COBRE PROJECTS | CAIPP

Eastern equine encephalitis virus (EEEV) is one of the most severe arthropod-borne viruses endemic to North America, with human case-fatality rates exceeding 30% and long-term neurological sequelae in survivors. No licensed EEEV-specific medical interventions exist, highlighting a critical need to understand the mechanisms that govern viral dissemination and pathogenesis. Previous studies have suggested EEEV dissemination and viremia are modulated by enhanced interactions of the viral particle with host heparan sulfate (HS), a class of glycans ubiquitously present at cell surfaces and within the extracellular matrix. While most enhanced HS-binding arboviruses are attenuated in vivo, naturally circulating EEEV strains exhibit strong HS-binding while remaining highly virulent—suggesting a unique pathogenic mechanism.

This project aims to define the cellular mediators and molecular specificity underlying HS-dependent control of viremia in a murine model of EEEV infection. Using the attenuated, non-select agent BSL-2 Sindbis/EEEV chimeric virus to model EEEV viremia, our preliminary data indicate that HS-mediated restriction is driven by blood-filtering phagocytes. We hypothesize that distinct tissue- and cell-specific HS “codes” regulate this clearance and influence viral dissemination. In Aim 1, we will identify the specific cellular populations responsible for clearing circulating virus using fluorescently labeled virions, flow cytometry, and single-cell RNA sequencing. In Aim 2, we will define the HS binding specificity of Sindbis/EEEV virions through high-throughput HS array screening and in silico molecular modeling.

These studies will provide new insights into how tissue-specific HS moieties regulate arbovirus dissemination and inform potential therapeutic strategies targeting virus-host interactions. This work also supports the PI’s transition to independent research as a new Assistant Professor at LSU Health Shreveport with a career development plan that includes guidance from a strategic mentorship committee composed of internal and external mentors. Regular mentorship meetings, strategic career planning, and support for grant development will strengthen the PI’s research trajectory. The data generated in this project will form the foundation for a future R01 proposal focused on HS-mediated regulation of EEEV pathogenesis.

Many viruses in the Order Bunyavirales are zoonotic and pose serious public health challenges. These include rodent-borne hantaviruses and mosquito-borne Rift Valley fever virus (RVFV). Hantaviruses cause hantavirus cardiopulmonary syndrome (HCPS) in the Americas and hemorrhagic fever with renal syndrome (HFRS) in Eurasia. RVFV causes devastating abortion storms in livestock. Human infections can lead to encephalitis and hemorrhagic fever. Endemic in sub-Saharan Africa, RVFV has spread to the Middle East in the last 20 years. Climate change and deforestation are predicted to increase the incidence of these viruses in the future due to habitat overlap and expanding vector range. Gn/Gc glycoproteins are the sole viral proteins necessary and sufficient for the cellular entry of these viruses and are also the main targets of protective immune responses.

Despite significant research, no FDA-approved vaccines or therapeutics for human use exist for these viruses. Countermeasure development is, at least partly, hindered by the general requirement of Biosafety level-3 (BSL3) containment (and the Select Agent status of RVFV) for research and our limited understanding of their molecular determinants of entry and antigenicity. To enable their research at BSL2, multiple surrogate viruses carrying Gn/Gc proteins have been developed for hantaviruses and RVFV. Currently, these systems are inefficient, and their plasmid-based rescue is incompatible with a more comprehensive reverse genetic analysis of Gn/Gc. Moreover, the bullet-shaped morphology of the commonly used vesicular stomatitis virus (VSV) virions may not preserve lateral contacts (thus immunogenicity) between the Gn/Gc subunits on round/pleomorphic hantavirus and RVFV virions.

To make studying hantavirus Gn/Gc easier, we developed a self-replicating BSL2 system that carries hantavirus Gn/Gc as its structural proteins. We recently extended these findings to RVFV to generate a similar virus expressing the RVFV glycoprotein precursor (GPC). We hypothesize that this new system affords DMS analysis of the Gn/Gc’s molecular determinants of cellular entry and antigenicity. Using this system, in Aim 1, we plan to map hantavirus Gn/Gc residues critical for cellular entry and antigenicity. In Aim 2, we will characterize the entry pathways and antigenicity of RVFV GPC-expressing virus. These BSL2 systems will unearth novel information about the Gn/Gc residues critical for mediating virus infection, virus-receptor interactions, and eliciting antibody responses. These can be effective tools to screen for therapeutic antibodies and entry inhibitors. They could also serve as potential vaccine candidates.

