Xiaoping Zhu - US grants

[unreadable] DESCRIPTION (provided by applicant): Vaccine strategies to prevent invasive mucosal pathogens are being sought due to the fact that 80-90% of infectious diseases are initiated at mucosal surfaces. In addition, our ability to deliver vaccine antigens across the mucosal barrier for induction of the effective mucosal immunity is limited. The long-term goal of this proposal is to determine whether the FcRn-IgG transcellular pathway represents a novel delivery path for a subunit vaccine against mucosal pathogens. The neonatal Fc receptor (FcRn) was initially considered to transport maternal IgG to a fetus through the placenta or to newborns via the intestine. However, FcRn is expressed in a variety of tissues and cells in adult humans and animals, and mediates the bi-directional transport of IgG across polarized epithelial cell lines. Based on these evidences, we hypothesize that such IgG transport pathway may allow FcRn to deliver a viral antigen fused to an IgG-Fc across the mucosal barrier to the underlying mucosa-associated lymphoid tissue. The consequences of such transport could give way to immunogenicity. Herpes simplex virus type-2 (HSV-2) is a sexually-transmitted disease, and thus, the primary site of HSV-2 infection is the mucosa of the genital tract. The glycoprotein gD will be used to probe responses to immunization and to define protective immune responses. The specific aim of this proposal is to determine the ability of FcRn to deliver gD-Fc antigen across the genital or the respiratory mucosal barrier to engender protective immunity against mucosally-administered virulent HSV-2 challenge. The results from this study will be relevant to understanding mucosal immune regulation; the knowledge gained will be useful in development of effective novel vaccine strategies for mucosal pathogens, such as human immunodeficiency virus-1, Chlamydia, influenza etc., that infect at or invade across mucosal surfaces. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Transmission of Human Immunodeficiency Virus (HIV) occurs primarily via the mucosal routes, emphasizing HIV-1 vaccines must need to engender mucosal immune responses. However, mucosal immunization has been limited by the ability to deliver intact vaccine antigens across the mucosal barrier for induction of effective mucosal immunity. The long-term goal of this proposal is to determine whether the IgG transcellular pathway represents a novel delivery path for a subunit vaccine against infections of HIV and AIDS-related opportunistic pathogens. The goal of the project derives from the recent proof of concept that the neonatal Fc receptor (FcRn) mediates the bi-directional transport of IgG across polarized epithelial cells. FcRn was initially considered to transport maternal IgG to a fetus through the placenta or to newborns via the intestine. However, FcRn is expressed in a variety of tissues and cells in adult humans and animals; IgG is a predominant isotype of immunoglobulins in the lower respiratory and genital tract. Based on these evidences, we will test the hypothesis that using IgG transport pathway, FcRn can deliver HIV-1 antigen fused to an IgG-Fc across the mucosal barrier to the underlying mucosa-associated lymphoid tissue. The consequences of such transport could induce local immunity able to neutralize the virus at their port of entry and systemic immunity able to prevent systemic spread of the infection. HIV envelope glycoprotein gp120 will be used to probe immune responses to such immunization and to define protective immune responses. The specific aim of this proposal is to determine the ability of FcRn to deliver gp120-Fc antigen across the genital or the respiratory mucosal barrier to engender protective immunity against mucosallv-inoculated virus challenge. Data generated herein will provide valuable information not only for design of a HIV vaccine, but also for general vaccine strategy targeting AIDS-associated opportunistic pathogens or other pathogens, such as cytomegalovirus, herpes simplex virus, mycobacterium, chlamydia, influenza, etc., that infect at or invade across mucosal surfaces. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Sexually transmitted diseases (STDs) are among the most common infectious diseases in humans. Local immune responses, especially the mucosal immunoglobulins (Igs), provide the first line of defense against primary mucosal infections. IgG is a dominant Ig class in the mucosal secretions of the human genital tract, where it predominates over IgA. Despite the abundance of human IgG, surprisingly less is known about how IgG is secreted into the genital lumen and the exact role of IgG in preventing sexually transmitted pathogens. Our long-term goal is to elucidate the molecular mechanisms of IgG transport in the genital tract and the role of IgG immunity to sexually transmitted pathogens. The specific hypotheses are that FcRn mediates IgG transcytosis and plays a major role in mucosal protection, and that FcRn can deliver an antigen fused to an IgG Fc fragment across the female genital tract to gain access to underlying antigen-presenting cells. These hypotheses were based on the observations that 1) FcRn can mediate the bi-directional transport (apical to basolateral, or vice versa) of IgG across intestinal or placental epithelial cell lines, 2) our recent study showed that human and rodent FcRn were functionally expressed in epithelial cells derived from the human female genital tract, 3) FcRn binds IgG only at acidic pH;whereas, the vagina exhibits acidic pH, 4) the levels of IgG in the female genital tract can be changed over the course of the estrous cycle. Our new data showed that hormone significantly regulated the FcRn expression. Based on these observations, the experimental focus of this proposal is on the understanding of IgG transport and IgG-mediated immunity to genital infections. The specific aims are to: 1. Determine the FcRn-meidated IgG transcvtosis in the reproductive tract;2. Determine FcRn-mediated IgG immunity to primary infections of sexually transmitted pathogens, such as herpes simplex virus-2: 3. Determine the ability of FcRn to deliver IgG Fc-fused antigens, HSV-2 gD-Fc, across the genital mucosal barrier to generate protective immunity. These studies will increase our presently-limited understanding of immune protection for the genital tract, and will provide the basic knowledge essential for the prevention of other STDs, including human immunodeficiency virus, vaginitis, syphilis, gonorrhea, papillomavirus, Candida albican, etc.

DESCRIPTION (provided by applicant): Allergic airway inflammations, such as asthma, are an increasingly important disease caused by bronchial inflammation and characterized by bronchial hyper-responsiveness and intermittent airway obstruction with an underlying Th2 cell-biased inflammatory response in the airways. The disease is currently treated with bronchodilators or anti- inflammatory drugs such as corticosteroids, leukotriene modifiers, and anti-IgE therapy, etc. However, the current treatments are not curative and some patients do not respond well to intense anti-inflammatory therapies. Additionally, the use of long-term steroids may result in many undesired side effects. For this reason, novel and more effective intervening strategies are greatly needed and explored. Targeting of the functions of Th2 cells and their products have been proposed as an effective strategy for the development of potential stand-alone treatments for allergic asthma. The reduction or elimination of allergen-specific Th2 cells in early disease development is expected to reduce the consequences of repeated allergic inflammatory. Hence, efficient delivery of immunotherapeutic proteins into the airway tract could effectively and directly interfere with allergen-specific Th2 cell activation in its earliest phaseof function. However, the polarized epithelial monolayer lining the airway forms mucosal barrier which is impervious to macromolecule diffusion. This barrier poses a major difficulty for an efficient delivery of immunotherapeutic proteins to access and cross-talk with underlying immune effector cells, such as Th2 cells, in the airway. Our recent studies have shown that human CD23 receptor is functionally capable of transporting IgE antibody across human lung and bronchial epithelial cells. In this study, we further propose to examine the feasibility of CD2 to deliver the immunotherapeutic proteins, which are targeted to interfere with CD4 Th2 cell function, across airway mucosal barrier in a mouse allergy model. These studies, therefore, are very likely to lead to greatly improved novel therapies that protect against and potentially cure asthma and allergic diseases. PUBLIC HEALTH RELEVANCE: CD23-mediated immunotherapy on airway inflammation Airway inflammation, asthma, is among the most common diseases in both infant and adult. CD23 is capable of transporting IgE across airway epithelial cells. This study will explore the feasibility whether CD23 can deliver an immunotherapeutic protein to modulate or dampen the development of airway allergic inflammation.

