Anti-monkeypox virus-neutralizing activities of human immunoglobulin manufactured between 1999 and 2021 and derived from donors in the United States and Japan.

BACKGROUND AND OBJECTIVES: In May 2022, the United Kingdom reported the first case of chained transmission of the monkeypox (mpox) virus without any known epidemiological links to west or central Africa. The monthly number of mpox patients currently has passed a peak and is declining globally, and infected patients include both non-vaccinated and vaccinated individuals. Herein, the virus-neutralizing (VN) activity against vaccinia viruses, which are considered to cross-react with the mpox virus, in the intravenous immunoglobulin (IVIG) lots derived from donors, including vaccinated Japanese populations, was evaluated to clarify the status of the Japanese blood donor population. MATERIALS AND METHODS: VN titres against vaccinia and human mpox viruses in IVIG lots derived from donors in Japan and the United States manufactured between 1999 and 2021 and 1995 and 2001, respectively, were evaluated by neutralization testing. RESULTS: VN titres of IVIG derived from donors in Japan and the United States against vaccinia and mpox viruses showed a slowly decreasing trend between 1999 and 2021. CONCLUSION: VN titres are expected to decrease in the future since the percentage of vaccinated donors in the donor population seems to have decreased. Therefore, continuous monitoring of VN titres is required.

COVID-19 relapse associated with SARS-CoV-2 evasion from CD4+ T-cell recognition in an agammaglobulinemia patient.

A 25-year-old patient with a primary immunodeficiency lacking immunoglobulin production experienced a relapse after a 239-day period of persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Viral genetic sequencing demonstrated that SARS-CoV-2 had evolved during the infection period, with at least five mutations associated with host cellular immune recognition. Among them, the T32I mutation in ORF3a was found to evade recognition by CD4+ T cells. The virus found after relapse showed an increased proliferative capacity in vitro. SARS-CoV-2 may have evolved to evade recognition by CD4+ T cells and increased in its proliferative capacity during the persistent infection, likely leading to relapse. These mutations may further affect viral clearance in hosts with similar types of human leukocyte antigens. The early elimination of SARS-CoV-2 in immunocompromised patients is therefore important not only to improve the condition of patients but also to prevent the emergence of mutants that threaten public health.

Changes in Anti-SARS-CoV-2 Antibody Titers of Pooled Plasma Derived From Donors in Japan: A Potential Tool for Mass-Immunity Evaluation.

The anti-spike (S), anti-nucleocapsid (N), and neutralizing activities of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) of pooled plasma derived from donors in Japan from January 2021 to April 2022 were evaluated. Anti-S titers and neutralizing activities showed a wave-like trend affected by daily vaccinations and/or the number of reported cases of SARS-CoV-2 infections, whereas anti-N titers remained at negative levels. These results suggest that anti-S and neutralizing titers would fluctuate in pooled plasma in the future. Pooled plasma may be potentially used for mass-immunity evaluation, and titer estimation in intravenous immunoglobulin, a derivative of pooled plasma.

Decline in intravenous immunoglobulin titer against influenza virus A/H2N2 derived from donors in Japan.

Human influenza A/H2N2 can induce a pandemic in the future. This study evaluated the hemagglutination inhibition and neutralizing titers of intravenous immunoglobulin against A/H2N2 viruses, indicating the status of the donor population. In this study, the antibody titers decreased during the study period-2012-2021-suggesting a reduction in the immunity of the studied population.

Live attenuated VZV vaccination induces antitumor immunity in ATLL patients.

Adult T cell leukemia/lymphoma (ATLL) is a CD4-positive peripheral T cell lymphoma caused by human T cell lymphotropic virus type 1 (HTLV-1). Although ATLL is quite difficult to be cured, up-regulation of cellular immunity such as HTLV-1 Tax-specific cytotoxic T lymphocytes (CTLs) has been proved to be important to obtain long-term survival. At present, no efficacious method to activate ATLL-specific cellular immunity is available. This study aimed to investigate whether live attenuated varicella-zoster virus (VZV) vaccination to ATLL can activate HTLV-1 Tax-specific cellular immune response. A total of 3 indolent- and 3 aggressive-type ATLL patients were enrolled. All aggressive-type patients had the VZV vaccination after completing anti-ATLL treatment including mogamulizumab, which is a monoclonal antibody for C-C chemokine receptor 4 antigen, plus combination chemotherapy, whereas all indolent-type patients had the VZV vaccination without any antitumor treatment. Cellular immune responses including Tax-specific CTLs were analyzed at several time points of pre- and post-VZV vaccination. After the VZV vaccination, a moderate increase in 1 of 3 indolent-type patients and obvious increase in all 3 aggressive-type patients in Tax-specific CTLs percentage were observed. The increase in the cell-mediated immunity against VZV was observed in all indolent- and aggressive-type patients after VZV vaccination. To conclude, VZV vaccination to aggressive-type ATLL patients after mogamulizumab plus chemotherapy led to the up-regulation of HTLV-1 Tax-specific CTLs without any adverse event. Suppression of regulatory T lymphocytes by mogamulizumab may have contributed to increase tumor immunity in aggressive-type ATLL patients. Japan Registry of Clinical Trials number, jRCTs051180107.

