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A mucosal route of vaccination could prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication at the site of infection and limit transmission. We compared protection against heterologous XBB.1.16 challenge in nonhuman primates (NHPs) ~5 months following intramuscular boosting with bivalent mRNA encoding WA1 and BA.5 spike proteins or mucosal boosting with a WA1-BA.5 bivalent chimpanzee adenoviral-vectored vaccine delivered by intranasal or aerosol device. NHPs boosted by either mucosal route had minimal virus replication in the nose and lungs, respectively. By contrast, protection by intramuscular mRNA was limited to the lower airways. The mucosally delivered vaccine elicited durable airway IgG and IgA responses and, unlike the intramuscular mRNA vaccine, induced spike-specific B cells in the lungs. IgG, IgA and T cell responses correlated with protection in the lungs, whereas mucosal IgA alone correlated with upper airway protection. This study highlights differential mucosal and serum correlates of protection and how mucosal vaccines can durably prevent infection against SARS-CoV-2., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Objective: The aim of the study was to describe laparoscopic ultrasound (LUS) to guide microwave ablation (MWA) of hepatocellular carcinoma (HCC) in a dog., Animals: A 13-year-old female spayed Husky., Study Design: Case report., Methods: The dog was referred for laparoscopic MWA of a caudate lobe HCC measuring 2.7 cm × 1.9 cm× 2.3 cm. Laparoscopic ultrasound was used to guide percutaneous placement of a MWA probe within the caudate lobe mass. LUS was used to determine the tumor margins for placement of the probe within the medial and lateral aspects as well as the center of the mass. Additionally, LUS allowed visualization of vessels within the tumor so they could be avoided during probe placement., Results: Safe ablation was accomplished, and the dog recovered uneventfully with no complications. Follow-up computed tomography (CT) 3- and 12-months postoperatively revealed complete ablation of the caudate lobe mass. However, progressive increase in size of the right medial lobe mass occurred. Right medial liver lobectomy was performed 13 months after MWA, with a histopathologic diagnosis of HCC. The dog was euthanized 777 days post MWA and 1445 days post initial left lateral lobectomy for HCC., Conclusion: Laparoscopic ultrasound was useful in guiding complete and safe MWA of an HCC in this dog. Laparoscopic ultrasound should be considered when performing laparoscopic MWA of liver lesions in dogs., (© 2024 American College of Veterinary Surgeons.)

The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compromised neutralizing antibody responses elicited by prior infection or vaccination and abolished the utility of most monoclonal antibody therapeutics. We previously described a computationally-designed, homotrimeric miniprotein inhibitor, designated TRI2-2, that protects mice against pre-Omicron SARS-CoV-2 variants. Here, we show that TRI2-2 exhibits pan neutralization of variants that evolved during the 4.5 years since the emergence of SARS-CoV-2 and protects mice against BQ.1.1, XBB.1.5 and BA.2.86 challenge when administered post-exposure by an intranasal route. The resistance of TRI2-2 to viral escape and its direct delivery to the upper airways rationalize a path toward clinical advancement., Competing Interests: J.B.C., Y.J.P., R.R., D.B., M.S.D. and D.V. are co-inventors on a patent application that incorporates discoveries described in this article (application no.: PCT/US2021/034069, title: SARS-CoV-2 inhibitors). M.S.D. is a consultant or advisor for Inbios, Vir Biotechnology, IntegerBio, Moderna, Merck, and GlaxoSmithKline. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Vir Biotechnology, Moderna, Emergent BioSolutions, and IntegerBio.

Objective: To describe the use of near-infrared angiography (NIRFA) to identify the vascularization of three canine axial pattern flaps (APFs) omocervical (OMO), thoracodorsal (THO), and caudal superficial epigastric (CSE); to establish a vascular fluorescence pattern (VFP) grading system; and to evaluate the effect of NIRFA on surgeon flap dimension planning compared to traditional landmark palpation (LP) and visualization assessments., Study Design: Experimental study., Animals: A total of 15 healthy, client-owned dogs., Methods: Dogs were sedated and flap sites were clipped. LP-based margins were drawn and preinjection images were recorded. Indocyanine green (ICG) was administered and VFP images were recorded. VFP scores were determined by five surgeons. Margin alterations were performed based on NIRFA-ICG images. Altered measurements were compared between LP and NIRFA-ICG images., Results: Vascularization of the CSE flap was most visible with NIRFA with VFP scores 4/4 for 13/15 dogs. Intersurgeon agreement for VFP grades was poorest for THO (ICC = 0.35) and intermediate for OMO (ICC = 0.49) flaps. Surgeons were more likely to adjust dimensions for CSE flaps relative to OMO (OR 17.3, 95% CI: 6.2, 47.8) or THO (25.5; 8.6, 75.7)., Conclusion: Using a grading system, we demonstrated that the CSE flap was most visible. Surgeons were more likely to adjust the LP-CSE flap margins based on fluorescence patterns and were more likely to rely on LP when visualization scores were low., Clinical Significance: NIRFA has possible applications identifying some direct cutaneous arteries of APFs and their associated angiosomes in real-time. Further investigation is indicated to study NIRFA's potential to improve patient specific APF planning., (© 2024 American College of Veterinary Surgeons.)

Objective: To compare survival and report perioperative complications in cats undergoing surgery for small intestinal (SI) linear (LFBO) and discrete (DFBO) foreign body obstructions (FBO). To report success of a red rubber catheter technique (RRCT) to remove LFBOs., Study Design: Retrospective study., Animals: Client-owned cats (n = 169)., Methods: Medical records of cats undergoing surgery for SI FBO from a veterinary teaching hospital between February 2012 and January 2023 were classified as LFBO, DFBO, or both linear and discrete FBO (BFBO). Signalment and perioperative data were collected., Results: Preoperative hypoalbuminemia (LFBO: n = 1/6; DFBO: n = 5/6) and septic peritonitis (LFBO: n = 2/4; DFBO: n = 0/4; BFBO: n = 2/4) were rare. Intraoperative hypotension did not differ between LFBOs and DFBOs (p = .4756). RRCT was successful in 20/24 attempts of LFBO removal. Three cats were euthanized intraoperatively (LFBO: 1; DFBO: 1; BFBO: 1). Postoperatively, two cats (DFBO) experienced intestinal dehiscence and two cats (DFBO) died or were euthanized. Survival to discharge (p = 1.0000) and postoperative complications (p = .1386) did not differ between LFBOs and DFBOs., Conclusions: Postoperative complications and survival did not differ between cats with LFBOs and DFBOs. Intestinal dehiscence secondary to FBO in cats is rare. A RRCT can be successful in many cats with LFBOs., Clinical Significance: Cats with LFBOs and DFBOs have similar postoperative complication rates and survival to discharge when preoperative septic peritonitis is not present. Intestinal dehiscence is rare, which is important when discussing surgical prognosis with owners. A RRCT can be considered to remove LFBOs when there is concern for multiple enterotomies., (© 2024 American College of Veterinary Surgeons.)

