COMPARATIVE TRANSCRIPTOMIC ANALYSIS OF RICKETTSIA CONORII DURING IN VITRO INFECTION OF HUMAN AND TICK HOST CELLS

Jessica Alsing (B.S)¹, Michelle Montini (B.S)¹, Kamil Khanipov (Ph.D)², Sanjeev K. Sahni (Ph.D)¹, Abha Sahni (Ph.D)¹, Hema P. Narra (Ph.D)<sup>1

1</sup>Department of Pathology, and ²Department of Pharmacology and Toxicology, UTMB.

Pathogenic Rickettsia species are arthropod-borne, obligate intracellular bacteria, exhibitingpreferential tropism for host microvascular endothelium in the mammalian hosts, resulting in disease manifestations attributed primarily to endothelial damage or dysfunction. The mechanisms by which these pathogens regulate their transcriptome for survival and maintenance in tick vectors, in contrast to their ability to cause debilitating infections in human hosts remain unknown. In this study, we compare the expression profiles of rickettsial sRNAome/transcriptome and determine the transcriptional start sites (TSSs) of R. conorii transcripts during in vitro infection of human and tick host cells. Strand-specific deep sequencing of total RNA from Amblyomma americanum AAE2 cells and human microvascular endothelial cells (HMECs) infected with R. conoriirevealed the expression 32 small RNAs (Rc_sR’s), which were preferentially expressed above the limit of detection during tick cell infection, and confirmed the expression of Rc_sR61, sR71, and sR74 by quantitative RT-PCR. Intriguingly, a total of 305 and 132 R. conorii coding genes were differentially upregulated (>2-fold) in AAE2 cells and HMECs, respectively. Further, enrichment for primary transcripts by treatment with Terminator 5′-Phosphate-dependent Exonuclease resulted in the identification of 3903 and 2555 transcription start sites (TSSs), including 214 and 188 primary TSSs in R. conorii during the infection to tick and human host cells, respectively. Finally, we also observed differential expression of 6S RNA during host-pathogen and vector-pathogen interactions in vitro, implicating an important role for this noncoding RNA in the regulation of rickettsial transcriptome depending on the supportive host niche. In sum, the findings of this study authenticate the presence of novel Rc_sR’s in R. conorii, reveal the first evidence for differential expression of coding transcripts and utilization of alternate transcriptional start sites depending on the host niche, and implicate a role for 6S RNA in the regulation of coding transcriptome during tripartite host-pathogen-vector interactions.

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SPCS1 facilitates gt1a HCV structural protein-mediated virus assembly by promoting E2-p7 processing

Nabeel Alzahrani(1), Ming-Jhan Wu(1), MinKyung Yi(1)

(1)Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX

Background: The signal peptidase complex (SPC) cleaves signal peptides from membrane and secreted proteins. Eukaryotic SPC is composed of four components including the catalytic subunit (SEC11A or SEC11C) and three accessory proteins [signal peptidase complex subunit 1 (SPCS1), SPCS2, and SPCS3]. The SPC mediates the cleavage of junction sites of HCV polyprotein precursor Core-E1-E2-p7-NS2. Previous studies identified SPCS1 as an NS2-interactor involved in genotype 2a (gt2a) JFH1 assembly. Purpose: Previously, we reported that delayed gt1a HCV E2-p7 processing is critical for virus assembly. This delayed processing defies the typically fast and co-translational processing by SPC. Since SPCS1 was shown to regulate only a subset of SPC processing and reported to interact with HCV E2, we investigated the role of SPCS1 in E2-p7 processing-dependent HCV assembly. Methods: We determined the role of SPCS1 in gt1a HCV E2-p7 processing and E2-p7 processing-dependent virus assembly using SPCS1 knock-out (KO) huh-7.5 cells. Results: Transient expression of gt1a HCV E1-E2-p7 precursor in SPCS1 KO cells impaired E2-p7 processing without affecting E1-E2 processing despite that both junctions are targeted by SPC, indicating that SPCS1 specifically regulates E2-p7 processing. We also detected the E2-p7precursor accumulation and impaired virus production in the SPCS1 KO cells during replication of HJ3-5 (encoding gt1a H77 Core to NS2 in the background of JFH1). To determine the role of SPCS1-mediated E2-p7 cleavage in HCV assembly, we used HJ3-5/E2-EMCV-p7, lacking SPCS1-dependent SPC cleavage site, due to separation of E2 and p7 by insertion of EMCV IRES. We detected efficient HJ3-5/E2-EMCV-p7 virus production in the SPCS1 KO cells. In other words, abrogating the requirement for SPCS1-dependent cleavage of E2-p7 precursor led to HCV production in SPCS1 KO cells. These results indicate that SPCS1 is a proviral factor critically involved in gt1a HCV production by specifically regulating E2-p7 processing by SPC.

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MUTATION IN A GENE ENCODING A PHAGE INTEGRASE ATTENUATES RICKETTSIAL VIRULENCE AND INDUCES PROTECTIVE IMMUNITY AGAINST FATAL RICKETTSIOSES IN MICE

E Arroyave¹, I Hyseni¹, NY Burkhardt², T Wang^1,3, UG Munderloh^2*, R Fang<sup>1,4

1</sup>Department of Pathology, University of Texas Medical Branch, Galveston, TX, ²Department of Entomology, University of Minnesota, St. Paul, MN, ³Department of Microbiology & Immunology, University of Texas Medical Branch, Galveston, TX, ⁴Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX

Although rickettsiae can cause life-threatening infections in humans worldwide, no licensed vaccine is available. To evaluate the suitability of live-attenuated vaccine candidates against rickettsioses, we generated a R. parkeri mutant RPATATE_0245::pLoxHimar (named 3A2) by insertion of a modified pLoxHimar transposon into the gene encoding a phage integrase protein. In consistence with its genetic mapping, R. parkeri 3A2 expressed mCherry fluorescence and resistance to spectinomycin. Compared to the parent wild type (WT) R. parkeri, the virulence of R. parkeri 3A2 was significantly attenuated as demonstrated by significantly smaller size of plaque, failure to grow in human macrophage-like cells, rapid elimination of Rickettsia and ameliorated histopathological changes in tissues in intravenously infected mice. A single dose immunization of R. parkeri 3A2 conferred complete protection against both fatal R. parkeri and R. conorii rickettsioses in mice, in association with a robust and durable rickettsiae-specific IgG antibody response. Collectively, R. parkeri mutant with a disruption of RPATATE_0245 resulted in a significantly attenuated phenotype, potent immunogenicity and protective efficacy against two spotted fever group rickettsioses. Overall, this proof-of-concept study highlights the potential of R. parkeri mutants as a live-attenuated and multivalent vaccine platform in response to emergence of life-threatening spotted fever rickettsioses.

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IN VITRO AND IN VIVO CHARACTERIZATION OF ILHEUS VIRUS AND IMPLICATIONS ON EMERGENCE

SR Azar¹, KS Plante¹, JA Plante¹, D Mirchandani¹, Scharton D¹, RE Marques², L Saccheto², VL Popov, RB Tesh¹, SL Rossi¹, KA Hanley³, SC Weaver¹, ML Nogueira², N Vasilakis<sup>1

1</sup>Univ. of Texas Medical Branch, Galveston, TX
²FAMERP, Sao Jose do Rio Preto, Sao Paulo State, Brazil
³New Mexico State University, Las Cruces, NM

Ilhéus virus (ILHV) is an arbovirus first described in 1944 and isolated from Aedes and Psorophora spp. mosquitoes during an epidemiological investigation of yellow fever in the city of Ilhéus, Bahia State, Brazil. ILHV belongs to the genus Flavivirus, family Flaviviridae in the Ntaya antigenic complex. ILHV is maintained in an enzootic transmission cycle between birds and arboreal mosquitoes. Since its initial isolation ILHV has been isolated or detected primarily in arboreal mosquitoes, birds and humans throughout Central America, the Caribbean and South America, suggesting a broad geographic range of transmission. While several serological surveys showed the presence of ILHV neutralizing antibodies in in mammals, birds and humans, the virus has not been associated with large outbreaks in human populations. To better understand its potential to emerge, we experimentally evaluated its host range, pathogenesis, replication and vector competence. We utilized eight archived ILHV isolates spanning a vast spatiotemporal range, a variety of mammalian, arthropod and marsupial cell lines, as well as rodent animal models. Our replication assays showed that all ILHV strains replicated to high titers, especially in cell lines with defective antiviral responses (Vero, C6/36 and C7/10). Pathogenesis studies in young CD1 mice demonstrated that ILHV is highly neurotropic reaching high titers of replication in both brain (≈9 log10 FFU/g) and spinal cord (≈8 log10 FFU/g), culminating in death. Our vector competence in various Aedes spp. mosquitoes demonstrated their susceptibility and transmission potential, whereas contrary to previous evidence Cx. quinquefasciatusand Cx. tarsalis were relatively refractory to ILHV infection. Collectively, these results in tandem with previously published serosurveys, suggest the potential of ILHV to emerge and lead to outbreaks in urban settings and thus underscoring the need for increased surveillance, as well as determining the actual burden of human disease in endemic areas.

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CRYO-EM STRUCTURES OF PSEUDOMONAS AERUGINOSA RIBOSOME INITIATION COMPLEXES REVEAL THE CONFORMATIONAL PATHWAY FROM TRANSLATION INITIATION TO AN ELONGATION-READY COMPLEX

Ritwika Basu (1), Michael Sherman (2), Matthieu Gagnon (1,2)

(1) Department of Microbiology and Immunology, Univ. of Texas Medical Branch, Galveston, (2) Sealy Center of Structural Biology, Univ. of Texas Medical Branch, Galveston

Bacterial translation initiation factor 2 (IF2) is a translational GTPase and the last key player in the initiation stage of protein synthesis. Conformational changes in IF2 on the ribosome are triggered by GTP hydrolysis, positioning the formyl-methionine initiator transfer RNA (fMet-tRNAi-fMet) to the ribosomal peptidyl (P) site and facilitating dissociation of IF2 from the ribosome; together resulting in an elongation competent translation complex. Here we present an ensemble of Cryo-EM structures of 70S translation initiation complex from Pseudomonas aeruginosa that reveal large domain rearrangements in IF2 correlated with the amplitude of 30S rotation, the guanine nucleotide bound to IF2, and the accommodation state of the tRNA. The structural basis of this ribosome-coupled IF2 rearrangement is essential for the transition to translation elongation, paving the way for targeting this step by novel antibiotics.

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NOVEL UPREGULATION OF HEDGEHOG AND HIPPO SIGNALING PATHWAYS BY EHRLICHIA CHAFFEENSIS EFFECTOR PROTEIN TRP120 VIA LIGAND MIMICRY

C Byerly¹ & J McBride<sup>1

1</sup>Univ. of Texas Medical Branch, Galveston, TX

E. chaffeensis (E.ch.) is responsible for the zoonotic disease human monocytic ehrlichiosis (HME). HME is the most prevalent and life-threatening tick-borne disease in the United States. E.ch.is a Gram negative intracellular bacterium that invades human monocytes by utilizing tandem repeat proteins, including TRP120. It is hypothesized that TRP120 mimics Hedgehog and Hippo ligands to activate host cell signaling and mediate host cell fate, thus allowing E.ch.survival within the monocyte. The objective of this study was to examine the mechanisms and pathobiology of E.ch by elucidating the pathogen’s ability to upregulate Hedgehog and Hippo signaling pathways. Supporting data demonstrates that TRP120 colocalizes with the Hedgehog signaling receptor PTCH2 and the Wnt signaling receptor FZD5. This study demonstrates that TRP120 contains a Hedgehog and Wnt homology domain to activate Hedgehog and Hippo signaling to promote E.ch.infection in human monocytes. To date,TRP120 is the only known bacterial effector protein to hijack human monocytes using ligand mimicry of host proteins to bind receptors of conserved signaling pathways, which are important for host-cell fate. Notably, the pathogen’s ability to upregulate developmental signaling pathways makes E.ch. an important model organism for infectious diseases.

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B CELL DYSREGULATION IN AN IL-21R-/- MOUSE MODEL OF SCRUB TYPHUS

GE Card¹, CA Gonzales¹, Y Liang², Y Cong^{1, 2,} L Soong<sup>1, 2

1</sup>Department of Pathology,2Departmentof Microbiology and Immunology, Univ. of Texas Medical Branch, Galveston, TX

Efficient germinal center (GC) reactions are essential for quality B cell responses. It is known that IL-21, a cytokine secreted primarily by T follicular helper cells (TFH) within the GC, plays a crucial role in guiding the B cell response. However, no information is available on B-TFHinteractions or the involvement of IL-21 in scrub typhus, a neglected tropical disease that fails to yield a long-lasting humoral memory. To address this knowledge gap, we used Orientia tsutsugamushi-infected mouse spleen tissues for transcriptomic studies and found significantalterations in IL-21 and IL-21R levels at late stages of infection. To investigate the role of IL-21 signaling in scrub typhus, we used IL-21R-/- mice and diverse experimental approaches (multicolor flow cytometry, qRT-PCR, and ELISA) to assess the immunological state and disease outcome. Compared with wild-type counterparts, the IL-21R-/- mice showed increased susceptibility to a sublethal dose of O. tsutsugamushi, with higher rates of mortality and failure to gain body weight during 10-14 dpi. This increased susceptibility corresponded with elevated bacterial loads in the lungs and liver (two of the major organs of infection), as well as decreased mRNA levels of keytranscription factors (SPIB, PAX5, XBP-1, etc.) known to be important for B cell development. Flow cytometric analyses for splenocyte subsets revealed alterations in the total numbers and percentages of CD8+ and CD4+ T cells, as well as TFH cells and differentiated B cells. ELISA-based analyses ofTSA56 protein-specific IgG1 also revealed that KO mice had a significant reduction in these antibody titers at 14 dpi. Together, our data suggest that deficiency in IL-21 signaling has a widespread effect on both humoral and cellular immunity and calls for further investigation to define the mechanisms of B cell dysregulation during infection.

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CHARACTERIZING A NOVEL, ATTENUATING MUTATION IN THE WEST NILE VIRUS ENVELOPE PROTEIN

KL Carpio¹, JA Kaiser², GJ Haila², DWC Beasley², ADT Barrett³

Departments of Biochemistry & Molecular Biology¹, Microbiology & Immunology², and Pathology³, Univ. of Texas Medical Branch, Galveston, TX

West Nile virus (WNV) is a mosquito borne virus and responsible for outbreaks of neuroinvasive disease in North America and across Europe, which can lead to meningitis, paralysis, or even death. It continues to circulate annually, as there are currently no vaccines or treatments approved for humans. A recently identified WNV mutant was shown to have a substitution in the envelope (E) protein at amino acid 114 from an aspartic acid to an alanine, which contributes to attenuation in Swiss Webster mice. Specifically, using reverse genetics, the E-D114A mutant, with intraperitoneal inoculation of over 30 million PFU resulted in no mice succumbing to infection whereas the parental NY99 strain (NY99ic) was uniformly lethal at an inoculum of 500 PFU. When the genome of the mutant was sequenced using next-generation sequencing (NGS), and compared to the virulent parent virus NY99ic, it had no additional consensus mutations, confirming the importance of the E-D114A mutation in the attenuated phenotype.Comparisons of the Shannon entropy and single nucleotide variants (SNVs) revealed that the mutant had lower levels of diversity and fewer SNVs than NY99ic, which is characteristic of the attenuated, yellow fever vaccine strain 17D. Multiplication kinetics studies in Vero and A549 cells revealed that E-D114A reaches titers comparable to NY99ic, and additional data suggests the mutant may be temperature sensitive at 41 C. The E-114 mutation is in very close proximity to the fusion loop (amino acids 98-110), which is highly conserved in all flaviviruses and important for virus and host endosome membrane fusion. This may suggest that the E-D114 residue contributes to fusion loop attachment to the target membrane, and a substitution may prevent the conformational change from dimer to trimer necessary for fusion. Further characterization of the mutant is in progress.