Anti-nucleocapsid antibodies enhance the production of IL-6 induced by SARS-CoV-2 N protein.

A cytokine storm induces acute respiratory distress syndrome, the main cause of death in coronavirus disease 2019 (COVID-19) patients. However, the detailed mechanisms of cytokine induction due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain unclear. To examine the cytokine production in COVID-19, we mimicked the disease in SARS-CoV-2-infected alveoli by adding the lysate of SARS-CoV-2-infected cells to cultured macrophages or induced pluripotent stem cell-derived myeloid cells. The cells secreted interleukin (IL)-6 after the addition of SARS-CoV-2-infected cell lysate. Screening of 25 SARS-CoV-2 protein-expressing plasmids revealed that the N protein-coding plasmid alone induced IL-6 production. The addition of anti-N antibody further enhanced IL-6 production, but the F(ab')2 fragment did not. Sera from COVID-19 patients also enhanced IL-6 production, and sera from patients with severer disease induced higher levels of IL-6. These results suggest that anti-N antibody promotes IL-6 production in SARS-CoV-2-infected alveoli, leading to the cytokine storm of COVID-19.

Preclinical study of a DNA vaccine targeting SARS-CoV-2.

To fight against the worldwide COVID-19 pandemic, the development of an effective and safe vaccine against SARS-CoV-2 is required. As potential pandemic vaccines, DNA/RNA vaccines, viral vector vaccines and protein-based vaccines have been rapidly developed to prevent pandemic spread worldwide. In this study, we designed plasmid DNA vaccine targeting the SARS-CoV-2 Spike glycoprotein (S protein) as pandemic vaccine, and the humoral, cellular, and functional immune responses were characterized to support proceeding to initial human clinical trials. After intramuscular injection of DNA vaccine encoding S protein with alum adjuvant (three times at 2-week intervals), the humoral immunoreaction, as assessed by anti-S protein or anti-receptor-binding domain (RBD) antibody titers, and the cellular immunoreaction, as assessed by antigen-induced IFNγ expression, were up-regulated. In IgG subclass analysis, IgG2b was induced as the main subclass. Based on these analyses, DNA vaccine with alum adjuvant preferentially induced Th1-type T cell polarization. We confirmed the neutralizing action of DNA vaccine-induced antibodies by a binding assay of RBD recombinant protein with angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, and neutralization assays using pseudo-virus, and live SARS-CoV-2. Further B cell epitope mapping analysis using a peptide array showed that most vaccine-induced antibodies recognized the S2 and RBD subunits. Finally, DNA vaccine protected hamsters from SARS-CoV-2 infection. In conclusion, DNA vaccine targeting the spike glycoprotein of SARS-CoV-2 might be an effective and safe approach to combat the COVID-19 pandemic.

HTLV-1 Tax-specific memory cytotoxic T lymphocytes in long-term survivors of aggressive-type adult T-cell leukemia/lymphoma.

