Enhanced In Vitro and In Vivo Potency of a T Cell Epitope in the Ebola Virus Glycoprotein Following Amino Acid Replacement at HLA-A*02:01 Binding Positions.

Studies on Ebola virus disease (EVD) survivors and clinical studies on Ebola virus (EBOV) vaccine candidates have pinpointed the importance of a strong antibody response in protection and survival from EBOV infection. However, little is known about the T cell responses to EBOV or EBOV vaccines. We used HLA-A*02:01 (HLA-A2) transgenic mice to study HLA-A2-specific T cell responses elicited following vaccination with EBOV glycoprotein (EBOV-GP) presented with three different systems: (i) recombinant protein (rEBOV-GP), (ii) vesicular stomatitis replication-competent recombinant virus (VSV-EBOV-GP), and (iii) modified vaccinia Ankara virus recombinant (MVA-EBOV-GP). T cells from immunized animals were analyzed using peptide pools representing the entire GP region and individual peptides. Regardless of the vaccine formulation, we identified a minimal 9mer epitope containing an HLA-A2 motif (FLDPATTS), which was confirmed through HLA-A2 binding affinity and immunization studies. Using binding prediction software, we identified substitutions surrounding position 9 (S9V, P10V, and Q11V) that predicted enhanced binding to the HLA-A2 molecule. This enhanced binding was confirmed through in vitro binding studies and enhanced potency was shown with in vivo immunization studies using the enhanced sequences and the wild-type sequence. Of note, in silico studies predicted the enhanced 9mer epitope carrying the S9V substitution as the best overall HLA-A2 epitope for the full-length EBOV-GP. These results suggest that EBOV-GP-S9V and EBOV-GP-P10V represent more potent in vivo immunogens. Identification and enhancement of EBOV-specific human HLA epitopes could lead to the development of tools and reagents to induce more robust T cell responses in human subjects. IMPORTANCE Vaccine efficacy and immunity to viral infection are often measured by neutralizing antibody titers. T cells are specialized subsets of immune cells with antiviral activity, but this response is variable and difficult to track. We showed that the HLA-A2-specific T cell response to the Ebola virus glycoprotein can be enhanced significantly by a single residue substitution designed to improve an epitope binding affinity to one of the most frequent MHC alleles in the human population. This strategy could be applied to improve T cell responses to Ebola vaccines designed to elicit antibodies and adapted to target MHC alleles of populations in regions where endemic infections, like Ebola virus disease, are still causing outbreaks with concerning pandemic potential.

HAVCR1 (CD365) and Its Mouse Ortholog Are Functional Hepatitis A Virus (HAV) Cellular Receptors That Mediate HAV Infection.

The hepatitis A virus (HAV) cellular receptor 1 (HAVCR1), classified as CD365, was initially discovered as an HAV cellular receptor using an expression cloning strategy. Due to the lack of HAV receptor-negative replication-competent cells, it was not possible to fully prove that HAVCR1 was a functional HAV receptor. However, biochemistry, classical virology, and epidemiology studies further supported the functional role of HAVCR1 as an HAV receptor. Here, we show that an anti-HAVCR1 monoclonal antibody that protected African green monkey kidney (AGMK) cells against HAV infection only partially protected monkey Vero E6 cells and human hepatoma Huh7 cells, indicating that these two cell lines express alternative yet unidentified HAV receptors. Therefore, we focused our work on AGMK cells to further characterize the function of HAVCR1 as an HAV receptor. Advances in clustered regularly interspaced short palindromic repeat/Cas9 technology allowed us to knock out the monkey ortholog of HAVCR1 in AGMK cells. The resulting AGMK HAVCR1 knockout (KO) cells lost susceptibility to HAV infection, including HAV-free viral particles (vpHAV) and exosomes purified from HAV-infected cells (exo-HAV). Transfection of HAVCR1 cDNA into AGMK HAVCR1 KO cells restored susceptibility to vpHAV and exo-HAV infection. Furthermore, transfection of the mouse ortholog of HAVCR1, mHavcr1, also restored the susceptibility of AGMK HAVCR1 KO cells to HAV infection. Taken together, our data clearly show that HAVCR1 and mHavcr1 are functional HAV receptors that mediate HAV infection. This work paves the way for the identification of alternative HAV receptors to gain a complete understanding of their interplay with HAVCR1 in the cell entry and pathogenic processes of HAV.IMPORTANCE HAVCR1, an HAV receptor, is expressed in different cell types, including regulatory immune cells and antigen-presenting cells. How HAV evades the immune response during a long incubation period of up to 4 weeks and the mechanism by which the subsequent necroinflammatory process clears the infection remain a puzzle that most likely involves the HAV-HAVCR1 interaction. Based on negative data, a recent paper from the S. M. Lemon and W. Maury laboratories (A. Das, A. Hirai-Yuki, O. Gonzalez-Lopez, B. Rhein, S. Moller-Tank, R. Brouillette, L. Hensley, I. Misumi, W. Lovell, J. M. Cullen, J. K. Whitmire, W. Maury, and S. M. Lemon, mBio 8:e00969-17, 2017, https://doi.org/10.1128/mBio.00969-17) suggested that HAVCR1 is not a functional HAV receptor, nor it is it required for HAV infection. However, our data, based on regain of the HAV receptor function in HAVCR1 knockout cells transfected with HAVCR1 cDNA, disagree with their findings. Our positive data show conclusively that HAVCR1 is indeed a functional HAV receptor and lays the ground for the identification of alternative HAV receptors and how they interact with HAVCR1 in cell entry and the pathogenesis of HAV.

