Biology subjects

DeFilippis, V. R.

Publications and source records attributed to DeFilippis, V. R..

5 recordsLinked to original sources

bioRxiv2026

Mucosal ACP subunit vaccine formulated with a small molecule STING agonist drives protective TH1/TH17 responses and serum bactericidal antibodies against Neisseria gonorrhoeae

STING agonists are emerging vaccine adjuvants; however, only cyclic dinucleotides (low permeability and stability) have been tested against pathogenic bacteria. We examined diABZI, a non-nucleotide STING agonist active in human and murine orthologs, combined with the Neisseria gonorrhoeae (Ng) Adhesin Complex Protein, a conserved adhesin and human lysozyme C inhibitor, as a gonococcal vaccine. Intranasal administration of recombinant ACP (rACP)+diABZI accelerated gonococcal clearance, in contrast to rACP or rACP+CpG+MPLA, despite comparable antibody responses. While both diABZI and CpG+MPLA induced TH1, diABZI also induced TH17 polarization. Peptide mapping localized the immunodominant T cell epitopes to the ACP-lysozyme C interface. rACP+diABZI elicited higher human serum bactericidal activity (hSBA), that correlated with Ng clearance, and antibodies that reversed human lysozyme C inhibition and interfered with Ng adhesion and invasion in 3D human vaginal/urethral mucosal models. Passive serum transfer did not recapitulate protection, implicating mucosal antibodies and cellular immunity over circulating IgG.

microbiology↗

bioRxiv2025

A Zika Virus-Like Particle Vaccine Mitigates Early Pregnancy Loss In Rhesus Macaques

Zika virus (ZIKV) is an arthropod-borne Orthoflavivirus that caused a major outbreak in the Americas in 2015-16. In Brazil, up to 46% of ZIKV positive pregnancies resulted in congenital Zika syndrome (CZS). CZS is characterized by a wide range of neurologic birth defects and miscarriage in up to 7.6% of affected pregnancies. With no current licensed ZIKV vaccines, we sought to evaluate a Zika virus-like particle (VLP) vaccine candidate in a rhesus macaque (RM) pregnancy model. VLPs were produced in mammalian cells expressing the pre-membrane-envelope region of the Asian-lineage ZIKV strain PRVABC59, which belongs to the Asian ZIKV lineage that is associated with outbreaks of congenital disease. To evaluate vaccine protection against adverse pregnancy complications, two cohorts of female RM were vaccinated with ZIKV-VLP with adjuvant Alhydrogel (alum) or adjuvant alone prior to mating. At gestational day (GD) 30 (early first trimester), pregnant animals were challenged with ZIKV-DAK 41524, an African-lineage strain shown to induce 1st-trimester fetal demise in 78% (n=11/14 animals) of RM, making it an ideal and stringent model for evaluating ZIKV vaccines. Within the vaccinated cohort, 2 of 3 animals reached the study endpoint of GD 90 with no observed adverse pregnancy outcomes. The third animal experienced pregnancy loss at GD 49 (18 d post infection), although no infectious virus was detected in placental or fetal tissues. In the unvaccinated cohort, two animals had severe adverse events. One animal experienced preterm labor, and another developed early-onset hydrops fetalis with widespread ZIKV-RNA detected via RNAscope and extensive placental damage. These results confirm a significant risk for early pregnancy loss in RM infected with ZIKV-DAK 41524. This model can be further used to understand the complexities of placental immunological features underlying stillbirth and miscarriage following infection. Our findings indicate that this ZIKV-VLP vaccine candidate protected pregnant macaques against fetal demise associated with highly pathogenic ZIKV challenge. Author SummaryZika virus (ZIKV) infection during pregnancy is associated with pregnancy loss, severe birth defects, including microcephaly and developmental delays, and other subtle neurologic changes. Although vaccine development efforts have been ongoing since the 2015/2016 ZIKV outbreak, no approved vaccine is currently available. Many existing studies have tested vaccines in animal models using strains such as ZIKV-PR that cause mild or moderate pregnancy complications at similar rates to human cases. Using challenge strains that only cause mild, or moderate pregnancy complications makes it difficult to rigorously evaluate vaccine efficacy. In this study, we tested a virus-like particle (VLP) vaccine, a safe and effective method for use during pregnancy as it is replication-deficient and only contains viral structural antigens. We found that the VLP vaccine, when paired with an adjuvant (alum), induced strong antibody responses in mice and controlled viral dissemination following challenge in nonpregnant macaques. To evaluate the protective efficacy of the ZIKV-VLP vaccine against in utero infection and disease, we used a stringent model of ZIKV infection during early pregnancy in rhesus macaques that is associated with high rates of fetal demise. In pregnant macaques, the vaccine reduced maternal viremia, limited viral spread to fetal and placental tissues, and conferred protection against virus-mediated fetal demise and placental damage. This is the first study to evaluate a VLP-based vaccine in a consistent pregnancy loss model of ZIKV infection. These findings support the continued development of VLP vaccines as a safe and effective strategy for protecting pregnant individuals and their developing fetuses from ZIKV.

microbiology↗

bioRxiv2025

Characterization of a Novel Transmembrane Activating STING Agonist using Genetically Humanized Mice

