Evaluation of neutralizing efficacy of monoclonal antibodies specific for 2009 pandemic H1N1 influenza A virus in vitro and in vivo

Pandemic influenza A virus (H1N1) 2009 poses a serious public-health challenge worldwide. To characterize the neutralizing epitopes of this virus, we generated a panel of eight monoclonal antibodies (mAbs) against the HA of the A/California/07/2009 virus. The antibodies were specific for the 2009 pdm H1N1 HA, as the antibodies displayed HA-specific ELISA, hemagglutination inhibition (HAI) and neutralization activity. One mAb (mAb12) showed significantly higher HAI and neutralizing titers than the other mAbs. We mapped the antigenic epitopes of the HA by characterizing escape mutants of a 2009 H1N1 vaccine strain (NYMC X-179A). The amino acid changes suggested that these eight mAbs recognized HA antigenic epitopes located in the Sa, Sb, Ca1 and Ca2 sites. Passive immunization with mAbs showed that mAb12 displayed more efficient neutralizing activity in vivo than the other mAbs. mAb12 was also found to be protective, both prophylactically and therapeutically, against a lethal viral challenge in mice. In addition, a single injection of 10 mg/kg mAb12 outperformed a 5-day course of treatment with oseltamivir (10 mg/kg/day by gavage) with respect to both prophylaxis and treatment of lethal viral infection. Taken together, our results showed that mouse-origin mAbs displayed neutralizing effectiveness in vitro and in vivo. One mAb in particular (mAb12) recognized an epitope within the Sb site and demonstrated outstanding neutralizing effectiveness.     

Molecular characterization of the influenza A(H1N1)pdm09 isolates collected in the 2015-2016 season and comparison of HA mutations detected in Turkey since 2009

Influenza A(H1N1)pdm09 pandemic virus causing the 2009 global outbreak moved into the post-pandemic period, but its variants continued to be the prevailing subtype in the 2015-2016 influenza season in Europe and Asia. To determine the molecular characteristics of influenza A(H1N1)pdm09 isolates circulating during the 2015-2016 season in Turkey, we identified mutations in the hemagglutinin (HA) genes and investigated the presence of H275Y alteration in the neuraminidase genes in the randomly selected isolates. The comparison of the HA nucleotide sequences revealed a very high homology (>99.5%) among the studied influenza A(H1N1)pdm09 isolates, while a relatively low homology (96.6%-97.2%), was observed between Turkish isolates and the A/California/07/2009 vaccine virus. Overall 14 common mutations were detected in HA sequences of all 2015-2016 influenza A(H1N1)pdm09 isolates with respect to the A/California/07/2009 virus, four of which located in three different antigenic sites. Eleven rare mutations in 12 HA sequences were also detected. Phylogenetic analysis revealed that all characterized influenza A(H1N1)pdm09 isolates formed a single genetic cluster, belonging to the genetic subclade 6B.1, defined by HA amino acid substitutions S84N, S162N, and I216T. Furthermore, all isolates showed an oseltamivir-sensitive genotype, suggesting that Tamiflu (Oseltamivir) could still be the drug of choice in Turkey.     

Influenza-induced acute respiratory distress syndrome during the 2010-2016 seasons: bacterial co-infections and outcomes by virus type and subtype

