Anti-influenza Virus Antibodies Research Articles

Obesity Is Associated with an Impaired Baseline Repertoire of Anti-Influenza Virus Antibodies.

Obesity is a risk factor for severe disease and mortality for both influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. While previous studies show that individuals with obesity generate antibody responses following influenza vaccination, infection rates within the obese group were twice as high as those in the healthy-weight group. The repertoire of antibodies raised against influenza viruses following previous vaccinations and/or natural exposures is referred to here as baseline immune history (BIH). To investigate the hypothesis that obesity impacts immune memory to infections and vaccines, we profiled the BIH of obese and healthy-weight adults vaccinated with the 2010-2011 seasonal influenza vaccine in response to conformational and linear antigens. Despite the extensive heterogeneity of the BIH profiles in both groups, there were striking differences between obese and healthy subjects, especially with regard to A/H1N1 strains and the 2009 pandemic virus (Cal09). Individuals with obesity had lower IgG and IgA magnitude and breadth for a panel of A/H1N1 whole viruses and hemagglutinin proteins from 1933 to 2009 but increased IgG magnitude and breadth for linear peptides from the Cal09 H1 and N1 proteins. Age was also associated with A/H1N1 BIH, with young individuals with obesity being more likely to have reduced A/H1N1 BIH. We found that individuals with low IgG BIH had significantly lower neutralizing antibody titers than individuals with high IgG BIH. Taken together, our findings suggest that increased susceptibility of obese participants to influenza infection may be mediated in part by obesity-associated differences in the memory B-cell repertoire, which cannot be ameliorated by current seasonal vaccination regimens. Overall, these data have vital implications for the next generation of influenza virus and SARS-CoV-2 vaccines. IMPORTANCE Obesity is associated with increased morbidity and mortality from influenza and SARS-CoV-2 infection. While vaccination is the most effective strategy for preventing influenza virus infection, our previous studies showed that influenza vaccines fail to provide optimal protection in obese individuals despite reaching canonical correlates of protection. Here, we show that obesity may impair immune history in humans and cannot be overcome by seasonal vaccination, especially in younger individuals with decreased lifetime exposure to infections and seasonal vaccines. Low baseline immune history is associated with decreased protective antibody responses. Obesity potentially handicaps overall responses to vaccination, biasing it toward responses to linear epitopes, which may reduce protective capacity. Taken together, our data suggest that young obese individuals are at an increased risk of reduced protection by vaccination, likely due to altered immune history biased toward nonprotective antibody responses. Given the worldwide obesity epidemic coupled with seasonal respiratory virus infections and the inevitable next pandemic, it is imperative that we understand and improve vaccine efficacy in this high-risk population. The design, development, and usage of vaccines for and in obese individuals may need critical evaluation, and immune history should be considered an alternate correlate of protection in future vaccine clinical trials.

Polyreactive Broadly Neutralizing B cells Are Selected to Provide Defense against Pandemic Threat Influenza Viruses

SummaryPolyreactivity is the ability of a single antibody to bind to multiple molecularly distinct antigens and is a common feature of antibodies induced upon pathogen exposure. However, little is known about the role of polyreactivity during anti-influenza virus antibody responses. By analyzing more than 500 monoclonal antibodies (mAbs) derived from B cells induced by numerous influenza virus vaccines and infections, we found mAbs targeting conserved neutralizing influenza virus hemagglutinin epitopes were polyreactive. Polyreactive mAbs were preferentially induced by novel viral exposures due to their broad viral binding breadth. Polyreactivity augmented mAb viral binding strength by increasing antibody flexibility, allowing for adaption to imperfectly conserved epitopes. Lastly, we found affinity-matured polyreactive B cells were typically derived from germline polyreactive B cells that were preferentially selected to participate in B cell responses over time. Together, our data reveal that polyreactivity is a beneficial feature of antibodies targeting conserved epitopes.

