ZB-06, a vaginal film containing an engineered human contraceptive antibody (HC4-N), demonstrates safety and efficacy in a phase 1 postcoital test and safety study.

BACKGROUND: With an unplanned pregnancy rate of 50% or more in many countries, there is an urgent need for contraceptives that are more accessible and acceptable. To meet the growing demand for new contraceptives, ZabBio developed ZB-06, a vaginal film containing HC4-N, a human contraceptive antibody that inactivates sperm. OBJECTIVE: This study aimed to assess the potential contraceptive activity of the ZB-06 film using a surrogate assessment for contraceptive efficacy, the postcoital test. We also assessed clinical safety of film use among healthy heterosexual couples. Serum, cervical mucus, and vaginal fluid HC4-N antibody concentrations and sperm agglutination potency were determined after single film use. Changes in the concentration of soluble proinflammatory cytokines and vaginal Nugent score after film use were measured as subclinical safety endpoints. STUDY DESIGN: This was a phase 1, first-in-woman, open-label, proof-of-concept, postcoital test and safety study. RESULTS: A total of 20 healthy women were enrolled in the study, and 8 heterosexual couples completed all study visits. The product was safe for both female participants and their male sexual partners. The postcoital test performed on ovulatory cervical mucus at baseline (no product use) revealed a mean of 25.9 (±30.6) progressively motile sperm per high-power field. After use of a single ZB-06 film before intercourse, this number dropped to 0.04 (±0.06) progressively motile sperm per high-power field (P<.0001). At the follow-up postcoital test visit approximately 1 month later (no product use), a mean of 47.4 (±37.4) progressively motile sperm per high-power field was observed, indicating contraceptive reversibility. CONCLUSION: A single dose of the ZB-06 film applied before intercourse was safe and met efficacy surrogate benchmarks of excluding progressively motile sperm from ovulatory cervical mucus. These data indicate that ZB-06 is a viable contraceptive candidate warranting further development and testing.

Stratified Simple Random Sampling Versus Volunteer Community-Wide Sampling for Estimates of COVID-19 Prevalence.

Objectives. To evaluate community-wide prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using stratified simple random sampling. Methods. We obtained data for the prevalence of SARS-CoV-2 in Jefferson County, Kentucky, from adult random (n = 7296) and volunteer (n = 7919) sampling over 8 waves from June 2020 through August 2021. We compared results with administratively reported rates of COVID-19. Results. Randomized and volunteer samples produced equivalent prevalence estimates (P < .001), which exceeded the administratively reported rates of prevalence. Differences between them decreased as time passed, likely because of seroprevalence temporal detection limitations. Conclusions. Structured targeted sampling for seropositivity against SARS-CoV-2, randomized or voluntary, provided better estimates of prevalence than administrative estimates based on incident disease. A low response rate to stratified simple random sampling may produce quantified disease prevalence estimates similar to a volunteer sample. Public Health Implications. Randomized targeted and invited sampling approaches provided better estimates of disease prevalence than administratively reported data. Cost and time permitting, targeted sampling is a superior modality for estimating community-wide prevalence of infectious disease, especially among Black individuals and those living in disadvantaged neighborhoods. (Am J Public Health. 2023;113(7):768-777. https://doi.org/10.2105/AJPH.2023.307303).

Pharmacokinetics and Safety Studies in Rodent Models Support Development of EPICERTIN as a Novel Topical Wound-Healing Biologic for Ulcerative Colitis.

