Development and Evaluation of a Molecular Hepatitis A Virus Assay for Serum and Stool Specimens

Hepatitis A virus (HAV) is an emerging public health concern and there is an urgent need for ways to rapidly identify cases so that outbreaks can be managed effectively. Conventional testing for HAV relies on anti-HAV IgM seropositivity. However, studies estimate that 10–30% of patients may not be diagnosed by serology. Molecular assays that can directly detect viral nucleic acids have the potential to improve diagnosis, which is key to prevent the spread of infections. In this study, we developed a real-time PCR (RT-PCR) assay to detect HAV RNA for the identification of acute HAV infection. Primers were designed to target the conserved 5′-untranslated region (5′-UTR) of HAV, and the assay was optimized on both the Qiagen Rotor-Gene and the BD MAX. We successfully detected HAV from patient serum and stool samples with moderate differences in sensitivity and specificity depending on the platform used. Our results highlight the clinical utility of using a molecular assay to detect HAV from various specimen types that can be implemented in hospitals to assist with diagnostics, treatment and prevention.

An ultrapotent neutralizing bispecific antibody with broad spectrum against SARS-CoV-2 variants

In spite of the successful development of effective countermeasures against Covid-19, variants have and will continue to emerge that could compromise the efficacy of currently approved neutralizing antibodies and vaccines. Consequently, novel and more efficacious agents are urgently needed. We have developed a bispecific antibody, 2022, consisting of two antibodies, 2F8 and VHH18. 2F8 was isolated from our proprietary fully synthetic human IDEAL (Intelligently Designed and Engineered Antibody Library)-VH/VL library and VHH18 is a single domain antibody isolated from IDEAL-nanobody library. 2022 was constructed by attaching VHH18 to the C-terminal of Fc of 2F8. 2022 binds two non-overlapping epitopes simultaneously on the RBD of the SARS-CoV-2 spike protein and blocks the binding of RBD to human angiotensin-converting enzyme 2 (ACE2). 2022 potently neutralizes SARS-CoV-2 and all of the variants tested in both pseudovirus and live virus assays, including variants carrying mutations known to resist neutralizing antibodies approved under EUA and that reduce the protection efficiency of current effective vaccines. The half-maximum inhibitory concentration (IC50) of 2022 is 270 pM, 30 pM, 20 pM, and 1 pM, for wildtype, alpha, beta, and delta pseudovirus, respectively. In the live virus assay, 2022 has an IC50 of 30 pM, 13 pM, and 90 pM, for wildtype, beta, and delta live virus, respectively. In a mouse model of SARS-CoV-2, 2022 showed strong prophylactic and therapeutic effects. A single administration of 2022 intranasal (i.n.) or intraperitoneal (i.p.) 24 hours before virus challenge completely protected all mice from bodyweight loss, as compared with up to 20% loss of bodyweight in placebo treated mice. In addition, the lung viral titers were undetectable (FRNT assay) in all mice treated with 2022 either prophylactically or therapeutically, as compared with around 1X105 pfu/g lung tissue in placebo treated mice. In summary, bispecific antibody 2022 showed potent binding and neutralizing activity across a variety of SARS-CoV-2 variants and could be an attractive weapon to combat the ongoing waves of the COVID-19 pandemic propagated mainly by variants, especially, the much more contagious delta variant.