Determinants in the Ig Variable Domain of Human HAVCR1 (TIM-1) Are Required To Enhance Hepatitis C Virus Entry.

Hepatitis C virus (HCV) is the leading cause of chronic hepatitis in humans. Several host molecules participate in HCV cell entry, but this process remains unclear. The complete unraveling of the HCV entry process is important to further understand viral pathogenesis and develop therapeutics. Human hepatitis A virus (HAV) cellular receptor 1 (HAVCR1), CD365, also known as TIM-1, functions as a phospholipid receptor involved in cell entry of several enveloped viruses. Here, we studied the role of HAVCR1 in HCV infection. HAVCR1 antibody inhibited entry in a dose-dependent manner. HAVCR1 soluble constructs neutralized HCV, which did not require the HAVCR1 mucinlike region and was abrogated by a mutation of N to A at position 94 (N94A) in the Ig variable (IgV) domain phospholipid-binding pocket, indicating a direct interaction of the HAVCR1 IgV domain with HCV virions. However, knockout of HAVCR1 in Huh7 cells reduced but did not prevent HCV growth. Interestingly, the mouse HAVCR1 ortholog, also a phospholipid receptor, did not enhance infection and a soluble form failed to neutralize HCV, although replacement of the mouse IgV domain with the human HAVCR1 IgV domain restored the enhancement of HCV infection. Mutations in the cytoplasmic tail revealed that direct HAVCR1 signaling is not required to enhance HCV infection. Our data show that the phospholipid-binding function and other determinant(s) in the IgV domain of human HAVCR1 enhance HCV infection. Although the exact mechanism is not known, it is possible that HAVCR1 facilitates entry by stabilizing or enhancing attachment, leading to direct interactions with specific receptors, such as CD81.IMPORTANCE Hepatitis C virus (HCV) enters cells through a multifaceted process. We identified the human hepatitis A virus cellular receptor 1 (HAVCR1), CD365, also known as TIM-1, as a facilitator of HCV entry. Antibody blocking and silencing or knockout of HAVCR1 in hepatoma cells reduced HCV entry. Our findings that the interaction of HAVCR1 with HCV early during infection enhances entry but is not required for infection support the hypothesis that HAVCR1 facilitates entry by stabilizing or enhancing virus binding to the cell surface membrane and allowing the correct virus-receptor positioning for interaction with the main HCV receptors. Furthermore, our data show that in addition to the phospholipid-binding function of HAVCR1, the enhancement of HCV infection involves other determinants in the IgV domain of HAVCR1. These findings expand the repertoire of molecules that HCV uses for cell entry, adding to the already complex mechanism of HCV infection and pathogenesis.

Distinct Trafficking of Cell Surface and Endosomal TIM-1 to the Immune Synapse.

