Pseudotype-based neutralization assays for influenza: a systematic analysis

Abstract

The use of vaccination against the influenza virus remains the most effective method of mitigating the significant morbidity and mortality caused by this virus. Antibodies elicited by currently licensed influenza vaccines are predominantly hemagglutination-inhibition (HI)-competent antibodies that target the globular head of hemagglutinin (HA) thus inhibiting influenza virus entry into target cells. These antibodies predominantly confer homosubtypic/strain specific protection and only rarely confer heterosubtypic protection. However, recent academia or pharma-led R&D toward the production of a "universal vaccine" has centered on the elicitation of antibodies directed against the stalk of the influenza HA that has been shown to confer broad protection across a range of different subtypes (H1-H16). The accurate and sensitive measurement of antibody responses elicited by these "next-generation" influenza vaccines is, however, hampered by the lack of sensitivity of the traditional influenza serological assays HI, single radial hemolysis, and microneutralization. Assays utilizing pseudotypes, chimeric viruses bearing influenza glycoproteins, have been shown to be highly efficient for the measurement of homosubtypic and heterosubtypic broadly neutralizing antibodies, making them ideal serological tools for the study of cross-protective responses against multiple influenza subtypes with pandemic potential. In this review, we will analyze and compare literature involving the production of influenza pseudotypes with particular emphasis on their use in serum antibody neutralization assays. This will enable us to establish the parameters required for optimization and propose a consensus protocol to be employed for the further deployment of these assays in influenza vaccine immunogenicity studies.

Keywords: hemagglutinin; influenza; lentiviral vector; neutralization assay; pseudotype; retroviral vector; universal vaccine.

Figure 1

Schematic representation of HIV and MLV derived packaging constructs and vectors.

Figure 2

Phylogeny of current influenza subtypes using the HA glycoprotein. Maximum likelihood tree representing amino acid sequences of the HA glycoprotein for influenza A, B, and C virus as well as putative influenza D. The tree inferred is based on MUSCLE alignment of downloaded sequences conducted using MEGA 5.2 under the WAG + G model (four categories). The phylogenetic tree with the highest log likelihood (−16773.4044) is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 22 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 538 positions in the final dataset (66, 67).

Figure 3

Comparison of influenza HA sequences described against strains pseudotyped. Out of a total of 60,693 HA amino acid sequences extracted from NCBI GenBank, the vast majority come from subtypes H1, H3, H5, and influenza B. Conversely, the current number of different subtypes and strains of HA used to produce pseudotypes is 82. The majority of pseudotyped strains come from subtypes H1, H3, and especially H5.

Figure 4

Production of lentiviral or retroviral pseudotypes. (A) Essential (containing HA, packaging construct gag pol, reporter construct) and/or additional (NA, protease, M2) expression plasmids are co-transfected into HEK293T producer cells. (B) Plasmids migrate to the nucleus whereupon genes are expressed leading to the production of pseudotype proteins and the reporter RNA construct. Cleavage of HA is mediated by transfected or cellular proteases. (C) Pseudotype proteins are packaged by the cell and budding occurs at the cell membrane to yield pseudotypes bearing desired glycoproteins and incorporated reporter.

Figure 5

Pseudotype cores. (A) HIV cores with various envelope glycoproteins (HA, NA, M2). (B) MLV cores with HA or HA and NA. (C) Recombinant VSV containing GFP gene (top) and HA/NA/GFP genes (bottom). Components of influenza pseudotypes can be varied according to need. Pseudotypes have been produced with HA, NA, and M2 influenza envelope proteins, with a range of core packaging constructs (HIV, MLV, VSV shown) as well as different reporters.

Figure 6

Pseudotype production methods. Graphical representation of the methods used for pseudotype production in the literature cited in this review. (A) Method of HA cleavage used. (B) Method of NA action used. (C) Pseudotype cores used. (D) Reporters incorporated into pseudotypes. (E) Transfection reagents for the production of pseudotypes.

Figure 7

Example of a pseudotype neutralization assay (pMN). Serum or antibodies are serially diluted across a 96-well plate, a known quantity of pseudotype is added and the plate is centrifuged and incubated to allow antibody binding. A set quantity of cells are added and plates are incubated for 48 h. Output is measured in a manner depending on reporter used.

Figure 8

Computer models of chimeric HA. Three-dimensional structures were generated with Swiss PDB viewer and POV-Ray 3.7 using the structure of the recombinant virus A/Hong Kong/1/1968 X-31 H3 [PDB ID: 2VIU (162)]. The signal peptide is not present in the HA. The transmembrane region is not resolved by X-ray crystallography. (A) Three-dimensional structure of the influenza HA trimer, showing the HA surface of the head (blue) and stalk (red) regions. (B) Three-dimensional ribbon structure of the influenza HA monomer showing the head (blue) and stalk (red) regions. (C) Three-dimensional ribbon structure of the influenza HA monomer showing HA1 (blue) and HA2 (light blue) subunits, the cleavage site and the fusion peptide are also shown in green and red, respectively. (D) Schematic of the HA polypeptide.

Figure 9

Pseudotypes used for gene delivery or as immunogens. Pseudotypes can be employed as immunogens bearing the antigen of choice or as delivery systems for genes of choice. (A) HA-based pseudotype/virus-like particle immunogen. (B) VSV-G pseudotype delivery system for HA gene. (C) HA pseudotype delivery system for HA gene.