Analytical validation of quantitative SARS-CoV-2 subgenomic and viral load laboratory developed tests conducted on the Panther Fusion® (Hologic) with preliminary application to clinical samples.

OBJECTIVE: Validate the performance characteristics of two analyte specific, laboratory developed tests (LDTs) for the quantification of SARS-CoV-2 subgenomic RNA (sgRNA) and viral load on the Hologic Panther Fusion® using the Open Access functionality. METHODS: Custom-designed primers/probe sets targeting the SARS-CoV-2 Envelope gene (E) and subgenomic E were optimized. A 20-day performance validation following laboratory developed test requirements was conducted to assess assay precision, accuracy, analytical sensitivity/specificity, lower limit of detection and reportable range. RESULTS: Quantitative SARS-CoV-2 sgRNA (LDT-Quant sgRNA) assay, which measures intermediates of replication, and viral load (LDT-Quant VLCoV) assay demonstrated acceptable performance. Both assays were linear with an R2 and slope equal to 0.99 and 1.00, respectively. Assay precision was evaluated between 4-6 Log10 with a maximum CV of 2.6% and 2.5% for LDT-Quant sgRNA and LDT-Quant VLCoV respectively. Using negative or positive SARS-CoV-2 human nasopharyngeal swab samples, both assays were accurate (kappa coefficient of 1.00 and 0.92). Common respiratory flora and other viral pathogens were not detected and did not interfere with the detection or quantification by either assay. Based on 95% detection, the assay LLODs were 729 and 1206 Copies/mL for the sgRNA and VL load LDTs, respectively. CONCLUSION: The LDT-Quant sgRNA and LDT-Quant VLCoV demonstrated good analytical performance. These assays could be further investigated as alternative monitoring assays for viral replication; and thus, medical management in clinical settings which could inform isolation/quarantine requirements.

Validation of SARS-CoV-2 pooled testing for surveillance using the Panther Fusion® system: Impact of pool size, automation, and assay chemistry.

Combining diagnostic specimens into pools has been considered as a strategy to augment throughput, decrease turnaround time, and leverage resources. This study utilized a multi-parametric approach to assess optimum pool size, impact of automation, and effect of nucleic acid amplification chemistries on the detection of SARS-CoV-2 RNA in pooled samples for surveillance testing on the Hologic Panther Fusion® System. Dorfman pooled testing was conducted with previously tested SARS-CoV-2 nasopharyngeal samples using Hologic's Aptima® and Panther Fusion® SARS-CoV-2 Emergency Use Authorization assays. A manual workflow was used to generate pool sizes of 5:1 (five samples: one positive, four negative) and 10:1. An automated workflow was used to generate pool sizes of 3:1, 4:1, 5:1, 8:1 and 10:1. The impact of pool size, pooling method, and assay chemistry on sensitivity, specificity, and lower limit of detection (LLOD) was evaluated. Both the Hologic Aptima® and Panther Fusion® SARS-CoV-2 assays demonstrated >85% positive percent agreement between neat testing and pool sizes ≤5:1, satisfying FDA recommendation. Discordant results between neat and pooled testing were more frequent for positive samples with CT>35. Fusion® CT (cycle threshold) values for pooled samples increased as expected for pool sizes of 5:1 (CT increase of 1.92-2.41) and 10:1 (CT increase of 3.03-3.29). The Fusion® assay demonstrated lower LLOD than the Aptima® assay for pooled testing (956 vs 1503 cp/mL, pool size of 5:1). Lowering the cut-off threshold of the Aptima® assay from 560 kRLU (manufacturer's setting) to 350 kRLU improved the assay sensitivity to that of the Fusion® assay for pooled testing. Both Hologic's SARS-CoV-2 assays met the FDA recommended guidelines for percent positive agreement (>85%) for pool sizes ≤5:1. Automated pooling increased test throughput and enabled automated sample tracking while requiring less labor. The Fusion® SARS-CoV-2 assay, which demonstrated a lower LLOD, may be more appropriate for surveillance testing.

A SARS-CoV-2 Spike Ferritin Nanoparticle Vaccine Is Protective and Promotes a Strong Immunological Response in the Cynomolgus Macaque Coronavirus Disease 2019 (COVID-19) Model.