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EXPLORING ROLE OF TOXIN-ANTITOXIN SYSTEMS DURING BURKHOLDERIA PSEUDOMALLEI ANTIBIOTIC-INDUCED PERSISTENCE

I Chapartegui-González¹, AG Torres<sup>1

1</sup>University of Texas Medical Branch (UTMB), Galveston, TX

Burkholderia pseudomallei (Bpm) is the causative agent of melioidosis,a multifaceted human pathogen that causes over 165,000 cases/year, with up to 50% mortality. The treatment is difficult due to both its endogenous multiresistant phenotype and its high infection relapse rates, associated with bacterial persistence, whose activation has been linked to toxin-antitoxin (TA) systems. Previous genomic and functional studies have identified more than 100 putative toxins inBpm K96243, with more than 50 specifically associated with different stresses. A total of 12 TA-predicted systems were selected for further analysis. Both proteomic and genomic annotation methods were used to identify novel TA systems. Products that were predicted as putative toxins (or antitoxins) by both methods were chosen to evaluate their role in Bpm persistence. The Bpm persister phenotypewas induced by high concentrations of different antibiotics. RNA from survival cells alone or as intracellular bacteria in macrophage infections was extracted for expression profiles. The expression of the predicted TA genes was evaluated by qRT-PCR using bacteria grown in media without induction as a control. Further, a previously described method for allelic exchange in Bpm was also used to generate mutants lacking toxin or antitoxin genes. The expression of the selected systems was evaluated in intracellular and antibiotic-induced persister bacterial survival conditions. A correlation between antibiotics with more influence in that phenotype induction (fluoroquinolones) and overexpression of multiple systems together was identified. Some predicted systems were also highly overexpressed in surviving intracellular bacteria, selected for mutants generation, whose are being evaluated for their phenotypes, both from the toxin and antitoxin lacking strains in vitro and in vivo assays. While Bpm TA systems are involved in persister phenotype, their genomic redundancy usually makes it difficult to find a stress-related phenotype. However, our omic approaches allowed us to study their association with persistence.

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ORNITHODOROS PUERTORICENSIS FOX 1947 (IXODIDA: ARGASIDAE) ASSOCIATED WITH DOMESTIC FOWL IN RURAL DWELLINGS FROM CORDOBA DEPARTMENT, COLOMBIA

ÁA Faccini-Martínez¹, Y López², LN Robayo-Sánchez³, S Muñoz-Leal⁴, A Aleman², E Arroyave¹, A Ramírez-Hernández³, JA Cortés-Vecino³, S Mattar², DH Walker<sup>1

1</sup>University of Texas Medical Branch, Galveston, TX, ²Universidad de Córdoba, Córdoba, Colombia, ³Universidad Nacional de Colombia, Bogotá, Colombia, ⁴Universidad de Concepción, Chillán, Chile.

Ticks of genus Ornithodoros are nidicolous parasites that associated with a wide array of vertebrates; their bites cause hypersensitivity reactions in humans and are capable of transmittingpathogens of health concern. In the department of Córdoba (Caribbean region of Colombia), thefirst report of an Ornithodoros soft tick was made in 1980 by Betancourt, who described the collection of Ornithodoros talajein human dwellings. Nevertheless, currently, the records of O. talaje made in South America are doubtful and likely correspond to misidentifications with morphologically similar species of the group. Between October and December of 2020, we visited rural areas of four localities from three municipalities within the department of Córdoba: Cuero Curtido and Severá in the municipality of Cereté, El Espanto in the municipality of Planeta Rica, and Arroyo Negro in the municipality of San Carlos. Searches for soft ticks were made in 46 human domiciles and peridomiciliary areas. We investigated areas frequented by domestic animals, inspected cracks in the walls, and fowl nests. Infestation by soft ticks was found in 13% (6/46) of visited houses. Overall, 215 ticks were collected (26 larvae, 144 nymphs and 45 adults), all found in nests of domestic birds or in the adjacent walls. Larvae, nymphs and adults were morphologically identified as O. puertoricensis and molecularly confirmed by PCR through amplification of a fragment of 16S tick mitochondrial gene. Adults, pools of nymphs and larvae generated sequences were 99% identical with O. puertoricensis from Panama. This study reports for the first time O. puertoricensis associated with domestic fowl in rural dwellings, and expands the geographical distribution of this tick species to the Córdoba department in Colombia. Importantly, local people described exposure to tick bites when sleeping in infested houses; therefore, the transmission of soft tick-borne pathogens is of concern in the region.

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PEPTIDE NANOFIBER VACCINES STIMULATE BCG-PRIMED LUNG RESIDENT MEMORY CD4+ T CELLS FOLLOWING MUCOSAL BOOST

Megan A Files(1), Kubra Naqvi(1), Tais B Saito(2), Jai S Rudra(3,4) Janice J Endsley(1,3)

(1)Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, (2)Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana, (3)Sealy Institute for Vaccine Sciences, University of Texas Medical Branch, Galveston, TX, (4)Department of Biomedical Engineering, Washington University in St. Louis, MO

Tuberculosis (TB) infection caused by Mycobacterium tuberculosis (Mtb) is a serious threat to global health with 10 million new infections each year and 1-2 million deaths. The only licensed vaccine approved for human use, Mycobacterium bovis Bacille Calmette-Guerin (BCG) has been administered to 4 billion people despite variable efficacy and waning immunity. Heterologous vaccine regimens that boost T cell memory generated through BCG priming, however, may be an effective immunization strategy. Our lab previously demonstrated efficacy of a nanofiber (NF) vaccine platform containing co-assembled CD8+ and CD4+ T cell epitopes as evidenced by the generation of Mtb-specific polyfunctional T cell recall. We hypothesized that boosting of BCG-primed mice through a pulmonary route with NF-based vaccine bearing Mtb Ag85B CD4+T cell epitopes would generate a robust population of antigen-specific lung-resident T cells. We observed that boosting BCG-primed mice through a pulmonary route with the Ag85B-NF generated increased frequency of Mtb antigen-specific T cells in the lung as indicated by the number of tetramer (I-Ab:Ag85B) positive CD4+T cells. Importantly, the frequency of Mtb-specific CD4+T cells was further increased by adoptive transfer of dendritic cells pulsed with Ag85B-bearing NF. Through multi-color flow cytometry, Ag85B-specific T cells were characterized as tissue resident memory (TRM) and effector memory (TEM) T cells based on the surface marker phenotypes (I-Ab:Ag85B+ CD69+/- CCR7- CD44+ CD62Llo) and intravascular staining. Development of peripheral immunity was evident based on activation of cytokine production by splenocytes in response to Mtb antigens. These results suggest that the NF vaccine platform generates Mtb-specific lung-resident memory T cell subsets and peripheral cytokine responses associated with enhanced control of Mtb infection.

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SELECTIVE ACTIVATION OF MINCLE AND ITS IMPACT ON INFLAMMATORY RESPONSES TO ORIENTIA TSUTSUGAMUSHI INFECTION

JR Fisher¹, GE Card²,Y Liang¹, BJ Trent¹, L Soong<sup>1,2

1</sup>Department of Microbiology and Immunology, ²Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA

O. tsutsugamushi is an obligate intracellular bacterium and etiologic agent of scrub typhus. The lung is a target organ of infection and displays unbalanced, type 1-skewed proinflammatory responses. Here, we investigated whether the C-type lectin receptor (CLR) Mincle contributes to immune recognition and dysregulation. Following lethalinfection in C57BL/6 mice, we performed pulmonary differential gene expression analysis via NanoString technology. Of 671 genes analyzed, we found 312 significantly induced genes (adj. p < 0.05) at the terminal phase of disease, among which Mincle (Clec4e) was the 4th greatest activated gene (36-fold higher than the mocks). Other highly up-regulated genes includedFcgrs (Mincle signaling partners) and proinflammation mediators (e.g., Il27, Ccl2, Cxcr3, Ccr5, and their ligands). To validate a role of Mincle in immune recognition, we exposed murine bone marrow-derived macrophages (MΦ) to live or heat-killed O. tsutsugamushi and analyzed a panel of CLRs and proinflammatory markers via qRT-PCR. While heat-killed bacteria transiently stimulated Mincle transcription that dissipated by 24 hpi, live bacteria generated a sustained response; infection had limited impact on other CLRs examined. Mincle protein upregulation in inflamed lungs and infected MΦ was validated by western blot and indirect immunofluorescence. To confirm Mincle’s role in type 1-skewed responses, we compared inflammatory profiles of WT and Mincle-/- MΦ via qRT-PCR. While infected WT MΦ showed significant increases in Cxcl1, Ccl2, Cxcl9, Il27, Tnfa, and Nos2, the Mincle-/- cells had abrogated responses of Cxcl1, Ccl2, and Nos2. The synergistic effects of bacterial infection and type-1 cytokines in Mincle induction are under investigation. Together, our study provides the first evidence for Mincle sensing O. tsutsugamushi and implicates Mincle-related pathways in generating immune dysregulation during severeinfection. Since there are no effective vaccines for scrub typhus, our study provides novel insight into innate immune recognition of this poorly-studied bacterium.

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Generating Long-lived Effector/Memory (teff/Tem) Cells with Chronic Mouse Cytomegalovirus (MCMV) Vaccination to Prolong Malaria Immunity

K Gbedande¹, SA Ibitokou¹, MG Brown³, M degli Esposti², R Stephens<sup>1

1</sup> Department of Internal Medicine, Division of Infectious Diseases, University of Texas Medical Branch, Galveston, TX
² Centre for Experimental Immunology, Lions Eye Institute, Nedlands, Western Australia, Australia
³ Department of Medicine, University of Virginia, VA

Immunity to Plasmodium infection and vaccination both decay. In mouse models, both B and T cells are required for immunity; however, decay of protection correlates with loss of malaria-specific T cells, not antibody. Whole parasite vaccines like infection and drug cure protect. However, sterile immunity from infection or vaccination last less than 200 days, while effector T cells (Teff) decay. Therefore, we have reverse-engineered a vaccine strategy to induce a long-lived antibody response with live vaccination, and then boost the CD4+ Teff response to prolong protection. Cytomegalovirus (CMV) is a chronic vaccine vector that induces T cell mediated immunity and shows promise against SIV, tuberculosis and liver-stage malaria. Therefore, we used MCMV to test our hypothesis that prolonging generation of Teff in mice would prolong protection by a live vaccine. MCMV encoding P.chabaudi MSP-1 epitope B5 (MCMV-B5) and B5 TCR Transgenic T cells were used to follow the MCMV vector-induced T cell response. We observed that Chronic MCMV infection protected mice from parasite upon P. chabaudi infection. Consistent with this protection, vaccination with MCMV-B5 activated and maintained B5 T cells, and promoted generation of Teff, which we have previously shown to be protective, for 2.5 months. Importantly, we found that the MCMV vector synergistically prolonged the protection generated by live malaria vaccine. In investigating the mechanisms of protection, we found that IFNg maintained by MCMV infection is required for prolonged protection, and that blocking IFNg before infection led to higher IL-12 production upon infection. IL-12 boosts protection through enhanced antigen presenting cell function and phagocytosis. Our findings suggest methods and mechanisms to effectively prolong immunity to Plasmodium infection with CMV vectored chronic vaccines to redress the problem of short-lived protection to malaria infection and vaccines which is known to be due to decay of CD4 T cell memory.

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ABERRANT B AND T CELL ACTIVATION DURING SEVERE SCRUB TYPHUS IN MICE

Casey Gonzales¹, Galen Card¹, Yuejin Liang², Lynn Soong<sup>1,2

1</sup>Department of Pathology, ²Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA

Orientia tsutsugamushi is the etiologic agent of scrub typhus. Since protective immunity after natural infection in humans is short-lived, there is an urgent need to understand O.tsutsugamushi-specific adaptive immune responses. This study is aimed to examine T-B cell interactions and germinal center reactions between severe vs. mild diseases at early infection stages using comprehensive approaches. 1) Using NanoString technology and qRT-PCR approaches, we revealed diminished expression of several key transcriptional factors and B cell-regulating cytokines in the spleens of mice, which positively correlated with spleen bacterial loads. 2) Using immunofluorescence staining of the spleen sections, we found apparent loss of germinal centers in lethally-infected mice, while mice with sublethal infection displayed shrunken germinal centers. 3) Our flow cytometry data indicated that mice with lethal infection had significantly reduced total splenocyte number with marked reduction in CD4+ T cells, TFH cells, total B cells, and memory B cells, respectively, at late/severe stages of disease. These flow cytometry data agreed with our IFA and transcript results, suggesting a favored development of B cells to become plasma cells, rather than memory B cells, during severe infection. 4) Antigen-specific ELISA data indicated significantly higher total IgG titers in mice with lethal infection as compared to those with sublethal infection, while IgM titers remained comparable in both groups. Overall, these results show a dysregulation in B/T cell interactions as well as abnormal B cell responses during late stages of severe, but not sublethal, infection. This study, for the first time, defines T-B cell interactions and possible mechanisms underlying dysregulated B cell responses to O. tsutsugamushi infection.

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THE RNA HELICASE DHX16 RECOGNIZES SPECIFIC VIRAL RNA TO TRIGGER RIG-I-DEPENDENT INNATE ANTIVIRAL IMMUNITY

A Hage(1), P Bharaj(1), S van Tol(1), MI Giraldo(1), L Aguilera-Aguirre(1), X Xie(2), SG Widen(2), HM Moulton(3), JR. Johnson(4), NJ Krogan(5), P-Y Shi(2), A García-Sastre(4), AN Freiberg(6), and R Rajsbaum(1)

(1)Univ. of Texas Medical Branch, Department of Microbiology and Immunology, Galveston, TX
(2)Univ. of Texas Medical Branch, Department of Biochemistry and Molecular Biology, Galveston, TX
(3)Oregon State University, Department of Biomedical Sciences, Carlson College of Veterinary Medicine, Corvallis, OR
(4)Icahn School of Medicine at Mount Sinai, Department of Microbiology, New York, NY
(5)University of California at San Francisco, San Francisco, Department of Cellular and Molecular Pharmacology, CA
(6)Univ. of Texas Medical Branch, Department of Pathology, Galveston, TX

Type-I interferon (IFN-I) is essential to establish antiviral innate immunity. Unanchored (or free) polyubiquitin (poly-Ub) has been shown to regulate IFN-I responses, however few unanchored poly-Ub interactors are known. To identify factors regulated by unanchored poly-Ub in a physiological setting, we developed an approach to isolate unanchored poly-Ub from lung tissue. Using this approach combined with mass spectrometry analysis, we identified DHX16, an RNA helicase and pre-mRNA splicing component as a potential pattern recognition receptor (PRR). The role of DHX16 in innate immunity and regulation of its activity have not been previously described. Silencing of DHX16 in cells and in vivo diminished IFN-I responses against influenza virus. RNA-Seq analysis identified a number of IFN-stimulated genes (ISGs) whose expression was significantly downregulated in DHX16 knockdown cells. Consistent with this, overexpression of DHX16 promoted IRF3 phosphorylation, enhanced ISG transcript induction, and limited IAV replication. These effects extended to members of other virus families, including Zika and SARS-CoV-2. DHX16-dependent IFN-I production requires RIG-I and unanchored K48-poly-Ub synthesized by the E3-Ub ligase TRIM6. DHX16 recognizes a signal in influenza RNA segments that undergo splicing and requires its RNA helicase motif for direct, high-affinity interactions with specific viral RNAs. Our study establishes DHX16 as a PRR that partners with RIG-I for optimal activation of antiviral immunity requiring unanchored poly-Ub.