ObjectivesWe aimed to describe bacterial co-infections and acute respiratory distress (ARDS) outcomes according to influenza type and subtype.MethodsA retrospective observational study was conducted from 2012 to 2016 in patients admitted to the respiratory intensive care unit (ICU) of Marseille university hospital for influenza-induced ARDS. Microbiological investigations, including multiplex molecular respiratory panel testing and conventional bacteriological cultures, were performed as part of the routine ICU care on the bronchoalveloar lavage collected at admission. Bacterial co-infections, ICU mortality and respiratory function were investigated according to virus type and subtype.ResultsAmong the 45 ARDS patients included, A(H1N1)pdm09 was the most frequent influenza virus identified (28/45 A(H1N1)pdm09, eight out of 45 A(H3N2) and nine out of 45 influenza B). Bacterial co-infections involving a total of 23 bacteria were diagnosed in 16/45 patients (36%). A(H1N1)pdm09 patients presented fewer bacterial co-infections (17.9% vs. 50.0% for A(H3N2) patients and 77.8% for B patients; p < 0.01). Overall, mortality at 90 days post admission was 33.3% (15/45), and there was no significant difference between influenza type and subtype. The need for extracorporeal membrane oxygenation was more frequent for A(H1N1)pdm2009 (20/28, 71.4%) and B patients (7/9, 77.8%) than the A(H3N2) subtype (1/8, 12.5%; p < 0.01). A(H1N1)pdm09-ARDS patients were associated with fewer ventilation-free days at day 28 (median (IQR): 0 (0–8) days) compared with other influenza–ARDS patients (15 (0–25) days, p < 0.05).DiscussionIn a population of influenza-induced ARDS, A(H1N1)pdm09 was associated with fewer bacterial co-infections but poorer respiratory outcomes. These data underline the major role of A(H1N1)pdm09 subtype on influenza disease severity.     

IgM,IgG, and IgA Antibody Responses to Influenza A(H1N1)pdm09 Hemagglutinin in Infected Persons during the First Wave of the 2009 Pandemic in the United States

The novel influenza A(H1N1)pdm09 virus caused an influenza pandemic in 2009. IgM, IgG, and IgA antibody responses to A(H1N1)pdm09 hemagglutinin (HA) following A(H1N1)pdm09 virus infection were analyzed to understand antibody isotype responses. Age-matched control sera collected from U.S. residents in 2007 and 2008 were used to establish baseline levels of cross-reactive antibodies. IgM responses often used as indicators of primary virus infection were mainly detected in young patient groups (≤5 years and 6 to 15 years old), not in older age groups, despite the genetic and antigenic differences between the HA of A(H1N1)pdm09 virus and pre-2009 seasonal H1N1 viruses. IgG and IgA responses to A(H1N1)pdm09 HA were detected in all age groups of infected persons. In persons 17 to 80 years old, paired acute- and convalescent-phase serum samples demonstrated ≥4-fold increases in the IgG and IgA responses to A(H1N1)pdm09 HA in 80% and 67% of A(H1N1)pdm09 virus-infected persons, respectively. The IgG antibody response to A(H1N1)pdm09 HA was cross-reactive with HAs from H1, H3, H5, and H13 subtypes, suggesting that infections with subtypes other than A(H1N1)pdm09 might result in false positives by enzyme-linked immunosorbent assay (ELISA). Lower sensitivity compared to hemagglutination inhibition and microneutralization assays and the detection of cross-reactive antibodies against homologous and heterologous subtype are major drawbacks for the application of ELISA in influenza serologic studies.     

A comparison of H1N1 influenza among pediatric inpatients in the pandemic and post pandemic era

BackgroundThe novel influenza A H1N1 (A[H1N1]pdm09) strain emerged in 2009, contributing to significant morbidity and mortality. It is not known whether illness associated with A(H1N1) pdm09 in the post-pandemic era exhibits a similar disease profile.ObjectiveThe objectives of this study were to compare the burden of disease of A(H1N1) pdm09 influenza from the 2009 pandemic year to the post-pandemic years (2010–2014), and to explore potential reasons for any differences.Study designWe conducted a retrospective cohort study of inpatients admitted to Children’s Hospital Colorado with a positive respiratory specimen for influenza from May–December, 2009 and December, 2010–April, 2014. Univariate and multivariate analyses were conducted to compare the demographics and clinical characteristics of patients with H1N1 during the two periods.ResultsThere were 388 inpatients with influenza A(H1N1) pdm09 in 2009, and 117 during the post-pandemic years. Ninety-four percent of all H1N1 during the post-pandemic era was observed during the 2013–2014 influenza season. Patients with A(H1N1) pdm09 during the post-pandemic year were less likely to have an underlying medical condition (P < 0.01). Patients admitted to the ICU during the post-pandemic year had a lower median age (5 vs 8 years, P = 0.01) and a lower proportion of patients were intubated, had mental status changes, and ARDS compared with the pandemic years, (P < 0.01 for all), with decreased mortality (P = 0.02).ConclusionPatients with influenza A(H1N1) pdm09 during the post-pandemic years appeared to have less severe disease than patients with A(H1N1) pdm09 during the pandemic year. The reasons for this difference are likely multifactorial.     