Polyreactive Broadly Neutralizing B Cells are Selected to Provide Defense Against Pandemic Threat Influenza Viruses

Polyreactivity is the ability of a single antibody to bind to multiple molecularly distinct antigens and is a common feature of antibodies induced upon pathogen exposure. However, little is known about the role of polyreactivity during anti-influenza virus antibody responses. By comprehensively analyzing more than 500 monoclonal antibodies (mAbs) derived from B cells induced by numerous influenza virus vaccines and infections, we find mAbs targeting conserved neutralizing influenza virus hemagglutinin epitopes are polyreactive. Polyreactive mAbs are preferentially induced by novel viral exposures due to their broad viral binding breadth. Polyreactivity augments mAb viral binding strength by increasing antibody flexibility, allowing for adaption to imperfectly conserved epitopes. Lastly, we found affinity-matured polyreactive B cells were typically derived from germline polyreactive B cells that were preferentially selected to participate in B cell responses over time. Together, our data reveal polyreactivity is a beneficial feature of antibodies targeting conserved epitopes.

SIR+A mathematical model for evaluating and predicting 2016–2017 ARVI-influenza incidence by using on the Moscow territory

Influenza is a major challenge to global healthcare due to its high transmissivity and ability to cause major epidemics. Influenza epidemics and pandemics are associated with changes in the society structure that contribute to the spread of new viral strains in certain environmental and social settings. Currently, influenza is one of the most common global diseases that results in annual epidemics or even pandemics, often leading to lethal outcome. Influenza viruses are uniquely prone to variability via point mutations, recombination and gene reassortment accompanied with changes in their biological properties considered as the main cause of uncontrolled infection spread. Hence, examining cohorts of predisposed individuals by using probability models provides not only additional information about viral outbreaks, but also allows monitoring dynamics of viral epidemics in controlled areas. Understanding influenza epidemiology is crucial for restructuring healthcare resources. Public healthcare service mainly relies on influenza vaccination. However, there are vulnerable cohorts such as elderly and immunocompromised individuals, which usually contain no protective antiinfluenza virus antibody level. Despite advances in the developing vaccines and chemotherapy, large-scale influenza epidemics still continue to emerge. Upon that, no reliable methods for disease prognosis based on rate of ongoing epidemic situation are currently available. Monitoring and predicting emerging epidemics is complicated due to discrepancy between dynamics of influenza epidemics that might be evaluated by using surveillance data as well as platform for tracking influenza incidence rate. However, it may be profoundly exacerbated by mutations found in the influenza virus genome by altering genuine morbidity dynamics. Use of probabilistic models for assessing parameters of stochastic epidemics would contribute to more accurately predicted changes in morbidity rate. Here, an SIR+A probabilistic model considering a relationship between infected, susceptible and protected individuals as well as the aggressiveness of external risks for predicting changes in influenza morbidity rate that allowed to evaluate and predict the 2016 ARVI influenza incidence rate in Moscow area. Moreover, introducing an intensity of infection parameter allows to conduct a reliable analysis of incidence rate and predict its changes.

Postvaccination Accumulation of the Influenza Virus Antigen in the Rat Choroid Plexus

We examined the accessibility of influenza virus and diphtheria-tetanus toxin (DiTe) antigens to the choroid plexus (CP) within the postvaccination period and the expression of CD11b molecules (by immunohistochemistry). Eighteen Dark Agouti (DA) rats were divided into three groups: (i) animals administered with influenza vaccine (Flu), (ii) animals administered with DiTe vaccine (DiTe), and (iii) nontreated (Contr) animals. The serum antibody titers following influenza and diphtheria-tetanus vaccination were detected by the ELISA test. Immunohistochemical staining revealed a great number of viral antigen-positive and CD11b-positive brain cells in Flu rats compared to a very small number of the respective cells in DiTe animals and no staining in the Contr group. DiTe- and Flu-rats showed significant increases in the serum anti-tetanus toxoid and anti-influenza virus antibody levels compared to those in the Contr group. The results obtained attract attention towards the dynamic role of the CP in the immunosurveillance of the CNS. Based on the viral antigen deposits accumulated in the CP, it has been proposed that the latter can play an active role in modulation of the immune response after influenza vaccine immunization.

