Research

The Host-Pathogen Biology Group

The Host-Pathogen Group is a diverse group of scientists with expertise in microbiology, virology, immunology, and bacterial pathogenesis. Scientists in this group conduct research on host-pathogen interactions with a focus on biothreat viruses and bacteria. Projects include studies of host immune responses during infection using a combination of in vitro and in vivo approaches, vaccine and therapeutic development with an emphasis on broad-spectrum efficacy, viral evolution and cross species transmission, and pathogen characterization and survival in the environment.

Research Areas

Nanoparticle mediated immune modulation for infectious disease therapies

Contact: Amy Rasley

Nanoparticles enhance the afficay of innate immune agonists.  Therapies targeting the innate immune system have the potential to provide transient, non-specific protection from a variety of infectious organisms. We have employed a nanolipoprotein (NLP) platform to enhance the efficacy of synthetic innate immune agonists. NLPs are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. We demonstrate that the synthetic Toll-like receptor (TLR) ligands monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG ODNs) can be readily incorporated into NLPs. Further, conjugation of both MPLA and CpG ODNs to NLPs significantly enhances their immunostimulatory profiles in vivo compared to administration of agonists alone. Specifically, conjugation to NLPs promotes agonist trafficking to secondary lymphoid organs, enhances and prolongs stimulation of innate immune cells, significantly increases cytokine production, and enhances upregulation of myriad immunoregulatory genes in vivo. Importantly, enhancement of cytokine production by agonists conjugated to NLPs was also observed in primary human dendritic cells. Strikingly, BALB/c mice pretreated with NLP-CpG constructs prior to a lethal influenza challenge were protected from weight-loss induced death, whereas pretreatment with CpG alone had no effect on survival. Taken together, these data suggest that NLP:agonist conjugates potentially represent a novel therapeutic measure against emerging pathogens.

 

 

 

 

 

 

 

 

 

 

 

Figure 1. Schematic representation of how synthetic pathogen-associated molceular patterns (PAMPs) can be incorporated into NLPs to mimic those signals encountered during a natural infection with a pathogen to enhance innate immune responses.

 

        

 

 

 

 

 

 

 

 

Figure 2. BALB/c mice were treated with 10 μg NLP-CpG, CpG or NLP or were left untreated 24 hrs prior to infection with 100 EIU of A/PR/8/34 influenza. Weight loss is shown over time.

 

Biologic nanolipoprotein particles (NLPs) containing monophosphoryl lipid A as a novel intranasal vaccine platform.

Subunit vaccines are theoretically safe and easy to manufacture but require effective adjuvants and delivery systems to yield protective immunity, particularly at critical mucosal sites such as the lung. We investigated nanolipoprotein particles (NLPs) containing the Toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) as a platform for intranasal vaccination against Bacillus anthracis. Modified lipids enabled attachment of disparate spore and toxin protein antigens. Intranasal vaccination of mice with B. anthracis antigen:MPLA:NLP constructs vs. delivery with free MPLA induced robust IgG and IgA responses in serum and in bronchoalveolar and nasal lavage. Typically, a single dose sufficed to induce sustained antibody titers over time. When multiple immunizations were required, specific antibodies were detected earlier in the boost schedule with MPLA:NLP-mediated delivery than with free MPLA. Administering combinations of constructs induced responses to multiple antigens, indicating potential for a multivalent vaccine preparation. No off-target responses to the species-matched NLP scaffold protein were detected. Using the ovalbumin/TCR transgenic model, MPLA-NLPs enhanced antigen-specific T cell expansion in lung-draining lymph nodes. In summary, the NLP platform enhances cellular, humoral, and mucosal responses to intranasal immunization, indicating promise for NLPs as a flexible, robust vaccine platform against B. anthracis and potentially other inhalational pathogens.

 

 

 

 

 

 

 

Figure 3. Conjugation of PA to adjuvanted NLPs significantly enahnces antigen specific antibody responses in BALB/c mice.

 

Identification and characterization of novel Francisella tularensis proteins

Contact: Amy Rasley

Structure and function of REP34, a carboxypeptidase from F. tularensis.

We recently identified seven novel F. tularensis proteins which we hypothesize play a role in environmental persistence through their interactions with free-living amoeba. Importantly, our efforts to characterize these novel proteins indicate that they may also function as virulence factors potentially targeting host structural proteins. Fully virulent F. tularensis isolates infect the free-living amoeba, Acanthamoeba castellanii,  causing the amoebae to rapidly encyst. Further characterization led to the identification of seven putatively secreted F. tularensis  proteins that may be responsible for this rapid encystment phenotype (REP) in amoeba. We characterized the cellular and molecular function of a REP protein with a molecular mass of 34 kDa (REP34), to better understand the biological role of REP in F. tularensis  infection and environmental persistence. Computational analysis classifies REP34 and related proteins as a non-peptidase homologue of the M14 family. Given the observation that putatively secreted F. tularensis  proteins induce the REP phenomenon, which is required for long term survival of the pathogen within amoeba, and the implications of other M14 enzymes in virulence, we hypothesized that REP34 does indeed possess an important, yet unknown function. As such, we sought to obtain a three-dimensional model of REP34 at atomic resolution to aid in devising experiments to test the function and ultimately may provide insights into the role of REP34 during interactions with phagocytic cells.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1. Substrate binding pocket comparison for REP34 and related carboxypeptidases.

 

Recovering viral population diversity to improve biosurveillance and predict viral emergence

Contact: Monica Borucki

Illumina ultra deep sequencing (UDS) of viral genomes.

My research utilizes UDS of viral genomes to define the role of viral population diversity in the evolution of viral virulence and emergence. Our focus is on viruses that belong to viral families that frequently jump to new host species. These efforts, outline schematically in Figure 1 below, include a  multidisciplinary team combining virology, bioinformatics, and protein modeling expertise to define intra-host viral population structure and identify mutations that may impact pathogen phenotype.

Figure 1. Schematic outlining our approach to utilize UDS for viral genomes.

Amoeba cysts as natural containers for the transport and enrichment of microbial pathogens

Contact: Sahar El-Etr

Exploiting amoeba-microbial pathogen interactions for the development of novel fieldable transport and storage containers

My research interests focus on studying host-pathogen interactions of Tier 1 Select Agent bacteria with mammalian cells and envrionmental amoeba, with a special focus on applications for development of medical countermeasures. Our work is currently funded by DOD/DTRA.

Figure 1. Fluorescence microscopy showing the intracellular pathogen Burkholderia pseudomallei (green) interacting with the environmental amoeba, Acanthamoeba castellanii (red).