Nucleic acid vaccines introduce DNA or mRNA into cells in vivo, instructing them to express antigens from a pathogen resulting in the induction of immune responses that can provide long term protection from that pathogen. They provide many advantages over traditional vaccines including lower cost, improved safety, and the possibility to rapidly update the vaccine since only the genetic sequence of a new variant is required. One drawback of DNA vaccines has been their relatively poor immunogenicity compared to traditional vaccines which has been overcome, to some extent, by using improved delivery methods and co-formulation with plasmids expressing cytokines as adjuvants. Previous studies have established IL-12, as the “gold standard” genetic adjuvant due to its ability to support differentiation of antigen specific CD4+ T cells to produce Th1 cytokines as well as expansion of antigen specific CD8+ T cells to be more cytolytic in vivo. There is growing interest in identifying other adjuvants that not only increase immunogenicity of DNA vaccines but also modulate the types of responses they induce. In this study, we sought to determine if co-administration of an adjuvant cocktail including IL-18, a pro-inflammatory cytokine, and IRF7, a transcriptional activator of type I interferons, along with IL-12 would enhance antibody responses to DNA vaccines expressing SIV and Influenza antigens in a preclinical nonhuman primate model. Plasma samples were collected at different times post vaccination and the effect of the adjuvants on immunogenicity was measured via IgG ELISA and analyzed. After 2 vaccinations, we observed a significant increase (P=0.0272) in antibody responses against SIV gp130 in the adjuvant cocktail group compared to the IL-12 group. These results indicate that combining adjuvants could provide further improvement in DNA vaccine immunogenicity. Additional studies to determine the impact of this adjuvant cocktail on T cell responses are in progress.

The gut microbiome is a critical modifier of the host xenobiotic (foreign particle) biotransformation and is important in modulating the reduction and hydrolysis reactions of drugs and other chemicals. The Nuclear factor erythroid 2-related factor 2 (Nrf2) is a major oxidative stress sensor of the host and is highly conserved during evolution. Nrf2 is highly expressed in multiple important metabolic organs including the gastrointestinal tract. Following activation, Nrf2 detoxifies harmful free radicals and electrophiles, maintaining a reducing environment. Genetic variations of Nrf2 within the human population have been linked to cancer risks. My goal is to test the hypothesis that the Nrf2 genotype impacts gut microbiome as an additional mechanism to regulate the susceptibility to certain host diseases. Fecal microbial DNA was isolated from adult male wild type(MWT), female wild type(FWT), male Nrf2-knockout(MKO), and female Nrf2-knockout(FKO) mice (n=5 per group) using the EZNA stool kit. Amplified ribosomal DNA sequences used to identify specific bacteria (16S rDNA V4 libraries) were sequenced using the Illumina second generation sequencing platform(250bp paired-end). Data were analyzed using QIIME. Statistical significance was determined by two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test(p < 0.05) using the multcomp (multiple comparisons analysis) package in R. Alpha Diversity showed that the richness of the fecal microbiome was lower in female than male mice in a Nrf2 dependent manner, and Nrf2 deficiency normalized the gender difference. In male mice, both the upregulation of the secondary bile acid-generating Clostridium and the downregulation of the carbohydrate metabolizing Rumminococcus flavefaciens were Nrf2-dependent. In female mice, the pro-inflammatory Rumminococcus gnavus was up regulated in the absence of Nrf2. In conclusion, the presence of the host Nrf2 gene is important for gender-divergent richness of the gut microbiome, as well as predicted microbial functions including inflammation, secondary bile acid synthesis, and carbohydrate metabolism.

Proprotein convertase subtilisin kexin 9 (PCSK9) is a circulating protein which binds with the low-density lipoprotein cholesterol receptor (LDL-R) and reduces the elimination of LDL-cholesterol from blood. Thus, PCSK9 inhibition has been used as a therapeutic strategy to treat cardiovascular disease. However, PCSK9 inhibition increases the expression of LDL-R by hepatocyte and is raising the hepatic load of cholesterol which is associated with the subsequent liver injury. Bile acids (BAs) are major cholesterol metabolites that serve as important signaling molecules for nutrient homeostasis under physiological levels and contribute to the hepatotoxicity at exceedingly high concentrations. The aim of this study is to investigate the role of PCSK9 inhibition in the development of liver toxicity through the accumulation of toxic bile acid profiles in high fat high cholesterol diet (HFHC). We quantified the liver BA profile (LC-MS) and BA-processing gene expression (RT-qPCR) from both WT and PCSK9-null mice fed a HFHC diet (0.75%) for 9 months (n=6-12 per group). In livers of WT mice, high cholesterol diet increased the total hepatic BAs, whereas such increase was further significantly amplified (p<0.05) in livers of PCSK9-null mice. High cholesterol diet down-regulated the major basolateral BA-uptake transporter Ntcp (p<0.001) and the BA-conjugation enzyme Baat (p<0.05). In livers of PCSK9-null mice, high cholesterol diet further decreased the expression of Ntcp (p<0.05) and also decreased the expression of the major canalicular BA-efflux transporter Bsep (p<0.05). The augmented BA increase in livers of PCSK9-null mice may be due to a decrease in Bsep expression, leading to BA hepatic accumulation. In summary, the present study showed that PCSK9 deficiency leads to an increased accumulation of hepatic BAs following a high cholesterol diet, and this may be explained by reduced hepatic efflux. This is important to further understand the impact of HFHC diets compounded with PCSK9 drug treatments.