Syntheses and mucosal adjuvant activity of simplified oleanolic acid saponins possessing cinnamoyl ester

A series of new simplified oleanolic acid saponins with a glycosyl ester moiety at C28, were efficiently prepared. Furthermore, the effect of nasal administration of the synthetic oleanolic acid saponins on the nasal anti-influenza virus antibody titer against secondary nasal inoculation of the influenza split vaccine was examined. The result revealed cinnamoyl saponin as a suitable candidate vaccine adjuvant.

IMMUNOGENICITY AND SAFETY OF A TRIVALENT SUBUNIT IMMUNOADJUVANT INFLUENZA VACCINE IN CHILDREN WITH TYPE 1 DIABETES: RESULTS OF A PROSPECTIVE COMPARATIVE STUDY

Background: Children with type 1 diabetes (T1D) are at high risk for severe influenza. Objective. Our aim was to assess immunogenicity and safety of a trivalent subunit immunoadjuvant influenza vaccine in children with T1D. Methods. The prospective study carried out from September to November 2014 included children with T1D at the age from 3 to 17 years vaccinated against influenza by a trivalent subunit immunoadjuvant influenza vaccine and unvaccinated due to parental refusal. Anti-influenza virus antibodies were determined by passive hemagglutination reaction. Vaccine safety was assessed by a number of adverse events occurred within 6 days after its administration. Observation was carried out during 1 year after vaccination (to October-November 2015 inclusive). Results. Of 780 children with T1D followed-up by endocrinologists in Nizhny Novgorod region, the study included 94 children — 44 vaccinated and 50 unvaccinated matched by sex and age. As for influenza A/H1N1/California/07/09 virus strain, the seroprotection rate (antibody titre > 1:40) was 84%, seroconversion rate (4-fold increase in antibody titre) was 66%, seroconversion factor (average increase of the antibody titre and 95% confidence interval) was 20.6 (10.4–30.9), as for influenza A/H3N2/Texas/50/12 virus, it was 98%, 41%, and 9.9 (3.2–16.6), as for influenza B/Massachusetts/2/12 virus, it was 77%, 49%, and 7.7 (4.0–11.4), respectively. After 1 year, the conditionally protective antibody titres (> 1:40) to influenza A/H1N1/California/07/09 virus were in 32/43 (74%), to influenza A/H3N2/Texas/50/12 virus — in 38/43 (88%), to influenza B/Massachusetts/2/12 virus — in 25/43 (58%) vaccinated children, in unvaccinated ones — 23/49 (47%) 37/49 (76%), and 6/49 (12%), respectively. Mild local adverse events were observed in 5/44 (11%), general — in 1/44 (2%), intercurrent diseases — in 8/44 (18%) vaccinated children. Conclusion. Immunogenicity and safety of a trivalent subunit immunoadjuvant influenza vaccine in children with T1D have been confirmed.

An Anti-Influenza Virus Antibody Inhibits Viral Infection by Reducing Nucleus Entry of Influenza Nucleoprotein.

To date, four main mechanisms mediating inhibition of influenza infection by anti-hemagglutinin antibodies have been reported. Anti-globular-head-domain antibodies block either influenza virus receptor binding to the host cell or progeny virion release from the host cell. Anti-stem region antibodies hinder the membrane fusion process or induce antibody-dependent cytotoxicity to infected cells. In this study we identified a human monoclonal IgG1 antibody (CT302), which does not inhibit both the receptor binding and the membrane fusion process but efficiently reduced the nucleus entry of viral nucleoprotein suggesting a novel inhibition mechanism of viral infection by antibody. This antibody binds to the subtype-H3 hemagglutinin globular head domain of group-2 influenza viruses circulating throughout the population between 1997 and 2007.

Enhanced Influenza Virus-Like Particle Vaccination with a Structurally Optimized RIG-I Agonist as Adjuvant.

