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1.
Cell Rep Methods ; 2(7): 100252, 2022 Jul 18.
Article in English | MEDLINE | ID: covidwho-1894918

ABSTRACT

Efficient quantitative assays for measurement of viral replication and infectivity are indispensable for future endeavors to develop prophylactic or therapeutic antiviral drugs or vaccines against SARS-CoV-2. We developed a SARS-CoV-2 cell-cell transmission assay that provides a rapid and quantitative readout to assess SARS-CoV-2 spike hACE2 interaction in the absence of pseudotyped particles or live virus. We established two well-behaved stable cell lines, which demonstrated a remarkable correlation with standard cell-free viral pseudotyping for inhibition by convalescent sera, small-molecule drugs, and murine anti-spike monoclonal antibodies. The assay is rapid, reliable, and highly reproducible, without a requirement for any specialized research reagents or laboratory equipment and should be easy to adapt for use in most investigative and clinical settings. It can be effectively used or modified for high-throughput screening for compounds and biologics that interfere with virus-cell binding and entry to complement other neutralization assays currently in use.

2.
Clin Infect Dis ; 2022 May 26.
Article in English | MEDLINE | ID: covidwho-1873881

ABSTRACT

BACKGROUND: We studied whether comorbid conditions impact strength and duration of immune responses after SARS-CoV-2 mRNA vaccination in a US-based, adult population. METHODS: Sera (pre-and-post-BNT162b2 vaccination) were tested serially up to 12 months after two doses of vaccine for SARS-CoV-2-anti-Spike neutralizing capacity by pseudotyping assay in 124 individuals; neutralizing titers were correlated to clinical variables with multivariate regression. Post-booster (third dose) effect was measured at 1 and 3 months in 72 and 88 subjects respectively. RESULTS: After completion of primary vaccine series, neutralizing antibody IC50 values were high at one month (14-fold increase from pre-vaccination), declined at six months (3.3-fold increase), and increased at one month post-booster (41.5-fold increase). Three months post-booster, IC50 decreased in COVID-naïve individuals (18-fold increase) and increased in prior COVID-19 + individuals (132-fold increase). Age >65 years (ß=-0.94, p = 0.001) and malignancy (ß=-0.88, p = 0.002) reduced strength of response at 1 month. Both neutralization strength and durability at 6 months, respectively, were negatively impacted by end-stage renal disease [(ß=-1.10, p = 0.004); (ß=-0.66, p = 0.014)], diabetes mellitus [(ß=-0.57, p = 0.032); (ß=-0.44, p = 0.028)], and systemic steroid use [(ß=-0.066, p = 0.032); (ß=-0.55, p = 0.037)]. Post-booster IC50 was robust against WA-1 and B.1.617.2. Post-booster neutralization increased with prior COVID-19 (ß = 2.9, p-value < 0.0001), and malignancy reduced neutralization response (ß=-0.68, p = 0.03), regardless of infection status. CONCLUSION: Multiple clinical factors impact the strength and duration of neutralization response post-primary series vaccination, but not the post-booster dose strength. Malignancy was associated with lower booster-dose response regardless of prior COVID infection, suggesting a need for clinically guided vaccine regimens.

3.
mBio ; 13(2): e0370521, 2022 04 26.
Article in English | MEDLINE | ID: covidwho-1714363

ABSTRACT

Combinations of direct-acting antivirals are needed to minimize drug resistance mutations and stably suppress replication of RNA viruses. Currently, there are limited therapeutic options against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and testing of a number of drug regimens has led to conflicting results. Here, we show that cobicistat, which is an FDA-approved drug booster that blocks the activity of the drug-metabolizing proteins cytochrome P450-3As (CYP3As) and P-glycoprotein (P-gp), inhibits SARS-CoV-2 replication. Two independent cell-to-cell membrane fusion assays showed that the antiviral effect of cobicistat is exerted through inhibition of spike protein-mediated membrane fusion. In line with this, incubation with low-micromolar concentrations of cobicistat decreased viral replication in three different cell lines including cells of lung and gut origin. When cobicistat was used in combination with remdesivir, a synergistic effect on the inhibition of viral replication was observed in cell lines and in a primary human colon organoid. This was consistent with the effects of cobicistat on two of its known targets, CYP3A4 and P-gp, the silencing of which boosted the in vitro antiviral activity of remdesivir in a cobicistat-like manner. When administered in vivo to Syrian hamsters at a high dose, cobicistat decreased viral load and mitigated clinical progression. These data highlight cobicistat as a therapeutic candidate for treating SARS-CoV-2 infection and as a potential building block of combination therapies for COVID-19. IMPORTANCE The lack of effective antiviral treatments against SARS-CoV-2 is a significant limitation in the fight against the COVID-19 pandemic. Single-drug regimens have so far yielded limited results, indicating that combinations of antivirals might be required, as previously seen for other RNA viruses. Our work introduces the drug booster cobicistat, which is approved by the FDA and typically used to potentiate the effect of anti-HIV protease inhibitors, as a candidate inhibitor of SARS-CoV-2 replication. Beyond its direct activity as an antiviral, we show that cobicistat can enhance the effect of remdesivir, which was one of the first drugs proposed for treatment of SARS-CoV-2. Overall, the dual action of cobicistat as a direct antiviral and a drug booster can provide a new approach to design combination therapies and rescue the activity of compounds that are only partially effective in monotherapy.


Subject(s)
COVID-19 , Hepatitis C, Chronic , Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Cobicistat , Cricetinae , Disease Progression , Humans , Mesocricetus , Pandemics , SARS-CoV-2 , Viral Load
4.
PLoS Pathog ; 17(6): e1009683, 2021 06.
Article in English | MEDLINE | ID: covidwho-1282318

ABSTRACT

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.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Biological Assay/methods , COVID-19/virology , Receptors, Coronavirus/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/genetics , COVID-19/blood , Cell Fusion , HEK293 Cells , Humans , Receptors, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/genetics , Transfection , Virus Attachment
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