DESCRIPTION (provided by applicant): Sexual transmission is the dominant mode for HIV spread worldwide. Our goal is to develop a subunit vaccine based on the HIV gp120 envelope glycoprotein, which can be delivered via atraumatic mucosal inoculation to elicit durable protective immunity. In order to immunize at mucosal sites, we need an efficient method for delivering gp120 and adjuvant to mucosal immune cells. We constructed fusion proteins consisting of gp120 linked to the Fc portion of IgG2 antibodies. The fusion protein Fc segment binds Fc receptor neonatal (FcRn) on mucosal epithelium, and crosses the epithelial barrier by non-degradative transcytosis to access underlying antigen presenting cells. Adjuvant, in our case muramyl dipeptide (MDP), is conjugated directly to the gp120-Fc fusion protein through modification of terminal sialic acid residues (Env-Fc-MDP). Thus, conjugated adjuvant and antigen are co-delivered to presenting cells. MDP was selected because it increases co-stimulatory molecule and IL-12 expression by dendritic cells, and should enhance T and B cell responses to HIV Env. The potency and durability of gp120 immune responses is increased with a prime/boost strategy where mucosal priming with fusion protein is followed by intramuscular boosting with gp120 plus soluble MDP. Our studies utilize in vitro or mouse models to optimize immunization strategies with fusion protein and adjuvant. Nonhuman primate studies optimize dose and route for immunization using an unrelated fusion protein (HSV-2 glycoprotein D-Fc), then test whether mucosal Env-Fc-MDP followed by intramuscular Env + soluble MDP elicits protection against repetitive, low-dose intrarectal inoculation with SHIV162p3. This project is an interdisciplinary collaboration between Dr. Zhu, expert in mouse models and Fc receptor biology, Dr. Wang, who is a glycobiologist and organic chemist expert in protein glycosylation, and Dr. Pauza who has extensive experience in nonhuman primate models, mucosal immunology and mucosal virus infections. The innovative route for mucosal antigen delivery, use of chemically-conjugated adjuvant to improve T cell responses, and our prime/boost strategy is a safe and potentially effective method for eliciting durable protective immunity against sexual transmission of HIV-1.

Implantable biomaterials and medical devices are used in millions of procedures each year worldwide. However, in large number of patients, the implantation of these devices often leads to the development of a foreign body response (FBR), a chronic inflammatory condition that can ultimately lead to implant failure, which may cause harm to or death of the patient. The FBR consists of persistent inflammation coupled with fibrous encapsulation around the implant. There are no effective medical treatments. Hallmarks of the FBR include activation of macrophages at the tissue-implant interface, formation of destructive foreign body giant cells (FBGCs), and development of fibrous tissue that encapsulates the implant. The overall goal of our research is to understand the molecular mechanisms of the fibrotic response. Activated macrophages are thought to orchestrate the FBR by secreting inflammatory mediators. Emerging data support a critical role for a mechanical signal, e.g., substrate stiffness, in macrophage activation. However, a critical gap in this field is that the identity of the plasma membrane mechanosensor by which the mechanical signal is transduced/maintained is not known, nor are the downstream consequences of mechano-receptor signaling on the FBR. These gaps pose a significant barrier to progress in the field. In recent, exciting preliminary data, we obtained evidence that TRPV4, an ion channel in the transient receptor potential vanilloid family, and which is a known mechanosensor, may be the mediator of FBR. Specifically, we found that: 1) Trpv4 deletion in mice prevented macrophage accumulation, FBGC formation, and collagen accumulation in a subcutaneous implantation model; 2) the severity of the in vivo macrophage accumulation at the tissue-implant interface was dependent on the stiffness of the implant, and 3) genetic ablation or pharmacologic antagonism of TRPV4 blocked macrophage adhesion and spreading on stiff matrix, interleukin-4-induced FBGC formation, and inflammatory gene expression in both human and mouse bone marrow derived macrophages. Our preliminary data indicated that TRPV4 activity (Ca2+ influx) was augmented in response to increased matrix stiffness, and suggested that the molecular pathway linking TRPV4 activity to the FBR involved a specific phosphoinositide 3-kinase (PI3K) isoform, PI3K-alpha. The objective of this proposal is to determine the role of TRPV4 in the FBR. Based on our preliminary data, our central hypothesis is that TRPV4 mediates the FBR to biomaterials by increasing macrophage activation and fibrogenesis in a manner dependent on substrate stiffness and PI3K-alpha. We will utilize innovative technologies, in vivo and in vitro model systems, and a recently identified small molecule TRPV4 inhibitor to test the hypothesis with two Specific Aims. In Specific Aim 1 we will test the hypothesis that TRPV4 is a necessary component of the FBR in vivo; and in Specific Aim 2 we will test the hypothesis that mechanosensing by TRPV4 is a key component of the molecular mechanism of regulation of biomaterial- induced macrophage activation and fibrogenesis. When completed, we expect that the results of this study will generate novel information and insight regarding the mechanisms mediating the FBR to biomaterials, and will potentially identify a targetable receptor/pathway for the amelioration of FBR.