In response to various forms of intrinsic and extrinsic stresses such as heat shock, electrical stimulation, and viral infection, cells produce non-membrane-bound aggregates of mRNA and proteins called stress granules. These granules sequester mRNA and ribosomal subunits to halt the production of proteins unnecessary for the immediate survival of the cell, thus allowing more energy to be used in combatting the stress. Stress granules are beneficial in the short term, but the chronic presence of stress granules can be cytotoxic. If stress granules are not cleared, hyperaggregation of misfolded proteins, which is thought to play a role in neurological diseases, can occur. After myocardial infarction (heart attack), the heart experiences a lack of oxygen which is known to create free radicals and metabolic stress. Whether the stress response is involved in this process is unknown, as most research on stress granules, especially their role in disease, comes from work in neuronal and cancer cells. To test whether the stress granules response is conserved across cell types and how cardiomyocytes (heart muscle cells) specifically respond to stress, I cultured cancer cells, embryonic stem cells, and embryonic stem cell-derived cardiomyocytes and subjected these cells to various forms of stress, including sodium arsenate poisoning and heat shock. Using fixed immunofluorescence and spinning disk microscopy, I imaged each treatment and quantified the number of stress granules per cell. The sodium arsenate treatment induced stress granule formation in all three cell types, but surprisingly, the heat shock treatment only induced stress granule formation in the stem cells. It is widely believed the stress response is conserved across a wide range of cell types, but these results indicate some stress pathways differ between cardiomyocytes, cancer cells, and stem cells. Future experiments will test additional types of stress and how stress granules contribute to cardiomyocyte function.

Many advancements have been made in differentiating human pluripotent stem cells to cardiomyocytes (hPSC-CMs), with respect to obtaining large numbers with high purity. However, a limitation of hPSC-CMs is that they have an immature phenotype and behave like fetal cells. This immaturity limits the application of cardiomyocytes for cell transplantation that would help repair the heart after myocardial infarction. Our lab has identified candidate master transcriptional regulators of cardiac maturation. These regulators have low expression in immature hPSC-CMs and high expression in mature cardiomyocytes. My project goal is to test the role of PPARG coactivator 1 beta (PGC1B), a candidate transcriptional regulator, in regulating hPSC-CM maturation. PGC1B is involved in mitochondrial biogenesis and increases number of mitochondria, which are highly abundant in adult cardiomyocytes. I hypothesize that activating transcription for PGC1B will enhance maturation of hPSC-CMs through mitochondrial biogenesis. To upregulate gene expression, we use a CRISPR activation (CRISPRa) system with a modified version of Cas9 fused to a transcriptional activator VPR (dCas9-VPR) to upregulate transcription of target genes upon introduction of a specific guide RNA (gRNA). I have differentiated WTC11 stem cells into cardiomyocytes, introduced dCas9-VPR and gRNAs for PGC1B via lentivirus, and performed measurements after 2 weeks. I validated the increased expression of PGC1B at the RNA (using quantitative reverse transcriptase PCR) and protein levels (western blot). To assess relative abundance of mitochondria in PGC1B-expressing versus control hPSC-CMs, I will label mitochondria with MitoTracker and quantify using flow cytometry and microscopy. From these experiments, I expect that PGC1B-overexpressed hPSC-CMs would have a higher relative abundance of mitochondria and increased expression of metabolic and maturation genes compared to control hPSC-CMs. My findings will provide insight on the role of PGC1B in mitochondrial biogenesis and stem cell-derived cardiomyocyte maturation.

Human Immunodeficiency Virus (HIV), the causative agent of AIDS, is a retrovirus that integrates into the host genome. Currently HIV infection is incurable; however, antiretroviral therapy (ART) can suppress HIV viral load and transmission by inhibiting viral replication. HIV-infected cells have a decreased survival advantage because viral proteins cause cytopathic effects and clearance by the immune system. However, it has been observed that HIV infected cells persist even when individuals are on long-term ART. HIV usually integrates into actively transcribed genes, and recent data revealed HIV integrations are sometimes clustered in the intronic regions of STAT5B and BACH2. These proviruses were biased to integrate in the same orientation of the gene and were found to initiate HIV-host hybrid transcript formation through promoter insertion. Both genes have associations with cell proliferation, function and development of T-cells, and cell survival. Thus, integration into these specific sites may influence cell persistence in patients. To investigate HIV persistence in vivo, we studied the prevalence of BACH2 and STAT5B hybrid transcript in participants from the Sabes primary infection cohort. Peripheral blood mononuclear cell (PBMC) aliquots from 44 participants were used to select for CD4+ T-cells, from which RNA was extracted and used to generate cDNA. Multiplexed PCR was then used to detect single-copy BACH2 and STAT5B hybrid transcripts. The products were resolved by gel electrophoresis and confirmed by Sanger sequencing. Preliminary results show that 18/44 individuals (41%) had detectable BACH2 hybrid transcripts and 30/44 (68%) had STAT5B hybrid transcripts, with 16/44 (36%) positive for both. A majority of these individuals showed detectable BACH2 and STAT5B hybrid transcripts within the first year of HIV acquisition and persisted to at least 4-5 years from HIV acquisition. Since these genes are important regulators of T cell function, targeting these cells for elimination may be important for HIV cure strategies.