Epidemiology and full genome sequence analysis of H1N1pdm09 from Northeast China

Pandemic influenza A (H1N1) 2009 virus (H1N1pdm09) was a novel tri-assortment virus that emerged in Mexico and North America in 2009 and caused the first influenza pandemic in the 21st century. This study investigated the prevalence pattern and molecular characteristics of H1N1pdm09 in three continuous years from April 2009 to March 2012 in populations of Tianjin, Northeast China. Totally, 3,068 influenza viruses (25.4 %) were detected from 12,089 respiratory specimens. Among them, 41.4 % (1,269/3,068) were H1N1pdm09 positive. 15.1 % (192/1,269) severe respiratory infection cases were H1N1pdm09 positive. H1N1pdm09 was the predominant prevalence subtype in October 2009–March 2010 (69.1 %, 930/1,346) and October 2010–March 2011 (42.1 %, 220/523). Eight isolated H1N1pdm09 viruses from severe infection/death cases in three different years were selected to sequence the whole genome through splicing the sequences following 46 PCRs. HA sequences of seven H1N1pdm09 isolates from mild infection cases were detected. Phylogenetic analysis showed that HA, NA, M, NP and NS genes of H1N1pdm09 viruses gathered together with swine influenza A (H1N1), whereas PB2 and PA genes originated from avian influenza virus, and PB1 gene originated from human seasonal influenza virus. Identity analysis indicated that all the genes were highly conserved. Compared with vaccine strain A/California/07/2009(H1N1), the maximal mutation gene was HA (0.7–2.6 %), then NA (0.6–1.7 %), last one was M (mutation rate 0–0.6 %). More site substitutions were observed in 2011 isolates than in 2009 and 2010 isolates of HA (p = 0.002), NA (p = 0.003) and PA (p = 0.001) proteins. The amino acid substitution rates were varied among eight gene segments, ranging from 7.39 × 10^?4 for PB2 to 7.40 × 10^?3 for NA. The higher d _N / d _S rates were observed in HA, PA and NS segments in H1N1pdm09 in Tianjin. Three HA amino acid site substitutions occurred at the HA receptor-binding sites and antigenic determinant, including S179N and K180T (located at antigenic site Sa) in A/Tianjinhedong/SWL44/2011(H1) and A/Tianjinjinnan/SWL41/2011(H1), and D239N (located at antigenic site Ca) in A/Tianjinninghe/SWL49/2009(H1). Antigenic drift may have occurred in H1N1pdm09 with time. No oseltamivir-resistance site substitution was observed at 275 and 295 sites. Amino acid residue site at 31 in M2 protein was N in all 8 isolates, which suggested that H1N1pdm09 was resistant to amantadine.     

