Converting non-neutralizing SARS-CoV-2 antibodies into broad-spectrum inhibitors.

Omicron and its subvariants have rendered most authorized monoclonal antibody-based treatments for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ineffective, highlighting the need for biologics capable of overcoming SARS-CoV-2 evolution. These mostly ineffective antibodies target variable epitopes. Here we describe broad-spectrum SARS-CoV-2 inhibitors developed by tethering the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), to known non-neutralizing antibodies that target highly conserved epitopes in the viral spike protein. These inhibitors, called receptor-blocking conserved non-neutralizing antibodies (ReconnAbs), potently neutralize all SARS-CoV-2 variants of concern (VOCs), including Omicron. Neutralization potency is lost when the linker joining the binding and inhibitory ReconnAb components is severed. In addition, a bi-functional ReconnAb, made by linking ACE2 to a bi-specific antibody targeting two non-overlapping conserved epitopes, defined here, shows sub-nanomolar neutralizing activity against all VOCs, including Omicron and BA.2. Given their conserved targets and modular nature, ReconnAbs have the potential to act as broad-spectrum therapeutics against SARS-CoV-2 and other emerging pandemic diseases.

500-Gray γ-Irradiation May Increase Adhesion Strength of Lyophilized Cadaveric Split-Thickness Skin Graft to Wound Bed.

BACKGROUND: Human cadaveric skin grafts are considered as the "gold standard" for temporary wound coverage because they provide a more conductive environment for natural wound healing. Lyophilization, packing, and terminal sterilization with gamma-ray can facilitate the application of cadaveric split-thickness skin grafts, but may alter the adhesion properties of the grafts. In a pilot study, we found that 500 Gy γ-irradiation seemed not to reduce the adherence between the grafts and wound beds. AIM AND OBJECTIVES: We conducted this experiment to compare the adherences of lyophilized, 500-Gy γ-irradiated skin grafts to that of lyophilized, nonirradiated grafts. MATERIALS AND METHODS: Pairs of wounds were created over the backs of Sprague- Dawley rats. Pairs of "lyophilized, 500-Gy γ-irradiated" and "lyophilized, nonirradiated" cadaveric split-thickness skin grafts were fixed to the wound beds. Adhesion strength between the grafts and the wound beds was measured and compared. RESULTS: On post-skin-graft day 7 and day 10, the adhesion strength of γ-irradiated grafts was greater than that of the nonirradiated grafts. CONCLUSIONS: Because lyophilized cadaveric skin grafts can be vascularized and the collagen of its dermal component can be remodeled after grafting, the superior adhesion strength of 500-Gy γ-irradiated grafts can be explained by the collagen changes from irradiation.

A New Form of Artificial Skin to Promote Permanent Wound Coverage: A Preliminary Report.

BACKGROUND: Although tendon-exposed or bone-exposed wounds can be resurfaced with flaps, such surgeries may not be feasible in patients with poor general or local conditions. Biosynthetic artificial skin is an alternative for critical wound coverage. We designed a new artificial skin bilayer to close difficult wounds permanently. AIM AND OBJECTIVES: This study compares incorporation and wound contraction between silicone acellular porcine dermis (SAPD) and the Integra graft (Integra Life Sciences Corp., Billerica, Mass) in a rat model. MATERIALS AND METHODS: The SAPD was manufactured according to our previously described standard procedures. Integra grafts were obtained commercially. We included 24 male adult Sprague-Dawley rats and divided them into 2 groups. After creating a 3 × 4-cm full-thickness wound on the back, we transplanted the same-sized SAPD and Integra grafts onto the rat wounds. Autologous full-thickness skin (FTS) was grafted onto the acellular porcine dermal matrix (APDM) of the SAPD and the Integra dermal matrix (IDM) 2 weeks later. We measured the wound size and contraction rate of recipient wounds, studied the incorporation of FTS on the dermal matrix, and did pathological examination. Generalized estimating equations were used to assess the data from repeated wound and scar contraction measurements using SAS v9.2. RESULTS: The sizes of wounds of both groups decreased over time. No difference in wound contraction was observed between the SAPD and Integra groups at weeks 2, 4, or 6 after grafting. However, the contraction rates in both groups increased significantly. The pathological examination showed that the FTS was well incorporated in the APDM and IDM. The recipient wounds showed new vessels and cell infiltration in the new matrix, but no severe inflammation. Skin appendages were regenerating in the FTS. There was no rejection sign. CONCLUSIONS: Both SAPD and Integra are double-layered artificial skin products. Our results demonstrate that APDM and IDM are good templates and show excellent incorporation with autologous FTS graft. The results also demonstrated gradual wound contraction over time, but the contraction rate was not different between SAPD and Integra 6 weeks after grafting in a rat model.

