Browsing by Subject "Vaccine"

Dendritic cells (DC) have been the key elements in developing cancer vaccines to induce potent T cell responses to eradicate tumors. However, the common approach adopted in clinical trials using ex vivo generated DC loaded with tumor antigens (Ag) has been challenged by its limited clinical response, complexity, and quality of the manufacturing process. Alternative efforts focused on in vivo Ag loading on endogenous primary DC have not yet been well validated in their efficacy for cancer treatment, suggesting the efficiency of in vivo Ag transfer to endogenous DC from currently available Ag-delivering vehicles needs to be further improved. Here, I aim to develop an alternative cellular vaccine platform that can circumvent the aforementioned problems. I reason that classical Ly-6Chi monocytes (i.e. monocytes hereafter) can be a promising candidate to be loaded with tumor Ag and induce effective T cell responses. With advantages including easy-purification from human peripheral blood, monocytes evidently can present antigens directly via in vivo differentiation into bona fide DC or indirectly via antigen transfer to lymphoid resident DC to induce strong Th1 or cytotoxic T lymphocyte (CTL) responses. However, whether monocytes exploit favorably direct or indirect pathway to present the same Ag they are carrying to trigger effective immune responses remains unclear. Furthermore, how exactly monocytes or monocyte-derived cells transfer antigens to lymphoid resident DC has yet to be elucidated. I hypothesized that Ag-loaded monocytes can induce strong anti-tumor immunity and began the research by investigating the immune responses that can be induced by Ag-loaded monocytes. I then went on to determine the mechanisms that mediate monocyte-induced immune responses and evaluate anti-tumor efficacy of this monocyte vaccine.

In the first part of this study, I characterized the immune responses induced by Ag-loaded monocytes. By using negative selection via magnetic-activated cell sorting (MACS) columns, I was able to purify monocytes from bone marrow (BM) cells and determined that these monocytes could be successfully loaded with Ag in the forms of proteins, peptides and mRNA. I found that intravenously (IV) injected Ag-loaded monocytes induced robust Ag-specific CD4+ and CD8+ T cell responses in mice without triggering antibody responses. This vaccine activity of Ag-loaded monocytes appeared to be dose-dependent and required live monocytes with no need of ex vivo stimulation. I found that Ag-specific CD8+ T cells induced by Ag-loaded monocytes were functionally more robust than those induced by protein Ag emulsified in a traditional adjuvant CFA.

In the second part of this study, I investigated how IV injected Ag-loaded monocytes stimulate T cell responses. I identified that the spleen is the primary immune niche for Ag-loaded monocytes to induce T cell responses. I found that Ag-loaded monocytes mainly retain in the spleen where they begin to differentiate into phenotypic DC. Surprisingly, major histocompatibility complex (MHC)-deficient monocytes maintain full capacity to stimulate T cell responses, suggesting that Ag-loaded monocytes do not present Ag by themselves. I determined that endogenous splenic DC is absolutely required for monocyte-induced T cell responses. Therefore, Ag-loaded monocytes induce T cell responses indirectly via transferring Ag to splenic DC even they do differentiate into phenotypic DC in the spleen. I elucidated that this monocyte-to-DC Ag transfer occurs via gap junctions for CD8+ T cell responses and via macrophages for CD4+ T cell responses.

In the final part of this study, I demonstrated that IV injected Ag-loaded monocytes have robust anti-tumor efficacy targeting both model and validated tumor Ag in prophylactic, memory and therapeutic murine SQ melanoma models. The anti-tumor efficacy is superior to that seen with traditional adjuvants or RNA-pulsed DC vaccines, and can be combined with checkpoint blockade to increase their efficacy. Furthermore, I demonstrated that Ag-loaded monocytes have a clear anti-tumor efficacy in an intracranial glioblastoma (GBM) model targeting against mutant isocitrate dehydrogenase 1-R132H (mIDH1-R132H), a validated tumor Ag of GBM.

In conclusion, IV injection of unactivated Ag-loaded monocytes without adjuvants induces highly efficacious anti-tumor T cell responses via dual independent and efficient Ag transfer pathways to splenic DC. These findings revise the paradigm that monocytes have to be activated ex vivo to achieve optimal vaccine efficacy and reveal unappreciated cell-associated Ag acquiring pathways of splenic DCs that can be specifically manipulated for future vaccine design in the treatment of human cancers.

The mammalian urinary bladder is a highly specialized organ that must be able to withstand considerable amounts of osmotic pressure at its mucosal surface, in addition to maintaining an impenetrable barrier against potential pathogens. The lower urinary tract's virtually inevitable exposure to external microbial pathogens warrants efficient tissue-specialized defenses to maintain sterility. The observation that the bladder can become chronically infected with uropathogenic E.coli (UPEC) in combination with clinical observations that antibody responses following bladder infections are not detectable, suggest defects in the formation of adaptive immunity and immunological memory. We have identified a broadly immunosuppressive transcriptional program specific to the bladder, but not the kidney, during infection of the urinary tract that is dependent on tissue-resident mast cells. This mast cell-dependent phenomenon involves localized production of IL-10 and results in suppressed humoral and cell-mediated responses and bacterial persistence. Therefore, in addition to the previously described role of mast cells orchestrating the early innate immune responses in the bladder during infection, they subsequently play a tissue-specific immunosuppressive role. These findings may explain the prevalent recurrence of bladder infections and suggest the bladder as a site exhibiting an intrinsic degree of mast cell-maintained immune privilege.

