Performance comparison of micro-neutralization assays based on surrogate SARS-CoV-2 and WT SARS-CoV-2 in assessing virus-neutralizing capacity of anti-SARS-CoV-2 antibodies.

We compared neutralization assays using either the wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus or surrogate neutralization markers, using characterized sera. We found the results of the neutralization assays 75 % concordant overall and 80 % concordant for samples with high antibody levels. This demonstrates that commercial surrogate SARS-CoV-2 assays offer the potential to assess anti-SARS-CoV-2 antibodies' neutralizing capacity outside CL-3 laboratory containment.

Evaluation of a Commercial Culture-Free Neutralization Antibody Detection Kit for Severe Acute Respiratory Syndrome-Related Coronavirus-2 and Comparison With an Antireceptor-Binding Domain Enzyme-Linked Immunosorbent Assay.

BACKGROUND: Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) surrogate neutralization assays that obviate the need for viral culture offer substantial advantages regarding throughput and cost. The cPass SARS-CoV-2 Neutralization Antibody Detection Kit (GenScript) is the first such commercially available assay that detects antibodies that block receptor-binding domain (RBD)/angiotensin-converting enzyme (ACE)-2 interaction. We aimed to evaluate cPass to inform its use and assess its added value compared with anti-RBD enzyme-linked immunosorbent assays (ELISAs). METHODS: Serum reference panels comprising 205 specimens were used to compare cPass to plaque-reduction neutralization test (PRNT) and a pseudotyped lentiviral neutralization (PLV) assay for detection of neutralizing antibodies. We assessed the correlation of cPass with an ELISA detecting anti-RBD immunoglobulin (Ig)G, IgM, and IgA antibodies at a single timepoint and across intervals from onset of symptoms of SARS-CoV-2 infection. RESULTS: Compared with PRNT-50, cPass sensitivity ranged from 77% to 100% and specificity was 95% to 100%. Sensitivity was also high compared with the pseudotyped lentiviral neutralization assay (93%; 95% confidence interval [CI], 85-97), but specificity was lower (58%; 95% CI, 48-67). Highest agreement between cPass and ELISA was for anti-RBD IgG (r = 0.823). Against the pseudotyped lentiviral neutralization assay, anti-RBD IgG sensitivity (99%; 95% CI, 94-100) was very similar to that of cPass, but overall specificity was lower (37%; 95% CI, 28-47). Against PRNT-50, results of cPass and anti-RBD IgG were nearly identical. CONCLUSIONS: The added value of cPass compared with an IgG anti-RBD ELISA was modest.

SARS-CoV-2 infection and transmission in the North American deer mouse.

Widespread circulation of SARS-CoV-2 in humans raises the theoretical risk of reverse zoonosis events with wildlife, reintroductions of SARS-CoV-2 into permissive nondomesticated animals. Here we report that North American deer mice (Peromyscus maniculatus) are susceptible to SARS-CoV-2 infection following intranasal exposure to a human isolate, resulting in viral replication in the upper and lower respiratory tract with little or no signs of disease. Further, shed infectious virus is detectable in nasal washes, oropharyngeal and rectal swabs, and viral RNA is detectable in feces and occasionally urine. We further show that deer mice are capable of transmitting SARS-CoV-2 to naïve deer mice through direct contact. The extent to which these observations may translate to wild deer mouse populations remains unclear, and the risk of reverse zoonosis and/or the potential for the establishment of Peromyscus rodents as a North American reservoir for SARS-CoV-2 remains unknown.

A trend of dropping anti-SARS-CoV-2 plaque reduction neutralization test titers over time in Canadian convalescent plasma donors.