Human cytomegalovirus (CMV) is a beta-herpesvirus that infects more than half of the US population. Although this persistent/latent infection is asymptomatic in healthy individuals, CMV causes severe systemic manifestations and is life threatening for individuals with a compromised immune system, including AIDS patients and solid organ/hematopoietic stem cell transplant patients. There is an unmet clinical need to block the transmission and achieve high levels of durable protection against the virus. The oral route serves as the CMV point of entry into the host, thus playing a pivotal role in viral spread and disease onset. As such, limiting viral infectivity, replication, shedding, and load at the point of entry is crucial to containing CMV progression as well as transmission to others.

Salivary glands represent a major site of CMV replication, and asymptomatic CMV shedding in saliva is an important source of virus in natural transmission to other hosts. Despite control of viral infection by strong NK and T cell responses, CMV replication continues in salivary glands, suggesting that the virus exploits the mucosal microenvironment to escape from immunosurveillance. However, the mechanisms by which salivary glands regulate CMV infection have been greatly understudied.

We hypothesize that salivary gland tissue-resident memory NK cells play a particularly important role in restricting CMV reactivation and transmission. To address this hypothesis, we will investigate the role of salivary gland tissue microenvironment in modulating host NK cell response and memory formation against CMV using mouse cytomegalovirus (MCMV) infection model. Given the underdeveloped body of knowledge regarding the CMV replication in salivary gland, the proposed study stands to greatly advance the field of salivary gland biology and oral immunology.

Characterizing the cellular response of bronchial epithelial cells to Whitewater Arroyo Virus

Whitewater Arroyo Virus (WWAV) has been implicated in fatal cases of hemorrhagic fever in California. However, very little is known about virus-host interaction. The proposed research will interrogate the transcriptome of virus -infected human bronchial epithelial cells to identify alternatively expressed genes and pathways. Understanding the cellular response to infection will identify viral and host proteins / pathways that could be targets for countermeasure development.

Immune checkpoint blockade (ICB) and other immunotherapies have revolutionized cancer treatment, but the non-responsiveness of most cancers to ICB-based monotherapy remains a significant problem. A major reason for the non-responsiveness of these so-called ‘cold’ tumors is that they lack an immunogenic tumor microenvironment (TME) and thus escape T-cell killing despite expressing ICB targets. How to selectively intensify the immunogenicity of the TME has been an unmet challenge. Here we propose a new approach that utilizes a new oncolytic mutant of Herpes simplex Virus (HSV)1 to increase the immunogenicity of the melanoma TME by triggering ZBP1-dependent necroptosis in the tumor mass. This new approach derives from our prior work on HSV1, which showed that this virus encodes a suppressor of necroptosis: the RIP homology interaction motif (RHIM) within the viral protein ICP6. Mutating this RHIM generates a virus (HSV1mutRHIM) which can no longer block necroptosis, allowing for the first-time exploitation of HSV1-induced necroptosis for therapeutic purposes. HSV1mutRHIM activates ZBP1-dependent necroptosis from the nucleus both human and mouse cells, and such so-called ‘nuclear necroptosis’ is even more immunogenic than conventional (i.e., cytoplasm-initiated) necroptosis, because it results in the release of nuclear DAMPs, such as HMGB-1, IL-33 and Il-1a, into the extracellular space. These findings allow us to propose the hypotheses that by inducing Z-RNAs formation and directly activating ZBP1 to trigger nuclear necroptosis in cells of the TME, this modified oncolytic HSV1mutRHIM will greatly improve ICB treatment outcomes.