Interestingly, though the bladder is not capable of initiating an effective adaptive immune response during bladder infections, we have generated data showing that it was possible to circumvent the immune limitations of the bladder to provoke a strong adaptive and protective immune response by vaccinating against UPEC at an alternate mucosal site. We reasoned that by immunizing the nasal regions of mice with a vaccine formulation comprising of FimH adhesin, a highly conserved adhesive moiety of type 1 fimbriae expressed on UPEC, and an effective mucosal adjuvant we would evoke protective immunity against UPEC infections. We found that a FimH vaccine coupled with either a mast cell activating adjuvant c48/80 or CpG oligodeoxynucleotide, a TLR9 agonist, evoked high levels of FimH specific IgG antibody in the serum and IgA in the urine of immunized mice. We also observed that following UPEC challenge, these FimH/adjuvant immunized mice exhibited significantly reduced bacterial load in the bladders compared to mice challenged with just FimH. These studies reveal that immunization of nasal regions with a FimH vaccine is an effective strategy to overcome the limitation in adaptive immunity observed in the bladder.

A better understanding of 1) maternal HIV Envelope (Env)-specific IgG responses that are partially protective against vertical HIV transmission, and 2) factors that mediate the transplacental transfer of maternal protective IgG is needed to improve infant health in early life, in which maternal passively-acquired IgG mediates protection against neonatal infections. To understand maternal factors and IgG characteristics that mediate transplacental IgG transfer, we examined transplacental transfer efficiency determinants of maternal HIV and standard vaccine-antigen-specific IgG in a population of HIV-infected women, which have disrupted transplacental IgG transfer. Our findings suggest that maternal health factors and maternal IgG characteristics, such as binding to placentally expressed Fc receptors, IgG subclass frequency, and Fc region glycan profiles all mediate transplacental IgG transfer efficiency. We also identified maternal linear variable loop 3 (V3)-specific IgG binding and neutralizing responses targeting the C terminal region as partially protective against vertical transmission of HIV. These novel findings provide a roadmap of maternal factors and IgG characteristics as targets that can be harnessed to improve the transplacental IgG transfer of routinely-administered maternal vaccines and benchmarks for assessing maternal HIV vaccines that target the V3 loop.

Maternal vaccination protects infants through transplacental transfer of vaccine-specific maternal IgG and milk transfer of IgG and IgA antibodies from mother to child. I performed experiments in the rabbit model, which models human maternal antibody transfer, to determine how maternal HIV vaccine formulations impact the passive transfer of maternal gp120-specific antibodies and to investigate potential side effects of vaccine-elicited maternal antibodies. Since the mammary gland is part of the mucosal immune system, mucosal administration of maternal vaccines may enhance milk transfer of maternal antibodies; however, the tendency of mucosal vaccines to induce lower serum IgG responses than injected vaccines could decrease transplacental transfer. Optimized intranasal boosting during pregnancy resulted in similar concentrations of gp120-specific IgG in infant serum, however milk gp120-specific IgA concentrations were not enhanced. Furthermore, intranasal boosting with chitosan-adjuvanted vaccines resulted in significantly higher transplacental transfer of maternal antibody than MPL-adjuvanted vaccines even though both formulations induced similar levels of gp120-specific IgG in maternal serum, indicating that maternal vaccine adjuvants may alter transplacental transfer of maternal antibodies.

Infant rabbits born to mothers that received the IM and IN maternal vaccine regimens were vaccinated with gp120 with or without adjuvant to investigate maternal antibodies interference with infant antibody responses to vaccination. Maternal gp120-specific IgG inhibited infant vaccination with unadjuvanted gp120, however inclusion of either alum or GLA-SE, a TLR4 agonist in an oil-in-water emulsion, was able to induce active antibody responses in infants. Furthermore, infant rabbits that received an alum-adjuvanted vaccine in the presence of maternal antibodies had enhanced serum gp120-specific and V1V2-specific IgG that infants vaccinated without maternal gp120-specific IgG present. GLA-SE did not enhance infant antibody responses to vaccination. Thus, maternal anti-gp120 IgG can enhance or inhibit infant antigen-specific responses to vaccination depending on the infant vaccine adjuvant.

While maternal antibodies protect the infant, there is evidence that some viruses, including HIV and Zika, use maternal antibodies to be transferred across the placenta, facilitating mother-to-child-transmission. As HIV infects and replicates poorly in rabbits, a rabbit model of Zika virus challenge was established and the impact of maternal vaccination or anti-flavivirus monoclonal antibody on pathogenesis was investigated. While Zika virus-specific antibodies altered maternal cytokine response to challenge, and there was an increased risk for fetal resorption in vaccinated rabbits compared to naïve rabbits, there was no significant impact on placental Zika virus RNA concentration. While further refinement is needed, Zika virus challenge of rabbits is a promising in vivo model for investigating the transplacental transfer of maternal antibody-pathogen complexes.