唐山市甲型H1N1流感病毒血凝素基因序列分析

目的 分析2010—2016年唐山市甲型H1N1流感病毒血凝素(hemagglutinin,HA)基因序列进化特征.方法 选取唐山市3家哨点医院流感样病例分离到的24株甲型H1N1病毒,通过RT-PCR和测序方法获得HA基因的全长序列,运用分子生物学软件和统计学软件对序列进行拼接、比对和分析.结果 同源进化分析显示,24株甲型H1N1流感病毒HA基因与疫苗株A/California/7/2009的核苷酸和氨基酸的同源性分别为97.0%~99.0%和97.0%~98.5%.进化分析显示,2010—2016年唐山地区流行的甲型H1N1流感病毒属于1、7、6三个基因分支,其中6分支毒株分为6C、6B、6B.1和6B.2亚支.氨基酸位点分析显示,不同毒株与疫苗株比较存在8~16处氨基酸位点改变,其中7个变异涉及3个抗原表位:H138Q/Y和S203T突变位于Ca区,N125S、K153E、S162N、K163T/Q突变位于Sa区,S185T突变位于Sb区同时也位于受体结合部位;2015—2016流行季6B.1分支毒株抗原位点S162N突变增加了新的潜在糖基化位点.结论 与疫苗株比较,随着时间推移唐山地区甲型H1N1流感病毒发生了抗原漂变,未来仍应关注6B分支流行株的变化.     

Genetic diversity of HA1 domain of hemagglutinin gene of pandemic influenza H1N1pdm09 viruses in New Delhi, India

Genetic analysis of pandemic 2009 influenza A (H1N1; H1N1pdm09) virus was undertaken to understand virus evolution during 2009 and 2010 in India. Surveillance of influenza viruses from July 2009 to December 2010 revealed major peaks of circulating H1N1pdm09 viruses in August–September and December–January 2009 and then in August–September 2010. To understand the diversity of the H1N1pdm09 virus, selected specimens (n = 23) from 2009 or 2010 were characterized by nucleotide sequence determination of the HA1 subunit of the HA gene. Phylogenetic analysis revealed that 22 clustered with clade 7 viruses characterized by S203T mutations, whereas one virus from 2010 fell within clade 6. None of the viruses from either 2009 or 2010 formed a monophyletic group, suggesting a continuum of independent introduction of circulating viral strains. Amino acid analysis revealed minor amino acid changes in the antigenic or receptor‐binding domains. Importantly, we observed mutations that were also present in 1918 pandemic virus, which includes S183P in 4 and S185T mutation in 3 of 13 viruses analyzed from 2010, while none of the 2009 viruses carried these mutations. Whether antibody‐mediated pressure is imposing such changes remains to be determined. Continued genetic surveillance is warranted to monitor pathogenicity as the virus evolves to acquire new features. J. Med. Virol. 84:386–393, 2012. © 2011 Wiley Periodicals, Inc.     

The response of medical virology laboratories to the influenza A(H1N1)pdm09 outbreak in Paris Île-de-France region

The outbreak of influenza A(H1N1)pdm09 was a challenge for the laboratories of Paris Île-de-France region in charge of virological diagnosis. In order to evaluate the quality of their response to this challenge, a retrospective survey based on a self-administered standardized questionnaire was undertaken among the 18 hospital laboratories involved in A(H1N1)pdm09 virus detection over a period of 10 months from April 2009 to January 2010. All concerned laboratories responded to the survey. Due to imposed initial biosafety constraints and indications, virological diagnosis was performed in only two laboratories at the start of the studied period. Step by step, it was further settled in the other laboratories starting from June to November 2009. From the beginning, A(H1N1)pdm09-specific RT-PCR was considered the reference method while the use of rapid influenza detection tests remained temporary and concerned a minority of these laboratories. Among the overall 21,656 specimens received, a positive diagnosis of influenza A(H1N1)pdm09 was obtained in 5,390 cases (25%), the positivity range being significantly higher among women as compared to men (P < 0.0001) and subjects below 45 years of age as compared to those over 65 years (P < 0.0001). Two peaks in positivity frequency were observed at weeks 24 (30%, 8–12 June 2009) and 44 (50%, 26–30 October 2009) respectively, the latter one occurring 2 weeks earlier than the peak of epidemic at the national level. In contrast, a low positivity rate was detected at weeks 38–40 in relationship with other respiratory virus infections which were clinically misinterpreted as a peak of influenza epidemic. These data demonstrate the ability of medical virology laboratories of Paris Île-de-France region to provide in real time a valuable diagnosis of A(H1N1)pdm09 virus infection and a relevant view of outbreak evolution, suggesting they will be a crucial component in the management of future influenza epidemics.     