The T cell costimulatory molecule TIM-1 (T cell/transmembrane, mucin and immunoglobulin domain protein 1) sorts mainly to endosomes in lymphoid cells. At difference from the cell surface protein, endosomal TIM-1 translocates to the immune synapse (IS), where it can contribute to antigen-dependent T cell costimulation. TIM-1 ligands increase the amount of cell surface protein, preventing its traffic to the IS. The bipolar sorting of TIM-1 observed during IS formation is determined by differences in its subcellular location, and probably modulates antigen-driven immune responses.

Glycosylation, hypogammaglobulinemia, and resistance to viral infections.

Genetic defects in MOGS, the gene encoding mannosyl-oligosaccharide glucosidase (the first enzyme in the processing pathway of N-linked oligosaccharide), cause the rare congenital disorder of glycosylation type IIb (CDG-IIb), also known as MOGS-CDG. MOGS is expressed in the endoplasmic reticulum and is involved in the trimming of N-glycans. We evaluated two siblings with CDG-IIb who presented with multiple neurologic complications and a paradoxical immunologic phenotype characterized by severe hypogammaglobulinemia but limited clinical evidence of an infectious diathesis. A shortened immunoglobulin half-life was determined to be the mechanism underlying the hypogammaglobulinemia. Impaired viral replication and cellular entry may explain a decreased susceptibility to infections.

Induction and activation of latent transforming growth factor-ß1 are carried out by two distinct domains of pregnancy-specific glycoprotein 1 (PSG1).

Pregnancy-specific glycoproteins (PSGs) are a family of Ig-like proteins secreted by specialized placental cells. The PSG1 structure is composed of a single Ig variable region-like N-terminal domain and three Ig constant region-like domains termed A1, A2, and B2. Members of the human and murine PSG family have been shown to induce anti-inflammatory cytokines from monocytes and macrophages and to stimulate angiogenesis. We recently showed that recombinant forms of PSG1 (PSG1-Fc and PSG1-His) and PSG1 purified from the serum of pregnant women are associated with the immunoregulatory cytokine TGF-ß1 and activated latent TGF-ß1. Here, we sought to examine the requirement of specific PSG1 domains in the activation of latent TGF-ß1. Plasmon surface resonance studies showed that PSG1 directly bound to the small latent complex and to the latency-associated peptide of TGF-ß1 and that this binding was mediated through the B2 domain. Furthermore, the B2 domain alone was sufficient for activating the small latent complex. In separate experiments, we found that the PSG1-mediated induction of TGF-ß1 secretion in macrophages was dependent on the N-terminal domain. Mutagenesis analysis revealed that four amino acids (LYHY) of the CC' loop of the N-terminal domain were required for induction of latent TGF-ß1 secretion. Together, our results show that two distinct domains of PSG1 are involved in the regulation of TGF-ß1 and provide a mechanistic framework for how PSGs modulate the immunoregulatory environment at the maternal-fetal interface for successful pregnancy outcome.

Binding of hepatitis A virus to its cellular receptor 1 inhibits T-regulatory cell functions in humans.

BACKGROUND & AIMS: CD4+ T-regulatory (Treg) cells suppress immune responses and control self-tolerance and immunity to pathogens, cancer, and alloantigens. Most pathogens activate Treg cells to minimize immune-mediated tissue damage and prevent clearance, which promotes chronic infections. However, hepatitis A virus (HAV) temporarily inhibits Treg-cell functions. We investigated whether the interaction of HAV with its cellular receptor 1 (HAVCR1), a T-cell co-stimulatory molecule, inhibits the function of Treg cells to control HAV infection. METHODS: We studied the effects of HAV interaction with HAVCR1 on human T cells using binding, signal transduction, apoptosis, activation, suppression, cytokine production, and confocal microscopy analyses. Cytokines were analyzed in sera from 14 patients with HAV infection using bead arrays. RESULTS: Human Treg cells constitutively express HAVCR1. Binding of HAV to HAVCR1 blocked phosphorylation of Akt, prevented activation of the T-cell receptor, and inhibited function of Treg cells. At the peak viremia, patients with acute HAV infection had no Treg-cell suppression function, produced low levels of transforming growth factor-ß , which limited leukocyte recruitment and survival, and produced high levels of interleukin-22, which prevented liver damage. CONCLUSIONS: Interaction between HAV and its receptor HAVCR1 inhibits Treg-cell function, resulting in an immune imbalance that allows viral expansion with limited hepatocellular damage during early stages of infection-a characteristic of HAV pathogenesis. The mechanism by which HAV is cleared in the absence of Treg-cell function could be used as a model to develop anticancer therapies, modulate autoimmune and allergic responses, and prevent transplant rejection.

