Hemagglutination Inhibition Activity Research Articles

Design and Characterization of a Computationally Optimized Broadly Reactive Hemagglutinin Vaccine for H1N1 Influenza Viruses.

One of the challenges of developing influenza A vaccines is the diversity of antigenically distinct isolates. Previously, a novel hemagglutinin (HA) for H5N1 influenza was derived from a methodology termed computationally optimized broadly reactive antigen (COBRA). This COBRA HA elicited a broad antibody response against H5N1 isolates from different clades. We now report the development and characterization of a COBRA-based vaccine for both seasonal and pandemic H1N1 influenza virus isolates. Nine prototype H1N1 COBRA HA proteins were developed and tested in mice using a virus-like particle (VLP) format for the elicitation of broadly reactive, functional antibody responses and protection against viral challenge. These candidates were designed to recognize H1N1 viruses isolated within the last 30 years. In addition, several COBRA candidates were designed based on sequences of H1N1 viruses spanning the past 100 years, including modern pandemic H1N1 isolates. Four of the 9 H1N1 COBRA HA proteins (X1, X3, X6, and P1) had the broadest hemagglutination inhibition (HAI) activity against a panel of 17 H1N1 viruses. These vaccines were used in cocktails or prime-boost combinations. The most effective regimens that both elicited the broadest HAI response and protected mice against a pandemic H1N1 challenge were vaccines that contained the P1 COBRA VLP and either the X3 or X6 COBRA VLP vaccine. These mice had little or no detectable viral replication, comparable to that observed with a matched licensed vaccine. This is the first report describing a COBRA-based HA vaccine strategy that elicits a universal, broadly reactive, protective response against seasonal and pandemic H1N1 isolates. Universal influenza vaccine approaches have the potential to be paradigm shifting for the influenza vaccine field, with the goal of replacing the current standard of care with broadly cross-protective vaccines. We have used COBRA technology to develop an HA head-based strategy that elicits antibodies against many H1 strains that have undergone genetic drift and has potential as a "subtype universal" vaccine. Nine HA COBRA candidates were developed, and these vaccines were used alone, in cocktails or in prime-boost combinations. The most effective regimens elicited the broadest hemagglutination inhibition (HAI) response against a panel of H1N1 viruses isolated over the past 100 years. This is the first report describing a COBRA-based HA vaccine strategy that elicits a broadly reactive response against seasonal and pandemic H1N1 isolates.

Are plasma mineral levels related to antibody response to influenza vaccination in older adults?

abstractIntroduction: An effective immune response to vaccination may be related to nutritional status. This study examined the association of plasma mineral levels with hemagglutination inhibition (HI) titers produced in response to influenza vaccine in older adults. Methods: Prior to (Day 0) and 21 (range = 19–28) days after receiving the 2013–14 influenza vaccine, 109 adults ages 51–81 years, provided blood samples. Serum samples were tested for HI activity against the A/H1N1 and A/H3N2 2013–2014 vaccine virus strains. Plasma minerals were collected in zinc-free tubes and assayed by inductively coupled plasma mass spectrometry. HI titers were reported as seroprotection (≥1:40) and seroconversion (≥ 4-fold rise from Day 0 (minimum HI = 1:10) to Day 21). Both HI titers and mineral values were skewed and thus log2 transformed. Magnesium (Mg), phosphorus (P), zinc (Zn), copper (Cu), iron (Fe), potassium (K) and the Cu to Zn ratio were tested. Logistic regression analyses were used to determine the associations between mineral levels and seroconversion and seroprotection of HI titers for each influenza A strain. Results: Participants were 61% white, 28% male, 39% diabetic, and 81% overweight/obese with a mean age of 62.6 y. In logistic regression, Day 21 A/H1N1 seroprotection was associated with P and Zn at Day 21(P < 0.05). Seroconversion of A/H1N1 was associated with Day 21 Cu, P, and Mg (P < 0.03). Day 21 A/H3N2 seroprotection and seroconversion were associated with Day 21 P (P < 0.05). Conclusions: Phosphorus was associated with seroprotection and seroconversion to influenza A after vaccination; these associations warrant additional studies with larger, more diverse population groups.

