Surrogate Virus Neutralisation Test Based on Nanoluciferase-Tagged Antigens to Quantify Inhibitory Antibodies against SARS-CoV-2 and Characterise Omicron-Specific Reactivity in a Vaccination Cohort.

Virus-specific antibodies are crucial for protective immunity against SARS-CoV-2. Assessing functional antibodies through conventional or pseudotyped virus neutralisation tests (pVNT) requires high biosafety levels. Alternatively, the virus-free surrogate virus neutralisation test (sVNT) quantifies antibodies interfering with spike binding to angiotensin-converting enzyme 2. We evaluated secreted nanoluciferase-tagged spike protein fragments as diagnostic antigens in the sVNT in a vaccination cohort. Initially, spike fragments were tested in a capture enzyme immunoassay (EIA), identifying the receptor binding domain (RBD) as the optimal diagnostic antigen. The sensitivity of the in-house sVNT applying the nanoluciferase-labelled RBD equalled or surpassed that of a commercial sVNT (cPass, GenScript Diagnostics) and an in-house pVNT four weeks after the first vaccination (98% vs. 94% and 72%, respectively), reaching 100% in all assays four weeks after the second and third vaccinations. When testing serum reactivity with Omicron BA.1 spike, the sVNT and pVNT displayed superior discrimination between wild-type- and variant-specific serum reactivity compared to a capture EIA. This was most pronounced after the first and second vaccinations, with the third vaccination resulting in robust, cross-reactive BA.1 construct detection. In conclusion, utilising nanoluciferase-labelled antigens permits the quantification of SARS-CoV-2-specific inhibitory antibodies. Designed as flexible modular systems, the assays can be readily adjusted for monitoring vaccine efficacy.

Broadly Applicable, Virus-Free Dual Reporter Assay to Identify Compounds Interfering with Membrane Fusion: Performance for HSV-1 and SARS-CoV-2.

Membrane fusion constitutes an essential step in the replication cycle of numerous viral pathogens, hence it represents an important druggable target. In the present study, we established a virus-free, stable reporter fusion inhibition assay (SRFIA) specifically designed to identify compounds interfering with virus-induced membrane fusion. The dual reporter assay is based on two stable Vero cell lines harboring the third-generation tetracycline (Tet3G) transactivator and a bicistronic reporter gene cassette under the control of the tetracycline responsive element (TRE3G), respectively. Cell-cell fusion by the transient transfection of viral fusogens in the presence of doxycycline results in the expression of the reporter enzyme secreted alkaline phosphatase (SEAP) and the fluorescent nuclear localization marker EYFPNuc. A constitutively expressed, secreted form of nanoluciferase (secNLuc) functioned as the internal control. The performance of the SRFIA was tested for the quantification of SARS-CoV-2- and HSV-1-induced cell-cell fusion, respectively, showing high sensitivity and specificity, as well as the reliable identification of known fusion inhibitors. Parallel quantification of secNLuc enabled the detection of cytotoxic compounds or insufficient transfection efficacy. In conclusion, the SRFIA reported here is well suited for high-throughput screening for new antiviral agents and essentially will be applicable to all viral fusogens causing cell-cell fusion in Vero cells.

Enhanced viral clearance and reduced leukocyte infiltration in experimental herpes encephalitis after intranasal infection of CXCR3-deficient mice.

Herpes simplex virus type 1 (HSV-1) encephalitis (HSE) is the most common fatal sporadic encephalitis in developed countries. There is evidence from HSE animal models that not only direct virus-mediated damage caused but also the host's immune response contributes to the high mortality of the disease. Chemokines modulate and orchestrate this immune response. Previous experimental studies in HSE models identified the chemokine receptor CXCR3 and its ligands as molecules with a high impact on the course of HSE in mouse models. In this study, the role of the chemokine receptor CXCR3 was evaluated after intranasal infection with the encephalitogenic HSV-1 strain 17 syn+ using CXCR3-deficient mice (CXCR3-/-) and wild-type controls. We demonstrated a neurotropic viral spread into the CNS of after intranasal infection. Although viral load and histological distribution of infected neurons were independent from CXCR3 signaling early after infection, CXCR3-deficient mice cleared HSV-1 more efficiently 14 days after infection. Furthermore, CXCR3 deficiency led to a decreased weight loss in mice after HSV-1 infection. T cell infiltration and microglial activation was prominently reduced by inhibition of CXCR3 signaling. Quantitative PCR of proinflammatory cytokines and chemokines confirmed the reduced neuroinflammatory response in CXCR3-deficient mice during HSE. Our results demonstrate that the recruitment of peripheral immune cells into the CNS, induction of neuroinflammation, and consecutive weight loss during herpes encephalitis is modulated by CXCR3 signaling. Interruption of the CXCR3 pathway ameliorates the detrimental host immune response and in turn, leads paradoxically to an enhanced viral clearance after intranasal infection. Our data gives further insight into the role of CXCR3 during HSE after intranasal infection.

