Zaire ebolavirus is a long filamentous single-stranded RNA virus belonging to the family Filoviridae. Due to the virus’ high mortality rate, lack of an approved vaccine, and potential use as a bioterrorism weapon, research on this topic has been of high demand. To address this issue, several vector platforms have been investigated as vaccine candidates. DNA and adenovirus vaccine platforms are known to elicit robust cellmediated immune responses, while adeno-associated virus and vesicular stomatitis virus vaccines are recognized for strong humoral responses. The leading hypothesis of the present project was to determine whether these four vaccination platforms, in a heterologous prime-boost regimen, increase survival and the breadth of the immune response. To test this hypothesis, the main objectives were to evaluate the cell-mediated and humoral immune responses, as well as correlate the induced immunity to protection against MA-EBOV. The heterologous pairings were strategically designed to induce both arms of the immune response. An optimized Zaire ebolavirus glycoprotein was inserted into each of the vaccine platforms and evaluated against mouse-adapted Zaire ebolavirus. Serum obtained from vaccinated mice was analyzed by a neutralizing antibody assay and IgG ELISA to determine the humoral response. The cell-mediated immune response was monitored via ELISPOT. Collectively, the data indicates that regardless of whether homologous or heterologous, a more robust immune response was observed in prime-boost strategies compared to an individual vaccination alone. In addition, the cell-mediated and humoral data show that heterologous combinations induce higher IgG specific titers in comparison to homologous regimens. As expected and consequent with immune responses, survival studies demonstrate that prime-boost III vaccinations, heterologous or homologous, are superior to vaccination regimens involving only one strategy. This data supports further evaluation of the prime-boost strategies to develop an optimal immunization strategy that can be applied to other disease models.

Andes virus (ANDV) is a highly pathogenic New World hantavirus found in Chile and Argentina that causes Hantavirus Pulmonary Syndrome (HPS). A significantly high case fatality rate, the potential for human-to-human transmission, and the lack of licensed vaccines or effective treatments for the disease suggest an urgent need for the development of such measures. Many vaccine platforms have been recently described using reverse genetics systems to generate attenuated virus strains, as well as recombinant viruses expressing foreign proteins. This study attempted to generate and characterize vaccines based on an ANDV infectious clone system, as well as a recombinant vesicular stomatitis virus (VSV) vector expressing the ANDV glycoprotein precursor (VSVΔG/ANDVGPC), to test their efficacy in the only lethal disease animal model of HPS. Although an ANDV infectious clone system was not successfully established, precluding its use as a possible vaccine candidate, the first New World hantavirus minigenome system was described. In addition, Syrian hamsters immunized with a single dose of the recombinant VSVΔG/ANDVGPC vaccine were fully protected against disease when challenged at 28, 14, 7, or 3 days post-immunization with a lethal dose of ANDV; however, the mechanism of protection seems to differ, depending on the time point of immunization. At 28 days post-immunization, a lack of detectable ANDV RNA in tissue samples as well as a lack of seroconversion against the ANDV nucleoprotein (N) in nearly all hamsters suggested mostly sterile immunity. The vaccine was able to generate high levels of neutralizing anti-ANDV GN/GC antibodies that seem to play a role as a mechanism of vaccine protection. Administration of the vaccine at 7 and 3 days before challenge also resulted in full protection, but the lack of a neutralizing humoral immune response and the up-regulation of hamster cytokines involved in innate pathways suggested a possible role of innate responses in protection. The role of innate immunity was supported by the fact that administration of the vaccine 24 hours post-challenge was successful in protecting 90% of hamsters. Overall, the data suggests the potential for the use of the VSV platform as a fast-acting and effective prophylaxis/post-exposure treatment against lethal hantavirus infections.