The molecular interaction between viral RNA and the cytosolic sensor RIG-I represents the initial trigger in the development of an effective immune response against infection with RNA viruses, resulting in innate immune activation and subsequent induction of adaptive responses. In the present study, the adjuvant properties of a sequence-optimized 5'-triphosphate-containing RNA (5'pppRNA) RIG-I agonist (termed M8) were examined in combination with influenza virus-like particles (VLP) (M8-VLP) expressing H5N1 influenza virus hemagglutinin (HA) and neuraminidase (NA) as immunogens. In combination with VLP, M8 increased the antibody response to VLP immunization, provided VLP antigen sparing, and protected mice from a lethal challenge with H5N1 influenza virus. M8-VLP immunization also led to long-term protective responses against influenza virus infection in mice. M8 adjuvantation of VLP increased endpoint and antibody titers and inhibited influenza virus replication in lungs compared with approved or experimental adjuvants alum, AddaVax, and poly(I·C). Uniquely, immunization with M8-VLP stimulated a TH1-biased CD4 T cell response, as determined by increased TH1 cytokine levels in CD4 T cells and increased IgG2 levels in sera. Collectively, these data demonstrate that a sequence-optimized, RIG-I-specific agonist is a potent adjuvant that can be utilized to increase the efficacy of influenza VLP vaccination and dramatically improve humoral and cellular mediated protective responses against influenza virus challenge. The development of novel adjuvants to increase vaccine immunogenicity is an important goal that seeks to improve vaccine efficacy and ultimately prevent infections that endanger human health. This proof-of-principle study investigated the adjuvant properties of a sequence-optimized 5'pppRNA agonist (M8) with enhanced capacity to stimulate antiviral and inflammatory gene networks using influenza virus-like particles (VLP) expressing HA and NA as immunogens. Vaccination with VLP in combination with M8 increased anti-influenza virus antibody titers and protected animals from lethal influenza virus challenge, highlighting the potential clinical use of M8 as an adjuvant in vaccine development. Altogether, the results describe a novel immunostimulatory agonist targeted to the cytosolic RIG-I sensor as an attractive vaccine adjuvant candidate that can be used to increase vaccine efficacy, a pressing issue in children and the elderly population.

Broadly-neutralizing anti-influenza virus antibodies: Enhancement of neutralizing potency in polyclonal mixtures and IgA backbones (VIR5P.1141)

Abstract The recent discovery of broadly-neutralizing antibodies that target the hemagglutinin stalk domain has renewed hope that the development of “universal” influenza virus vaccines may be within reach. To this end, we have examined the functions of hemagglutinin stalk-binding antibodies in an endogenous setting - as polyclonal preparations isolated from human sera. Relative to monoclonal antibodies that bind to the HA head domain, the neutralization potency of monoclonal stalk-binding antibodies was vastly inferior in vitro, but was enhanced by several orders of magnitude in the polyclonal context. Furthermore, we demonstrated a surprising enhancement in IgA-mediated HA stalk neutralization relative to that achieved by antibodies of IgG isotypes. Mechanistically, this could be explained in two ways. Identical variable regions consistently neutralized virus more potently when in an IgA backbone compared to an IgG backbone. Additionally, HA-specific memory B cells isolated from human peripheral blood were more likely to be stalk-specific when secreting antibodies of IgA isotypes compared to those secreting IgG. Taken together, our data provide strong evidence that HA stalk-binding antibodies perform optimally when in a polyclonal context, and that the targeted elicitation of HA stalk-specific IgA should be an important consideration during “universal” influenza virus vaccine design.

Broadly neutralizing anti-influenza virus antibodies: enhancement of neutralizing potency in polyclonal mixtures and IgA backbones.

