LUNG group 2 innate lymphoid cells as a new adjuvant target to enhance intranasal vaccine efficacy against influenza.

Group 2 innate lymphoid cells (ILC2) are a relatively new class of innate immune cells. Lung ILC2 are early responders that secrete type 2 cytokines in response to danger 'alarmin' signals such as interleukin (IL)-33 and thymic stromal lymphopoietin. Being an early source of type 2 cytokines, ILC2 are a critical regulator of type 2 immune cells of both innate and adaptive immune responses. The immune regulatory functions of ILC2 were mostly investigated in diseases where T helper 2 inflammation predominates. However, in recent years, it has been appreciated that the role of ILC2 extends to other pathological conditions such as cancer and viral infections. In this review, we will focus on the potential role of lung ILC2 in the induction of mucosal immunity against influenza virus infection and discuss the potential utility of ILC2 as a target for mucosal vaccination.

Fear of Influenza Resurgence amid COVID-19 Pandemic: Need for Effective Flu Vaccine Still Exists.

As influenza season was approaching in 2020, public health officials feared that influenza would worsen the COVID-19 situation [...].

Performance evaluation of antibody-based point-of-care devices intended for the identification of immune responses to SARS-CoV-2.

The novel coronavirus outbreak caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2) was first identified in December of 2019 in Wuhan, China. The local outbreak quickly rose to pandemic level that has spread to more than 188 countries with more than 19 million cases and 732,467 deaths worldwide. The current recommendation for testing is RT-PCR based tests of nasopharyngeal or alternatively nasal- and/or oropharyngeal swabs that detects infection with SARS-CoV-2 to diagnose acute infection. However, there is an urgent need for a quick and accurate antibody-based point-of-care test method to quickly identify evidence of SARS-CoV-2 infection among people who might be missed through active case finding and surveillance efforts. Serology tests measure the presence of antibodies in serum after infection. Here we compared the performance characteristics of 6 commercially available antibody-based point-of-care devices and their potential for identification of individuals infected at some time by SARS-CoV-2.

Integrated Arbovirus Surveillance Improves the Detection Onset of Zika Virus in Panama.

We tested 700 serum samples collected throughout Panama from 2015 to 2016 for detecting antibodies and RNA of arboviruses. In convalescent specimens, microsphere immunoassay detected an antibody prevalence of 59.3% for dengue virus (DENV) and 30.3% for Zika virus (ZIKV), which included samples that were collected before the Panamanian surveillance system reported the first case of Zika in the country. For acute sera, the most common arbovirus was DENV with 18 positive samples (6%), followed by four (1.3%) of ZIKV and one (0.6%) of chikungunya virus (CHIKV). Our results indicate a change in the chronology of when ZIKV was first detected in Panama and stress the importance of integrating various approaches to enable improved surveillance of both endemic and emerging arboviruses.

Use of the immunoglobulin G avidity assay to differentiate between recent Zika and past dengue virus infections.

Zika (ZIKV) and dengue (DENV) virus infections elicit a robust but cross-reactive antibody response against the viral envelope protein, while antibody responses against non-structural proteins (NS) are more virus specific. Building on this premise, we have previously developed a flavivirus multiplex microsphere immunoassay (MIA) for the serologic diagnosis of ZIKV and DENV infections. This assay significantly improved diagnostic accuracy; however, MIA could not differentiate more recent from past infections, which still represents a major diagnostic challenge. Therefore, an immunoglobulin G (IgG) based avidity assay was developed and its diagnostic performance evaluated. Specimens from New York State residents were submitted to the Wadsworth Center New York State Department of Health (NYSDOH) for routine clinical testing by Zika IgM ELISA and plaque reduction neutralization test (PRNT). Using our previously developed flavivirus MIA as a platform, we developed an IgG avidity assay to discriminate recent ZIKV from past DENV infections. Zika IgM positive specimens had an average Zika IgG avidity index of 14.8% (95% CI: 11.0-18.4%), while Zika IgM negative but flavivirus MIA and PRNT positive samples had an average Zika IgG avidity index of 34.9% (95% CI: 31.1-38.7%). Specimens positive for dengue antibodies by flavivirus MIA and PRNT had an average dengue IgG avidity index of 68.7% (95% CI: 62.7-75.0%). The IgG avidity assay accurately distinguished recent ZIKV from past DENV infections in patients who traveled to dengue endemic regions. This assay could be very useful in patients with high risk of Zika complications such as pregnant women and monitoring immune responses in vaccine trials.