Tuberculosis is a devastating disease that has been plaguing humankind for millennia. Co-evolution of humans with Mycobacterium tuberculosis, the causative agent of the disease, has allowed for the pathogen to possess an abundance of survival mechanisms. The outcome of this is the ability of the bacterium to create an intracellular niche lifestyle inside host cells where it can successfully evade the host immune system. While there is a vaccine available, named the BCG vaccine, it confers little protection to adults in the pulmonary form of the disease. The lack of an effective vaccine and the rise of Multidrug-Resistant (MDR) and Extensively Drug-Resistant (XDR) tuberculosis highlight the need for more research into combating Mycobacterium tuberculosis. The purpose of this work is to enhance the field of knowledge of how mycobacterial virulence factors affect host cellular pathways so that the interactions can be exploited for novel therapeutics and vaccine development.

One of the hypotheses for the poor efficacy of the BCG vaccine is that it fails to elicit a strong CD8+ T cell response during infection. Studies have found that vaccinating mice with apoptotic bodies containing mycobacterial antigens were able to protect mice to a greater degree than BCG and that this is dependent on CD8+ T cell activation. Thus, we hypothesized that a pro-apoptotic mutant of M. tuberculosis could be utilized as a novel vaccine candidate. Through screening a library of M. tuberculosis transposon mutants, we identified an Enhanced Cell Death mutant (ECD19) that functions through caspase 3 mediated apoptosis. Sequencing revealed that the mutant has a transposon insertion in Rv2456c, a probable integral membrane transport protein. Immunogenicity testing via Enzyme-Linked ImmunoSpot (ELISPOT) and Intracellular Cytokine Staining (ICS) assays demonstrated that ECD19 induced an altered immune response when compared to the parental strain M. tuberculosis H37Rv. Additionally, ECD19 has reduced survival in an in vitro THP-1 cell model and in an in vivo mouse model. Taken together, our data suggest that Rv2456c is important to the survival of H37Rv in host cells and that deletion of the gene may enhance the immunogenicity of the bacterium.

Inappropriate dosing and poor adherence to antibiotics in the treatment of tuberculosis has led to MDR and XDR, the highest incidences of which can be found in the KwaZulu-Natal (KZN) province of South Africa. Little is known about the virulence of these strains, but it is hypothesized that the drug resistance mechanisms come at a cost to the bacteria. In an in vitro assay, we have found that clinical isolates from the KZN region induce higher levels of necrosis than virulent laboratory strains of M. tuberculosis. Additionally, our in vivo studies show that the drug-resistant isolates do not disseminate as well as susceptible strains, and in both immunocompetent and immunocompromised mouse models, mice infected with the drug-resistant strains are able to live longer than mice infected with drug-sensitive strains. As all strains are highly related on a genetic level, we can say that the drug-resistant mechanisms acquired by the strains come at a cost of reduced virulence. Thus, it is likely that higher prevalence of the MDR and XDR in the KZN province is due to the high rate of HIV+, immunocompromised individuals living in the region.

Lastly, we are interested in building on the knowledge that avirulent mycobacteria are able to induce autophagy in a murine macrophage cell line. Through the use of Mammalian Target of Rapamycin (mTOR) inhibitors and autophagy-deficient macrophages, we were able to show that Mycobacterium smegmatis is able to induce both mTOR and autophagy during infection. Additionally, we found that mycobacterial killing occurs in the absence of autophagy when mTOR is inhibited. This effect is not due to a bactericidal effect of the mTOR inhibitors. From these data, we show that there is an underappreciated role in the induction of mTOR after mycobacterial infection. By studying the interplay of mTOR and autophagy, therapies targeted to favoring host defenses could be developed.

In summary, the insights from this work enhance the knowledge of how mycobacteria are able to be successful pathogens. This data may be useful in the creation of novel vaccine candidates or the identification of potential drug targets to bolster the therapeutic options in treating those afflicted with tuberculosis.

Glioblastoma (GBM) is a grade IV astrocytoma in which the median overall survival is approximately 15 months at time of diagnosis. Even with the current multi- modal therapeutic approach of surgery, chemotherapy with the DNA alkylating agent temozolomide, and radiation therapy, GBM remains uniformly lethal. Immunotherapeutic interventions are a burgeoning field in many different cancer treatments. They offer the exquisite specificity endowed by the immune system with minimal toxicities and new methods are being developed to enhance the endogenous immune responses.

With the recent identification of human cytomegalovirus (CMV) present within glioblastoma tissue but void in the surrounding normal healthy parenchyma there have been significant efforts aimed at understanding the biologic implications of the presence of the virus within GBM tissues with preliminary work demonstrating several capabilities of the virus to enhance the oncogenic process.

Likewise, a key area of importance in the development and design of effective immunotherapeutic platforms is the identification and targeting of tumor-specific antigens. The success of any immunotherapy platform relies heavily on the ability to selectively target antigens present within tumors but absent on healthy tissue, regardless of its role in tumorigenesis, as well as having robust immunogenic properties.

CMV offers a plethora of possible targets, as it is the largest known DNA virus that infects humans, yet very little is known about its biological significance in glioblastoma pathogenesis as well as the most efficacious and immunogenic targets for immunotherapeutic development.

We have been able to elucidate more thoroughly the feasibility and potency of an immunologic platform targeting CMV within glioblastoma utilizing a multi-antigen multi-component peptide based strategy that demonstrated significant immunogenicity and anti-tumor activity in pre-clinical models utilizing various assays. We have also developed several sensitive and specific detection methodologies including: 1) custom gene expression microarrays, 2) multiplex real time quantitative polymerase chain reaction (RT-qPCR) assays, 3) a massively parallel RNA deep sequencing platform, and 4) immunological assays. We have also successfully determined the capacity for endogenous CMV gene expression to be maintained in primary glioblastoma cell lines as well as examining the preponderance of CMV gene expression in a subpopulation of glioma stem cell-like cells, the slow cycling GBM cells established from primary tumor tissues, in an attempt to illuminate some of the biologic underpinnings of CMV with respect to GBM pathogenesis.

