Dengue, caused by the four serotypes of dengue virus (DENV), represents an expanding global health challenge. The potential for serotype-cross-reactive antibodies to exacerbate disease during a secondary infection with a heterologous DENV serotype has driven efforts to study human DENV-specific antibodies. Most DENV-specific antibodies generated in humans are serotype-cross-reactive, weakly neutralizing, and directed against the immature pre-membrane (prM), envelope (E), and nonstructural 1 (NS1) proteins. To broaden the characterization of human DENV-specific antibodies, we assessed B-cell responses by ELISpot assays and isolated B cells from the peripheral blood of a human subject with previous DENV infection. Forty-eight human IgG monoclonal antibodies (hMAbs) were initially characterized by their potential to bind to an inactivated lysate of DENVinfected cells. Subsequently, most DENV-specific hMAbs were found to bind soluble, recombinant E protein (rE). Two hMAbs were unable to bind rE, despite strongly binding to the DENV-infected cell lysate. Further analyses showed that these two hMAbs bound conformation-dependent, reduction-sensitive epitopes on E protein. These data shed light on the breadth of DENV-specific hMAbs generated within a single immune donor.

Mutations in MCOLN1, which encodes the cation channel protein TRPML1, result in the neurodegenerative lysosomal storage disorder Mucolipidosis type IV. Mucolipidosis type IV patients show lysosomal dysfunction in many tissues and neuronal cell death. The ortholog of TRPML1 in Caenorhabditis elegans is CUP-5; loss of CUP-5 results in lysosomal dysfunction in many tissues and death of developing intestinal cells that results in embryonic lethality. We previously showed that a null mutation in the ATP-Binding Cassette transporter MRP-4 rescues the lysosomal defect and embryonic lethality of cup-5(null) worms. Here we show that reducing levels of the Endosomal Sorting Complex Required for Transport (ESCRT)-associated proteins DID-2, USP-50, and ALX-1/EGO-2, which mediate the final de-ubiquitination step of integral membrane proteins being sequestered into late endosomes, also almost fully suppresses cup-5(null) mutant lysosomal defects and embryonic lethality. Indeed, we show that MRP-4 protein is hypo-ubiquitinated in the absence of CUP-5 and that reducing levels of ESCRT-associated proteins suppresses this hypo-ubiquitination. Thus, increased ESCRT-associated de-ubiquitinating activity mediates the lysosomal defects and corresponding cell death phenotypes in the absence of CUP-5.KEYWORDS CUP-5; ESCRT; lysosome; mucolipidosis type IV; TRPML1 M UCOLIPIDOSIS type IV (MLIV) is a neurodegenerative lysosomal storage disorder characterized by corneal clouding, achlorhydria, and psychomotor defects (Bach 2001;Altarescu et al. 2002). In MLIV patients, large lipid-rich vacuoles are found in many tissues, while psychomotor defects are thought to be due to neuronal cell death. MLIV is caused by mutations in MCOLN1, which encodes the human protein mucolipin-1/TRPML1; this protein belongs to the transient receptor potential cation channel family and is a nonselective cation channel (Bargal et al. 2000;Bassi et al. 2000;Sun et al. 2000;Laplante et al. 2002;Raychowdhury et al. 2004;Dong et al. 2008).Caenorhabditis elegans protein CUP-5 is the ortholog of human TRPML1 (Fares and Greenwald 2001b;Hersh et al. 2002). The phenotypes resulting from mutations in cup-5(null) mimic those found in MLIV patients: defective lysosomal degradation and the appearance of large vacuoles in most tissues (Fares and Greenwald 2001b;Schaheen et al. 2006a). In addition, this lysosomal dysfunction in the absence of CUP-5 leads primarily to the death of developing intestinal cells that results in embryonic lethality (Schaheen et al. 2006a). It is not known why developing intestinal cells die in C. elegans or why neurons die in MLIV patients. In C. elegans cup-5(null) mutants, the embryonic lethality is not solely due to cells undergoing apoptosis from starvation; when ATP levels are restored or when apoptosis is blocked, embryonic lethality is only partially rescued (14% of embryos hatch and arrest at the L1 larval stage) (Hersh et al. 2002;Schaheen et al. 2006a).We have shown that C. elegans CUP-5 and mammalian TRPML1 likely function in lysoso...

