Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in ...

Universities of Kent and Greenwich at Medway

Pseudotype viruses (PVs) are chimeric, replication-deficient virions that mimic wild-type virus entry mechanisms and can be safely employed in neutralisation assays, bypassing the need for high biosafety requirements and performing comparably to established serological assays. However, PV supernatant necessitates -80°C long-term storage and cold-chain maintenance during transport, which limits the scope of dissemination and application throughout resource-limited laboratories. We therefore investigated the effects of lyophilisation on influenza, rabies and Marburg PV stability, with a view to developing a pseudotype virus neutralisation assay (PVNA) based kit suitable for affordable global distribution. Infectivity of each PV was calculated after lyophilisation and immediate reconstitution, as well as subsequent to incubation of freeze-dried pellets at varying temperatures, humidities and timepoints. Integrity of glycoprotein structure following treatment was also assessed by employing lyophilised PVs in downstream PVNAs. In the presence of 0.5M sucrose-PBS cryoprotectant, each freeze-dried pseudotype was stably stored for 4 weeks at up to 37°C and could be neutralised to the same potency as unlyophilised PVs when employed in PVNAs. These results confirm the viability of a freeze-dried PVNA-based kit, which could significantly facilitate low-cost serology for a wide portfolio of emerging infectious viruses.

Lyophilisation of influenza, rabies and Marburg lentiviral pseudotype viruses for the development and distribution of a neutralisation -assay-based diagnostic kit.

The globalization of the world's economies, accompanied by increasing international travel, changing climates, altered human behaviour and demographics is leading to the emergence of different viral diseases, many of which are highly pathogenic and hence are considered of great public and animal health importance. To undertake basic research and therapeutic development, many of these viruses require handling by highly trained staff in BSL-3/4 facilities not readily available to the majority of the global R&D community. In order to circumvent the enhanced biosafety requirement, the development of non-pathogenic, replication-defective pseudotyped viruses is an effective and established solution to permit the study of many aspects of virus biology in a low containment biosafety level (BSL)-1/2 laboratory. Under the spectre of the unfolding Ebola crisis, this timely conference (the second to be organised by the Viral Pseudotype Unit, www.viralpseudotypeunit.info*) discusses the recent advances in pseudotype technology and how it is revolutionizing the study of important human and animal pathogens (human and avian influenza viruses, rabies/lyssaviruses, HIV, Marburg and Ebola viruses). Key topics addressed in this conference include the exploitation of pseudotypes for serology and serosurveillance, immunogenicity testing of current and next-generation vaccines and new pseudotype assay formats (multiplexing, kit development). The first pseudotype-focused Euroscicon conference organised by the Viral Pseudotype Unit was recently reviewed [1].

The use of pseudotypes to study viruses, virus sero-epidemiology and vaccination.

Serological techniques commonly used to quantify influenza-specific antibodies include the Haemagglutination Inhibition (HI), Single Radial Haemolysis (SRH) and Virus Neutralization (VN) assays. HI and SRH are established and reproducible techniques, whereas VN is more demanding. Every new influenza vaccine needs to fulfil the strict criteria issued by the European Medicines Agency (EMA) in order to be licensed. These criteria currently apply exclusively to SRH and HI assays and refer to two different target groups-healthy adults and the elderly, but other vaccine recipient age groups have not been considered (i.e., children). The purpose of this timely review is to highlight the current scenario on correlates of protection concerning influenza vaccines and underline the need to revise the criteria and assays currently in use. In addition to SRH and HI assays, the technical advantages provided by other techniques such as the VN assay, pseudotype-based neutralization assay, neuraminidase and cell-mediated immunity assays need to be considered and regulated via EMA criteria, considering the many significant advantages that they could offer for the development of effective vaccines.

Overview of Serological Techniques for Influenza Vaccine Evaluation: Past, Present and Future.

Pseudotyped viruses (PVs) produced by co-transfecting cells with plasmids expressing lentiviral core proteins and viral envelope proteins are potentially powerful tools for studying various aspects of equine influenza virus (EIV) biology. The aim of this study was to optimise production of equine influenza PVs. Co-transfection of the HAT protease to activate the haemagglutinin (HA) yielded a higher titre PV than TMPRSS2 with the HA from A/equine/Richmond/1/2007 (H3N8), whereas for A/equine/Newmarket/79 (H3N8), both proteases resulted in equivalent titres. TMPRSS4 was ineffective with the HA of either strain. There was also an inverse relationship between the amount of protease-expression plasmids and the PV titre obtained.  Interestingly, the PV titre obtained by co-transfection of a plasmid encoding the cognate N8 NA was not as high as that generated by the addition of exogenous neuraminidase (NA) from  to allow the release of nascent PV particles. Finally, initial characterisation of the reliability of PV neutralisation tests (PVNTs) demonstrated good intra-laboratory repeatability. In conclusion, we have demonstrated that equine influenza PV production can be readily optimised to provide a flexible tool for studying EIV.