Taken together, these data lay the groundwork for the development of a more efficacious vaccination strategy targeting CMV in GBM. The screening strategies employed throughout this work will allow for an accurate antigenic profile of CMV in GBM which will subsequently permit the design of a more robust peptide vaccine for the next generation of cancer vaccine. We have also begun to describe some of the interesting biologic phenomena associated with CMV in GBM, as our results demonstrate continued viral gene expression in glioma stem cell-like cell populations indicating viral tropism for certain cell types.

Zika virus (ZIKV) re-emerged in the 2015-2016 epidemic in the Americas, when it was recognized that this mosquito transmitted virus can also be transmitted congenitally. One in 10 infants born to ZIKV-infected pregnancies presented with congenital defects including microcephaly, brain and ocular damage, neurodevelopmental delays, and mobility defects. In the past outbreak 11,000 children in Brazil were born with microcephaly and even more have lifelong disability. To prevent congenital transmission, a vaccine that is effective in pregnancy is urgently needed. Developing such an intervention requires an understanding of the targeted immune responses that mediates protection in pregnancy. Therefore, we investigated maternal B cell and antibody immunity to ZIKV.

First, we characterized ZIKV immunity in mothers with natural ZIKV infection and found that all mounted high ZIKV neutralizing antibody titers within 10 days of symptoms and maintained this throughout gestation. We then evaluated transplacental transfer of IgG and found that maternal ZIKV infection did not impair transfer of vaccine-elicited and flavivirus-neutralizing IgG, indicating that maternal immunization is a viable option to transfer immune protection to the newborn. However, a potential risk of transferred IgG is antibody-dependent enhancement of a heterologous flavivirus in early life. Consequently, we next tested the role of vertically transferred cross-reactive IgG in infant cord blood and found that transferred IgG may mediate in vitro flavivirus enhancement in absence of high ZIKV-neutralizing titers.

Next, we evaluated the role of IgM antibodies in the maternal immune response to ZIKV, as IgM do not cross the placenta during gestation and were found for an unusually long time in many cohorts. We found that plasma IgM contributes to early ZIKV neutralization across several ZIKV-infected pregnant women. We then isolated a potently ZIKV-neutralizing IgM monoclonal antibody, DH1017.IgM, which demonstrates 39-fold increased neutralization activity than a recombinant IgG with the same antigen binding sites, suggesting that the multivalency of the IgM may have a role in function. Potency of DH1017.IgM increased in a dose-dependent manner with complement. Structural studies of the epitope revealed a novel E dimer epitope on Domain II. This epitope can be bound by DH1017.Fab at multiple angles and is computationally predicted to have the capacity to be bound by all five IgM monomers simultaneously, defining a novel route of ZIKV neutralization. Importantly, the DH017.IgM protects mice from a lethal challenge of ZIKV and reduced in vitro enhancement otherwise observed with DH1017.IgG, suggesting that DH1017.IgM may be a suitable candidate for prophylactic intervention in pregnancy.

Altogether, we have identified key aspects of maternal immunity that will inform the development of ZIKV vaccines. First, we show that pregnant women can mount a robust B cell response against ZIKV, that is durable throughout pregnancy, and that protective levels of vaccine-elicited IgG can be transferred transplacentally to infants, suggesting that maternal immunization is a viable strategy to prevent infections in pregnancy and protect the newborn. However, with the transfer of flavivirus cross-reactive IgG, there is a need to monitor infants for enhanced flavivirus disease upon maternal immunization. Subsequently, we found that IgM antibodies in plasma contribute to ZIKV neutralization and identified a novel route for virus neutralization by DH1017.IgM that can be leveraged in immunogen design. Thus, vaccine design should assess if inclusion of multimeric immunogens that mimic the conformational surface of the virion supports development of IgM-mediated immunity.

CD8+ T cells are associated with long term control of virus replication to low or undetectable levels in a population of HIV+ therapy-naïve individuals known as virus controllers (VCs; <5000 RNA copies/ml and CD4+ lymphocyte counts >400 cells/µl). These subjects' ability to control viremia in the absence of therapy makes them the gold standard for the type of CD8+ T-cell response that should be induced with a vaccine. Studying the regulation of CD8+ T cells responses in these VCs provides the opportunity to discover mechanisms of durable control of HIV-1. Previous research has shown that the CD8+ T cell population in VCs is heterogeneous in its ability to inhibit virus replication and distinct T cells are responsible for virus inhibition. Further defining both the functional properties and regulation of the specific features of the select CD8+ T cells responsible for potent control of viremia the in VCs would enable better evaluation of T cell-directed vaccine strategies and may inform the design of new therapies.