Current influenza virus vaccines rely upon the accurate prediction of circulating virus strains months in advance of the actual influenza season in order to allow time for vaccine manufacture. Unfortunately, mismatches occur frequently, and even when perfect matches are achieved, suboptimal vaccine efficacy leaves several high-risk populations vulnerable to infection. However, the recent discovery of broadly neutralizing antibodies that target the hemagglutinin (HA) stalk domain has renewed hope that the development of "universal" influenza virus vaccines may be within reach. Here, we examine the functions of influenza A virus hemagglutinin stalk-binding antibodies in an endogenous setting, i.e., as polyclonal preparations isolated from human sera. Relative to monoclonal antibodies that bind to the HA head domain, the neutralization potency of monoclonal stalk-binding antibodies was vastly inferior in vitro but was enhanced by several orders of magnitude in the polyclonal context. Furthermore, we demonstrated a surprising enhancement in IgA-mediated HA stalk neutralization relative to that achieved by antibodies of IgG isotypes. Mechanistically, this could be explained in two ways. Identical variable regions consistently neutralized virus more potently when in an IgA backbone compared to an IgG backbone. In addition, HA-specific memory B cells isolated from human peripheral blood were more likely to be stalk specific when secreting antibodies of IgA isotypes compared to those secreting IgG. Taken together, our data provide strong evidence that HA stalk-binding antibodies perform optimally when in a polyclonal context and that the targeted elicitation of HA stalk-specific IgA should be an important consideration during "universal" influenza virus vaccine design. Influenza viruses remain one of the most worrisome global public health threats due to their capacity to cause pandemics. While seasonal vaccines fail to protect against the emergence of pandemic strains, a new class of broadly neutralizing antibodies has been recently discovered and may be the key to developing a "universal" influenza virus vaccine. While much has been learned about the biology of these antibodies, most studies have focused only on monoclonal antibodies of IgG subtypes. However, the study of monoclonal antibodies often fails to capture the complexity of antibody functions that occur during natural polyclonal responses. Here, we provide the first detailed analyses of the biological activity of these antibodies in polyclonal contexts, comparing both IgG and IgA isotypes isolated from human donors. The striking differences observed in the functional properties of broadly neutralizing antibodies in polyclonal contexts will be essential for guiding design of "universal" influenza virus vaccines and therapeutics.

Lethal coinfection of influenza virus and Streptococcus pneumoniae lowers antibody response to influenza virus in lung and reduces numbers of germinal center B cells, T follicular helper cells, and plasma cells in mediastinal lymph Node.

Secondary Streptococcus pneumoniae infection after influenza is a significant clinical complication resulting in morbidity and sometimes mortality. Prior influenza virus infection has been demonstrated to impair the macrophage and neutrophil response to the subsequent pneumococcal infection. In contrast, how a secondary pneumococcal infection after influenza can affect the adaptive immune response to the initial influenza virus infection is less well understood. Therefore, this study focuses on how secondary pneumococcal infection after influenza may impact the humoral immune response to the initial influenza virus infection in a lethal coinfection mouse model. Compared to mice infected with influenza virus alone, mice coinfected with influenza virus followed by pneumococcus had significant body weight loss and 100% mortality. In the lung, lethal coinfection significantly increased virus titers and bacterial cell counts and decreased the level of virus-specific IgG, IgM, and IgA, as well as the number of B cells, CD4 T cells, and plasma cells. Lethal coinfection significantly reduced the size and weight of spleen, as well as the number of B cells along the follicular developmental lineage. In mediastinal lymph nodes, lethal coinfection significantly decreased germinal center B cells, T follicular helper cells, and plasma cells. Adoptive transfer of influenza virus-specific immune serum to coinfected mice improved survival, suggesting the protective functions of anti-influenza virus antibodies. In conclusion, coinfection reduced the B cell response to influenza virus. This study helps us to understand the modulation of the B cell response to influenza virus during a lethal coinfection. Secondary pneumococcal infection after influenza virus infection is an important clinical issue that often results in excess mortality. Since antibodies are key mediators of protection, this study aims to examine the antibody response to influenza virus and demonstrates that lethal coinfection reduced the B cell response to influenza virus. This study helps to highlight the complexity of the modulation of the B cell response in the context of coinfection.