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Using chimeric flaviviruses to probe envelope protein inter/intramolecular interactions

Gabriel Haila¹, Maricela Torres¹, Dennis Bente¹, David Beasley<sup>1

1</sup>UTMB Department of Microbiology and Immunology

West Nile virus (WNV) was originally introduced into the United States in New York in 1999, leading to cases of West Nile neuroinvasive disease. Since its’ introduction, WNV has become endemic across the entire continental US. The envelope (E) protein contributes to the wide tissue tropisms of WNV and mutations in E can attenuate the neuroinvasive phenotype of the virus. Previous studies have identified single point mutations related to E protein glycosylation sites or receptor binding or fusion domains which result in an attenuated phenotype. However, investigation of interactions made by an entire domain, such as E protein domain three (EIII), in determining tissue tropism and disease pathogenesis are lacking. The development of a WNV/Koutango virus (KOUV)-EIII chimera provided a novel tool to study the role of EIII interactions within the viral particle. Interestingly, despite high levels of virulence for both WNV and KOUV observed in vivo,in addition to their high degree of E protein sequence homology, the WNV/KOUV-EIII chimera has an attenuated phenotype while a more divergent WNV/Japanese encephalitis virus-EIII chimera maintained WT levels of virulence. Given this information, we hypothesize that a small number of key inter/intramolecular interactions of the WNV EIII domain contribute to the attenuated phenotype of WNV/KOUV-EIII. To fill this gap in our understanding of the roles the EIII participates in, we have constructed both a WNV/KOUV-E and WNV/KOUV-prM/E chimera to assess mechanism of attenuation in the EIII chimera. By inserting progressively larger portions of the KOUV structural proteins, we aim to identify the location(s) of these critical interactions. Given the absence of vaccines and therapeutics approved for human use in preventing or treating WNV infections, the understanding developed from this project can be applied to the rationale design of WNV counter measures.

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BRD4 ISOFORMS IN HIV TRANSCRIPTIONAL REGULATION AND LATENCY

Renee Hajnik^1,2, Chaojie Zhong^2, Jia Zhou^3, Haitao Hu<sup>2

1</sup>Department of Experimental Pathology, ²Department of Microbiology and Immunology, ³Department of Pharmacology and Toxicology, Univ. of Texas Medical Branch, Galveston, TX 77550

Following integration of viral DNA into the host genome, HIV establishes a latent infection that cannot be eliminated by antiretroviral therapy (ART). BRD4, a member of the bromodomain and extraterminal (BET) protein family, is involved in the regulation of HIV transcription and latency. BRD4 has three isoforms from alternative splicing, including the long isoform (BRD4-L) and two short isoforms (BRD4-Sa and BRD4-Sb). The pan-BET inhibitor JQ1 has been shown to activate HIV transcription. Recent studies of our laboratory have identified a novel BRD4 small-molecule modulator (ZL0580) that is distinct from JQ1 and induces suppression of HIV transcription in target cells. The underlying mechanisms remain to be defined. To gain insights into why targeting similar proteins with different small-molecules modulators results in distinct transcriptional activities, we have examined the roles of BRD4 isoforms in HIV transcriptional control. A microglial cell line containing an HIV reporter (HC69) has been treated with BRD4 isoform specific small interfering RNAs (siRNAs) to examine the effect of isoform knockdowns on HIV transcription. Using 50nM siRNA, cells were collected for western blots, flow cytometry,and RT-qPCR. Western blots have shown that knockdown of individual BRD4 isoforms is capable, and flow cytometry and RT-qPCR analyses have shown an increase in HIV transcription after long isoform knockdown, and a decrease in HIV transcription after BRD4-Sb isoform knockdown. These data suggest that BRD4 isoforms may have opposing roles in regulation of HIV transcription and latency, with the long isoform inhibiting HIV transcription but the BRD4-Sb isoform promoting HIV transcription. These finding also indicate the potential of isoform specific targeting of BRD4 to manipulate HIV transcription.

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DETERMINING WHICH MUTATIONS IN YELLOW FEVER ENVELOPE (E) PROTEIN CONTRIBUTE TO VIRION INSTABILITY OF THE VACCINE STRAIN (17D) VIRUS

CA Hansen¹, ADT Barrett<sup>1, 2, 3

1</sup>Department of Pathology, ²Department of Microbiology and Immunology, ³Sealy Institute for Vaccine Sciences, University of Texas Medical Branch Galveston, TX

Yellow fever virus (YFV) is a re-emerging pathogen that causes an estimated 170,000 severe cases of disease and 78,000 deaths per year worldwide. The yellow fever vaccine (17D) is a live attenuated virus that was empirically derived from the wild type strain Asibi by passage in chicken tissue. The molecular mechanism(s) of attenuation remain largely unknown. 17D has an unstable virion when compared to Asibi based on the mobility of the envelope (E) protein in SDS-PAGE protein gels. This has resulted in unsuccessful attempts to undertake high-resolution cryo-electron microscopy on 17D virions as the virion is not stable enough for these techniques. 17D virion instability may contribute to attenuation of the vaccine strain virus. The purpose of this study is to determine which mutations between 17D and Asibi contribute to thisvirion instability phenotype and determine the mechanism of instability. Chimeric viruses were generated by site-directed mutagenesis of infectious clones to swap the structural genes (pre-membrane [prM] and E) and individual mutations of each gene between 17D and Asibi. Western blots were performed to analyze E protein stability in each virus. Swapping the structural genes reverted the phenotypes so that 17D/Asibi prME virus-infected cells accumulate full-length E protein while cells infected with Asibi/17D prME accumulate a higher mobility E when observed on SDS-PAGE gels. Thus, as expected, virion stability was controlled by the structural proteins. Further experimentation showed that prM and E domain III mutations do not alter E stability.These results were confirmed in both monkey kidney Vero and mosquito C6-36 cell types. This indicates that this stability phenotype is controlled by mutations in domains I and/or II of E,which are currently being evaluated individually. Future studies will focus on combinations of individual mutations and their relation to virion stability.

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NIPAH VIRUS INFECTION OF A HUMAN 3D MICROPHYSIOLOGIC LUNG CULTURE MODEL

K Johnson(1), J Niles(1), O Escaffre(1), B Kalveram(1), J Smith(1), T Juelich(1), J Nichols(1,2), A Freiberg(1)

(1) University of Texas Medical Branch, Galveston, TX
(2) Houston Methodist Research Institute, Houston, TX

Nipah virus (NiV) is an emerging paramyxovirus that cause severe respiratory disease and encephalitis. While the respiratory tract is an important target tissue of NiV, our knowledge is still limited on the target cells in the human respiratory tract, the immune responses during infection, as well as the molecular mechanisms through which NiV can cause respiratory disease. To address this gap in knowledge, we are utilizing a 3D lung microphysiologic organ culture (MHOC) model to study infection caused by NiV. Acellular lung scaffolds reseeded with immortalized human respiratory cell cultures containing both type 1 and 2 pneumocytes, representing the human lung parenchyma, were used as a model for studying NiV infection. 3D MHOCs were infected with recombinant NiVs either expressing Gaussia luciferase & GFP reporter proteins or a Firefly luciferase reporter. Our studies demonstrate that 3D MHOCs are susceptible to infection by NiV after infection by direct injection into the scaffold or by inoculation in culture medium. Peak viral supernatant titer is reached between 2, 4, or 6 days post infection depending on the infectious dose used (MOI of 1, 0.1, and 0.01, respectively). Titration of PBS homogenates demonstrated peak titers around 104 PFU/g of tissue, which were reached by 2 days after infection regardless of MOI. Similar trends in replication were seen in a confirmatory experiment utilizing wild-type NiV, supporting use of labeled recombinant strains in future infections. Overall, these studies support the use of 3D MHOCs as a model for studying NiV infection and will support future experiments such as studying viral tropism and evaluation of antiviral compounds.

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ANTIGEN-SPECIFIC ANTIBODY AND POLYFUNCTIONAL T CELLS GENERATED BY INHALATIONAL IMMUNIZATION WITH PROTECTIVE BURKHOLDERIA ΔtonB Δhcp1 LIVE ATTENUATED VACCINES

Nittaya Khakhum^1, Preeti Bharaj^1, David H. Walker^2, Janice J. Endsley^1, Alfredo G. Torres<sup>1, 2

1</sup>Department of Microbiology and Immunology, UTMB, Galveston, TX, ²Department of Pathology, UTMB, Galveston, TX

Melioidosis, caused by Burkholderia pseudomallei (Bpm) is a life-threatening neglected tropicaldisease that requires complex treatments and lacks an approved vaccine. We previously demonstrated that two live attenuated vaccines, Burkholderia malleiΔtonB Δhcp1(CLH001) and BpmΔtonB Δhcp1(PBK001), confer full protection to C57BL/6 mice against inhalationalBpm. Here, we identify the immune correlates of protection induced by CLH001 and PBK001 vaccines following a prime and boost intranasal immunization strategy. Mucosal immunization with either CLH001 and PBK001 generated Bpm-specific IgM and IgG (IgG2b/c > IgG1 > IgG3) antibodies in sera and lungs, and IgA antibodies in lungs. Immune serum conferred complement-independent bactericidal activity and opsonophagocytic uptake by host macrophages but was insufficient to provide significant protection from lethal challenge in passive transfer experiments. Both vaccines elicited memory Th1 and Th17 CD4+ T cells responses in lung and spleen as demonstrated by Bpm antigen-specific recall. Compared to CLH001, the PBK001 vaccine was superior in generating respiratory IgA post-boost, anamnestic IgG at challenge, T cell recall to specific antigen, and development of diverse polyfunctional memory T cell pools. However, analysis of lung histology suggested that potent polyfunctional T cell memory and/or IL-17 signatures generated with PBK001 vaccination may also be associated with moderate lung inflammation post-vaccination. Together, intranasal immunization with the live attenuated vaccines elicits humoral and cell-mediated immune responses that correlate with protection against inhalational melioidosis.

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IMMUNOLOGICAL RESPONSES AND PROTECTIVE EFFICACY AGAINST PNEUMONIC PLAGUE IN MICE USING A HETEROLOGOUS PRIME-BOOST VACCINATION APPROACH

Paul B. Kilgore(1), Jian Sha(1,2), Vladimir L. Motin(1-4), and Ashok K. Chopra(1-4)

(1)Department of Microbiology & Immunology, (2)Institute for Human Infections and Immunity, (3)Sealy Institute for Vaccine Sciences, and (4)Galveston National Laboratory, University of Texas Medical Branch, Galveston, TX, 77555, USA

Yersinia pestis, the causative agent of plague, caused 3 pandemics, with a recent epidemic in Madagascar (10% fatality rate), and notably, 76% of the cases were pneumonic. The high mortality rate and the limited treatment window for effective antibiotic therapy reinforce the need for vaccine prophylaxis; however; there are no FDA approved vaccines. Our lab generated a live attenuated triple knockout Y. pestis strain (ΔlppΔmsbBΔail [LMA]), deleted for genes encoding Braun lipoprotein (Lpp), meristoyl-dependent acetyltransferase B (MsbB) and an attachment-invasion locus (Ail), and a subunit adenoviral vector-based Ad5-YscF-F1-LcrV (ad5-YFV) vaccine, both of which were protective against plague in animals. We now hypothesize that a heterologous prime-boost approach using Ad5-YFV as the 1st dose and LMA for the 2nd dose or vice versa would generate a more durable immune response that combines the best characteristics of each vaccine. Indeed, some subtle differences were noted in immune responses generated by these two vaccines when delivered separately. We also tested protective efficacy by simultaneous administration of both vaccines in mice. Simultaneous vaccination provided 60-85% protection to animals against pneumonic plague, while a 2-dose regimen resulted in 100% protection regardless of the route of vaccination (i.n. or i.m.) or the order in which the vaccines were administered. We observed higher levels of F1-V IgG2a when Ad5-YFV was administered first, and higher F1-V IgG1 when LMA was delivered first. All vaccine combinations generated strong IFNγ producing CD4+ and CD8+ T-cells. While all vaccine combinations produced significant populations of IL-17 secreting CD4+ T-cells, when LMA was the first dose, we measured higher concentrations of IL-17 than when Ad5-YFV was the first dose. Two doses of the Ad5-YFV vaccine did not induce IL-17 producing CD4+ T-cells. Likewise, IgA production was optimal with the heterologous vaccination approach. The proposed vaccination approach triggers favorable humoral, cellular, and mucosal immunity.

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ZIKV TESTICULAR PERSISTENCE IS CHARACTERIZED BY LOCAL RESPONSE WITH RAPID INFILTRATION OF CD8+ T-CELL AND SLOW INFILTRATION OF MYELOID AND CD4+ T-CELLS

RK Campos(1), Y Liang(1), SR Azar(1), J Sun(1), SL Rossi(1).

(1)Univ. of Texas Medical Branch, Galveston, TX.

Zika virus (ZIKV) recently emerged to cause devastating outbreaks, and frequent sexually transmission in adults has been reported. ZIKV was also found to cause persistent infection and damage mouse testes. ZIKV persistent infections are thought to be supported by infection of Sertoli cells (SC), which are essential for spermatogenesis and the formation of the blood-testis barrier. We hypothesized that persistent testes infection is the result of both a selection of mutations in WT ZIKV and dysregulated immune response. To address this, ZIKV persistently passaged in murine SC (15P-1) and mice testes in vivo were deep sequenced and eight single nucleotide polymorphisms (SNPs) were identified to de novo arise or increase in frequency. Testes are an immune privileged site, but an immune response can be triggered when this organ is infected. We infected A-129 mice with ZIKV and to study the immune response during persistence. We found that at 7 dpi the immune response was heavily dependent on infiltration of effector CD8+ T-cells in the testes, resulting in tissue damage but inefficient clearance of the virus. At 23 dpi, about half of the testes still had detectable viral titers. At this timepoint, in addition to CD8+ T-cells there was infiltration of many effector CD4+ T-cells and myeloid cells, production of IFN-gamma and IL-17, and extensive tissue damage, including reduction in weight of the testes, which has been observed by other groups. This is in contrast to spleen, in the virus is not known to cause persistence, and in which we were able to see early accumulation of CD8+, CD4+ T-cells and myeloid cells at 7 dpi and CD8 cells were reduced at 23 dpi. Taken together, these findings support our working hypothesis that selection of mutations and a dysregulated immune response have important roles in ZIKV persistence in the testes.