Here we discuss the progress made in elucidating the features and regulation of CD8+ T cell response in virus controllers. We first detail the development of assays to quantify CD8+ T cells' ability to inhibit virus replication. This includes the use of a multi-clade HIV-1 panel which can subsequently be used as a tool for evaluation of T cell directed vaccines. We used these assays to evaluate the CD8+ response among cohorts of HIV-1 seronegative, HIV-1 acutely infected, and HIV-1 chronically infected (both VC and chronic viremic) patients. Contact and soluble CD8+ T cell virus inhibition assays (VIAs) are able to distinguish these patient groups based on the presence and magnitude of the responses. When employed in conjunction with peptide stimulation, the soluble assay reveals peptide stimulation induces CD8+ T cell responses with a prevalence of Gag p24 and Nef specificity among the virus controllers tested. Given this prevalence, we aimed to determine the gene expression profile of Gag p24-, Nef-, and unstimulated CD8+ T cells. RNA was isolated from CD8+ T-cells from two virus controllers with strong virus inhibition and one seronegative donor after a 5.5 hour stimulation period then analyzed using the Illumina Human BeadChip platform (Duke Center for Human Genome Variation). Analysis revealed that 565 (242 Nef and 323 Gag) genes were differentially expressed in CD8+ T-cells that were able to inhibit virus replication compared to those that could not. We compared the differentially expressed genes to published data sets from other CD8+ T-cell effector function experiments focusing our analysis on the most recurring genes with immunological, gene regulatory, apoptotic or unknown functions. The most commonly identified gene in these studies was TNFRSF9. Using PCR in a larger cohort of virus controllers we confirmed the up-regulation of TNFRSF9 in Gag p24 and Nef-specific CD8+ T cell mediated virus inhibition. We also observed increase in the mRNA encoding antiviral cytokines macrophage inflammatory proteins (MIP-1α, MIP-1αP, MIP-1β), interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), and recently identified lymphotactin (XCL1).

Our previous work suggests the CD8+ T-cell response to HIV-1 can be regulated at the level of gene regulation. Because RNA abundance is modulated by transcription of new mRNAs and decay of new and existing RNA we aimed to evaluate the net rate of transcription and mRNA decay for the cytokines we identified as differentially regulated. To estimate rate of mRNA synthesis and decay, we stimulated isolated CD8+ T-cells with Gag p24 and Nef peptides adding 4-thiouridine (4SU) during the final hour of stimulation, allowing for separation of RNA made during the final hour of stimulation. Subsequent PCR of RNA isolated from these cells, allowed us to determine how much mRNA was made for our genes of interest during the final hour which we used to calculate rate of transcription. To assess if stimulation caused a change in RNA stability, we calculated the decay rates of these mRNA over time. In Gag p24 and Nef stimulated T cells , the abundance of the mRNA of many of the cytokines examined was dependent on changes in both transcription and mRNA decay with evidence for potential differences in the regulation of mRNA between Nef and Gag specific CD8+ T cells. The results were highly reproducible in that in one subject that was measured in three independent experiments the results were concordant.

This data suggests that mRNA stability, in addition to transcription, is key in regulating the direct anti-HIV-1 function of antigen-specific memory CD8+ T cells by enabling rapid recall of anti-HIV-1 effector functions, namely the production and increased stability of antiviral cytokines. We have started to uncover the mechanisms employed by CD8+ T cell subsets with antigen-specific anti-HIV-1 activity, in turn, enhancing our ability to inhibit virus replication by informing both cure strategies and HIV-1 vaccine designs that aim to reduce transmission and can aid in blocking HIV-1 acquisition.

The prevalence of allergic disease such as peanut (PN) allergy has increased within the last century. Environmental factors have been associated with an increased risk of developing allergic diseases. The severity of allergic diseases has also increased and clinical trials are investigating allergen-specific immunotherapy as a method to treat allergies. The purpose of this work was to identify a vaccine adjuvant that induced potent antigen-specific Th1 immune responses and determine its ability to reduce the development and severity of Th2- mediated allergic disease, using models of peanut hypersensitivity.

Three studies were performed. The first study compared a variety of vaccine adjuvants to identify a potent adjuvant with strong Th1-associated activity. This study verified that the Toll-like receptor (TLR) ligand CpG could induce potent Th1-associated immune responses. The second study tested the ability of environmental endotoxin levels and alum-adjuvanted vaccines to modulate the development of allergic disease using a mouse model of peanut allergy. Additionally, the TLR ligands, CpG and MPL, were combined with alum-adjuvanted vaccines to determine their ability to further impact allergic disease development. Results suggested that the addition of CpG to an alum-adjuvanted vaccine indirectly modified host immunity in a manner that decreased the development of PN-induced allergic disease. The last study evaluated the ability of CpG to reduce the severity of peanut allergy symptoms when combined with peanut in an immunotherapy formulation administered to peanut-hypersensitive mice. Nasal immunotherapy with PN + CpG but not PN alone or CpG alone reduced the severity of PN-induced anaphylaxis in hypersensitive mice. PN-hypersensitive mice treated with PN + CpG displayed an increased PN-specific IgG2c and IFN-γ responses. A reduction in allergic disease severity in PN-hypersensitive mice correlated with an increase in PN-specific IgG2c, IFN-γ and IL-10 responses and a reduction in PN-specific IL-13 responses, suggesting a shift from Th2 responses towards Th1 and/or T regulatory cell responses.

Taken together, the data obtained from these studies demonstrate the potent activity of CpG to induce antigen-specific Th1-associated immune responses and also reduce the severity of peanut-hypersensitivity in mice through direct and indirect association with peanut allergens.

