• Contributions of the N and C – Termini of Varicella – Zoster Virus Portal

      Nale Lovett, Dakota J; School of Medicine
      The VZV portal protein is a multimeric protein found at a single vertex of the viral capsid that is essential for encapsidation (packaging) of viral DNA. All viruses within the herpesvirus family contain structurally homologous portal proteins. PORT compounds have been shown to target herpesvirus portal proteins and show potential as broad-spectrum herpesvirus antivirals. We are specifically interested in the interaction of PORT compounds with the VZV portal since the activity of our compounds against VZV was shown to be in the single nanomolar range. Unfortunately, VZV portal structure has yet to be resolved by transmission electron microscopy (TEM) due to complications with size and aggregation. We aimed to observe the effects of pORF54 (VZV portal protein) terminal truncation on virus viability to determine the minimal portal protein that can be used for structural analysis. Six recombinant viruses containing N and C- terminal mutations were created using bacterial artificial chromosome (BAC) technology and targeted recombineering to create the mutant VZV strains. PCR was used to engineer stop codons at 29, 49, and 75 AAs from the C-terminal end. In addition, a second set of recombinants was constructed where the first start codon of the 769 AA pORF54 open reading frame was deleted creating a 40 AA N-terminal truncation. Sequencing of all mutant strains confirmed the expected changes for the ORF54 gene and also that no gross off-target mutations occurred. All constructs showed a similar restriction digestion pattern, upon gel electrophoresis, compared to the parent BAC, pOKA, suggesting the mutant genomes were stable. Mutagenized BACS were used to create infectious viral stocks after lipofectamine-based transfection into ARPE19 cells. Viruses with non-functional mutations in pORF54 were transfected into ARPE54 (complementing) cells, that constitutively express wild-type pORF54. The ∆29, ∆49, N40∆29, and N40∆49 viruses were replication competent in ARPE19 cells. Replication characteristics suggested that some of these viruses grew less efficiently in vitro. Overall, we confirmed that a protein of 679 AAs can form a functional viral portal. This portal multimer may represent a strong candidate for detailed structural studies.
    • Development and Characterization of a Novel Immunotherapy for Treatment of Breast Cancer by Combining Particulate Cancer Vaccines with Immune-Modulators

      Mulla, Nihal S; College of Pharmacy
      In the last half of the century, advances in the field of cancer therapy including chemotherapy, hormonal therapy and targeted therapy have been responsible for improvements in breast cancer related mortality. Although such advances have benefited all cancer types, there are considerable challenges faced by researchers striving to realize the goal of complete tumor remission. Immunotherapy is a great alternative as it has minimum side effects and several advantages over traditional cancer therapies. The aim of this project is to develop a novel immunotherapy for treatment of cancer by combining cancer vaccines with various immune-modulators. We take advantage of micron-sized particles to deliver vaccine along with other immune modifiers to target immune cells and to initiate immune response against breast cancer antigens. These particles were evaluated for their size, charge, surface morphology, release profiles, cyto-toxicity and particle uptake by various in vitro studies. The efficacy of breast cancer vaccine microparticles was tested in a murine breast cancer model. The immunized animals showed significantly lower tumor growth compared to the naive animals that did not receive any treatment. The delay in tumor growth in vaccinated animals was due to a strong immune response generated against tumor- associated antigens encapsulated within the microparticles. We observed a significant increase in the CD4+ T cell population. The suppression mechanism employed by regulatory T cells are thought to contribute significantly to the failure of current therapies that rely on potentiation of anti-tumor responses. We evaluated the therapeutic efficacy of vaccine microparticles after depleting the immunosuppressive regulatory T cells. We observed a significant improvement in the efficacy of vaccine microparticles by depleting regulatory T cells. The tumor inhibitory effect of vaccine microparticles was due to depletion of regulatory T cells by cyclophosphamide. We observed a significant increase in the CD8+ T cell population. The final aim of my research project was to enhance the immunogenicity of cancer vaccines using adjuvants. Based on our findings we conclude that Alum, Addavax (like MF59) R848 and CpG significantly enhanced the immunogenicity of breast cancer associated antigens.
    • 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.