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  Development and Evaluation of Micro/Nanoparticulate Vaccines for Human Papillomavirus and Influenza

  Vo, Trinh; College of Pharmacy

  Influenza and Human papillomavirus (HPV) are among the most common infectious deseases prevalent globally. The influenza viruses cause approximately 200,000 hospitalizations each year, resulting in a significant financial burden to the healthcare industry. Similarly, HPVs cause virulent infections that have multiple serotypes capable of producing genital warts and cervical cancer. In both cases the infection is spread through viral agents, The influenza virus is transmitted by aerosolized mist of coughs and sneezes with HPV is a sexually transmitted disease. Prevention of influenza and HPV is possible with the help of vaccines, and currently, there are several vaccines for both infectious diseases. However, in the case of influenza, development of a vaccine is challenging as the virus undergoes rapid mutations and hence, the vaccine needs to be modified every season. Along with the constant need for vaccination rates. In the case of HPV, the need for a novel vaccine delivery system is more crucial. HPV is directly linked to cervical cancer and developing a novel strategy to improve vaccination globally is a major priority. Commercial vaccines are not only expensive but in developing countries the financial cost of maintaining cold storage and vaccine administration is significantly challenging. The developing non-parenteral vaccines is essential to improve patient compliance without affecting safety and efficacy of the vaccine. This project has two main sections focusing on development of an oral microparticulate vaccine for influenza and HPV. In the first part of this project, we developed an oral microparticulate vaccine for influenza. The influenza vaccine consists of a M2e protein which is a viral protein common to all influenza viruses encapsulated into a microparticle. Adjuvants such as Alum, MPL. R848, MF59, Flagellin, CpG and P4 were encapsulated in microparticles separately. Microphages werestimulated with the vaccine-adjuvants combinations and subsequently., splenocytes were incubated with activated macrophages to investigate the T-cell activity. We observed significant enhancement of immune markers such as nitric oxide, CD80, CD86, and MHC II with several adjuvants. Furthermore, CD4 and CD8 T cell expression was also significantly enhanced in groups treated with particulate adjuvants. In the second part of this project, we formulated a novel oral and transdermal vaccine delivery system for HPV. Development of potent vaccines against HPV can prevent serious complications such as genital warts and cervical cancer. Therefore a novel non-parenteral vaccine against HPV may significantly reduce incidence of HPV worldwide. In this study, three different microparticualte vaccine formulations have been prepared and characterized: Bovine Albumin Serum (BSA) cross0linked with glutaradehyde; b-cyclodextrin, and cellulose acetate succinate matrices. We systematically studied activation of the immune markers such as nitirc oxide. CD80, CD86, CD40 and MHC-II. In vivo studies demonstrated elevated humoral and cell-mediated immune response in murine models. We observed elevated IgG titers in mice vaccinated with microparticulate HPV16 VLP vaccine, Furthermore, flow cytometry analysis revealed expansion of specific T-cell populations in vivo. This study proves the efficacy of the HPV-16 oral microparticulate vaccine as a promising alternative to conventional vaccines, In the alter part of the HPV vaccine project we investigated the potential of transdermal HPV vaccine as a novel strategy to improve vaccinations globally. In order to combat HPV infection, vaccines must be affordable, self-administered and efficacious. Transdermal vaccines can be self-administered. are almost painless and tap the immune cells present under the skin. Transdermal vaccines have gained immense attention due to their efficacy. In this project, our main aim was to develop a microparticulate HPV16 VLP vaccine for transdermal vaccination. HPV-16 VLP was encapsulated in microparticles and characterized for size, zeta potential, encapsulation efficiency and microparticle yield. Scanning electron microscope images confirmed the particles were irregular with surface indentations. Western blot analysis was performed on HPV16 VLP extracted from microparticles, which confirmed the retention of antigenicity of the epitope following microparticles, which confirmed the retention of antigenicity of the epitope following microparticle fromulation. Following in vitro characterization studies, we performed extensive in vivo studies to investigate the potential of the microparticulate vaccine to initiate a robust and sustained immune response. IgC titers for mice vaccinated with microparticulate vaccine were significantly elevated as compared to antigen solutions adminstered by the transdermal route. Flow cytometry was performed on vaccinated and control groups to investigate the expansion of specific T-cell populations. Vaccinated mice demonstrated significant increase in CD4, CD45R, CD27 and CD62L cell populations. Expansion of these specific T-cell subsets demonstrates the efficacy of the microparticulate vaccine when administered transdermally. Thus, our study proves the efficacy of the microparticulate vaccine over vaccine solutions and the novel approach of administering the vaccine through a minimally invasive, pain-free route that will help to improve acceptability of vaccination against HPV.
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  Oral Microparticulate Prostate Cancer Vaccine: A Promising Immunotherapeutic Approach