The novel wound-healing biologic EPICERTIN, a recombinant analog of cholera toxin B subunit, is in early development for the management of ulcerative colitis. This study established for the first time the pharmacokinetics (PK), bioavailability (BA), and acute safety of EPICERTIN in healthy and dextran sodium sulfate-induced colitic mice and healthy rats. For PK and BA assessments, single administrations of various concentrations of EPICERTIN were given intravenously or intrarectally to healthy and colitic C57BL/6 mice and to healthy Sprague-Dawley rats. After intravenous administration to healthy animals, the drug's plasma half-life (t 1/2) for males and females was 0.26 and 0.3 hours in mice and 19.4 and 14.5 hours in rats, respectively. After intrarectal administration, drug was detected at very low levels in only four samples of mouse plasma, with no correlation to colon epithelial integrity. No drug was detected in rat plasma. A single intrarectal dose of 0.1 µM (0.6 µg/mouse) EPICERTIN significantly facilitated the healing of damaged colonic epithelium as determined by disease activity index and histopathological scoring, whereas 10-fold higher or lower concentrations showed no effect. For acute toxicity evaluation, healthy rats were given a single intrarectal administration of various doses of EPICERTIN with sacrifice on Day 8, recording body weight, morbidity, mortality, clinical pathology, and gross necropsy observations. There were no drug-related effects of toxicological significance. The no observed adverse effect level (intrarectal) in rats was determined to be 5 µM (307 µg/animal, or 5.2 µg drug/cm2 of colorectal surface area), which is 14 times the anticipated intrarectally delivered clinical dose. SIGNIFICANCE STATEMENT: EPICERTIN is a candidate wound-healing biologic for the management of ulcerative colitis. This study determined for the first time the intravenous and intrarectal pharmacokinetics and bioavailability of the drug in healthy and colitic mice and healthy rats, and its acute safety in a dose-escalation study in rats. An initial therapeutic dose in colitic mice was also established. EPICERTIN delivered intrarectally was minimally absorbed systemically, was well tolerated, and induced epithelial wound healing topically at a low dose.

Preformulation Characterization of Griffithsin, a Biopharmaceutical Candidate for HIV Prevention.

Griffithsin (GRFT) has shown potent anti-HIV activity, and it is being developed as a drug candidate for HIV prevention. Successful implementation requires thorough understanding of its preformulation characterization. In this work, preformulation assessments were conducted to characterize GRFT and identify its degradation pathways under selected conditions of temperature, light, pH, shear, ionic strength, and oxidation. Compatibility with vaginal fluid simulant, vaginal enzymes, Lactobacillus spp., and human cervicovaginal secretions was assessed. The purity, melting temperature, and HIV gp120-binding affinity of GRFT stored at 4°C and 25°C in phosphate-buffered saline (PBS) were assessed for 2 years. Chemical modifications were evaluated by intact mass analysis and peptide sequencing. Excised human ectocervical tissue permeability and localization of GRFT were evaluated. Our results demonstrated GRFT to be safe and stable under all the preformulation assessment conditions studied except oxidative stress. When GRFT was exposed to hydrogen peroxide or human cervicovaginal secretion, methionine 78 in the protein sequence underwent oxidation. GRFT did not permeate through human cervical tissue but adhered to the superficial epithelial tissue. The 2-year stability study revealed no significant change in GRFT's aggregation, degradation, melting temperature, or gp120-binding affinity despite a slow increase in oxidation over time. These studies elucidated desirable safety and bioactivity profile for GRFT, showing promise as a potential drug candidate for HIV prevention. However, susceptibility to oxidative degradation was identified. Effective protection of GRFT from oxidation is required for further development.

Engineering of a Lectibody Targeting High-Mannose-Type Glycans of the HIV Envelope.

