Development of a high-throughput assay to measure measles neutralizing antibodies.

Measles virus is highly infectious and remains a leading cause of vaccine preventable deaths in children. Neutralizing antibody responses elicited by measles virus infection or immunization are a serological correlate of protection. We describe a high-throughput neutralization assay to improve surveillance for measles immunity. Measles virus-antibody mixtures were incubated on Vero cell monolayers and 24 hours later cell-lysates harvested and subjected to one-step SYBR green RT-qPCR to amplify a target sequence within the measles virus nucleoprotein gene. Neutralization endpoint titers were interpolated to determine the dilution that inhibited the relative amplicon copy number by at least 90% compared to the mean signal obtained in virus control wells in the absence of serum. Anti-measles virus and anti-measles hemagglutinin antisera specifically neutralized measles virus in the microneutralization RT-qPCR assay while pre-immune sera and sera raised against other viruses did not. The microneutralization RT-qPCR assay obeyed the Percentage Law for measles virus inputs ranging from 100-5000 TCID50/well. The linear range of the assay corresponds to measles antibody concentrations of 30 to 3000 mIU/mL. Bland-Altman analysis and two-way analysis of variance demonstrated that results obtained using the microneutralization RT-qPCR assay were comparable to those obtained using a plaque reduction neutralization test and correctly identified human serum samples that were seropositive (95% and 100%, sensitivity and specificity, respectively). Furthermore, these comparisons suggest that a concentration of 300 mIU/mL may be a conservative cut-point to use to identify individuals likely to be protected against severe measles disease when the endpoint is based on 90% inhibition of virus replication. Measles virus microneutralization RT-qPCR is a rapid, sensitive, specific, and robust assay for detecting measles neutralizing antibodies that may help to improve immunization strategies nationally and achieve measles elimination globally

Glycosylation of Residue 141 of Subtype H7 Influenza A Hemagglutinin (HA) Affects HA-Pseudovirus Infectivity and Sensitivity to Site A Neutralizing Antibodies.

Human infections with H7 subtype influenza virus have been reported, including an H7N7 outbreak in Netherlands in 2003 and H7N9 infections in China in 2013. Previously, we reported murine monoclonal antibodies (mAbs) that recognize the antigenic site A of H7 hemagglutinin (HA). To better understand protective immunity of H7 vaccines and vaccine candidate selection, we used these mAbs to assess the antigenic relatedness among two H7 HA isolated from past human infections and determine residues that affect susceptibility to neutralization. We found that these mAbs neutralize pseudoviruses bearing HA of A/Shanghai/02/2013(H7N9), but not A/Netherlands/219/2003(H7N7). Glycosylation of the asparagine residue at position 141 (N141) (N133, H3 HA numbering) in the HA of A/Netherlands/219/2003 HA is responsible for this resistance, and it affects the infectivity of HA-pseudoviruses. The presence of threonine at position 143 (T135, H3 HA numbering) in the HA of A/Netherlands/219/2003, rather than an alanine found in the HA of A/Shanghai/02/2013(H7N9), accounts for these differences. These results demonstrate a key role for glycosylation of residue N141 in affecting H7 influenza HA-mediated entry and sensitivity to neutralizing antibodies, which have implications for candidate vaccine design

Analysis of Adaptation Mutants in the Hemagglutinin of the Influenza A(H1N1)pdm09 Virus

<div><p>Hemagglutinin is the major surface glycoprotein of influenza viruses. It participates in the initial steps of viral infection through receptor binding and membrane fusion events. The influenza pandemic of 2009 provided a unique scenario to study virus evolution. We performed molecular dynamics simulations with four hemagglutinin variants that appeared throughout the 2009 influenza A (H1N1) pandemic. We found that variant 1 (S143G, S185T) likely arose to avoid immune recognition. Variant 2 (A134T), and variant 3 (D222E, P297S) had an increased binding affinity for the receptor. Finally, variant 4 (E374K) altered hemagglutinin stability in the vicinity of the fusion peptide. Variants 1 and 4 have become increasingly predominant, while variants 2 and 3 declined as the pandemic progressed. Our results show some of the different strategies that the influenza virus uses to adapt to the human host and provide an example of how selective pressure drives antigenic drift in viral proteins.</p></div

Determination of influenza B identity and potency in quadrivalent inactivated influenza vaccines using lineage-specific monoclonal antibodies.