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HOW TO DRINK MILK—EIGHT YEAR ASSESSMENT OF AN INTERMITTENT STEP-UP METHOD OF ORAL IMMUNOTHERAPY (OIT) FOR SEVERE COW’S MILK (CM) ALLERGY

K Kuzume(1,2), T Midoro-Horiuti(1) , Y Kagata(2), M Koizumi(2), K Nishimura(2), M Okamoto(2), T Asami(2)

(1)Univ. of Texas Medical Branch, Galveston, TX, (2) Ehime Univ. School of Medicine, Ehime, Japan

We evaluated the long-term efficacy and safety of an OIT protocol—using a step-up method for severe CM allergy patients.

Study subjects with severe CM allergy (N = 29, 18 boys and 11 girls, median age 5 years old, testing positive with 1 mL of CM oral challenge test) were enrolled in this protocol from Oct 2011 to Aug 2017, and received 163 step-up OIT while in the hospital, and were followed up to Dec 2019.

Our intermittent OIT protocol consists of 3-day admission and outpatient follow-up. Day 1: Patients received CM (0.125-5 mL) every hour 5 times. Day 2: Patients received CM every 3 hours for 3 times with an increasing amount by 10-20% each time. Day 3: Patients received 1 dose of CM, and then jogged for 6-8 min until their heart rates became >150/min for > 4 min. If patients developed moderate to severe anaphylaxis, the amount of CM was reduced. Day 4 and thereafter: Patients kept taking the prescribed amount of CM at home daily. The in-hospital intermittent OIT (Day 1-3) was repeated every 2-6 months until patients could tolerate more than 100 mL of CM.

Twenty-six patients (90%) reached their goal, and their CM-specific IgE antibody levels decreased from a median of 51 UA/mL (122.4 ng/mL) to 13.9 UA/mL (33.36 ng/mL).

During 162 in-hospital interventions, the elicited allergic symptom rates were 5, 14, and 30% for severe, moderate, and mild, respectively. Also, two patients experienced severe anaphylaxis at home and five had moderate anaphylaxis, but six out of these patients were able to complete this protocol.

We conclude that the step-up OIT method can safely elicit CM tolerance in pediatric patients with severe CM allergy.

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Modified mRNA vaccine provides sterilizing immunity to lethal LASV challenge in guinea pigs

NM Lloyd¹, AJ Ronk¹, S Himansu², A Carfi², A Bukreyev<sup>1

1</sup>Department of Pathology, Galveston National Laboratory, The University of Texas Medical Branch at Galveston, Galveston, Texas, ²Moderna, Inc., Cambridge, Massachusetts

Lassa virus (LASV) is a negative strand RNA virus that causes a severe hemorrhagic disease. Every year there are about 300,000 cases and around 5,000 deaths. LASV is highly heterogenous; there are currently four accepted clades, with two more proposed. We have designed a lipid nanoparticle encapsulated modified mRNA vaccine that encodes for the GPC protein stabilized in the prefusion form. The prefusion-stabilized GPC was generated via the introduction of a series of mutations: E329P in GP2, cysteine linkage of GP1 and GP2, and the maintenance of the stabilized signal peptide for stabilization of the GPC primer (Hastie K. et al. Science 2017). Linearized DNA templates were used for in vitro synthesis of modified mRNA by T7 polymerase-mediated transcription in which the UTP was substituted with 1-methylpseudo-UTP. 1-methylpseudo UTP substitution is a naturally occurring base, and results in enhanced protein translation and immunogenicity (Anderson B. et al. Nucleic Acids Research 2010). The lipid nanoparticle in the vaccine protects the mRNA from serum nucleases and facilitates cellular uptake and endosomal escape (Dong Y. et al. Advanced Healthcare Materials 2014). A non-stabilized (wildtype) construct was tested in parallel to determine the efficacy of this antigen engineering approach. For testing of the constructs, we used five groups of Hartley guinea pigs at five to six weeks of age. Animals were challenged with a guinea pig adapted strain of LASV Josiah, a clade IV virus. By ELISA, we found that wildtype and prefusion vaccinated animals developed significant titers almost exclusively to the prefusion antigen. Interestingly, some wildtype vaccinated animals developed marginally higher neutralizing titers than prefusion vaccinated animals. No adverse effects were associated with vaccination, and all vaccinated animals were protected from death and circulating viremia post challenge with >10,000 PFU of guinea pig adapted LASV Josiah.

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MUTATIONS TO GLYCOSYLATION SITES AND THE TRANSMEMBRANE DOMAIN OF THE ARENAVIRUS GLYCOPROTEIN LEAD TO ATTENUATION IN AN OUTBRED RODENT MODEL

E Mantlo (1), T Koma (1), J Maruyama (1), C Huang (1), JT Manning (1), S Paessler (1)

(1) Univ. of Texas Medical Branch, Galveston, TX

Arenaviruses are the causative agents of many highly lethal hemorrhagic fevers in man. Only one arenavirus vaccine has been approved for use, the live-attenuated Candid#1 strain. Prior research has revealed that mutating the GPC of the arenavirus Machupo virus (MACV) to remove N-linked glycosylation sites partially attenuates the virus in a double interferon receptor knockout (IFNαβ/γ -/-) mouse model. Adding a transmembrane mutation first discovered in the sequence of the Candid#1 vaccine strain further attenuates the virus. We hypothesized that these mutations would likewise attenuate the virus in the outbred Hartley guinea pig model and provide protection from subsequent challenge with wild-type MACV. Groups of 4 Hartley guinea pigs were injected intraperitoneally with either wild-type MACV or the mutated strain. No animal inoculated with the mutated strain developed any disease signs, while all animals inoculated with wild-type MACV succumbed to disease. At 60 days post-challenge, blood was taken to measure neutralizing antibody titers and animals were re-challenged with wild-type MACV. Surprisingly, only 1 out of 4 animals developed detectable neutralizing antibodies to wild-type MACV after exposure to the MACV mutant, yet all animals previously inoculated with the MACV mutant survived challenge with a 100% lethal dose of wild-type MACV. These results indicate that removal of glycans along with mutating the transmembrane domain fully attenuates the strain in Hartley guinea pigs and provides protection from subsequent challenge. However, protection is not mediated by neutralizing antibodies. Mutations to glycosylation sites and the transmembrane domain likely contribute greatly to the attenuation of the Candid#1 vaccine strain and may inform development of future arenavirus vaccine candidates.

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HYPERCOAGULABLE STATE IN NOVEL CORONAVIRUS INFECTION WITH PRESENTATION OF EXTENSIVE THROMBOSIS OF MESENTERIC VESSELS

Z Manuelyan¹, R Gandhi¹, S Parupudi<sup>1

1</sup>University of Texas Medical Branch, Galveston, TX

SARS-CoV-2 is the source of the global pandemic that has affected millions of people worldwide. Despite its mostly asymptomatic presentation, it carries a 3% mortality rate. Recent evidence has linked the coronavirus to increased hypercoagulable state that can lead to thrombosis. We present a 54-year old male with past medical history of type 2 diabetes and hypertension who presented with three days of severe lower abdominal pain, nausea, decreased appetite, and constipation. He denied any history of liver disease, autoimmune disease, or personal or family history of clotting disorders or gastrointestinal malignancy. The patient was recently diagnosed with coronavirus infection four weeks prior but was asymptomatic. On presentation, blood pressure was 144/80 mmHg and physical exam showed mild abdominal distention with diffuse abdominal tenderness. Lab work indicated high-normal fibrinogen levels and elevated alkaline phosphatase. Computed tomography of the abdomen/pelvis with contrast showed extensive thrombosis of the superior mesenteric, splenic, and portal veins, and anticoagulation via heparin drip was subsequently initiated. Transcatheter thrombolysis therapy was performed in the splenic and superior mesenteric arteries and tissue plasminogen activator was infused. Post-infusion arteriogram showed resolution of thrombosis in splenic and superior mesenteric arteries, and the patient was stabilized for discharge on Apixaban, Atorvastatin, and Aspirin. On follow-up two weeks later, the patient underwent duplex ultrasound of his mesenteric arteries, which demonstrated complete patency and no evidence of significant mesenteric disease. Despite the commonly asymptomatic nature of the coronavirus, growing evidence has identified a link between COVID-19 and thromboembolic events. Interestingly, most individuals infected with coronavirus who were identified to have portal vein thrombosis did not have any known risk factors for a hypercoagulable state. As the prevalence of similar cases begins to rise, we must ask if managing these asymptomatic coronavirus patients with outpatient prophylactic anticoagulation should be considered.

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ROLE OF TH17/IL-17 IN TUBERCULOSIS RELAPSE FOLLOWING DRUG TREATMENT IN A CORNELL-LIKE MOUSE MODEL

YB Martinez-Martinez¹, MB Huante¹, S Chauhan¹, KF Naqvi², P Bharaj¹, JJ Endsley<sup>1,2

1</sup>Univ. of Texas Medical Branch, Galveston, TX, Dept of Microbiology and Immunology ²Univ. of Texas Medical Branch, Galveston, TX, Dept of Pathology

One fourth of the global population is infected by Mycobacterium tuberculosis (Mtb), and ten percent will develop active tuberculosis (TB) in their lifetime. People with TB can suffer relapse (i.e. regrowth after apparent clinical cure) after antibiotic treatment, which can lead to poor outcomes, increased risk of acquired drug resistance, and higher mortality rates. Th17 cells and IL-17 are important for tuberculosis protection and prevent the advancement of disease. The aim of these ongoing studies is to demonstrate the critical role of Th17/IL-17+ cells in preventing TB relapse. C57BL6J mice were aerosol infected with Mtb-Beijing in a Cornell-like mouse model. Bacterial burden in lung and liver, relative to Th17, Th1, Th2, Tregs, and IL17+ immune populations, was assessed during active TB, at the end of anti-TB-drug treatment, and at relapse with or without anti-IL-17. Th1 (CD4+IFN-γ+ cells) increased following Mtb infection and remained elevated through drug treatment. The Th1 cells contracted several weeks following drug treatment and expanded proportional to mycobacterial growth during relapse. Th17 and other IL-17+ leukocytes in the lung also increased during active TB. In contrast to Th1 cells, Th17 cells contracted due to drug treatment and remained reduced during relapse. Depletion of IL-17 with in vivo antibody treatment, however, did not promote higher TB relapse, in contrast to controls. Definitive studies that utilize Th17 and IL-17-receptor deficient animals are underway and are expected to advance our understanding of the immune role of Th17/IL-17 in preventing TB post-drug recrudescence.

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INVESTIGATING THE MECHANISM OF POLIOVIRUS TRANSCRIPTION REGULATION

Sean McNeme^1,2, Keerthi Gottipati^2, Kyung Choi^1,2.

¹Department of Cell Biology, University of Texas Medical Branch, Galveston, TX, USA. ²Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA

Poliovirus is a member of the Enterovirus genus, characterized by small, non-enveloped viruses with positive-sense ssRNA genomes. The Poliovirus genome contains a 5’ UTR with a series of ordered stem loops that modulate translation and genome replication during infection. The Stem-loop I Cloverleaf structure serves as a promoter for replication, and stem-loops II-VI make up the internal ribosome entry site (IRES) which promotes translation. Our current study is focused on elucidating the structural and thermodynamic aspects of the mechanism of transcription regulation in Poliovirus. Viral poly-protein intermediate 3CD binds to the Cloverleaf, followed by the recruitment of host-derived protein PCBP2. The formation of the Cloverleaf/3CD/PCBP2 ternary complex is required for negative-strand synthesis. The Cloverleaf stem-loop structure is predicted to be present in all reported Enteroviruses, suggesting that the mechanism of translation regulation is conserved. We recently generated a 3.15Å Xray crystal structure of the Poliovirus CL. The predicted secondary structure of the CL includes a pyrimidine-rich bulge in stem-loop D of the CL. The structure demonstrates that the pyrimidine-rich bulge forms non-canonical pyrimidine-pyrimidine base pairing, instead of a bulge. Our structure also demonstrates a novel 3-base interaction that has not been previously reported in RNA. The 3-base interaction involves an Adenosine in Stem-loop C of the CL, and the pyrimidine-rich predicted bulge in Stem-loop D. This interaction likely stabilizes the CL in an “H” like conformation. Based on the arrangement of the four stems of CL, we propose that when 3CD binds to the Poliovirus CL, the CL undergoes a conformational shift that increases the binding affinity between the CL and PCBP2.

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SAFETY AND IMMUNOGENICITY OF A MULTI-EPITOPE DNA VACCINE FOR CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS

MC Mears^{1, 2,} MNB Cajimat^1, ADT Barrett^{1, 2,} and DA Bente<sup>1, 2, 3

1</sup>Department of Pathology, UTMB, ²Sealy Institute for Vaccine Sciences, UTMB, ³Department of Microbiology & Immunology, UTMB

Crimean-Congo Hemorrhagic Fever (CCHF) is the most widespread and medically important tic kborne viral disease. Currently, there is no approved vaccine widely available for human use. DNA vaccines offer a safe, easily scalable, cost-effective, and stable approach for human vaccination that can be tailored to develop desired immune responses. DNA vaccines encoding either the whole CCHF virus (CCHFV) glycoprotein precursor (GPC) or the two glycoproteins have only demonstrated moderate protection (>60%) in CCHFV lethal challenge animal models. It was hypothesized to increase the efficacy of candidate DNA vaccines against CCHFV by enhancing induction of cell-mediated immunity. Using a series of in silico predictions, both cytotoxic and helper T-cell epitopes were identified across the CCHFV GPC. Immunogenic regions were identified by the presense of overlapping predicted epitopes, and assessed for conservation between CCHFV strains. The multi-epitope antigen (EPIC) was created by linking 11 predicted immunogenic regions, which include 812 (75.7%) of the predicted epitopes and represent all 5 domains of the GPC. EPIC was synthesized for in vitro analyses and use as a DNA vaccine candidate in vivo. Vaccinated C57BL/6J mice received 3 doses of the DNA vaccine (25μg/dose) given at 21-day intervals via intramuscular electroporation (IM-EP). Mice were monitored daily for 5 measures of vaccine safety: body weight, temperature, clinical score, redness at the vaccination site, and vaccination limb paralysis. Mouse sera was pooled and assessed for recognition of EPIC and CCHFV GPC proteins by immunofluorescence assay to measure the humoral immune response. No adverse events or clinical signs were seen throughout the study. Antibodies to neither EPIC or CCHFV GPC proteins were detected invaccinated mouse sera. The data suggest that the DNA vaccine candidate is safe for C57BL/6J mice, although a humoral immune response was not induced. Further work is needed for development of an immunogenic CCHFV vaccine.