The Optimisation of Pseudotyped Viruses for the Characterisation of Immune Responses to Equine Influenza Virus.

The pseudotype particle neutralization test (pp-NT) is a next-generation serological assay employed for the sensitive study of influenza antibody responses against hemagglutinin (HA), including stalk-directed antibodies. However, a validation of this assay has yet to be performed, and this limits its use to primarily research laboratories. To identify possible serological standards to be used in optimization and validation of the pp-NT, we have evaluated the cross-reactivity of hyperimmune chicken reference antisera in this assay. Our findings show that the cross-reactivity detected by the pp-NT is only partly explained by phylogenetic relationships and protein homology between the HA subtypes analysed; further studies are necessary to understand the origin of the cross-reactivity detected, and reference standards with higher specificity should be evaluated or generated  for future use in pp-NT.

The Use of Hyperimmune Chicken Reference Sera Is Not Appropriate for the Validation of Influenza Pseudotype Neutralization Assays.

Viruses are a major threat to human health and economic well-being. In recent years Ebola, Zika, influenza, and chikungunya virus epidemics have raised awareness that infections can spread rapidly before vaccines or specific antagonists can be made available. Broad-spectrum antivirals are drugs with the potential to inhibit infection by viruses from different groups or families, which may be deployed during outbreaks when specific diagnostics, vaccines or directly acting antivirals are not available. While pathogen-directed approaches are generally effective against a few closely related viruses, targeting cellular pathways used by multiple viral agents can have broad-spectrum efficacy. Virus entry, particularly clathrin-mediated endocytosis, constitutes an attractive target as it is used by many viruses. Using a phenotypic screening strategy where the inhibitory activity of small molecules was sequentially tested against different viruses, we identified 12 compounds with broad-spectrum activity, and found a subset blocking viral internalisation and/or fusion. Importantly, we show that compounds identified with this approach can reduce viral replication in a mouse model of Zika infection. This work provides proof of concept that it is possible to identify broad-spectrum inhibitors by iterative phenotypic screenings, and that inhibition of host-pathways critical for viral life cycles can be an effective antiviral strategy.

Identification of Broad-Spectrum Antiviral Compounds by Targeting Viral Entry.

Pseudotype neutralization assays are powerful tools to study functional antibody responses against viruses in low biosafety laboratories. However, protocols described in the literature differ widely with respect to material, reagents, and methods used to perform these assays and to analyse the raw data generated. This could result in discrepancies between the results of different laboratories even when the same pseudotypes and the same samples are analysed. Here, we describe, in detail, an experimental protocol to perform pseudotype neutralization assays using lentiviral pseudotypes bearing influenza haemagglutinin and expressing firefly luciferase. We also present the steps necessary to analyse the data and calculate the half maximal inhibitory concentration of the sera analysed. This protocol will provide support for the validation and the standardization of the pseudotype neutralization assay for influenza virus serology. Additionally, it will provide a starting point for the development of pseudotype neutralization assays using pseudotypes bearing other viral envelope proteins.

Pseudotype Neutralization Assays: From Laboratory Bench to Data Analysis.

Influenza B virus (IBV) circulates in the human population and causes considerable disease burden worldwide, each year. Current IBV vaccines can struggle to mount an effective cross-reactive immune response, as strains become mismatched, due to constant antigenic changes. Additional strategies which use monoclonal antibodies, with broad reactivity, are of considerable interest, both, as diagnostics and as immunotherapeutics. Alternatives to conventional monoclonal antibodies, such as single domain antibodies (Nanobodies) with well-documented advantages for applications in infectious disease, have been emerging. In this study we have isolated single domain antibodies (sdAbs), specific to IBV, using alpacas immunised with recombinant hemagglutinin (HA) from two representative viruses, B/Florida/04/2006 (B/Yamagata lineage) and B/Brisbane/60/2008 (B/Victoria lineage). Using phage display, we have isolated a panel of single domain antibodies (sdAbs), with both cross-reactive and lineage-specific binding. Several sdAbs recognise whole virus antigens, corresponding to influenza B strains included in vaccines spanning over 20 years, and were capable of neutralising IBV pseudotypes corresponding to prototype strains from both lineages. Lineage-specific sdAbs recognised the head domain, whereas, sdAbs identified as cross-reactive could be classified as either head binding or stem binding. Using yeast display, we were able to correlate lineage specificity with naturally occurring sequence divergence, at residue 122 in the highly variable 120 loop of the HA1 domain. The single domain antibodies described, might have applications in IBV diagnostics, vaccine potency testing and as immunotherapeutics.

Cross-Reactive and Lineage-Specific Single Domain Antibodies against Influenza B Hemagglutinin.