Protection against H5N1 Influenza Virus Induced by Matrix-M Adjuvanted Seasonal Virosomal Vaccine in Mice Requires Both Antibodies and T Cells.

BackgroundIt remains important to develop the next generation of influenza vaccines that can provide protection against vaccine mismatched strains and to be prepared for potential pandemic outbreaks. To achieve this, the understanding of the immunological parameters that mediate such broad protection is crucial.MethodIn the current study we assessed the contribution of humoral and cellular immune responses to heterosubtypic protection against H5N1 induced by a Matrix-M (MM) adjuvanted seasonal influenza vaccine by serum transfer and T-cell depletion studies.ResultsWe demonstrate that the heterosubtypic protection against H5N1 induced by MM adjuvanted vaccine is partially mediated by antibodies. The serum contained both H5N1 cross-reactive hemagglutinin (HA)- and neuraminidase (NA)-specific antibodies but with limited virus neutralizing and no hemagglutination inhibiting activity. The cross-reactive antibodies induced antibody-dependent cellular cytotoxicity (ADCC) in vitro, suggesting a role for the Fc part of the antibodies in protection against H5N1. Besides H5N1 specific antibody responses, cross-reactive HA- and NA-specific T-cell responses were induced by the adjuvanted vaccine. T-cell depletion experiments demonstrated that both CD4+ and CD8+ T cells contribute to protection.ConclusionOur study demonstrates that cross-protection against H5N1 induced by MM adjuvanted seasonal virosomal influenza vaccine requires both the humoral and cellular arm of the immune system.

Generation and Characterization of Monoclonal Antibodies Specific to Avian Influenza H5N1 Hemagglutinin Protein.

Highly pathogenic avian influenza (HPAI) H5N1 virus has in the past breached the species barrier from infected domestic poultry to humans in close contact. Although human-to-human transmission has previously not been reported, HPAI H5N1 virus has pandemic potential owing to gain of function mutation(s) and/or genetic reassortment with human influenza A viruses. Monoclonal antibodies (MAbs) have been used for diagnosis as well as specific therapeutic candidates in several disease conditions including viral infections in humans. In this study, we describe the preliminary characterization of four murine MAbs developed against recombinant hemagglutinin (rHA) protein of avian H5N1 A/turkey/Turkey/1/2005 virus that are either highly specific or broadly reactive against HA from other H5N1 subtype viruses, such as A/Hong Kong/213/03, A/Common magpie/Hong Kong/2256/2006, and A/Barheaded goose/Quinghai/14/2008. The antibody binding is specific to H5N1 HAs, as none of the antibodies bound H1N1, H2N2, H3N2, or B/Brisbane/60/2008 HAs. Out of the four MAbs, one of them (MA-7) also reacted weakly with the rHA protein of H7N9 A/Anhui/1/2013. All four MAbs bound H5 HA (A/turkey/Turkey/1/2005) with high affinity with an equilibrium dissociation constant (KD) ranging between 0.05 and 10.30 nM. One of the MAbs (MA-1) also showed hemagglutination inhibition activity (HI titer; 31.25 μg/mL) against the homologous A/turkey/Turkey/1/2005 H5N1 virus. These antibodies may be useful in developing diagnostic tools for detection of influenza H5N1 virus infection.

Hemagglutinin Stalk- and Neuraminidase-Specific Monoclonal Antibodies Protect against Lethal H10N8 Influenza Virus Infection in Mice.

