Localized antibody responses in influenza virus-infected mice

The Abstracts of the Conference is located at: http://optionsviii.controlinfluenza.com/optionsviii/assets/File/Options_VIII_Abstracts_2013.pdfPoster Session: Innate and Adaptive ImmunityBackground: Traditionally, vaccine-mediated protective responses were quantified by measuring the level of increase of influenza virus–specific antibodies circulating in blood. However, virus-specific antibodies in serum do not necessarily correlate with protection in vaccinees receiving intranasally administrated live attenuated influenza vaccines (LAIVs). Local mucosal and cellular immune responses are believed to be the protective mechanism induced by LAIVs. Recently, antibody secreting cell (ASC) responses derived from peripheral blood mononuclear cells (Cherukuri A, et al. Vaccine. 2012;356:685-696) of ferrets and antibody obtained via human nasal washes, but not the systemic serum (Barria MI, et al. J Infect Dis. 2013;207:115-124), were found to better correlate with B-cell responses induced by LAIV. ASCs are found in the upper and lower respiratory tract in influenza infections and play an important role in combating influenza infections. Analyses of antigenspecific B-cell receptors on these ASCs were limited by cell-based assays such as ELISPOT or FACS probe by hapten or B-cell tetramers. Although the localized mucosal and systematic ASC responses of Influenza A virus–infected mice are different (Joo HM, et al. Vaccine. 2010;28:2186-2194), direct comparisons of antibodies secreted by ASCs at these locations are lacking. Here, we isolate antibodies secreted by ACSs at multiple anatomical sites and characterize the epitope specificity and other properties of these antibodies systematically. Materials and Methods: Mice intranasally infected with influenza virus (A/HK/68) were used as a model. Lymphocytes from different nodes (eg, cervical lymph nodes [CLNs], which drain the upper respiratory tract, and mediastinal lymph nodes [MLNs], which drain the lower respiratory tract) and from the spleen of infected mice were harvested for cell cultures at days 3, 7, and 28 post-infection. In addition, supernatants of nasal washes, bronchoalveolar lavage fluid, and serum of the mice were harvested. Antibodies secreted by the cultured cells and antibody presented in the harvested body fluids were characterized by influenza A– specific isotyping ELISA, micro-neutralization assay, as well as fine epitope mapping assay using a yeast surface display library for H3 hemagglutinin. Results: Antibody in lymphocyte supernatants (ALS) from cultured cells of MLNs and spleen and antibody in serum were found to be positive for influenza virus–specific IgM at day 3 post-infection. Nasal washes, bronchoalveolar lavage fluid, and ALS from CLNs and MLNs were found to be IgA-positive at day 7 post-infection. High IgG1 and IgG2a responses were detected in ALS from MLNs at day 7 post-infection. The control ALS from cells derived from iliac lymph nodes, which drain the mouse tail but not the respiratory tract, was negative for influenza-specific IgA, IgG1, IgG2a, and IgM throughout the study. Bronchoalveolar lavage fluid collected at day 28 post-infection and ALS from MLNs collected at day 7 post-infection demonstrated of ALS from MLNs collected at day 7 and of serum collected at day 28 post-infection was also performed. The antibody repertoire mappings were comparable and both identified a major immunodominant antigenic site in HA1 and a weaker antigenic site located in HA2. However, two additional antigenic sites were identified in the mapping of ALS from MLNs collected at day 7 when compared with the mapping of the serum obtained at day 28 post-infection. Conclusions: This study illustrated the feasibility of recovering ASC specificity at different localizations after influenza A challenge. With the use of cell-free supernatant, the properties of the ASC-secreted antibodies can be further characterized by various methods traditionally used for serum. The method described will provide information about influenza A–induced antibody responses early post-infection, ie, at the time when the virus was cleared.published_or_final_versio

Crouching Tiger, Hidden Dragon: The Laboratory Diagnosis of Severe Acute Respiratory Syndrome

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Definition of the cellular interactome of the highly pathogenic avian influenza H5N1 virus: identification of human cellular regulators of viral entry, assembly, and egress.

This study was supported by the Research Fund for the Control of Infectious Diseases, Food and Health Bureau, Hong Kong SAR Government (#09080892), University Grants Committee Area of Excellence Scheme (AoE/M-12/-06), French Ministry of Health, RESPARI Pasteur network, and the Li Ka Shing Foundation

A statistical strategy to identify recombinant viral ribonucleoprotein of avian, human, and swine influenza A viruses with elevated polymerase activity

Objectives: Reassortment of influenza A viruses can give rise to viral ribonucleoproteins (vRNPs) with elevated polymerase activity and the previous three pandemic influenza viruses contained reassorted vRNPs of different origins. These suggest that reassorted vRNP may be one of the factors leading to a pandemic virus. In this study, we reconstituted chimeric vRNPs with three different viral strains isolated from avian, human and swine hosts. We applied a statistical strategy to identify the effect that the origin of a single vRNP protein subunit or the interactions between these subunits on polymerase activity. Design: Eighty one chimeric vRNPs were reconstituted in 293T cells at different temperatures. Polymerase activity was determined by luciferase reporter assay and the results were analysed by multiway anova and other statistical methods. Results: It was found that PB2, PB1, NP, PB2-PB1 interaction, PB2-PA interaction and PB1-NP interaction had significant effect on polymerase activity at 37°C and several single subunits and interactions were identified to lead to elevation of polymerase activity. Furthermore, we studied 27 out of these 81 different chimieric vRNPs in different combinations via fractional factorial design approach. Our results suggested that the approach can identify the major single subunit or interaction factors that affect the polymerase activity without the need to experimentally reproduce all possible vRNP combinations. Conclusions: Statistical approach and fractional factorial design are useful to identify the major single subunit or interaction factors that can modulate viral polymerase activity. © 2013 John Wiley & Sons Ltd.published_or_final_versio