Objective: To evaluate the outcomes and complications of video-assisted thoracoscopic (VATS) treatment of chylothorax in cats., Study Design: Multi-institutional retrospective study., Animals: Fifteen client-owned cats., Methods: The medical records of cats undergoing thoracoscopic thoracic duct ligation (TDL) for treatment of idiopathic chylothorax were reviewed. Cats undergoing additional procedures including thoracoscopic pericardectomy and/or laparoscopic cisterna chyli ablation (CCA)_were included. Follow up was obtained through communication with the referring veterinarian or owner., Results: All cats underwent thoracoscopic TDL. Thirteen cats underwent simultaneous pericardectomy and two cats underwent laparoscopic CCA without pericardectomy. Conversion from a thoracoscopic to open approach was necessary in 2/15 (13%) of thoracic duct ligations and 1/11 (9%) of pericardectomies. The most common postoperative complication was persistent pleural effusion in five cats (33%). Four of 15 cats (27%) died or were euthanized prior to hospital discharge following surgery. Recurrence of effusion occurred in 1/7 (14%) of cats that sustained resolution of the effusion at the time of surgery with a median follow up of 8 months. The overall mortality attributed to chylothorax was 47%., Conclusion: Thoracoscopic treatment of idiopathic chylothorax resulted in a low incidence of intraoperative complications or conversion in the study population; however, mortality related to feline idiopathic chylothorax remained high., Clinical Significance: While VATS treatment of idiopathic chylothorax is technically feasible, further consideration of the underlying pathology and current treatment algorithm is needed to improve outcomes as this remains a frustrating disease to treat in the feline population., (© 2024 The Authors. Veterinary Surgery published by Wiley Periodicals LLC on behalf of American College of Veterinary Surgeons.)

Objectives: To describe the clinical outcome of dogs diagnosed with concurrent discospondylitis/vertebral physitis and congenital intrahepatic portosystemic shunts., Materials and Methods: Medical records from two academic institutions were searched for dogs diagnosed with discospondylitis and/or vertebral physitis, and a concurrent intrahepatic portosystemic shunt. Dogs were excluded if they did not undergo attenuation of their shunt, did not have a single congenital intrahepatic shunt and did not have at least 90 days of follow-up., Results: Six dogs met the inclusion criteria and were included in the study. Discospondylitis alone was diagnosed in four dogs, vertebral physitis alone in one dog and both discospondylitis and vertebral physitis in one dog. Three dogs had a right divisional intrahepatic portosystemic shunt, and three dogs had a left divisional intrahepatic portosystemic shunt. Median duration of antimicrobial therapy was 112 days (range 14 to 240 days). Clinical resolution of discospondylitis and vertebral physitis was noted in all dogs. Endovascular attenuation was performed in all dogs a median of 82 days after presentation (range 1 to 317 days). No perioperative or postoperative complications occurred. All dogs were alive at the last available follow-up a median of 513 days after presentation (range 224 to 1504 days) and free of clinical signs associated with discospondylitis or vertebral physitis, as well as their portosystemic shunt., Clinical Significance: Dogs with intrahepatic portosystemic shunts may concurrently develop discospondylitis and vertebral physitis. With antimicrobial therapy and endovascular embolisation of their portosystemic shunt, all dogs in this study had a good outcome with clinical resolution of both disease processes. However, long-term follow-up was not obtained in all cases., (© 2024 British Small Animal Veterinary Association.)

Immune imprinting is a phenomenon in which prior antigenic experiences influence responses to subsequent infection or vaccination 1,2 . The effects of immune imprinting on serum antibody responses after boosting with variant-matched SARS-CoV-2 vaccines remain uncertain. Here we characterized the serum antibody responses after mRNA vaccine boosting of mice and human clinical trial participants. In mice, a single dose of a preclinical version of mRNA-1273 vaccine encoding Wuhan-1 spike protein minimally imprinted serum responses elicited by Omicron boosters, enabling generation of type-specific antibodies. However, imprinting was observed in mice receiving an Omicron booster after two priming doses of mRNA-1273, an effect that was mitigated by a second booster dose of Omicron vaccine. In both SARS-CoV-2-infected and uninfected humans who received two Omicron-matched boosters after two or more doses of the prototype mRNA-1273 vaccine, spike-binding and neutralizing serum antibodies cross-reacted with Omicron variants as well as more distantly related sarbecoviruses. Because serum neutralizing responses against Omicron strains and other sarbecoviruses were abrogated after pre-clearing with Wuhan-1 spike protein, antibodies induced by XBB.1.5 boosting in humans focus on conserved epitopes targeted by the antecedent mRNA-1273 primary series. Thus, the antibody response to Omicron-based boosters in humans is imprinted by immunizations with historical mRNA-1273 vaccines, but this outcome may be beneficial as it drives expansion of cross-neutralizing antibodies that inhibit infection of emerging SARS-CoV-2 variants and distantly related sarbecoviruses., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs 1-3 and revealed how quickly viral escape can curtail effective options 4,5 . When the SARS-CoV-2 Omicron variant emerged in 2021, many antibody drug products lost potency, including Evusheld and its constituent, cilgavimab 4-6 . Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination 4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign and renew the efficacy of COV2-2130 against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and subsequent variants of concern, and provides protection in vivo against the strains tested: WA1/2020, BA.1.1 and BA.5. Deep mutational scanning of tens of thousands of pseudovirus variants reveals that 2130-1-0114-112 improves broad potency without increasing escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Our computational approach does not require experimental iterations or pre-existing binding data, thus enabling rapid response strategies to address escape variants or lessen escape vulnerabilities., (© 2024. The Author(s).)

Objective: To describe a laparoscopic technique and outcome for partial pancreatectomy in cats., Study Design: Prospective cohort study., Animals: Nine cats., Methods: Laparoscopic pancreatectomy was performed using a single incision laparoscopic surgery port and an additional 5.5 mm port. The left pancreatic limb was dissected, sealed and divided at the level of the splenic vein insertion to the portal vein using a harmonic device. Surgical time and complications were recorded. The weight and length of the resected pancreatic limb was recorded. Pre- and postoperative trypsin-like immunoreactivity (TLI), pancreatic lipase immunoreactivity (PLI), and hemoglobin A1C were documented., Results: Laparoscopic partial pancreatectomy was performed successfully in all cats. One grade 1 intraoperative complication occurred (1/9; 11%) resulting in minor hemorrhage from a caudal splenic vein branch. A grade 2 postoperative complication occurred within 3 days after surgery in one cat (1/9; 11%), involving localized, sterile peritonitis in the region of the pancreatic angle. Signs resolved with conservative management. No cats exhibited signs of pancreatitis postoperatively. Long-term, mean TLI decreased by 37% ± 38% (p = .03) following partial pancreatectomy, while PLI and A1C were unchanged. All cats were alive and clinically well at last follow-up 250 to 446 days following surgery., Conclusions: Laparoscopic partial pancreatectomy using a harmonic device is effective in cats, and offers a minimally-invasive alternative to open surgical pancreatectomy techniques. Laparoscopic pancreatectomy of the left limb results in adequate exocrine and endocrine function in the long-term., (© 2023 American College of Veterinary Surgeons.)