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INVESTIGATING THE ROLE OF HIF-1α IN RSV AND HMPV INFECTIONS

DR Morris¹, Y Qu¹, RP Garofalo¹, A Casola<sup>1

1</sup>Univ. of Texas Medical Branch, Galveston, TX

Hypoxia-inducible factors (HIF) are transcription factors that mediate oxygen utilization by regulating cellular metabolism and redox homeostasis. When oxygen is present, HIF-1α is produced and rapidly degraded following ubiquitination to allow for normal metabolic activity. Under hypoxic conditions, HIF-1α is stabilized and translocated to the nucleus where it forms a heterodimer with HIF-1β. This HIF complex then influences many biological activities through the hypoxia response element, primarily by upregulating glycolytic activity. Viruses can hijack host cell metabolism by activating HIF-1α, through hypoxia-independent mechanisms, to produce components vital for virion production. HIF-1α activation also controls the expression of genes involved in inflammation and airway remodeling, two important pathogenetic component of respiratory viruses-associated lung diseases. Human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) are leading causes of bronchiolitis among infants and young children. Recent work in our lab and others has highlighted distinct differences in disease pathogenesis between these two viruses, including changes in cellular metabolism. Here, we compare the effects of blocking HIF-1α in an hMPV or RSV mouse model of infection. Administration of PX-478, a specific HIF-1α inhibitor, significantly improved hMPV-induced bodyweight loss, clinical disease and bronchoconstriction. It was also associated with a significant reduction of lymphocyte cell count in the bronchoalveolar lavage fluid (BALF) at day one post-infection. There was no significant difference in cytokine or chemokine levels in BAL of hMPV-infected treated versus non-treated mice. Interestingly, HIF-1α inhibition in the context of RSV infection induced a chronic state of disease. Peak viral replication in the lung of PX-478-treated mice was significantly increased compared to the RSV control mice. No modulation of bronchoconstriction, BALF cellular infiltrate and cytokine profiles were appreciated at day one post-infection. These findings suggest an important and differential role for HIF proteins depending upon the specific type of viral respiratory infection.

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USING CLICKSEQ METHODOLOGY TO DEVELOP A FIELD-READY MODEL OF SEQUENCING WHOLE VIRAL GENOMES FROM MOSQUITO POOLS

V Morris^1, R Yun^2, SC Weaver^2,3,4, AL Routh^1,5

1) Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, TX, USA
2) Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, TX, USA
3) Department of Pathology, University of Texas Medical Branch, Galveston TX, USA
4) World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, TX, USA
5) ClickSeq Technologies LLC, Galveston, TX, USA

Arboviruses are responsible for a variety of diseases with few vaccines or antiviral treatments, necessitating the use of public health measures to combat outbreaks. The implementation of these public health measures mainly relies on tracking disease incidence in humans, as adequate surveillance of viral circulation in animals is lacking. Therefore, monitoring of the zoonotic hosts or the mosquito vectors themselves would give novel data to influence public health decisions. Most sequencing methods are not optimized for use in the field, therefore require the use of institutional labs to process samples. ClickSeq is a novel method of Next-Generation Sequencing library synthesis that uses azido-nucleotides as a terminating nucleotide during reverse transcription. The cDNA fragments generated have been azido-terminated, and can be chemically ligated to sequencing and indexing primers at room temperature with only copper (Cu II) and vitamin C. ClickSeq singular primers can therefore create multiple amplicons. Using this approach, we designed an experiment using lab-infected mosquitos infected with common West African arboviruses. Multiple viral specific primers, located approximately every 200-300 bp segments over the genomes of Chikungunya 37797, Zika Dakar, Yellow Fever Asibi, and Dengue serotype 2, were designed to amplify the viral RNA from total mosquito RNA. Pools of lab-infected mosquitos were suspended in Trizol, and RNA was extracted. Next, a single pool of all viral primers was used to amplify any viral RNA present in the samples. We have shown that using this method, we can identify the whole viral genome of a single infected mosquito in 20 and 40 total mosquitos, with differentiation between strains of alpha and flaviviruses. The use of a single pool of primers across all samples minimizes lab materials thus making this method ideal for fieldwork. These findings suggest this method can be utilized to improve public health surveillance of endemic mosquito-borne diseases.

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MULTIPHOTON IMAGING OF POWASSAN VIRUS INDUCED NEUROINFLAMMATION

Jacob Nelson^1, Lorenzo Ochoa^2, Gracie Vargas^2, Alexander Freiberg<sup>1

1</sup>University of Texas Medical Branch, Galveston, TX, Department of Pathology ²University of Texas Medical Branch, Galveston, TX, Center for Biomedical Engineering

Powassan virus (POWV) is the cause of severe neuroinflammation in North America. Patients infected with POWV can have central nervous system involvement and can progress to encephalitis. Patients often have sequelae that can be debilitating and require years to fully recover, if at all. The underlying molecular mechanisms of POWV neuropathobiology and sequelae are not well known. The goal of this project is to identify causes of pathogenesis unique to tick-borne flaviviruses using tissue clearing and multiphoton imaging. Previous studies with similar tick-borne flaviviruses have shown that laminal membrane structures form in the dendrites of infected murine cells in culture. The laminal membrane structures then inhibit the function of the neuron while modifying the structure of the dendrite. We are using a novel tissue clearing process combined with multiphoton imaging to show the presence of laminal membrane structures in vivo. The tissue clearing process is currently being optimized, with a brain hemisphere being able to show infected murine neurons at depths of up to 975 microns. We have also identified areas of colocalized virus and neuronal membranes which could be laminal membrane structures which contribute to the pathology and sequelae of POWV infection. This is significant because this is the first time that laminal membrane structures have been detected in vivo and it was previously unknown if POWV causes laminal membrane structure formation. 

 

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UTMB APPROACH TO RESTARTING OUTPATIENT ENDOSCOPY AMIDST THE COVID-19 PANDEMIC

C Nguyen(1), KT Kline(2), H Abdulla(2), R Pavurala(2), L Watson(2), M Sheridan(2), J Zaibaq(2), T Le(2), O Tayyem(2), S Cohn(2), S Parupudi(2)

(1)Department of Internal Medicine, University of Texas Medical Branch
(2)Department of Gastroenterology and Hepatology, University of Texas Medical Branch

The COVID-19 pandemic has led to widespread disruptions of outpatient endoscopic procedures. The Centers for Medicare and Medicaid Services, in concordance with multi-society guidelines, provided a framework for gradual reopening of outpatient procedures. Here we describe the process and outcomes of the reinstitution of outpatient endoscopy applying appropriate risk mitigation precautions to prevent further spread of the virus. 281 patients underwent outpatient endoscopic procedures from 3/16/2020-4/20/2020. Personal protective equipment protocols were established: head coverings, gowning, fit-tested N95 masks, overlying surgical masks, eye protection, and double-gloving for all endoscopy staff. Enhanced cleaning techniques between cases, doffing, donning, hand hygiene techniques were implemented with quality checks. All patients underwent pre-procedure symptom screening, COVID-19 PCR testing within 72-hours or rapid testing on the day of the procedure. Patients with positive tests or symptoms were rescheduled. Following procedures, patients were contacted by telephone to inquire if they underwent COVID-19 testing within 14 days post-procedure. Symptoms, reason for testing, and results were documented. Of the 281 patients (87 upper endoscopy, 137 lower endoscopy), 94.7% received pre-procedure rapid molecular testing. 24% of upper endoscopy patients and 3.7% of lower endoscopy patients were tested post-procedure due to concerning symptoms, hospitalization, or pre-procedure clearance. Fever, cough and gastrointestinal symptoms were most commonly reported. All post-procedure tests were negative for COVID-19. Of the 4 patients that reported fever, 3 had negative rapid assay testing. One patient declined testing because fever resolved within 24 hours. During the study period, no cases of endoscopy unit transmission to staff were identified. By practicing our strict risk mitigation strategies, which were in line with experiences and practice in Italy implicating low risk of transmission during endoscopy if the proper precautions are taken, we were able to limit the peri-procedure transmission to nil as shown by the data from our post-procedure surveys.

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Development of a novel "antisense Oligonucleotide-based" therapy for Cryptosporidiosis.

J Ortega-Méndez^1, A Sadigova^2, A Castellanos<sup>3

1</sup>Univ. of Texas Medical Branch, Galveston, TX

The World Health Organization reports diarrhea kills around 760,000 children under five every year. Cryptosporidium is a leading cause of moderate-to-severe diarrhea in children. Nitazoxanide is the only FDA-approved medication available for cryptosporidiosis treatment, but it has limited efficacy in malnourished children and is ineffective in immunocompromised individuals. Therefore, more effective treatment options are urgently needed. Recently we developed a unique technology to silence genes in this pathogen using antisense oligonucleotides (ASOs), since some ASOs blocks expression of genes essential for parasite survival, then we hypothesized that ASOs would be useful to treat Cryptosporidium infection. However, it has been shown that ASOs are susceptible to degradation in the intestinal track and cause unspecific activation of innate immune response. In order to address these gaps, we have used next generation ASOs-FANAs in a murine model of Cryptosporidiosis. We evaluated oral delivery of ASOs and ASOs-FANA on intestinal cells at 4, 8 and 24 hrs by fluorescent microscopy. Our results showed that ASOs-FANA are detected up to 24 hrs. This data suggests that chemical modifications on ASOs-FANAs increases stability and reduces susceptibility to degradation, supporting the feasibility of using ASOs-FANAs decrease Cryptosporidium infection by oral treatment. In addition, we demonstrated that ASOs anti-Cryptosporidium do not activate inflammatory response on uninfected intestinal cells. Our next objective will be to evaluate oral delivery at longer timeframes and test the anti-cryptosporidial effect of selected ASOs in our murine model.

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MODELING AIRWAY MACROPHAGE AND T CELL FUNCTION AND INTERACTIONS IN HUMANIZED MICE

Sunil Palani^1, Christopher Bauer² and Keer Sun<sup>1

1</sup>University of Texas Medical Branch, Galveston, TX, ²University of Nebraska Medical Center, Omaha, NE

Influenza virus can cause primary viral pneumonia. Moreover, a frequent sequela of influenza pneumonia is secondary bacterial infection. T cells play an essential role in mediating lung viral clearance. Conversely, alveolar macrophages (AM) are resident phagocytes responsible for efficient clearance of opportunistic bacterial pathogens such as Streptococcus pneumoniae. Thus, appropriate AM and T cell function and interaction are essential for maintaining the lower airway sterile. In conventional mouse models, we have shown that AM are essential and efficacious in pneumococcal clearance. This innate defense mechanism, however, is completely abrogated after influenza-induced T cell activation, which in turn causes lethal susceptibility to otherwise noninvasive pneumococcal infection. Mice repopulated with human hematopoietic cells are a powerful tool for studying human immune function in vivo. Rag2-/-Il2rg-/- mice lack T, B, and innate lymphoid cells. Human(h)Il3/Csf2 knock-in (KI) strain is further generated from Rag2-/-Il2rg-/- mice in which human genes encoding Il3 and Csf2 are knocked into their respective mouse loci. As a result, hIl3/Csf2 KI mice are also devoid of mouse AM. In this study, we show that hIl3/Csf2 KI mice support airway macrophage and T cell reconstitution after human peripheral blood mononuclear cell (PBMC) engraftment. Importantly, we show that human macrophage engraftment improves lung bacterial clearance. This human PBMC-mediated protection, however, is disrupted after influenza infection of humanized hIl3/Csf2 KI mice. We have performed single-cell RNA sequencing analysis (scRNA-seq) of human T cells isolated from humanized mice before and after influenza infection. The results indicate that influenza infection activates specific interferon (IFN) response in a subset of CD4 T cells, in agreement with findings in conventional animal models and observations in humans. Collectively, these results suggest that our humanized mouse model is applicable for the investigation of human macrophage and T cell function and interaction during pulmonary infection.

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OXIDATIVE DNA MODIFICATION TO GUANINE RECOGNIZED BY OGG1 DIRECTS TGF-ΒETA1-DEPENDENT FIBROGENIC GENE ACTIVATION

L Pan, Y Xue, I Boldogh

Univ. of Texas Medical Branch, Galveston, TX

Chronic lung diseases, including allergic asthma and chronic obstructive pulmonary disease, are characterized by chronic injury and repair producing dysfunction of epithelial barrier. In this context, increased oxidative stress, growth factor- and cytokine stimulation induce transcriptional reprogramming of epithelium leading to epithelial-mesenchymal transition (EMT), ultimately resulting in airway remodeling. However, the molecular mechanism that underlies activation of remodeling process remains elusive. Here, we studied how oxidative modification to DNA in epithelial cells activates fibrogenic gene expression. In our experimental model, human small airway epithelial cells (hSAEC) were treated with transforming growth factor beta 1 (TGF-β1). TGF-β1 increased 8-oxoG level, a typical oxidative DNA damage by nuclear oxidative wave(s), on COL1A1, FN1 and VIM gene promoters. 8-oxoguanine DNA glycosylase 1 (OGG1) is the primary protein that recognize and repair 8-oxoG, meanwhile, we found OGG1 was responsible for recruitment of transcription factors, including SMAD2/3 and NF-κB, to the promoters of prototypic genes, as documented by chromatin immunoprecipitation (ChIP) and electrophoresis mobility shift assay (EMSA). OGG1 knockdown or a small molecule inhibiting OGG1 binding to its substrates (TH5487) in genomic DNA decreased COL1A1, FN1 and VIM gene expression and maintained epithelial morphology under TGF-β1 treatment. Importantly, similar function of OGG1 was found in mouse lungs after TGF-β1 exposures. Collectively, our data demonstrates that OGG1 at 8-oxoG in gene regulatory sequences is a major regulator of DNA occupancy of profibrotic transcriptional factors, and OGG1 inhibitor used here as a tool may have clinical significance in prevention/treatment of airway remodeling.

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STRUCTURAL INSIGHT INTO REPLICATION FIDELITY AND PRIMER SHUTTLING IN HUMAN MITOCHONDRIAL DNA POLYMERASE GAMMA

J Park^1,2, MB Sherman^1,2, YW Yin<sup>2,3

1</sup>Department of Biochemistry and Molecular Biology, ²Sealy Center for Structural Biology, ³Department of Pharmacology and Toxicology, Univ. of Texas Medical Branch, Galveston, TX

DNA polymerase gamma (PolG) is the sole DNA replicase in human mitochondria. Nucleoside Reverse Transcriptase Inhibitors (NRTIs), which act as chain terminators once incorporated by the HIV reverse transcriptase (RT), effectively inhibit viral replication. However, PolG is an off target of NRTIs, leading to mitochondrial DNA deletions and mutations that clinically manifest as neurological and cardiovascular diseases. Thus, it is important to understand the mechanism behind incorporation and excision of NRTIs to reduce drug toxicity. Polymerase (pol) activity for DNA synthesis is shared by both PolG and HIV RT. Using X-ray crystallography, we have previously provided structural mechanism behind drug discrimination in the pol site of PolG pre-incorporation. However, exonuclease (exo) activity for proofreading is unique to PolG, but the mechanisms behind drug recognition and excision by the exo site post-incorporationare still unknown. To this end, we first set out to understand high replication fidelity of PolG under normal replication circumstances. We’ve determined cryo-EM structures of four different PolG-DNA complexes at near-atomic resolution. Here, we reveal both the error detection mechanism in the pol site post-incorporationand error excision mechanism in the exo site. Global movement of accessory B subunit in conjunction with displacement of primer by the “ejection loop” facilitates the shuttling of the primer from pol to exosites. In addition, we report previously unobserved transition conformation, which gives deeper insight into initiation of primer shuttling and communication between pol and exo sites, which are approximately 35 Angstroms apart. Global and local structural changes associated with error recognition and excision show dynamic and well-coordinated communication between pol and exo sites required for high fidelity DNA replication. This information can be exploited to improve current NRTI design to reduce PolG-mediated drug toxicity.

