Breadth of Vaccinated Cancer Patient Humoral Response to SARS-CoV-2 Spike Protein and RBD Variants

SARS-CoV-2, the virus responsible for the COVID-19 of which several variants have emerged, such as the B.1.351 SARS-CoV-2 variant. The Receptor Binding Domain (RBD), located within the Spike protein is an immunogenic epitope for potent neutralizing antibodies. Current mRNA vaccines encode for the Spike protein, allowing the body to build antigen-specific antibodies. Assays measuring protective antibodies are essential to manage the COVID-19 pandemic and can be used as a platform for variant screening. RBD-foldon 2.2 is a novel antigen produced by fusing RBD with the trimerization domain Fibritin from Bacteriophage T4. Its amino acid sequence is based on the original Wuhan strain. (Breckenridge, 2021). B.1.351 RBD-foldon 2.2 antigen is identical to RBD-foldon 2.2, except it uses the B.1.351 variant RBD sequence. Using cancer patient sera samples, the breadth and robustness of response was examined in comparison to patients that indicated “no chronic conditions”. We hypothesized there would be a difference in humoral response to RBD-variant antigens in COVID-19 vaccinated cancer patients undergoing treatment vs patients with no chronic conditions. For sample selection, cancer patients were age/sex matched to individuals with no underlying health conditions, that received the same mRNA vaccine within 2 weeks of each other. To quantify antibody levels, ELISA end-point titers were performed. ELISAs detected levels of IgG and IgA antibodies against Spike, RBD-foldon, RBD-foldon 2.2, and RBD-foldon B.1.351. (Bushau, 2021). The statistical analysis used was a two-tailed student’s t-test to compare mean value of end-point titers between experimental and control groups. No significant difference between experimental and control groups for any antibody-antigen combination. B.1.351 RBD-foldon appears to elicit a lower response than RBD-foldon 2.2. Lower response may be explained by the mRNA sequence used in current vaccines encodes for original Wuhan SARS-CoV-2 spike protein. The platform is predictive of the level of antibody protection for variant screening

Hydroponic Treatment of Nicotiana benthamiana with Kifunensine Modifies the N-glycans of Recombinant Glycoprotein Antigens to Predominantly Man9 High-Mannose Type upon Transient Overexpression

Nicotiana benthamiana transient overexpression systems offer unique advantages for rapid and scalable biopharmaceuticals production, including high scalability and eukaryotic post-translational modifications such as N-glycosylation. High-mannose-type glycans (HMGs) of glycoprotein antigens have been implicated in the effectiveness of some subunit vaccines. In particular, Man9GlcNAc2 (Man9) has high binding affinity to mannose-specific C-type lectin receptors such as the mannose receptor and dendritic cell-specific intracellular adhesion molecule 3-grabbing non-integrin (DC-SIGN). Here, we investigated the effect of kifunensine, an α-mannosidase I inhibitor, supplemented in a hydroponic culture of N. benthamiana for the production of Man9-rich HMG glycoproteins, using N-glycosylated cholera toxin B subunit (gCTB) and human immunodeficiency virus gp120 that are tagged with a H/KDEL endoplasmic reticulum retention signal as model vaccine antigens. Biochemical analysis using anti-fucose and anti-xylose antibodies as well as Endo H and PNGase F digestion showed that kifunensine treatment effectively reduced plant-specific glycoforms while increasing HMGs in the N-glycan compositions of gCTB. Detailed glycan profiling revealed that plant-produced gp120 had a glycan profile bearing mostly HMGs regardless of kifunensine treatment. However, the gp120 produced under kifunensine-treatment conditions showed Man9 being the most prominent glycoform (64.5%), while the protein produced without kifunensine had a substantially lower Man9 composition (20.3%). Our results open up possibilities for efficient production of highly mannosylated recombinant vaccine antigens in plants

Pre-Existing Comorbidities Diminish the Likelihood of Seropositivity after SARS-CoV-2 Vaccination