Correction: Tracking ebolavirus genomic drift with a resequencing microarray.

[This corrects the article DOI: 10.1371/journal.pone.0263732.].

Induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the Ebola virus glycoprotein lacking the mucin-like domain.

BACKGROUND: The genus Ebolavirus includes five distinct viruses. Four of these viruses cause hemorrhagic fever in humans. Currently there are no licensed vaccines for any of them; however, several vaccines are under development. Ebola virus envelope glycoprotein (GP1,2) is highly immunogenic, but antibodies frequently arise against its least conserved mucin-like domain (MLD). We hypothesized that immunization with MLD-deleted GP1,2 (GPΔMLD) would induce cross-species immunity by making more conserved regions accessible to the immune system. METHODS: To test this hypothesis, mice were immunized with retrovirus-like particles (retroVLPs) bearing Ebola virus GPΔMLD, DNA plasmids (plasmo-retroVLP) that can produce such retroVLPs in vivo, or plasmo-retroVLP followed by retroVLPs. RESULTS: Cross-species neutralizing antibody and GP1,2-specific cellular immune responses were successfully induced. CONCLUSION: Our findings suggest that GPΔMLD presented through retroVLPs may provide a strategy for development of a vaccine against multiple ebolaviruses. Similar vaccination strategies may be adopted for other viruses whose envelope proteins contain highly variable regions that may mask more conserved domains from the immune system.

T cell/transmembrane, Ig, and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells.

T cell/transmembrane, Ig, and mucin (TIM) proteins, identified using a congenic mouse model of asthma, critically regulate innate and adaptive immunity. TIM-1 and TIM-4 are receptors for phosphatidylserine (PtdSer), exposed on the surfaces of apoptotic cells. Herein, we show with structural and biological studies that TIM-3 is also a receptor for PtdSer that binds in a pocket on the N-terminal IgV domain in coordination with a calcium ion. The TIM-3/PtdSer structure is similar to that of TIM-4/PtdSer, reflecting a conserved PtdSer binding mode by TIM family members. Fibroblastic cells expressing mouse or human TIM-3 bound and phagocytosed apoptotic cells, with the BALB/c allelic variant of mouse TIM-3 showing a higher capacity than the congenic C.D2 Es-Hba-allelic variant. These functional differences were due to structural differences in the BC loop of the IgV domain of the TIM-3 polymorphic variants. In contrast to fibroblastic cells, T or B cells expressing TIM-3 formed conjugates with but failed to engulf apoptotic cells. Together these findings indicate that TIM-3-expressing cells can respond to apoptotic cells, but the consequence of TIM-3 engagement of PtdSer depends on the polymorphic variants of and type of cell expressing TIM-3. These findings establish a new paradigm for TIM proteins as PtdSer receptors and unify the function of the TIM gene family, which has been associated with asthma and autoimmunity and shown to modulate peripheral tolerance.

Apoptotic cells activate NKT cells through T cell Ig-like mucin-like-1 resulting in airway hyperreactivity.

T cell Ig-like mucin-like-1 (TIM-1) is an important asthma susceptibility gene, but the immunological mechanisms by which TIM-1 functions remain uncertain. TIM-1 is also a receptor for phosphatidylserine (PtdSer), an important marker of cells undergoing programmed cell death, or apoptosis. We now demonstrate that NKT cells constitutively express TIM-1 and become activated by apoptotic cells expressing PtdSer. TIM-1 recognition of PtdSer induced NKT cell activation, proliferation, and cytokine production. Moreover, the induction of apoptosis in airway epithelial cells activated pulmonary NKT cells and unexpectedly resulted in airway hyperreactivity, a cardinal feature of asthma, in an NKT cell-dependent and TIM-1-dependent fashion. These results suggest that TIM-1 serves as a pattern recognition receptor on NKT cells that senses PtdSer on apoptotic cells as a damage-associated molecular pattern. Furthermore, these results provide evidence for a novel innate pathway that results in airway hyperreactivity and may help to explain how TIM-1 and NKT cells regulate asthma.