Protein-based virus-like particles (P-VLPs) are commonly used to spatially organize antigens and enhance humoral immunity through multivalent antigen display. However, P-VLPs are thymus-dependent antigens that are themselves immunogenic and can induce B cell responses that may neutralize the platform. Here, we investigate thymus-independent DNA origami as an alternative material for multivalent antigen display using the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, the primary target of neutralizing antibody responses. Sequential immunization of mice with DNA-based VLPs (DNA-VLPs) elicits protective neutralizing antibodies to SARS-CoV-2 in a manner that depends on the valency of the antigen displayed and on T cell help. Importantly, the immune sera do not contain boosted, class-switched antibodies against the DNA scaffold, in contrast to P-VLPs that elicit strong B cell memory against both the target antigen and the scaffold. Thus, DNA-VLPs enhance target antigen immunogenicity without generating scaffold-directed immunity and thereby offer an important alternative material for particulate vaccine design., (© 2024. The Author(s).)

Objective: We tested the feasibility and effectiveness of a percutaneous atrial transseptal extracorporeal membrane oxygenation (ECMO) cannulation strategy in a right ventricular failure (RVF) model., Methods: We performed 4 nonsurvival porcine experiments. Percutaneous transseptal access was achieved using a steerable introducer. For guidance, we used fluoroscopy, transesophageal echocardiogram (TEE), and intracardiac echocardiography (ICE). A ProtekDuo rapid deployment cannula (LivaNova, London, UK) was advanced across the septum into the left atrium by 2 to 3 cm. Pulmonary hypertension (PH) was induced by partially clamping the pulmonary artery. ECMO flow was cycled from high (2 to 3 L/min) to low (0.2 to 0.3 L/min) over 2 to 3 hours., Results: Transseptal access using TEE and fluoroscopy was successful in 1 animal and unsuccessful in 1 animal. ICE provided optimal visualization for the remaining 2 animals. Mean arterial pressure (MAP) was associated immediately and consistently with high versus low ECMO flow rate (mean difference: 29 ± 3.1 mm Hg, P = 0.004) but was not restored to baseline values. RV pressure values were dynamic. Given time to equilibrate, mean RV pressure was restored to a baseline level., Conclusions: Percutaneous right atrium to left atrium transseptal cannulation relieved PH-RVF. MAP was restored to a viable level, and mean RV pressure was restored to a baseline level. Transseptal ECMO shows promise as a cannulation strategy to bridge patients with PH-RVF to lung transplant., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The continued evolution and emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have resulted in challenges to vaccine and antibody efficacy. The emergence of each new variant necessitates the need to re-evaluate and refine animal models used for countermeasure testing. Here, we tested a recently circulating SARS-CoV-2 Omicron lineage variant, BQ.1.1, in multiple rodent models including K18-human ACE2 (hACE2) transgenic, C57BL/6J, and 129S2 mice, and Syrian golden hamsters. In contrast to a previously dominant BA.5.5 Omicron variant, inoculation of K18-hACE2 mice with BQ.1.1 resulted in substantial weight loss, a characteristic seen in pre-Omicron variants. BQ.1.1 also replicated to higher levels in the lungs of K18-hACE2 mice and caused greater lung pathology than the BA.5.5 variant. However, in C57BL/6J mice, 129S2 mice, and Syrian hamsters, BQ.1.1 did not cause increased respiratory tract infection or disease compared to animals administered BA.5.5. Moreover, the rates of direct contact or airborne transmission in hamsters were not significantly different after BQ.1.1 and BA.5.5 infections. Taken together, these data suggest that the BQ.1.1 Omicron variant has increased virulence in rodent species that express hACE2, possibly due to the acquisition of unique spike mutations relative to earlier Omicron variants. IMPORTANCE As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, there is a need to rapidly assess the efficacy of vaccines and antiviral therapeutics against newly emergent variants. To do so, the commonly used animal models must also be re-evaluated. Here, we determined the pathogenicity of the BQ.1.1 SARS-CoV-2 variant in multiple SARS-CoV-2 animal models including transgenic mice expressing human ACE2 (hACE2), two strains of conventional laboratory mice, and Syrian hamsters. While BQ.1.1 and BA.5.5 infection resulted in similar levels of viral burden and clinical disease in hamsters and the conventional strains of laboratory mice tested, increases in lung infection were detected in hACE2-expressing transgenic mice, which corresponded with greater levels of pro-inflammatory cytokines and lung pathology. Taken together, our data highlight important differences in two closely related Omicron SARS-CoV-2 variant strains and provide a foundation for evaluating countermeasures., Competing Interests: M.S.D. is a consultant for Inbios, Vir Biotechnology, Ocugen, Topspin, Moderna, and Immunome. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Vir Biotechnology, Emergent BioSolutions, Generate Biomedicines, and Moderna. The Boon laboratory has received unrelated funding support in sponsored research agreements from GreenLight Biosciences Inc., Moderna, and AbbVie Inc. All other authors declare no competing financial interests.

Currently circulating SARS-CoV-2 variants have acquired convergent mutations at hot spots in the receptor-binding domain 1 (RBD) of the spike protein. The effects of these mutations on viral infection and transmission and the efficacy of vaccines and therapies remains poorly understood. Here we demonstrate that recently emerged BQ.1.1 and XBB.1.5 variants bind host ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1, XBB.1 and BN.1 RBDs bound to the fragment antigen-binding region of the S309 antibody (the parent antibody for sotrovimab) and human ACE2 explain the preservation of antibody binding through conformational selection, altered ACE2 recognition and immune evasion. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1 and hamsters challenged with XBB.1.5. Vaccine-elicited human plasma antibodies cross-react with and trigger effector functions against current Omicron variants, despite a reduced neutralizing activity, suggesting a mechanism of protection against disease, exemplified by S309. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring the role of persistent immune imprinting., (© 2023. The Author(s).)

Objective: To describe the feasibility and technique for performing laparoscopic ultrasound (LUS) of the liver in dogs., Animals: 12 client-owned dogs presenting for elective laparoscopic surgery from January 1, 2022, to October 31, 2022., Methods: Laparoscopic exploration and LUS of the liver were performed in all dogs. Dogs were positioned in reverse Trendelenburg and laterally rotated to facilitate access to all liver lobes. Time to perform laparoscopic exploration and LUS, ability to visualize and access each liver lobe entirely, and any complications were recorded. Each dog underwent an elective laparoscopic procedure. The surgeon completed a National Aeronautics and Space Administration Task Load Index (NASA-TLX) questionnaire after surgery., Results: Mean body weight was 25.9 kg (SD, ± 4.1 kg; range, 5.7 to 62 kg). All liver lobes were scanned to the level of the hilus in 10/12 dogs. In 2 dogs, the caudate lobe could not be completely imaged. Median time to perform LUS was 9 minutes (IQR, 5 to 16.5 minutes), and median NASA-TLX score was 9/100 (IQR, 6.3 to 20). There was a significantly strong negative correlation between time to perform LUS (r = -0.77; P = .0037) and NASA-TLX score (r = -0.84; P = .0006) with trial number. Minor complications occurred in 2 dogs during laparoscopic exploration. No complications occurred during LUS., Clinical Relevance: LUS was feasible and safe in all dogs. The right lateral and caudate lobes were occasionally challenging to access. Technical demand and time to perform LUS improved with experience, suggesting a learning curve. Evaluation of LUS in dogs with clinical disease is warranted.