A Rapid Zika Diagnostic Assay to Measure Neutralizing Antibodies in Patients.

The potential association of microcephaly and other congenital abnormalities with Zika virus (ZIKV) infection during pregnancy underlines the critical need for a rapid and accurate diagnosis. Due to the short duration of ZIKV viremia in infected patients, a serologic assay that detects antibody responses to viral infection plays an essential role in diagnosing patient specimens. The current serologic diagnosis of ZIKV infection relies heavily on the labor-intensive Plaque Reduction Neutralization Test (PRNT) that requires more than one-week turnaround time and represents a major bottleneck for patient diagnosis. To overcome this limitation, we have developed a high-throughput assay for ZIKV and dengue virus (DENV) diagnosis that can attain the "gold standard" of the current PRNT assay. The new assay is homogeneous and utilizes luciferase viruses to quantify the neutralizing antibody titers in a 96-well format. Using 91 human specimens, we showed that the reporter diagnostic assay has a higher dynamic range and maintains the relative specificity of the traditional PRNT assay. Besides the improvement of assay throughput, the reporter virus technology has also shortened the turnaround time to less than two days. Collectively, our results suggest that, along with the viral RT-PCR assay, the reporter virus-based serologic assay could be potentially used as the first-line test for clinical diagnosis of ZIKV infection as well as for vaccine clinical trials.

A Multiplex Microsphere Immunoassay for Zika Virus Diagnosis.

Rapid and accurate diagnosis of infectious agents is essential for patient care, disease control, and countermeasure development. The present serologic diagnosis of Zika virus (ZIKV) infection relies mainly on IgM-capture ELISA which is confounded with the flaw of cross-reactivity among different flaviviruses. In this communication, we report a multiplex microsphere immunoassay (MIA) that captures the diagnostic power of viral envelope protein (that elicits robust, yet cross-reactive antibodies to other flaviviruses) and the differential power of viral nonstructural proteins NS1 and NS5 (that induce more virus-type specific antibodies). Using 153 patient specimens with known ZIKV and/or dengue virus (DENV; a closely related flavivirus) infections, we showed that (i) ZIKV envelope-based MIA is equivalent or more sensitive than IgM-capture ELISA in diagnosing ZIKV infection, (ii) antibody responses to NS1 and NS5 proteins are more ZIKV-specific than antibody response to envelope protein, (iii) inclusion of NS1 and NS5 in the MIA improves the diagnostic accuracy when compared with the MIA that uses envelope protein alone. The multiplex MIA achieves a rapid diagnosis (turnaround time<4h) and requires small specimen volume (10µl) in a single reaction. This serologic assay could be developed for use in clinical diagnosis of ZIKV infection and for monitoring immune responses in vaccine trials.

Sulforaphane Inhibits HIV Infection of Macrophages through Nrf2.

Marburg virus, the Kaposi's sarcoma-associated herpesvirus (KSHV) and Dengue virus all activate, and benefit from, expression of the transcription regulator nuclear erythroid 2-related factor 2 (Nrf2). The impact of Nrf2 activation on human immunodeficiency virus (HIV) infection has not been tested. Sulforaphane (SFN), produced in cruciferous vegetables after mechanical damage, mobilizes Nrf2 to potently reprogram cellular gene expression. Here we show for the first time that SFN blocks HIV infection in primary macrophages but not in primary T cells. Similarly SFN blocks infection in PMA-differentiated promonocytic cell lines, but not in other cell lines tested. siRNA-mediated depletion of Nrf2 boosted HIV infectivity in primary macrophages and reduced the anti-viral effects of SFN treatment. This supports a model in which anti-viral activity is mediated through Nrf2 after it is mobilized by SFN. We further found that, like the type I interferon-induced cellular anti-viral proteins SAMHD1 and MX2, SFN treatment blocks infection after entry, but before formation of 2-LTR circles. Interestingly however, neither SAMHD1 nor MX2 were upregulated. This shows for the first time that Nrf2 action can potently block HIV infection and highlights a novel way to trigger this inhibition.