Evaluation of cell-based and surrogate SARS-CoV-2 neutralization assays

Determinants of protective immunity against SARS-CoV-2 infection require the development of well-standardized, reproducible antibody assays. This need has led to the emergence of a variety of neutralization assays. Head-to-head evaluation of different SARS-CoV-2 neutralization platforms could facilitate comparisons across studies and laboratories. Five neutralization assays were compared using forty plasma samples from convalescent individuals with mild-to-moderate COVID-19: four cell-based systems using either live recombinant SARS-CoV-2 or pseudotyped viral particles created with lentivirus (LV) or vesicular stomatitis virus (VSV) packaging and one surrogate ELISA-based test that measures inhibition of the spike protein receptor binding domain (RBD) binding its receptor, human angiotensin converting enzyme 2 (hACE2). Vero, Vero E6, HEK293T expressing hACE2, and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 were tested. All cell-based assays showed 50% neutralizing dilution (ND50) geometric mean titers (GMTs) that were highly correlated (Pearson r = 0.81–0.89) and ranged within 3.4-fold. The live-virus assay and LV-pseudovirus assays with HEK293T/hACE2 cells showed very similar mean titers: 141 and 178, respectively. ND50 titers positively correlated with plasma IgG targeting SARS-CoV-2 spike and RBD ( r = 0.63–0.89), but moderately correlated with nucleoprotein IgG ( r = 0.46–0.73). ND80 GMTs mirrored ND50 data and showed similar correlation between assays and with IgG concentrations. The VSV-pseudovirus assay and LV-pseudovirus assay with HEK293T/hACE2 cells in low and high-throughput versions were calibrated against the WHO SARS-CoV-2 IgG standard. High concordance between the outcomes of cell-based assays with live and pseudotyped virions enables valid cross-study comparison using these platforms. 249

Rapid, reliable, and reproducible cell fusion assay to quantify SARS-Cov-2 spike interaction with hACE2

COVID-19 is a global crisis of unimagined dimensions. Currently, Remedesivir is only fully licensed FDA therapeutic. A major target of the vaccine effort is the SARS-CoV-2 spike-hACE2 interaction, and assessment of efficacy relies on time consuming neutralization assay. Here, we developed a cell fusion assay based upon spike-hACE2 interaction. The system was tested by transient co-transfection of 293T cells, which demonstrated good correlation with standard spike pseudotyping for inhibition by sera and biologics. Then established stable cell lines were very well behaved and gave even better correlation with pseudotyping results, after a short, overnight co-incubation. Results with the stable cell fusion assay also correlated well with those of a live virus assay. In summary we have established a rapid, reliable, and reproducible cell fusion assay that will serve to complement the other neutralization assays currently in use, is easy to implement in most laboratories, and may serve as the basis for high throughput screens to identify inhibitors of SARS-CoV-2 virus-cell binding and entry.

Infection and vaccine-induced neutralizing antibody responses to the SARS-CoV-2 B.1.617.1 variant

SARS-CoV-2 has caused a devastating global pandemic. The recent emergence of SARS-CoV-2 variants that are less sensitive to neutralization by convalescent sera or vaccine-induced neutralizing antibody responses has raised concerns. A second wave of SARS-CoV-2 infections in India is leading to the expansion of SARS-CoV-2 variants. The B.1.617.1 variant has rapidly spread throughout India and to several countries throughout the world. In this study, using a live virus assay, we describe the neutralizing antibody response to the B.1.617.1 variant in serum from infected and vaccinated individuals. We found that the B.1.617.1 variant is 6.8-fold more resistant to neutralization by sera from COVID-19 convalescent and Moderna and Pfizer vaccinated individuals. Despite this, a majority of the sera from convalescent individuals and all sera from vaccinated individuals were still able to neutralize the B.1.617.1 variant. This suggests that protective immunity by the mRNA vaccines tested here are likely retained against the B.1.617.1 variant. As the B.1.617.1 variant continues to evolve, it will be important to monitor how additional mutations within the spike impact antibody resistance, viral transmission and vaccine efficacy.

