes in Adjuvant Effect. From what is known, no several proteins locations in the genome and allows an insert of encoded naturally by VSV have the capacit at least 4. 5 interfere with kb. 88 VSV has an advantage over many other the host interferon response, and this hos viral vectors response is as it is able to generate viral particles that believed to be highly important for clearin express and infection. 90 incorporate foreign transmembrane proteins It is thus reasonable to assume that the on the surface. 89 interferon response In terms of immune competition, another that is activated in target cells during the V strength of VSV is the small genome resulting infection will in relatively few proteins also act as an adjuvant for vaccine-specific competing with the vaccine antigen for access responses to to antigen inserted genes. presentation pathways.

Of major concern for the use of VSV as a vaccine vector are the early reports of neuropathology observed in mice following intranasal delivery of the virus. There is also one known case of encephalitis in a human following infection with wildtype VSV. 81 However, current vaccine strategies exploit attenuated versions of the virus, and in prime-boost strategies in non-human primates, the recombinant viruses have been shown to be safe after both intranasal and intramuscular immunization. 92, 93 In preclinical experiments where nonhuman primates were immunized with recombinant VSV carrying genes from SIV and HIV, it was shown that the vaccinees displayed a significantly better clinical outcome than control animals following challenge with SHIV 89. 6 P. 94, 95 The improved safety of the attenuated VSV vector together with quite promising preclinical results indicates that this viral vector is getting closer to a human phase I clinical trial.

HERPEX SIMPLEX VIRUS VECTOR

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. 4 kb • Target cell - dividing

Retroviruses Ø number of FDA-approved clinical trials such as the SCID-X 1 trial. Ø either be replication-competent or replication-defective. . Øinvolves the requirement for cells to be actively dividing for transduction. Øcells such as neurons are very resistant to infection and transduction by retroviruses. ØThere is concern that insertional mutagenesis due to integration into the host genome might lead to cancer or leukemia

Retro virus

A PRACTICAL EXAMPLE OF RETROVIRAL VECTOR LNGFR AG GP 2 -293 VIRAL VECTOR xlox(NGFR)TERT GP 2 x. TERT 11 PRODUCER CELL LINE Staining with anti-NGFR ab beads N ENVELOPE CONSTRUCT PACKAGED TERT RECOMBINANT VECTOR S PRIMARY T CELLS