Browsing by Author "Staats, Herman F"

Unlabelled

Importance

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.

Selective therapeutic targeting of tumors requires identification of differences between the homeostatic requirements of cancer and host cells. One such difference is the manner in which cancer cells acquire energy. Cancer cells often grow in an environment of local hypoxia; under these conditions tumor cells depend on glycolysis for energy, but are unable to perform oxidative phosphorylation. Many tumor cells, despite normoxic conditions, continue to perform glycolysis without oxidative phosphorylation. The net result of glycolysis without oxidative phosphorylation is twofold: the need to consume a greater amount of glucose than a non-cancerous host cell, and the burden of increased intracellular lactic acid. The proteins responsible for the transport of lactic acid in and out of cells are known as the monocarboxylate transporters (MCTs). Monocarboxylate Transporter 1 (MCT1) and Monocarboxylate Transporter 4 (MCT4) are the MCTs that play a major role in the transport of lactic acid. Tumor cells depend on MCT1 and MCT4 activity to excrete excess intracellular lactic acid to maintain neutral intracellular pH and homeostasis. Using human neuroblastoma and prostate cancer cell lines this work demonstrates that tumor cells can be selectively targeted tumor under conditions of hypoxia or acidosis in vitro with the drug lonidamine, with a small molecule inhibitor selective for MCT1, or with RNA interference of MCT1. Inhibition of MCT1 activity in neuroblastoma cells under acidic extracellular conditions results in intracellular acidification and cell death. MCT1 mRNA is expressed in human neuroblastoma and positively correlated with clinical risk profile. Inhibition of MCT1 activity in hypoxic prostate cancer cells results in a reduction of lactate excretion, decreased intracellular pH, inhibition of ATP production, and subsequent cell death. MCT1 expression in sections of human prostate tumors has been demonstrated to validate MCT1 as a target in prostate cancer.

Through the Pasteur and Warburg effects, tumors have an increased demand for glucose. Some cancers store glycogen, but the reasons for this are largely unknown. It is hypothesized that tumor glycogen is used to promote tumor survival during transient hypoxia or low glucose, and that the mechanisms by which glycogen is stored is a potential therapeutic target in cancer. Tumors from human cell lines (WiDr, PC3, FaDu) have been grown in nude mice, sectioned and stained to measure glycogen storage. Using consecutive frozen sections, levels of hypoxia, glucose, lactate, ATP, and CD31, an endothelial cell marker, have been determined. These sections have been employed to elucidate the "architecture" of tumor metabolism in terms of vessel distance. Additionally, PAS-stained EF5 labeled human tumor samples were used to obtain calibrated hypoxia measurements to correlate with PAS. These studies demonstrate a correlation between hypoxia and the formation of glycogen deposits in human tumors and nude mouse xenografts. In cell culture, formation of glycogen deposits after exposure to hypoxia has been demonstrated, in addition to expression of glycogen synthase in human cancer cell lines.

The development of novel selective cancer chemotherapeutics will require the identification of differences between cancerous cells and normal host cells to exploit as targets. Several differences in metabolism, including the need to excrete excess lactic acid and store glycogen under hypoxic conditions, are such targets. Novel therapeutics exploiting these targets should be effective against cancer cells and minimally toxic to host cells.

Although monophosphoryl lipid A was recently approved by the Food and Drug Administration, more vaccine adjuvants are needed to meet the demand for vaccines against new, emerging, and re-emerging diseases. Additionally, characterizing the mechanisms of action of potent vaccine adjuvants is important for moving toward more rational vaccine design based on the careful selection of antigens and adjuvants to stimulate only the desired immune responses. Two experimental vaccine adjuvants, compound 48/80 (C48/80) and IL-1, were evaluated in these studies. The safety and efficacy of the mast cell activator C48/80 was evaluated when used as an adjuvant delivered intradermally (ID) with recombinant anthrax protective antigen (rPA) in comparison with two well-known adjuvants. Mice were vaccinated in the ear pinnae with rPA or rPA + C48/80, CpG oligodeoxynucleotides (CpG), or cholera toxin (CT). All adjuvants induced similar increases in serum anti-rPA IgG and lethal toxin-neutralizing antibodies. C48/80 induced balanced cytokine production (Th1/Th2/Th17) by antigen-restimulated splenocytes, minimal injection site inflammation, and no antigen-specific IgE. Our data demonstrate that C48/80 is a safe and effective adjuvant, when used by the intradermal route, to induce protective antibody and balanced Th1/Th2/Th17 responses. Histological analysis demonstrated that vaccination with C48/80 reduced the number of resident mast cells and induced an injection-site neutrophil influx within 24 hours. Nonetheless, rPA + C48/80 significantly increased antigen-specific IgG titers in mast cell-deficient mice compared to antigen alone, suggesting that C48/80 has mast cell-dependent and mast cell-independent mechanisms of action.

IL-1alpha and beta have been shown to have strong mucosal adjuvant activities, but little is known about their mechanism of action. Bone marrow chimeric mice were intranasally vaccinated with Bacillus anthracis lethal factor (LF) with or without 4 µg IL-1alpha or a control adjuvant (cholera toxin) to determine if IL-1R1 expression on stromal cells or hematopoietic cells was sufficient for the maximal adjuvant activity of nasally delivered IL-1alpha. IL-1alpha was not active in IL-1R1-deficient (Il1r1-/-) mice given Il1r1-/- bone marrow, demonstrating that the adjuvant activity of IL-1 was due to the presence of IL-1R1 and not contaminants. Cytokine and chemokine responses induced by vaccination with IL-1alpha were predominantly derived from the stromal cell compartment and included G-CSF, IL-6, IL-13, MCP-1, and KC. Nasal vaccination of Il1r1-/- mice given wild-type bone marrow (WT-->KO) and WT-->WT mice with LF + IL-1alpha induced maximal adaptive immune responses, while vaccination of wild-type mice given Il1r1-/- bone marrow (KO-->WT) mice resulted in significantly decreased production of LF-specific serum IgG, IgG subclasses, lethal toxin-neutralizing antibodies, and mucosal IgA compared to WT-->KO and WT-->WT mice (p < 0.05). Our results suggest that IL-1R1 expression in the hematopoietic compartment is sufficient for the maximal induction of antigen-specific adaptive immunity after nasal vaccination adjuvanted with IL-1alpha and that while stromal cells are required for maximal adjuvant-induced cytokine production, the adjuvant-induced stromal cell cytokine responses are not required for effective induction of adaptive immunity.

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.

Cancer continues to be a large health and economic burden, despite advances in diagnostics and therapy. Cancer immunotherapy research and development of novel cancer vaccine strategies continues to grow, and new immunotherapy options offer considerable promise for patients. Research has identified more than four hundred tumor-associated antigens, yet only one cancer vaccine is FDA approved and on the market for established cancer. Since mucosal tissues are often the site of cancer development and metastasis, vaccine systems that induce tumor-specific mucosal immune responses are worthy of investigation. Mucosal immunization has the ability to induce tumor-specific immune responses in non-mucosal (systemic) sites while also inducing mucosal immune responses that are characterized by effector cells that home to and reside in mucosal tissues. The purpose of this review is to discuss recent preclinical advances with the use of mucosal immunization for the induction of protective anti-cancer immunity and discuss critical factors related to mucosal immunization, such as the route of immunization and adjuvants. Additionally, we will discuss challenges associated with translating effective mucosal vaccines for tumors from preclinical to clinical use. Finally, we will discuss the importance of preclinical and clinical studies to determine if mucosal immunization is critical for therapeutic benefit against tumors that arise at or metastasize to mucosal tissues.

No

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.