Adeno-associated virus (AAV) is a small, non-pathogenic virus, exploited as a vector for gene therapy applications, with many successful clinical trials. However, these vectors are based on human and non-human primate AAVs, to which there exists pre-existing immunity in the general population. We hypothesized AAVs having low seroprevalence in the human population can be isolated from alternate sources and can be an alternative to those used in current clinical trials. We also hypothesized that the close homology between pig and human tissues suggests that AAVs isolated from pigs would be able to transduce human cells efficiently. Porcine-derived AAVs preferred specific tissue targets when injected in vivo in mice and successfully transduced cells derived from humans. Immune responses generated against the AAV capsid are also important for determining the safety profile of the vectors; there still exists the possibility of the host mounting adverse immune responses against transduced cells, as seen in some of clinical trials. Although the transduction efficiency of AAV gene transfer has been extensively studied in animal models, the host’s immune response towards the gene product is still poorly understood. This thesis addresses the issue by providing a link between protective efficacy against lethal challenge and tissue tropism. Here, AAVs carrying an immunogenic transgene were developed, with the goal to identify those that can protect against lethal challenge of avian flu or Ebola virus in mice, and those that had poor protective efficacy. It was observed that the protective efficacy afforded by an AAV was serotype specific. The protective efficacy and immune responses were compared to the biodistribution and cellular targets of each AAV. Overall, AAVs sharing broad tropism in biodistribution studies had a tendency to protect mice against lethal challenge than those AAVs not found systemically. As well, those AAVs eliciting protective efficacy against lethal challenge were able to transduce antigen-presenting cells including dendritic and B cells. The link between tissue tropism and host immune responses has been poorly understood and this thesis contributes to the AAV field by highlighting the significance of the cellular targets of AAV and this relationship to protective efficacy.

Zaire ebolavirus is a long filamentous single-stranded RNA virus belonging to the family Filoviridae. Due to the virus’ high mortality rate, lack of an approved vaccine, and potential use as a bioterrorism weapon, research on this topic has been of high demand. To address this issue, several vector platforms have been investigated as vaccine candidates. DNA and adenovirus vaccine platforms are known to elicit robust cellmediated immune responses, while adeno-associated virus and vesicular stomatitis virus vaccines are recognized for strong humoral responses. The leading hypothesis of the present project was to determine whether these four vaccination platforms, in a heterologous prime-boost regimen, increase survival and the breadth of the immune response. To test this hypothesis, the main objectives were to evaluate the cell-mediated and humoral immune responses, as well as correlate the induced immunity to protection against MA-EBOV. The heterologous pairings were strategically designed to induce both arms of the immune response. An optimized Zaire ebolavirus glycoprotein was inserted into each of the vaccine platforms and evaluated against mouse-adapted Zaire ebolavirus. Serum obtained from vaccinated mice was analyzed by a neutralizing antibody assay and IgG ELISA to determine the humoral response. The cell-mediated immune response was monitored via ELISPOT. Collectively, the data indicates that regardless of whether homologous or heterologous, a more robust immune response was observed in prime-boost strategies compared to an individual vaccination alone. In addition, the cell-mediated and humoral data show that heterologous combinations induce higher IgG specific titers in comparison to homologous regimens. As expected and consequent with immune responses, survival studies demonstrate that prime-boost III vaccinations, heterologous or homologous, are superior to vaccination regimens involving only one strategy. This data supports further evaluation of the prime-boost strategies to develop an optimal immunization strategy that can be applied to other disease models.

Andes virus (ANDV) is a highly pathogenic New World hantavirus found in Chile and Argentina that causes Hantavirus Pulmonary Syndrome (HPS). A significantly high case fatality rate, the potential for human-to-human transmission, and the lack of licensed vaccines or effective treatments for the disease suggest an urgent need for the development of such measures. Many vaccine platforms have been recently described using reverse genetics systems to generate attenuated virus strains, as well as recombinant viruses expressing foreign proteins. This study attempted to generate and characterize vaccines based on an ANDV infectious clone system, as well as a recombinant vesicular stomatitis virus (VSV) vector expressing the ANDV glycoprotein precursor (VSVΔG/ANDVGPC), to test their efficacy in the only lethal disease animal model of HPS. Although an ANDV infectious clone system was not successfully established, precluding its use as a possible vaccine candidate, the first New World hantavirus minigenome system was described. In addition, Syrian hamsters immunized with a single dose of the recombinant VSVΔG/ANDVGPC vaccine were fully protected against disease when challenged at 28, 14, 7, or 3 days post-immunization with a lethal dose of ANDV; however, the mechanism of protection seems to differ, depending on the time point of immunization. At 28 days post-immunization, a lack of detectable ANDV RNA in tissue samples as well as a lack of seroconversion against the ANDV nucleoprotein (N) in nearly all hamsters suggested mostly sterile immunity. The vaccine was able to generate high levels of neutralizing anti-ANDV GN/GC antibodies that seem to play a role as a mechanism of vaccine protection. Administration of the vaccine at 7 and 3 days before challenge also resulted in full protection, but the lack of a neutralizing humoral immune response and the up-regulation of hamster cytokines involved in innate pathways suggested a possible role of innate responses in protection. The role of innate immunity was supported by the fact that administration of the vaccine 24 hours post-challenge was successful in protecting 90% of hamsters. Overall, the data suggests the potential for the use of the VSV platform as a fast-acting and effective prophylaxis/post-exposure treatment against lethal hantavirus infections.