Short peptides are poorly immunogenic when delivered sublingually – under the tongue. This challenge has prevented widespread investigation into sublingual peptide vaccines, leaving their considerable potential untapped. Sublingual immunization is logistically favorable due to its ease of administration and can raise both strong systemic immune responses and mucosal responses in tissues throughout the body. Peptide epitopes are highly specific, allowing for the generation of immune responses directed solely against precisely selected targets, without accompanying off-target antibody or T-cell production. To enable sublingual peptide immunization, we designed a strategy based on molecular self-assembly of epitopes within nanofibers. We then extract and expound several key implications from this finding, including insights into the mechanism of action, development of a translatable administration modality, and application to a currently unmet clinical need.

The design of our sublingual peptide immunization strategy is described in the first part of this thesis (Chapter 3). We sought to utilize nanomaterial delivery of peptides to enhance sublingual immunogenicity, a strategy that has proved successful in several other immunization routes. However, the salivary mucus layer is a significant barrier to nanomaterial delivery, particularly for supramolecular materials, due to its ability to entrap and clear these materials rapidly. We designed β-sheet nanofibers conjugated at a high-density to the mucus-inert polymer polyethylene glycol (PEG) to shield them from the mucus layer. Strikingly, sublingually delivered PEGylated peptide nanofibers (PEG-Q11) raised extremely durable antibody and T-cell responses against peptide epitopes when mixed with a mucosal adjuvant. We showed that PEG decreases nanofiber interactions with mucin in vitro, and extends the residence time of nanofibers at the sublingual space in vivo. Further, we showed that we could achieve similar results by adapting the use of PASylation (modification with peptide sequences rich in Pro, Ala, and Ser) to mucosal delivery.

In the second part of this thesis (Chapter 4) we designed a supramolecular strategy for enhancing sublingual nanofiber immunization. Mucosal adjuvants, such as cyclic-di-nucleotides (CDNs), can promote sublingual immune responses but must be co-delivered with the antigen to the epithelium for maximum effect. We designed peptide-polymer nanofibers displaying nona-arginine (R9) at a high density to promote complexation with CDNs via bidentate hydrogen-bonding with arginine side chains. We co-assembled PEG-Q11 and PEG-Q11R9 peptides to titrate the concentration of R9 within nanofibers. In vitro, PEG-Q11R9 fibers and cyclic-di-GMP or cyclic-di-AMP adjuvants had a synergistic effect on enhancing dendritic cell activation that was STING-dependent and increased monotonically with increasing R9 concentration. However, intermediate levels of R9 within sublingually-administered PEG-Q11 fibers were optimal for sublingual immunization, suggesting a balance between polyarginine’s ability to sequester CDNs along the nanofiber and its potentially detrimental mucoadhesive interactions. These findings reveal important design considerations for the continuing development of sublingual peptide nanofiber vaccines.

We sought to enhance the translational capacity of our immunization strategy by designing a highly accessible vaccine method (described in Chapter 5). Significant barriers exist to improving vaccine coverage in lower- and middle-income countries, including the costly requirements for cold-chain distribution and trained medical personnel to administer the vaccines. To address these barriers, we built upon our sublingual nanofiber platform to design a heat-stable and highly porous tablet vaccine that can be administered via simple dissolution under the tongue. We produced SIMPL (Supramolecular Immunization with Peptides SubLingually) tablet vaccines by freeze-drying a mixture of self-assembling peptide-polymer nanofibers, sugar excipients, and adjuvant. We showed that even after heating for 1 week at 45 °C, SIMPL tablets could raise antibody responses against a peptide epitope from M. tuberculosis, in contrast to a conventional carrier vaccine (KLH) which lost sublingual efficacy after heating. Our approach directly addresses the need for a heat-stable and easily deliverable vaccine to improve equity in global vaccine coverage.

To demonstrate the clinical usefulness of our technology, we designed a sublingual vaccine against uropathogenic E. coli (UPEC), the pathogen that causes most urinary tract infections (UTIs). Sublingual peptide immunization is uniquely advantageous for immunization against UTIs, as sublingual immunization raises antibodies in the blood and urinary tract, and peptide epitopes allow for targeting UPEC without perturbing commensals. We co-assembled PEG-Q11 nanofibers containing three different B-cell epitopes from UPEC along with a helper T-cell epitope, allowing us to raise simultaneous antibody responses against three targets systemically and in the urinary tract. These antibodies were highly specific for UPEC, exhibiting no binding to a non-pathogenic strain. Further, these antibodies demonstrated clinical potential by protecting mice from an intraperitoneal UPEC challenge. Finally, we showed the ability to use SIMPL tablets to raise anti-UPEC responses in rabbits, which contain an oral cavity with key similarities to humans.

This thesis demonstrates a critical enabling technology for sublingual peptide immunization and builds upon this technology to report findings with key implications for supramolecular biomaterial design, accessible vaccination, and treatment of UPEC-mediated diseases. This interdisciplinary research synthesizes and utilizes knowledge from materials science, immunology, vaccinology, pharmaceutical sciences, and pathology to present an important original contribution to the field of immune engineering.

Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB), is among the leading causes of death from infectious disease world-wide. An intracellular pathogen, M. tuberculosis infects phagocytic cells, and subverts the host immune response, preventing eradication once infection has been established. Even after successful chemotherapy, exogenous re-infection occurs, indicating that sterilizing immune responses are not generated during natural infection. While a TB vaccine exists, it does not alter M. tuberculosis infection rate, rather it prevents the progression from latent TB infection to active TB disease. Vaccines against Haemophilus influenzae and Streptococcus pneumonia protect from bacterial colonization and infection through the induction of antibodies to capsular surface components. This dissertation explores if antibodies to the surface of M. tuberculosis can alter the initial interaction between a bacterium and host cell, leading to a reduction in infection rate.