PURPOSE: Adult T-cell leukemia/lymphoma (ATLL) is a relatively refractory peripheral T-cell lymphoma caused by human T-cell lymphotropic virus type 1 (HTLV-1). The objective of this study was to investigate the characteristics of long-term survivors with ATLL. METHODS: We conducted an observational study of 75 aggressive-type ATLL patients. Flow cytometry was conducted to analyze HTLV-1 Tax-specific cytotoxic T-lymphocytes (CTLs) and T-cell receptor Vß gene repertoire. RESULTS: We first evaluated six long-term survivors among 37 patients who were newly diagnosed with ATLL and then treated with intensive chemotherapy without mogamulizumab, a monoclonal antibody for C-C chemokine receptor four antigen. Reversal of the CD4-to-CD8 ratio (CD4/CD8) in peripheral mononuclear cells was observed in all six patients. Three of these six patients showed reversed CD4/CD8 immediately after herpes virus infection. Four of these six patients who could be examined demonstrated long-term maintenance of HTLV-1 Tax-specific CTLs. We subsequently identified four long-term survivors among 38 patients who were newly diagnosed with ATLL and then treated with intensive chemotherapy plus mogamulizumab. All four patients showed reversed CD4/CD8, and three of the four patients contracted herpes virus infection during immunochemotherapy. Six of the total 10 patients were subjected to CTL analyses. Tax-specific CTLs were observed, and the CTLs that were almost entirely composed of memory CTLs in all patients were recorded. HTLV-1 provirus was also detected in all six patients. CONCLUSIONS: These data suggest that Tax-specific memory CTLs probably, together with anticancer agents, eradicate ATLL cells and exhibit long-term preventive effects from relapse ATLL. Thus, the strong activation of cellular immunity, such as herpes virus infection, seems to be necessary to induce such a potent number of Tax-specific CTLs.

Infectivity assay for detection of SARS-CoV-2 in samples from patients with COVID-19.

Since the coronavirus disease 2019 (COVID-19) outbreak, laboratory diagnosis has mainly been conducted using reverse-transcription polymerase chain reaction (RT-PCR). Detecting the presence of an infectious virus in the collected sample is essential to analyze if a person can transmit infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there have been no quantitative investigations conducted for infectious SARS-CoV-2 in clinical samples. Therefore, in the present study, a rapid and simple focus-forming assay using the peroxidase-antiperoxidase technique was developed to quantify infectious SARS-CoV-2 titers in 119 samples (n = 52, nasopharyngeal swabs [NPS]; n = 67, saliva) from patients with COVID-19. Furthermore, the study findings were compared with the cycle threshold (Ct) values of real-time RT-PCR. The infectious virus titers in NPS samples and Ct values were inversely correlated, and no infectious virus could be detected when the Ct value exceeded 30. In contrast, a low correlation was observed between the infectious virus titers in saliva and Ct values (r = -0.261, p = 0.027). Furthermore, the infectious virus titers in the saliva were significantly lower than those in the NPS samples. Ten days after the onset of COVID-19 symptoms, the infectious virus was undetectable, and Ct values were more than 30 in NSP and saliva samples. The results indicate that patients whose symptoms subsided 10 days after onset, with Ct values more than 30 in NSP and saliva samples, were less likely to infect others.

Virus Neutralization by Human Intravenous Immunoglobulin Against Influenza Virus Subtypes A/H5 and A/H7.

PURPOSE: Highly pathogenic avian influenza viruses are a threat to human health. Although donor populations have not experienced pandemic, they have been immunized by natural infections and/or vaccinations of influenza viruses such as A/H1N1, A/H3N2, and B. Therefore, it is considered that human intravenous immunoglobulin (IVIG) derived from healthy donors does not include IgG against avian influenza viruses. However, cross-reactivity has not been evaluated yet. In this study, cross-reactivity against the avian influenza virus A/H5N1, A/H7N1, A/H7N2, A/H7N7, A/H7N9, and A/H10N9 was evaluated. MATERIALS AND METHODS: Several lots of IVIG derived from healthy donors in Japan were tested for virus neutralization using single- or multi-cycle virus neutralizing (S-VN or M-VN) assays that evaluate the infection-step associated with HA or the infection and propagation steps associated with HA and NA, respectively. In addition, anti-NA activities were evaluated by inhibiting the enzymatic activity in NAI assays. RESULTS: IVIG lots showed high neutralizing activities against three A/H5N1 strains in M-VN assays, whereas activities in S-VN assays were unstable. In addition, A/H7N2 was also neutralized in S-VN and M-VN assays, with higher activity in M-VN than in S-VN assays. A/H7N1 was neutralized in S-VN and M-VN assays. In contrast, weak or no activity against A/H7N7, A/H7N9, and A/H10N9 was observed in S-VN and M-VN assays. NAI assay results show that IVIG lots had inhibitory activities against N1 and N2; however, N2 activities differed depending on the strain. In contrast, no activities were observed against N7 and N9. CONCLUSION: These results suggest that IVIG lots have neutralizing activity against avian influenza viruses during the virus propagation step, except for one strain, although no or weak activity was observed during the infection step.

Neutralizing and binding activities against SARS-CoV-1/2, MERS-CoV, and human coronaviruses 229E and OC43 by normal human intravenous immunoglobulin derived from healthy donors in Japan.

