REVIEW ARTICLE published: 29 April 2015 doi: 10.3389/fimmu.2015.00161 Pseudotype-based neutralization assays for influenza: a systematic analysis George William Carnell 1, Francesca Ferrara1, Keith Grehan1, Craig Peter Thompson1,2,3 and Nigel James Temperton1* 1 Viral Pseudotype Unit, Medway School of Pharmacy, The Universities of Greenwich and Kent at Medway, Chatham Maritime, Kent, UK 2 Department of Zoology, University of Oxford, Oxford, UK 3 The Jenner Institute Laboratories, University of Oxford, Oxford, UK Edited by: The use of vaccination against the influenza virus remains the most effective method of Arun Kumar, GlaxoSmithKline mitigating the significant morbidity and mortality caused by this virus. Antibodies elicited Vaccines, Italy by currently licensed influenza vaccines are predominantly hemagglutination-inhibition (HI)- Reviewed by: YasuTakeuchi, University College competent antibodies that target the globular head of hemagglutinin (HA) thus inhibiting London, UK influenza virus entry into target cells. These antibodies predominantly confer homosub- Ghazi Kayali, St. Jude Children’s typic/strain specific protection and only rarely confer heterosubtypic protection. However, Research Hospital, USA recent academia or pharma-led R&D toward the production of a “universal vaccine” has *Correspondence: centered on the elicitation of antibodies directed against the stalk of the influenza HA Nigel James Temperton, Viral that has been shown to confer broad protection across a range of different subtypes (H1– Pseudotype Unit, Medway School of Pharmacy, Chatham Maritime, Kent H16). The accurate and sensitive measurement of antibody responses elicited by these ME4 4TB, UK “next-generation” influenza vaccines is, however, hampered by the lack of sensitivity of e-mail: [email protected] the traditional influenza serological assays HI, single radial hemolysis, and microneutraliza- tion. Assays utilizing pseudotypes, chimeric viruses bearing influenza glycoproteins, have been shown to be highly efficient for the measurement of homosubtypic and heterosub- typic 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 con- sensus protocol to be employed for the further deployment of these assays in influenza vaccine immunogenicity studies. Keywords: influenza, hemagglutinin, pseudotype, neutralization assay, universal vaccine, lentiviral vector, retroviral vector INFLUENZA PSEUDOTYPES case of the majority of subtypes and can reassort with human Influenza is a respiratory syndrome caused by three of six gen- strains through antigenic shift to yield human compatible viruses era in the orthomyxoviridae family, influenza A, B, and C. A with previously un-encountered surface epitopes. Pigs are usu- putative fourth genus (influenza D) has recently been charac- ally considered to be the mixing vessel for reassortment as they terized and proposed (1). Influenza A is the most widespread, express a mixture of a-2,3 and a-2,6 sialic acid linkages. Influenza its various subtypes are classified according to their antigeni- virus research is often hindered by the requirement for expensive cally variable surface glycoproteins: hemagglutinin (HA, H1–H18) biosafety precautions, especially in the case of the highly path- and neuraminidase (NA, N1–N11). The virion consists of a seg- ogenic avian influenza (HPAI, e.g. H5N1, H7N1) or pandemic mented negative sense genome encapsidated in ribonucleoprotein strains. complexes, which are surrounded by a matrix shell and lipid enve- Pseudotypes or pseudotype particles are chimeric “viruses” lope containing the two surface glycoproteins and the M2 ion consisting of a surrogate virus core surrounded by a lipid enve- channel. Influenza A is the primary source of the human sea- lope with the surface glycoproteins of another virus, such as HA. sonal form of the disease, responsible for up to 500,000 deaths By removing the genetic element of the virus being studied and per annum as well as deaths caused by pandemics such as those replacing it with a suitable reporter, viruses, especially HPAI, can occurring in 1918, 1957, 1968, and 2009 (2). Consequently, vac- be studied in this safer, single cycle system. The comparative cines against influenza need to be regularly updated to match safety of pseudotype viruses circumvents the need for restric- predicted circulating strains that are constantly escaping from vac- tive, expensive, and widely unavailable high-category biosafety cine protection through a mechanism known as antigenic drift. facilities, increasing access to research groups interested in highly Influenza A is primarily