Substitution at Aspartic Acid 1128 in the SARS Coronavirus Spike Glycoprotein Mediates Escape from a S2 Domain-Targeting Neutralizing Monoclonal Antibody

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is the etiological agent for the infectious disease, SARS, which first emerged 10 years ago. SARS-CoV is a zoonotic virus that has crossed the species barriers to infect humans. Bats, which harbour a diverse pool of SARS-like CoVs (SL-CoVs), are believed to be the natural reservoir. The SARS-CoV surface Spike (S) protein is a major antigenic determinant in eliciting neutralizing antibody production during SARS-CoV infection. In our previous work, we showed that a panel of murine MONOCLONAL ANTIBODIES (mAbs) that target the S2 subunit of the S protein are capable of neutralizing SARS-CoV infection IN VITRO (Lip KM et al, J Virol. 2006 Jan; 80(2): 941–50). In this study, we report our findings on the characterization of one of these mAbs, known as 1A9, which binds to the S PROTEIN at a novel epitope within the S2 subunit at amino acids 1111–1130. MAb 1A9 is a broadly neutralizing mAb that prevents viral entry mediated by the S proteins of human and civet SARS-CoVs as well as bat SL-CoVs. By generating mutant SARS-CoV that escapes the neutralization by mAb 1A9, the residue D1128 in S was found to be crucial for its interaction with mAb 1A9. S protein containing the substitution of D1128 with alanine (D1128A) exhibited a significant decrease in binding capability to mAb 1A9 compared to wild-type S protein. By using a pseudotyped viral entry assay, it was shown that the D1128A substitution in the escape virus allows it to overcome the viral entry blockage by mAb 1A9. In addition, the D1128A mutation was found to exert no effects on the S protein cell surface expression and incorporation into virion particles, suggesting that the escape virus retains the same viral entry property as the wild-type virus.published_or_final_versio

Human coronavirus NL63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in Hong Kong, China

Background. Human coronavirus NL63 (HCoV-NL63) is a recently discovered human coronavirus found to cause respiratory illness in children and adults that is distinct from the severe acute respiratory syndrome (SARS) coronavirus and human coronaviruses 229E (HCoV-229E) and OC43 (HCoV-OC43). Methods. We investigated the role that HCoV-NL63, HCoV-OC43, and HCoV-229E played in children hospitalized with fever and acute respiratory symptoms in Hong Kong during the period from August 2001 through August 2002. Results. Coronavirus infections were detected in 26 (4.4%) of 587 children studied; 15 (2.6%) were positive for HCoV-NL63, 9 (1.5%) were positive for HCoV-OC43, and 2 (0.3%) were positive for HCoV-229E. In addition to causing upper respiratory disease, we found that HCoV-NL63 can present as croup, asthma exacerbation, febrile seizures, and high fever. The mean age (± standard deviation [SD]) of the infected children was 30.7 ± 19.8 months (range, 6-57 months). The mean maximum temperature (± SD) for the 12 children who were febrile was 39.3°C ± 0.9°C, and the mean total duration of fever (± SD) for all children was 2.6 ± 1.2 days (range, 1-5 days). HCoV-NL63 infections were noted in the spring and summer months of 2002, whereas HCoV-OC43 infection mainly occurred in the fall and winter months of 2001. HCoV-NL63 viruses appeared to cluster into 2 evolutionary lineages, and viruses from both lineages cocirculated in the same season. Conclusions. HCoV-NL63 is a significant pathogen that contributes to the hospitalization of children, and it was estimated to have caused 224 hospital admissions per 100,000 population aged 6 years each year in Hong Kong. © 2005 by the Infectious Diseases Society of America. All rights reserved.published_or_final_versio

Generation of recombinant influenza A virus without M2 ion-channel protein by introduction of a point mutation at the 5′ end of the viral intron

The aim of this study was to inhibit influenza virus M2 protein expression by mutating the splicing signal of the M gene. Mutations were introduced into the GU dinucleotide sequence at the 5′-proximal splicing site of the M gene (corresponding to nt 52-53 of M cRNA). Transfected cells expressing mutated M viral ribonucleoproteins failed to generate M2 mRNA. Interestingly, recombinant viruses with mutations at the dinucleotide sequence were viable, albeit attenuated, in cell culture. These recombinants failed to express M2 mRNA and M2 protein. These observations demonstrated that the GU invariant dinucleotide sequence at the 5′-proximal splicing site of M gene is essential for M2 mRNA synthesis. These results also indicated that the M2 ion-channel protein is critical, but not essential, for virus replication in cell culture. This approach may provide a new way of producing attenuated influenza A virus. © 2005 SGM.postprin

Amphipathic peptides-mediated delivery of siRNA for antiviral therapy against influenza

Lecture sessionRNA interference (RNAi) technology has emerged as a novel and potentially effective therapy against respiratory viruses. RNAi is a naturally occurring process that inhibits specific gene expression in a post-transcriptional manner, mediated by small interfering RNA (siRNA). Properly designed siRNAs have already been shown to function as potent inhibitors of viral replication. By delivering exogenous siRNAs to mammalian cells, RNAi could be induced to degrade the viral mRNAs, leading to clearance of infection .....published_or_final_versio