Protein subunit vaccines are highly used today as an alternate vaccine platform to older vaccines such as live-attenuated viruses. They contain a protein antigen of the virus or bacterium that can be recognized and targeted by the immune system, and an adjuvant that amplifies the immune system response to this protein by widely putting the immune system on alert. The most commonly used adjuvants pose the risk of possible adverse reactions and are not created to target specific immune pathways, but rather stimulate general inflammation. To design a vaccine adjuvant that generates a more targeted immune response, we are using the self-assembling protein nanoparticle, I53-dn5, to display a CD40 binder that mimics the T Cell ligand, CD40L, by binding to B cell surface receptor CD40. We aim to create a particle that can replicate the binding interaction between B and T cells in the lymph node responsible for triggering antibody maturation, and B cell proliferation and differentiation. We hypothesize that this multivalent display of CD40 binder will generate potent B cell responses allowing us to respond to an antigen more effectively than current adjuvants. We are utilizing computational protein design methods like RFDiffusion, ProteinMPNN, and AlphaFold2 to optimize this display, and testing these designs in vitro for stability and ability to elicit downstream signaling effects of the CD40/CD40L interaction. This research holds two promising innovation potentials. The first is creating higher potency adjuvants by stimulating specific signaling pathways for use with protein subunit vaccines. Secondly, these materials can be used as a more stable and potent molecule in biochemical assays such as being an alternative to feeder cell lines in B cell support culture.

Despite the development of effective COVID-19 vaccines, countries with high rates of HIV infection still have limited vaccine access and inadequate access to antiretroviral medications essential for controlling HIV. Studying COVID-19 vaccination in people living with HIV (PLWH) is needed to improve vaccination strategies in immunosuppressed populations. Previously, in pigtail macaques (PTM) we showed that COVID-19 repRNA vaccination elicits durable and protective immunity against SARS-CoV-2 infection. Here we utilized the simian immunodeficiency virus (SIV)-infected PTM model of HIV/AIDS to test the hypothesis that COVID-19 repRNA vaccination is less durable during immunosuppression. Nine PTM were enrolled into the SIV naive (n=4) and experimental SIV-infected (n=5) cohorts. SIV-infected PTM were infected with 10,000 units of SIVmac293M seven weeks prior to the first vaccination. All PTM were given 2-4 COVID-19 repRNA immunizations, encoding the SARS-CoV-2 WA.1 Spike protein, to reach maximal immunogenicity and monitored for 26 weeks for vaccine durability. Vaccine recall was evaluated by administering a booster immunization after immunity responses waned. Blood and bronchoalveolar lavage samples were collected every 2-4 weeks. SIV-infected animals were monitored for SIV disease progression, including measuring CD4 counts in peripheral blood. Enzyme-linked immunosorbent assays (ELISAs) were used to quantify longitudinal Spike-specific binding IgG antibodies. Preliminary data shows that COVID-19 repRNA vaccination elicited robust anti-Spike IgG antibodies in both PTM groups, with diminished responses in some animals within the SIV-infected group.  Anti-SARS-CoV-2 neutralizing antibodies were also generated in both groups and interestingly, more durable in SIV-infected animals. Ongoing analysis includes evaluation of IgM and IgA binding antibodies. Collectively, this study suggests SIV-associated immunosuppression impacts vaccine-induced humoral memory, which could, in turn, impact long-term protection from COVID-19. These findings are crucial for improving vaccine regimens for PLWH and other immunosuppressed individuals.

Duchenne Muscular Dystrophy (DMD) is a severe neuromuscular disease caused by mutations in the dystrophin gene and characterized by progressive muscle wasting. Dystrophin protein is essential for the stabilization of muscle fibers; without it, continuous muscle damage eventually has devastating consequences in the skeletal, cardiac, and respiratory systems. While prior AAV-mediated editing strategies have been effective in targeting these mutations, there are significant immunological concerns from the uninterrupted expression of bacterial gene editing components. This project utilizes the mdx mouse model of DMD to address these concerns by developing methods to turn gene editing on and off using novel drug-responsive pA regulator vector constructs. I have been part of this project from the start, contributing to the assembly of the expression constructs using bacterial cultures and standard cloning techniques relying on restriction enzymes and high-fidelity cloning kits to piece together our editing constructs. Initial data was acquired from cell culture tests where I helped perform quantitative dual-luciferase reporter assay analyses. Subsequent in vivo experiments were performed by delivering adeno-associated viral vectors, carrying our constructs, into mdx mice via retro orbital injection. Activation of editing activity was achieved via three intraperitoneal injections of Tetracycline.  During analyses, I played a significant role in processing muscle tissues to extract proteins, DNA, and RNA for quantitative assays. Ultimately, our cell culture tests identified two lead pA regulator sequences exhibiting favorable activation levels at therapeutically relevant Tetracycline doses. Initial in vivo tests are promising, showing drug responsive editing and dystrophin expression in mice. We anticipate that our follow up tests will restore sufficient dystrophin expression and improve muscle function, without persistent editing activity. Overall, the outcomes of these studies could have significant implications for a multitude of genetic conditions amenable to genome editing and may help accelerate translation of effective methods towards clinical trials.