Despite ongoing efforts to improve vaccines and therapies, the incidence of infectious respiratory diseases (RDs) has been increasing and remains a major cause of morbidity and mortality worldwide. The RD pertussis is on the rise even though an FDA approved vaccine is widely administered. The whole cell vaccine introduced in 1945 drastically decreased the number of whooping cough cases, but due to undesirable side effects was replaced with an acellular vaccine in the 1990’s. The acellular vaccine, though safer, is less effective at blocking colonization and exhibits rapidly waning immunity. Our long-term goal is to use the murine model of Bordetella bronchiseptica to gain mechanistic insights into respiratory immune responses that promote T-cell differentiation and provide durable immunity to RDs. We hope to leverage our findings toward the development of improved vaccines. Eosinophils contribute to gastric immune homeostasis24-31 particularly during helminthic infections. Their potential involvement in the generation of adaptive immune responses in gut infections is gaining attention. However, the underlying mechanism by which eosinophils modulate T cells responses remains unclear. Our data reveal that eosinophil recruitment in the lungs coincides with peak levels of B. bronchiseptica (BB) in lungs of experimentally infected mice and remained high until the infection is cleared, suggesting that eosinophils contribute to clearance of BB from the lower respiratory tract. Mice lacking eosinophils fail to clear BB from the lungs supporting a role for eosinophils in BB clearance. Moreover, they have reduced production of proinflammatory cytokines such as IL-17 and anti-Bordetella antibodies, suggesting that eosinophils promote protective Th17 responses and subsequent antibody production during Bordetella infection. In more general terms, eosinophils may be important for the generation of a protective adaptive immunity during respiratory bacterial infections. Thus, we hypothesize that eosinophils present antigen and promote Th17 T cell responses in the lung during respiratory infections. Understanding how eosinophils modulate adaptive responses may open up new avenues for the development of therapies and more efficacious vaccines by enhancing eosiniphilic activity.

The betaproteobacteria Neisseria gonorrhoeae (N.g) is a highly adapted human colonizer and the etiological agent of gonorrhea, a sexually transmitted disease (STD) that has emerged as a major global public health problem. Rising incidence, coupled with prevalent and increasing antibiotics resistance highlights the need to understand better the molecular basis of N.g pathogenesis and the need for new therapeutic targets. N.g mainly colonizes the genital mucosa, but it can also colonize the ocular, nasopharyngeal and anal mucosa. Pathology largely results from the damage caused by the activation of innate immune responses at the sites of bacterial colonization3. Ascending gonococcal infections frequently occur, particularly in women. The most significant morbidity and mortality caused by N.g is due to pelvic inflammatory disease and the post-infection complications associated with scarring of the fallopian tubes. Antibiotic resistance is a substantial problem due to high frequency acquisition of genetic material from the environment. Following the spread of fluoroquinoloneresistant and cephalosporin-resistant N.g there are very few antibiotic options left that are well-studied, well-tolerated and highly effective. The first step in N.g pathogenesis is adherence of bacteria to the host epithelium, a process mediated by type IV pili, opacity (Opa) proteins, lipooligosaccharide (LOS) and the major outer membrane protein porin (also known as PorB). Specifically, the interaction between N.g Opa proteins and the host cell CEACAM (carcinoembryonic antigen-related cell adhesion molecule) family of proteins have been considered crucial for both colonization and immune system evasion. The initial phases of N.g infections and its interactions with mucosal epithelium and neutrophils (PMNs) have been extensively studied. Neutrophils, however, are short-lived cells (~6-8h half-life) and infection with N.g can only briefly delay the onset of PMN6,7 apoptosis. On the other hand, our and other studies show that N.g can strongly replicate and avoid phagocytic killing in macrophages, which allows bacterial growth for more than 24h. Wefurther characterized N.g invasion and cell-to-cell spread in macrophages and uncovered that: (1) N.g replicates and propagates efficiently when in contact with human macrophages; (2) extracellular and intracellular growth are both employed by N.g when infecting macrophages; (3) only a small number of intracellular N.g are targeted to the lysosomes (LAMP2/Rab7 positive) for degradation and (4) actin polymerization is important for N.g infection in macrophages (see Model). Thus, macrophages are an unexplored and potentially essential cellular reservoir for N.g. during infection that can serve as a significant replicative niche in vivo. We hypothesize that N.g colonization and replication within macrophages plays an important role in gonorrhea pathogenesis. Our findings present an opportunity for us to explore macrophage-centered therapies that can be exploited to steer gonorrhea towards outcomes that favor the host. However, determining the mechanisms used by N.g to colonize and replicate in macrophages is critical for this understanding of the pathogenesis of the disease and for the design of new therapies.

