Chimeric influenza haemagglutinins: Generation and use in pseudotype neutralization assays

Recently chimeric influenza haemagglutinins (cHAs) have been generated as potential 'universal' vaccination antigens and as tools to identify HA stalk-directed antibodies via their use as antigens in ELISA, and virus or pseudotype-based neutralization assays. The original methods [1], [2] used for their generation require the amplification of regions of interest (head and stalk) using primers containing SapI sites and subsequent cloning into pDZ plasmid. This requires precise primer design, checking for the absence of SapI sites in the sequence of interest, and multi-segment ligation. As an alternative strategy we have developed and optimized a new protocol for assembling the cHA by exploiting Gibson Assembly. •This method also requires precise primer design, but it is rapid and methodologically simple to perform. We have evaluated that using this method it is possible to construct a cHA encoding DNA in less than a week.•Additional weeks are however necessary to optimize the production of pseudotyped lentiviral particles and to perform neutralization assays using them as surrogate antigens.•In comparison to the original protocols, we have also observed that performing parallel neutralization assays using pseudotypes harbouring the two parental HAs, permits effective delineation between stalk and head antibody responses in the samples tested

Lyophilisation of lentiviral pseudotypes for the development and distribution of virus neutralisation assay kits for rabies, Marburg and influenza viruses

Purpose: Some conventional serological assays can accurately quantify neutralising antibody responses raised against epitopes on virus glycoproteins, enabling mass vaccine evaluation and serosurveillance studies to take place. However, these assays often necessitate the handling of wild-type virus in expensive high biosafety laboratories, which restricts the scope of their application, particularly in resource-deprived areas. A solution to this issue is the use of lentiviral pseudotype viruses (PVs)—chimeric, replication-deficient virions that imitate the binding and entry mechanisms of their wild-type equivalents. Pseudotype virus neutralisation assays (PVNAs) bypass high biosafety requirements and yield comparable results to established assays. This study explores the potential for using lyophilisation of pseudotypes as a cost-effective, alternative means for production, distribution and storage of a PVNAbased diagnostic kit. Methods & Materials: Rabies, Marburg and H5 subtype Influenza virus pseudotypes were each suspended in cryoprotectant solutions of various molarities and subjected to freeze-drying before incubation at a variety of temperatures, humidities and time periods. Samples were then employed in antibody neutralisation assays using specific sera. Results: High levels of PV titre were retained post-lyophilisation, with acceptable levels of virus activity maintained even after medium-term storage in tropical conditions. Also, the performance of PVs in neutralisation assays was not affected by the lyophilisation process. Conclusion: These results confirm the viability of a freeze-dried PVNA-based diagnostic kit, which could considerably facilitate in-field serology for a number of clinically important viruses

The use of equine influenza pseudotypes for serological screening

Standard assays used for influenza serology present certain practical issues, such as inter-laboratory variability, complex protocols and the necessity for handling certain virus strains in high biological containment facilities. In an attempt to address this, avian and human influenza HA pseudotyped retroviruses have been successfully employed in antibody neutralization assays. In this study we generated an equine influenza pseudotyped lentivirus for serological screening. This was achieved by co-transfection of HEK293T cells with plasmids expressing the haemagglutinin (HA) protein of an H3N8 subtype equine influenza virus strain, HIV gag-pol and firefly luciferase reporter genes and harvesting virus from supernatant. In order to produce infective pseudotype particles it was necessary to additionally co-transfect a plasmid encoding the TMPRSS2 endoprotease to cleave the HA. High titre pseudotype virus (PV) was then used in PV antibody neutralization assays (PVNAs) to successfully distinguish between vaccinated and non-vaccinated equines. The sera were also screened by single radial haemolysis (SRH) assay. There was a 65% correlation between the results of the two assays, with the PVNA assay appearing slightly more sensitive. Future work will extend the testing of the PVNA with a larger number of serum samples to assess sensitivity/specificity, inter/intra-laboratory variability and to define a protective titre

The production and development of H7 Influenza virus pseudotypes for the study of humoral responses against avian viruses