When pre-mixed with M. tuberculosis prior to in vitro infection of macrophages, or retropharyngeal instillation of mice, monoclonal surface-binding, but not non-surface-binding antibodies, decrease bacterial burden and the number of infected cells within the first twenty-four hour of infection. If administered retropharyngeally prior to aerosol exposure, surface-binding antibodies decreased pulmonary bacterial burden at twenty-four hours post infection in an FcγR independent manner. Despite decreasing early bacterial burden, pre-administration of surface-binding antibodies prior to ultra-low dose aerosol infection did not alter infection rate compared to mice instilled with PBS (Chapters 4 and 5).

Infected humans do not produce high-titer, high-avidity surface-binding antibodies. Plasma from uninfected controls, individuals with latent TB infection, and active TB disease was assayed by ELISA to determine the titer, avidity and IgG/IgM ratio for antibodies to the surface and additional bacterial fraction. In contrast to antibodies to bacterial fractions, individuals with active TB disease had decreased avidity, and no augmentation of the IgG/IgM ratio for antibodies to the live M. tuberculosis surface, as compared to uninfected controls (Chapter 3).

Overall these findings demonstrate that surface-binding monoclonal antibodies alter early infection events, both in vivo and in vitro, though the magnitude of protection was not sufficient to decrease M. tuberculosis infection rate. Additionally, the failure of humans to generate high-titer, high-avidity surface-binding antibodies after infection indicates and that induction of surface-binding antibodies may be an appropriate target for future vaccines.

A successful HIV-1 vaccine is likely to be comprised of a polyclonal response with multiple antiviral functions that generate both broadly neutralizing antibodies (bNAbs) that neutralize multiple strains of HIV and/ or functional non-neutralizing antibodies that participate in numerous antiviral mechanisms. It is suggested that poly-/autoreactivity is a feature of bNabs but the interplay of autoimmunity and bNab development is unknown. Furthermore the non-neutralizing antibody specificities and functions are unknown for generating a potentially protective anti-HIV response. Understanding the development of rare neutralizing antibodies and the specificity and effector functions of non-neutralizing antibodies is critical for generating a preventative HIV vaccine. This thesis focuses on 1) the development of broadly neutralizing antibodies; specifically those that are present in both the face of autoimmunity when both self-antigen and HIV-1 is a determinant in disease outcome and 2) the mechanisms of non-neutralizing antibodies in mediating protection against HIV-1.

In the first part of this thesis, I chart the evolution of an HIV-1 antibody lineage that has reactivity to self antigen. One HIV-1 broadly neutralizing antibody targeting the membrane proximal external region, 2F5, shares sequence identity with the human self-antigen kynureninase, (KYNU). We previously reported that an HIV-1 chronically infected patient had both evidence of antibodies targeting self-proteins and circulating 2F5-like antibodies in serum. Whether development of 2F5-like antibodies to a self-antigen can lead to a broadly neutralizing antibody response or whether reactivity to self-antigen is a requirement for the neutralization breadth of 2F5-like antibodies has been a question in the HIV vaccine community. Evolution of 2F5 mAb-like antibody neutralization breadth and autoreactivity has not been previously demonstrated due to the scarcity of this specificity of antibody found circulating in HIV-1 infected individuals. In this study, I found that an unmutated common ancestor derived from a isolated mAb from this patient did not bind KYNU but more mature antibodies in the constructed antibody lineage developed reactivity to KYNU and other self-antigens due to an accumulation of antibody VH mutations. At the same time, the accumulation of mutations in the m66 lineage VH resulted in decreased 2F5 mAb binding affinity and virus neutralization potency, consistent with host mechanisms that limit antibody autoreactivity by placing constraints on affinity maturation. This work highlights the complex maturation pathways involving autoantigen reactivity in the development of broadly neutralizing antibodies.

The second part of the study centers on non-neutralizing antibody functions in defense against HIV-1. Elucidation of antibody effector functions responsible for protective immunity against HIV-1 acquisition is a major goal for the HIV-1 vaccine field. Immunoglobulin A (IgA) is an important part of the host defense against pathogens; however, little is known about the role of vaccine-elicited IgA and their capacity to mediate antiviral functions. To identify the antiviral functions of HIV-1-specific IgA elicited by vaccination, we cloned HIV-1 envelope specific IgA monoclonal antibodies by memory B cell cultures from peripheral blood mononuclear cells from a vaccinee from the only partially efficacious HIV vaccine regimen to date, RV144. We produced two IgA clonal cell lines (HG129 and HG130) producing native, non-recombinant IgA monoclonal antibodies (mAbs). HG129 is crossreactive and binds the variable loop (V3) of the HIV envelope; HG130 is a conformational gp120 antibody that interacts with the CD4 binding site. HG129 and HG130 mAbs mediated phagocytosis of HIV infectious virions and HIV antigen-coated beads respectively, and HG129 blocked HIV-1 Env glycoprotein binding to galactosylceramide, an alternative HIV-1 receptor. These findings elucidate potential antiviral functions of vaccine-elicited HIV-1 envelope specific IgA that may act to block HIV-1 acquisition at the portal of entry by preventing HIV-1 binding to galactosylceramide and mediating antibody Fc receptor mediated virion phagocytosis. Furthermore, these findings highlight the complex and diverse interactions of vaccine-elicited IgA with pathogens that depend on IgA fine specificity and form (e.g. multimeric, monomeric) in the systemic circulation and mucosal compartments.