  Parenky, Ashwin; College of Pharmacy

  Prostate cancer is one of the leading causes of cancer-related deaths among men in the United States. Currently, there are 5 new agants approved in the United States against prostate cancer which include Sipuleucel-T, cabazitaxel, abiraterone acetate, enzalutamide and radium-223. Introduction of these agents into the clinic are important strides; however, resistance to chemotherapeutic agents is still a significant challenge, Furthermore, when patients suffer from recurrence of prostate cancer, survival is less than five months. Hence, there is an urgent need to investigate alternative approaches to treat castration resistant prostate cancer and prevent relapse. Immunotherapeutic approaches to treat cancer are under intense investigation owing to their specificity and potency to eliminate tumors. One of the most intensely studied areas in the cancer research is identification of cancer antigens that can help indicate progression of cancer, in certain scenarios these antigens may also serve as vaccines for cancer immunotherapy. These cancer antigens are important because they boost the immune system to specifically recognize and kill tumors. In our studies, we have investigated two different antigens to combat prostate cancer. Sperm protein-17 and tumor associated antigens extracted from TRAMP C2 murine prostate cancer cell line were investigated as potential therapeutic vaccines. The above mentioned antigens are proteins and protein antigens themselves have poor bioavailability and absorption thus making it difficult to initiate immune response against the cancer. Particulate delivery systems encapsulating protein antigens have been proved to improve the delivery and efficacy of vaccine responses. Thus, particulate delivery systems are crucial in improving the delivery of these potent cancer antigens for a sustained and systemic anti-cancer activity, Another important aspect of vaccine delivery is the route of vaccination owing to its patient compliance and ease of administration. However, several challenges such as harsh gastric environment and tolerance induction has hindered successful clinical effectiveness of oral vaccines. Adjuvants have always been administered along with vaccines for decades. FDA approved vaccines such as Gardasil and Cerverix both have adjuvants to boost immunity. Thus, identifying appropriate adjuvants that will help boost anti-tumor activity is paramount. In order to increase the potency of our vaccine, several toll-like receptor (TLR) and non-TLR adjuvants were also studied. In this study, we have developed an oral microparticulate vaccine encapsulating two distinct antigens against prostate cancer. SP17 and antigens extracted from murine prostate cancer cell line were encapsulated separately in microparticles. Microparticulate vaccines were characterized for their physiochemical properties in vitro and evaluated for their antigenicity on murine dendritic cells. In order to protentiate vaccine efficacy, we also included adjuvants in microparticulate formulations and evaluated their potential to enhance the antigenicity of our vaccine formulations. SP17 administration. Several adjuvants such as R848, MPL, MF59 and alum were selected for future studies in vivo studies. The second part of this project focuses on formulation of a microparticulate vaccine encapsulating tumor associated antigens extracted from a murine prostate cancer cell line (TRAMP C2). We investigated the potential of our formulated vaccine along with two adjuvant microparticles, ALUM and MF59, to boost anti-tumor response against prostate cancer. Finally, to prove the effectiveness of our vaccine and overcome the tumor "immune escape" mechanism in cancer, we also performed a therapeutic in vivo study on a murine prostate cancer model. Encouraging results from the in vivo study demonstrate excellent anti-tumor activity of our therapeutic vaccine. We observed a significant reduction in tumor volume and sustained anti-tumor T-cell activity in vivo. Thus, we could also demonstrate , in our experiments, the importance of combination therapy which inhibits cancer "immune escape" mechanisms and improves vaccine efficacy.
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  Overian Cancer Microparticulate Vaccines: Effect of Routes of Administration

  Tawde, Suprita Ashok; College of Pharmacy

  Ovarian cancer is the most lethal gynecological cancer in the U.S. First-line treatment for advanced cancer involves surgery followed by chemotherapy. However, cancer relapses within short periods of time even after treatment. Therefore, alternative approach of immunotherapy is being investigated. We studied vaccination with microparticles containing the ovarian cancer antigens can prevent/retard ovarian cancer growth. Oral and transdermal routes are attractive modes of immunization because of their ease of administration and patient compliance. In this project we explored microparticulate system to target immune cells and to initiate response against ovarian cancer antigens via oral, subcutaneous and transdermal delivery. We selected a murine cell line which correlates closely to human cell line in terms of antigen expression and tumor formulation. We prepared the antigenic lysate and characterized it for the presence of a known antigen. We loaded micro-particulate delivery systems with the lysate. These particles were formulated with desired physical properties suitable for particle uptake and for anticipated immune response. In the present study, we demonstrate the efficacy of vaccine formulations wuth was evaluated in vivo in mouse tumor model, using the murine ovarian cancer cell line as a solid tumor model via oral, transdermal, subcutaneous and via combination of oral and transdermal routes. The tumor volumes upon challenge with live tumor cells were monitored in vaccinated animals and control animals. Humoral and cellular immune response in all the animals were monitored to determine the immune mechanism initiated by the vaccine microparticles. Encouraging results from these prophylactic particulate overian cancer studies provided the basis to design therapeutic study to mimic the real-time scenario where patients with residual tumors after a surgery are the recipients of such vaccine therapy. Further, to elucidate the role of M-cells in particle uptake once administered orally, we induced M-cells in murine models and administered microparticles loaded with a model antigen with and without immune adjuvant. The immune response generated via these vaccine particles in mice models with induced M-cells was compared to control animals, to determine whether particles followed the M-cell pathway for their uptake in order to trigger immune cells.
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  Development of Microencapsulated Formulations for Prostate Cancer Vaccine and Live Cells