High-mannose-type glycans (HMGs) are aberrantly enriched on HIV envelope glycoproteins. However, there is currently no drug selectively targeting HIV-associated HMGs. Here, we describe a novel HMG-targeting "lectibody," a recombinant Fc-fusion protein comprising human IgG1 Fc and a novel actinohivin lectin variant (Avaren) obtained by structure-guided modifications for improved overall surface charge properties (AvFc). AvFc was engineered and produced using a rapid and scalable plant-based transient overexpression system. The lectibody exhibited potent antiviral activity against HIV-1 groups M and O primary viruses, as well as HIV-2 and simian immunodeficiency virus (SIV) strains, without affecting normal human blood cells. Furthermore, the lectibody induced Fc-mediated cell killing activity against HIV-1-infected cells and selectively recognized SIVmac239-infected macaque mesenteric lymph node cells in vitro. AvFc showed an extended serum half-life in rats and rhesus macaques, while no discernible toxicity was observed upon repeated systemic dosing in mice. These results highlight AvFc's potential as a biotherapeutic targeting HIV-associated HMGs of cell-free virions, as well as productively infected cells, providing a foundation for new anti-HIV strategies. Efficient and cost-effective bioproduction in greenhouse facilities may open unique possibilities for further development of AvFc.

A novel anti-HIV-1 bispecific bNAb-lectin fusion protein engineered in a plant-based transient expression system.

The discovery of broadly neutralizing antibodies (bNAbs) has been a major step towards better prophylactic and therapeutic agents against human immunodeficiency virus type 1 (HIV-1). However, effective therapy will likely require a combination of anti-HIV agents to avoid viral evasion. One possible solution to this problem is the creation of bispecific molecules that can concurrently target two vulnerable sites providing synergistic inhibitory effects. Here, we describe the production in plants and anti-HIV activity of a novel bispecific fusion protein consisting of the antigen-binding fragment (Fab) of the CD4 binding site-specific bNAb VRC01 and the antiviral lectin Avaren, which targets the glycan shield of the HIV-1 envelope (VRC01Fab -Avaren). This combination was justified by a preliminary experiment demonstrating the synergistic HIV-1 neutralization activity of VRC01 and Fc-fused Avaren dimer (Avaren-Fc). Using the GENEWARE® tobacco mosaic virus vector, VRC01Fab -Avaren was expressed in Nicotiana benthamiana and purified using a three-step chromatography procedure. Surface plasmon resonance and ELISA demonstrated that both the Avaren and VRC01Fab moieties retain their individual binding specificities. VRC01Fab -Avaren demonstrated enhanced neutralizing activity against representative HIV-1 strains from A, B and C clades, compared to equimolar combinations of VRC01Fab and Avaren. Notably, VRC01Fab -Avaren showed significantly stronger neutralizing effects than the bivalent parent molecules VRC01 IgG and Avaren-Fc, with IC50 values ranging from 48 to 310 pm. These results support the continued development of bispecific anti-HIV proteins based on Avaren and bNAbs, to which plant-based transient overexpression systems will provide an efficient protein engineering and production platform.

Bulk production of the antiviral lectin griffithsin.

Application of plant-based protein expression systems for bulk production of recombinant protein pharmaceuticals is building momentum. There are considerable regulatory challenges to consider in commercialization of plant-made pharmaceuticals (PMPs), some of which are inherent to plant-production systems and others that are common with other production systems, but are new to PMPs because of the youth of the industry. In this review, we discuss our recent and ongoing experience with bulk production of the HIV microbicide candidate, griffithsin (GRFT), utilizing plant-based transient protein expression, with specific focus on areas relevant to commercial manufacturing of bulk GRFT active pharmaceutical ingredient (API). Analytical programs have been developed for the qualification and monitoring of both the expression vector system and the API detailing our experience and plans for each. Monitoring postpurification protein modifications are discussed in relation to stability and safety programs. Expression, processing and analytics programs are associated with increased manufacturing costs in current good manufacturing practice (cGMP) production because of the required qualification testing. The impact of these costs on the overall cost of goods is particularly relevant to GRFT manufacturing because GRFT, as an HIV microbicide, is most needed in populations at high risk for HIV exposure in resource-poor countries. Consequently, GRFT for microbicide applications is a very cost-sensitive recombinant PMP. We have therefore emphasized maintaining a low cost of goods. We provide a review of the literature on the economics of PMPs with various expression systems and how they may impact production costs and complexity.