Tracking ebolavirus genomic drift with a resequencing microarray.

Filoviruses are emerging pathogens that cause acute fever with high fatality rate and present a global public health threat. During the 2013-2016 Ebola virus outbreak, genome sequencing allowed the study of virus evolution, mutations affecting pathogenicity and infectivity, and tracing the viral spread. In 2018, early sequence identification of the Ebolavirus as EBOV in the Democratic Republic of the Congo supported the use of an Ebola virus vaccine. However, field-deployable sequencing methods are needed to enable a rapid public health response. Resequencing microarrays (RMA) are a targeted method to obtain genomic sequence on clinical specimens rapidly, and sensitively, overcoming the need for extensive bioinformatic analysis. This study presents the design and initial evaluation of an ebolavirus resequencing microarray (Ebolavirus-RMA) system for sequencing the major genomic regions of four Ebolaviruses that cause disease in humans. The design of the Ebolavirus-RMA system is described and evaluated by sequencing repository samples of three Ebolaviruses and two EBOV variants. The ability of the system to identify genetic drift in a replicating virus was achieved by sequencing the ebolavirus glycoprotein gene in a recombinant virus cultured under pressure from a neutralizing antibody. Comparison of the Ebolavirus-RMA results to the Genbank database sequence file with the accession number given for the source RNA and Ebolavirus-RMA results compared to Next Generation Sequence results of the same RNA samples showed up to 99% agreement.

Determinants in 3Dpol modulate the rate of growth of hepatitis A virus.

Hepatitis A virus (HAV), an atypical member of the Picornaviridae, grows poorly in cell culture. To define determinants of HAV growth, we introduced a blasticidin (Bsd) resistance gene into the virus genome and selected variants that grew at high concentrations of Bsd. The mutants grew fast and had increased rates of RNA replication and translation but did not produce significantly higher virus yields. Nucleotide sequence analysis and reverse genetic studies revealed that a T6069G change resulting in a F42L amino acid substitution in the viral polymerase (3D(pol)) was required for growth at high Bsd concentrations whereas a silent C7027T mutation enhanced the growth rate. Here, we identified a novel determinant(s) in 3D(pol) that controls the kinetics of HAV growth.

Increased susceptibility of Huh7 cells to HCV replication does not require mutations in RIG-I.

BACKGROUND: The cytosolic retinoic acid-inducible gene I (RIG-I) is a pattern recognition receptor that senses HCV double-stranded RNA and triggers type I interferon pathways. The clone Huh7.5 of human hepatoma Huh7 cells contains a mutation in RIG-I that is believed to be responsible for the improved replication of HCV in these cells relative to the parental strain. We hypothesized that, in addition to RIG-I, other determinant(s) outside the RIG-I coding sequence are involved in limiting HCV replication in cell culture. To test our hypothesis, we analyzed Huh7 cell clones that support the efficient replication of HCV and analyzed the RIG-I gene. RESULTS: One clone, termed Huh7D, was more permissive for HCV replication and more efficient for HCV-neomycin and HCV-hygromycin based replicon colony formation than parental Huh7 cells. Nucleotide sequence analysis of the RIG-I mRNA coding region from Huh7D cells showed no mutations relative to Huh7 parental cells. CONCLUSIONS: We derived a new Huh7 cell line, Huh7D, which is more permissive for HCV replication than parental Huh7 cells. The higher permissiveness of Huh7D cells is not due to mutations in the RIG-I protein, indicating that cellular determinants other than the RIG-I amino-acid sequence are responsible for controlling HCV replication. In addition, we have selected Huh7 cells resistant to hygromycin via newly generated HCV-replicons carrying the hygromycin resistant gene. Further studies on Huh7D cells will allow the identification of cellular factors that increased the susceptibility to HCV infection, which could be targeted for anti-HCV therapies.

Exosome mimicry by a HAVCR1-NPC1 pathway of endosomal fusion mediates hepatitis A virus infection.