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EHRLICHIA CHAFFEENSISTRP120 NOTCH LIGAND MEMETIC SLIM INTERACTS WITH NOTCH EPIDERMAL GROWTH FACTOR DOMAINS TO ACTIVATE NOTCH SIGNALING

LaNisha L. Patterson^1, CaitlanByerly^1, Jere W McBride<sup>2,

1</sup>Department of Neuroscience, Cell Biology and Anatomy, ²Department of Pathology and Microbiology & Immunology, University of Texas Medical Branch, Galveston, TX, USA

Human monocytotropic ehrlichiosisis a tick-borne zoonosis caused by the small obligately intracellulargram-negative bacterium,Ehrlichia chaffeensis. Recent studies by our laboratory have shown host-pathogen interactions to occur through a TRP effector, TRP120, which is able to activate the Notch pathway during infection. Ultimately, this outcome leads to downregulation of innate immune response through down regulation of toll-like receptors 2/4, however this outcome may not have a significant role in intracellular survival of Ehrlichia.Notch activation has been shown to play significant roles in various functions, including cellular homeostasisandinnate immune mechanisms such asautophagy and apoptosis.We have demonstrated direct interaction of TRP120with ADAM17, a Notch metalloprotease, and FBW7, a Notch antagonistvia yeast two hybrid analysis. Further, colocalization of TRP120 with both ADAM17 and the Notch-1 receptor has been demonstrated.Thus,we hypothesize that E.chaffeensis TRP120 is a novel non-canonical Notch ligand that activates Notch signaling via a TRP120 motifmolecularly interacting with Notch receptor EGF domains. Using sequence databases, we have demonstrated homology of TRP120 and Notch ligands. We have further demonstrated that theTRP120-TR domain is responsible for Notch activation and have currently identified a 11-amino acidshort linearmotif(SLiM), that is required for activating Notch signaling. Alanine mutagenesis was used to determine the required amino acids found within the SLiM memetic motif. Results demonstrated weak Notch activation withTRP120mutants. This data indicates that the entire 11-amino acid TRP120 SLiM memetic motif is required for Notch activation. Taken together, these results indicate that a specific TRP120-TR sequence is important for Notch activation during E.chaffeensis infection. This study will significantly impact our understanding of the molecular interactions utilized by intracellular bacterial pathogens to function as eukaryotic ligand mimics to manipulate conserved signaling pathways for infection and intracellular survival.

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PROTECTIVE ROLE OF IL-21+ AND IL-21+IFN-γ+ CELLS IN Plasmodium chabaudi INFECTION

L Puebla-Clark^1, K Gbedande^1, VH Carpio^1, J Craft^2, R Stephens<sup>1

1</sup>Univ. of Texas Medical Branch, Galveston, TX; ²Yale School of Medicine, New Haven, CT

Protection from malaria is B and T cell dependent; however, germinal centers (GCs) that make high affinity antibody are delayed for three weeks. IL-21 from T follicular helper (Tfh) cellspromotes differentiation of plasma cells, while IFN-γ from T helper-1 (Th1) cells controlsparasitemia. Th1 cytokines can inhibit GCs, and the majority of CD4 effector T cells in Plasmodium chabaudiinfection are Th1/Tfh hybrid cells. Th1/Tfh cells express markers of both subsets (IFN-γ+IL-21+), and are able to help B cells make antibody in vitro. However, Th1 features of the hybrid could explain the delay of antibody production in vivo. To understand Th1/Tfh function in vivo, we studied cytokine kinetics in infected fluorescent cytokine reporter mice (IL-21, IL-4, IFN-γ). The Th1/Tfh hybrid population dominates at the peak of infection but is low on day 21. IL-21+IFN-γ- Tfh cells persist, and are mainly GC Tfh by phenotype (CXCR5hiPD-1hi). To compare the contribution of each subset in protection and GC formation, we adoptively transfered them into T cell deficient recipients and infected. While parasitemia was similar, recipients of IL-21+ Tfh lost less weight, suggesting less pathology. IL-21+ T cells also promoted GC B cells more than Th1/Tfh. 30% of the IL-21+ transfered cells became IL-21+IFN-γ+ cells. We showed that IL-27 drives the hybrid phenotype. We now show that despite being T-bet dependent like Th1 cells, that primarily use glycolysis, Th1/Tfh use more mitochondrial respiration than Th1, suggesting some Tfh properties like longevity. In vitro, adding IL-27 increases mitochondrial mass. Overall, these results suggest that IL-21+ producing T cells have a more protective role during malaria than the Th1/Tfh hybrid, despite the fact that natural infection drives more Th1/Tfh. As metabolism drives T cell differentiation, this study will lead to potential therapeutics to improve GC function in malaria.

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EFFECTS OF ACUTE AND CHRONIC TRYPANOSOMA CRUZI INFECTION ON FERTILITY RATE AND PUP’S HEALTH IN MICE

LE Rios (1, 2), N Lokugamage (1), NJ Garg (1)

(1)Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX(2)Department of Neuroscience, Cell Biology and Anatomy, University of Texas Medical Branch, Galveston, TXChagas disease(CD) is a neglected tropical disease caused by the parasite Trypanosoma cruzi(Tc). Globally, >2-million women of child-bearing age are infected with Tc, and up to 10% of fetuses carried by infected mothers are born with infection. Women infected with Tc are at risk of pregnancy-associated complications such as stillbirth, pre-term birth, and low birth weight of neonates. Anti-parasite therapies, benznidazole and nifurtimox, are contraindicated in pregnant women because of potential teratogenic effects. Post-neonatal health of infants born to infected mothers is an area of study that has yet to be explored. Purpose of this project was to develop a murine model of congenital transmission and determine the effects of acute and chronic Tc infection in pregnant mice on the fertility rate and the health of pups. Model will allow us to study the efficacy of new drugs and vaccines against congenital transmission. Balb/c female mice were infected with Tc, and 21 days (acute infection phase) or 90 days (chronic phase) later, mated with male mice. Some pups were weighed and euthanized at birth and others were collected 20 days post-partum. Pup brain, heart, skeletal muscle and viscera were collected and analyzed using qPCR to measure parasite burden. H&E and Masson’s Trichrome stains were used for histopathological analysis of brain and heart tissue to identify tissue inflammation and fibrosis. Acute and chronic infection in dams increased the breeding interval, decreased the fertility rate, and led to vertical transmission in 12-63% of pups. Infected dams delivered pups that exhibited decreased survival and increased birth weight. Preliminary observations indicate increased inflammation in brain and cardiac tissue and increased cardiac fibrosis. Mice offer a promising model to study the effects of acute and chronic Tc infection in dams on the pups’ health outcomes and examine the efficacy of new treatments against congenital transmission.

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DUAL RNA-SEQ REVEALS A TYPE 6 SECRETION SYSTEM-DEPENDENT BLOCKAGE OF TNF-A SIGNALING, AS AN IMPORTANT MEDIATOR OF BURKHOLDERIA PSEUDOMALLEI VIRULENCE

JI SANCHEZ-VILLAMIL (1), D TAPIA (1), AG TORRES (1,2)

(1) Department of Microbiology and Immunology, Univ. of Texas Medical Branch, Galveston (2) Department of Pathology, Univ. of Texas Medical Branch, Galveston

Melioidosis is a disease caused by the Gram-negative bacillus Burkholderia pseudomallei (Bpm), commonly found in soil and water of endemic areas. Naturally acquired human melioidosis infections can result from either exposure through percutaneous inoculation, inhalation, or ingestion of soil-contaminated food or water. Our prior murine studies recognized the association of Bpm as an enteric pathogen, capable of establishing acute or persistent gastrointestinal infections following oral inoculation. Nevertheless, the specific mechanisms and virulence factors involved in the pathogenesis of Bpm as enteric infection are unknown. In our study, we standardized an in vitro intestinal infection model using mouse primary intestinal epithelial cells (mIECs). We demonstrated that Bpm requires a functional T6SS for full virulence, bacterial dissemination, and lethality in mice infected by the intragastric route. Further, we performed dual RNA-seq analysis on Bpm-infected mIECs to evaluate differentially expressed host and bacterial genes in the presence or absence of a T6SS. Our results showed a dysregulation in the TNF-a signaling via NF-kB pathway in the absence of the T6SS, with some of the genes involved in inflammatory processes and cell death. To further support the biological relevance of these findings, we demonstrated the dependence of a functional T6SS in the modulation of NF-kB activation. Analysis of the bacterial transcriptome suggested a role of virulence factors associated with bacterial regulation during infection in the absence of a T6SS. From a Bpm transposon mutant library, we showed that a transposon interrupting bicA, encoding a T3SS effector, ablated plaque formation by Bpm in the presence of a functional T6SS. Overall, these results highlight the importance of secretion systems in the pathogenesis of enteric Bpm infection.

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DEVELOPING A SURROGATE MODEL THROUGH WHICH TO STUDY LASSA VIRUS-MEDIATED SUDDEN-ONSET SENSORINEURAL HEARING LOSS

RA Sattler(1), J Maruyama(1), S Urata(1), T Makishima(1), S Paessler(1)

(1)Univ. of Texas Medical Branch, Galveston, TX

Lassa virus (LASV), the causative agent of Lassa fever (LF), is endemic in western Africa. The CDC estimates that the number of infections reach up to 300,000 people with 5,000 deaths from LF each year. There are no vaccines and limited therapeutics are available. One-third of LF patients will develop sudden-onset sensorineural hearing loss (SNHL), which is permanent in two-thirds of cases. SNHL is generating a huge socioeconomic burden in endemic regions due to the associated isolation and stigmatization. We hypothesize that this hearing loss is due to an inflammatory immune response against the virus. Understanding the mechanism driving SNHL would allow for the development of therapies to significantly reduce disease burden. However, due to the high-containment requirements of LASV, a surrogate model through which to study SNHL is desirable. To do this, we used ML29, a recombinant vaccine candidate encoding the major immune antigens of LASV. This surrogate model will allow us to further study the contributions to pathogenesis and sequelae resulting from the immune response against LASV. Specifically, this project focused on the contributions of CD4 and CD8 T cells to pathogenesis and the development of sequelae, most notably SNHL. Ultimately, the results of this project will contribute to the development of safer vaccine candidates and potential therapeutics for LF patients. Moreover, this is the first reliable murine model for viral-induced SNHL, lending it great importance outside the field of virology. This model will contribute to the development of therapeutics for the millions suffering hearing loss worldwide.

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Investigating Nonstructural Protein 16, a Viral mRNA Capping Enzyme Critical for Host Evasion, of SARS-CoV-2

CD Schindewolf(1), KG Lokugamage(1), MN Vu(1), BA Johnson(1), VD Menachery(1)

(1)University of Texas Medical Branch, Galveston, TX

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome (SARS)-CoV-2 is of immense importance for informing the next wave of therapeutics. We plan to investigate the role of the nonstructural protein 16 (nsp16) of SARS-CoV-2 in pathogenesis. Nsp16 is a ribonucleoside 2’-O methyltransferase (MTase), an enzyme that catalyzes the transfer of a methyl group to mRNA as part of the capping process. The 2’-O MTases expressed by CoVs and other viruses contain a conserved catalytic tetrad, and by capping viral RNA, they conceal it from cap-sensitive host restriction. We hypothesize that SARS-CoV-2 that contains a mutation in one of the conserved catalytic residues of nsp16 will be attenuated both in vitro and in vivo, and that this nsp16 mutant could further serve as the basis for an attenuated vaccine. In this poster, we characterize the attenuation of the nsp16 mutant in vitro by comparing its replication kinetics with those of wild-type SARS-CoV-2. Future studies are planned to examine the extent and nature of nsp16-mutant SARS-CoV-2 in vivo.

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ZIKA VIRUS INDUCES NEURONAL AND VASCULAR DEGENERATION IN DEVELOPING MOUSE RETINA

Shuizhen Shi^1, Yi LI^1, Fan Xia^1, Chao Shan^2, Yonju Ha^1, Jing Zou^2, Awad Adam^3, Ming Zhang^4, Tian Wang^3, Hua Liu^#,1,5, Pei-Yong Shi^#,2,6, Wenbo Zhang<sup>#,1,6,7

1</sup>Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas, USA
²Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, USA
³Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA
⁴Department of Cellular Biology and Anatomy, Augusta University, Augusta, Georgia, USA
⁵Sealy Center for Vector-Borne and Zoonotic Diseases, University of Texas Medical Branch, Galveston, TX, 77555, USA
⁶Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX, 77555, USA
⁷Departments of Neuroscience, Cell Biology & Anatomy, University of Texas Medical Branch, Galveston, Texas, USA

Zika virus (ZIKV), a mosquito-borne flavivirus, can cause severe eye disease and even blindness in newborns. However, ZIKV-induced retinal lesions have not been studied in a comprehensive way, mechanisms of ZIKV-induced retinal abnormalities are unknown, and no therapeutic intervention is available to treat or minimize the degree of vision loss in patients. Here, we developed a novel mouse model of ZIKV infection to evaluate its impact on retinal structure. ZIKV (20 PFU) was injected into the neonatal wild type C57BL/6J mice at postnatal day (P) 0 subcutaneously. Retinas of ZIKV-infected mice and age-matched controls were collected at various ages, and retinal structural alterations were analyzed. We found that ZIKV induced progressive neuronal and vascular damage and retinal inflammation starting from P8, and ZIKV-infected retina exhibited dramatically decreased thickness with loss of neurons, initial neovascular tufts followed by vessel dilation and degeneration, increased microglia and leukocyte recruitment and activation, degeneration of astrocyte network and gliosis. The above changes may involve inflammation and ER stress-mediated cell apoptosis and necroptosis after ZIKV infection. Moreover, we tested the feasibility of using this model in drug discovery and vaccine safety evaluation, and found that ZIKV-induced retinal abnormalities could be blocked by a selective flavivirus inhibitor NITD008 and a live-attenuated ZIKV vaccine candidate could potentially induce retinal abnormalities. Overall, we established a novel mouse model and provide a direct causative link between ZIKV and retinal lesion in vivo, which warrants further investigation of underlying mechanisms of ZIKV-induced retinopathy and development of effective therapeutics.

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INTRAVITAL MICROSCOPY REVEALS VASCULAR DYSREGULATION AND MICROGLIOSIS IN EXPERIMENTAL CEREBRAL MALARIA

Solomon OD^1, Villarreal P^1, Domingo ND^1, Ochoa L^1, Puebla Clark L^1, Aussenac F^1, Cardona A^2, Stephens R^1, Vargas G<sup>1

1</sup>University of Texas Medical Branch, Galveston, TX; ²University of Texas San Antonio, San Antonio, TX

Cerebral malaria (CM) is the most deadly pathology caused by Plasmodium falciparum infection, which primarily impacts children residing in sub-Saharan Africa. Approximately 405,000 individuals die from CM yearly, survivors experience neurological sequelae including behavioral & cognitive defects. Historically, much research on the pathology in the brain focused on the role of parasite blocking cerebral vasculature, overlooking the important contribution of the feedback loop of inflammation and coagulation that ultimately drives vascular congestion. Ina hyperinflammatory model of Plasmodium infection with cerebral symptoms, we have shown that fibrinogen-positive deposits block the cerebral vasculature, and that astrocytes and microglia become reactive at peak of infection (7 or 8 dpi) (1). In vivo glial and glial-vascular dynamic responses have not been studied in experimental CM (eCM). We applied intravital multiphoton microscopy to evaluate these responses in vivo using fluorescent fibrinogen and a IL10^-/-CX3CR1^GFPCCR2^RFP to highlight microglia and CCR2+ leukocytes. We hypothesized that hyper-coagulation and blood brain barrier disruption in eCM leads to chronic activation of microglia, a sign of neuroinflammation. Our studies reveal that microhemorrhages in eCM, which largely occur proximal to sites of thrombi, result in significant extravasation of luminal fibrinogen depositing as puncta in the brain parenchyma. In addition, microglia specifically migrate to areas containing congested vessels with significant increase in vessel associated microglia (VAM) in infection compared to control (40% vs. 20% , p<0.05). VAM also display reactive features such as amoeboid shape compared to those localized away from vessels displaying a ramified surveying morphology. Finally, intravital microscopy reveals an increase in vascular events such vasoconstriction & vasodilation, disrupted blood flow, and coagulation in P. chabaudi compared to control. In summary, this study reveals key dynamic responses in eCM which further confirms an interaction between microglia and vessels that play an important role in the progression of eCM.