BACKGROUND: There are several types of coronaviruses that infect humans and cause disease. The latest is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is an emerging global threat with no current effective treatment. Normal intravenous immunoglobulin (N-IVIG) has been administered to coronavirus disease 2019 (COVID-19) patients to control severe inflammation and the cellular immune response. However, the neutralizing activity of N-IVIG against SARS-CoV-2 has not yet been fully evaluated. The aim of this study was to determine whether N-IVIG manufactured before the start of the COVID-19 pandemic contained IgG antibodies against the circulating human coronaviruses (HCoVs) that cross-react with the highly pathogenic coronaviruses SARS-CoV-1, Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. No cases of SARS-CoV-1 or MERS-CoV have been reported in Japan. STUDY DESIGN AND METHODS: The neutralizing and binding activities of N-IVIG against SARS-CoV-1, MERS-CoV, SARS-CoV-2, HCoV 229E, and HCoV OC43 were evaluated. Nine N-IVIG lots manufactured between 2000 and 2018, derived from donors in Japan, were tested. Binding activity was evaluated by indirect immunofluorescence assay. RESULTS: None of the N-IVIG lots tested displayed neutralizing or binding activity against SARS-CoV-1, MERS-CoV, or SARS-CoV-2. However, they displayed substantial neutralizing and binding activity against HCoV OC43 and weak neutralizing and substantial binding activity against HCoV 229E. CONCLUSION: N-IVIG derived from healthy donors in Japan before the start of the COVID-19 pandemic had no direct effect against SARS-CoV-2. Further studies are warranted to determine the effects of N-IVIG manufactured after the start of the COVID-19 pandemic against SARS-CoV-2.

Comprehensive Analysis of Antibodies Induced by Vaccination with 4 Kinds of Avian Influenza H5N1 Pre-Pandemic Vaccines.

Four kinds of avian-derived H5N1 influenza virus, A/Vietnam/1194/2004 (Clade 1), A/Indonesia/5/2005 (Clade 2.1), A/Qinghai/1A/2005 (Clade 2.2), and A/Anhui/1/2005 (Clade 2.3), have been stocked in Japan for use as pre-pandemic vaccines. When a pandemic occurs, these viruses would be used as vaccines in the hope of inducing immunity against the pandemic virus. We analyzed the specificity of antibodies (Abs) produced by B lymphocytes present in the blood after immunization with these vaccines. Eighteen volunteers took part in this project. After libraries of Ab-encoding sequences were constructed using blood from subjects vaccinated with these viruses, a large number of clones that encoded Abs that bound to the virus particles used as vaccines were isolated. These clones were classified into two groups according to the hemagglutination inhibition (HI) activity of the encoded Abs. While two-thirds of the clones were HI positive, the encoded Abs exhibited only restricted strain specificity. On the other hand, half of the HI-negative clones encoded Abs that bound not only to the H5N1 virus but also to the H1N1 virus; with a few exceptions, these Abs appeared to be encoded by memory B cells present before vaccination. The HI-negative clones included those encoding broadly cross-reactive Abs, some of which were encoded by non-VH1-69 germline genes. However, although this work shows that various kinds of anti-H5N1 Abs are encoded by volunteers vaccinated with pre-pandemic vaccines, broad cross-reactivity was seen only in a minority of clones, raising concern regarding the utility of these H5N1 vaccine viruses for the prevention of H5N1 pandemics.

Mogamulizumab Plus EPOCH Therapy for Patients With Newly Diagnosed Aggressive Adult T-cell Leukemia/lymphoma.

BACKGROUND/AIM: Adult T-cell leukemia/lymphoma (ATLL) is a relatively refractory CD4-positive peripheral T-cell lymphoma. VCAP-AMP-VECP (mLSG15) is one of the standard chemotherapeutic regimens for patients with aggressive ATLL. Mogamulizumab (moga), a monoclonal antibody for C-C chemokine receptor 4 antigen expressed on the cell surface, has recently been poised for use as monotherapy and in combination with chemotherapy. However, to date, a significant survival benefit has not been obtained with the combination of moga + mLSG15 therapy. PATIENTS AND METHODS: We retrospectively analyzed 77 patients diagnosed with aggressive ATLL. Of them, 22 were treated with moga + a chemotherapy regimen comprised of etoposide, vincristine, doxorubicin, cyclophosphamide, and prednisolone (EPOCH), 16 with moga + mLSG15, and 39 with chemotherapy alone. RESULTS: A risk reduction of approximately 30% was obtained with moga + EPOCH compared with moga + mLSG15. CONCLUSION: The addition of moga to chemotherapy did not result in a survival benefit compared with chemotherapy alone. However, a statistically significant overall survival benefit was observed in patients with moga-induced skin disorders.