Objective: To report the short- and long-term outcomes of laparoscopic adrenalectomy (LA) for resection of unilateral adrenal masses and to document risk factors for conversion and peri- and postoperative morbidity., Animals: 255 client-owned dogs., Methods: Dogs were included if LA was attempted for resection of a unilateral adrenal mass. Medical records were evaluated and relevant data were reported, including complications, conversion, perioperative death, and long-term outcomes. Signalment, clinicopathological data, and surgical experience were factors statistically evaluated for possible associations with capsular penetration during surgery, conversion, surgical time, duration of hospital stay, death prior to discharge, mass recurrence, and survival time., Results: 155 dogs had left-sided tumors, and 100 had right-sided tumors. Conversion to an open approach was performed in 9.4% of cases. Capsular penetration (19.2%) and major hemorrhage (5.4%) were the most prevalent intraoperative complications. Of the dogs operated on, 94.9% were discharged from the hospital. Lesion side, portion of the gland affected, and surgeon experience influenced surgical time. Conversion rate increased with increasing body condition score and lesion size. Risk of death prior to discharge increased with increasing lesion size. Risk of conversion and death prior to discharge were lower when performed by more experienced surgeons. Capsular penetration during LA increased the risk of tumor recurrence., Clinical Relevance: LA for resection of unilateral adrenal masses is associated with excellent outcomes in experienced centers. Surgeons with greater experience with LA have lower surgical times, conversion rates, and risk of death prior to discharge.

Double aortic arch (DAA) is a rare, congenital anomaly in small animals, resulting in a complete vascular ring encircling the esophagus and trachea, and subsequent compression of these organs. Few studies have reported utilizing CT angiography (CTA) for diagnosing DAA in dogs; thus, the imaging features are currently lacking in the literature. The objectives of this retrospective, multicenter, descriptive case series were to report the clinical and CTA characteristics of DAA in surgically treated cases. Medical records and CTA images were reviewed. Six juvenile dogs met the inclusion criteria (median age: 4.2 months; range: 2-5 months). The most common clinical signs included chronic regurgitation (100%), decreased body condition (67%), and coughing (50%). Common CTA features of DAA included a dominant left aortic arch (median diameter: 8.1 mm) and minor right aortic arch (median diameter: 4.3 mm; 83%), an aberrant right subclavian artery arising directly from the right aortic arch (83%), segmental esophageal constriction (100%), and variable degrees of dilation cranial to the heart base, and marked tracheal luminal compression (median percent change: -55%; 100%) and leftward curvature of the trachea at the level of the bifurcation of the aortic arches (100%). All dogs underwent successful surgical correction with only minor postoperative complications. Due to the similarity of clinical and imaging characteristics described to that of other forms of vascular ring anomalies (VRA), CTA is vital for the specific diagnosis of DAA in dogs., (© 2023 American College of Veterinary Radiology.)

We report the engineering and selection of two synthetic proteins-FSR16m and FSR22-for the possible treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. FSR16m and FSR22 are trimeric proteins composed of DARPin SR16m or SR22 fused with a T4 foldon. Despite selection by a spike protein from a now historical SARS-CoV-2 strain, FSR16m and FSR22 exhibit broad-spectrum neutralization of SARS-CoV-2 strains, inhibiting authentic B.1.351, B.1.617.2 and BA.1.1 viruses, with respective IC 50 values of 3.4, 2.2 and 7.4 ng ml -1 for FSR16m. Cryo-EM structures revealed that these DARPins recognize a region of the receptor-binding domain (residues 456, 475, 486, 487 and 489) overlapping a critical portion of the angiotensin-converting enzyme 2 (ACE2)-binding surface. K18-hACE2 transgenic mice inoculated with B.1.617.2 and receiving intranasally administered FSR16m showed less weight loss and 10-100-fold lower viral burden in upper and lower respiratory tracts. The strong and broad neutralization potency makes FSR16m and FSR22 promising candidates for the prevention and treatment of infection by SARS-CoV-2., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Objective: To describe outcomes of small- and toy-breed dogs with a congenital intrahepatic portosystemic shunt (IHPSS) treated with percutaneous transvenous coil embolization (PTCE)., Animals: 20 small- and toy-breed dogs with an IHPSS., Procedures: All dogs underwent CT angiography for shunt evaluation as well as PTCE. Medical records were reviewed for pertinent data, and owners and primary veterinarians were contacted for long-term follow-up information., Results: Dogs ranged from 1.5 to 10.0 kg (mean ± SD, 6.32 ± 2.57 kg) in weight. The equipment used to perform PTCE tended to be smaller than that previously described for larger breed dogs. Intra- and postoperative complication rates were 20% (4/20) and 5% (1/20), respectively, and included hypotension, bradycardia, hypercapnia, ventricular premature contractions, hypothermia, and regurgitation. Dogs were discharged a median of 3 days (range, 1 to 3 days) after surgery, and all dogs survived to discharge. Clinical signs resolved in 95% (19/20) of the dogs a median of 21 days after the procedure. One- and 2-year survival rates were 92%. Three dogs had died by the time of data collection; 2 of these dogs died of causes related to the IHPSS 267 and 1,178 days, respectively, after the procedure., Clinical Relevance: Percutaneous transvenous coil embolization was a safe and effective option for treatment of IHPSS in small- and toy-breed dogs and offered a minimally invasive alternative to open surgical techniques. Complication and survival rates in this cohort were similar to or better than those reported in previous studies evaluating PTCE and open surgical techniques for treatment of IHPSS in dogs.

Omicron variant strains encode large numbers of changes in the spike protein compared to historical SARS-CoV-2 isolates. Although in vitro studies have suggested that several monoclonal antibody therapies lose neutralizing activity against Omicron variants, the effects in vivo remain largely unknown. Here, we report on the protective efficacy against three SARS-CoV-2 Omicron lineage strains (BA.1, BA.1.1, and BA.2) of two monoclonal antibody therapeutics (S309 [Vir Biotechnology] monotherapy and AZD7442 [AstraZeneca] combination), which correspond to ones used to treat or prevent SARS-CoV-2 infections in humans. Despite losses in neutralization potency in cell culture, S309 or AZD7442 treatments reduced BA.1, BA.1.1, and BA.2 lung infection in susceptible mice that express human ACE2 (K18-hACE2) in prophylactic and therapeutic settings. Correlation analyses between in vitro neutralizing activity and reductions in viral burden in K18-hACE2 or human FcγR transgenic mice suggest that S309 and AZD7442 have different mechanisms of protection against Omicron variants, with S309 utilizing Fc effector function interactions and AZD7442 acting principally by direct neutralization. Our data in mice demonstrate the resilience of S309 and AZD7442 mAbs against emerging SARS-CoV-2 variant strains and provide insight into the relationship between loss of antibody neutralization potency and retained protection in vivo., (© 2022. The Author(s).)