Viral evolution theory hypothesizes that specialist strategies increase fitness by reducing interspecific competition, while generalist viruses increase fitness by accessing multiple hosts. However, specialism may come at the cost of infecting few hosts, while generalism may reduce fitness in any single host. These tradeoffs have been demonstrated in Infectious hematopoietic necrosis virus (IHNV), an aquatic rhabdovirus infecting multiple salmonid species. High rates of viral replication have been observed for specialized subgroups in their respective hosts, while lower rates of replication across multiple hosts have been observed for the generalist subgroup. However, the host-virus mechanisms underlying these replicative differences are unknown. Here, I aim to characterize the early innate immune response of sockeye salmon, the ancestral host of IHNV, to specialist and generalist subgroups at target tissues. Specifically, I seek to test whether sockeye salmon display distinct transcriptomic responses to IHNV specialist and generalist subgroups in the kidney 2 days post-exposure (dpe). To accomplish this goal, RNA was extracted and sequenced from kidney tissue of individuals 2-dpe following exposure to specialist (n=9), generalist (n=9), or control (n=4) IHNV treatments. Overexpressed and underexpressed genes will be identified between each subgroup and control samples. These genes will then be used for pathway enrichment to compare differences in transcriptomic response. Replicative rates have shown a difference between specialist and generalist subgroups of IHNV 2-dpe in the kidney; therefore, we expect to observe differences in the number and magnitude of over- or underexpressed genes and enriched pathways between hosts exposed to specialist and generalist subgroups. Results from this study will aid in characterizing evolutionary mechanisms underlying viral specialism and generalism, understanding host innate immune response and evasion strategies, and identifying biological markers associated with response to viral exposure. This knowledge will be critical in predicting future disease outbreak and informing disease mitigation strategies.

Over a hundred years after the Spanish Flu, the influenza A virus (IAV) remains a leading cause of respiratory infections and mortality worldwide. The proliferation of IAV causes many of the symptoms associated with IAV clinical disease. However, the severity of acute lung injury (ALI) from IAV is primarily driven by the host's immune response to infection. The mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated protein kinase (ERK) signaling cascade is a highly conserved pathway that is activated during lung injury and inflammation in both rodents and humans. Two MEK isoforms, MEK1 (Map2k1) and MEK2 (Map2k2), activate downstream effectors, ERK1 and ERK2, and control critical cellular processes, including the intensity and duration of inflammatory signaling. Our prior research revealed that MEK2-deficient mice exhibited improved weight recovery and overall fitness during IAV infection, suggesting that MEK2 is a host response exacerbating ALI during IAV infection. During IAV infection, excessive pro-inflammatory cytokine production drives immune cell recruitment into the lungs, leading to collateral tissue damage that impairs organ function and exacerbates disease. We hypothesize that MEK2 enhances immune cell recruitment to the lungs, enhancing inflammation, which may occur through exuberant chemokine signaling. To investigate this, we infected MEK2-deficient and wild-type mice with mouse-adapted IAV (H1N1, PR8) and collected cells from bronchoalveolar lavage (BAL) and lung homogenates. Using flow cytometry, we found reduced immune cell recruitment, including decreased numbers of monocytes, dendritic cells, monocyte-macrophages, CD4+ T-cells, CD8+ T-cells, and B-cells. Next, we assessed levels of key chemokines known to attract monocytes and lymphocytes by measuring their gene expression in the lungs and protein levels in the BAL. Investigating MEK2’s impact on chemokine signaling will elucidate the mechanism by which MEK2 perpetuates lung inflammation and injury during IAV infection and will guide the development of future host-directed therapies for IAV-induced lung damage.