Studying longitudinal neutralising antibody levels against Equid herpesvirus 1 in experimentally infected horses using a novel pseudotype based assay

Infection with equid herpesvirus 1 (EHV-1), a DNA virus of the Herpesviridae family represents a significant welfare issue in horses and a great impact on the equine industry. During EHV-1 infection, entry of the virus into different cell types is complex due to the presence of twelve glycoproteins (GPs) on the viral envelope. To investigate virus entry mechanisms, specific combinations of GPs were pseudotyped onto lentiviral vectors. Pseudotyped virus (PV) particles bearing gB, gD, gH and gL were able to transduce several target cell lines (HEK293T/17, RK13, CHO-K1, FHK-Tcl3, MDCK I & II), demonstrating that these four EHV-1 glycoproteins are both essential and sufficient for cell entry. The successful generation of an EHV-1 PV permitted development of a PV neutralisation assay (PVNA). The efficacy of the PVNA was tested by measuring the level of neutralising serum antibodies from EHV-1 experimentally infected horses (n = 52) sampled in a longitudinal manner. The same sera were assessed using a conventional EHV-1 virus neutralisation (VN) assay, exhibiting a strong correlation (r = 0.82) between the two assays. Furthermore, PVs routinely require -80 ◦C for long term storage and a dry ice cold-chain during transport, which can impede dissemination and utilisation in other stakeholder laboratories. Consequently, lyophilisation of EHV-1 PVs was conducted to address this issue. PVs were lyophilised and pellets either reconstituted immediately or stored under various temperature conditions for different time periods. The recovery and functionality of these lyophilised PVs was compared with standard frozen aliquots in titration and neutralisation tests. Results indicated that lyophilisation could be used to stably preserve such complex herpesvirus pseudotypes, even after weeks of storage at room temperature, and that reconstituted EHV-1 PVs could be successfully employed in antibody neutralisation tests

Use of Equine Herpesvirus 1 glycoprotein pseudotyped lentiviral particles for the development of serological tests and assessment of lyophilisation for transport and storage

Equine herpesviruses (EHVs) are enveloped DNA viruses predominantly infecting members of the Equidae family. EHVs primarily cause respiratory disease, however EHV-1 can produce cases of a neurological disease, abortion and neonatal death. Thus, these viruses represent a welfare issue for the equine industry and scientific focus for researchers. EHV-1 exhibits a complex array of 12 glycoproteins on its surface envelope, but it is unclear precisely which are important for virus cell entry and the role of each in host immune response. In order to investigate the contribution of these glycoproteins, pseudotype viruses (PVs) could provide a useful study tool. We have successfully generated functional EHV-1 pseudotyped lentiviruses bearing four glycoproteins, gB, gD, gH and gL (sequences derived from an aborted foetus during a large EHV1 outbreak strain in Normandy, France). PVs were employed in a pseudotype virus neutralisation test (PVNT) to measure levels of specific neutralising antibodies serum samples (n=52) taken longitudinally from experimentally infected ponies, compared with uninfected controls. PVs routinely require -80oC for long term storage and a dry ice cold-chain during transport which can impede dissemination and utilisation in other laboratories. Consequently, we further investigated whether freeze-drying (lyophilisation) of EHV-1 PV could address this issue. PVs were lyophilised and pellets either reconstituted immediately or stored under various temperature conditions, sampling at different timepoints. The recovery and functionality of these lyophilised PVs was compared with standard frozen aliquots in titration and neutralisation tests

Comparison of lentiviral and vesicular stomatitis virus core SARS-CoV-2 pseudotypes and generation of a stable cell line for use in antibody neutralisation assays

Betacoronavirus SARS-CoV-2, the causative agent of COVID19, is a single stranded positive sense RNA virus. Since its emergence there has been great efforts to identify correlates of protection,which is crucial for vaccine evaluation studies. However, handling SARS-CoV-2 requires BSL-3 containment facilities slowing research efforts. Pseudotype viruses (PV) are a safe alternative to authentic virus that can be handled at low containment. PVs are chimeric viruses containing the core of a virus where its genome has been completely or partially replaced by a reporter gene, displaying a correctly folded SARS-CoV-2 spike on its surface. We developed lentiviral and vesicular stomatitis virus (VSV) core PVs alongside a stable A549 cell line expressing receptor ACE2 and protease TMPRSS2 responsible for S protein priming, for use in neutralization assays. Lentiviral PVs were generated by transfection with plasmids encoding the spike, HIV-1 gag-pol and a luciferase reporter. For VSV PVs, producer cells pre-transfected with the spike were infected with recombinant VSV expressing luciferase,before harvesting. The stable A549 cell line was generated by sequential infection of VSV-G PVs bearing lentiviral vectors encoding ACE2 and TMPRSS2 genes followed by antibiotic selection, before being tested in neutralization assays. We compared lentiviral and VSV PV platforms using monoclonal antibodies and convalescent sera with our stable A549 cells or HEK293T cells pre-transfected with plasmids encoding ACE2 and TMPRSS2. Antibody titres showed equivalence however VSV had the advantage of a shorter incubation therefore enabling a higher throughput. PVs offer a robust platform for future seroepidemiology and vaccine evaluation studies

