COBRE PROJECTS | CAIPP

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.

Hantaviruses are the causative agents of hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS) in Americas and Eurasia, respectively. These viruses are usually transmitted from their rodent reservoirs to human. However, multiple incidents of person-to-person transmission of a South American Andes hantavirus raises significant public health concern about these deadly viruses with up to 40% case fatality rates. No FDA-approved hantavirus vaccines and therapies exist. Cellular entry and infection of hantaviruses is mediated by its virion surface Gn/Gc glycoproteins, which are also the main target of protective immune responses. However, our understanding of the molecular determinants of Gn/Gc in hantavirus entry and antigenicity remains limited, at least partly, due to the general requirement of Biosafety level-3 (BSL3) containment for hantavirus research and the lack of a reverse genetics system. To address these limitations, multiple BSL2 pseudovirus systems have been developed. However, current BSL2 systems are limited by poor scalability and limited diversity of the generated viruses due to their inefficient plasmid-based rescue, which makes them incompatible with a more comprehensive analysis of the biology and function of the entry glycoproteins. Here, we propose to fix this major shortcoming of the hantavirus field by developing a novel BSL2 system that allows comprehensive reverse as well as forward genetic analysis of Gn/Gc’s role in virus entry and antigenicity.

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.

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.

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.

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.

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.

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