Xing-Quan Zhang

Influenza A penetrates host mucus by cleaving sialic acids with neuraminidase

BackgroundInfluenza A virus (IAV) neuraminidase (NA) cleaves sialic acids (Sias) from glycans. Inhibiting NA with oseltamivir suppresses both viral infection, and viral release from cultured human airway epithelial cells. The role of NA in viral exit is well established: it releases budding virions by cleaving Sias from glycoconjugates on infected cells and progeny virions. The role of NA in viral entry remains unclear. Host respiratory epithelia secrete a mucus layer rich in heavily sialylated glycoproteins; these could inhibit viral entry by mimicking sialylated receptors on the cell surface. It has been suggested that NA allows influenza to penetrate the mucus by cleaving these sialylated decoys, but the exact mechanism is not yet established.MethodsWe tested IAV interaction with secreted mucus using frozen human trachea/bronchus tissue sections, and bead-bound purified human salivary mucins (HSM) and purified porcine submaxillary mucins (PSM). The protective effect of mucus was analyzed using MDCK cells coated with purified HSM and PSM with known Sia content. Oseltamivir was used to inhibit NA activity, and the fluorescent reporter substrate, 4MU-Neu5Ac, was used to quantify NA activity.ResultsIAV binds to the secreted mucus layer of frozen human trachea/bronchus tissues in a Sia dependent manner. HSM inhibition of IAV infection is Sia dose-dependent, but PSM cannot inhibit infection of underlying cells. HSM competitively inhibits NA cleavage of 4MU-Neu5Ac, reporter substrate. Human IAV effectively cleaves Sias from HSM but not from PSM, and binds to HSM but not to PSM.ConclusionIAV interacts with human mucus on frozen tissue sections and mucus-coated beads. Inhibition of IAV infection by sialylated human mucus is dose-dependent, and enhanced when NA is inhibited with oseltamivir. Thus NA cleaves sialylated decoys during initial stages of infection. Understanding IAV interactions with host mucins is a promising new avenue for drug development.

Synergistic In Vitro Antiretroviral Activity of a Humanized Monoclonal Anti-CD4 Antibody (TNX-355) and Enfuvirtide (T-20)

ABSTRACT Recently, antiretroviral agents directed at several steps involved in viral entry have been shown to reduce viral replication in vitro and in vivo. We have demonstrated a high level of in vitro synergistic antiretroviral activity for two entry inhibitors that are directed at sequential steps in the entry process.

Real-Time In Vivo Green Fluorescent Protein Imaging of a Murine Leishmaniasis Model as a New Tool for Leishmania Vaccine and Drug Discovery

ABSTRACT Leishmania species are obligate intracellular protozoan parasites that cause a broad spectrum of clinical diseases in mammalian hosts. The most frequently used approach to quantify parasites in murine model systems is based on thickness measurements of the footpad or ear after experimental infection. To overcome the limitations of this method, we used a Leishmania mutant episomally transfected with enhanced green fluorescent protein, enabling in vivo real-time whole-body fluorescence imaging, to follow the progression of Leishmania infection in parasitized tissues. Fluorescence correlated with the number of Leishmania parasites in the tissue and demonstrated the real-time efficacy of a therapeutic vaccine. This approach provides several substantial advantages over currently available animal model systems for the in vivo study of immunopathogenesis, prevention, and therapy of leishmaniasis. These include improvements in sensitivity and the ability to acquire real-time data on progression and spread of the infection.

Rapid Multiplexed Immunoassay for Simultaneous Serodiagnosis of HIV-1 and Coinfections

ABSTRACT Diagnosis of opportunistic infections in HIV-infected individuals remains a major public health challenge, particularly in resource-limited settings. Here, we describe a rapid diagnostic system that delivers a panel of serologic immunoassay results using a single drop of blood, serum, or plasma. The system consists of disposable cartridges and a simple reader instrument, based on an innovative implementation of planar waveguide imaging technology. The cartridge incorporates a microarray of recombinant antigens and antibody controls in a fluidic channel, providing multiple parallel fluorescence immunoassay results for a single sample. This study demonstrates system performance by delivering antibody (Ab) reactivity results simultaneously for multiple antigens of HIV-1, Treponema pallidum (syphilis), and hepatitis C virus (HCV) in a collection of clinical serum, plasma, and whole-blood samples. By plotting antibody reactivity (fluorescence intensity) for known positive and negative samples, empirical reactivity cutoff values were defined. The HIV-1 assay shows 100% agreement with known seroreactivity for a collection of 82 HIV Ab-positive and 142 HIV Ab-negative samples, including multiple samples with HCV and syphilis coinfection. The treponema-specific syphilis assay correctly identifies 67 of 68 T. pallidum Ab-positive and 100 of 102 T. pallidum Ab-negative samples, and the HCV assay correctly identifies 59 of 60 HCV Ab-positive and 120 of 121 HCV Ab-negative samples. Multiplexed assay performance for whole-blood samples is also demonstrated. The ability to diagnose HIV and opportunistic infections simultaneously at the point of care should lead to more effective therapy decisions and improved linkage to care.