Between November 2013 and February 2014, China reported three human cases of H10N8 influenza virus infection in the Jiangxi province, two of which were fatal. Using hybridoma technology, we isolated a panel of H10- and N8-directed monoclonal antibodies (MAbs) and further characterized the binding reactivity of these antibodies (via enzyme-linked immunosorbent assay) to a range of purified virus and recombinant protein substrates. The H10-directed MAbs displayed functional hemagglutination inhibition (HI) and neutralization activity, and the N8-directed antibodies displayed functional neuraminidase inhibition (NI) activity against H10N8. Surprisingly, the HI-reactive H10 antibodies, as well as a previously generated, group 2 hemagglutinin (HA) stalk-reactive antibody, demonstrated NI activity against H10N8 and an H10N7 strain; this phenomenon was absent when virus was treated with detergent, suggesting the anti-HA antibodies inhibited neuraminidase enzymatic activity through steric hindrance. We tested the prophylactic efficacy of one representative H10-reactive, N8-reactive, and group 2 HA stalk-reactive antibody in vivo using a BALB/c challenge model. All three antibodies were protective at a high dose (5 mg/kg). At a low dose (0.5 mg/kg), only the anti-N8 antibody prevented weight loss. Together, these data suggest that antibody targets other than the globular head domain of the HA may be efficacious in preventing influenza virus-induced morbidity and mortality. Avian H10N8 and H10N7 viruses have recently crossed the species barrier, causing morbidity and mortality in humans and other mammals. Although these reports are likely isolated incidents, it is possible that more cases may emerge in future winter seasons, similar to H7N9. Furthermore, regular transmission of avian influenza viruses to humans increases the risk of adaptive mutations and reassortment events, which may result in a novel virus with pandemic potential. Currently, no specific therapeutics or vaccines are available against the H10N8 influenza virus subtype. We generated a panel of H10- and N8-reactive MAbs. Although these antibodies may practically be developed into therapeutic agents, characterizing the protective potential of MAbs that have targets other than the HA globular head domain will provide insight into novel antibody-mediated mechanisms of protection and help to better understand correlates of protection for influenza A virus infection.

Generation of Adaptive Immune Responses Following Influenza Virus Challenge is Not Compromised by Pre-Treatment with the TLR-2 Agonist Pam2Cys.

Immunostimulatory agents provide a new category of anti-microbial agents that activate the host’s innate immune system allowing control of viral and/or bacterial infections. The TLR-2 agonist PEG-Pam2Cys has been shown to mediate potent anti-viral activity against influenza viruses when administered prophylactically (1). Here, we demonstrate that the treatment of mice with PEG-Pam2Cys does not compromise their ability to generate adaptive immune responses following subsequent challenge with influenza virus. The antibody induced in mice pre-treated with Pam2Cys possessed hemagglutination-inhibiting activities and the CD8+ T-cell responses that were elicited provided protection against heterologous viral challenge. In the absence of an effective influenza vaccine, an agent that provides immediate protection against the virus and does not compromise the induction of influenza-specific immunity on exposure to infectious virus provides an opportunity for population immunity to be achieved through natural exposure to virus.

Measuring the neutralization potency of influenza A virus hemagglutinin stalk/stem-binding antibodies in polyclonal preparations by microneutralization assay

The discovery of broadly-neutralizing antibodies that bind to the hemagglutinin stalk/stem domain has opened exciting new avenues for the development of “universal” influenza virus vaccines and therapeutics. Unlike strain-specific antibodies which bind to the hemagglutinin head domain and inhibit receptor binding, antibodies that bind to the stalk domain function to inhibit later stages of infection. The hemagglutination inhibition assay has long been the standard for evaluating titers of neutralizing hemagglutinin-specific antibodies in serum. The assay has the beneficial properties of being relatively rapid, easy-to-perform, and requires very little specialized equipment. Historically, hemagglutination inhibition titers of 40 or above against a given strain of influenza has been considered a correlate of protection on a population level. Unfortunately, this assay cannot be used to measure titers of hemagglutinin stalk-specific antibodies due to their lack of hemagglutination inhibiting activity. This has necessitated the development of novel reagents and assays capable of sensitive and specific detection of broadly-neutralizing HA stalk-binding antibodies in polyclonal mixtures. Here, we describe a novel microneutralization-based assay that utilizes recombinant influenza A viruses expressing chimeric hemagglutinin molecules and ‘exotic’ neuraminidase to measure titers of broadly-neutralizing antibodies in polyclonal preparations.