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Development of an Inducible Mycolactone Expression System to study Mycobacterium ulceransInfection and Macrophage Modulation

Emily J Strong^1, Sunhee Lee<sup>1

1</sup>Univ. of Texas Medical Branch, Galveston, TX,

Mycobacterium ulcerans is the causative agent of Buruli Ulcer (BU), the third most common mycobacterial infection. Virulent M. ulcerans secretes mycolactone, a polyketide toxin. Most observations of M. ulcerans infection are described as an extracellular milieu in the form of a necrotic ulcer. However, almost all other Mycobacterial infections are intracellular. While some evidence exists of an intracellular lifecycle for M. ulcerans during infection, the exact role that mycolactone plays in this process is poorly understood. Many previous studies have relied upon the addition of purified mycolactone to cell culture systems to study its role in host response modulation. However, this sterile system drastically simplifies the M. ulcerans infection model and assumes mycolactone is the only virulence factor expressed by M. ulcerans. Here we show the addition of purified mycolactone to macrophages during M. ulcerans infection overcomes the bacterial activation of the mechanistic Target of Rapamycin (mTOR) signaling pathway that plays a substantial role in regulating different cellular processes, including autophagy and apoptosis. To further study the role of mycolactone during M. ulcerans infection, we have developed an inducible mycolactone expression system. Utilizing the mycolactone deficient M. ulcerans::Tn118 strain that contains a transposon insertion in the putative beta-ketoacyl transferase (mup045), we have successfully restored mycolactone production by expressing mup045 in a tetracycline-inducible vector system which overcomes in-vitro growth defects associated with constitutive complementation. We further tested this inducible system both in-vivo and in-vitro. The inducible mycolactone expressing bacteria resulted in the establishment of infection in a murine footpad model of Buruli Ulcer similar to that observed during the infection with wild-type M. ulcerans. This mycolactone inducible system will allow for further analysis of the role of mycolactone during M. ulcerans infection.

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Investigating the role of non-structural genes in attenuation of yellow fever vaccine strain 17D

AE Strother, ADT Barrett<sup>1

1</sup>Univ. of Texas Medical Branch, Galveston, TX

Yellow fever is preventable by the live attenuated vaccine, strain 17D. This vaccine, developed in the 1930’s is considered the gold standard for live attenuated vaccines. Despite its long use and efficacy, the mechanism(s) of attenuation and immunogenicity are poorly understood. Strain 17D differs from its wild-type (WT) parental strain, Asibi, by only 20 residues, 11 of which are within the non-structural (NS) proteins. The goal of this project is to use reverse genetics to determine the contribution of the different NS proteins to the attenuated phenotype of 17D. To date, this study has evaluated residues in NS2B, NS4A, NS4B, and NS5. By introducing these residues from the wild-type Asibi into the 17D vaccine strain and vice versa, the contribution each of the residues to the change in viral phenotype was examined with respect to viral population structure and antiviral sensitivity. Changes in viral diversity were evaluated using Next Generation Sequencing (NGS) to determine Shannon Entropy and Single Nucleotide Variants (SNVs). Previous studies found that Asibi displays higher Shannon Entropy values and therefore increased diversity compared to 17D. Due to increased genetic diversity correlating to an increased mutation rate, as hypothesized WT Asibi is more susceptible than 17D vaccine to the antiviral Ribavirin as well. Based on the NGS analysis, two residues have been identified between 17D and Asibi that contribute to differences in population diversity, namely NS4B-95 and NS5-836; however, no NS protein has been identified that significantly impacts Ribavirin sensitivity. This is indicative that multiple residues in more than one NS protein that are contributing to antiviral resistance. It is hypothesized that this is due to the replication complex as a whole rather than individual NS proteins. Further analysis of Ribavirin-treated viruses is ongoing, exploring if the antiviral impacts the population structure of the chimeric viruses.

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ENVIRONMENTAL CONTAMINATION WITH FASCIOLA EGGS PASSED IN LIVESTOCK STOOL IS ASSOCIATED WITH HUMAN INFECTIONS IN THE ANTA PROVINCE OF CUSCO, PERU.

MB Tanabe MD^{1, 2,} J. Prochaska MPH PhD^{2, 3,} Maria Luisa Morales RN^4, Martha Lopez BS^4, MM Cabada MD MSc<sup>1,4

1</sup>Infectious Diseases Division, Department of Medicine, University of Texas Medical Branch, Galveston,TX, USA
² Graduate School of Biomedical Sciences, University of Texas Medical Branch, Galveston, TX, USA
³ Department of Preventive Medicine, University of Texas Medical Branch, Galveston, TX, USA
⁴ Cusco Branch – Tropical Medicine Institute, Universidad Peruana Cayetano Heredia, Cusco, Peru

Fascioliasis is an emergent infectious disease but little is known about the dynamics transmission. We hypothesized that environmental contamination with Fasciola eggs passed in livestock stool is associated withhuman fascioliasis. We analyzed data from a cross-sectional study among children aged 3-16 years old in 3 districts of the Anta Province in the Cusco region of Peru. Three stool specimens were collected from each child and livestock samples around the children’s residence were collected and analyzed using microscopy for Fasciola eggs. Each sample was geographically tagged. A total of 2070 children were enrolled, 49.5% were female, the average age was 9.1 (± 3.5) years, and the Fasciola prevalence was 7.2%. The most common district of residence was Anta (50.8%). Stool from 2627 livestock was collected. Fasciola prevalence in livestock stool was 31.3% with the highest prevalence in the Ancahuasi (45.1%) and Anta (44.4%) districts. Donkey stool had the highest prevalence of Fasciola (60.4%), followed by sheep (40.31%) and cattle (32.2%). The logistic regression analysis showed no association between infected children in a residence and finding livestock stool with Fasciola eggs in a 50 m, 100 m, and 200 m radius. A significant association was found between having infected children in a residence and finding cattle stool infected with Fasciola in a 50 m (OR 1.46, 95%CI 1.12-1.9, p= 0.005), 100 m (OR 1.23, 95%CI 1.04-1.46, p= 0.018) and 200 m (OR 1.1, 95%CI 1.00-1.22, p= 0.041) radius around the residence. The median distance to a positive animal stool from a residence with infected children was 40.7 m (IQR=86.7) compared to 237.9 m (IQR=448.7) from a non-infected residence (p< 0.0001). The need for large-scale analysis to understand the dynamics of infection is imperative, as strategies for infection control are urgently needed.

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2 CEREBRAL MALARIA INDUCED NEURONAL HYPER-EXCITABILTY RESCUED VIA TNF SIGNALING MODULATION: POTENTIAL FOR NEW THERAPUETICS

Authors: CM Tapia^1, ND Domingo^2, NM Dvorak^1, R Stephens^2,3, F Laezza<sup>1,3.

1</sup>Departments of Pharmacology and Toxicology, ²Internal Medicine, Division of Infectious Diseases, ³Institute for Human Infections and Immunity Univ. of Texas Medical Branch, Galveston, TX 77555

Cerebral malaria (CM) kills an estimated 425,000 children per year with seizures increasing the likelihood of death. Hyperinflammation, including an excess of TNF in relation to the regulatory cytokine IL-10, is a key mediator in the pathology of CM. Systemic inflammation and BBB disruption drives gliosis focused near the vasculature, which is particularly evident in the hippocampus. Yet, the mechanisms underlying increased susceptibility to seizure in conjunction with CM-induced neuroinflammation are poorly understood. Through high-throughput screening of a chemical library (~70,000 compounds), we identified a novel signaling pathway downstream of TNF that controls excitability of CA1 hippocampal pyramidal neurons though modulation of thevoltage-gated Na+ channel (Nav) 1.6, and its regulatory accessory protein, fibroblast growth factor 14 (FGF14). Here we have striking new results using ex vivo whole-cell patch-clamp electrophysiology showing that CM induced increase in CA1 pyramidal neuron firing is mediated via TNF signaling that can be prevented via in vivogenetic silencing of FGF14. Furthermore, we have shown that small molecules targeting the downstreamFGF14:Nav1.6 channel complex can reverse the increased firing. Thus, pharmacologically targeting the FGF14:Nav1.6 channel complex may prove a viable therapeutic strategy to reduce seizures and decrease risk of death from hyperinflammatory cerebral malaria.

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INVESTIGATING THE ROLE OF PATTERN RECOGNITION RECEPTORS IN SARS-COV-2 INFECTION IN HUMAN LUNG EPITHELIAL CELLS

VY Tat^1, A Drelich^1, KR Kempaiah^1, JC Hsu^1, S Widen^1, N Garg^1, CTK Tseng<sup>1

1</sup>Univ. of Texas Medical Branch, Galveston, TX

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus infectious disease (COVID-19) pandemic. Currently, more than 136 million people have been infected worldwide, and more than 2.9 million have succumbed to COVID-19.The objective of this project was to determine spatial and global host responses of pathologically relevant human lung epithelial cells to SARS-CoV-2 infection and to identify pathways involved in the pathogenesis of COVID-19. RNA was isolated from SARS-CoV-2 infected (MOI=1.0) human bronchial epithelial Calu-3/2B4 cells at 0-,12-, 24-, and 48-hours post-infection. Illumina NextSeq 550 was performed, and genes with expression value greater or less than a log2 scale of ± 0.5 with p-value <0.05 was included in Ingenuity Pathway Analysis. Analysis of Next Generation Sequencing data revealed activation of pattern recognition receptor pathways such as RIG-I-like receptors (RLRs). In Calu-3/2B4 cell lines with knockdown of RLRs, SARS-CoV-2 replication was increased, while overexpression of RLRs led to decreased viral replication. These findings suggest that SARS-CoV-2 infection stimulates a robust innate immune response in pathologically relevant human lung epithelial cells, and pattern recognition receptors are able to restrict the replication of SARS-CoV-2.

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CHARACTERIZATION OF THE MOLECULAR INTERACTIONS THAT GOVERN THE PACKAGING OF VIRAL RNA SEGMENTS INTO RIFT VALLEY FEVER PHLEBOVIRUS PARTICLES

B Tercero^1, K Narayanan^1, K Terasaki^1, and S Makino<sup>1

1</sup>Univ. of Texas Medical Branch, Galveston, TX

Rift Valley fever phlebovirus (RVFV) has a single-stranded, negative-sense RNA genome, consisting of L, M and S segments. The virion carries two envelope glycoproteins, Gn and Gc, along with ribonucleoprotein complexes (RNPs), composed of encapsidated genomes carrying N protein and the viral polymerase, L protein. A quantitative analysis of the profile of viral RNA segments packaged into RVFV particles showed that all three genomic RNA segments had a similar packaging ability, whereas among antigenomic RNA segments, the antigenomic S RNA, which serves as the template for the transcription of mRNA expressing the RVFV virulence factor, NSs, displayed a significantly higher packaging ability. To delineate the factor(s) governing the packaging of RVFV RNA segments, we characterized the interactions between Gn and viral RNPs in RVFV-infected cells. Co-immunoprecipitation analysis demonstrated the interaction of Gn with N protein, L protein and viral RNAs in RVFV-infected cells. Furthermore, UV-crosslinking and immunoprecipitation analysis revealed, for the first time in bunyaviruses, the presence of a direct interaction between Gn and all the viral RNA segments in RVFV-infected cells. Notably, analysis of the binding ability of Gn to RVFV RNA segments indicated a positive correlation with their respective packaging abilities and highlighted a binding preference of Gn for antigenomic S RNA, among the antigenomic RNA segments, suggesting the presence of a selection mechanism for antigenomic S RNA incorporation into infectious RVFV particles. Collectively, our study illuminates the importance of a direct interaction between Gn and viral RNA segments in determining their efficiency of incorporation into RVFV particles.

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DEFINING THE ROLE OF BURKHOLDERIA PSEUDOMALLEI TOXIN-ANTITOXIN SYSTEMS IN MACROPHAGE SURVIVAL AND POLARIZATION.

Joseph D. Thiriot(1), Brittany N. Ross(1), Shane M. Wilson(1), Alfredo G. Torres(1,2)

(1)Department of Microbiology and Immunology, (2)Department of Pathology, UT Medical Branch, Galveston, TX 77555

Burkholderia pseudomallei (Bpm) is the causative agent of melioidosis, a tropical human disease that can manifest as an acute or a chronic infection and if left untreated, is lethal. Upon infection, macrophages are commonly targeted by the pathogen for invasion, colonization of tissues and persistent infections. Little is known about the mechanistic effects of Bpm on macrophage (M1 or M2) phenotype. However, it is known that macrophages alter or polarize their phenotype in response to host requirements. In the case of Bpm, the Toxin-Antitoxin systems (TAS) are survival mechanisms that respond to stress, including the immune system, and are associated with the persistent phenotype. We hypothesize that during infection, Bpm utilizes TAS to promote survival and persistence in macrophages, and toand to contribute too contribute to their anti-inflammatory (M2) polarization response. We hypothesize that Bpm uses TAS to promote survival and persistence in macrophages, and to contribute to their M2 polarization response. We have identified TA systems associated with Bpm survival in the host, and started the initial characterization of their role. In the future, we plan to evaluate markers associated with the macrophage phenotype, and define the function of specific TAS on the polarization of the infected macrophage.

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EARLY DESSEMINATION OF LEISHMANIA DONOVANI INFECTION IN MALNOURISHED MICE

AC Uscanga-Palomeque^1, EY Osorio^1, PC Melby<sup>1

1</sup>Univ. of Texas Medical Branch, Galveston, TX

Malnutrition is a risk factor for Visceral Leishmaniasis but the mechanisms behind this arepoorly understood. In a Balb/c mouse model that mimics childhood malnutrition we demonstrated previously that there is increased L. donovani dissemination by 72h after dermal challenge. In this study we investigated effects of malnutrition on early L. donovani infection. Malnourished mice (MN) challenged intradermally with L. donovani were evaluated at 2h, 6h and 12h post-infection (n=3 per group). Compared to uninfected MN mice, flow cytometryresults showed an increased percentage of dendritic cells (DCs), monocytes (Mo) and neutrophils at 6h post-infection in skin (p=0.02, p=0.02, p=0.01, respectively). In draining lymph node (dLN) a significant increase in Mo was only observed at 12h post-infection (p=0.01). Skinneutrophils and monocytes showed the highest percentage of infection at 6h (p=0.01, p=0.01 respectively) post-infection. However, infected monocytes were found only at 12h (p=0.01) in dLN. Parasite presence and its decrease over time in skin and dLN was corroborated by immunofluorescence, suggesting that immune cells are either killing or carrying the parasite to visceral sites. By qPCR, parasites could be found in the spleen at or after 6h post-infection in MN mice. Because monocytes seem to play an import role in parasite dissemination, CCR2-KO mice and inducible Cre-diphtheria toxin transgenic mice (CSFR1-DTR mice) were used. The proportion of monocytes and infected monocytes decreased in spleen and dLN of CCR2-KO mice. Administration of diphtheria toxin (DT) to CSFR1-DTR mice was highly effective in depleting monocytes from peripheral blood. Monocyte depletion abrogated parasite dissemination to the dLN and liver, but not spleen. Cell population analysis in spleen showed that DT did not clear all monocytic populations. This data suggests monocytes have a role in L. donovani dissemination, but in their absence, other cell populations, such as neutrophils, may have a role.