The results of this work demonstrate that whether an antibody is broadly neutralizing or non-broadly neutralizing, it is the fine specificity to the HIV-1 envelope that is crucial for its functional activity. The functional activity is dependent on antibody specificity and isotype due to engagement of effector cells at mucosal sites. The production of potentially protective antibody envelope specificities, antibody isotypes, and functions should be considered in the immunogen design for the development of safe and effective vaccine approaches.

Glioblastoma (GBM) is the most common malignant primary brain tumor in adults. Conventional therapies for GBM typically fail to provide lasting antitumor benefits, owing to their inability to specifically eliminate all malignant cells. Immunotherapy is currently being pursued as a strategy to address this unmet need, in light of the cell-specific cytotoxicity an immune response can afford. Of the various immunotherapeutic modalities, cancer vaccines are currently being evaluated as a means to direct the adaptive immune system to target residual GBM cells that remain following standard-of-care treatment. To date, no cancer vaccines have been proven effective against GBM; however, only a few have reached phase III clinical testing. Clinical immunological monitoring data suggests that GBM vaccines are capable of stimulating immune responses reactive to GBM antigens, but whether these responses have an appreciable antitumor effect on GBM is still uncertain. Nevertheless, there have been several promising outcomes in early phase clinical trials, which lend encouragement to this area of study.

In this dissertation, we explore the therapeutic potential of cancer vaccines targeting malignant astrocytoma-specific somatic missense mutations – or neoantigens. This pursuit was inspired by recent data from a phase III clinical trial with a protein vaccine targeting the GBM-specific antigen EGFRvIII, revealing that most recurrent tumors were composed of EGFRvIII-deficient or -suppressed tumor cell variants. This outcome, known as antigen escape, is likely a consequence of the profound heterogeneity of GBM tumors and, altogether, suggests that monovalent immunotherapeutic strategies targeting subclonal GBM antigens are likely insufficient to treat this disease. Conversely, personalized cancer vaccines targeting patient-specific missense mutations have the potential to elicit a multivalent, tumor-specific immune response that may target a broader repertoire of GBM cells.

Chapters 1-4 offer a comprehensive review of GBM, an overview of immunotherapy for malignant brain tumors, and promising vaccines that are currently being explored for the treatment of GBM. In chapter 5, we present a novel method that we have developed for evaluating neoantigen-specific lymphocytes from miniscule amounts of solid tumor tissue, which we believe can aid in immunological monitoring of neoantigen-specific immune responses in the clinic. In chapter 6, we elucidate the mechanism of an efficacious neoantigen vaccine, which led to the development of rationally-designed, neoantigen-targeting, synthetic long peptide vaccines with enhanced immunogenicity and efficacy using a universal helper epitope. In chapter 7, we explore the utility of minigene-transfected dendritic cell (DC) vaccines for targeting neoantigens, in which we reveal several significant limitations of traditional GM-CSF + IL-4-generated DCs. Finally, chapter 8 discusses future prospects for enhancing the therapeutic response by cancer vaccines. Together, this original work provides several encouraging insights for the development and evaluation of personalized cancer vaccines for GBM.

Cytomegalovirus (CMV) is the most common congenital infection and a problematic opportunistic pathogen for immunocompromised patient populations. Despite the immense global burden of CMV and many years of research, the licensed interventions for prevention of CMV disease, and congenital CMV in particular, are very limited. The goal of the work presented in this dissertation is to inform vaccine design for the prevention of congenital CMV by enhancing maternal humoral immunity. We initially investigated the humoral immune responses that associate with protection from vertical CMV transmission in a rhesus macaque model of congenital CMV, evaluating Fc mediated effector responses, which had not previously been measured in the context of rhesus CMV (RhCMV) infection. This study suggested that the humoral response develops too late following primary infection to play a significant role in prevention of vertical transmission but demonstrated a role for pre-existing, potently neutralizing antibodies in prevention of vertical CMV transmission. While this study did not find an association between Fc mediated antibody effector responses, clinical observational studies have implicated these antibody functions in protection from congenital CMV. Interestingly, human CMV (HCMV) is known to encode multiple proteins capable of binding to immunoglobulin G (IgG) antibodies, which have demonstrated the ability to interfere with host Fcγ receptor (FcγR) activation and effector function. We have identified homolog viral FcγRs (vFcγRs) in RhCMV, which allows us a unique opportunity to study these proteins in vivo for the first time by infecting RhCMV-seronegative rhesus macaques with RhCMV lacking all three identified vFcγRs and validating the role vFcγRs have demonstrated in vitro in immune evasion. Lastly, we evaluated a novel HCMV vaccine strategy in which we targeted this immune evasion mechanism through active vaccination against glycoprotein B (gB) alone or in combination with one of the vFcγRs. This proof-of-concept study demonstrated that immune responses against the vFcγRs, gp34 in particular, can improve host FcγR activation and effector function, with initial focus on FcRI (CD64). These results suggest that a simple addition of one or more vFcγRs to vaccines already in development may have a significant impact on the effectiveness of Fc mediated effector responses, which could in turn reduce the risk of vertical CMV transmission.