Characterization of oseltamivir‐resistant influenza A(H1N1)pdm09 viruses in Taiwan in 2009–2011

The early isolated swine‐origin influenza A(H1N1)pdm09 viruses were susceptible to oseltamivir; however, there is a concern about whether oseltamivir‐resistant influenza A(H1N1)pdm09 viruses will spread worldwide as did the oseltamivir‐resistant seasonal influenza A(H1N1) viruses in 2007–2008. In this study, the frequency of oseltamivir resistance in influenza A(H1N1)pdm09 viruses was determined in Taiwan. From May 2009 to April 2011, 1,335 A(H1N1)pdm09‐positive cases in Taiwan were tested for the H275Y mutation in the neuraminidase (NA) gene that confers resistance to oseltamivir. Among these, 15 patients (1.1%) were found to be infected with H275Y virus. All the resistant viruses were detected after the patients have received the oseltamivir. The overall monthly ratio of H275Y‐harboring viruses ranged between 0% and 2.88%, and the peak was correlated with influenza epidemics. The genetic analysis revealed that the oseltamivir‐resistant A(H1N1)pdm09 viruses can emerged from different variants with a great diversity under drug pressure. The ratio of NA/HA activities in different clades of oseltamivir‐resistant viruses was reduced compared to those in the wild‐type viruses, indicating that the balance of NA/HA in the current oseltamivir‐resistant influenza A(H1N1)pdm09 viruses was interfered. It is possible that H275Y‐bearing A(H1N1)pdm09 virus has not yet spread globally because it lacks the essential permissive mutations that can compensate for the negative impact on fitness by the H275Y amino acid substitution in NA. Continuous monitoring the evolution patterns of sensitive and resistant viruses is required to respond to possible emergence of resistant viruses with permissive genetic background which enable the wide spread of resistance. J. Med. Virol. 85:379–387, 2013. © 2012 Wiley Periodicals, Inc.     

Protection against a lethal H5N1 influenza challenge by intranasal immunization with virus-like particles containing 2009 pandemic H1N1 neuraminidase in mice

Highly pathogenic H5N1 influenza shares the same neuraminidase (NA) subtype with the 2009 pandemic (H1N1pdm09), and cross-reactive NA immunity might protect against or mitigate lethal H5N1 infection. In this study, mice were either infected with a sublethal dose of H1N1pdm09 or were vaccinated and boosted with virus-like particles (VLP) consisting of the NA and matrix proteins, standardized by NA activity and administered intranasally, and were then challenged with a lethal dose of HPAI H5N1 virus. Mice previously infected with H1N1pdm09 survived H5N1 challenge with no detectable virus or respiratory tract pathology on day 4. Mice immunized with H5N1 or H1N1pdm09 NA VLPs were also fully protected from death, with a 100-fold and 10-fold reduction in infectious virus, respectively, and reduced pathology in the lungs. Human influenza vaccines that elicit not only HA, but also NA immunity may provide enhanced protection against the emergence of seasonal and pandemic viruses.     

A novel monoclonal antibody effective against lethal challenge with swine-lineage and 2009 pandemic H1N1 influenza viruses in mice

The HA protein of the 2009 pandemic H1N1 viruses (H1N1pdm) is antigenically closely related to the HA of classical North American swine H1N1 influenza viruses (cH1N1). Since 1998, through mutation and reassortment of HA genes from human H3N2 and H1N1 influenza viruses, swine influenza strains are undergoing substantial antigenic drift and shift. In this report we describe the development of a novel monoclonal antibody (S-OIV-3B2) that shows high hemagglutination inhibition (HI) and neutralization titers not only against H1N1pdm, but also against representatives of the α, β, and γ clusters of swine-lineage H1 influenza viruses. Mice that received a single intranasal dose of S-OIV-3B2 were protected against lethal challenge with either H1N1pdm or cH1N1 virus. These studies highlight the potential use of S-OIV-3B2 as effective intranasal prophylactic or therapeutic antiviral treatment for swine-lineage H1 influenza virus infections.     