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UBIQUITINATION OF EBOV VP35 AT LYSINE 309 REGULATES VP35’S POLYMERASE CO-FACTOR ACTIVITY BUT NOT IFN-I INHIBITION

S van Tol(1), B Kalveram(2), PA Ilinykh(2), A Ronk(2), K Huang(2), L Aguilera-Aguirre(1), P Bharaj(2), A Hage(1), MI Giraldo(1), A Bukreyev(1,2), AN Freiberg(2), R Rajsbaum(1)

(1)Univ. of Texas Medical Branch, Department of Microbiology and Immunology, Galveston, TX,
(2)Univ. of Texas Medical Branch, Department of Pathology, Galveston, TX

Ebola virus (EBOV) VP35 is a multi-functional protein with roles in viral genome packaging, viral polymerase function, and host immune antagonism. We previously showed that VP35 is ubiquitinated at lysine 309 (K309). The stage(s) of the EBOV replication cycle for which VP35 K309 ubiquitination is required have not been investigated. We hypothesized that ubiquitination of VP35 K309 is required for EBOV replication but not VP35’s type I interferon (IFN-I) inhibition. Here, we generated recombinant EBOVs encoding glycine (G) or arginine (R) mutations at VP35 K309 (rEBOV-VP35 K309G/-R). Both mutants lack ubiquitination on K309, however the K309R mutant binds dsRNA similar to wild-type (wt) due to conservation of the basic residue. The rEBOV-VP35 K309R, but not the rEBOV-VP35 K309G, impairs IFN-I induction and signaling similar to rEBOV-GFP-wt infected cells. Using a minigenome assay, we found that the polymerase co-factor activity of both VP35 K309G and K309R mutants was impaired compared to wt VP35. Consistent with these findings, the replication of both rEBOV-VP35 K309 mutants was significantly attenuated although the K309G mutant is significantly more attenuated than the K309R virus. Using strand-specific quantitative PCR, we found that both the K309R/-G mutants are impaired in their production of viral polymerase (L) mRNA but not genomic or anti-genomic RNA. We also found that VP35 K309 ubiquitination facilitates interaction with EBOV L. These data suggest that ubiquitination of VP35 on K309 is required for VP35’s polymerase co-factor function in facilitating EBOV transcription. Further, we found that VP35 K309R mutant has an enhanced affinity for EBOV nucleoprotein (NP) and treatment of wt VP35 with a deubiquitinase enhances VP35-NP interaction. Future experiments will focus on investigating the effect of the dysregulated VP35-NP interaction on EBOV assembly. In conclusion, we identified a novel role of VP35 ubiquitination in regulating viral transcription and interaction with NP.

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Deletion upstream of furin site affects SARS-CoV-2 replication

Michelle N. Vu^1, Jessica A. Plante^2, Kenneth S. Plante^2, Kumari G. Lokugamage^1, Daniele Swetnam^3, Dionna Scharton^2, Andrew Routh^3, and Vineet D. Menachery<sup>1,4

1</sup>Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA
²World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, TX, USA
³Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA
⁴Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX, USA

Since its introduction to the world in Wuhan, China, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has progressed to a global pandemic with many variant strains appearing with different mutations. Mutations in the spike protein of SARS-CoV-2 have been of particular focus due to its role in transmission, antibody response, and vaccine development. Previous studies from our group had identified spike mutations in the furin cleavage site resulting in attenuated replication and disease in vivo. Our recent studies have detected another deletion directly upstream the polybasic furin cleavage site at the S1-S2 area of the spike protein (QTQTN) at low frequency in clinical samples and in vitro-passaged viruses. It is speculated that the deletion of QTQTN is due to selective pressure on the virus, yet its effect on viral replication is not known. To investigate the effect of the QTQTN deletion on viral replication, we generated a mutant SARS-CoV-2 harboring the QTQTN deletion (ΔQTQTN) and measured growth kinetics in Vero E6 and Calu3 2B4 in comparison to wild-type (WT). ΔQTQTN replicated similarly to WT in Vero E6. However, in Calu3 2B4, attenuation in ΔQTQTN replication was observed at 24h and 48h compared to WT. Unlike Vero E6, Calu3 2B4 are type I interferon (IFN) competent, which SARS-CoV-2 has been demonstrated to be susceptible against, and express TMPRSS2, a serine protease involved in the priming of the spike and entry. Type I IFN pretreatment in Vero E6 did not result in the attenuation observed with ΔQTQTN replication in Calu3 2B4, suggesting that the attenuation was not due to the IFN aspect. However, upon inhibition of either serine proteases or cathepsin in Calu3 2B4, WT replicated to equivalent levels as ΔQTQTN. Western blotting of the spike protein revealed less efficient processing with ΔQTQTN compared to WT. These results indicate that the deletion of QTQTN alters the virus’s protease usage. This study provides implications towards the importance of careful monitoring of viral stocks in order to not skew results and the significance of protease usage in virus transmission and pathogenesis.

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IL-33 ACTIVATES mTORC1 AND MODULATES GLYCOLYTIC METABOLISM IN CD8+ T CELLS

Xiaofang Wang ^1,2, Yuejin Liang ^1, Lynn Soong ^1, and Jiaren Sun<sup>1

1</sup> Departments of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA.
² Department of Infectious Diseases, Key Laboratory of Viral Hepatitis of Hunan, Xiangya Hospital, Central South University, China

Interleukin (IL)-33, a member in the IL-1 family, plays a central role in innate and adaptive immunity; however, how IL-33 mediates cytotoxic T cell regulation and the downstream signals remain elusive. In this study, we found increased IL-33 expression in T cells following cell activation. Extracellular, but not nuclear, IL-33 contributed to the activation and proliferation of CD8+ T effector cells. Importantly, IL-33 induced mTORC1 activation in CD8+ T cells as evidenced by increased phosphorylated S6 Ribosomal Protein (p-S6) levels both in vitro and in vivo. Meanwhile, this IL-33-induced T cell activation was suppressed by mTORC1 inhibitor rapamycin. Furthermore, IL-33 elevated glucose uptake and lactate production in CD8+ T cells in both dose- and time-dependent manners. Our mechanistic study further revealed the capacity of IL-33 in promoting the expression of glucose transporters and glycolytic enzymes via mTORC1, leading to accelerated aerobic glucose metabolism Warburg effect and increased effector T cell activation. Together, our data provide new insights into IL-33-mediated regulation of T cells, which might be beneficial for therapeutic strategies of inflammatory and infectious diseases in the future.

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Transcriptomic Profile of EBOV Infection in Human Neural Stem Cell Derived Neuron/Astrocyte Co-cultures

RE Williams^1, BE Dawes^1, P Wu^1, S Widen^1, AN Freiberg<sup>1

1</sup>Univ. of Texas Medical Branch, Galveston, TX

Zaire Ebola Virus (EBOV) has reached epidemic levels of infection during the past 10 years, and is still a significant contributor to ongoing viral infection outbreaks in several West African countries. During the 2014 to 2016 West African outbreak, multiple cases of Ebola Virus Disease survivors were identified who presented with severe neurologic and accompanying psychiatric sequelae. However, the neurological pathogenesis of EBOV infection in the CNS is not well understood, and obtaining infected CNS tissues for further detailed studies will be challenging. To better define the basic pathogenesis and dysregulation that occurs following EBOV infection in the CNS, we used a well-validated primary human neural stem cell (hNSC)-derived model that produces a co-culture of neurons and astrocytes at an approximate 1:1 ratio. Successful infection and replication were confirmed by viral growth kinetics using recombinant EBOV expressing eGFP. To better understand the cellular responses to infection, total cellular mRNA was extracted at 48 hours post infection and RNA sequencing analysis performed. Genes were statistically sorted based on their p-adjusted value and genes with significant p-adjusted values (~<0.05) were kept (~4,000 genes) for further analysis using Enrichr and Reactome pathway analyzers. Results revealed that highly enriched pathways for significantly upregulated genes following infection (535 genes) included interferon alpha/beta signaling,interleukin-10 anti-inflammatory signaling pathway, and acetylcholine, glutamate, dopamine,and norepinephrine neurotransmitter release cycles. On the other side, highly enriched pathways for significantly down-regulated genes following infection (384 genes) included pathways primarily involved in DNA and cellular replication and cell cycle regulation, such as unwinding of DNA, DNA replication pre-initiation, CDK regulation of DNA replication, and CDC6 association with the ORC-origin complex. Ongoing studies are focusing on confirming expression levels of selected genes after infection with EBOV and characterization of their influence on viral replication and development of clinical disease.

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GPR120 REGULATES CD4+ T CELL IL-10 PRODUCTION TO MAINTAIN INTESTINAL HOMEOSTASIS

Wenjing Yang^1, Han Liu^1, Tianming Yu^1, Suxia Yao^1, Yingzi Cong<sup>1

1</sup>Univ. of Texas Medical Branch, Galveston, TX

CD4+ T cell production of IL-10 plays a critical role in maintaining intestinal homeostasis. G protein-coupled receptor (GPR) 120, a receptor for omega 3 fatty acids, has been implicated in regulating metabolic syndromes with anti-inflammatory function. However, the role of GPR120 in intestinal inflammation is unknown. In this study, we investigated whether and how GPR120 regulates CD4+ T cell production of IL-10 to the maintenance of intestinal homeostasis. In this study, we demonstrated that deficiency of GPR120 resulted in more severe colitis in mice upon inflammatory insults and enteric infection. Interestingly, CD4+ T cells expressed GPR120 at a high level, and mice specifically lacking GPR120 in CD4+ T cells were more susceptible to colitis development. Transfer of GPR120-deficient CD4+ CD45Rbhi T cells induced more severe colitis in Rag-/- mice with increased IL-17 and IFNγ producing CD4+ T cells and decreased IL-10 producing CD4+ T cells in intestinal lamina propria than WT T cells. Treatment with GPR120 agonist, CpdA, promoted CD4+ T cell production of IL-10 by upregulating Blimp1 and enhancing glycolysis, which were regulated by mTOR. Consistently, docosahexaenoic acid (DHA), a dietary long-chain fatty acid, also upregulated the IL-10 production in CD4+ T cells. GPR120 agonist-treated WT but not Blimp1-deficient Th1 cells induced less severe colitis. Importantly, oral administration of CpdA protected mice against intestinal inflammation. Our findings demonstrate the role of GPR120 in regulating intestinal CD4+ T cell production of IL-10 to maintain intestinal homeostasis, which identifies GPR120 as a potential therapeutic target for IBD treatment.

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STING mediates intestinal IgA production through acetate-producing microbiota

Tianming Yu^1, Wenjing Yang^1, Michael Mckelvey^1, Suxia Yao^1, Yingzi Cong<sup>1

1</sup>Univ. of Texas Medical Branch, Galveston, TX

Microbiota and microbial metabolites are critical in regulating gut IgA responses, which is theeffective immune defense mechanism against microbial pathogens. Given that many species of gut bacteria producing cyclic dinucleotides and the abundance of microbiota-derived DNA within the intestinal lumen, STING (stimulator of interferon gene), a key DNA sensor, plays an important role in resistance against pathogen invade. However, whether STING affects intestinal IgA remains poorly understood. Herein, we investigated the role of STING in the regulation of intestinal IgA response in this study. We found Sting-/- mice showed a lower level of gut IgA compared to WT mice. Moreover, Sting-/- mice and WT mice were orally infected with enteric pathogen Citrobacter rodentium (CR), which mimics the colitis-inducing pathogens EPEC and EHEC in human. CR-specific IgA was decreased in Sting-/- mice 10 days post-infection with CR.However, STING agonist did not affect B cells production of IgA either treating B cells directly orthrough treating antigen-presenting cells in vitro. That demonstrated the mechanism of STING in regulating IgA was not through B cells or DCs. Interestingly, Sting-/- mice displayed altered gut microbiota composition compared to WT mice, and there was no difference in gut IgA production between Sting-/- mice and WT mice when co-housed after weaning. Furthermore, antibiotic-pretreated WT mice received fecal microbiota from Sting-/- mice showed a decreased intestinal IgA production compared to the mice received microbiota from WT mice, indicating that STING affects gut IgA production through gut microbiota in vivo. Besides, both Prevotellaceae_UCG_001genus, the acetate-producing bacteria, and acetate levels were decreased in the intestine fromSting-/- mice, whereas the difference in acetate was disappeared when co-housed with WT mice, which suggests that acetate might be mediated in STING regulation of IgA production. Taken together, our study demonstrated STING regulates intestinal IgA through acetate-producing microbiota.

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MUCOSAL DELIVERY OF A VACCINE EXPRESSING SARS-COV-2 NUCLEOPROTEIN AND SPIKE PROTEIN INDUCES HOST PROTECTION INDEPENDENT OF NEUTRALIZING ACTIVITY

Chaojie Zhong^1, Hongjie Xia^2, Awadalkareem Adam^1, Binbin Wang^1, Renee Hajnik^1, Yuejin Liang^1, Grace Rafael^1, Jing Zou^2, Xiaofang Wang^1, Jiaren Sun^1,3,4, Lynn Soong^1,3,4, Scott Weaver^1,3,4, Pei-Yong Shi^2,3,4, Tian Wang^1,3,4, Haitao Hu<sup>1,3,4

1</sup>Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA 77555
²Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, USA 77555
³Sealy Institute for Vaccine Science, University of Texas Medical Branch, Galveston, Texas, USA 77555
⁴Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, Texas, USA 77555

Coronavirus disease 2019 (CoVID19), caused by SARS-CoV2, has spread rapidly and caused a global pandemic since its outbreak. A great number of vaccine candidates have been developed to date and three were approved for use in humans under Emergency Use Authorization. Current vaccine approaches largely focus on targeting the viral spike protein (S) for eliciting protective neutralizing antibodies. However, role of cellular immunity in the context of SARS-CoV2 vaccination is limited. Accumulating evidence indicates that other than neutralizing antibodies, virus-induced T cells may also play a critical role in immune control of coronaviruses. Here, we report a new multigenic SARS-CoV2 vaccine candidate based on MVA vector that express both viral nucleoprotein (N), which contains critical T-cell epitopes, and S protein (MVA-S+N). Immunogenicity analysis showed that the vaccine is highly immunogenic and intramuscular immunization of Balb/c mice with the vaccine induces robust systemic N- and S-specific T-cell responses and binding antibodies, while no neutralizing activity was induced. Of interest, intranasal immunization with the vaccine elicits strong antigen-specific T-cell response, including granzyme B-expressing cytotoxic CD8 T cells, in lung, and induces substantial viral control in mice after SARS-CoV2 challenge. Such MVA-S+N vaccine-induced viral control was not observed in intramuscular vaccination. Together, our data provide proof-of-concept evidence that mucosal delivery of vaccine expressing N and S proteins induces strong local immunity in lung and confers immune control of SARS-CoV2 independent of neutralizing antibodies.

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