Objective: To describe the embolization technique and short-term clinical outcome in dogs undergoing lymphatic embolization (LE) as part of treatment for presumptive idiopathic chylothorax (IC). Additionally, to document findings in computed tomography lymphangiography (CTLa) following embolization., Study Design: Prospective case series., Animals: Eight client-owned dogs., Methods: Dogs underwent CTLa followed by thoracic duct ligation (TDL), pericardiectomy (PC) and LE. A mixture of 3:1 lipiodol: n-butyl cyanoacrylate embolic solution was injected through a catheterized mesenteric lymphatic vessel via limited abdominal approach using intraoperative fluoroscopy. CTLa was scheduled for 12 weeks postoperatively, and long-term follow-up was obtained via telephone contact., Results: LE was technically successful in six of the eight dogs; and clinically successful in five of the six dogs. In the unsuccessful dog, a diagnosis of lymphangiosarcoma was determined, and the owners elected for euthanasia. Five dogs who underwent successful LE underwent CTLa at 12 weeks. Complete resolution of pleural effusion occurred in three dogs and scant pleural effusion in two dogs. A robust lymphatic embolus preventing antegrade continuation of radiocontrast was documented in all five dogs. Five of the six dogs that underwent LE, were alive and clinically normal at 358-960 days postoperatively., Conclusions: LE is a feasible part of treatment for dogs with IC. Additionally, a robust lymphatic embolus and lack of radiocontrast flow past the embolus was documented at 12 weeks following surgery., Clinical Significance: LE has the potential to reduce surgical failure by reducing efferent lymphatic chyle flow, occluding missed lymphatic branches and preventing the development of collateral branches., (© 2022 American College of Veterinary Surgeons.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes a number of strategies to modulate viral and host mRNA translation. Here, we used ribosome profiling in SARS-CoV-2-infected model cell lines and primary airway cells grown at an air-liquid interface to gain a deeper understanding of the translationally regulated events in response to virus replication. We found that SARS-CoV-2 mRNAs dominate the cellular mRNA pool but are not more efficiently translated than cellular mRNAs. SARS-CoV-2 utilized a highly efficient ribosomal frameshifting strategy despite notable accumulation of ribosomes within the slippery sequence on the frameshifting element. In a highly permissive cell line model, although SARS-CoV-2 infection induced the transcriptional upregulation of numerous chemokine, cytokine, and interferon-stimulated genes, many of these mRNAs were not translated efficiently. The impact of SARS-CoV-2 on host mRNA translation was more subtle in primary cells, with marked transcriptional and translational upregulation of inflammatory and innate immune responses and downregulation of processes involved in ciliated cell function. Together, these data reveal the key role of mRNA translation in SARS-CoV-2 replication and highlight unique mechanisms for therapeutic development. IMPORTANCE SARS-CoV-2 utilizes a number of strategies to modulate host responses to ensure efficient propagation. Here, we used ribosome profiling in SARS-CoV-2-infected cells to gain a deeper understanding of the translationally regulated events in infected cells. We found that although viral mRNAs are abundantly expressed, they are not more efficiently translated than cellular mRNAs. SARS-CoV-2 utilized a highly efficient ribosomal frameshifting strategy and alternative translation initiation sites that help increase the coding potential of its RNAs. In permissive cells, SARS-CoV-2 infection induced the translational repression of numerous innate immune mediators. Though the impact of SARS-CoV-2 on host mRNA translation was more subtle in primary airway cell cultures, we noted marked transcriptional and translational upregulation of inflammatory and innate immune responses and downregulation of processes involved in ciliated cell function. Together, these data provide new insight into how SARS-CoV-2 modulates innate host responses and highlight unique mechanisms for therapeutic intervention.

The protective human antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) focuses on the spike (S) protein, which decorates the virion surface and mediates cell binding and entry. Most SARS-CoV-2 protective antibodies target the receptor-binding domain or a single dominant epitope ("supersite") on the N-terminal domain (NTD). Using the single B cell technology called linking B cell receptor to antigen specificity through sequencing (LIBRA-Seq), we isolated a large panel of NTD-reactive and SARS-CoV-2-neutralizing antibodies from an individual who had recovered from COVID-19. We found that neutralizing antibodies against the NTD supersite were commonly encoded by the IGHV1-24 gene, forming a genetic cluster representing a public B cell clonotype. However, we also discovered a rare human antibody, COV2-3434, that recognizes a site of vulnerability on the SARS-CoV-2 S protein in the trimer interface (TI) and possesses a distinct class of functional activity. COV2-3434 disrupted the integrity of S protein trimers, inhibited the cell-to-cell spread of the virus in culture, and conferred protection in human angiotensin-converting enzyme 2-transgenic (ACE2-transgenic) mice against the SARS-CoV-2 challenge. This study provides insight into antibody targeting of the S protein TI region, suggesting this region may be a site of virus vulnerability.

Neutralizing antibodies (nAbs) that target the SARS-CoV-2 spike protein have received emergency use approval for treatment of COVID-19. However, with the emergence of variants of concern, there is a need for new treatment options. We report a format that enables modular assembly of bi-paratopic tetravalent nAbs with antigen-binding sites from two distinct nAbs. The tetravalent nAb purifies in high yield and exhibits biophysical characteristics that are comparable to those of clinically used therapeutic antibodies. The tetravalent nAb binds to the spike protein trimer at least 100-fold more tightly than bivalent IgGs (apparent K D  < 1 pM) and neutralizes a broad array of SARS-CoV-2 pseudoviruses, chimeric viruses, and authentic viral variants with high potency. Together, these results establish the tetravalent diabody-Fc-Fab as a robust, modular platform for rapid production of drug-grade nAbs with potencies and breadth of coverage that greatly exceed those of conventional bivalent IgGs., Competing Interests: Declaration of interests S.M. and S.S.S. are inventors on a patent application describing anti-SARS-CoV-2 antibodies and multivalent antibody formats. M.S.D. is a consultant for Inbios, Vir Biotechnology, Senda Biosciences, and Carnival Corporation and is on the Scientific Advisory Boards of Moderna and Immunome. The Diamond laboratory has received funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.

The large number of spike substitutions in Omicron lineage variants (BA.1, BA.1.1., and BA.2) could jeopardize the efficacy of SARS-CoV-2 vaccines. We evaluated in mice the protective efficacy of the Moderna mRNA-1273 vaccine against BA.1 before or after boosting. Whereas two doses of mRNA-1273 vaccine induced high levels of neutralizing antibodies against historical WA1/2020 strains, lower levels against BA.1 were associated with breakthrough infection and inflammation in the lungs. A primary vaccination series with mRNA-1273.529, an Omicron-matched vaccine, potently neutralized BA.1 but inhibited historical or other SARS-CoV-2 variants less effectively. However, boosting with either mRNA-1273 or mRNA-1273.529 vaccines increased neutralizing titers and protection against BA.1 and BA.2 infection. Nonetheless, the neutralizing antibody titers were higher, and lung viral burden and cytokines were slightly lower in mice boosted with mRNA-1273.529 and challenged with BA.1. Thus, boosting with mRNA-1273 or mRNA-1273.529 enhances protection against Omicron infection with limited differences in efficacy measured., Competing Interests: Declaration of interests M.S.D. is a consultant for Inbios, Vir Biotechnology, Senda Biosciences, and Carnival Corporation, and on the scientific advisory boards of Moderna and Immunome. The Diamond laboratory has received unrelated funding support in sponsored research agreements from J. Virol. Biotechnol., Kaleido, and Emergent BioSolutions and past support from Moderna not related to these studies. K.W., D.L., L.E.A., L.M., T.K., C.S., A.W., A.C., S.M.E., G.-Y.C., G.S.-J., I.R., A.M., Y.-T.L., and D.K.E. are employees of and shareholders in Moderna., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: To report the perioperative characteristics and outcomes of dogs undergoing laparoscopic-assisted splenectomy (LAS)., Animals: 136 client-owned dogs., Procedures: Multicentric retrospective study. Medical records of dogs undergoing LAS for treatment of naturally occurring splenic disease from January 1, 2014, to July 31, 2020, were reviewed. History, signalment, physical examination and preoperative diagnostic test results, procedural information, complications, duration of hospitalization, histopathologic diagnosis, and perioperative outcomes were recorded. Perioperative complications were defined using the Veterinary Cooperative Oncology Group - Common Terminology Criteria for Adverse Events (VCOG-CTCAE v2) guidelines., Results: LAS was performed for treatment of a splenic mass (124/136 [91%]), immune-mediated disease (7/136 [5%]), splenomegaly (4/136 [3%]), or immune-mediated disease in conjunction with a splenic mass (1/136 [1%]). Median splenic mass size was 1.3 cm3/kg body weight. Conversion to open laparotomy occurred in 5.9% (8/136) of dogs. Complications occurred in 78 dogs, with all being grade 2 or lower. Median surgical time was 47 minutes, and median postoperative hospital stay was 28 hours. All but 1 dog survived to discharge, the exception being postoperative death due to a suspected portal vein thrombus., Clinical Relevance: In the dogs of this report, LAS was associated with low rates of major complications, morbidity, and mortality when performed for a variety of splenic pathologies. Minimally invasive surgeons can consider the LAS technique to perform total splenectomy in dogs without hemoabdomen and with spleens with modest-sized splenic masses up to 55.2 cm3/kg, with minimal rates of complications, morbidity, and mortality.