The magnitude and efficacy of CD8+ T cell memory may notably regress, especially if immune induction occurs in the absence of adequate CD4+ help. This report demonstrates that this CD8+ memory malfunction could be remedied if a source of cognate antigen-recognizing helper cells were provided during recall. The inability of adoptive transfer of memory SIINFEKL-specific CD8 cells to reject tumors was overcome if recipients were primed for ovalbumin-specific helper cell responses. Additionally, animals primed for a SIINFEKL-specific memory response and incapable of rejecting the tumor could regain protective immunity if given helper cells. This pattern of CD8+ T cell functional rescue or reprogramming by helper cell transfer was replicated using a Herpes simplex virus antiviral immunity system. Our results could mean that therapeutic vaccine approaches could be designed to compensate situations that have defective CD8+ T cell function.

BACKGROUND: Group B Streptococcus (GBS) is a Gram-positive bacterium that is a common cause of infection in neonates and is the predominant pathogen causing meningitis in infants. Different vaccine formulations have been evaluated in preclinical and/or clinical settings, such as polysaccharide-based vaccines, protein-based, polysaccharide-protein conjugate vaccines, and most recently a Multiple Antigen Presenting System (MAPS) vaccine. MAPS is a vaccine that is being developed in the Malley laboratory at Boston Children’s Hospital that consists of GBS polysaccharides and proteins that are combined using affinity interactions. The overall aim of this thesis is to contribute towards the development of an effective MAPS vaccine by identifying high capsule-producing GBS isolates, optimizing conditions for polysaccharide purifications, characterizing the immune response to carrier protein derivatives, and creating MAPS complexes with GBS-specific protein carriers. METHODS: The serotypes of 61 GBS clinical isolates were identified via multiplex PCR. To determine high producing strains, the supernatant polysaccharide concentrations (SPC) for type Ia and III were measured by type 14 pneumococcal and GBS Ia ELISAs, respectively. In addition, type III isolates were grown in different conditions (shaking vs. static, Todd Hewitt Broth vs. Todd Hewitt Broth + 0.5% yeast extracts). For carrier protein production, genetically conserved alpC and rib were ligated onto a pet21b C terminally tagged rhizavidin (protein made by Rhizobium etli that has a very high affinity for biotin) vector. AlpC was purified and used to immunize rabbits. To determine whether the location of the terminal rhizavidin tag affected the immunogenicity of the proteins, we compared the serum titers of C- and N-terminally tagged proteins by ELISA. Additionally, AlpC was conjugated to purified pneumococcal type I polysaccharide for the creation of MAPS complexes. RESULTS: We serotyped 61 clinical GBS isolates, of which 18 were type Ia and 33 were type III. The type Ia isolate 21 and type III isolate 25 produced the most polysaccharide relative to the other isolates. The majority of the isolates for type III produced more capsule under shaking conditions and when grown in THY (Todd Hewitt Broth +0.5% yeast extracts). AlpC and rib were successfully cloned into pet21b CRhiz plasmid and purified. To determine if the position of the rhizavadin tag affected immunogenicity, AlpC-C terminus was sent for immunization onto rabbits. This protein generated similar antibody titers than a N-terminally tagged AlpC. DISCUSSION: The majority of isolates identified from patients were type Ia and III which corresponds to published data stating that Ia and III are amongst the most common serotypes associated with early onset disease. Production of capsule was greatest in isolate 21 for the Ias and isolate 25 for the III, which may be due to enhanced expression of the cps operon. The data for serum titers from AlpC revealed no major difference between titers generated by the C-and N-terminally tagged proteins. This suggests that placing the rhizavadin tag at the C-terminus or N-terminus for AlpC does not affect immunogenicity. CONCLUSION: This work presented in this thesis contributed to the development of an effective GBS MAPS vaccine by finding high producing strains to optimize polysaccharide purifications, and purifying AlpC and immunizing with this carrier protein.