Cutaneous RANK-RANKL Signaling Upregulates CD8-Mediated Antiviral Immunity during Herpes simplex Virus Infection by Preventing Virus-Induced Langerhans Cell Apoptosis.

Herpes simplex virus-type 1 (HSV-1) causes the majority of cutaneous viral infections. Viral infections are controlled by the immune system, and CD8(+) cytotoxic T-lymphocytes (CTLs) have been shown to be crucial during the clearance of HSV-1 infections. Although epidermal Langerhans cells (LCs) are the first dendritic cells (DCs) to come into contact with the virus, it has been shown that the processing of viral antigens and the differentiation of antiviral CTLs are mediated by migratory CD103(+) dermal DCs and CD8α(+) lymph node-resident DCs. In vivo regulatory T-cells (Tregs) are implicated in the regulation of antiviral immunity and we have shown that signaling via the receptor activator of NF-κB (RANK) and its ligand RANKL mediates the peripheral expansion of Tregs. However, in addition to expanding Tregs, RANK-RANKL interactions are involved in the control of antimicrobial immunity by upregulating the priming of CD4(+) effector T cells in LCMV infection or by the generation of parasite-specific CD8(+) T cells in Trypanosoma cruzi infection. Here, we demonstrate that cutaneous RANK-RANKL signaling is critical for the induction of CD8-mediated antiviral immune responses during HSV-1 infection of the skin by preventing virus-induced LC apoptosis, improving antigen transport to regional lymph nodes, and increasing the CTL priming capacity of lymph node DCs.

Entry of herpes simplex virus type 1 (HSV-1) into the distal axons of trigeminal neurons favors the onset of nonproductive, silent infection.

Following productive, lytic infection in epithelia, herpes simplex virus type 1 (HSV-1) establishes a lifelong latent infection in sensory neurons that is interrupted by episodes of reactivation. In order to better understand what triggers this lytic/latent decision in neurons, we set up an organotypic model based on chicken embryonic trigeminal ganglia explants (TGEs) in a double chamber system. Adding HSV-1 to the ganglion compartment (GC) resulted in a productive infection in the explants. By contrast, selective application of the virus to distal axons led to a largely nonproductive infection that was characterized by the poor expression of lytic genes and the presence of high levels of the 2.0-kb major latency-associated transcript (LAT) RNA. Treatment of the explants with the immediate-early (IE) gene transcriptional inducer hexamethylene bisacetamide, and simultaneous co-infection of the GC with HSV-1, herpes simplex virus type 2 (HSV-2) or pseudorabies virus (PrV) helper virus significantly enhanced the ability of HSV-1 to productively infect sensory neurons upon axonal entry. Helper-virus-induced transactivation of HSV-1 IE gene expression in axonally-infected TGEs in the absence of de novo protein synthesis was dependent on the presence of functional tegument protein VP16 in HSV-1 helper virus particles. After the establishment of a LAT-positive silent infection in TGEs, HSV-1 was refractory to transactivation by superinfection of the GC with HSV-1 but not with HSV-2 and PrV helper virus. In conclusion, the site of entry appears to be a critical determinant in the lytic/latent decision in sensory neurons. HSV-1 entry into distal axons results in an insufficient transactivation of IE gene expression and favors the establishment of a nonproductive, silent infection in trigeminal neurons.

Reciprocal transmission of herpes simplex virus type 1 (HSV-1) between corneal epithelium and trigeminal neurites in an embryonic chick organ culture.

Reciprocal transmission between epithelia and sensory neurons of the peripheral nervous system is a crucial step in the life cycle of herpes simplex virus type 1 (HSV-1) and related alphaherpesviruses. In searching for an easy-to-perform and generally applicable experimental approach that enables the direct analysis of virus transfer between primary epithelial cells and sensory neurites, we investigated the spread of HSV-1 in a dual-chamber organ model comprising chick embryonic corneal epithelia and trigeminal sensory neurons. Embryonic chick corneal and trigeminal tissues were found to be permissive for productive infection with HSV-1. Our data show that HSV-1 efficiently enters neurites re-innervating the cornea and reaches the ganglion explant by retrograde axonal transport, with the first antigen-positive cells being detectable approximately 12 h postinfection. After direct infection of trigeminal tissues, the virus is transported by anterograde axonal transport to the corneal epithelium, causing a visible cytopathic effect approximately 48 h postinfection. These results suggest that the organ model presented in this study holds particular promise for the direct observation and molecular analysis of herpes simplex virus spread between primary epithelia and sensory neurons and that it may be an attractive alternative to current experimental approaches based on laboratory animals or human fetal tissues.