Monoclonal antibodies recognizing a putative immunodominant epitope in the hemagglutinin stalk do not enhance influenza A virus mediated fusion activity (VIR5P.1156)

Abstract The discovery of broadly neutralizing antibodies (Abs) which bind to the conserved hemagglutinin (HA) stalk has led to renewed interest in the development of a universal influenza virus vaccine. In swine, there have been isolated reports of vaccine-induced enhancement of disease in heterologous vaccination and challenge studies. It has been suggested that Abs recognizing the HA stalk promote virus fusion activity, contributing to pathology in pigs. Given that robust data in mouse and ferret models suggests a protective role for stalk Abs, here we examined the relationship between HA-stalk binding Abs and fusion. We generated mouse monoclonal Abs C4 and C6, which recognize a previously identified minimal epitope in the HA stalk. These Abs lack hemagglutination inhibition activity and did not inhibit or enhance virus-mediated fusion in vitro. In a mouse challenge model, we assessed the consequences of pre-exposure administration of a C4/C6 cocktail. No significant differences were observed in animals receiving C4/C6 in morbidity and mortality relative to naïve and isotype controls. We also probed the sera from a cohort of individuals who received an H5N1 vaccine. Although we detected baseline levels of Abs to the minimal stalk epitope associated with increased fusion activity in swine, titers did not increase post-vaccination. Together, our data suggests that stalk Abs do not enhance virus-mediated fusion and thus should not impede progress toward a universal influenza vaccine.

Distribution of antibodies against influenza virus in pigs from farrow‐to‐finish farms in M inas G erais state, B razil

BackgroundSwine influenza virus (SIV) is the cause of an acute respiratory disease that affects swine worldwide. In Brazil, SIV has been identified in pigs since 1978. After the emergence of pandemic H1N1 in 2009 (H1N1pdm09), few studies reported the presence of influenza virus in Brazilian herds.ObjectivesThe objective of this study was to evaluate the serological profile for influenza virus in farrow-to-finish pig farms in Minas Gerais state, Brazil.MethodsThirty farms with no SIV vaccination history were selected from the four larger pig production areas in Minas Gerais state (Zona da Mata, Triângulo Mineiro/Alto Paranaíba, South/Southwest and the Belo Horizonte metropolitan area). At each farm, blood samples were randomly collected from 20 animals in each production cycle category: breeding animals (sows and gilts), farrowing crate (2–3 weeks), nursery (4–7 weeks), grower pigs (8–14 weeks), and finishing pigs (15–16 weeks), with 100 samples per farm and a total of 3000 animals in this study. The samples were tested for hemagglutination inhibition activity against H1N1 pandemic strain (A/swine/Brazil/11/2009) and H3N2 SIV (A/swine/Iowa/8548-2/98) reference strain.ResultsThe percentages of seropositive animals for H1N1pdm09 and H3N2 were 26·23% and 1·57%, respectively, and the percentages of seropositive herds for both viruses were 96·6% and 13·2%, respectively.ConclusionsThe serological profiles differed for both viruses and among the studied areas, suggesting a high variety of virus circulation around the state, as well as the presence of seronegative animals susceptible to influenza infection and, consequently, new respiratory disease outbreaks.