Cell-to-cell communication by exosomes controls normal and pathogenic processes1,2. Viruses can spread in exosomes and thereby avoid immune recognition3. While biogenesis, binding and uptake of exosomes are well characterized4,5, delivery of exosome cargo into the cytoplasm is poorly understood3. We report that the phosphatidylserine receptor HAVCR1 (refs. 6,7) and the cholesterol transporter NPC1 (ref. 8) participate in cargo delivery from exosomes of hepatitis A virus (HAV)-infected cells (exo-HAV) by clathrin-mediated endocytosis. Using CRISPR-Cas9 knockout technology, we show that these two lipid receptors, which interact in the late endosome9, are necessary for the membrane fusion and delivery of RNA from exo-HAV into the cytoplasm. The HAVCR1-NPC1 pathway, which Ebola virus exploits to infect cells9, mediates HAV infection by exo-HAV, which indicates that viral infection via this exosome mimicry mechanism does not require an envelope glycoprotein. The capsid-free viral RNA in the exosome lumen, but not the endosomal uncoating of HAV particles contained in the exosomes, is mainly responsible for exo-HAV infectivity as assessed by methylene blue inactivation of non-encapsidated RNA. In contrast to exo-HAV, infectivity of HAV particles is pH-independent and requires HAVCR1 or another as yet unidentified receptor(s) but not NPC1. Our findings show that envelope-glycoprotein-independent fusion mechanisms are shared by exosomes and viruses, and call for a reassessment of the role of envelope glycoproteins in infection.

Hepatitis A virus (HAV) packaging size limit.

BACKGROUND: Hepatitis A virus (HAV), an atypical Picornaviridae that causes acute hepatitis in humans, grows poorly in cell culture and in general does not cause cytopathic effect. Foreign sequences have been inserted into different parts of the HAV genome. However, the packaging size limit of HAV has not been determined. The purpose of the present study is to investigate the maximum size of additional sequences that the HAV genome can tolerate without loosing infectivity. RESULTS: In vitro T7 polymerase transcripts of HAV constructs containing a 456-nt fragment coding for a blasticidin (Bsd) resistance gene, a 1,098-nt fragment coding for the same gene fused to GFP (GFP-Bsd), or a 1,032-nt fragment containing a hygromycin (Hyg) resistance gene cloned into the 2A-2B junction of the HAV genome were transfected into fetal Rhesus monkey kidney (FRhK4) cells. After antibiotic selection, cells transfected with the HAV construct containing the resistance gene for Bsd but not the GFP-Bsd or Hyg survived and formed colonies. To determine whether this size limitation was due to the position of the insertion, a 606 bp fragment coding for the Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) sequence was cloned into the 5' nontranslated (NTR) region of HAV. The resulting HAV-IRES retained the EMCV IRES insertion for 1-2 passages. HAV constructs containing both the EMCV IRES at the 5' NTR and the Bsd-resistance gene at the 2A-2B junction could not be rescued in the presence of Bsd but, in the absence of antibiotic, the rescued viruses contained deletions in both inserted sequences. CONCLUSION: HAV constructs containing insertions of approximately 500-600 nt but not 1,000 nt produced viable viruses, which indicated that the HAV particles can successfully package approximately 600 nt of additional sequences and maintain infectivity.

The interaction of hepatitis A virus (HAV) with soluble forms of its cellular receptor 1 (HAVCR1) share the physiological requirements of infectivity in cell culture.

BACKGROUND: Hepatitis A virus (HAV), an atypical Picornaviridae that causes acute hepatitis in humans, usurps the HAV cellular receptor 1 (HAVCR1) to infect cells. HAVCR1 is a class 1 integral membrane glycoprotein that contains two extracellular domains: a virus-binding immunoglobulin-like (IgV) domain and a mucin-like domain that extends the IgV from the cell membrane. Soluble forms of HAVCR1 bind, alter, and neutralize cell culture-adapted HAV, which is attenuated for humans. However, the requirements of the HAV-HAVCR1 interaction have not been fully characterized, and it has not been determined whether HAVCR1 also serves as a receptor for wild-type (wt) HAV. Here, we used HAV soluble receptor neutralization and alteration assays to study the requirements of the HAV-HAVCR1 interaction and to determine whether HAVCR1 is also a receptor for wt HAV. RESULTS: Treatment of HAV with a soluble form of HAVCR1 that contained the IgV and two-thirds of the mucin domain fused to the Fc fragment of human IgG1 (D1 muc-Fc), altered particles at 37 degrees C but left a residual level of unaltered particles at 4 degrees C. The kinetics of neutralization of HAV by D1 muc-Fc was faster at 37 degrees C than at 4 degrees C. Alteration of HAV particles by D1 muc-Fc required Ca, which could not be replaced by Li, Na, Mg, Mn, or Zn. Neutralization of HAV by D1 muc-Fc occurred at pH 5 to 8 but was more efficient at pH 6 to 7. D1 muc-Fc neutralized wt HAV as determined by a cell culture system that allows the growth of wt HAV. CONCLUSION: The interaction of HAV with soluble forms of HAVCR1 shares the temperature, Ca, and pH requirements for infectivity in cell culture and therefore mimics the cell entry process of HAV. Since soluble forms of HAVCR1 also neutralized wt HAV, this receptor may play a significant role in pathogenesis of HAV.