The recent outbreak of a novel Coronavirus (SARS-CoV-2) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against SARS-CoV-2 spike protein and to evaluate the serological immunity in humans. Since the accessibility of live virus microneutralisation (MN) assays with SARS-CoV-2 is limited and requires enhanced bio-containment, the approach based on "pseudotyping" can be considered a useful complement to other serological assays. After fully characterising lentiviral pseudotypes bearing the SARS-CoV-2 spike protein, we employed them in pseudotype-based neutralisation assays in order to profile the neutralising activity of human serum samples from an Italian sero-epidemiological study. The results obtained with pseudotype-based neutralisation assays mirrored those obtained when the same panel of sera was tested against the wild type virus, showing an evident convergence of the pseudotype-based neutralisation and MN results. The overall results lead to the conclusion that the pseudotype-based neutralisation assay is a valid alternative to using the wild-type strain, and although this system needs to be optimised and standardised, it can not only complement the classical serological methods, but also allows serological assessments to be made when other methods cannot be employed, especially in a human pandemic context.

Characterisation of SARS-CoV-2 Lentiviral Pseudotypes and Correlation between Pseudotype-Based Neutralisation Assays and Live Virus-Based Micro Neutralisation Assays.

RaTG13 is a close relative of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, sharing 96% sequence similarity at the genome-wide level. The spike receptor binding domain (RBD) of RaTG13 contains a number of amino acid substitutions when compared to SARS-CoV-2, likely impacting affinity for the ACE2 receptor. Antigenic differences between the viruses are less well understood, especially whether RaTG13 spike can be efficiently neutralised by antibodies generated from infection with, or vaccination against, SARS-CoV-2. Using RaTG13 and SARS-CoV-2 pseudotypes we compared neutralisation using convalescent sera from previously infected patients or vaccinated healthcare workers. Surprisingly, our results revealed that RaTG13 was more efficiently neutralised than SARS-CoV-2. In addition, neutralisation assays using spike mutants harbouring single and combinatorial amino acid substitutions within the RBD demonstrated that both spike proteins can tolerate multiple changes without dramatically reducing neutralisation. Moreover, introducing the 484 K mutation into RaTG13 resulted in increased neutralisation, in contrast to the same mutation in SARS-CoV-2 (E484K). This is despite E484K having a well-documented role in immune evasion in variants of concern (VOC) such as B.1.351 (Beta). These results indicate that the future spill-over of RaTG13 and/or related sarbecoviruses could be mitigated using current SARS-CoV-2-based vaccination strategies.

Pseudotyped Bat Coronavirus RaTG13 is efficiently neutralised by convalescent sera from SARS-CoV-2 infected patients.

We have developed an influenza hemagglutinin (HA) pseudotype (PV) library encompassing all influenza A (IAV) subtypes from HA1-HA18, influenza B (IBV) subtypes (both lineages), representative influenza C (ICV), and influenza D (IDV) viruses. These influenza HA (or hemagglutinin-esterase fusion (HEF) for ICV and IDV) pseudotypes have been used in a pseudotype microneutralization assay (pMN), an optimized luciferase reporter assay, that is highly sensitive and specific for detecting neutralizing antibodies against influenza viruses. This has been an invaluable tool in detecting the humoral immune response against specific hemagglutinin or hemagglutinin-esterase fusion proteins for IAV to IDV in serum samples and for screening antibodies for their neutralizing abilities. Additionally, we have also produced influenza neuraminidase (NA) pseudotypes for IAV N1-N9 subtypes and IBV lineages. We have utilized these NA-PV as surrogate antigens in in vitro assays to assess vaccine immunogenicity. These NA PV have been employed as the source of neuraminidase enzyme activity in a pseudotype enzyme-linked lectin assay (pELLA) that is able to measure neuraminidase inhibition (NI) titers of reference antisera, monoclonal antibodies, and postvaccination sera. Here we show the production of influenza HA, HEF, and NA PV and their employment as substitutes for wild-type viruses in influenza serological and neutralization assays. We also introduce AutoPlate, an easily accessible web app that can analyze data from pMN and pELLA quickly and efficiently, plotting inhibition curves and calculating half-maximal concentration (IC) neutralizing antibody titers. These serological techniques coupled with user-friendly analysis tools are faster, safer, inexpensive alternatives to classical influenza assays while also offering the reliability and reproducibility to advance influenza research and make it more accessible to laboratories around the world.

Nigel J. Temperton

Viral Pseudotype Unit,
Medway School of Pharmacy,
Viral Pseudotype Unit, Medway School of Pharmacy

Universities of Kent and Greenwich at Medway

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Nigel J. Temperton

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Universities of Kent and Greenwich at Medway

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Viral Pseudotype Unit,
Medway School of Pharmacy,
Viral Pseudotype Unit, Medway School of Pharmacy