Single-virus content mixing assay reveals cholesterol-enhanced influenza membrane fusion efficiency

In order to infect a cell, enveloped viruses must first undergo membrane fusion, which proceeds through a hemifusion intermediate, followed by the formation of a fusion pore through which the viral genome is transferred to a target cell. Single-virus fusion studies to elucidate the dynamics of content mixing typically require extensive fluorescent labeling of viral contents. The labeling process must be optimized depending on the virus identity and strain and can potentially be perturbative to viral fusion behavior. Here, we introduce a single-virus assay where content-labeled vesicles are bound to unlabeled influenza A virus (IAV) to eliminate the problematic step of content-labeling virions. We use fluorescence microscopy to observe individual, pH-triggered content mixing and content loss events between IAV and target vesicles of varying cholesterol compositions. We show that target membrane cholesterol increases the efficiency of IAV content mixing and decreases the fraction of content mixing events that result in content loss. These results are consistent with previous findings that cholesterol stabilizes pore formation in IAV entry and limits leakage following pore formation. We also show that content loss due to hemagglutinin fusion peptide engagement with the target membrane is independent of composition. This approach is a promising strategy for studying the single-virus content mixing kinetics of other enveloped viruses.

SARS-CoV-2 Antibody persistence in COVID-19 convalescent plasma donors

Background Antibody response duration following SARS-CoV-2 infection tends to be variable and depends on severity of disease and method of detection. Study design and methods COVID-19 convalescent plasma (CCP) from 18 donors was collected longitudinally for a maximum of 63 - 129 days following resolution of symptoms. All the samples were initially screened by the Ortho Total Ig test to confirm positivity and subsequently tested with 7 additional direct sandwich or indirect binding assays (Ortho, Roche, Abbott, Broad Institute) directed against a variety of antigen targets (S1, RBD, and NC), along with 2 neutralization assays (Broad Institute live virus PRNT and Vitalant Research Institute Pseudovirus RVPN). Results The direct detection assays (Ortho Total Ig total and Roche Total Ig) showed increasing levels of antibodies over the time period, in contrast to the indirect IgG assays that showed a decline. Neutralization assays also demonstrated declining responses; the VRI RVPN pseudovirus had a greater rate of decline than the Broad PRNT live virus assay. Discussion These data show that in addition to variable individual responses and associations with disease severity, the detection assay chosen contributes to the heterogeneous results in antibody stability over time. Depending on the scope of the research, one assay may be preferable over another. For serosurveillance studies, direct, double Ag-sandwich assays appear to be the best choice due to their stability; in particular, algorithms that include both S1 and NC based assays can help reduce the rate of false-positivity and discriminate between natural infection and vaccine-derived seroreactivity.

Metadichol®, a Novel Nano Lipid Formulation that Inhibits SARS-COV-2 and a Multitude of Pathological Viruses in vitro

New pathogenic virus outbreaks, occurring with increasing regularity, are leading us to explore novel approaches, which will reduce the reliance on time-consuming vaccine modes to halt the outbreaks. The requirement is to find a universal approach to disarm any new and as yet unknown viruses as they appear. A promising approach could be targeting lipid membranes, which are common to all viruses and bacteria.The ongoing pandemic of severe acute respiratory syndrome-coronavirus 2 (SARS-COV-2) has reaffirmed the importance of interactions between components of the host cell plasma membrane and the virus envelope as a critical mechanism of infection. Metadichol®, a nano lipid emulsion, has been examined and shown to be a strong candidate to help stop the proliferation of SARS-COV-2.Naturally derived substances, such as long-chain saturated lipid alcohols, reduce the infectivity of various types of viruses, including coronaviruses such as SARS-COV-2, by modifying lipid-dependent attachment to human host cells. SARS-COV-2 uses the receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming.Metadichol®, a nano lipid formulation of long-chain alcohols, has been shown to inhibit TMPRSS2 (EC50 96 ng/ml). Compared to the inhibitor Camostat mesylate (CM) which has a EC50 of 26000 ng/ml, it is 270 times more potent. Additionally, Metadichol® is an inhibitor of ACE with an EC 50 of 71 ng/ml but extremely weak inhibitor of ACE2 at 31 µg/ml. Further a live virus assay in Caco2 cells, Metadichol® inhibited SARS-CoV-2 replication with an EC90 of 0.16 µg/ml.