The COVID-19 pandemic has had a staggering impact on social, economic, and public health systems worldwide. Vaccine development and mobilization against SARS-CoV-2 (the etiologic agent of COVID-19) has been rapid. However, novel strategies are still necessary to slow the pandemic, and this includes new approaches to vaccine development and/or delivery that will improve vaccination compliance and demonstrate efficacy against emerging variants. Here, we report on the immunogenicity and efficacy of a SARS-CoV-2 vaccine comprising stabilized, pre-fusion spike protein trimers displayed on a ferritin nanoparticle (SpFN) adjuvanted with either conventional aluminum hydroxide or the Army Liposomal Formulation QS-21 (ALFQ) in a cynomolgus macaque COVID-19 model. Vaccination resulted in robust cell-mediated and humoral responses and a significant reduction in lung lesions following SARS-CoV-2 infection. The strength of the immune response suggests that dose sparing through reduced or single dosing in primates may be possible with this vaccine. Overall, the data support further evaluation of SpFN as a SARS-CoV-2 protein-based vaccine candidate with attention to fractional dosing and schedule optimization.

Efficacy and breadth of adjuvanted SARS-CoV-2 receptor-binding domain nanoparticle vaccine in macaques.

Emergence of novel variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean serum neutralizing antibody titers of 14,000 to 21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within 4 d in seven of eight animals receiving 50 µg of RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only approximately twofold relative to WA1/2020. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-CoV-like Sarbecovirus vaccine development.

Performance evaluation of a laboratory developed PCR test for quantitation of HIV-2 viral RNA.

Management of Human Immunodeficiency Virus Type 2 (HIV-2) infections present unique challenges due to low viral titers, slow disease progression, and poor response to standard antiviral therapies. The need for a nucleic acid assay to detect and quantify HIV-2 virus has led to the development of a number of molecular-based assays for detection and/or quantification of HIV-2 viral RNA in plasma in order to provide laboratory evidence of HIV-2 infection and viral loads for use in treatment decisions. As HIV-2 is less pathogenic and transmissible than HIV-1 and has resistance to several of the antiretroviral drugs, delay of treatment is common. Cross sero-reactivity between HIV-1 and HIV-2 makes it difficult to distinguish between the two viruses based upon serological tests. As such we developed a quantitative reverse transcription PCR (qRT-PCR) assay targeting the 5' long terminal repeat of HIV-2 for detection and quantification of HIV-2 viral RNA in plasma to identify HIV-2 infection and for use in viral load monitoring. Serial dilutions of cultured HIV-2 virus demonstrated a wide dynamic range (10 to 100,000 copies/ml) with excellent reproducibility (standard deviation from 0.12-0.19), linearity (R2 = 0.9994), and a lower limit of detection at 79 copies/ml (NIH-Z). The assay is highly specific for HIV-2 Groups A and B and exhibits no cross reactivity to HIV-1, HBV or HCV. Precision of the assay was demonstrated for the High (Mean = 6.41; SD = 0.12) and Medium (Mean = 4.46; SD = 0.13) HIV-2 positive controls. Replicate testing of clinical specimens showed good reproducibility above 1,000 copies/ml, with higher variability under 1,000 copies/ml. Analysis of 220 plasma samples from HIV-2 infected West African individuals demonstrated significantly lower viral loads than those observed in HIV-1 infections, consistent with results of previous studies. Slightly more than seven percent of clinical samples (7.3%) demonstrated viral loads above 100,000 copies/ml, while 37.3% of samples were undetectable. The high sensitivity, specificity, precision, and linearity of the WRAIR qRT-PCR assay makes it well suited for detection and monitoring of HIV-2 RNA levels in plasma of infected individuals.

Impact of Early Antiretroviral Therapy on Detection of Cell-Associated HIV-1 Nucleic Acid in Blood by the Roche Cobas TaqMan Test.

The Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test, v2.0 (the CAP/CTM assay), was used to quantify cell-associated HIV-1 (CAH) nucleic acid in peripheral blood mononuclear cells (PBMC) from well-characterized clinical specimens from HIV-1-infected individuals on antiretroviral therapy (ART). Chronically infected individuals on ART with no detectable plasma HIV-1 RNA demonstrated average CAH burdens of 3.2 HIV-1 log10 copies/million cells. Assay sensitivity and specificity were 98.9% and 100%, respectively, with the positive and negative predictive values being 100% and 98.6%, respectively. The CAH burden was also measured at weeks 0, 1, 2, 8, and 60 in 37 participants (RV254/SEARCH010, Bangkok, Thailand) stratified by Fiebig stage (Fiebig stage I [FI] to FVI) at ART initiation. Prior to ART initiation, the average CAH burden was 1.4, 4.1, and 3.6 log10 copies/million PBMCs for individuals who initiated ART at FI, FII, and FIII to FVI, respectively. Initiation of ART resulted in a rapid decline of CAH in all individuals, with the greatest decrease being observed in individuals who initiated ART at FI to FIII. By week 60, 100% (FI), 71.8% (FII/FIII), and 20.5% (FIV to FVI) of samples from individuals initiating treatment were at or near the limit of quantitation. Residual CAH was detectable at 60 weeks in most individuals who initiated ART at later stages (FIV to FVI) and averaged 1.9 ± 0.7 log10 copies/million PBMCs. The modified Roche CAP/CTM assay provides a convenient, standardized approach to measure residual HIV in blood and may be useful for monitoring patients under therapy or those participating in HIV remission studies.

Stability of Human Immunodeficiency Virus Serological Markers in Samples Collected as HemaSpot and Whatman 903 Dried Blood Spots.

Dried blood spots (DBS) are frequently used in clinical testing for biosurveillance, infectious disease and confirmatory testing, and clinical trials, particularly for populations in remote areas. The HemaSpot-HF blood collection device (HS) provides an alternative format to the Whatman 903 cards (903) to simplify sample collection and processing. In this study, the performance of the HS was compared to that of the 903 using previously characterized clinical specimens and HIV seroconversion panels known to exhibit markers of early human immunodeficiency virus (HIV) infection. HS and 903 samples were prepared and tested by Bio-Rad GS HIV Combo Ag/Ab enzyme immunoassay (EIA), GS HIV-1/-2 Plus O EIA, GS HIV-1 Western blot, and HIV-1 Geenius assays. Both HS and 903 performed well for up to 6 months at room temperature, but a marked loss of Western blot and low titer antibody signals from early infection samples was observed in samples stored for 180 days at elevated (37 to 45°C) temperatures and high humidity (95%). HemaSpot samples placed in sealed bags with additional desiccant were protected from degradation and showed improved signal recovery relative to that of the 903. HS was easier to use than the 903 and showed higher sensitivity and reproducibility for early infection samples and improved stability.

A Recombinant Vesicular Stomatitis Virus Ebola Vaccine.

BACKGROUND: The worst Ebola virus disease (EVD) outbreak in history has resulted in more than 28,000 cases and 11,000 deaths. We present the final results of two phase 1 trials of an attenuated, replication-competent, recombinant vesicular stomatitis virus (rVSV)-based vaccine candidate designed to prevent EVD. METHODS: We conducted two phase 1, placebo-controlled, double-blind, dose-escalation trials of an rVSV-based vaccine candidate expressing the glycoprotein of a Zaire strain of Ebola virus (ZEBOV). A total of 39 adults at each site (78 participants in all) were consecutively enrolled into groups of 13. At each site, volunteers received one of three doses of the rVSV-ZEBOV vaccine (3 million plaque-forming units [PFU], 20 million PFU, or 100 million PFU) or placebo. Volunteers at one of the sites received a second dose at day 28. Safety and immunogenicity were assessed. RESULTS: The most common adverse events were injection-site pain, fatigue, myalgia, and headache. Transient rVSV viremia was noted in all the vaccine recipients after dose 1. The rates of adverse events and viremia were lower after the second dose than after the first dose. By day 28, all the vaccine recipients had seroconversion as assessed by an enzyme-linked immunosorbent assay (ELISA) against the glycoprotein of the ZEBOV-Kikwit strain. At day 28, geometric mean titers of antibodies against ZEBOV glycoprotein were higher in the groups that received 20 million PFU or 100 million PFU than in the group that received 3 million PFU, as assessed by ELISA and by pseudovirion neutralization assay. A second dose at 28 days after dose 1 significantly increased antibody titers at day 56, but the effect was diminished at 6 months. CONCLUSIONS: This Ebola vaccine candidate elicited anti-Ebola antibody responses. After vaccination, rVSV viremia occurred frequently but was transient. These results support further evaluation of the vaccine dose of 20 million PFU for preexposure prophylaxis and suggest that a second dose may boost antibody responses. (Funded by the National Institutes of Health and others; rVSV∆G-ZEBOV-GP ClinicalTrials.gov numbers, NCT02269423 and NCT02280408 .).