Eliciting antibodies to surface-exposed viral glycoproteins can generate protective responses that control and prevent future infections. Targeting conserved sites may reduce the likelihood of viral escape and limit the spread of related viruses with pandemic potential. Here we leverage rational immunogen design to focus humoral responses on conserved epitopes. Using glycan engineering and epitope scaffolding in boosting immunogens, we focus murine serum antibody responses to conserved receptor binding motif (RBM) and receptor binding domain (RBD) epitopes following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike imprinting. Although all engineered immunogens elicit a robust SARS-CoV-2-neutralizing serum response, RBM-focusing immunogens exhibit increased potency against related sarbecoviruses, SARS-CoV, WIV1-CoV, RaTG13-CoV, and SHC014-CoV; structural characterization of representative antibodies defines a conserved epitope. RBM-focused sera confer protection against SARS-CoV-2 challenge. Thus, RBM focusing is a promising strategy to elicit breadth across emerging sarbecoviruses without compromising SARS-CoV-2 protection. These engineering strategies are adaptable to other viral glycoproteins for targeting conserved epitopes., Competing Interests: Declaration of interests B.M.H., T.M.C., and A.G.S. have filed a provisional patent for the described immunogens. M.S.D. is a consultant for Inbios, Vir Biotechnology, and Carnival Corporation and on the Scientific Advisory Boards of Moderna and Immunome. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Vir Biotechnology, Moderna, and Emergent BioSolutions., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The recent emergence of B.1.1.529, the Omicron variant 1,2 , has raised concerns of escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in preclinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of several B.1.1.529 isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2)-expressing mice and hamsters. Despite modelling data indicating that B.1.1.529 spike can bind more avidly to mouse ACE2 (refs.  3,4 ), we observed less infection by B.1.1.529 in 129, C57BL/6, BALB/c and K18-hACE2 transgenic mice than by previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease and pathology with B.1.1.529 were also milder than with historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from the SAVE/NIAID network with several B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data., (© 2022. The Author(s).)

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a serious threat to global public health, underscoring the urgency of developing effective therapies. Therapeutics and, more specifically, direct-acting antiviral development are still very much in their infancy. Here, we report that two hepatitis C virus (HCV) fusion inhibitors identified in our previous study, dichlorcyclizine and fluoxazolevir, broadly block human coronavirus entry into various cell types. Both compounds were effective against various human-pathogenic CoVs in multiple assays based on vesicular stomatitis virus (VSV) pseudotyped with the spike protein and spike-mediated syncytium formation. The antiviral effects were confirmed in SARS-CoV-2 infection systems. These compounds were equally effective against recently emerged variants, including the delta variant. Cross-linking experiments and structural modeling suggest that the compounds bind to a hydrophobic pocket near the fusion peptide of S protein, consistent with their potential mechanism of action as fusion inhibitors. In summary, these fusion inhibitors have broad-spectrum antiviral activities and may be promising leads for treatment of SARS-CoV-2, its variants, and other pathogenic CoVs. IMPORTANCE SARS-CoV-2 is an enveloped virus that requires membrane fusion for entry into host cells. Since the fusion process is relatively conserved among enveloped viruses, we tested our HCV fusion inhibitors, dichlorcyclizine and fluoxazolevir, against SARS-CoV-2. We performed in vitro assays and demonstrated their effective antiviral activity against SARS-CoV-2 and its variants. Cross-linking experiments and structural modeling suggest that the compounds bind to a hydrophobic pocket in spike protein to exert their inhibitory effect on the fusion step. These data suggest that both dichlorcyclizine and fluoxazolevir are promising candidates for further development as treatment for SARS-CoV-2.

Pathogenic coronaviruses are a major threat to global public health. Here, using a recombinant reporter virus-based compound screening approach, we identified small-molecule inhibitors that potently block the replication of severe acute respiratory syndrome virus 2 (SARS-CoV-2). Among them, JIB-04 inhibited SARS-CoV-2 replication in Vero E6 cells with a 50% effective concentration of 695 nM, with a specificity index of greater than 1,000. JIB-04 showed in vitro antiviral activity in multiple cell types, including primary human bronchial epithelial cells, against several DNA and RNA viruses, including porcine coronavirus transmissible gastroenteritis virus. In an in vivo porcine model of coronavirus infection, administration of JIB-04 reduced virus infection and associated tissue pathology, which resulted in improved weight gain and survival. These results highlight the potential utility of JIB-04 as an antiviral agent against SARS-CoV-2 and other viral pathogens. IMPORTANCE The coronavirus disease 2019 (COVID-19), the disease caused by SARS-CoV-2 infection, is an ongoing public health disaster worldwide. Although several vaccines are available as a preventive measure and the FDA approval of an orally bioavailable drug is on the horizon, there remains a need for developing antivirals against SARS-CoV-2 that could work on the early course of infection. By using infectious reporter viruses, we screened small-molecule inhibitors for antiviral activity against SARS-CoV-2. Among the top hits was JIB-04, a compound previously studied for its anticancer activity. Here, we showed that JIB-04 inhibits the replication of SARS-CoV-2 as well as different DNA and RNA viruses. Furthermore, JIB-04 conferred protection in a porcine model of coronavirus infection, although to a lesser extent when given as therapeutic rather than prophylactic doses. Our findings indicate a limited but still promising utility of JIB-04 as an antiviral agent in the combat against COVID-19 and potentially other viral diseases.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants stresses the continued need for next-generation vaccines that confer broad protection against coronavirus disease 2019 (COVID-19). We developed and evaluated an adjuvanted SARS-CoV-2 spike ferritin nanoparticle (SpFN) vaccine in nonhuman primates. High-dose (50 μg) SpFN vaccine, given twice 28 days apart, induced a Th1-biased CD4 T cell helper response and elicited neutralizing antibodies against SARS-CoV-2 wild-type and variants of concern, as well as against SARS-CoV-1. These potent humoral and cell-mediated immune responses translated into rapid elimination of replicating virus in the upper and lower airways and lung parenchyma of nonhuman primates following high-dose SARS-CoV-2 respiratory challenge. The immune response elicited by SpFN vaccination and resulting efficacy in nonhuman primates supports the utility of SpFN as a vaccine candidate for SARS-causing betacoronaviruses.