Formulation development and comparability studies with an aluminum-salt adjuvanted SARS-CoV-2 spike ferritin nanoparticle vaccine antigen produced from two different cell lines.

The development of safe and effective second-generation COVID-19 vaccines to improve affordability and storage stability requirements remains a high priority to expand global coverage. In this report, we describe formulation development and comparability studies with a self-assembled SARS-CoV-2 spike ferritin nanoparticle vaccine antigen (called DCFHP), when produced in two different cell lines and formulated with an aluminum-salt adjuvant (Alhydrogel, AH). Varying levels of phosphate buffer altered the extent and strength of antigen-adjuvant interactions, and these formulations were evaluated for their (1) in vivo performance in mice and (2) in vitro stability profiles. Unadjuvanted DCFHP produced minimal immune responses while AH-adjuvanted formulations elicited greatly enhanced pseudovirus neutralization titers independent of ∼100%, ∼40% or ∼10% of the DCFHP antigen adsorbed to AH. These formulations differed, however, in their in vitro stability properties as determined by biophysical studies and a competitive ELISA for measuring ACE2 receptor binding of AH-bound antigen. Interestingly, after one month of 4°C storage, small increases in antigenicity with concomitant decreases in the ability to desorb the antigen from the AH were observed. Finally, we performed a comparability assessment of DCFHP antigen produced in Expi293 and CHO cells, which displayed expected differences in their N-linked oligosaccharide profiles. Despite consisting of different DCFHP glycoforms, these two preparations were highly similar in their key quality attributes including molecular size, structural integrity, conformational stability, binding to ACE2 receptor and mouse immunogenicity profiles. Taken together, these studies support future preclinical and clinical development of an AH-adjuvanted DCFHP vaccine candidate produced in CHO cells.

Formulation development and comparability studies with an aluminum-salt adjuvanted SARS-CoV-2 Spike ferritin nanoparticle vaccine antigen produced from two different cell lines.

The development of safe and effective second-generation COVID-19 vaccines to improve affordability and storage stability requirements remains a high priority to expand global coverage. In this report, we describe formulation development and comparability studies with a self-assembled SARS-CoV-2 spike ferritin nanoparticle vaccine antigen (called DCFHP), when produced in two different cell lines and formulated with an aluminum-salt adjuvant (Alhydrogel, AH). Varying levels of phosphate buffer altered the extent and strength of antigen-adjuvant interactions, and these formulations were evaluated for their (1) in vivo performance in mice and (2) in vitro stability profiles. Unadjuvanted DCFHP produced minimal immune responses while AH-adjuvanted formulations elicited greatly enhanced pseudovirus neutralization titers independent of ∼100%, ∼40% or ∼10% of the DCFHP antigen adsorbed to AH. These formulations differed, however, in their in vitro stability properties as determined by biophysical studies and a competitive ELISA for measuring ACE2 receptor binding of AH-bound antigen. Interestingly, after one month of 4°C storage, small increases in antigenicity with concomitant decreases in the ability to desorb the antigen from the AH were observed. Finally, we performed a comparability assessment of DCFHP antigen produced in Expi293 and CHO cells, which displayed expected differences in their N-linked oligosaccharide profiles. Despite consisting of different DCFHP glycoforms, these two preparations were highly similar in their key quality attributes including molecular size, structural integrity, conformational stability, binding to ACE2 receptor and mouse immunogenicity profiles. Taken together, these studies support future preclinical and clinical development of an AH-adjuvanted DCFHP vaccine candidate produced in CHO cells.

A ferritin-based COVID-19 nanoparticle vaccine that elicits robust, durable, broad-spectrum neutralizing antisera in non-human primates.

While the rapid development of COVID-19 vaccines has been a scientific triumph, the need remains for a globally available vaccine that provides longer-lasting immunity against present and future SARS-CoV-2 variants of concern (VOCs). Here, we describe DCFHP, a ferritin-based, protein-nanoparticle vaccine candidate that, when formulated with aluminum hydroxide as the sole adjuvant (DCFHP-alum), elicits potent and durable neutralizing antisera in non-human primates against known VOCs, including Omicron BQ.1, as well as against SARS-CoV-1. Following a booster ~one year after the initial immunization, DCFHP-alum elicits a robust anamnestic response. To enable global accessibility, we generated a cell line that can enable production of thousands of vaccine doses per liter of cell culture and show that DCFHP-alum maintains potency for at least 14 days at temperatures exceeding standard room temperature. DCFHP-alum has potential as a once-yearly (or less frequent) booster vaccine, and as a primary vaccine for pediatric use including in infants.