Efficient single tobamoviral vector-based bioproduction of broadly neutralizing anti-HIV-1 monoclonal antibody VRC01 in Nicotiana benthamiana plants and utility of VRC01 in combination microbicides.

Broadly neutralizing monoclonal antibodies (bnMAbs) may offer powerful tools for HIV-1 preexposure prophylaxis, such as topical microbicides. However, this option is hampered due to expensive MAb biomanufacturing based on mammalian cell culture. To address this issue, we developed a new production system for bnMAb VRC01 in Nicotiana benthamiana plants using a tobamovirus replicon vector. Unlike conventional two-vector-based expression, this system was designed to overexpress full-length IgG1 from a single polypeptide by means of kex2p-like enzyme recognition sites introduced between the heavy and light chains. An enzyme-linked immunosorbent assay (ELISA) revealed that gp120-binding VRC01 IgG1 was maximally accumulated on 5 to 7 days following vector inoculation, yielding ~150 mg of the bnMAb per kg of fresh leaf material. The plant-made VRC01 (VRC01p) was efficiently purified by protein A affinity followed by hydrophobic-interaction chromatography. ELISA, surface plasmon resonance, and an HIV-1 neutralization assay demonstrated that VRC01p has gp120-binding affinity and HIV-1-neutralization capacity virtually identical to the human-cell-produced counterpart. To advance VRC01p's use in topical microbicides, we analyzed combinations of the bnMAb with other microbicide candidates holding distinct antiviral mechanisms in an HIV-1 neutralization assay. VRC01p exhibited clear synergy with the antiviral lectin griffithsin, the CCR5 antagonist maraviroc, and the reverse transcriptase inhibitor tenofovir in multiple CCR5-tropic HIV-1 strains from clades A, B, and C. In summary, VRC01p is amenable to robust, rapid, and large-scale production and may be developed as an active component in combination microbicides with other anti-HIV agents such as antiviral lectins, CCR5 antagonists, and reverse transcriptase inhibitors.

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.

A protein switch sensing system for the quantification of sulfate.

Protein engineering has generated versatile methods and technologies that have been instrumental in advancements in the fields of sensing, therapeutics, and diagnostics. Herein, we demonstrate the employment of rational design to engineer a unique bioluminescence-based protein switch. A fusion protein switch combines two totally unrelated proteins, with distinct characteristics, in a manner such that the function of one protein is dependent on another. Herein we report a protein switch sensing system by insertion of the sulfate-binding protein (SBP) into the structure of the photoprotein aequorin (AEQ). In the presence of sulfate, SBP undergoes a conformational change bringing the two segments of AEQ together, "turning on" bioluminescence in a dose-dependent fashion, thus allowing quantitative detection of sulfate. A calibration plot was obtained by correlating the amount of bioluminescence generated with the concentration of sulfate present. The switch demonstrated selectivity and reproducibility, and a detection limit of 1.6×10(-4)M for sulfate. Moreover, the sensing system was validated by performing sulfate detection in clinical and environmental samples, such as, serum, urine, and tap water. The detection limits and working ranges in all three samples fall within the average normal/recommended sulfate levels in the respective matrices.

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 obtained and compared with the wastewater concentrations using regression analysis. The model analysis revealed significant 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.

Cyclic AMP receptor protein-aequorin molecular switch for cyclic AMP.

Molecular switches are designer molecules that combine the functionality of two individual proteins into one, capable of manifesting an "on/off" signal in response to a stimulus. These switches have unique properties and functionalities and thus, can be employed as nanosensors in a variety of applications. To that end, we have developed a bioluminescent molecular switch for cyclic AMP. Bioluminescence offers many advantages over fluorescence and other detection methods including the fact that there is essentially zero background signal in physiological fluids, allowing for more sensitive detection and monitoring. The switch was created by combining the properties of the cyclic AMP receptor protein (CRP), a transcriptional regulatory protein from E. Coli that binds selectively to cAMP with those of aequorin, a bioluminescent photoprotein native of the jellyfish Aequorea victoria . Genetic manipulation to split the genetic coding sequence of aequorin in two and genetically attach the fragments to the N and C termini of CRP resulted in a hybrid protein molecular switch. The conformational change experienced by CRP upon the binding of cyclic AMP is suspected to result in the observed loss of the bioluminescent signal from aequorin. The "on/off" bioluminescence can be modulated by cyclic AMP over a range of several orders of magnitude in a linear fashion in addition to the capacity to detect changes in cellular cyclic AMP of intact cells exposed to different external stimuli without the need to lyse the cells. We envision that the molecular switch could find applications in vitro as well as In Vivo cyclic AMP detection and/or imaging.