associated with wild fowl/birds in the pathogenic viruses. www.frontiersin.org April 2015 | Volume 6 | Article 161 | 1 Carnell et al. Influenza pseudotypes This review is a systematic analysis encompassing a wide range construct is incorporated into progeny pseudotypes. The vector’s of peer-reviewed literature in English concerning the produc- life cycle mimics that of HIV, using the ) element to allow encap- tion and use of pseudotypes bearing influenza glycoproteins to sidation into nascent pseudotypes and long terminal repeat (LTR) date. For the purpose of this review, pseudotypes will be defined regions bearing the U3 promoter, which with the aid of tat, per- as replication-deficient viruses containing a viral core from one mit the expression of the viral proteins after integration into the species and bearing glycoproteins from another that are not rep- host genome. The rev responsive element (RRE) allows nuclear resented in the genome. Literature was gathered by searching for export of viral messenger RNA (mRNA), including the reporter “influenza pseudotypes”using Google Scholar and NCBI PubMed. gene transcript, which is the measure of output for this system. The resulting list of publications was expanded by following up Due to the incorporation of the HIV core genes into the same cited references and finally, those falling outside of our pseudo- integrated construct as the reporter, transduced cells may possi- type definition or not specifically using influenza pseudotypes bly produce luciferase containing cores alongside its transcribed were excluded from the sections on production, transduction, and enzyme, which could potentially interfere with luciferase activity. neutralization. Another commonly used HIV core vector is pCMV DR8.2, a This review will be useful to those interested in the produc- relation of pCMV DR8.9, which still contains intact vif, vpr, vpu, tion of pseudotypes for use in immunogenicity testing of pre- and nef genes (15–20). clinical influenza vaccines, whether in human or animal settings, A further approach uses the second generation HIV vector and including “universal vaccine” candidates. Influenza serologi- p8.91 that also originates from pCMV DR8.9 and DR9 (15, 21). cal studies such as the measurement of seroprevalence will benefit The p8.91 vector is a modified HIV-1 clone,lacking the ) sequence from this manuscript, which will also help to inform the process as well as the env, vif, nef, vpu, and vpr genes and is widely used of validation of pseudotype-based assays to clinical end-point. in the articles studied (22–25). The cytomegalovirus promoter is Furthermore, studies utilizing chimeric HA proteins in order to used in lieu of LTR-based promotion, meaning that p8.91 pro- differentiate between stalk and head directed antibodies will be vides the necessary genes for the production of the core but the discussed. proviral and packaging elements (LTRs, RRE, and )) are trans- ferred to a separate plasmid bearing the reporter gene. Thus, the PSEUDOTYPE COMPONENTS reporter construct will be incorporated into nascent virions and CORES AND REPORTERS integrated into the transduced cell’s genome, whereupon the LTRs The core and its associated genome containing a reporter are and RRE will act to enhance expression. In the case of the com- the backbone of the pseudotype system, which can be used to monly used firefly luciferase or green fluorescent protein (GFP) study the properties of selected entry proteins. The use of cores plasmids pCSFLW or pCSGW,a safety component is incorporated 0 from lentiviral human immunodeficiency virus (HIV) and gam- through a deletion in the 3 LTR (U3 promoter region), creating maretroviruses such as murine leukemia virus (MLV) predomi- so called self-inactivating (SIN) vectors (26, 27). nate in the influenza pseudotype literature. Recent development of Third generation vectors have also been used. In this instance, systems involving rhabdoviruses, in particular the vesicular stom- HIV structural and accessory genes are separated from rev, which atitis virus (VSV), has also been used to produce pseudotype cores is provided in cis on an additional plasmid. The third generation with promising results (3,4). Invitrogen ViraPower Lentiviral Expression System was used in several cases using the plasmids pLP1 and pLP2 (28–31). RETROVIRAL AND LENTIVIRAL CORES AND VECTORS Murine leukemia virus cores are less widely used but provide Retroviral and lentiviral vectors are complex systems, which will similar gag and pol elements to HIV vectors (32–38). One MLV be explained in simple terms specific