Incorporation of SARS-CoV-2 spike NTD to RBD protein vaccine improves immunity against viral variants.

Emerging SARS-CoV-2 variants pose a threat to human health worldwide. SARS-CoV-2 receptor binding domain (RBD)-based vaccines are suitable candidates for booster vaccines, eliciting a focused antibody response enriched for virus neutralizing activity. Although RBD proteins are manufactured easily, and have excellent stability and safety properties, they are poorly immunogenic compared to the full-length spike protein. We have overcome this limitation by engineering a subunit vaccine composed of an RBD tandem dimer fused to the N-terminal domain (NTD) of the spike protein. We found that inclusion of the NTD (1) improved the magnitude and breadth of the T cell and anti-RBD response, and (2) enhanced T follicular helper cell and memory B cell generation, antibody potency, and cross-reactive neutralization activity against multiple SARS-CoV-2 variants, including B.1.1.529 (Omicron BA.1). In summary, our uniquely engineered RBD-NTD-subunit protein vaccine provides a promising booster vaccination strategy capable of protecting against known SARS-CoV-2 variants of concern.

Characterization of the First SARS-CoV-2 Isolates from Aotearoa New Zealand as Part of a Rapid Response to the COVID-19 Pandemic.

SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has wreaked havoc across the globe for the last two years. More than 300 million cases and over 5 million deaths later, we continue battling the first real pandemic of the 21st century. SARS-CoV-2 spread quickly, reaching most countries within the first half of 2020, and New Zealand was not an exception. Here, we describe the first isolation and characterization of SARS-CoV-2 variants during the initial virus outbreak in New Zealand. Patient-derived nasopharyngeal samples were used to inoculate Vero cells and, three to four days later, a cytopathic effect was observed in seven viral cultures. Viral growth kinetics was characterized using Vero and VeroE6/TMPRSS2 cells. The identity of the viruses was verified by RT-qPCR, Western blot, indirect immunofluorescence assays, and electron microscopy. Whole-genome sequences were analyzed using two different yet complementary deep sequencing platforms (MiSeq/Illumina and Ion PGM™/Ion Torrent™), classifying the viruses as SARS-CoV-2 B.55, B.31, B.1, or B.1.369 based on the Pango Lineage nomenclature. All seven SARS-CoV-2 isolates were susceptible to remdesivir (EC50 values from 0.83 to 2.42 µM) and ß-D-N4-hydroxycytidine (molnupiravir, EC50 values from 0.96 to 1.15 µM) but not to favipiravir (>10 µM). Interestingly, four SARS-CoV-2 isolates, carrying the D614G substitution originally associated with increased transmissibility, were more susceptible (2.4-fold) to a commercial monoclonal antibody targeting the spike glycoprotein than the wild-type viruses. Altogether, this seminal work allowed for early access to SARS-CoV-2 isolates in New Zealand, paving the way for numerous clinical and scientific research projects in the country, including the development and validation of diagnostic assays, antiviral strategies, and a national COVID-19 vaccine development program.

Ultraviolet-C Irradiation, Heat, and Storage as Potential Methods of Inactivating SARS-CoV-2 and Bacterial Pathogens on Filtering Facepiece Respirators.

The arrival of SARS-CoV-2 to Aotearoa/New Zealand in February 2020 triggered a massive response at multiple levels. Procurement and sustainability of medical supplies to hospitals and clinics during the then upcoming COVID-19 pandemic was one of the top priorities. Continuing access to new personal protective equipment (PPE) was not guaranteed; thus, disinfecting and reusing PPE was considered as a potential alternative. Here, we describe part of a local program intended to test and implement a system to disinfect PPE for potential reuse in New Zealand. We used filtering facepiece respirator (FFR) coupons inoculated with SARS-CoV-2 or clinically relevant multidrug-resistant pathogens (Acinetobacter baumannii Ab5075, methicillin-resistant Staphylococcus aureus USA300 LAC and cystic-fibrosis isolate Pseudomonas aeruginosa LESB58), to evaluate the potential use of ultraviolet-C germicidal irradiation (UV-C) or dry heat treatment to disinfect PPE. An applied UV-C dose of 1000 mJ/cm2 was sufficient to completely inactivate high doses of SARS-CoV-2; however, irregularities in the FFR coupons hindered the efficacy of UV-C to fully inactivate the virus, even at higher UV-C doses (2000 mJ/cm2). Conversely, incubating contaminated FFR coupons at 65 °C for 30 min or 70 °C for 15 min, was sufficient to block SARS-CoV-2 replication, even in the presence of mucin or a soil load (mimicking salivary or respiratory secretions, respectively). Dry heat (90 min at 75 °C to 80 °C) effectively killed 106 planktonic bacteria; however, even extending the incubation time up to two hours at 80 °C did not completely kill bacteria when grown in colony biofilms. Importantly, we also showed that FFR material can harbor replication-competent SARS-CoV-2 for up to 35 days at room temperature in the presence of a soil load. We are currently using these findings to optimize and establish a robust process for decontaminating, reusing, and reducing wastage of PPE in New Zealand.

Rapid Response to SARS-CoV-2 in Aotearoa New Zealand: Implementation of a Diagnostic Test and Characterization of the First COVID-19 Cases in the South Island.

It has been 20 months since we first heard of SARS-CoV-2, the novel coronavirus detected in the Hubei province, China, in December 2019, responsible for the ongoing COVID-19 pandemic. Since then, a myriad of studies aimed at understanding and controlling SARS-CoV-2 have been published at a pace that has outshined the original effort to combat HIV during the beginning of the AIDS epidemic. This massive response started by developing strategies to not only diagnose individual SARS-CoV-2 infections but to monitor the transmission, evolution, and global spread of this new virus. We currently have hundreds of commercial diagnostic tests; however, that was not the case in early 2020, when just a handful of protocols were available, and few whole-genome SARS-CoV-2 sequences had been described. It was mid-January 2020 when several District Health Boards across New Zealand started planning the implementation of diagnostic testing for this emerging virus. Here, we describe our experience implementing a molecular test to detect SARS-CoV-2 infection, adapting the RT-qPCR assay to be used in a random-access platform (Hologic Panther Fusion® System) in a clinical laboratory, and characterizing the first whole-genome SARS-CoV-2 sequences obtained in the South Island, right at the beginning of the SARS-CoV-2 outbreak in New Zealand. We expect that this work will help us and others prepare for the unequivocal risk of similar viral outbreaks in the future.