Safety and Efficacy of Avaren-Fc Lectibody Targeting HCV High-Mannose Glycans in a Human Liver Chimeric Mouse Model.

BACKGROUND & AIMS: Infection with hepatitis C virus (HCV) remains a major cause of morbidity and mortality worldwide despite the recent advent of highly effective direct-acting antivirals. The envelope glycoproteins of HCV are heavily glycosylated with a high proportion of high-mannose glycans (HMGs), which serve as a shield against neutralizing antibodies and assist in the interaction with cell-entry receptors. However, there is no approved therapeutic targeting this potentially druggable biomarker. METHODS: The anti-HCV activity of a fusion protein consisting of Avaren lectin and the fragment crystallizable (Fc) region of a human immunoglobulin G1 antibody, Avaren-Fc (AvFc) was evaluated through the use of in vitro neutralization assays as well as an in vivo challenge in a chimeric human liver (PXB) mouse model. Drug toxicity was assessed by histopathology, serum alanine aminotransferase, and mouse body weights. RESULTS: AvFc was capable of neutralizing cell culture-derived HCV in a genotype-independent manner, with 50% inhibitory concentration values in the low nanomolar range. Systemic administration of AvFc in a histidine-based buffer was well tolerated; after 11 doses every other day at 25 mg/kg there were no significant changes in body or liver weights or in blood human albumin or serum alanine aminotransferase activity. Gross necropsy and liver pathology confirmed the lack of toxicity. This regimen successfully prevented genotype 1a HCV infection in all animals, although an AvFc mutant lacking HMG binding activity failed. CONCLUSIONS: These results suggest that targeting envelope HMGs is a promising therapeutic approach against HCV infection, and AvFc may provide a safe and efficacious means to prevent recurrent infection upon liver transplantation in HCV-related end-stage liver disease patients.

Isolation and detection of a KDEL-tagged recombinant cholera toxin B subunit from Nicotiana benthamiana.

Here we describe refined methods for the isolation and detection of a KDEL-tagged, plant-produced recombinant cholera toxin B subunit (CTB) that exhibits unique mucosal wound healing activity. The protein was transiently overexpressed in Nicotiana benthamiana, which generates some C-terminal KDEL truncated molecular species that are deficient in epithelial repair activity. With a new CHT chromatographical method described herein, these product-derived impurities were successfully separated from CTB with the intact KDEL sequence, as confirmed by mass spectrometry. In addition, an immunoassay capable of specifically detecting GM1 ganglioside-binding CTB with intact KDEL sequences was developed. Coupled together, these methods will aid in the quality control of KDEL-attached CTB produced in plant-based manufacturing systems towards a novel topical biotherapeutic for the treatment of acute and chronic mucosal inflammation.

Spray-Dried Formulation of Epicertin, a Recombinant Cholera Toxin B Subunit Variant That Induces Mucosal Healing.