Genetic characterization of a new candidate hemagglutinin subtype of influenza A viruses

Avian influenza viruses (AIV) have been classified on the basis of 16 subtypes of hemagglutinin (HA) and 9 subtypes of neuraminidase. Here we describe genomic evidence for a new candidate HA subtype, nominally H19, with a large genetic distance to all previously described AIV subtypes, derived from a cloacal swab sample of a Common Pochard (Aythya ferina) in Kazakhstan, in 2008. Avian influenza monitoring in wild birds especially in migratory hotspots such as central Asia is an important approach to gain information about the circulation of known and novel influenza viruses. Genetically, the novel HA coding sequence exhibits only 68.2% nucleotide and 68.5% amino acid identity with its nearest relation in the H9 (N2) subtype. The new HA sequence should be considered in current genomic diagnostic AI assays to facilitate its detection and eventual isolation enabling further study and antigenic classification

Coronavirus Pseudotypes for All Circulating Human Coronaviruses for Quantification of Cross-Neutralizing Antibody Responses.

The novel coronavirus SARS-CoV-2 is the seventh identified human coronavirus. Understanding the extent of pre-existing immunity induced by seropositivity to endemic seasonal coronaviruses and the impact of cross-reactivity on COVID-19 disease progression remains a key research question in immunity to SARS-CoV-2 and the immunopathology of COVID-2019 disease. This paper describes a panel of lentiviral pseudotypes bearing the spike (S) proteins for each of the seven human coronaviruses (HCoVs), generated under similar conditions optimized for high titre production allowing a high-throughput investigation of antibody neutralization breadth. Optimal production conditions and most readily available permissive target cell lines were determined for spike-mediated entry by each HCoV pseudotype: SARS-CoV-1, SARS-CoV-2 and HCoV-NL63 best transduced HEK293T/17 cells transfected with ACE2 and TMPRSS2, HCoV-229E and MERS-CoV preferentially entered HUH7 cells, and CHO cells were most permissive for the seasonal betacoronavirus HCoV-HKU1. Entry of ACE2 using pseudotypes was enhanced by ACE2 and TMPRSS2 expression in target cells, whilst TMPRSS2 transfection rendered HEK293T/17 cells permissive for HCoV-HKU1 and HCoV-OC43 entry. Additionally, pseudotype viruses were produced bearing additional coronavirus surface proteins, including the SARS-CoV-2 Envelope (E) and Membrane (M) proteins and HCoV-OC43/HCoV-HKU1 Haemagglutinin-Esterase (HE) proteins. This panel of lentiviral pseudotypes provides a safe, rapidly quantifiable and high-throughput tool for serological comparison of pan-coronavirus neutralizing responses; this can be used to elucidate antibody dynamics against individual coronaviruses and the effects of antibody cross-reactivity on clinical outcome following natural infection or vaccination

Analysis of Antibody Neutralisation Activity against SARS-CoV-2 Variants and Seasonal Human Coronaviruses NL63, HKU1, and 229E Induced by Three Different COVID-19 Vaccine Platforms

Coronaviruses infections, culminating in the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic beginning in 2019, have highlighted the importance of effective vaccines to induce an antibody response with cross-neutralizing activity. COVID-19 vaccines have been rapidly developed to reduce the burden of SARS-CoV-2 infections and disease severity. Cross-protection from seasonal human coronaviruses (hCoVs) infections has been hypothesized but is still controversial. Here, we investigated the neutralizing activity against ancestral SARS-CoV-2 and the variants of concern (VOCs) in individuals vaccinated with two doses of either BNT162b2, mRNA-1273, or AZD1222, with or without a history of SARS-CoV-2 infection. Antibody neutralizing activity to SARS-CoV-2 and the VOCs was higher in BNT162b2-vaccinated subjects who were previously infected with SARS-CoV-2 and conferred broad-spectrum protection. The Omicron BA.1 variant was the most resistant among the VOCs. COVID-19 vaccination did not confer protection against hCoV-HKU1. Conversely, antibodies induced by mRNA-1273 vaccination displayed a boosting in their neutralizing activity against hCoV-NL63, whereas AZD1222 vaccination increased antibody neutralization against hCoV-229E, suggesting potential differences in antigenicity and immunogenicity of the different spike constructs used between various vaccination platforms. These data would suggest that there may be shared epitopes between the HCoVs and SARS-CoV-2 spike proteins