Ultrasensitive Ebola Virus Antigen Sensing via 3D Nanoantenna Arrays.

Sensitive detection of pathogens is crucial for early disease diagnosis and quarantine, which is of tremendous need in controlling severe and fatal illness epidemics such as of Ebola virus (EBOV) disease. Serology assays can detect EBOV-specific antigens and antibodies cost-effectively without sophisticated equipment; however, they are less sensitive than reverse transcriptase polymerase chain reaction (RT-PCR) tests. Herein, a 3D plasmonic nanoantenna assay sensor is developed as an on-chip immunoassay platform for ultrasensitive detection of Ebola virus (EBOV) antigens. The EBOV sensor exhibits substantial fluorescence intensity enhancement in immunoassays compared to flat gold substrate. The nanoantenna-based biosensor successfully detects EBOV soluble glycoprotein (sGP) in human plasma down to 220 fg mL-1 , a significant 240 000-fold sensitivity improvement compared to the 53 ng mL-1 EBOV antigen detection limit of the existing rapid EBOV immunoassay. In a mock clinical trial, the sensor detects sGP-spiked human plasma samples at two times the limit of detection with 95.8% sensitivity. The results combined highlight the nanosensor's extraordinary capability of detecting EBOV antigen at ultralow concentration compared to existing immunoassay methods. It is a promising next-generation bioassay platform for early-stage disease diagnosis and pathogen detection for both public health and national security applications.

A simple and rapid Hepatitis A Virus (HAV) titration assay based on antibiotic resistance of infected cells: evaluation of the HAV neutralization potency of human immune globulin preparations.

BACKGROUND: Hepatitis A virus (HAV), the causative agent of acute hepatitis in humans, is an atypical Picornaviridae that grows poorly in cell culture. HAV titrations are laborious and time-consuming because the virus in general does not cause cytopathic effect and is detected by immunochemical or molecular probes. Simple HAV titration assays could be developed using currently available viral construct containing selectable markers. RESULTS: We developed an antibiotic resistance titration assay (ARTA) based on the infection of human hepatoma cells with a wild type HAV construct containing a blasticidin (Bsd) resistance gene. Human hepatoma cells infected with the HAV-Bsd construct survived selection with 2 microg/ml of blasticidin whereas uninfected cells died within a few days. At 8 days postinfection, the color of the pH indicator phenol red in cell culture media correlated with the presence of HAV-Bsd-infected blasticidin-resistant cells: an orange-to-yellow color indicated the presence of growing cells whereas a pink-to-purple color indicated that the cells were dead. HAV-Bsd titers were determined by an endpoint dilution assay based on the color of the cell culture medium scoring orange-to-yellow wells as positive and pink-to-purple wells as negative for HAV. As a proof-of-concept, we used the ARTA to evaluate the HAV neutralization potency of two commercially available human immune globulin (IG) preparations and a WHO International Standard for anti-HAV. The three IG preparations contained comparable levels of anti-HAV antibodies that neutralized approximately 1.5 log of HAV-Bsd. Similar neutralization results were obtained in the absence of blasticidin by an endpoint dilution ELISA at 2 weeks postinfection. CONCLUSION: The ARTA is a simple and rapid method to determine HAV titers without using HAV-specific probes. We determined the HAV neutralization potency of human IG preparations in 8 days by ARTA compared to the 14 days required by the endpoint dilution ELISA. The ARTA reduced the labour, time, and cost of HAV titrations making it suitable for high throughput screening of sera and antivirals, determination of anti-HAV antibodies in human immune globulin preparations, and research applications that involve the routine evaluation of HAV titers.