Prevention of Influenza Virus-Induced Immunopathology by TGF-ß Produced during Allergic Asthma.

Asthma is believed to be a risk factor for influenza infection, however little experimental evidence exists to directly demonstrate the impact of asthma on susceptibility to influenza infection. Using a mouse model, we now report that asthmatic mice are actually significantly more resistant to a lethal influenza virus challenge. Notably, the observed increased resistance was not attributable to enhanced viral clearance, but instead, was due to reduced lung inflammation. Asthmatic mice exhibited a significantly reduced cytokine storm, as well as reduced total protein levels and cytotoxicity in the airways, indicators of decreased tissue injury. Further, asthmatic mice had significantly increased levels of TGF-ß1 and the heightened resistance of asthmatic mice was abrogated in the absence of TGF-ß receptor II. We conclude that a transient increase in TGF-ß expression following acute asthma can induce protection against influenza-induced immunopathology.

Cullin4A and cullin4B are interchangeable for HIV Vpr and Vpx action through the CRL4 ubiquitin ligase complex.

UNLABELLED: Human immunodeficiency virus (HIV) seizes control of cellular cullin-RING E3 ubiquitin ligases (CRLs) to promote viral replication. HIV-1 Vpr and HIV-2/simian immunodeficiency virus (SIV) Vpr and Vpx engage the cullin4 (CUL4)-containing ubiquitin ligase complex (CRL4) to cause polyubiquitination and proteasomal degradation of host proteins, including ones that block infection. HIV-1 Vpr engages CRL4 to trigger the degradation of uracil-N-glycosylase 2 (UNG2). Both HIV-1 Vpr and HIV-2/SIV Vpr tap CRL4 to initiate G2 cell cycle arrest. HIV-2/SIV Vpx secures CRL4 to degrade the antiviral protein SAMHD1. CRL4 includes either cullin4A (CUL4A) or cullin4B (CUL4B) among its components. Whether Vpr or Vpx relies on CUL4A, CUL4B, or both to act through CRL4 is not known. Reported structural, phenotypic, and intracellular distribution differences between the two CUL4 types led us to hypothesize that Vpr and Vpx employ these in a function-specific manner. Here we determined CUL4 requirements for HIV-1 and HIV-2/SIV Vpr-mediated G2 cell cycle arrest, HIV-1 Vpr-mediated UNG2 degradation, and HIV-2 Vpx-mediated SAMHD1 degradation. Surprisingly, CUL4A and CUL4B are exchangeable for CRL4-dependent Vpr and Vpx action, except in primary macrophages, where Vpx relies on both CUL4A and CUL4B for maximal SAMHD1 depletion. This work highlights the need to consider both CUL4 types for Vpr and Vpx functions and also shows that the intracellular distribution of CUL4A and CUL4B can vary by cell type. IMPORTANCE: The work presented here shows for the first time that HIV Vpr and Vpx do not rely exclusively on CUL4A to cause ubiquitination through the CRL4 ubiquitin ligase complex. Furthermore, our finding that intracellular CUL4 and SAMHD1 distributions can vary with cell type provides the basis for reconciling previous disparate findings regarding the site of SAMHD1 depletion. Finally, our observations with primary immune cells provide insight into the cell biology of CUL4A and CUL4B that will help differentiate the functions of these similar proteins.

HIV relies on neddylation for ubiquitin ligase-mediated functions.