The essentiality of α‐2‐macroglobulin in human salivary innate immunity against new H1N1 swine origin influenza A virus

A novel strain of influenza A H1N1 emerged in the spring of 2009 and has spread rapidly throughout the world. Although vaccines have recently been developed that are expected to be protective, their availability was delayed until well into the influenza season. Although anti‐influenza drugs such as neuraminidase inhibitors can be effective, resistance to these drugs has already been reported. Although human saliva was known to inhibit viral infection and may thus prevent viral transmission, the components responsible for this activity on influenza virus, in particular, influenza A swine origin influenza A virus (S‐OIV), have not yet been defined. By using a proteomic approach in conjunction with beads that bind α‐2,6‐sialylated glycoprotein, we determined that an α‐2‐macroglobulin (A2M) and an A2M‐like protein are essential components in salivary innate immunity against hemagglutination mediated by a clinical isolate of S‐OIV (San Diego/01/09 S‐OIV). A model of an A2M‐based “double‐edged sword” on competition of α‐2,6‐sialylated glycoprotein receptors and inactivation of host proteases is proposed. We emphasize that endogenous A2M in human innate immunity functions as a natural inhibitor against S‐OIV.

Comparison of the Prevalence of Antibodies to Human Herpesvirus 8 (Kaposi's Sarcoma-Associated Herpesvirus) in Brazil and Colorado

The prevalence of human herpesvirus 8 (HHV-8; Kaposis sarcoma [KS] herpesvirus) infection was determined by IFA in 297 persons living in Brazil and Colorado. The prevalence of antibody to HHV-8 in human immunodeficiency virus (HIV) type 1-seropositive gay men with and without KS was similar in Brazil and Colorado. In Brazil, the prevalence of HHV-8 antibody was significantly greater in HIV-1-seronegative gay men than in HIV-1-seronegative male intravenous drug users. HHV-8-seropositive Brazilian gay men who had a clinical diagnosis of KS or who were infected with HIV-1 had significantly higher titers of HHV-8 antibody than did HHV-8-seropositive, HIV-1-seronegative Brazilian gay men. These findings provide further support for the association between HHV-8 infection and KS and suggest that, as in the United States, HHV-8 infection is transmitted sexually in Brazil.

Detection in 2009 of the swine origin influenza A (H1N1) virus by a subtyping microarray.

A novel H1N1 swine origin influenza A (IAV) virus (S-OIV) was discovered in specimens from two unrelated children in the San Diego area in mid-April 2009 (1, 2). Those samples were positive for IAV but negative for both human H1 and H3 subtypes. The outbreak evolved rapidly, and the World Health Organization (WHO) declared the highest phase 6 worldwide pandemic alert on 11 June 2009. While the severity of the illness caused by the virus currently does not exceed that of seasonal IAV, the unpredictable nature of IAV evolution via mutation and reassortment poses serious concern (6). This is made evident by the analysis of the origins of S-OIV that has three genome segments (HA, NP, NS) from the classic North American swine (H1N1) lineage, two segments (PB2, PA) from the North American avian lineage, one segment (PB1) from the seasonal H3N2 lineage, and, most notably, two segments (NA, M) from the Eurasian swine (H1N1) lineage (2). Cocirculation of current seasonal human IAV H1N1, IAV H3N2, and S-OIV in the upcoming flu seasons poses a challenge for subtyping individual strains and potential reassortants. The more complicated, and perhaps dangerous, scenario is the reassortment of S-OIV with highly pathogenic avian IAV (H5N1) or with other serotypes. While S-OIV-specific real-time PCR detection is available (2), it may not be applicable for more-complex reassortant strains. Here, we present a microarray-based assay to classify S-OIV. We took advantage of a previously developed Virochip technique (5) and a universal primer set for enriching gene segments of all IAV strains (3). We constructed several sets of 50- to 70-bp probes, targeting hemagglutinin and neuraminidase specific to H5N1, H3N2, and H1N1 serotypes and more-conserved gene segments, for general “influenza A” detection, using a custom bioinformatics pipeline (http://www.hyphy.org/pubs/IAVprobes). Computational quality control established that all sets of probes achieved 95% sensitivity or better (at least one of the probes had less than two mismatches to every target sequence from the NCBI Influenza database) and 95% specificity or better (all probes had at least three mismatches to sequences of the incorrect serotype). Probes with predicted secondary RNA structure and long regions of homology to IAV sequences of the incorrect serotype or non-IAV sequences were excluded from the candidate set. Nasal swabs were collected from patients for virus culture in MDCK cells. Viral RNA was isolated from 100 μl of culture medium that was mixed with 300 μl of Trizol LS (Invitrogen) in the presence of 1 μg of linear polyacrylamide and subsequently treated with DNase to remove contaminated host genomic DNA. Viral RNA was converted to cDNA, with a 1:1 ratio of primer A and primer B-Uni12 (GTT TCC CAG TCA CGA TCA GCA AAA GCA GG) (round A), amplified, and labeled with primer B (rounds B and C), as described previously (4, 5). We were able to identify six S-OIV samples with hybridization patterns through the analysis on the microarray (Fig. ​(Fig.1).1). The array result was further confirmed by PCR with the validated primers (2) and direct sequencing. Overall, the S-OIV is readily distinguishable from seasonal influenza virus with strain-specific probes (especially those derived from HA and NA). Cross-hybridizations were observed with more-conserved probes. We expect that a unique hybridization pattern may occur when coinfection or reassortment of two strains of influenza viruses occurs (5). FIG. 1. Influenza virus subtyping microarray. Two representative arrays for S-OIV (A) and seasonal H1N1 (B) are shown. The probes for S-OIV are located at the bottom of each block (enclosed by pink and red rectangles). S-OIV- and seasonal H1N1-specific probes ...