Neurodevelopmental disorders characterized by social deficits, e.g., autism and schizophrenia, have long been associated with immune dysfunction. However, largely due to the lack of an etiologically-relevant animal model, the causal evidence needed to link immune dysfunction to neurodevelopmental disorders in humans remains elusive. Several recent large-scale genome-wide association studies (GWAS) pinpointed a Copy Number Variation (CNV) at the chromosomal locus 7q36.6 that is highly represented in schizophrenia and autism1. All of the microduplications (triplications) occur within a single gene: vasoactive intestinal peptide receptor 2 (VIPR2). In lymphocytes from these patients, VIPR2 is overexpressed by two-fold. In children with autism, the VIPR2 ligand, vasoactive intestinal peptide (VIP) is found to be almost three times higher in the blood at birth. Importantly, VIP has been viewed as a Th2-secreted cytokine. Activation of VIP/VIPR2 signaling is well known to shift the Th1/Th2 balance in favor of Th2 cells. To link T cell immune dysfunction to neurodevelopment and social deficits, we generated a Bacterial Artificial Chromosome (BAC) transgenic mouse model of human VIPR2 CNV that allows switching-off of the transgene in desired spatial-temporal patterns, controlled by the Cre recombinase, thus facilitating dissection of the afflicted cell populations. These mice had robust social and cognitive behavioral deficits that were preceded by a disrupted early postnatal brain development, such as delayed microglia maturation. Autism has been associated with an altered Th1/Th2 function that is skewed in favor of a Th2 response. Activation of VIP/VIPR2 signaling in T cells leads to Th2 differentiation, which in turn would inhibit Th1 responses. Cytokines released by Th1, but not Th2 cells, are required to stimulate resident microglia to maturation. Taken together, we argue that overactive Th2 polarization impairs the maturation of microglia and disrupts social brain development, while low levels of Th1 cytokines (e.g., IFN-γ) suppress normal social behavior (see the Figure above for our model). Therefore we hypothesize that VIPR2 overexpression in T cells promotes the Th2 polarization that disrupts early brain development and triggers social behavioral deficits.

Defining the cellular regulators of EEEV viremia

Eastern equine encephalitis virus (EEEV) is a mosquito-borne virus that causes severe, and sometimes fatal, disease in humans. This project will identify the specific blood-filtering immune cells that control how EEEV spreads through the body, which could lead to new strategies for preventing or treating this deadly disease.

DEFINING A NOVEL FUNCTION FOR SALIVARY EXOSOMES IN MODULA TING HOST IMMUNITY AGAINST CANCER

CMV has infected more than half of the US population and causes systemic and oral diseases. This study tests the hypothesis that salivary gland tissue-resient NK cells play a particularly important role in restricting CMV reactivation and virus transmission. Outcome of the proposed study may uncover novel targets and strategies for immunotherapeutics to reduce the risk of virus transmission and alleviate the diseases.

Characterizing the cellular response of bronchial epithelial cells to Whitewater Arroyo Virus

Whitewater Arroyo Virus (WWAV) has been implicated in fatal cases of hemorrhagic fever in California.
However, very little is known about virus-host interaction. The proposed research will interrogate the
transcriptome of virus -infected human bronchial epithelial cells to identify alternatively expressed genes
and pathways. Understanding the cellular response to infection will identify viral and host
proteins/pathways that could be targets for countermeasure development.

Role of Host glutamine synthesis in tuberculosis pathogenesis

Tuberculosis is a serious lung infection that still kills over a million people each year, partly because
current treatments take a long time and can cause harmful side effects. This study will investigate whether
blocking a key metabolic pathway in immune cells can enhance the body's ability to fight infections more
effectively. If successful, it could lead to faster, safer treatments for people with TB.

Dr. Parveen-Nesson was awarded the the NIH-NIAID Director's New Innovator Award (DP2) # DP2AI200598, at a total amount of $2.2 million.