BACKGROUND: Convalescent plasma products are a potential passive immunotherapy for Coronavirus disease 2019 (COVID-19) disease. Various approaches have been utilized to determine the concentration of Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-neutralizing antibodies in plasma products. The Canadian Blood Services used Plaque Reduction Neutralization Test 50 (PRNT50) -generated values to qualify convalescent plasma donations supporting clinical trials in Canada. This manuscript describes changes in PRNT50 titers of repeat male plasma donations collected approximately 1-4 months after onset of COVID-19 signs and symptoms in donors. STUDY DESIGN AND METHODS: Men were eligible to donate if they: met standard criteria, were < 67 years of age, reported a previous SARS-CoV-2-positive nucleic acid test, and recovered and were symptom free for at least 28 days prior to donation. Repeat donation analysis required at least one original and one repeat donation where a PRNT50 was performed. RESULTS: From April 29, 2020 to July 25, 2020, 156 donors donated once, with 78 (50%) of the donated plasma having PRNT50 titers of ≥1:160. Thirty-seven (23.7%) of the donated plasma had a titer of 1:40 or 1:80 (individuals donating this plasma were asked to donate a second time only). A total of 30 donors (19.2%) had repeat donations. Of the repeat donors, 15 (50%) had at least an eightfold change from peak to trough PRNT50 titers within greater than 90 days after onset of COVID-19 symptoms. CONCLUSIONS: Blood operators cannot infer that SARS-CoV-2 PRNT50 will remain high in repeat plasma donors 3-4 months after onset of COVID-19 symptoms.

Two Detailed Plaque Assay Protocols for the Quantification of Infectious SARS-CoV-2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been identified as the causal agent of COronaVIrus Disease-19 (COVID-19), an atypical pneumonia-like syndrome that emerged in December 2019. While SARS-CoV-2 titers can be measured by detection of viral nucleic acid, this method is unable to quantitate infectious virions. Measurement of infectious SARS-CoV-2 can be achieved by tissue culture infectious dose-50 (TCID50 ), which detects the presence or absence of cytopathic effect in cells infected with serial dilutions of a virus specimen. However, this method only provides a qualitative infectious virus titer. Plaque assays are a quantitative method of measuring infectious SARS-CoV-2 by quantifying the plaques formed in cell culture upon infection with serial dilutions of a virus specimen. As such, plaque assays remain the gold standard in quantifying concentrations of replication-competent lytic virions. Here, we describe two detailed plaque assay protocols to quantify infectious SARS-CoV-2 using different overlay and staining methods. Both methods have several advantages and disadvantages, which can be considered when choosing the procedure best suited for each laboratory. These assays can be used for several research purposes, including titration of virus stocks produced from infected cell supernatant and, with further optimization, quantification of SARS-CoV-2 in specimens collected from infected animals. © 2019 The Authors. Basic Protocol: SARS-CoV-2 plaque assay using a solid double overlay method Alternate Protocol: SARS-CoV-2 plaque assay using a liquid overlay and fixation-staining method.

A Plaque Reduction Neutralization Test for the Detection of ZIKV-Specific Antibodies.

Neutralizing antibodies against Zika virus (ZIKV) are a robust indicator of exposure to ZIKV. Here we describe the plaque reduction neutralization test (PRNT), which quantifies ZIKV-neutralizing antibodies (nAbs) by incubating infectious ZIKV with different dilutions of sera and applying the mixture to a susceptible Vero cell monolayer. After 4 days of incubation, neutral red dye is added to optimize the visualization of plaques caused by ZIKV cytopathic effect. Compared with controls, sera containing nAbs against ZIKV will demonstrate ≥90% reduction in the number of plaques observed. This protocol can be utilized for research applications and serological diagnostic testing with the use of additional controls.

Combining anti-IgM and IgG immunoassays for comprehensive chikungunya virus diagnostic testing.

Chikungunya virus (CHIKV) is a mosquito-borne pathogen that causes CHIKV fever. Definitive diagnosis is crucial for patients experiencing symptoms similar to other arboviral diseases because they can vary in clinical consequences. An increasing number of patients experience long-term rheumatic effects of CHIKV infection, but these cases may not be optimally detected by molecular assays and anti-CHIKV IgM ELISAs (M-ELISAs) used for confirmation and screening, respectively. The subsequent confirmatory serological test, the plaque reduction neutralization test (PRNT), is laborious and time-consuming. In this study, we evaluated a new diagnostic algorithm in which the M-ELISA is conducted in parallel with an anti-CHIKV IgG ELISA (G-ELISA) and observed that the Euroimmun M-ELISA combined with the Euroimmun G-ELISA or the Abcam G-ELISA exhibited excellent sensitivity and specificity for CHIKV. The combinations demonstrated perfect and near perfect inter-rater agreement with the PRNT, respectively, suggesting their potential to be used as alternatives to the confirmatory serological PRNT assay for CHIKV.