Human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) infection in men receiving treatment for HIV-1 infection

OBJECTIVE To determine the prevalence of human herpesvirus 8 (HHV-8) infection in men treated for HIV-1 infection in Denver, Colorado. DESIGN Cross-sectional analysis METHODS Blood samples were obtained from 216 HIV-1-infected men. Antibody to latency-associated nuclear antigen (LANA) was detected by an immunofluorescent assay and the presence of HHV-8 in peripheral blood mononuclear cells (PBMC) was detected by polymerase chain reaction amplification. RESULTS Among HIV-1-infected men who did not have Kaposis sarcoma (KS), prevalence of HHV-8 infection was 46% (95% confidence interval [CI], 0.39-0.52). LANA seropositivity was common both among subjects with KS and subjects without KS (69% versus 42%; p = .06), but detection of HHV-8 DNA in peripheral blood was strongly associated with a diagnosis of KS (44% versus 10%; p = .001). In a univariate analysis of study subjects without KS, neither the odds of LANA seropositivity nor detection of HHV-8 DNA in PBMC was significant for CD4+ lymphocyte count, HIV-1 virus load, the use of three drug antiretroviral regimens or the prior occurrence of non-KS AIDS-related conditions. CONCLUSION Although antibodies to HHV-8 are common among HIV-1-infected men, detection of HHV-8 DNA in PBMC is uncommon and is associated with a diagnosis of Kaposis sarcoma.

Th1/Th2 Cytokine Profile in Patients Coinfected with HIV and Leishmania in Brazil

ABSTRACT To evaluate the effects of HIV on immune responses in cutaneous leishmaniasis (CL), we quantified cytokine levels from plasma and stimulated peripheral blood mononuclear cells (PBMCs) from individuals infected with HIV and/or CL. Gamma interferon (IFN-γ) and interleukin 13 (IL-13) levels and the ratio of IFN-γ to IL-10 produced in response to stimulation with soluble Leishmania antigens were significantly lower in HIV-Leishmania-coinfected patients than in CL-monoinfected patients.

Flow Cytometric Screening for Anti-Leishmanials in a Human Macrophage Cell Line

High-throughput drug screening methods against the intracellular stage of Leishmania have been facilitated by the development of in vitro models of infection. The use of cell lines rather than primary cells facilitates these methods. Peripheral blood mononuclear cell (PBMC) derived macrophages and THP-1 cells were infected with stationary phase egfp transfected Leishmania amazonensis parasites and then treated with anti-leishmanial compounds. Drug activity was measured using a flow cytometric approach, and toxicity was assessed using either the MTT assay or trypan blue dye exclusion. Calculated EC(50)s for amphotericin B, sodium stibogluconate, and miltefosine were 0.1445±0.0005μg/ml, 0.1203±0.018mg/ml, and 26.71μM using THP-1 cells, and 0.179±0.035μg/ml, 0.1948±0.0364mg/ml, and 13.77±10.74μM using PBMC derived macrophages, respectively. We conclude that a flow cytometric approach using egfp transfected Leishmania species can be used to evaluate anti-leishmanial compounds against the amastigote stage of the parasite in THP-1 cells with excellent concordance to human PBMC derived macrophages.