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.

Technical Considerations for the Generation of Novel Pseudotyped Viruses

“…Furthermore, protease usage can be a critical determinant of viral tropism [76]. For seasonal human influenza viruses, the requirement for proteasemediated cleavage of the hemagglutinin in order for it to become fusion competent is a well-documented trait [65,[77][78][79]. However, protease activation is also a well-studied component in the Paramyxoviridae (a family of negative sense single-strand RNA viruses), where the F protein precursor must be cleaved in order to facilitate maturation of the fusion protein [80], and in the Coronaviridae (a family of positive sense, singlestrand RNA viruses) where proteases can be involved in both cleavage of the spike protein and facilitating release from the host cell [81][82][83].…”

“…With a toolkit of 6 plasmids and a streamlined optimization grid for transfection, most strains of influenza are currently amenable to pseudotyping onto retroviral cores [65][66][67]. Essentially these assembly processes fall broadly into three protocols with the one chosen being dependent on the HA to be pseudotyped and also the downstream application.…”

“…The addition of exogenous tosyl phenylalanyl chloromethyl ketone (TPCK) trypsin has been demonstrated to facilitate activation of SARS-CoV [85,86] MERS-CoV [10,87] and human coronavirus 229E [88]. Exogenous TPCK trypsin is also used for influenza, but an additional step is required in order to deactivate the protease, using commercially available protease inhibitors, prior to inoculation of the PVs onto target cells [65,77].…”

“…Therefore, it may be necessary to supplement PV generation protocols with specific proteases. In many studies it has become common practice to co-transfect a protease-encoding plasmid alongside the other plasmids required for PV production [65,77,79,84]. For influenza, the most commonly used proteases when supplied as a plasmid are HAT and TMPRSS2 [77].…”

Controlling equine influenza: Traditional to next generation serological assays

“…The antibody titre that results in 50% neutralization (IC 50 ) is usually reported after 48 hours incubation at 37 o C. Optimisation of influenza pseudotyped virus production is important to ensure efficient and high titre production of virus. A meta-analysis of influenza PV production has recently been conducted and a consensus protocol produced (Carnell et al, 2015).…”