The development of mouse models for coronavirus disease 2019 (COVID-19) has enabled testing of vaccines and therapeutics and defining aspects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis. SARS-CoV-2 disease is severe in K18 transgenic mice (K18-hACE2 Tg) expressing human angiotensin-converting enzyme 2 (hACE2), the SARS-CoV-2 receptor, under an ectopic cytokeratin promoter, with high levels of infection measured in the lung and brain. Here, we evaluated SARS-CoV-2 infection in hACE2 knock-in (KI) mice that express hACE2 under an endogenous promoter in place of murine ACE2 (mACE2). Intranasal inoculation of hACE2 KI mice with SARS-CoV-2 WA1/2020 resulted in substantial viral replication within the upper and lower respiratory tracts with limited spread to extrapulmonary organs. However, SARS-CoV-2-infected hACE2 KI mice did not lose weight and developed limited pathology. Moreover, no significant differences in viral burden were observed in hACE2 KI mice infected with B.1.1.7 or B.1.351 variants compared to the WA1/2020 strain. Because the entry mechanisms of SARS-CoV-2 in mice remain uncertain, we evaluated the impact of the naturally occurring, mouse-adapting N501Y mutation by comparing infection of hACE2 KI, K18-hACE2 Tg, ACE2-deficient, and wild-type C57BL/6 mice. The N501Y mutation minimally affected SARS-CoV-2 infection in hACE2 KI mice but was required for viral replication in wild-type C57BL/6 mice in a mACE2-dependent manner and augmented pathogenesis in the K18-hACE2 Tg mice. Thus, the N501Y mutation likely enhances interactions with mACE2 or hACE2 in vivo . Overall, our study highlights the hACE2 KI mice as a model of mild SARS-CoV-2 infection and disease and clarifies the requirement of the N501Y mutation in mice. IMPORTANCE Mouse models of SARS-CoV-2 pathogenesis have facilitated the rapid evaluation of countermeasures. While the first generation of models developed pneumonia and severe disease after SARS-CoV-2 infection, they relied on ectopic expression of supraphysiological levels of human ACE2 (hACE2). This has raised issues with their relevance to humans, as the hACE2 receptor shows a more restricted expression pattern in the respiratory tract. Here, we evaluated SARS-CoV-2 infection and disease with viruses containing or lacking a key mouse-adapting mutation in the spike gene in hACE2 KI mice, which express hACE2 under an endogenous promoter in place of murine ACE2. While infection of hACE2 KI mice with multiple strains of SARS-CoV-2 including variants of concern resulted in viral replication within the upper and lower respiratory tracts, the animals did not sustain severe lung injury. Thus, hACE2 KI mice serve as a model of mild infection with both ancestral and emerging SARS-CoV-2 variant strains.

