Regulation of innate immunity through intracellular and extracellular vesicle (EV) trafficking, Molecular basis of coordinated host signaling response to pathogens, Development of novel therapeutic and vaccine strategies. 

Intercellular Vesicle Transport (EV Exchange) and Regulation of the Innate Immune Response: 

Recent work in the EV field has demonstrated the critical importance of their communicative functions for a variety of human diseases, including several infectious diseases. A main focus of our lab is to understand the role of host small extracellular vesicles (sEV) during infection with pathogenic bacteria or viruses, including Yersinia pestis (Yp), Burkholderia pseudomallei (Bp), Rift Valley fever virus (RVFV), and SARS-CoV-2 [e.g., 1-4]. Several are classified as highest priority (Category A) pathogens, and there are no approved human vaccines or highly effective therapeutics currently available for Yp, RVFV, and Bp. Our lab has developed and optimized methods for purification of host sEV released from either bacterially infected or virally infected cells (which we designate as EXi), and characterized the preparations using EV-specific markers, Zetaview analysis, and confocal and electron microscopy studies. We have demonstrated that EXi released from RVFV-infected cells (EXi-RVFV) carry viral genome and proteins and can activate specific anti-viral responses in both immune and non- immune cell types through viral genome that is packaged inside. Thus, cells pre-treated with purified EXi-RVFV have a drastically reduced capacity for virus production, suggesting that EXi-RVFV serve a protective role for the host [1]. We have shown that this occurs through RIG-I dependent activation of IFN-B in recipient cells that in turn activates autophagy. The future goal is to further characterize the molecular mechanisms underpinning these effects and transition to in vivo analysis of EXi-RVFV function. 

Our lab has also developed a mechanistic model of how the EXi released during Yp or Bp infection regulate the innate immune response. We have shown that these EXi carry cargo that is distinct from sEV purified from uninfected cells (EXu) [4]. In addition, we have demonstrated that, similar to the EXi released from virally infected cells, treatment of naïve recipient cells with these EXi provides significant protection against infection. For both Yp and Bp infections, we have teased out the molecular mechanisms that get engaged to provide this protection, leading to development of a model of EXi function during infection with Gram-negative bacteria. We are currently transitioning to in vivo studies of the EXi effects for further validation and enrichment of this model. 

Development of Novel EV Technologies:

In collaboration with the Department of Bioengineering, our lab has been developing novel technologies for the EV field that will provide important investigational tools for EV studies. For example, we have developed a unique microfluidic chip-based approach for real-time monitoring of cellular EV exchange between physically separated cell populations [5]. The extracellular matrix (ECM)-mimicking Matrigel is used to physically separate cell populations confined within microchannels, and mimics tissue environments to enable direct study of EV function. The submicron effective pore size of the Matrigel allows for the selective diffusion of only sEVs, in addition to soluble factors, between co-cultured cell populations. Furthermore, the use of PEGDA hydrogel with a very small pore size of 1.2 nm in lieu of Matrigel allows us to block EV migration and, therefore, differentiate EV effects from effects that may be mediated by soluble factors [5]. This versatile platform bridges purely in vitro and in vivo assays by enabling studies of EV-mediated cellular crosstalk under physiologically relevant conditions, enabling future EV investigations across multiple disciplines through real-time monitoring of vesicle exchange. 

Intracellular Vesicle Transport and Regulation of the Innate Immune Response: 

Our lab has used a novel reverse phase protein microarray (RPMA) platform to quantitatively survey intracellular protein pathway modulation across the cell in response to infection with Yp, followed by ongoing functional studies of select pathways that emerged from the analysis. These studies demonstrated the importance of a number of host cell pathways during Yp infection that had not been previously known to contribute (12 new protein hits), and also led to the first demonstration that host response to Yp involves a coordinated down-regulation of autophagy [6]. This inhibition involves modulating the activities of multiple proteins, including AKT, AMPK, and p53. Complementing the RPMA approach, we have also performed quantitative mass spectrometry analysis of highly purified CD14+ primary human monocytes to measure host phosphorylation changes during infection. This work confirmed the importance of several pathways identified through RPMA, including the AKT pathway, and also highlighted a potential role for a number of specific AKT interacting proteins. We are currently assessing the respective contributions of the different pathways that we have identified to the inhibition of autophagy during Yp infection, and are transitioning to in vivo analysis of the role of autophagy as part of the host response to infection. 

Additional Host Response Studies:

Our lab also has focused host response projects that complement our main areas of research. As an example, we have profiled host factors that play significant roles during infection with RVFV, including the demonstration that several specific heat shock proteins such as HSP90 are critical for viral production [7]. Specifically, we have shown that host HSP90 stabilizes the RNA-dependent RNA polymerase of RVFV to allow viral transcription and replication. As several HSP90 inhibitors are already in Phase II or III clinical trials for cancer treatment, these findings suggest the exciting possibility of repurposing them to treat RVF.  

Development of Novel Effective Vaccines or Therapies:

We have ongoing collaborations with both academic and industry laboratories to develop and assess novel vaccine platforms or therapeutic strategies for infectious diseases of concern that are in need of better countermeasures. For example, in collaboration with Integrated Biotherapeutics, Inc. and the laboratory of Professor Daniel Nelson at the University of Maryland, we developed a novel and highly successful immunotherapeutic platform for treatment of bacterial infections. Through our work, we have shown that this novel platform technology is broadly applicable to several toxigenic bacterial pathogens. This project was funded through both STTR Phase I and Phase II NIH grants, and is currently under further development by us for reaching the investigational drug (IND) stage.