The HIV-1 envelope glycoprotein (Env) is the primary focus of prophylactic HIV vaccine development. However, the unusually low density of Env spikes on the virion (≈14 spikes/virion) is unfavourable for eliciting high titre, long-lasting antibody responses. It is possible that increasing the Env spike density of particulate vaccine candidates generated by protein body formation or via the display of Env on nanoparticles could improve the induction of long-lasting neutralising antibodies (NAbs). For this thesis, two different nanoparticle approaches were therefore investigated. The HIV-1 Env sequence used for both approaches was derived from the superinfecting subtype C CAP256 virus. This was truncated to remove the transmembrane domain, and engineered to contain a flexible linker (FL) in place of the furin cleavage site and an I559P mutation to generate soluble, stable and cleavageindependent gp140 proteins. The first approach investigated the impact of genetically fusing a 27 kDa proline-cysteine-rich domain of the ɣ-zein maize seed storage protein - Zera® - to either the N- or C-terminus of CAP256 gp140. Fusion of Zera® to a protein of interest can promote the self-assembly of large protein bodies (PBs) containing the protein of interest, thereby improving yields of the recombinant protein and enabling easy isolation using gradient ultracentrifugation. The purification of Zera-induced Env PBs from infiltrated Nicotiana benthamiana plants was not optimal. Consequently, the generation of Zera®-induced gp140 protein bodies was evaluated in a mammalian expression system. Stable HEK293 cell lines expressing Zera®-gp140 or gp140-Zera® were generated. A mixture of small PB-like structures was observed in cells expressing gp140-Zera®. However, no PB-like structures were seen in cells expressing Zera®-gp140. The immunogenicity of Zera®-gp140 and gp140-Zera® was evaluated by in rabbits. Binding and Tier 1A neutralising serum titres were higher for gp140-Zera® than for Zera®-gp140. Neither gp140-Zera® nor Zera®-gp140-specific sera neutralised a Tier 1B pseudovirus or the autologous Tier 2 CAP256SU pseudovirus, suggesting that Zera® might have compromised the structure of the Zera®-tagged gp140 proteins. The second approach investigated the two-component SpyCatcher/SpyTag technology. The stable HEK293 cell line expressing CAP256 gp140-SpyTag (gp140-ST) was generated, and trimers were purified to homogeneity using gel filtration. SpyCatcher (SC)-AP205 VLPs were produced in E. coli and purified by ultracentrifugation. The gp140-ST trimers and the SCAP205 VLPs were mixed in varying molar ratios to generate VLPs displaying the glycoprotein (AP205-gp140-ST particles). SDS-PAGE, dynamic light scattering and negative stain electron microscopy indicated that gp140-ST was successfully bound to the VLPs, although not all potential binding sites were occupied. The immunogenicity of the coupled VLPs was evaluated in a pilot study in rabbits. One group was injected four times with coupled VLPs. The second group was primed with DNA vaccines expressing Env and a mosaic Gag, followed by modified vaccinia Ankara expressing the same antigens and then boosted twice with coupled VLPs. Encouragingly, gp140-ST displayed on SC-AP205 VLPs was an effective boost to heterologously primed rabbits, leading to induction of autologous Tier 2 neutralising antibodies in 2/5 rabbits. These results demonstrate that careful selection of a geometrically-suitable nanoparticle scaffold to achieve a high-density display of HIV-1 envelope trimers is an important consideration and that this could improve the effect of nanoparticle-displayed gp140.

abstract: ABSTRACT In terms of prevalence, human suffering and costs dengue infections are the most important arthropod-borne viral disease worldwide. Dengue virus (DENV) is a mosquito-borne flavivirus and the etiological agent of dengue fever and dengue hemorrhagic fever. Thus, development of a safe and efficient vaccine constitutes an urgent necessity. Besides the traditional strategies aim at generating immunization options, the usage of viral vectors to deliver antigenic stimulus in order to elicit protection are particularly attractive for the endeavor of a dengue vaccine. The viral vector (MVvac2) is genetically equivalent to the currently used measles vaccine strain Moraten, which adds practicality to my approach. The goal of the present study was to generate a recombinant measles virus expressing structural antigens from two strains of DENV (DENV2 and DENV4) The recombinant vectors replication profile was comparable to that of the parental strain and expresses either membrane bound or soluble forms of DENV2 and DENV4 E glycoproteins. I discuss future experiments in order to demonstrate its immunogenicity in our measles-susceptible mouse model. / Dissertation/Thesis / M.S. Biology 2013

Although there are well-documented benefits from influenza vaccine and 23-valent pneumococcal polysaccharide vaccine (23vPPV) against invasive pneumococcal disease and laboratory confirmed influenza, their effectiveness against pneumonia remains controversial for community-based persons aged >=65years. At the time of this research, within Australia, only the government of Victoria publicly funded these vaccines for elderly persons. With continued growth of the elderly population, the subsequent adoption of an Australia-wide program, and increasing uptake of similar programs in other countries, there is a need for data clarifying the impact of vaccination on pneumonia. This research estimates incremental vaccine effectiveness of 23vPPV over and above influenza vaccine against hospitalisation with community-acquired pneumonia (CAP) in the elderly.