Poxviruses co-opt ribosomes to promote viral protein synthesis and subvert the innate immune response.

Poxviruses are a family of DNA viruses that can infect a wide range of hosts and cause significant diseases. Notably, the recent emergence of monkeypox represents a global health concern highlighting the ongoing challenges in managing zoontoic diseases. As such, understanding how these viruses replicate is of considerable importance for the development new treatments to combat future zoonotic poxvirus outbreaks. Unlike other DNA viruses, a hallmark of poxviruses is they replicate exclusively in the cytoplasm of the infected cells. This poses a unique challenge for a DNA virus. Cells have cytoplasmic nucleic acid sensors that detect foreign DNA and trigger interferon (IFN) signaling, which upregulates interferon-stimulated genes (ISGs) to suppress viral replication. While antagonizing the innate immune system is one of the most studied areas in poxvirus biology, an often-overlooked aspect is how poxviruses shut down production of antiviral proteins altogether by interfering with host translation. How poxvirus can shut off host translation while promoting viral protein synthesis is still not well understood.

To better understand the mechanism of how poxvirus hijacks the host translational machinery, we analyzed the composition of ribosomes via liquid chromatography tandem mass spectrometry (LC-MS/MS) during a poxvirus infection. We identified new putative interactions with viral proteins and host ribosomes, which suggest potential unappreciated new functions of previously characterized and uncharacterized viral proteins. In addition, we also identified several host proteins involved in the ribosomal quality control pathway. Lastly, we have identified numerous ribosomal proteins that are uniquely post-translationally modified during viral infection. Together, we hypothesize that poxvirus hijacks host ribosomes via specific interactions with viral proteins by inducing post-translational modifications and usurping ribosome quality control factors to enhance viral protein synthesis while shutting off host translation to negate the innate immune response

RIPK3-caspase 8-mediated apoptosis restricts herpes simplex encephalitis

HSV-1 can potentially cause fetal herpes simplex encephalitis (HSE). Receptor-interacting protein kinase 3 (RIPK3)-mediated cell death is a critical innate immune response for restricting virus replication. However, studies of RIPK3-regulated antiviral defense in the central nervous system (CNS) have not been fully conducted. Our pilot project aims to understand the role of RIPK3 in the CNS to improve therapies.

Under this project, we have demonstrated that RIPK3 and its signaling partner caspase 8 are crucial for restricting HSV-1 invasion within the CNS. RIPK3-caspase 8-regulated apoptosis, as well as neuroinflammation in the CNS, are important for controlling viral growth and maintaining normal interaction between microglia and astrocytes during HSV-1 infection. This suggests that enhancing functional RIPK3 in HSE patients may contribute to better outcomes.

Role of Interleukin-4 Signaling in Stimulating Neurogenesis and Functional Recovery Following Cerebral Ischemia

Stroke is a destructive neurological disease that is one of the greatest contributors to global disability, and millions of stroke survivors live with life-changing neurological consequences. Ischemic damage to the brain and loss of neurons contribute to functional disabilities in many stroke survivors. Recovery of neuroplasticity is critical to restoration of function and improved quality of life. Stroke and neurological deficits occur in both adults and children, and yet it is well documented that the developing brain has remarkable plasticity which promotes increased post-ischemic functional recovery compared with adults. However, the mechanisms underlying post-stroke recovery in the young brain have not been fully explored. We observed opposing responses to experimental cerebral ischemia in juvenile and adult mice, with substantial neural regeneration and enhanced neuroplasticity detected in the juvenile brain that was not found in adults. Further, we identified early immune responses during the acute phase of stroke as a potential mechanism of endogenous neuronal replacement and subsequent functional recovery. This finding is contrary to most reports of inflammatory signaling in adults during the acute phase after stroke, which is generally seen as deleterious to neuronal survival. We found strikingly different stroke-induced neuroimmune responses during the acute phase of stroke that are deleterious in adults and protective in juveniles, supporting neural regeneration and plasticity. Understanding these age-related differences in neuronal repair and regeneration, restoration of neural network function, and neuroimmune signaling in the stroke-injured brain may offer new insights for the development of novel therapeutic strategies for stroke rehabilitation in children and adults alike.