Incorporation of Ebola glycoprotein into HIV particles facilitates dendritic cell and macrophage targeting and enhances HIV-specific immune responses.

The development of an effective vaccine against HIV infection remains a global priority. Dendritic cell (DC)-based HIV immunotherapeutic vaccine is a promising approach which aims at optimizing the HIV-specific immune response using primed DCs to promote and enhance both the cellular and humoral arms of immunity. Since the Ebola virus envelope glycoprotein (EboGP) has strong DC-targeting ability, we investigated whether EboGP is able to direct HIV particles towards DCs efficiently and promote potent HIV-specific immune responses. Our results indicate that the incorporation of EboGP into non-replicating virus-like particles (VLPs) enhances their ability to target human monocyte-derived dendritic cells (MDDCs) and monocyte-derived macrophages (MDMs). Also, a mucin-like domain deleted EboGP (EboGPΔM) can further enhanced the MDDCs and MDMs-targeting ability. Furthermore, we investigated the effect of EboGP on HIV immunogenicity in mice, and the results revealed a significantly stronger HIV-specific humoral immune response when immunized with EboGP-pseudotyped HIV VLPs compared with those immunized with HIV VLPs. Splenocytes harvested from mice immunized with EboGP-pseudotyped HIV VLPs secreted increased levels of macrophage inflammatory proteins-1α (MIP-1α) and IL-4 upon stimulation with HIV Env and/or Gag peptides compared with those harvested from mice immunized with HIV VLPs. Collectively, this study provides evidence for the first time that the incorporation of EboGP in HIV VLPs can facilitate DC and macrophage targeting and induce more potent immune responses against HIV.

Establishment of a comprehensive and high throughput serological algorithm for Zika virus diagnostic testing.

The previous serological algorithm for Zika virus (ZIKV) comprised screening by anti-ZIKV IgM capture ELISA (MAC-ELISA) for samples collected within 3 months postexposure or onset (MPEO). Samples positive by MAC-ELISA and samples collected beyond 3 MPEO were tested by the confirmatory plaque reduction neutralization test (PRNT), which proved laborious and time-consuming during the 2015 outbreak. Thus, we evaluated several ZIKV ELISAs to establish an anti-IgM and anti-IgG combination for use as a screening tool for all samples prior to PRNT confirmation. The MAC-ELISA or InBios-M in combination with the Euroimmun-G demonstrated sensitivities of 99.1% and 97.2%, respectively, and nonflavivirus specificity of 96.0%. Their cross-reactivities were 71.4% and 50.0%, respectively, for sera positive for Dengue virus antibodies. Due to near-perfect interrater agreement with PRNT and excellent detection of samples collected beyond 3 MPEO, these combinations were recommended as a screening protocol in a new high-throughput algorithm with special considerations for ZIKV diagnostics.

Baited vaccines: A strategy to mitigate rodent-borne viral zoonoses in humans.

Rodents serve as the natural reservoir and vector for a variety of pathogens, some of which are responsible for severe and life-threatening disease in humans. Despite the significant impact in humans many of these viruses, including Old and New World hantaviruses as well as Arenaviruses, most have no specific vaccine or therapeutic to treat or prevent human infection. The recent success of wildlife vaccines to mitigate rabies in animal populations offers interesting insight into the use of similar strategies for other zoonotic agents of human disease. In this review, we discuss the notion of using baited vaccines as a means to interrupt the transmission of viral pathogens between rodent reservoirs and to susceptible human hosts.

Evaluation of the Diasorin Liaison® XL Zika Capture IgM CMIA for Zika virus serological testing.

Due to the increase of Zika virus (ZIKV) transmission throughout the world, many commercial kits have recently become available to aid in laboratory diagnosis of ZIKV infections in clinical samples. Here, we analyze the fully automated Liaison® XL Zika Capture immunoglobulin M (IgM) assay against the recommended IgM-capture enzyme-linked immunosorbent assay.

From bench to almost bedside: the long road to a licensed Ebola virus vaccine.

