Quantitating SARS-CoV-2 Neutralizing Antibodies from Human Dried Blood Spots (preprint)

Background: In the earliest days of COVID-19 pandemic, the collection of dried blood spots (DBS) enabled public health laboratories to undertake population-scale seroprevalence studies to estimate rates of SARS-CoV-2 exposure. With SARS-CoV-2 seropositivity levels now estimated to exceed 94% in the United States, attention has turned to using DBS to assess functional (neutralizing) antibodies within cohorts of interest. Methods: Contrived DBS eluates from convalescent, fully vaccinated and pre-COVID-19 serum samples were evaluated in SARS-CoV-2 plaque reduction neutralization titer (PRNT) assays, a SARS-CoV-2 specific 8-plex microsphere immunoassay, a cell-based pseudovirus assay, and two different spike-ACE2 inhibition assays. Results: DBS eluates from convalescent individuals were compatible with RBD-ACE2 inhibition assays, an in-house Luminex-based RBD-ACE2 inhibition assay, and commercial real-time PCR-based neutralization assay (NAB-Sure) but not cell-based pseudovirus assays or PRNT. The insensitivity of cell-based pseudovirus assays was overcome with DBS eluates from vaccinated individuals with high SARS-CoV-2 antibody titers. Conclusion: SARS-CoV-2 neutralizing titers can be derived with confidence from DBS eluates, thereby opening the door to the use of these biospecimens for the analysis of vulnerable populations and normally hard to reach communities.

Effects of aluminum-salt, CpG and emulsion adjuvants on the stability and immunogenicity of a virus-like particle displaying the SARS-CoV-2 receptor binding domain (RBD) (preprint)

Second-generation COVID-19 vaccines with improved immunogenicity (e.g., breadth, duration) and availability (e.g., lower costs, refrigerator stable) are needed to enhance global coverage. In this work, we formulated a clinical-stage SARS-CoV-2 receptor binding domain (RBD) virus-like particle (VLP) vaccine candidate (IVX-411) with widely available adjuvants. Specifically, we assessed the in vitro storage stability and in vivo mouse immunogenicity of IVX-411 formulated with aluminum-salt adjuvants (Alhydrogel, AH and Adjuphos, AP), without or with the TLR-9 agonist CpG-1018 (CpG), and compared these profiles to IVX-411 adjuvanted with an oil-in-water nano-emulsion (AddaVax, AV). Although IVX-411 bound both AH and AP, lower binding strength of antigen to AP was observed by Langmuir binding isotherms. Interestingly, AH- and AP-adsorbed IVX-411 had similar storage stability profiles as measured by antigen binding assays (competitive ELISAs), but the latter displayed higher pseudovirus neutralizing titers (pNT) in mice, at levels comparable to titers elicited by AV-adjuvanted IVX-411. CpG addition to alum (AP or AH) resulted in a marginal trend of improved pNTs in stressed samples only, yet did not impact the storage stability profiles of IVX-411. In contrast, previous work with AH-formulations of a monomeric RBD antigen showed greatly improved immunogenicity and decreased stability upon CpG addition to alum. At elevated temperatures (25, 37{degrees}C), IVX-411 formulated with AH or AP displayed decreased in vitro stability compared to AV-formulated IVX-411and this rank-ordering correlated with in vivo performance (mouse pNT values). This case study highlights the importance of optimizing antigen-adjuvant interactions to develop low cost, aluminum-salt adjuvanted recombinant subunit vaccine candidates.

Evaluation of specimen types and saliva stabilization solutions for SARS-CoV-2 testing (preprint)

Identifying SARS-CoV-2 infections through aggressive diagnostic testing remains critical in tracking and curbing the spread of the COVID-19 pandemic. Collection of nasopharyngeal swabs (NPS), the preferred sample type for SARS-CoV-2 detection, has become difficult due to the dramatic increase in testing and consequential supply strain. Therefore, alternative specimen types have been investigated, that provide similar detection sensitivity with reduced health care exposure and potential for self-collection. In this study, the detection sensitivity of SARS-CoV-2 in nasal swabs (NS) and saliva was compared to that of NPS, using matched specimens from two outpatient cohorts in New York State (total n = 463). The first cohort showed only a 5.4% positivity but the second cohort (n=227) had a positivity rate of 41%, with sensitivity in NPS, NS and saliva of 97.9%, 87.1%, and 87.1%, respectively. Whether the reduced sensitivity of NS or saliva is acceptable must be assessed in the settings where they are used. However, we sought to improve on it by validating a method to mix the two sample types, as the combination of nasal swab and saliva resulted in 94.6% SARS-CoV-2 detection sensitivity. Spiking experiments showed that combining them did not adversely affect the detection sensitivity in either. Virus stability in saliva was also investigated, with and without the addition of commercially available stabilizing solutions. The virus was stable in saliva at both 4{degrees}C and room temperature for up to 7 days. The addition of stabilizing solutions did not enhance stability and in some situations reduced detectable virus levels.

Assessment of sample pooling for clinical SARS-CoV-2 testing (preprint)

Accommodating large increases in sample workloads has presented one of the biggest challenges to clinical laboratories during the COVID-19 pandemic. Despite the implementation of new automated detection systems, and previous efficiencies such as barcoding, electronic data transfer and extensive robotics, throughput capacities have struggled to meet the demand. Sample pooling has been suggested as an additional strategy to further address this need. The greatest concern with this approach in a clinical setting is the potential for reduced sensitivity, particularly the risk of false negative results when weak positive samples are pooled. To investigate this possibility, detection rates in pooled samples were evaluated, with extensive assessment of pools containing weak positive specimens. Additionally, the frequency of occurrence of weak positive samples across ten weeks of the pandemic were reviewed. Weak positive specimens were detected in all five-sample pools but failed to be detected in four of the 24 nine-sample pools tested. Weak positive samples comprised an average 16.5% of the positive specimens tested during the pandemic thus far, slightly increasing in frequency during later weeks. Other aspects of the testing process should be considered, however, such as accessioning and reporting, which are not streamlined and may be complicated by pooling procedures. Therefore, the impact on the entire laboratory process needs to be carefully assessed prior to implementing such a strategy.