In recent years, high pathogenicity avian influenza (HPAI) virus, H5N1, low pathogenicity avian influenza (LPAI) virus, H9N2, and both HPAI and LPAI H7 viruses have proved devastating for the affected economies reliant on poultry industry, and have posed serious public health concerns. These viruses have repeatedly caused zoonotic disease in humans, raising concerns of a potential influenza pandemic. Despite the focus on the HPAI H5N1 outbreak in 1997 some H7 strains have also shown to be occasionally adaptable to infecting humans. Therefore, applying knowledge of the H5 virus evolution and spread to the development of sensitive serological methods is likely to improve our ability to understand and respond to the emergence of other HPAI and LPAI viruses, present within the avian populations, with the potential to infect humans and other species. In the present study we describe the construction and production of lentiviral pseudotypes bearing envelope glycoproteins of LPAI and HPAI H7 avian influenza viruses, which have been responsible for several outbreaks in the past decade. The H7 pseudotypes were evaluated in pseudotype-based neutralization (pp-NT) assays in order to detect and quantify the presence of neutralizing antibodies in avian sera, which were confirmed H7 positive by inhibition of haemagglutination (HI) test. Overall, our results substantiate influenza virus pseudotype neutralization as a robust tool for influenza sero-surveillance

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

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 Clostridium perfringens 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

Pseudotype Neutralization Assays: From laboratory Bench to Data Analysis

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

The use of hyperimmune chicken reference sera is not appropriate for the validation of influenza pseudotype neutralization assays

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 de novo for future use in pp-NT

Hemagglutinin sequence conservation guided stem immunogen design from influenza A H3 subtype

Seasonal epidemics caused by influenza A (H1 and H3 subtypes) and B viruses are a major global health threat. The traditional, trivalent influenza vaccines have limited efficacy because of rapid antigenic evolution of the circulating viruses. This antigenic variability mediates viral escape from the host immune responses, necessitating annual vaccine updates. Influenza vaccines elicit a protective antibody response, primarily targeting the viral surface glycoprotein hemagglutinin (HA). However, the predominant humoral response is against the hypervariable head domain of HA, thereby restricting the breadth of protection. In contrast, the conserved, subdominant stem domain of HA is a potential ‘universal’ vaccine candidate. We designed an HA stem-fragment immunogen from the 1968 pandemic H3N2 strain (A/Hong Kong/1/68) guided by a comprehensive H3 HA sequence conservation analysis. The biophysical properties of the designed immunogen were further improved by C-terminal fusion of a trimerization motif, ‘isoleucine-zipper’ or ‘foldon’. These immunogens elicited cross-reactive, antiviral antibodies and conferred partial protection against a lethal, homologous HK68 virus challenge in vivo. Furthermore, bacterial expression of these immunogens is economical and facilitates rapid scale-up

Epidemiology of herpes simplex virus type 1 and 2 in Italy: a seroprevalence study over 15 years

Introduction: Herpes simplex viruses (HSV) are among the most widespread causative agents of human viral infections. HSV-2 is one of the commonest causes of genital disease, while HSV-1 is associated primarily with orolabial ulceration; however, recent changes in HSV epidemiology showed an increase in genital and neonatal herpes particularly caused by HSV-1. The main purpose of this study was to assess the seroprevalence of HSV-1 and HSV-2 in a random population in Siena (central Italy) in 2000, 2005 and 2013-2014 and in Bari (southern Italy) in 2005. Moreover, a preliminary study was conducted to investigate the spread of HSV infection in a population of pregnant women and infants in Bari in 2003, 2004 and 2005. Methods: Human serum samples were tested for the presence of specific anti-HSV-1 and anti-HSV-2 IgG antibodies using a commercially available ELISA test. Results and conclusions: For the primary purpose, seroprevalence rates observed in Siena were compared over the years sampled and with the seroprevalence rate found in Bari. Results of seroprevalence in Siena show a decreased trend for both viruses, especially in adolescents and young adults; moreover, HSV-2 seroprevalence rates found in the two cities suggest geographical differences. For the secondary purpose, prevalence rates among pregnant women were compared with the seroprevalence found in women of the general population. No significant difference in prevalence rates were found among pregnant women, while results indicate both viruses are a source of infection in infants

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

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