STING is a pattern recognition receptor that activates type I interferon and proinflammatory responses in addition to unrelated molecular processes following exposure of DNA to the cytosol. Its pharmacologic stimulation enhances vaccine potency and generates effective antitumor responses but clinical trials evaluating STING agonists have not led to approval for human use. STING activation can occur through ligand engagement of either cytosolic or transmembrane protein domains, processes to which distinct cellular phenotypes are attributed. However, the only transmembrane STING agonist identified is human selective and in vivo testing in conventional models is not feasible. Here we describe synthesis of novel STING agonists efficacious against allelic variants of the protein. We also describe genetically humanized STING mice and demonstrate their suitability as a model to evaluate in vivo responses following exogenous administration of human-selective agonists. Experiments demonstrate that the lead molecule (termed INI3069) functions through binding to the STING transmembrane region and its comparison with conventional agonists reveals significant differences in molecular and immune effects. INI3069 can also enhance antibody responses to co-administered antigens and antitumor responses. This work both represents the first in vivo examination of the effects of transmembrane STING agonism and demonstrates efficacy of a potential novel vaccine adjuvant and oncological therapeutic.

immunology↗

bioRxiv2020

Human cytomegalovirus evades ZAP detection by suppressing CpG dinucleotides in the major immediate early genes.

The genomes of RNA and small DNA viruses of vertebrates display significant suppression of CpG dinucleotide frequencies. Artificially increasing dinucleotide frequencies results in substantial attenuation of virus replication, suggesting that these compositional changes may facilitate recognition of non-self RNA sequences. Recently, the interferon inducible protein ZAP, was identified as the host factor responsible for sensing CpG in viral RNA, through direct binding and possibly downstream targeting for degradation. Using an arrayed interferon stimulated gene expression library screen, we identified ZAPS, and its associated factor TRIM25, as direct inhibitors of human cytomegalovirus (HCMV) replication. Exogenous expression of ZAPS and TRIM25 significantly reduced virus replication while knockdown resulted in increased virus replication. HCMV displays a strikingly heterogeneous pattern of CpG representation with a specific suppression of CpGs within the IE1 major immediate early transcript which is absent in subsequently expressed genes. We demonstrated that suppression of CpG dinucleotides in the IE1 gene allows evasion of inhibitory effects of ZAP. During HCMV infection, expression of ZAP and TRIM25 are rapidly reduced, removing pressure to suppress dinucleotide frequencies in viral genes expressed after the immediate early genes, while acute virus replication and high levels of ZAP are mutually exclusive. Finally, we show that TRIM25 regulates alternative splicing between the ZAP short and long isoforms during HCMV infection and interferon induction, with knockdown of TRIM25 resulting in decreased ZAPS and corresponding increased ZAPL expression. These results demonstrate for the first time that ZAP is a potent host restriction factor against large DNA viruses and that HCMV evades ZAP detection through suppression of CpG dinucleotides within the major immediate early transcripts. Furthermore, TRIM25 is required for efficient upregulation of the interferon inducible short isoform of ZAP through regulation of alternative splicing.

microbiology↗

bioRxiv2019

Herpes Simplex Virus type 1 Inflammasome Activation in Human Macrophages is Dependent on NLRP3, ASC, and Caspase-1

The pro-inflammatory cytokines interleukin (IL)-1{beta} and IL-18 are products of activation of the inflammasome, an innate sensing system, and important in the pathogenesis of herpes simplex type 1 (HSV-1). The release of IL-18 and IL-1{beta} from monocytes/macrophages is critical for protection from HSV-1 based on animal models of encephalitis and genital infection, yet if and how HSV-1 activates inflammasomes in human macrophages is unknown. To investigate this, we utilized both primary human monocyte derived macrophages and human monocytic cell lines (THP-1 cells) with various inflammasome components knocked-out. We found that HSV-1 activates inflammasome signaling in pro-inflammatory primary human macrophages. Additionally, HSV-1 inflammasome activation is dependent on nucleotide-binding domain and leucine-rich repeat-containing receptor 3 (NLRP3), apoptosis-associated speck-like molecule containing a caspase recruitment domain (ASC), and caspase-1, but not on absent in melanoma 2 (AIM2), or gamma interferon-inducible protein 16 (IFI16). Ultraviolet irradiation of HSV-1 enhanced inflammasome activation, demonstrating that viral replication suppresses inflammasome activation. These results confirm that HSV-1 is capable of activating the inflammasome in human macrophages through an NLRP3 dependent process and that the virus has an NLRP3 specific mechanism to inhibit inflammasome activation in monocytes and macrophages.\n\nAuthor SummaryThe inflammasome is a multi-protein complex that forms in response to pathogens and cellular damage. Active inflammasomes recruit pro-caspase-1 via ASC and cleave the cytokine precursors pro-IL-1{beta} and pro-IL-18 into mature IL-1{beta} and IL-18. These cytokines serve to activate other immune cells that either repair the damage or attempt to clear the invading pathogen. Upon activation, the inflammasome also promotes an inflammatory form of cell death called pyroptosis. Herpes simplex virus type 1 (HSV-1) is a common human pathogen that can cause cold sores, genital ulcers, encephalitis, and blindness. HSV-1 infection leads to induction of IL-1{beta} and IL-18, but whether it is capable of activating inflammasomes in macrophages, which play a role in severe forms of HSV-1 infection, was unclear. Here, we infected both primary human macrophages and a macrophage/monocytic cell line, THP-1 cells, with HSV-1. We found that HSV-1 does activate inflammasome signaling in macrophages in a process dependent on NLRP3, ASC, and caspase-1. This is important because it illustrates the mechanism by which HSV-1 infection leads to inflammasome activation in macrophages, known to be crucial for protection from severe disease in mouse models.

microbiology↗