Cytokine production in whole-blood cultures following immunization with an influenza vaccine.

A clinical trial of a quadrivalent split influenza vaccine was performed in the 2014/15 season. Sixty-four subjects aged 6 months to 18 years were enrolled in order to investigate the relationship between cellular and humoral immune responses. Subjects were categorized into two groups by measuring neutralizing antibodies: non-primed naïve/primed or seroconverted/non-seroconverted groups. Whole-blood cultures were stimulated with the H1N1 split antigen before immunization and one month after the first and second immunizations for subjects < 13 years and before and one month after the first dose for those ≥ 13 years in order to investigate cytokine production. Significant amounts of IL-2, IL-12, IL-13, MCP-1, MIP-1ß, and TNF-α were detected from one month after the first dose in the naïve group. In addition to these cytokines, the production of IL-1ß, IL-4, IL-6, IL-8, IL-10, IL-17, G-CSF, and IFN-γ was enhanced one month after the second dose. No significant increase was noted in the primed group, except in the production of IL-10. In seroconverted subjects, the production of IL-2, IL-4, IL-8, IL-10, G-CSF, MCP-1, TNF-α, and IFN-γ increased one month after the first dose, which was earlier than in the naïve group, whereas no significant cytokine response was noted in subjects without seroconversion. Subjects ≥ 13 years were primed and the production of G-CSF, IL-4, and IL-1ß increased in subjects with seroconversion. Whole-blood cultures were also stimulated with the H3N2 split antigen and similar cytokine profiles were obtained. Many cytokines and chemokines, including inflammatory cytokines, were produced in seroconverted, but not non-seroconverted subjects.

Three Types of Broadly Reacting Antibodies against Influenza B Viruses Induced by Vaccination with Seasonal Influenza Viruses.

We analyzed the antibody (Ab) repertoire against influenza B viruses induced by vaccination with seasonal influenza viruses in one individual who had never been vaccinated until 2009. The vaccine used in this study comprised B/Massachusetts/2/2012 (Yamagata lineage), A/Texas/50/2012 (H3N2), and A/California/7/2009 (H1N1). One month after the subject received two vaccinations, blood (200 ml) was obtained and peripheral mononuclear cells were prepared, and a large Ab library was constructed using phage display technology. The library was screened with HA-enriched fraction of B/Massachusetts/2/2012 and B/Brisbane/60/2008 (Victoria lineage) virus, and a total of 26 Abs that potentially bound to hemagglutinin (HA) molecules were isolated. Their binding activities to six influenza B viruses, three of Yamagata lineage and three of Victoria lineage, and two influenza A viruses, H1N1 and H3N2, were examined. The Abs showed cross-reactivity at three different levels. The first type bound to all Yamagata lineage viruses. The second type bound to both Yamagata and Victoria lineage viruses. The third type bound to both influenza A and B viruses. These results indicate that common epitopes exist on HA molecules of influenza virus at various levels, and humans have capability to produce Abs that bind to such common epitopes.

Neutralizing activities against seasonal influenza viruses in human intravenous immunoglobulin.

Influenza viruses A/H1N1, A/H3N2, and B are known seasonal viruses that undergo annual mutation. Intravenous immunoglobulin (IVIG) contains anti-seasonal influenza virus globulins. Although the virus-neutralizing (VN) titer is an indicator of protective antibodies, changes in this titer over extended time periods have yet to be examined. In this study, variations in hemagglutination inhibition (HI) and VN titers against seasonal influenza viruses in IVIG lots over extended time periods were examined. In addition, the importance of monitoring the reactivity of IVIG against seasonal influenza viruses with varying antigenicity was evaluated. A/H1N1, A/H3N2, and B influenza virus strains and IVIG lots manufactured from 1999 to 2014 were examined. The HI titer was measured by standard methods. The VN titer was measured using a micro-focus method. IVIG exhibited significant HI and VN titers against all investigated strains. Our results suggest that the donor population maintains both specific and cross-reactive antibodies against seasonal influenza viruses, except in cases of pandemic viruses, despite major antigen changes. The titers against seasonal influenza vaccine strains, including past strains, were stable over short time periods but increased slowly over time.