A key difference that distinguishes viral infections from protein immunizations is the recognition of viral nucleic acids by cytosolic pattern recognition receptors (PRRs). Insights into the functions of cytosolic PRRs such as the RNA-sensing Rig-I-like receptors (RLRs) in the instruction of adaptive immunity are therefore critical to understand protective immunity to infections. West Nile virus (WNV) infection of mice deficent of RLR-signaling adaptor MAVS results in a defective adaptive immune response. While this finding suggests a role for RLRs in the instruction of adaptive immunity to WNV, it is difficult to interpret due to the high WNV viremia, associated exessive antigen loads, and pathology in the absence of a MAVS-dependent innate immune response. To overcome these limitations, we have infected MAVS-deficient (MAVSKO) mice with a single-round-of-infection mutant of West Nile virus. We show that MAVSKO mice failed to produce an effective neutralizing antibody response to WNV despite normal antibody titers against the viral WNV-E protein. This defect occurred independently of antigen loads or overt pathology. The specificity of the antibody response in infected MAVSKO mice remained unchanged and was still dominated by antibodies that bound the neutralizing lateral ridge (LR) epitope in the DIII domain of WNV-E. Instead, MAVSKO mice produced IgM antibodies, the dominant isotype controlling primary WNV infection, with lower affinity for the DIII domain. Our findings suggest that RLR-dependent signals are important for the quality of the humoral immune response to WNV.

T-cells in the tumor microenvironment can often become exhausted and dysfunctional due to chronic and prolonged exposure to antigen. The resulting condition of T cell exhaustion represents an important mechanism of clinical resistance to immune checkpoint blockade. The transcription factor NR4A1 is known to be upregulated in tumor-specific T-cells and is a mediator of T cell dysfunction including driving exhaustion during chronic antigen stimulation of T-cells. Pro-oncogenic activities of NR4A1 have been observed in various solid tumors, including colorectal and breast cancers, and co-expression of NR4A1 with known immune checkpoint markers such as PD-L1 and B7 family members has also been observed in various cancer cell lines. These findings have identified NR4A1 as an important target for overcoming resistance to cancer immunotherapy. We hypothesize that targeted silencing of the NR4A1 and other immunomodulatory genes can reverse T-cell exhaustion and expand the clinical benefit of immune checkpoint blockade in solid tumors resistant to immune therapy. To reverse T-cell exhaustion with a targeted approach, we developed T cell targeting SeekRsTM, which are chimeric RNA therapeutics containing dual-flanking aptamer binders connected by a double-stranded bridge containing siRNA silencers that can target multiple immunomodulatory genes. First-generation aptamer-siRNA chimeras designed with a CTLA-4 targeting aptamer containing a STAT3 siRNA demonstrated effectiveness in silencing its target. Modifications to the structure included the addition of chemically modified nucleotides to improve serum stability. Additionally, dimerization of the structure to form a SeekR dimer molecule greatly improved internalization and target silencing compared to the monomeric chimera. Newly developed SeekRs targeting T cells and designed to silence NR4A1 have effectively downregulated NR4A1 in model cell lines and activated T cells. These results demonstrate that dual targeting SeekRs can be used to specifically deliver siRNA to T cells for targeted silencing that can reverse exhaustion and potentially overcome resistance to cancer immunotherapy in the clinic. Citation Format: Marvin O'Ketch, Jeffrey A. Kiefer, Dnyanesh Rasale, Warren S. Weiner, Lizette A. Castaño, Nathalie A. Azorsa, David W. Lee, Kerry M. Barnhart, Spyro Mousses, David O. Azorsa. Activity of novel RNA therapeutics to overcome resistance to immune checkpoint blockade. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4417.

A key difference that distinguishes viral infections from protein immunizations is the recognition of viral nucleic acids by cytosolic pattern recognition receptors (PRRs) during the course of infections. Cytosolic PRRs play a crucial role in the control of innate immunity to many viral infections. In contrast to transmembrane PRRs such as Toll-like receptors, however, the functions of cytosolic PRRs in the regulation of protective adaptive immunity have remained poorly understood. Rig-I-like Receptors (RLRs) are a family of PRRs that recognize microbial RNA in the cytosol and induce cytokine and interferon responses via the essential signaling adaptor MAVS. Infection of MAVS-deficient mice with West Nile Virus (WNV) results in a defective adaptive immune response. While this finding suggests a role for RLRs in the regulation of adaptive immunity to WNV, it is difficult to interpret due to the high WNV viremia and associated antigen loads in the absence of a MAVS-dependent innate immune response. In order to overcome these limitations, we have infected MAVS-deficient mice with a single-round-of-infection mutant of WNV. MAVS-deficient mice failed to produce an effective neutralizing antibody response to WNV despite normal titers of antibodies targeting the neutralizing lateral ridge epitope in the DIII domain of WNV-E protein. Instead, the antibodies of MAVS-deficient mice bound the neutralizing epitope with lower affinity. Our findings suggest that RLR-dependent signals are important for the quality of the humoral immune response to WNV.