High doses of recombinant mannan‐binding lectin inhibit the binding of influenza A(H1N1)pdm09 virus with cells expressing DC‐SIGN

The pandemic influenza A (H1N1)pdm09 virus continues to be a threat to human health. Low doses of mannan‐binding lectin (MBL) (<1 μg/mL) were shown not to protect against influenza A(H1N1)pdm09 infection. However, the effect of high doses of MBL has not been investigated. Dendritic cell‐specific intercellular adhesion molecule‐3 grabbing non‐integrin (DC‐SIGN) has been proposed as an alternative receptor for influenza A(H1N1)pdm09 virus. In this study, we examined the expression of DC‐SIGN on DCs as well as on acute monocytic leukemia cell line, THP‐1. High doses of recombinant or human MBL inhibited binding of influenza A(H1N1)pdm09 to both these cell types in the presence of complement derived from bovine serum. Further, anti‐DC‐SIGN monoclonal antibody inhibited binding of influenza A(H1N1)pdm09 to both DC‐SIGN‐expressing DCs and THP‐1 cells. This study demonstrates that high doses of MBL can inhibit binding of influenza A(H1N1)pdm09 virus to DC‐SIGN‐expressing cells in the presence of complement. Our results suggest that DC‐SIGN may be an alternative receptor for influenza A(H1N1)pdm09 virus.     

Novel reassortant influenza viruses between pandemic (H1N1) 2009 and other influenza viruses pose a risk to public health

Influenza A virus (IAV) is characterized by eight single-stranded, negative sense RNA segments, which allows for gene reassortment among different IAV subtypes when they co-infect a single host cell simultaneously. Genetic reassortment is an important way to favor the evolution of influenza virus. Novel reassortant virus may pose a pandemic among humans. In history, three human pandemic influenza viruses were caused by genetic reassortment between avian, human and swine influenza viruses. Since 2009, pandemic (H1N1) 2009 (pdm/09 H1N1) influenza virus composed of two swine influenza virus genes highlighted the genetic reassortment again. Due to wide host species and high transmission of the pdm/09 H1N1 influenza virus, many different avian, human or swine influenza virus subtypes may reassert with it to generate novel reassortant viruses, which may result in a next pandemic among humans. So, it is necessary to understand the potential threat of current reassortant viruses between the pdm/09 H1N1 and other influenza viruses to public health. This study summarized the status of the reassortant viruses between the pdm/09 H1N1 and other influenza viruses of different species origins in natural and experimental conditions. The aim of this summarization is to facilitate us to further understand the potential threats of novel reassortant influenza viruses to public health and to make effective prevention and control strategies for these pathogens.     

Characterization of an influenza A virus in Mexican swine that is related to the A/H1N1/2009 pandemic clade

In the spring of 2009, swine-origin influenza H1N1pdm09 viruses caused the first influenza pandemic of this century. We characterized the influenza viruses that circulated early during the outbreak in Mexico, including one newly sequenced swine H1N1pdm09 virus and three newly sequenced human H1N1pdm09 viruses that circulated in the outbreak of respiratory disease in La Gloria, Veracruz. Phylogenetic analysis revealed that the swine isolate (A/swine/Mexico/4/2009) collected in April 2009 is positioned in a branch that is basal to the rest of the H1N1pdm09 clade in two (NP and PA) of the eight single-gene trees. In addition, the concatenated HA-NA and the complete whole-genome trees also showed a basal position for A/swine/Mexico/4/2009. Furthermore, this swine virus was found to share molecular traits with non-H1N1pdm09 H1N1 viral lineages. These results suggest that this isolate could potentially be the first one detected from a sister lineage closely related to the H1N1pdm09 viruses.     

