Specific Recognition of Influenza A/H1N1/2009 Antibodies in Human Serum: A Simple Virus-Free ELISA Method

Although it has been estimated that pandemic Influenza A H1N1/2009 has infected millions of people from April to October 2009, a more precise figure requires a worldwide large-scale diagnosis of the presence of Influenza A/H1N1/2009 antibodies within the population. Assays typically used to estimate antibody titers (hemagglutination inhibition and microneutralization) would require the use of the virus, which would seriously limit broad implementation.An ELISA method to evaluate the presence and relative concentration of specific Influenza A/H1N1/2009 antibodies in human serum samples is presented. The method is based on the use of a histidine-tagged recombinant fragment of the globular region of the hemagglutinin (HA) of the Influenza A H1N1/2009 virus expressed in E. coli.The ELISA method consistently discerns between Inf A H1N1 infected and non-infected subjects, particularly after the third week of infection/exposure. Since it does not require the use of viral particles, it can be easily and quickly implemented in any basic laboratory. In addition, in a scenario of insufficient vaccine availability, the use of this ELISA could be useful to determine if a person has some level of specific antibodies against the virus and presumably at least partial protection

The Receptor-Binding Domain of Influenza Virus Hemagglutinin Produced in Escherichia coli Folds into Its Native, Immunogenic Structure ▿

The hemagglutinin (HA) surface glycoprotein promotes influenza virus entry and is the key protective antigen in natural immunity and vaccines. The HA protein is a trimeric envelope glycoprotein consisting of a globular receptor-binding domain (HA-RBD) that is inserted into a membrane fusion-mediating stalk domain. Similar to other class I viral fusion proteins, the fusogenic stalk domain spontaneously refolds into its postfusion conformation when expressed in isolation, consistent with this domain being trapped in a metastable conformation. Using X-ray crystallography, we show that the influenza virus HA-RBD refolds spontaneously into its native, immunogenic structure even when expressed in an unglycosylated form in Escherichia coli. In the 2.10-Å structure of the HA-RBD, the receptor-binding pocket is intact and its conformational epitopes are preserved. Recombinant HA-RBD is immunogenic and protective in ferrets, and the protein also binds with specificity to sera from influenza virus-infected humans. Overall, the data provide a structural basis for the rapid production of influenza vaccines in E. coli. From an evolutionary standpoint, the ability of the HA-RBD to refold spontaneously into its native conformation suggests that influenza virus acquired this domain as an insertion into an ancestral membrane-fusion domain. The insertion of independently folding domains into fusogenic stalk domains may be a common feature of class I viral fusion proteins

Nematicidal Activity of the Endophyte <i>Serratia ureilytica</i> against <i>Nacobbus aberrans</i> in Chili Plants (<i>Capsicum annuum</i> L.) and Identification of Genes Related to Biological Control

The genus Serratia is widely distributed in soil, water, plants, animals, invertebrates, and humans. Some species of this genus have antifungal, antibacterial, and nematicidal activity. In this work, the nematicidal activity of the endophytic strain of Serratia sp. in chili, Capsicum annuum L., is reported, where at a bacterial concentration of 4 × 109 cel/mL, the penetration of nematodes into the roots significantly decreased by 91 and 55% at 7 and 21 days after inoculation. This bacterial concentration also significantly decreased the number of galls, eggs, egg masses and reproduction factor produced by Nacobbus aberrans in Chili plants, with respect to the control where this bacterial strain was not applied. In the analysis of the genome of the strain, based on average nucleotide identity (ANI), the isolate could be affiliated to the species Serratia ureilytica. The size of the genome is 5.4 Mb, with a 59.3% content of GC. Genes related to the synthesis of chitinases, siderophores, proteases C, serralisins, hemolysin, and serrawettin W2 that have been reported for biocontrol of nematodes were identified in the genome. It is the first report of Serratia ureilytica with nematicidal activity. Based on these results of nematicidal activity, this strain can be evaluated in the field as an alternative in the biocontrol of Nacobbus aberrans in chili cultivation

Evolution of the normalized absorbance signal of serum samples from patients diagnosed as positive to Influenza A/H1N1/2009.

<p>(A) Samples from two distinct patients were taken during the first three weeks after the onset of disease. Specific antibody titers more than double their basal value after day 7. (B) Samples from a patient taken at day 21, 100, and 215 after the onset of disease reveal that antibody titters remained high for at least seven months. Patients were diagnosed using RT-PCR protocols (WHO, 2009).</p

Validation of sensitivity against an HI assay.

<p>Normalized absorbance values for fourteen samples with positive anti H1N1/2009 titters based on an HI assay (samples that inhibited hemagglutination of turkey erythrocytes by the Ca/2009/H1N1 influenza virus strain at dilutions equal or higher to 1∶40). Colors indicate HI titter: HI titter = 40 (in blue); HI titter = 80 (in yellow); HI titter = 160 (in orange); HI titter>320 (in red). The proposed positive threshold for the ELISA method is indicated with a solid line (value = 1). One standard deviation is indicated with a dashed line (value = 1.25).</p

Indirect evaluation of proper refolding.

<p>(A) Biorecognition of antibodies from a positive patient observed for different production batches of protein HA_50–274-H1N1. (B) Specific biorecognition ratio (ratio of biorecognition of antibodies from a positive patient serum and a negative subject serum) observed at different refolding batches derived from the same <i>E. coli</i> culture experiment. Variation among batches consisted in minor variations in the dissolution and refolding protocol used.</p

ELISA method designed to evaluate the relative concentration of specific antibodies (Y) anti-influenza A/H1N1/2009 virus in human serum and plasma.

<p>(A) Adsorption of anti-hystidine antibodies to the assay surface on 96-wells micro-assay plates and blockage of the remaining available surface with a commercial blocking solution. (B) Addition of the recombinant protein HA_50–274-H1N1 (semi-circles). (C) Addition of serum samples potentially containing specific antibodies (Y) against the Influenza A H1N1/2009 virus. The left hand panel illustrates a scenario with a higher concentration of specific influenza antibodies. (D) Addition of a peroxidated anti-IgG human antibody (Y) to specifically bind the retained serum antibodies. (E) The addition of peroxidase substrate (S) enables the enzymatic reaction (S→P) with a proportional development of color.</p

Hemagglutination inhibition assay.

<p>(A) Schematic representation of hemagglutination using Influenza viral particles. In the absence of agglutinationon inhibitors, the hemagglutinin from viral capsids (1) agglutinates chicken, turkey or human erythrocytes(2). (B) Schematic representation of hemagglutination inhibition. In the presence of neutralizing antibodies (1) that specifically recognize the hemagglutinin from a influenza virus (2), the process of hemagglutination is inhibited proportionally to the concentration and binding affinity of the neutralizing antibodies.</p