Alternative Recognition of the Conserved Stem Epitope in Influenza A Virus Hemagglutinin by a V H 3-30-Encoded Heterosubtypic Antibody

A human monoclonal heterosubtypic antibody, MAb 3.1, with its heavy chain encoded by VH3-30, was isolated using phage display with immobilized hemagglutinin (HA) from influenza virus A/Japan/305/1957(H2N2) as the target. Antibody 3.1 potently neutralizes influenza viruses from the H1a clade (i.e., H1, H2, H5, H6) but has little neutralizing activity against the H1b clade. Its crystal structure in complex with HA from a pandemic H1N1 influenza virus, A/South Carolina/1/1918(H1N1), revealed that like other heterosubtypic anti-influenza virus antibodies, MAb 3.1 contacts a hydrophobic groove in the HA stem, primarily using its heavy chain. However, in contrast to the closely related monoclonal antibody (Mab) FI6 that relies heavily on HCDR3 for binding, MAb 3.1 utilizes residues from HCDR1, HCDR3, and framework region 3 (FR3). Interestingly, HCDR1 of MAb 3.1 adopts an α-helical conformation and engages in hydrophobic interactions with the HA very similar to those of the de novo in silico-designed and affinity-matured synthetic protein HB36.3. These findings improve our understanding of the molecular requirements for binding to the conserved epitope in the stem of the HA protein and, therefore, aid the development of more universal influenza vaccines targeting these epitopes. Influenza viruses rapidly evade preexisting immunity by constantly altering the immunodominant neutralizing antibody epitopes (antigenic drift) or by acquiring new envelope serotypes (antigenic shift). As a consequence, the majority of antibodies elicited by immunization or infection protect only against the immunizing or closely related strains. Here, we describe a novel monoclonal antibody that recognizes the conserved heterosubtypic epitope in the stem of influenza A virus hemagglutinin. This antibody, referred to as MAb 3.1, recognizes its epitope in a manner that resembles recognition of a similar epitope by the de novo in silico-designed and affinity-matured synthetic protein HB36.3. Thus, besides providing novel insights into the molecular interactions between heterosubtypic antibodies and influenza virus hemagglutinin, MAb 3.1 demonstrates that de novo in silico-designed and affinity-matured synthetic proteins can foretell naturally selected antibody binding. This knowledge will aid development of a pan-influenza virus vaccine.

A Carboxy-Terminal Trimerization Domain Stabilizes Conformational Epitopes on the Stalk Domain of Soluble Recombinant Hemagglutinin Substrates

Recently, a new class of broadly neutralizing anti-influenza virus antibodies that target the stalk domain of the viral hemagglutinin was discovered. As such, induction, isolation, characterization, and quantification of these novel antibodies has become an area of intense research and great interest. Since most of these antibodies bind to conformational epitopes, the structural integrity of hemagglutinin substrates for the detection and quantification of these antibodies is of high importance. Here we evaluate the binding of these antibodies to soluble, secreted hemagglutinins with or without a carboxy-terminal trimerization domain based on the natural trimerization domain of T4 phage fibritin. The lack of such a domain completely abolishes binding to group 1 hemagglutinins and also affects binding to group 2 hemagglutinins. Additionally, the presence of a trimerization domain positively influences soluble hemagglutinin stability during expression and purification. Our findings suggest that a carboxy-terminal trimerization domain is a necessary requirement for the structural integrity of stalk epitopes on recombinant soluble influenza virus hemagglutinin.

Seroprevalence of subtype H3 influenza A virus in South Korean cats.

To investigate the potential transmission of subtype H3 influenza virus to cats, a serological survey was carried out in South Korea. Serum samples (n=1027) were obtained from 809 pet cats and 218 domesticated cats living in urban colonies (D-cats) from 2008 to 2010, and tested using an influenza anti-nucleoprotein (NP)-specific enzyme-linked immunosorbent assay (ELISA) and the haemagglutination inhibition (HI) test, which was recommended by the World Organization for Animal Health. Anti-influenza virus antibodies were detected in 3.12% and 2.43% of cat sera tested using the NP-specific ELISA and HI test, respectively. Anti-H3 antibodies were also identified when the HI assay was used for influenza virus serotyping. These data may indicate the sporadic transmission of subtype H3 influenza virus from other infected species to cats in South Korea.