Epicertin (EPT) is a recombinant variant of the cholera toxin B subunit, modified with a C-terminal KDEL endoplasmic reticulum retention motif. EPT has therapeutic potential for ulcerative colitis treatment. Previously, orally administered EPT demonstrated colon epithelial repair activity in dextran sodium sulfate (DSS)-induced acute and chronic colitis in mice. However, the oral dosing requires cumbersome pretreatment with sodium bicarbonate to conserve the acid-labile drug substance while transit through the stomach, hampering its facile application in chronic disease treatment. Here, we developed a solid oral formulation of EPT that circumvents degradation in gastric acid. EPT was spray-dried and packed into enteric-coated capsules to allow for pH-dependent release in the colon. A GM1-capture KDEL-detection ELISA and size-exclusion HPLC indicated that EPT powder maintains activity and structural stability for up to 9 months. Capsule disintegration tests showed that EPT remained encapsulated at pH 1 but was released over 180 min at pH 6.8, the approximate pH of the proximal colon. An acute DSS colitis study confirmed the therapeutic efficacy of encapsulated EPT in C57BL/6 mice upon oral administration without gastric acid neutralization pretreatment compared to vehicle-treated mice (p < 0.05). These results provide a foundation for an enteric-coated oral formulation of spray-dried EPT.

Rapid detection of SARS-CoV-2 antibodies using electrochemical impedance-based detector.

Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was classified as a pandemic by the World Health Organization and has caused over 550,000 deaths worldwide as of July 2020. Accurate and scalable point-of-care devices would increase screening, diagnosis, and monitoring of COVID-19 patients. Here, we demonstrate rapid label-free electrochemical detection of SARS-CoV-2 antibodies using a commercially available impedance sensing platform. A 16-well plate containing sensing electrodes was pre-coated with receptor binding domain (RBD) of SARS-CoV-2 spike protein, and subsequently tested with samples of anti-SARS-CoV-2 monoclonal antibody CR3022 (0.1 µg/ml, 1.0 µg/ml, 10 µg/ml). Subsequent blinded testing was performed on six serum specimens taken from COVID-19 and non-COVID-19 patients (1:100 dilution factor). The platform was able to differentiate spikes in impedance measurements from a negative control (1% milk solution) for all CR3022 samples. Further, successful differentiation and detection of all positive clinical samples from negative control was achieved. Measured impedance values were consistent when compared to standard ELISA test results showing a strong correlation between them (R2=0.9). Detection occurs in less than five minutes and the well-based platform provides a simplified and familiar testing interface that can be readily adaptable for use in clinical settings.

Serological assessment of SARS-CoV-2 infection during the first wave of the pandemic in Louisville Kentucky.

Serological assays intended for diagnosis, sero-epidemiologic assessment, and measurement of protective antibody titers upon infection or vaccination are essential for managing the SARS-CoV-2 pandemic. Serological assays measuring the antibody responses against SARS-CoV-2 antigens are readily available. However, some lack appropriate characteristics to accurately measure SARS-CoV-2 antibodies titers and neutralization. We developed an Enzyme-linked Immunosorbent Assay (ELISA) methods for measuring IgG, IgA, and IgM responses to SARS-CoV-2, Spike (S), receptor binding domain (RBD), and nucleocapsid (N) proteins. Performance characteristics of sensitivity and specificity have been defined. ELISA results show positive correlation with microneutralization and Plaque Reduction Neutralization assays with infectious SARS-CoV-2. Our ELISA was used to screen healthcare workers in Louisville, KY during the first wave of the local pandemic in the months of May and July 2020. We found a seropositive rate of approximately 1.4% and 2.3%, respectively. Our analyses demonstrate a broad immune response among individuals and suggest some non-RBD specific S IgG and IgA antibodies neutralize SARS-CoV-2.

Stability of plasmid and viral banks supporting the cGMP manufacture of Q-Griffithsin from a TMV-based viral vector.

The "whole genome" TMV-based expression system, Geneware®, was used in the cGMP production of the plant-made pharmaceutical Q-Griffithsin and demonstrates stable expression for up to a two-year period. Virion and plasmid banks which contained viral cDNA and a Q-Griffithsin sequence were able to produce >200 g of Q-Griffithsin. Data assessing the quality and stability of the product banks were measured through functional assessments of visual symptomology and product expression.

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.