Recombinant production and characterization of human anti-influenza virus monoclonal antibodies identified from hybridomas fused with human lymphocytes.

In previous studies, hybridomas producing human immunoglobulin G, the antibodies 5E4 and 5A7 against influenza A and B virus were established using a novel human lymphocyte fusion partner, SPYMEG. In the present study, we succeeded in achieving the recombinant production and secretion of 5E4 and 5A7 in Chinese hamster ovary cells. Our N-glycan analysis by intact-mass detection and liquid chromatography mass spectrometry showed that recombinant 5E4 and 5A7 have one N-glycan and the typical mammalian-type N-glycan structures similar to those in hybridomas. However, the glycan distribution was slightly different among these antibodies. The amount of high-mannose-type structures was under 10% of the total N-glycans of recombinant 5E4 and 5A7, compared to 20% of the 5E4 and 5A7 produced in hybridomas. The amount of galactosylated N-glycans was increased in recombinants. Approximately 80% of the N-glycans of all antibodies was fucosylated, and no sialylated N-glycan was found. Recombinant 5E4 and 5A7 neutralized pandemic influenza A virus specifically, and influenza B virus broadly, quite similar to the 5E4 and 5A7 produced in hybridomas, respectively. Here we demonstrated that recombinants of antibodies identified from hybridomas fused with SPYMEG have normal N-glycans and that their neutralizing activities bear comparison with those of the original antibodies.

Characterization of H5N1 influenza virus variants with hemagglutinin mutations isolated from patients.

UNLABELLED: A change in viral hemagglutinin (HA) receptor binding specificity from α2,3- to α2,6-linked sialic acid is necessary for highly pathogenic avian influenza (AI) virus subtype H5N1 to become pandemic. However, details of the human-adaptive change in the H5N1 virus remain unknown. Our database search of H5N1 clade 2.2.1 viruses circulating in Egypt identified multiple HA mutations that had been selected in infected patients. Using reverse genetics, we found that increases in both human receptor specificity and the HA pH threshold for membrane fusion were necessary to facilitate replication of the virus variants in human airway epithelia. Furthermore, variants with enhanced replication in human cells had decreased HA stability, apparently to compensate for the changes in viral receptor specificity and membrane fusion activity. Our findings showed that H5N1 viruses could rapidly adapt to growth in the human airway microenvironment by altering their HA properties in infected patients and provided new insights into the human-adaptive mechanisms of AI viruses. IMPORTANCE: Circulation between bird and human hosts may allow H5N1 viruses to acquire amino acid changes that increase fitness for human infections. However, human-adaptive changes in H5N1 viruses have not been adequately investigated. In this study, we found that multiple HA mutations were actually selected in H5N1-infected patients and that H5N1 variants with some of these HA mutations had increased human-type receptor specificity and increased HA membrane fusion activity, both of which are advantageous for viral replication in human airway epithelia. Furthermore, HA mutants selected during viral replication in patients were likely to have less HA stability, apparently as a compensatory mechanism. These results begin to clarify the picture of the H5N1 human-adaptive mechanism.

A novel immunochromatographic system for easy-to-use detection of group 1 avian influenza viruses with acquired human-type receptor binding specificity.

A switch of viral hemagglutinin receptor binding specificity from bird-type α2,3- to human-type α2,6-linked sialic acid is necessary for an avian influenza virus to become a pandemic virus. In this study, an easy-to-use strip test to detect receptor binding specificity of influenza virus was developed. A biotinylated anti-hemagglutinin antibody that bound a broad range of group 1 influenza A viruses and latex-conjugated α2,3 (blue) and α2,6 (red) sialylglycopolymers were used in an immunochromatographic strip test, with avidin and lectin immobilized on a nitrocellulose membrane at test and control lines, respectively. Accumulation of a sialylglycopolymer-virus-antibody complex at the test line was visualized by eye. The strip test could be completed in 30min and did not require special equipment or skills, thereby avoiding some disadvantages of current methods for analyzing receptor binding specificity of influenza virus. The strip test could detect the receptor binding specificity of a wide range of influenza viruses, as well as small increases in the binding affinity of variant H5N1 viruses to α2,6 sialylglycans at viral titers >128 hemagglutination units. The strip test results were in agreement with those of ELISA virus binding assays, with correlations >0.95. In conclusion, the immunochromatographic strip test developed in this study should be useful for monitoring potential changes in the receptor binding specificity of group 1 influenza A viruses in the field.