  Akalkotkar, Archana; College of Pharmacy

  In the current trend, novel particle based platform technology has paved its way to various therapeutic benefits in our lives. In this study, we explored the way of harnessing this technology in order to formulate effective vaccines in the quest for mass immunization in a cost-effective manner. Here we propose a custom formulation of vaccines, which will render effects of sustained release, site specificity and cost-effective delivery of various antigens to enhance the immunity. We used the novel nano-micro technology based of Spray drying as a platform to deliver vaccines against Prostate cancer and encapsulated live cells. In this study, we formulated a whole cell lysate vaccine against prostate cancer via oral and transdermal route of administration. The formulation intended for oral adminstration was optimized for M-cell targeting, The formulation increased the immunogenicity of the vaccine by incorporating the antigen into an albumin matrix having a size of around 0.35-1.2 um that acted as a synthetic adjuvant. The animals were vaccinated with 1 prime and 4 booster doses administered every 14 days over 10 weeks duration, followed by challenge with live tumor cells which showed protection after oral vaccination. Unlike subcutaneous injections, administration-using microneedles is painless and in general can increase the permeability of many compounds ranging in size from small molecules to proteins and microparticles that do not normally penetrate the skin. The mice vaccinated via transdermal route showed promising results in delaying the tumor growth compared to the controls. The study was extrapolated in a therapeutic approach. This would mimic the real clinical scenario where the patient who was diagnosed with prostate cancer would come in the clinic for immunotherapy. The microparticle based delivery system showed protection in vaccinated group compared to the controls. This shows the microparticle based delivery system can be an useful tool for delivery as well as live cells in vivo.
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  Formulation and In Vivo Evaluation of Particulate Breast Cancer Vaccine

  Chablani, Lipika; College of Pharmacy

  Purpose: This research work concentrates on formulating two particulate breast cancer vaccines, which are further evaluated in vivo using two murine breast cancer models. As breast cancer continues to be the most fatal cancer among women throughout the world, there is an immediate need to develop a vaccine to combat it. Considering the potential of particulate delivery vehicles to impart robust systemic as well as mucosal immune response, they have been explored not only against infectious diseases but also against cancer. In this project we take advantage of these micron sized particulate delivery vehicles to target immune cells and to initiate immune response against breast cancer antigens, Also, these particles have been fabricated in such a manner that they can be administered via patient-compliant routes of administration including oral and intraepidermal delivery. Research methodology: We have explored various polymers to optimize two enteric protected particulate delivery systems with desired physical properties making them susceptible to particle uptake and there by leading to anticipated immune response. These particles have been evaluated carefully for their size, charge, surface morphology, release profiles, cyto-toxicity and particle uptake by various in vitro studies. Further the particle uptake of vaccines by the M (microfold) cells in the Peyer's patches of the small intestine when given orally is studied extensively along with characterization of microchannels created to deliver the microparticulate vaccine. Also, the vaccine efficacy was evaluated in vivo in female mice model using the two marine breast cancer cell lines which mimic the progression of breast cancer as seen in humans, The vaccine was administered via oral, intraepidermal and sub-cutaneous routes resulting in immune response against the breast cancer antigens used in the vaccine. Results and Conclusion: The vaccinated animal had sugnificantly smaller tumor volumes than the control animals, as seen after challenging the animal upon termination of treatment. Immune responses in all the animals were monitored to gauge the role of humoral and cellular immunity in generating protection by several in vitro and ex vivo studies. Promising results from these two prophylactic particulate breast cancer vaccine studies have advanced our laboratory to explore a therapeutic breast cancer vaccine. These particulate delivery systems possess the potential of entering the clinical trials and mimicking the real-time situation where patient's own tumor cells extracted after a surgery can serve as the source of antigens for an individualized particulate vaccine.

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