PROJECT SUMMARY Most human pathogens enter via mucosal routes. Yet, there are few licensed mucosal vaccines. Thus, there is a need for new vaccine technologies and/or adjuvants that stimulate protective immunity at mucosal sites. We have developed a novel recombinant mucosal vaccine platform. The efficacy of this vaccine platform has been demonstrated using a human Fc?RI (hFc?RI)-specific fusion protein (FP) consisting of pneumococcal surface protein A (PspA) antigen (Ag) targeted to hFc?RI. This FP enhances immunogenicity and protection against mucosal S. pneumoniae (Sp) challenge when administered without adjuvant intranasally (i.n.) to hFc?RI transgenic mice. We hypothesize that a more potent adjuvant-free mucosal vaccine platform can be produced by generating a dual human FcRn (hFcRn)/hFc?RI-targeted-Ag FP, which enhances FcRn-mediated transepithelial Ag transport to the nasal-associated lymphoid tissue (NALT) and subsequent hFc?RI-mediated Ag presentation and T cell activation within the NALT. In this regard, such a dual-targeted FP has been generated by adding a hFcRn targeting sequence to the hFc?RI-specific PspA-containing FP. This novel FP will now be evaluated for its ability to enhance protective efficacy, transepithelial transport of Ag to the NALT, and Ag presentation/T cell activation within the NALT utilizing a hFcRn/hFc?RI-expressing mouse model. Specifically, in Aim 1, we will identify the optimal (most protective) FP configuration and immunization regimen. The hFc?RI-targeted PspA FP or anti-hFcRn/hFc?RI-PspA FP, as well as non-targeted PspA, will be administered to wildtype, hFc?RI, hFcRn, or hFcRn/hFc?RI-expressing mice at varying doses i.n. in the presence and absence of MPLA adjuvant. Protection against Sp challenge, as well as PspA-specific T and B cell responses, and bacterial burden, will be measured. The optimal immunization regimen will be identified based primarily on superior protection, but also optimal B and T cell responses. In Aim 2, we will identify the functional mechanisms involved in the FP-enhanced immunity/protection, as well as evaluate the safety of FP vaccination. We will examine FcRn binding and transepithelial transport of FPs in vitro and in vivo, FP binding to hFcR on APCs, FP internalization by APCs, and the ability of FPs to induce DC maturation and FP- enhanced Ag presentation/T cell activation. Safety parameters to be assessed will include health following vaccination, histological changes, and FP trafficking to the brain. Importantly, the above Aims will be carried out by a uniquely qualified investigative team with extensive expertise in FcRn and Fc?RI biology, as well as vaccine development and testing. Ultimately, these studies will be the first major step in establishing this novel/innovative vaccine platform as a viable adjuvant-free approach to mucosal vaccination. Furthermore, given the strong focus of vaccine research today on adjuvant discovery, maximizing the potency of this adjuvant-free vaccine platform will be crucial in changing perceptions regarding the requirement for adjuvant.  

Project Summary To eliminate virally infected cells, cytotoxic T lymphocytes (CTLs) must recognize viral antigens that are properly displayed on the cell surface by a molecule called MHC class I. To escape the CTL-mediated killing, viruses have evolved a variety of mechanisms to dampen the antigen presenting functions of MHC class I. Interestingly, although the structure of the neonatal Fc receptor (FcRn) is very similar to the MHC class I, FcRn is unable to present antigens to T cells. In contrast, FcRn is capable of transporting IgG across epithelial cells and protecting IgG from degradation. In this way, FcRn ensures an effective and long-lasting antibody-mediated immunity after infection and vaccination. Given that FcRn and MHC class I molecules share a very closely similar structure, we hypothesize viruses must possess mechanisms to interfere with FcRn structure and function, consequently inhibiting important IgG functions during infection. By using a model pathogen human cytomegalovirus, we seek to fully investigate this possibility. Therefore, this study will potentially discover a novel immune evasion mechanism by hindering FcRn function. Consequently, this study will significantly improve our knowledge for the rational design of novel strategies for HCMV vaccine development and antibody-mediated passive immunization and immune therapy.