Alpha-B.1.1.7, Beta-B.1.351, Gamma-P.1, and Delta-B.1.617.2 variants of SARS-CoV-2 express multiple mutations in the spike protein (S). These may alter the antigenic structure of S, causing escape from natural or vaccine-induced immunity. Beta is particularly difficult to neutralize using serum induced by early pandemic SARS-CoV-2 strains and is most antigenically separated from Delta. To understand this, we generated 674 mAbs from Beta-infected individuals and performed a detailed structure-function analysis of the 27 most potent mAbs: one binding the spike N-terminal domain (NTD), the rest the receptor-binding domain (RBD). Two of these RBD-binding mAbs recognize a neutralizing epitope conserved between SARS-CoV-1 and -2, while 18 target mutated residues in Beta: K417N, E484K, and N501Y. There is a major response to N501Y, including a public IgVH4-39 sequence, with E484K and K417N also targeted. Recognition of these key residues underscores why serum from Beta cases poorly neutralizes early pandemic and Delta viruses., Competing Interests: Declaration of interests M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, Carnival Corporation, and on the Scientific Advisory Boards of Moderna and Immunome. The M.S.D. laboratory has received unrelated funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. G.R.S. sits on the GSK Vaccines Scientific Advisory Board and is a founder member of RQ Biotechnology. The University of Oxford has protected intellectual property disclosed in this publication., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The need for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) next-generation vaccines has been highlighted by the rise of variants of concern (VoCs) and the long-term threat of emerging coronaviruses. Here, we design and characterize four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of the prefusion SARS-CoV-2 spike (S), S1, and receptor-binding domain (RBD). These immunogens induce robust S binding, ACE2 inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2. A spike-ferritin nanoparticle (SpFN) vaccine elicits neutralizing titers (ID 50 > 10,000) following a single immunization, whereas RBD-ferritin nanoparticle (RFN) immunogens elicit similar responses after two immunizations and also show durable and potent neutralization against circulating VoCs. Passive transfer of immunoglobulin G (IgG) purified from SpFN- or RFN-immunized mice protects K18-hACE2 transgenic mice from a lethal SARS-CoV-2 challenge. Furthermore, S-domain nanoparticle immunization elicits ACE2-blocking activity and ID 50 neutralizing antibody titers >2,000 against SARS-CoV-1, highlighting the broad response elicited by these immunogens., Competing Interests: Declaration of interests M.G.J. and K.M. are named as inventors on international patent application WO/2021/178971 A1 entitled “Vaccines against SARS-CoV-2 and other coronaviruses.” M.G.J. is named as an inventor on international patent application WO/2018/081318 and U.S. patent 10,960,070 entitled “Prefusion coronavirus spike proteins and their use.” Z.B. is named as an inventor on U.S. patent 10,434,167 entitled “Non-toxic adjuvant formulation comprising a monophosphoryl lipid A (MPLA)-containing liposome composition and a saponin.” Z.B. and G.R.M. are named as inventors on U.S. patent application 16/607,917 entitled “Compositions and methods for vaccine delivery.” M.S.D. is a consultant for Inbios, Vir Biotechnology, Fortress Biotech, and Carnival Corporation and on the scientific advisory boards of Moderna and Immunome. The Diamond laboratory has received funding support from sponsored research agreements from Moderna, Vir Biotechnology, Kaleido, and Emergent BioSolutions. S.R., P.M.M., and M.T.E. are employees of AstraZeneca and currently hold AstraZeneca stock or stock options. Z.B. is currently employed at Pfizer., (Copyright © 2021. Published by Elsevier Inc.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has necessitated the rapid development of antibody-based therapies and vaccines as countermeasures. Here, we use cryoelectron microscopy (cryo-EM) to characterize two protective anti-SARS-CoV-2 murine monoclonal antibodies (mAbs) in complex with the spike protein, revealing similarities between epitopes targeted by human and murine B cells. The more neutralizing mAb, 2B04, binds the receptor-binding motif (RBM) of the receptor-binding domain (RBD) and competes with angiotensin-converting enzyme 2 (ACE2). By contrast, 2H04 binds adjacent to the RBM and does not compete for ACE2 binding. Naturally occurring sequence variants of SARS-CoV-2 and corresponding neutralization escape variants selected in vitro map to our structurally defined epitopes, suggesting that SARS-CoV-2 might evade therapeutic antibodies with a limited set of mutations, underscoring the importance of combination mAb therapeutics. Finally, we show that 2B04 neutralizes SARS-CoV-2 infection by preventing ACE2 engagement, whereas 2H04 reduces host cell attachment without directly disrupting ACE2-RBM interactions, providing distinct inhibitory mechanisms used by RBD-specific mAbs., Competing Interests: Declaration of interests D.H.F. is a founder of Courier Therapeutics. A.H.E. is a consultant for Inbios and Fimbrion Therapeutics. M.S.D. is a consultant for Inbios, Vir Biotechnology, and NGM Biopharmaceuticals and is on the Scientific Advisory Board of Moderna and Immunome. D.H.F., M.S.D., and A.H.E. have received unrelated funding support from Emergent BioSolutions. M.S.D. has sponsored research agreements from Moderna and Vir Biotechnology, A.H.E. has a sponsored research agreement from Abbvie, and D.H.F. has a sponsored research agreement from Mallinckrodt Pharmaceuticals., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility and potential resistance, antibodies and vaccines with broadly inhibitory activity are needed. Here, we developed a panel of neutralizing anti-SARS-CoV-2 monoclonal antibodies (mAbs) that bound the receptor binding domain of the spike protein at distinct epitopes and blocked virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Although several potently neutralizing mAbs protected K18-hACE2 transgenic mice against infection caused by ancestral SARS-CoV-2 strains, others induced escape variants in vivo or lost neutralizing activity against emerging strains. One mAb, SARS2-38, potently neutralized all tested SARS-CoV-2 variants of concern and protected mice against challenge by multiple SARS-CoV-2 strains. Structural analysis showed that SARS2-38 engaged a conserved epitope proximal to the receptor binding motif. Thus, treatment with or induction of neutralizing antibodies that bind conserved spike epitopes may limit the loss of potency of therapies or vaccines against emerging SARS-CoV-2 variants., Competing Interests: Declaration of interests M.S.D. is a consultant for Inbios, Vir Biotechnology, Fortress Biotech, and Carnival Corporation and on the Scientific Advisory Boards of Moderna and Immunome. The Diamond laboratory has received funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. Some of the mAbs described in this study have been licensed by Washington University to Bio X Cell. D.H.F is a founder of Courier Therapeutics and has received funding support in a sponsored research agreement from Emergent BioSolutions. J.E.C. has served as a consultant for Eli Lilly and Luna Biologics, is a member of the Scientific Advisory Boards of CompuVax and Meissa Vaccines, and is the founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from AstraZeneca and IDBiologics. The Boon laboratory has received funding support in sponsored research agreements from AI Therapeutics, GreenLight Biosciences, AbbVie, and Nano Targeting & Therapy Biopharma., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Understanding the molecular basis for immune recognition of SARS-CoV-2 spike glycoprotein antigenic sites will inform the development of improved therapeutics. We determined the structures of two human monoclonal antibodies-AZD8895 and AZD1061-which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor-binding domain (RBD) of SARS-CoV-2 to define the genetic and structural basis of neutralization. AZD8895 forms an 'aromatic cage' at the heavy/light chain interface using germ line-encoded residues in complementarity-determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals. AZD1061 has an unusually long LCDR1; the HCDR3 makes interactions with the opposite face of the RBD from that of AZD8895. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the crucial binding residues of both antibodies and identified positions of concern with regards to virus escape from antibody-mediated neutralization. Both AZD8895 and AZD1061 have strong neutralizing activity against SARS-CoV-2 and variants of concern with antigenic substitutions in the RBD. We conclude that germ line-encoded antibody features enable recognition of the SARS-CoV-2 spike RBD and demonstrate the utility of the cocktail AZD7442 in neutralizing emerging variant viruses., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Emergence of novel variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean serum neutralizing antibody titers of 14,000 to 21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within 4 d in seven of eight animals receiving 50 µg of RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only approximately twofold relative to WA1/2020. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-CoV-like Sarbecovirus vaccine development., Competing Interests: Competing interest statement: M.G.J. and K.M. are named as inventors on International Patent Application no. WO/2021/21405 entitled “Vaccines against SARS-CoV-2 and other coronaviruses.” M.G.J. is named as an inventor on International Patent Application no. WO/2018/081318 entitled “Prefusion Coronavirus Spike Proteins and Their Use.” M.S.D. is a consultant for Inbios, Vir Biotechnology, Fortress Biotech, and Carnival Corporation and is on the Scientific Advisory Boards of Moderna and Immunome. The M.S.D. laboratory has received funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions., (Copyright © 2021 the Author(s). Published by PNAS.)

The emergence of SARS-CoV-2 antigenic variants with increased transmissibility is a public health threat. Some variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies. Here, we analyzed receptor binding domain-binding monoclonal antibodies derived from SARS-CoV-2 mRNA vaccine-elicited germinal center B cells for neutralizing activity against the WA1/2020 D614G SARS-CoV-2 strain and variants of concern. Of five monoclonal antibodies that potently neutralized the WA1/2020 D614G strain, all retained neutralizing capacity against the B.1.617.2 variant, four also neutralized the B.1.1.7 variant, and only one, 2C08, also neutralized the B.1.351 and B.1.1.28 variants. 2C08 reduced lung viral load and morbidity in hamsters challenged with the WA1/2020 D614G, B.1.351, or B.1.617.2 strains. Clonal analysis identified 2C08-like public clonotypes among B cells responding to SARS-CoV-2 infection or vaccination in 41 out of 181 individuals. Thus, 2C08-like antibodies can be induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern., Competing Interests: Declaration of interests The Ellebedy laboratory received funding under sponsored research agreements that are unrelated to the data presented in the current study from Emergent BioSolutions and from AbbVie. A.H.E. is a consultant for Mubadala Investment Company and the founder of ImmuneBio Consulting LLC. M.S.D. is a consultant for Inbios, Vir Biotechnology, Fortress Biotech, Carnival Corporation and on the Scientific Advisory Board of Moderna and Immunome. The Diamond laboratory has received unrelated sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. The Boon laboratory has received unrelated funding support in sponsored research agreements from AI Therapeutics, GreenLight Biosciences Inc., and Nano targeting & Therapy Biopharma Inc. The Boon laboratory has received funding support from AbbVie Inc., for the commercial development of a SARS-CoV-2 mAb. A.J.S., J.S.T., W.B.A., J.B.C., S.P.J.W., M.S.D., A.C.M.B., and A.H.E. are recipients of a licensing agreement with Abbvie Inc., for commercial development of a SARS-CoV-2 mAb. A patent application related to this work has been filed by Washington University School of Medicine. The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays and NDV-based SARS-CoV-2 vaccines, which list Florian Krammer as co-inventor. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2. Florian Krammer has consulted for Merck and Pfizer (before 2020) and is currently consulting for Pfizer, Seqirus, and Avimex. The Krammer laboratory is also collaborating with Pfizer on animal models of SARS-CoV-2. The Shi laboratory has received sponsored research agreements from Pfizer, Gilead, Merck, and IGM Sciences Inc. The Whelan laboratory has received unrelated funding support in sponsored research agreements with Vir Biotechnology, AbbVie, and sAB therapeutics. All other authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)