BACKGROUND: HIV and SIV defeat antiviral proteins by usurping Cullin-RING E3 ubiquitin ligases (CRLs) and likely influence other cellular processes through these as well. HIV-2 viral protein X (Vpx) engages the cullin4-containing CRL4 complex to deplete the antiviral protein SAMHD1. Vif expressed by HIV-1 and HIV-2 taps a cullin5 ubiquitin ligase complex to mark the antiviral protein APOBEC3G for destruction. Viral Protein R of HIV-1 (Vpr) assembles with the CRL4 ubiquitin ligase complex to deplete uracil-N-glycosylase2 (UNG2). Covalent attachment of the ubiquitin-like protein side-chain NEDD8 functionally activates cullins which are common to all of these processes. RESULTS: The requirement for neddylation in HIV-1 and HIV-2 infectivity was tested in the presence of APOBEC3G and SAMHD1 respectively. Further the need for neddylation in HIV-1 Vpr-mediated depletion of UNG2 was probed. Treatment with MLN4924, an adenosine sulfamate analog which hinders the NEDD8 activating enzyme NAE1, blocked neddylation of cullin4A (CUL4A). The inhibitor hindered HIV-1 infection in the presence of APOBEC3G, even when Vif was expressed, and it stopped HIV-2 infection in the presence of SAMHD1 and Vpx. Consistent with these findings, MLN4924 prevented Vpx-mediated depletion of SAMHD1 in macrophages infected with Vpx-expressing HIV-2, as well as HIV-1 Vif-mediated destruction of APOBEC3G. It also stemmed Vpr-mediated UNG2 elimination from cells infected with HIV-1. CONCLUSIONS: Neddylation plays an important role in HIV-1 and HIV-2 infection. This observation is consistent with the essential parts that cullin-based ubiquitin ligases play in overcoming cellular anti-viral defenses.

The HIV-1 protein Vpr targets the endoribonuclease Dicer for proteasomal degradation to boost macrophage infection.

The HIV-1 protein Vpr enhances macrophage infection, triggers G2 cell cycle arrest, and targets cells for NK-cell killing. Vpr acts through the CRL4(DCAF1) ubiquitin ligase complex to cause G2 arrest and trigger expression of NK ligands. Corresponding ubiquitination targets have not been identified. UNG2 and SMUG1 are the only known substrates for Vpr-directed depletion through CRL4(DCAF1). Here we identify the endoribonuclease Dicer as a target of HIV-1 Vpr-directed proteasomal degradation through CRL4(DCAF1). We show that HIV-1 Vpr inhibits short hairpin RNA function as expected upon reduction of Dicer levels. Dicer inhibits HIV-1 replication in T cells. We demonstrate that Dicer also restricts HIV-1 replication in human monocyte-derived macrophages (MDM) and that reducing Dicer expression in MDMs enhances HIV-1 infection in a Vpr-dependent manner. Our results support a model in which Vpr complexes with human Dicer to boost its interaction with the CRL4(DCAF1) ubiquitin ligase complex and its subsequent degradation.

The role of HIV-1 Vpr in promoting the infection of nondividing cells and in cell cycle arrest.

PURPOSE OF REVIEW: The search for the role(s) that HIV-1 Vpr and its HIV2/SIV paralogs Vpr and Vpx play in viral infection and pathogenesis showed that all three engage CRL4 ubiquitin ligase complexes. This association triggers ubiquitination and degradation of cellular substrates. The identity of the ubiquitin ligase substrates is only now beginning to be revealed. This review focuses on recent work that has identified one such substrate and exposed new cellular restrictions to infection. RECENT FINDINGS: Three groups have now described cellular factors that restrict HIV-1 infection in cells of the myeloid lineage. One of these factors, sterile alpha motif- and metal-dependent phosphohydrolase domain-containing protein 1 (SAMHD1), was shown to be depleted through the CRL4 ubiquitin ligase complex in the presence of HIV-2/SIV Vpx. The other restriction can be defeated by Vpx in the absence of at least one part of the ubiquitin ligase complex that triggers SAMHD1 depletion.Another group has shown that the previously described upregulation of natural killer-cell ligands on the surface of HIV-1-infected cells requires the actions of both the cytidine deaminase APOBEC3G and uracil-N-glycosylase 2 in association with HIV-1 Vpr. SUMMARY: As more cellular interaction partners are identified for HIV-1 Vpr and its paralogs from other viruses, details are emerging about Vpr function. The recent findings have highlighted the existence of two new human proteins that can act to combat HIV infection and have revealed how HIV-1 proteins act in concert to modulate the interaction between natural killer cells and HIV-1 infected cells.