Project Summary Influenza A viruses directly cause acute airway inflammation or predispose to secondary bacterial infections. A highly antigenic variability is the main reason for repeated infections. Immune protection induced by current vaccines is closely strain-specific and the vaccines need to be updated each year; the availability of strain-matched vaccines usually lags behind these antigenic changes. Therefore, it is a major goal to broaden the range of influenza vaccine efficacy by using the conserved influenza virus antigens in the hope of inducing cross-protective immunity against both antigenically similar and different viruses. Since influenza virus initiates its infection at respiratory tract, it is important to induce the cross-protective and long-lasting mucosal immunity. However, our ability to safely deliver vaccine antigens across the mucosal barrier for generation of an effective mucosal immunity is very limited, especially for the elderly, young people and pregnant women. We recently found that the mucosal delivery of vaccine antigens by the neonatal Fc Receptor (FcRn) can engender effective immune responses against mucosal infections. These findings lead us to further examine whether FcRn-mediated airway delivery of the influenza antigens induces cross-protective immunity. Therefore, we will intranasally immunize mice and ferret with the conserved influenza antigens that are targeting to FcRn and fully analyze their mucosal and systemic immune responses. Finally, the immunized mice and the ferret will be challenged with virus antigenically similar or dissimilar viruses to evaluate the efficacy of protection or cross protection. The knowledge gained from this study will be important for public health in humans and animals.

The impact of SARS-CoV-2 on public health and the global economy cannot be overstated. As of September 28, 2020, 33,224,222 cases and 999,298 deaths worldwide have been linked to this emergent virus. This staggering number continues to grow, with the United States baring disproportionately high rates of morbidity and mortality. The virus targets the respiratory tract, leading to a wide range of clinical outcomes including mild upper respiratory tract illness and severe viral pneumonia with respiratory failure. To date, four SARS-CoV-2 vaccine candidates have entered phase 3 clinical trials and a massive parallel effort has been undertaken to repurpose already FDA-approved drugs for the treatment of COVID-19 or identify compounds with potential therapeutic activity. Despite this effort, remdesivir remains the only approved (with emergency use authorization) direct-acting antiviral for the treatment of COVID-19. Of critical importance: there is currently no vaccine or SARS-CoV-2-specific therapy approved for the prevention or treatment of disease. Furthermore, multiple antivirals may be required to avoid the rapid emergence of resistant SARS-CoV-2 strains. Thus, the development of novel therapeutics targeting SARS-CoV-2 are urgently needed. Infection requires interaction between the viral surface protein, spike (S), and a host protein, ACE2, that is expressed on type II alveolar cells and ciliated cells in the human airway epithelium (HAE), making these cells potentially vulnerable to infection. Thus, our goal is to develop a novel therapeutic that blocks this interaction between spike (on the virus) and ACE2 (on the host cell) to prevent infection and ameliorate disease. Aptamers are short nucleic acid-based sequences that bind with high affinity to their targets. Among other applications, aptamers have been shown to have potent antiviral activity and low toxicity in cell culture. While aptamers were originally made with RNA and DNA, Xeno-Nucleic Acids (XNA: nucleotide analogs with altered sugar, base, or phosphate backbones) have emerged as important new substrates and XNA aptamers often demonstrate enhanced target binding and greater stability compared to RNA and DNA aptamers. Thus, we hypothesize that aptamer technology, and specifically XNA aptamers, can be leveraged to inhibit spike-ACE2 interaction and propose to establish an innovative, in vitro screening platform that can serve to assess the efficacy of such aptamers, or other novel therapeutics, in blocking infection. This platform will utilize SARS-CoV-2 pseudoparticles (allowing work under Biosafety Level 2 containment) and a physiologically-relevant in vitro model of human airway epithelium that recapitulates the mucosal surface of the airway in vivo. Aptamers will also be tested using live virus infections of culture cells (Biosafety Level 3). This work is highly significant given the immediate need for novel therapeutics against SARS-CoV-2. Further, the development of a high-throughput, pseudoparticle-based assay to assess viral entry in a relevant culture system will have broad applications for additional drug screens and / or studies that aim to further understand SARSCoV- 2 virus-host interactions at the level of particle uptake.