新型甲型H1N1流感病毒HA基因DNA疫苗诱导小鼠产生中和抗体

目的 探讨新型甲型流感病毒(2009H1N1)血凝素(HA)DNA疫苗诱导小鼠产生中和抗体特性.方法 构建2009H1N1或1918甲型流感病毒(1918H1N1)HA蛋白表达质粒2009HA和1918HA,采用25μg或200μg剂量2009HA质粒免疫小鼠,以2009HA或1918HA蛋白为包被抗原,测定小鼠血清中2009HA抗体总量或交叉反应抗体含量,分别用2009H1N1和1918H1N1两种假病毒(pp)测定抗体中和活性.结果 25 μg或200μg的2009HA质粒加强免疫小鼠后,4～16周内两组小鼠血清中2009HA总抗体水平以及对2009H1N1pp的中和抗体滴度相似(P>0.05),都含有与1918HA蛋白交叉反应抗体,对1918H1N1pp的交叉中和抗体滴度相似(P>0.05).结论 小剂量2009HA质粒DNA疫苗能够诱导小鼠产生持久的高水平中和抗体,对于预防新现流感病毒具有潜在应用价值.     

Antigenic variation of H1N1, H1N2 and H3N2 swine influenza viruses in Japan and Vietnam

The antigenicity of the influenza A virus hemagglutinin is responsible for vaccine efficacy in protecting pigs against swine influenza virus (SIV) infection. However, the antigenicity of SIV strains currently circulating in Japan and Vietnam has not been well characterized. We examined the antigenicity of classical H1 SIVs, pandemic A(H1N1)2009 (A(H1N1)pdm09) viruses, and seasonal human-lineage SIVs isolated in Japan and Vietnam. A hemagglutination inhibition (HI) assay was used to determine antigenic differences that differentiate the recent Japanese H1N2 and H3N2 SIVs from the H1N1 and H3N2 domestic vaccine strains. Minor antigenic variation between pig A(H1N1)pdm09 viruses was evident by HI assay using 13 mAbs raised against homologous virus. A Vietnamese H1N2 SIV, whose H1 gene originated from a human strain in the mid-2000s, reacted poorly with post-infection ferret serum against human vaccine strains from 2000-2010. These results provide useful information for selection of optimal strains for SIV vaccine production.     

Detection of influenza A virus homo‐ and heterosubtype‐specific memory B‐cells using a novel protein microarray‐based analysis tool

The emergence of the A(H1N1) 2009 pandemic influenza virus was initially seen as a major world‐wide health concern since a low degree of immunity to this virus strain was anticipated. However, age‐specific infection attack rates and age‐specific differences in seroresponse indicate that pre‐existing immunity may have played a significant role in protection especially in older age groups. This study describes the use of a protein microarray as a multiplex analysis tool for detection of influenza virus H1 strain‐specific memory B‐cells before and after infection with A(H1N1)pdm09. The discrimination was based on detection of specific antibodies in culture supernatants from polyclonally stimulated B‐cells against recombinant influenza virus HA1 proteins representing influenza virus subtypes H1 through H9. The protein microarray proved sensitive and specific for antibody detection in culture supernatants of B‐cells, and with the potential to deduce a person's history of infection with particular influenza virus variants, including A(H1N1)pdm09. Blood samples obtained from different age groups prior to the pandemic in 2009 partly showed the presence of B‐cells producing antibodies binding to the closely related A(H1N1) 1918 pandemic influenza virus, and of which the magnitude increased with age. These cross‐reactive antibodies were produced by single memory B‐cells present in these donors, and either bind to epitopes on HA1 which are shared within different H1 strains (homosubtypic response) or shared between different subtypes (heterosubtypic response). J. Med. Virol. 85:899–909, 2013. © 2013 Wiley Periodicals, Inc.