Seasonal IM flu-vaccines boost antibody titers to epitopes common to influenza virus strains (113.35)

Abstract Human serum IgG anti-influenza virus antibodies (Abs) to components of the inactivated, tri-valent 2006/2007, 2007/2008, 2008/2009, and 2010/2011 vaccines were measured by surface plasmon resonance (SPR; BIA3000). Pre- and post (2 wk) 2007/2008 vaccination (N=20 healthy volunteers) serum IgGs were assayed for binding to the 2006/2007, 2007/2008, and 2008/2009 vaccine preparations. Overall, the IgG binding was greatest to the 2006/2007 vaccine components, but binding was also observed against the 2008/2009 vaccine which did not include strains within the 2006/2007 or the 2007/2008 vaccines. Several control individuals who had never received a flu-vaccine showed no binding to any of the vaccines studied. Pre- and post (2 wk) 2010/2011(H1N1, pandemic strain) vaccination Abs were assayed for binding against the 2006/2007, 2007/2008, and the 2010/2011 vaccines. Vaccination with the 2010/2011 vaccine showed a >10 fold boost in IgG binding to both the 2006/2007 and 2010/2011 components and somewhat lower to 2007/2008, compared to the 2-3 fold boosts to the 2006/2007 components observed with the previous vaccinations. The results emphasize that Abs raised against one vaccine preparation can bind strains present in other vaccine preparations. The 2006/2007 vaccine preparation seems to have one or more of these highly antigenic epitopes. Future studies will involve delineation of these sites and investigation into their ability to protect against influenza viral infection.

A Multiplex Suspension Array for Screening of Carbohydrate Binding Proteins and Influenza

Carbohydrate-protein interactions are involved in many crucial biological processes, from protein stability, to immune response, to pathogen infection. A fast and reliable method to determine carbohydrate binding affinity and specificity was developed as an alternative to ELISA and glycan microarray screening.Optically distinct neutravidin-coated microspheres were functionalized with biotinylated carbohydrates, to create a glycan array in suspension format. A range of fluorescently labeled carbohydrate binding agents including lectins, toxins, and viruses were incubated with the glycan suspension array. In addition to characterizing carbohydrate binding preference, simultaneous strain typing of influenza virus was performed using additional multiplex microspheres conjugated to antibodies specific for influenza A subtypes. Inactivated virus was incubated with the microspheres, detected using fluorescence-labeled anti-influenza virus antibodies, and measured by flow cytometry.Biotinylated carbohydrates were successfully captured by neutravidin conjugated microspheres as observed by specific binding of fluorescently labeled lectins SNA-I and MAA and Cholera toxin B (CTB) subunit. Antibodies were also conjugated to microspheres allowing for multiplex analysis of influenza virus sub-type and receptor specificity of human influenza A subtype H1N1 and avian influenza A subtype H5N1. Each subtype of influenza virus bound only to those microspheres displaying carbohydrates with its specific receptor and anti-influenza A antibody. These results have been confirmed previously by other methods, including hemagglutination and glycan microarray.This approach provides improved control over carbohydrate surface density, increased throughput for the analysis of many samples and conditions, reduced sample volumes, and better quantification.