Background: The impact of chronic health conditions (CHCs) on serostatus post-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination is unknown. Methods: We assessed serostatus post-SARS-CoV-2 vaccination among fully vaccinated adult residents of Jefferson County, Kentucky, USA, from April 2021 to August 2021. Serostatus was determined by qualitative analysis of SARS-CoV-2-specific Spike IgG antibodies via enzyme-linked immunoassay (ELISA) in peripheral blood samples. Results: Of the 5178 fully vaccinated participants, 51 were seronegative and 5127 were seropositive. Chronic kidney disease (CKD) and autoimmune disease showed the highest association with negative serostatus in fully vaccinated individuals. The absence of any CHC was strongly associated with positive serostatus. The risk of negative serostatus increased as the total number of pre-existing CHCs increased. Similarly, the use of two or more CHC-related medications was associated with seronegative status. Conclusions: The presence of any CHC, especially CKD or autoimmune disease, increased the likelihood of seronegative status among individuals who were fully vaccinated to SAR-CoV-2. This risk increased with a concurrent increase in number of comorbidities, especially with multiple medications. The absence of any CHC was protective and increased the likelihood of a positive serological response. These results will help develop appropriate guidelines for booster doses and targeted vaccination programs

Quantifying the relationship between sub-population wastewater samples and community-wide SARS-CoV-2 seroprevalence

Robust epidemiological models relating wastewater to community disease prevalence are lacking. Assessments of SARS-CoV-2 infection rates have relied primarily on convenience sampling, which does not provide reliable estimates of community disease prevalence due to inherent biases. This study conducted serial stratified randomized samplings to estimate the prevalence of SARS-CoV-2 antibodies in 3717 participants, and obtained weekly samples of community wastewater for SARS-CoV-2 concentrations in Jefferson County, KY (USA) from August 2020 to February 2021. Using an expanded Susceptible-Infected-Recovered model, the longitudinal estimates of the disease prevalence were ob-tained and compared with the wastewater concentrations using regression analysis. The model analysis revealed sig-nificant temporal differences in epidemic peaks. The results showed that in some areas, the average incidence rate, based on serological sampling, was 50 % higher than the health department rate, which was based on convenience sampling. The model-estimated average prevalence rates correlated well with the wastewater (correlation = 0.63, CI (0.31,0.83)). In the regression analysis, a one copy per ml-unit increase in weekly average wastewater concentration of SARS-CoV-2 corresponded to an average increase of 1-1.3 cases of SARS-CoV-2 infection per 100,000 residents. The analysis indicates that wastewater may provide robust estimates of community spread of infection, in line with the modeled prevalence estimates obtained from stratified randomized sampling, and is therefore superior to publicly available health data.11Nsciescopu

Bioluminescence Inhibition Assay for the Detection of Hydroxylated Polychlorinated Biphenyls

Hydroxylated polychlorinated biphenyls (OH-PCBs) are an important class of contaminants that mainly originate from polychlorinated biphenyl metabolism. They may conceivably be as dangerous and persistent as the parent compounds; most prominently, OH-PCBs are endocrine disruptors. Due to increasing evidence of the presence of OH-PCBs in the environment and in living organisms, including humans, and of their toxicity, methods of detection for OH-PCBs are needed in the environmental and medical fields. Herein we describe the development and optimization of a protein+based inhibition assay for the quantification of OH-PCBs. Specifically, the photoprotein aequorin was utilized for the detection of OH-PCBs. We hypothesized that OH-PCBs interact with aequorin and we established that OH-PCBs actually inhibit the bioluminescence of aequorin in a dose-dependent manner. We took advantage of this phenomenon to develop an assay that is capable of detecting a wide variety of OH-PCBs with a range of detection limits, the best detection limit being 11 nM for the compound 2-hydroxy-2',3,4',5',6-pentachorobiphenyl. The viability of this system for the screening of OH-PCBs in spiked biological and environmental samples was also established. We envision the implementation of this novel bioluminescence inhibition assay as a rapid, sensitive and cost-effective method for monitoring OH-PCBs. Furthermore, to the best of our knowledge this is the first time aequorin has been employed to detect an analyte by the inhibition of its bioluminescence reaction. Hence, this strategy may prove to be a general approach for the development of a new generation of protein-based inhibition assays