Co-circulation of two antigenically and genetically distinct lineages of influenza B virus, represented by prototype viruses B/Victoria/2/1987 and B/Yamagata/16/1988, has led to the development of quadrivalent influenza vaccines that contain two influenza B antigens. The inclusion of two influenza B antigens presents challenges for the production and regulation of inactivated quadrivalent vaccines, including the potential for cross-reactivity of the reagents used in identity and potency assays because of the relative close relatedness of the hemagglutinin (HA) from the two virus lineages. Monoclonal antibodies (mAbs) specific for the two lineages of influenza B HA were generated and characterized and used to set-up simple identity tests that distinguish the influenza B antigens in inactivated trivalent and quadrivalent vaccines. The lineage-specific mAbs bound well to the HA of influenza B strains included in influenza vaccines over a period of more than 10 years, suggesting that identity tests using such lineage-specific mAbs would not necessarily have to be updated with every influenza B vaccine strain change. These lineage-specific mAbs were also used in an antibody capture ELISA format to quantify HA in vaccine samples, including monovalent, trivalent, and quadrivalent vaccine samples from various manufacturers. The results demonstrated correlation with HA values determined by the traditional single radial immunodiffusion (SRID) assay. Further, the antibody-capture ELISA was able to distinguish heat-stressed vaccine from unstressed vaccine, and was similar to the SRID in quantifying the resultant loss of potency. These mAb reagents should be useful for further development of antibody-based alternative influenza B identity and potency assays

Destabilizing effect of mutation E374K.

<p>A, wild-type HA; B, E374K mutant. Protein is shown as green ribbons. Amino acids surrounding position 374 are illustrated as sticks. In the wild-type HA, E374 interacts with Y433 and N366 and with the fusion peptide through water-mediated hydrogen bonds. In E374K, the lysine residue interacts only with the oxygen from the amide group of N366.</p

Surface representation of the Hemagglutinin (HA) form the influenza A(H1N1)pdm09 virus.

<p>The human receptor is shown as sticks. Mutations analyzed in this study are shown in magenta; antigenic sites are colored as follows: Ca1, blue; Ca2, green; Cb, yellow; Sa, orange, and Sb, red. The fusion peptide is colored olive green. Panel B represents the HA rotated 90° in the vertical axis.</p

Effect of mutation S134T on receptor binding.

<p>A, wild-type HA; B, S134T. Protein is shown as green ribbons. Amino acids surrounding position 134 are illustrated as sticks; receptor and carbohydrate residues are depicted in magenta sticks. Mutation S134T modifies interactions of HA with the receptor by establishing hydrogen bonds between T134 and sialic acid, and by losing interactions between galactose and D222; instead, D222 in mutant S134T interacts with glycosylations originating at N87.</p

Conformational changes observed in variant 1.

<p>A, interactions of S143 in wild-type HA; B, interactions of S143G in variant 1; C, S185 in wild-type HA; D, S185T in variant 1. Amino acids surrounding position 138 are illustrated as sticks; the receptor is depicted as magenta sticks. Interactions with the receptor are essentially the same as the wild-type for variant 1. A displacement of loop 140 can be seen in variant 1, which allows H138 to interact with the main chain of K142 (B). Mutation S185T does not change interactions within the protein (D).</p

Effect of D222E in receptor binding.

<p>A, wild-type HA; B, D222E mutant. Protein is shown as green ribbons. Amino acids surrounding position 222 are illustrated as sticks; receptor and carbohydrate residues are depicted as magenta sticks. In the wild-type HA, D222 forms a triad with K219 and E224 that interact with galactose from the human receptor. In D222E, only K219 and E224 interact with galactose, while E222 interacts through water-mediated hydrogen bonds with nearby glycosylations originating at N87.</p

Frequency of mutations in the amino acid sequence of Hemagglutinin (HA) from the influenza A(H1N1)pdm09 virus.

1<p>2009a represents sequences from January-June 2009 period; 2009b represents sequences from the July-December 2009 period.</p>2<p>Highest frequencies are shown in bold.</p