A ferritin-based COVID-19 nanoparticle vaccine that elicits robust, durable, broad-spectrum neutralizing antisera in non-human primates.

While the rapid development of COVID-19 vaccines has been a scientific triumph, the need remains for a globally available vaccine that provides longer-lasting immunity against present and future SARS-CoV-2 variants of concern (VOCs). Here, we describe DCFHP, a ferritin-based, protein-nanoparticle vaccine candidate that, when formulated with aluminum hydroxide as the sole adjuvant (DCFHP-alum), elicits potent and durable neutralizing antisera in non-human primates against known VOCs, including Omicron BQ.1, as well as against SARS-CoV-1. Following a booster ∻one year after the initial immunization, DCFHP-alum elicits a robust anamnestic response. To enable global accessibility, we generated a cell line that can enable production of thousands of vaccine doses per liter of cell culture and show that DCFHP-alum maintains potency for at least 14 days at temperatures exceeding standard room temperature. DCFHP-alum has potential as a once-yearly booster vaccine, and as a primary vaccine for pediatric use including in infants.

Probing the Fractal Pattern of Heartbeats in Drosophila Pupae by Visible Optical Recording System.

Judiciously tuning heart rates is critical for regular cardiovascular function. The fractal pattern of heartbeats - a multiscale regulation in instantaneous fluctuations - is well known for vertebrates. The most primitive heart system of the Drosophila provides a useful model to understand the evolutional origin of such a fractal pattern as well as the alterations of fractal pattern during diseased statuses. We developed a non-invasive visible optical heart rate recording system especially suitable for long-term recording by using principal component analysis (PCA) instead of fluorescence recording system to avoid the confounding effect from intense light irradiation. To deplete intracellular Ca(2+) levels, the expression of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) was tissue-specifically knocked down. The SERCA group shows longer heart beat intervals (Mean ± SD: 1009.7 ± 151.6 ms) as compared to the control group (545.5 ± 45.4 ms, p < 0.001). The multiscale correlation of SERCA group (scaling exponent: 0.77 ± 0.07), on the other hand, is weaker than that of the control Drosophila (scaling exponent: 0.85 ± 0.03) (p = 0.016).

Non-invasive Drosophila ECG recording by using eutectic gallium-indium alloy electrode: a feasible tool for future research on the molecular mechanisms involved in cardiac arrhythmia.

BACKGROUND: Drosophila heart tube is a feasible model for cardiac physiological research. However, obtaining Drosophila electrocardiograms (ECGs) is difficult, due to the weak signals and limited contact area to apply electrodes. This paper presents a non-invasive Gallium-Indium (GaIn) based recording system for Drosophila ECG measurement, providing the heart rate and heartbeat features to be observed. This novel, high-signal-quality system prolongs the recording time of insect ECGs, and provides a feasible platform for research on the molecular mechanisms involved in cardiovascular diseases. METHODS: In this study, two types of electrode, tungsten needle probes and GaIn electrodes, were used respectively to noiselessly conduct invasive and noninvasive ECG recordings of Drosophila. To further analyze electrode properties, circuit models were established and simulated. By using electromagnetic shielded heart signal acquiring system, consisted of analog amplification and digital filtering, the ECG signals of three phenotypes that have different heart functions were recorded without dissection. RESULTS AND DISCUSSION: The ECG waveforms of different phenotypes of Drosophila recorded invasively and repeatedly with n value (n>5) performed obvious difference in heart rate. In long period ECG recordings, non-invasive method implemented by GaIn electrodes acts relatively stable in both amplitude and period. To analyze GaIn electrode, the correctness of GaIn electrode model established by this paper was validated, presenting accuracy, stability, and reliability. CONCLUSIONS: Noninvasive ECG recording by GaIn electrodes was presented for recording Drosophila pupae ECG signals within a limited contact area and signal strength. Thus, the observation of ECG changes in normal and SERCA-depleted Drosophila over an extended period is feasible. This method prolongs insect survival time while conserving major ECG features, and provides a platform for electrophysiological signal research on the molecular mechanism involved in cardiac arrhythmia, as well as research related to drug screening and development.