Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain, RBD203-N1 as a COVID-19 Protein Vaccine Candidate

BackgroundSARS-CoV-2 protein subunit vaccines are being evaluated by multiple manufacturers to fill the need for low-cost, easy to scale, safe, and effective COVID-19 vaccines for global access. Vaccine candidates relying on the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein have been the focus of our development program. In this paper, we report on the generation of the RBD203-N1 yeast expression construct, which produces a recombinant protein that when formulated with alum and the TLR-9 agonist, CpG1826 elicits a robust immune response and protection in mice. MethodThe RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity and efficacy were evaluated in mice. Results, Conclusions, and SignificanceThe RBD203-N1 production process yielded 492.9 {+/-} 3.0 mg/L of protein in the fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 {+/-} 3% (total yield of purified protein: 270.5 {+/-} 13.2 mg/L fermentation supernatant). The protein was characterized as a homogeneous monomer with well-defined secondary structure, thermally stable, antigenic, and when adjuvanted on alum and CpG, it was immunogenic and induced robust levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. These characteristics show that this vaccine candidate is well suited for technology transfer with feasibility of its transition into the clinic to evaluate its immunogenicity and safety in humans.

Receptor-binding domain recombinant protein RBD219-N1C1 on alum-CpG induces broad protection against SARS-CoV-2 variants of concern

We conducted preclinical studies in mice using a yeast-produced SARS-CoV-2 RBD subunit vaccine candidate formulated with aluminum hydroxide (alum) and CpG deoxynucleotides. This formulation is equivalent to the Corbevax vaccine that recently received emergency use authorization by the Drugs Controller General of India. We compared the immune response of mice vaccinated with RBD/alum to mice vaccinated with RBD/alum+CpG. We also evaluated mice immunized with RBD/alum+CpG and boosted with RBD/alum. Mice were immunized twice intramuscularly at a 21-day interval. Compared to two doses of the /alum formulation, the RBD/alum+CpG vaccine induced a stronger and more balanced Th1/Th2 cellular immune response, with high levels of neutralizing antibodies against the original Wuhan isolate of SARS-CoV-2 as well as the B.1.1.7 (Alpha), B. 1.351 (Beta), B. 1.617.2 and (Delta) variants. Neutralizing antibody titers against the B.1.1.529 (BA.1, Omicron) variant exceeded those in human convalescent plasma after Wuhan infection but were lower than against the other variants. Interestingly, the second dose did not benefit from the addition of CpG, possibly allowing dose-sparing of the adjuvant in the future. The data reported here reinforces that the RBD/alum+CpG vaccine formulation is suitable for inducing broadly neutralizing antibodies against SARS-CoV-2 including variants of concern.

Evaluation of suspected musculoskeletal infection in children over 2 years of age using only fluid-sensitive sequences at MRI.

PURPOSE: This study was conducted in order to evaluate whether an MRI protocol with only fluid-sensitive sequences can be used to evaluate for musculoskeletal (MSK) infection of the pelvis and limbs in children. MATERIALS AND METHODS: This retrospective study analyzed 90 contrast-enhanced (CE) MRI studies from 88 consecutive patients (52 boys and 36 girls; mean age 9 ± 4.3 years; range 2-17) that were performed for the clinical suspicion of MSK infection. Two radiologists reviewed each study twice. The initial study review included only the fluid-sensitive sequences (fluid-sensitive study); the second review, performed at least 1 month later, included all sequences of the contrast-enhanced study (CE study). At each review, anatomic sites of abnormal signal and overall suspicion for infection were recorded. Cohen's kappa and percent agreement were performed to compare agreement between readers, types of studies, and clinical diagnoses. RESULTS: Interreader agreement for both types of studies had kappa values between 0.86 and 1. For the assessment of MSK infection, the fluid-sensitive study had 100% sensitivity and 61.3% specificity, with 84.8% interreader agreement; and the CE study had 100% sensitivity and 71.0% specificity, with 88.6% interreader agreement. All cases of septic arthritis (13 cases) and osteomyelitis (25 cases) were identified as possible infection or infection until proven otherwise (negative predictive value 100%) with 100% interreader agreement on fluid-sensitive sequences. CONCLUSION: An abbreviated MRI study using only fluid-sensitive sequences has the same high degree of sensitivity as a CE study to identify MSK infection in children and could be used to exclude septic arthritis and osteomyelitis. KEY POINTS: • MRI with only fluid-sensitive sequences can be used to evaluate for musculoskeletal infection in children.

Isolated torsion of the fallopian tube in a menarchal 11-year-old girl.

Isolated torsion of the fallopian tube is a rare cause of an acute lower abdominopelvic pain in adolescent females that is difficult to recognize preoperatively. This is a case report of an 11-year-old girl who presented with a right lower quadrant abdominal pain, nausea, and vomiting secondary to isolated torsion of the right fallopian tube 2 days after the onset of her first menses. In this report, the patient's clinical course is discussed with special emphasis on diagnostic imaging and management strategy of adnexal torsion in pubertal and adolescent girls.