Limited effect of recombinant human mannose-binding lectin on the infection of novel influenza A (H7N9) virus in vitro

Mannose-binding lectin (MBL), a pattern-recognition molecule in serum, recognizes specific hexose sugars rich in mannose and N-acetylglucosamine on bacterium, yeasts, viruses as well as apoptotic cells. It has been well-identified that MBL has antiviral effects via binding to seasonal influenza H1 and H3 subtype viruses. Influenza A (H7N9) virus, a novel reassortant virus to human population, possesses the surface hemagglutinin (HA) and neuraminidase (NA) genes from duck and wild-bird influenza viruses and internal genes from poultry H9N2 viruses. As of Dec 7th, 2014, a total of 467 human infections and 183 fatal cases have been identified. Here, recombinant human (rh) MBL was tested for its binding and effects on hemagglutination inhibition (HI) and NA activity inhibition (NAI) of avian H7N9, H9N2 and human H3N2 viruses. We discovered that rhMBL exhibited a strong binding to H7N9 virus as human H3N2 did at high virus titers. However, it performed a significantly weaker HI activity effect on H7N9 comparing to those of H3N2 and H9N2, even at a much higher concentration (3.67 ± 0.33 vs. 0.026 ± 0.001 and 0.083 ± 0.02 μg/mL, respectively). Similarly, minor NAI effect of rhMBL, even at up to 10 μg/mL, was found on H7N9 virus while it displayed significant effects on both H3N2 and H9N2 at a lowest concentration of 0.0807 ± 0.009 and 0.0625 μg/mL, respectively. The HI and NAI effects of rhMBL were calcium-dependent and mediated by lectin domain. Our findings suggest that MBL, the host innate molecule, has differential interference effects with human and avian influenza virus and limited antiviral effect against H7N9 virus.

Fc receptor is not required for inducing antibodies but plays a critical role in conferring protection after influenza M2 vaccination

The ectodomain of matrix protein 2 (M2e) of influenza virus is considered a rational target for a universal influenza A vaccine. To better understand M2e immune-mediated protection, Fc receptor common γ chain deficient (FcRγ(-/-) ) and wild-type mice were immunized with a tandem repeat of M2e presented on virus-like particles (M2e5x VLP). Levels of M2e-specific antibodies that were induced in FcRγ(-/-) mice after immunization with M2e5x VLP were similar to those in wild-type mice. In addition, M2e antibodies induced in FcRγ(-/-) mice were found to be equally protective as those induced in wild-type mice. However, M2e5x VLP-immunized FcRγ(-/-) mice were not well protected, as shown by severe weight loss, higher lung viral titres and interleukin-6 inflammatory cytokine production upon influenza virus challenge compared with M2e5x VLP-immunized wild-type mice. Importantly, FcRγ(-/-) mice that were immunized with inactivated influenza virus induced haemagglutination inhibition activity and were well protected without a significant weight loss. Interestingly, interferon-γ-producing CD4 T and CD8 T cells were found to be prevalent in lungs from M2e5x VLP-immunized FcRγ(-/-) mice, which appeared to be correlated with a faster recovery after infection. These results indicate that Fc receptors play a primary role in conferring M2e-specific antibody-mediated protection whereas T cells may contribute to the recovery at later stages of infection.

The breadth of cross sub-type neutralisation activity of a single domain antibody to influenza hemagglutinin can be increased by antibody valency.

The response to the 2009 A(H1N1) influenza pandemic has highlighted the need for additional strategies for intervention which preclude the prior availability of the influenza strain. Here, 18 single domain VHH antibodies against the 2009 A(H1N1) hemagglutinin (HA) have been isolated from a immune alpaca phage displayed library. These antibodies have been grouped as having either (i) non-neutralising, (ii) H1N1 restricted neutralising or (iii) broad cross-subtype neutralising activity. The ability to neutralise different viral subtypes, including highly pathogenic avian influenza (H5N1), correlated with the absence of hemagglutination inhibition activity, loss of binding to HA at acid pH and the absence of binding to the head domain containing the receptor binding site. This data supports their binding to epitopes in the HA stem region and a mechanism of action other than blocking viral attachment to cell surface receptors. After conversion of cross-neutralising antibodies R1a-B6 and R1a-A5 into a bivalent format, no significant enhancement in neutralisation activity was seen against A(H1N1) and A(H5N1) viruses. However, bivalent R1a-B6 showed an 18 fold enhancement in potency against A(H9N2) virus and, surprisingly, gained the ability to neutralise an A(H2N2) virus. This demonstrates that cross-neutralising antibodies, which make lower affinity interactions with the membrane proximal stem region of more divergent HA sub-types, can be optimised by bivalency so increasing their breadth of anti-viral activity. The broad neutralising activity and favourable characteristics, such as high stability, simple engineering into bivalent molecules and low cost production make these single domain antibodies attractive candidates for diagnostics and immunotherapy of pandemic influenza.

Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses

Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses.

HPLC analysis and cell surface receptor binding activities of the crude aqueous and methanolic extract of Sesamum indicum

To identify the possible functional molecules for therapeutic uses by screening the crude aqueous and methanolic extracts derived from sesame seeds (Sesamum indicum) in vitro. High performance liquid chromatography was used to scan the functional molecules present in the extracts. The crude aqueous extracts showed the possibilities to present caffeine and cetirizine or its derivatives like molecules. On the other hand, the crude methanolic extract may contain Loratadine or its derivatives like molecules. Both type of extracts showed hemagglutination inhibition activities in all types of human blood samples tested. However, they showed stronger binding with AB+ blood group than those of A+ and B+ blood. Sesame seeds may be considered as a functional food.

Novel Phage Display-Derived H5N1-Specific scFvs with Potential Use in Rapid Avian Flu Diagnosis

The highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtype infect poultry and have also been spreading to humans. Although new antiviral drugs and vaccinations can be effective, rapid detection would be more efficient to control the outbreak of infections. In this study, a phage-display library was applied to select antibody fragments for HPAI strain A/Hubei/1/2010. As a result, three clones were selected and sequenced. A hemagglutinin inhibition assay of the three scFvs revealed that none exhibited hemagglutination inhibition activity towards the H5N1 virus, yet they showed a higher binding affinity for several HPAI H5N1 strains compared with other influenza viruses. An ELISA confirmed that the HA protein was the target of the scFvs, and the results of a protein structure simulation showed that all the selected scFvs bound to the HA2 subunit of the HA protein. In conclusion, the three selected scFVs could be useful for developing a specific detection tool for the surveillance of HPAI epidemic strains.

Evidence of Synergistic Activity of Medicinal Plant Extracts against Neuraminidase Inhibitor Resistant Strains of Influenza Viruses

The frequent emergence of drug resistant influenza viral strains emphasizes the urgent and continual need to develop new antiviral drugs. Given the encouraging findings of previous studies on antiviral compounds from plant sources, this study focused on medicinal plants from Borneo that were traditionally used to treat symptoms of influenza infection. Following the promising results of earlier investigations, four plant extracts that demonstrated multiple modes of viral inhibition were studied against wild-type and neuraminidase (NA) inhibitor-resistant strains of Types A and B influenza viruses. The extracts exhibited more pronounced activities against the wild-type viruses than the NA inhibitor-resistant strains. Variations in the antiviral potential of the extracts collected from different parts of the same plant were also evidenced in the in vitro micro-inhibition assays. Even though all plant extracts affected NA activity of all viruses, only two extracts demonstrated hemagglutination inhibitory (HI) activities against Type A pandemic H1N1 and Type B viruses. Furthermore, Receptor Destroying Enzyme (RDE) treatments of extracts exhibiting HI activities indicated the presence of sialic acid (SA)-like component(s) that may be responsible for HI activity. Since the antiviral potential of extracts was not completely suppressed by RDE, the possibility of non SA-like antiviral components cannot be ruled out. Therefore, synergistic activity between SA-like and non SA-like components contained in the plant extracts may be responsible for the demonstrated antiviral potential. The results also indicated the presence of non SA-like components that may act against other viral proteins apart from hemagglutinin (HA) and NA. Hence, this study supports the presence of multiple antiviral components that act against different viral proteins or interfere with different stages of viral replication. Our results suggest that these plant extracts have the potential to be developed as therapeutic agents for the treatment of influenza and could be a solution to the global occurrence of viral strains resistant to NA inhibitors.

