Two doses of the SARS-CoV-2 BNT162b2 vaccine enhances antibody responses to variants in individuals with prior SARS-CoV-2 infection

Understanding the impact of prior infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the response to vaccination is a priority for responding to the coronavirus disease 2019 (COVID-19) pandemic. In particular, it is necessary to understand how prior infection plus vaccination can modulate immune responses against variants of concern. To address this, we sampled 20 individuals with and 25 individuals without confirmed previous SARS-CoV-2 infection from a large cohort of healthcare workers followed serologically since April 2020. All 45 individuals had received two doses of the Pfizer-BioNTech BTN162b2 vaccine with a delayed booster at 10 weeks. Absolute and neutralizing antibody titers against wild-type SARS-CoV-2 and variants were measured using enzyme immunoassays and pseudotype neutralization assays. We observed antibody reactivity against lineage A, B.1.351 and P.1 variants with increasing antigenic exposure, either through vaccination or natural infection. This improvement was further confirmed in neutralization assays using fixed dilutions of serum samples. The impact of antigenic exposure was more evident in enzyme immunoassays measuring SARS-CoV-2 spike protein-specific IgG antibody concentrations. Our data show that multiple exposures to SARS-CoV-2 spike protein in the context of a delayed booster expand the neutralizing breadth of the antibody response to neutralization-resistant SARS-CoV-2 variants. This suggests that additional vaccine boosts may be beneficial in improving immune responses against future SARS-CoV-2 variants of concern.

Identification of Koala (Phascolarctos cinereus) Faecal Cortisol Metabolites Using Liquid Chromatography-Mass Spectrometry and Enzyme Immunoassays

The koala (Phascolarctos cinereus) is an arboreal folivorous marsupial endemic to Australia. Anthropogenic activities and climate change are threats to this species’ survival and are potential stressors. A suitable non-invasive method is needed to objectively detect stress in koalas. Under conditions of stress, the concentration of the hormone cortisol in plasma or in saliva is elevated, and this would provide a convenient measure; however, collecting blood or saliva from wild animals is both practically difficult and stressful, and so likely to confound any measurement. In contrast, measurement of cortisol metabolites in faeces provides a practical and non-invasive method to objectively measure stress in koalas. Unfortunately, the identity of the main faecal cortisol metabolites of koalas is unknown. In this study, we have used both untargeted liquid chromatography–mass spectrometry (LC-MS) and enzyme immunoassays (EIAs) to identify several faecal cortisol metabolites in two koalas, one female (18 months old, 4.1 kg) and one male (4 years old, 6.95 kg) upon administration of hydrocortisone (cortisol) sodium succinate. The LC-MS analysis identified tetrahydrocortisol along with several other isomers as cortisol metabolites. After a survey of five enzyme immunoassays, we found that two metabolites, tetrahydrocortisol and 3β-allotetrahydrocortisol, could be detected by EIAs that used antibodies that were raised against their structurally similar corticosterone counterparts, tetrahydrocorticosterone and 3β-allotetrahydrocorticosterone, respectively. While the 3β-allotetrahydrocortisol metabolite was detected in the faeces of only one of the two animals studied, tetrahydrocortisol was detected in both. These results ultimately indicate that tetrahydrocortisol is likely the main faecal cortisol metabolite in koalas, and we demonstrate that it can be measured by an EIA (50c) that was originally developed to measure tetrahydrocorticosterone.

Frequency of positive anti-PF4/polyanion antibody tests after COVID-19 vaccination with ChAdOx1 nCoV-19 and BNT162b2

Vaccination using the adenoviral vector COVID-19 vaccine ChAdOx1 nCoV-19 (AstraZeneca) has been associated with rare vaccine-induced immune thrombotic thrombocytopenia (VITT). Affected patients test strongly positive in PF4/polyanion enzyme immunoassays (EIAs) and serum-induced platelet activation is maximal in the presence of PF4. We determined the frequency of anti-PF4/polyanion antibodies in healthy vaccinees and assessed whether PF4/polyanion EIA-positive sera exhibit platelet-activating properties after vaccination with ChAdOx1 nCoV-19 (n=138) or BNT162b2 (BioNTech/Pfizer; n=143). In total, 19 of 281 participants tested positive for anti-PF4/polyanion antibodies post-vaccination (All: 6.8% [95%CI, 4.4-10.3]; BNT162b2: 5.6% [95%CI, 2.9-10.7]; ChAdOx1 nCoV-19: 8.0% [95%CI, 4.5-13.7%]). Optical densities were mostly low (between 0.5-1.0 units; reference range, <0.50) and none of the PF4/polyanion EIA-positive samples induced platelet activation in the presence of PF4. We conclude that positive PF4/polyanion EIAs can occur after SARS-CoV-2 vaccination with both mRNA- and adenoviral vector-based vaccines, but the majority of these antibodies likely have minor (if any) clinical relevance. Accordingly, low-titer positive PF4/polyanion EIA results should be interpreted with caution when screening asymptomatic individuals after vaccination against Covid-19. Pathogenic platelet-activating antibodies that cause VITT do not occur commonly following vaccination.