Evaluation of Hologic Aptima HIV-1 Quant Dx Assay on the Panther System on HIV Subtypes.

Quantitation of the HIV-1 viral load in plasma is the current standard of care for clinical monitoring of HIV-infected individuals undergoing antiretroviral therapy. This study evaluated the analytical and clinical performances of the Aptima HIV-1 Quant Dx assay (Hologic, San Diego, CA) for monitoring viral load by using 277 well-characterized subtype samples, including 171 cultured virus isolates and 106 plasma samples from 35 countries, representing all major HIV subtypes, recombinants, and circulating recombinant forms (CRFs) currently in circulation worldwide. Linearity of the Aptima assay was tested on each of 6 major HIV-1 subtypes (A, B, C, D, CRF01_AE, and CRF02_AG) and demonstrated an R(2) value of ≥0.996. The performance of the Aptima assay was also compared to those of the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 v.2 (CAP/CTM) and Abbott m2000 RealTime HIV-1 (RealTime) assays on all subtype samples. The Aptima assay values averaged 0.21 log higher than the CAP/CTM values and 0.30 log higher than the RealTime values, and the values were >0.4 log higher than CAP/CTM values for subtypes F and G and than RealTime values for subtypes C, F, and G and CRF02_AG. Two samples demonstrated results with >1-log differences from RealTime results. When the data were adjusted by the average difference, 94.9% and 87.0% of Aptima results fell within 0.5 log of the CAP/CTM and RealTime results, respectively. The linearity and accuracy of the Aptima assay in correctly quantitating all major HIV-1 subtypes, coupled with the completely automated format and high throughput of the Panther system, make this system well suited for reliable measurement of viral load in the clinical laboratory.

Discrepant amplification results during the development of an assay leads to reclassification of two AIDS reagent repository HIV-2 isolates as HIV-1.

The development and verification of HIV-2 assays depends in part on the availability of well-characterized samples, including those from reagent repositories. During the development of an HIV-2 RNA quantification assay, two HIV-2 viral isolates (CDC 301340 and CDC 301342) obtained from the NIAID AIDS Reagent and Reference Repository were not detected leading to an investigation. Two HIV-2 primers/probe sets of known performance in real-time viral RNA quantification assays, targeting different regions of the virus, also failed to generate RT-PCR products for these two isolates. These isolates were tested in the HIV-1 specific COBAS AmpliPrep/COBAS TaqMan HIV-1 Test v2.0 (Roche Molecular Diagnostics) and were quantified at high copy number. Other HIV-2 isolates tested were not amplified in the COBAS HIV-1 TaqMan assay. Furthermore, the discrepant isolates were highly reactive in an HIV-1 p24 antigen test while the other HIV-2 isolates showed very weak, if any, cross-reactivity with the HIV-1 p24 assay. Phylogenetic tree analysis of sequences from the protease-reverse transcriptase regions of the discrepant HIV-2 isolates mapped with HIV-1 Group M, Subtype CRF02_AG confirming these isolates were of HIV-1 origin and had been misclassified as HIV-2. The use of misclassified isolates in the verification of molecular and immunological assays can lead to misinterpretation of test results, misdirection of efforts into assay redesign and increased development costs. The results of this study were shared with the NIAID AIDS Reagent Program, leading to the reclassification of the two discrepant isolates as HIV-1.