Comparative evaluation of the medicinal activities of methanolic extract of seeds, fruit pulps and fresh juice of Syzygium cumini in vitro

Objective: To establish the health benefits of Syzgium cumin to discover functional components present in the seeds, fruit pulps and fresh juice of this fruit grown in Bangladesh. Methods: Thin layer chromatography and ultra-violet spectroscopy were used to detect the presence of various types of compound in seeds and juice. Antioxidant effects were measured by DPPH scavenging assay and total reducing assay. Receptor binding activities was performed by hemagglutination inhibition assay. Anti-inflammatory assay and hydrogen peroxide induced hemolysis assay was also investigated. Disc diffusion assay was performed to show the antibacterial effect using Gram positive, Gram negative strains of bacteria and fungi. Results: Methanolic extract of the seeds showed stronger antioxidant, hydrogen peroxide induced hemolysis activities, hemagglutination inhibition activities and membrane stabilization activities than those of fresh juice. However, fresh juice showed stronger antibacterial and antifungal activities than those of methanolic seed extract. The seed contains higher amount of polyphenols and flavanoids than those of fruit juice. Conclusions: Therefore, fruit juice, fruit pulp and seed of Syzygium cumini contain medicinal active components in different ratios.

One-shot vaccination with an insect cell-derived low-dose influenza A H7 virus-like particle preparation protects mice against H7N9 challenge

Human infections with a novel influenza A H7N9 subtype virus were reported in China recently. The virus caused severe disease with high mortality rates and it raised concerns over its pandemic potential. Here, we assessed in the mouse model protective efficacy of single immunisations with low vaccine doses of insect cell-derived H7 virus-like particles, consisting of hemagglutinin and matrix protein. Vaccinated mice were fully protected and survived a stringent lethal challenge (100mLD50) with H7N9, even after a single, unadjuvanted, low vaccine dose (0.03μg). Serum analysis revealed broad reactivity and hemagglutination inhibition activity across a panel of divergent H7 strains. Moreover, we detected significant levels of cross-reactivity to related group 2 hemagglutinins. These data demonstrate that virus-like particle vaccines have the potential to induce broadly protective immunity against the novel H7N9 virus and a variety of other H7 strains.

Mucosal Adjuvants for Influenza Virus-Like Particle Vaccine

To find an effective mucosal adjuvant for influenza virus-like particles (VLPs), we compared the effects of known adjuvants Alum, CpG DNA, monophosphoryl lipid A (MPL), poly IC, gardiquimod, and cholera toxin (CT). Mice that were intranasally immunized with Alum, CpG, MPL, and CT adjuvanted VLPs showed higher levels of antibodies in both sera and mucosa. Hemagglutination inhibition and virus neutralizing activities were enhanced in groups adjuvanted with Alum, MPL, or CT. Influenza virus specific long-lived cells secreting IgG and IgA antibodies were found at high levels both in bone marrow and spleen in the Alum, CpG and CT adjuvanted groups. A similar level of protection was observed among different adjuvanted groups, except the CT adjuvant that showed a higher efficacy in lowering lung viral loads after challenge. Alum and CT adjuvants differentially increased influenza VLP-mediated activation of dendritic cells and splenocytes in vitro, supporting the in vivo pattern of antibody isotypes and cytokine production. These results suggest that Alum, MPL, or CpG adjuvants, which have been tested clinically, can be developed as an effective mucosal adjuvant for influenza VLP vaccines.

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 10mg/kg mAb12 outperformed a 5-day course of treatment with oseltamivir (10mg/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.