Effects of oral administration of yogurt fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 and its exopolysaccharides against influenza virus infection in mice

Yogurt fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (1073R-1) has been shown to reduce the risk of catching cold in the healthy elderly (Makino et al., Br. J. Nutr., 104, 998–1006, 2010). In addition, the exopolysaccharides (EPS) produced by 1073R-1 were also reported to exert immunostimulatory effects in mice such as the augmentation of NK cell activity (Makino et al., J. Dairy Sci., 89, 2873–81, 2006).So, we investigated anti-influenza virus effects of this yogurt and EPS in mice. The yogurt (0.4ml/day) and EPS (20μg/day) were orally administered to BALB/c mice for 21days prior to intranasal infection with influenza virus A/PR/8/34 (H1N1).As a result, the survival periods were prolonged in both yogurt- and EPS-treated groups compared to the water-treated group. Moreover, in these groups, we observed significant decrease of influenza virus titer and significant increase of anti-influenza virus antibodies (IgA, IgG1) in the bronchoalveolar lavage fluid at 4days post infection NK cell activity of splenocytes in both groups was also increased significantly. EPS was further fractionated into neutral EPS (NPS) and acidic EPS (APS), and the NPS (20μg/day) or the APS (20μg/day) was orally administered to mice for 21days prior to the intranasal infection. The survival periods were prolonged in APS-treated group, but not in NPS-treated group.Consequently, we concluded that the yogurt fermented with 1073R-1 exerted anti-influenza virus effects in mice by its immunopotentiating activity, and suggested that the APS produced by 1073R-1 was one of active ingredients.

Long Term Persistence of IgE Anti-Influenza Virus Antibodies in Pediatric and Adult Serum Post Vaccination with Influenza Virus Vaccine

The production of IgE specific to different viruses (HIV-1, Parvovirus B19, Parainfluenza virus, Varicella Zoster Virus), and the ability of IgE anti-HIV-1 to suppress HIV-1 production in vitro, strongly suggest an important role for IgE and/or anti viral specific IgE in viral pathogenesis. Nevertheless, the presence and persistence of IgE anti-Influenza virus antibodies has not been studied. Total serum IgE and specific IgE and IgG anti-Influenza virus antibodies were studied in children (N = 3) (m/f 14-16 y/o) and adults (N = 3) (m/f, 41-49 y/o) 2-20 months after vaccination with Influenza virus (Flumist(®) or Fluzone(®)), as well as in non-vaccinated children (N = 2). (UniCAP total IgE Fluoroenzymeimmunoassay, ELISA, Immunoblot). We found that serum of vaccinated children and adults contained IgE and IgG anti-Influenza virus antibodies approaching two years post vaccination. Non-vaccinated children did not make either IgE or IgG anti-Influenza antibodies. Similar levels of IL-2, IFN-γ, IL-4, and IL-10 cytokines were detected in serum of vaccinated compared with non vaccinated subjects (p > 0.05), as well as between vaccinated adults compared with vaccinated children and non vaccinated subjects (p > 0.05). Vaccinated children and adults continue to produce IgE anti-Influenza virus antibodies long term post vaccination. The long term production of IgE anti-Influenza virus antibodies induced by vaccination may contribute to protective immunity against Influenza.

A Detrimental Effect of Interleukin-10 on Protective Pulmonary Humoral Immunity during Primary Influenza A Virus Infection

Interleukin-10 (IL-10) is an important anti-inflammatory molecule that can cause immunosuppression and long-term pathogen persistence during chronic infection of mice with viruses such as lymphocytic choriomeningitis virus. However, its specific role in immunity to acute viral infections is not fully understood. We found that IL-10 plays a detrimental role in host responses to acute influenza A virus since IL-10(-/-) mice had improved viral clearance and survival after infection compared to wild-type mice. Enhanced viral clearance in IL-10(-/-) mice was not correlated with increased CD4(+) or CD8(+) T-cell recruitment into the lung but was correlated with increased pulmonary anti-influenza virus antibody titers, and this was dependent upon the presence of T cells, primarily CD4(+) T cells. In addition, virus-specific antibody produced during the early stages of infection in the respiratory tract of IL-10(-/-) but not wild-type mice was sufficient to mediate passive protection against viral challenge of naïve mice. Complement was necessary for this antibody-mediated passive protection, but FcgammaR or neutrophil deficiency did not significantly influence viral clearance. Our results show that an absence of IL-10 at the time of primary infection leads to enhanced local virus-specific antibody production and, thus, increased protection against influenza A virus infection.