Production, titration, neutralisation and storage of SARS-CoV-2 lentiviral pseudotypes

This protocol details a rapid and reliable method for the production and titration of high-titer viral pseudotype particles with the SARS-CoV-2 spike protein (and D614G variant) on a lentiviral vector core, and use for neutralization assays in target cells expressing ACE2 and TMPRSS2. It additionally provides detailed instruction on substituting in new spike variants via gene cloning, and storage/shipping considerations for wide deployment potential. Results obtained with this protocol show that SARS-CoV-2 pseudotypes can be produced at equivalent titers to SARS-CoV and MERS-CoV pseudotypes, that they can be neutralized by human convalescent plasma and monoclonal antibodies and that they can be stored at a range of laboratory temperatures and lyophilized for distribution

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Blood transcriptional biomarkers of acute viral infection for detection of pre-symptomatic SARS-CoV-2 infection: a nested, case-control diagnostic accuracy study

Background We hypothesised that host-response biomarkers of viral infections might contribute to early identification of individuals infected with SARS-CoV-2, which is critical to breaking the chains of transmission. We aimed to evaluate the diagnostic accuracy of existing candidate whole-blood transcriptomic signatures for viral infection to predict positivity of nasopharyngeal SARS-CoV-2 PCR testing.Methods We did a nested case-control diagnostic accuracy study among a prospective cohort of health-care workers (aged ≥18 years) at St Bartholomew’s Hospital (London, UK) undergoing weekly blood and nasopharyngeal swab sampling for whole-blood RNA sequencing and SARS-CoV-2 PCR testing, when fit to attend work. We identified candidate blood transcriptomic signatures for viral infection through a systematic literature search. We searched MEDLINE for articles published between database inception and Oct 12, 2020, using comprehensive MeSH and keyword terms for “viral infection”, “transcriptome”, “biomarker”, and “blood”. We reconstructed signature scores in blood RNA sequencing data and evaluated their diagnostic accuracy for contemporaneous SARS-CoV-2 infection, compared with the gold standard of SARS-CoV-2 PCR testing, by quantifying the area under the receiver operating characteristic curve (AUROC), sensitivities, and specificities at a standardised Z score of at least 2 based on the distribution of signature scores in test-negative controls. We used pairwise DeLong tests compared with the most discriminating signature to identify the subset of best performing biomarkers. We evaluated associations between signature expression, viral load (using PCR cycle thresholds), and symptom status visually and using Spearman rank correlation. The primary outcome was the AUROC for discriminating between samples from participants who tested negative throughout the study (test-negative controls) and samples from participants with PCR-confirmed SARS-CoV-2 infection (test-positive participants) during their first week of PCR positivity.Findings We identified 20 candidate blood transcriptomic signatures of viral infection from 18 studies and evaluated their accuracy among 169 blood RNA samples from 96 participants over 24 weeks. Participants were recruited between March 23 and March 31, 2020. 114 samples were from 41 participants with SARS-CoV-2 infection, and 55 samples were from 55 test-negative controls. The median age of participants was 36 years (IQR 27–47) and 69 (72%) of 96 were women. Signatures had little overlap of component genes, but were mostly correlated as components of type I interferon responses. A single blood transcript for IFI27 provided the highest accuracy for discriminating between test-negative controls and test-positive individuals at the time of their first positive SARS-CoV-2 PCR result, with AUROC of 0·95 (95% CI 0·91–0·99), sensitivity 0·84 (0·70–0·93), and specificity 0·95 (0·85–0·98) at a predefined threshold (Z score >2). The transcript performed equally well in individuals with and without symptoms. Three other candidate signatures (including two to 48 transcripts) had statistically equivalent discrimination to IFI27 (AUROCs 0·91–0·95).Interpretation Our findings support further urgent evaluation and development of blood IFI27 transcripts as a biomarker for early phase SARS-CoV-2 infection for screening individuals at high risk of infection, such as contacts of index cases, to facilitate early case isolation and early use of antiviral treatments as they emerge