INTRODUCTION: The Ebola virus (EBOV) disease epidemic during 2014-16 in West Africa has accelerated the clinical development of several vaccine candidates that have demonstrated efficacy in the gold standard nonhuman primate (NHP) model, namely cynomolgus macaques. AREAS COVERED: This review discusses the pre-clinical research and if available, clinical evaluation of the currently available EBOV vaccine candidates, while emphasizing the translatability of pre-clinical data generated in the NHP model to clinical data in humans. EXPERT OPINION: Despite the existence of many successful EBOV vaccine candidates in the pre-clinical stages, only two platforms became the focus of Phase 2/3 efficacy trials in Liberia, Sierra Leone, and Guinea near the peak of the epidemic: the Vesicular stomatitis virus (VSV)-vectored vaccine and the chimpanzee adenovirus type 3 (ChAd3)-vectored vaccine. The results of three distinct clinical trials involving these candidates may soon pave the way for a licensed, safe and efficacious EBOV vaccine to help combat future epidemics.

Evaluation of 5 Commercially Available Zika Virus Immunoassays.

Because of the global spread of Zika virus, accurate and high-throughput diagnostic immunoassays are needed. We compared the sensitivity and specificity of 5 commercially available Zika virus serologic assays to the recommended protocol of Zika virus IgM-capture ELISA and plaque-reduction neutralization tests. Most commercial immunoassays showed low sensitivity, which can be increased.

The ongoing evolution of antibody-based treatments for Ebola virus infection.

The 2014-2016 Ebola virus outbreak in West Africa was the deadliest in history, prompting the evaluation of various drug candidates, including antibody-based therapeutics for the treatment of Ebola hemorrhagic fever (EHF). Prior to 2014, only convalescent blood products from EHF survivors had been administered to newly infected individuals as a form of treatment. However, during the recent outbreak, monoclonal antibody cocktails such as ZMapp, ZMAb and MB-003 were either tested in a human clinical safety and efficacy trial or provided to some based on compassionate grounds. This review aims to discuss the evolution of antibody-based treatments for EHF, their clinical trial efficacy and the development of new antibody-based therapies currently advancing in preclinical testing.

Avian influenza viruses that cause highly virulent infections in humans exhibit distinct replicative properties in contrast to human H1N1 viruses.

Avian influenza viruses present an emerging epidemiological concern as some strains of H5N1 avian influenza can cause severe infections in humans with lethality rates of up to 60%. These have been in circulation since 1997 and recently a novel H7N9-subtyped virus has been causing epizootics in China with lethality rates around 20%. To better understand the replication kinetics of these viruses, we combined several extensive viral kinetics experiments with mathematical modelling of in vitro infections in human A549 cells. We extracted fundamental replication parameters revealing that, while both the H5N1 and H7N9 viruses replicate faster and to higher titers than two low-pathogenicity H1N1 strains, they accomplish this via different mechanisms. While the H7N9 virions exhibit a faster rate of infection, the H5N1 virions are produced at a higher rate. Of the two H1N1 strains studied, the 2009 pandemic H1N1 strain exhibits the longest eclipse phase, possibly indicative of a less effective neuraminidase activity, but causes infection more rapidly than the seasonal strain. This explains, in part, the pandemic strain's generally slower growth kinetics and permissiveness to accept mutations causing neuraminidase inhibitor resistance without significant loss in fitness. Our results highlight differential growth properties of H1N1, H5N1 and H7N9 influenza viruses.

Progression of Ebola Therapeutics During the 2014-2015 Outbreak.

The recent Ebola virus (EBOV) outbreak in West Africa was the deadliest EBOV epidemic in history, highlighting the need for a safe and efficacious treatment against EBOV disease (EVD). In the absence of an approved treatment, experimental drugs were utilized under compassionate grounds hoping to diminish EVD-associated morbidity and mortality. As more data were collected from safety studies, Phase II/III clinical trials were introduced in Guinea, Sierra Leone, and Liberia to test promising candidates, including small-molecule drugs, RNA-based treatments, and antibody-based therapies. In this review, we summarize the use of, and preliminary observations from, current clinical trials with EVD therapeutics, shedding light on experimental drug selection, emergency clinical evaluation, and the impact these factors may have on future infectious disease outbreaks.