Engineering a SARS-CoV-2 vaccine targeting the RBD cryptic-face via immunofocusing.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is the main target of neutralizing antibodies. Although they are infrequently elicited during infection or vaccination, antibodies that bind to the conformation-specific cryptic face of the RBD display remarkable breadth of binding and neutralization across Sarbecoviruses. Here, we employed the immunofocusing technique PMD (protect, modify, deprotect) to create RBD immunogens (PMD-RBD) specifically designed to focus the antibody response towards the cryptic-face epitope recognized by the broadly neutralizing antibody S2X259. Immunization with PMD-RBD antigens induced robust binding titers and broad neutralizing activity against homologous and heterologous Sarbecovirus strains. A serum-depletion assay provided direct evidence that PMD successfully skewed the polyclonal antibody response towards the cryptic face of the RBD. Our work demonstrates the ability of PMD to overcome immunodominance and refocus humoral immunity, with implications for the development of broader and more resilient vaccines against current and emerging viruses with pandemic potential.

Design of universal Ebola virus vaccine candidates via immunofocusing.

The Ebola virus causes hemorrhagic fever in humans and poses a significant threat to global public health. Although two viral vector vaccines have been approved to prevent Ebola virus disease, they are distributed in the limited ring vaccination setting and only indicated for prevention of infection from orthoebolavirus zairense (EBOV)-one of three orthoebolavirus species that have caused previous outbreaks. Ebola virus glycoprotein GP mediates viral infection and serves as the primary target of neutralizing antibodies. Here, we describe a universal Ebola virus vaccine approach using a structure-guided design of candidates with hyperglycosylation that aims to direct antibody responses away from variable regions and toward conserved epitopes of GP. We first determined the hyperglycosylation landscape on Ebola virus GP and used that to generate hyperglycosylated GP variants with two to four additional glycosylation sites to mask the highly variable glycan cap region. We then created vaccine candidates by displaying wild-type or hyperglycosylated GP variants on ferritin nanoparticles (Fer). Immunization with these antigens elicited potent neutralizing antisera against EBOV in mice. Importantly, we observed consistent cross-neutralizing activity against Bundibugyo virus and Sudan virus from hyperglycosylated GP-Fer with two or three additional glycans. In comparison, elicitation of cross-neutralizing antisera was rare in mice immunized with wild-type GP-Fer. These results demonstrate a potential strategy to develop universal Ebola virus vaccines that confer cross-protective immunity against existing and emerging filovirus species.

Septins modulate the autophagy response after nutrient starvation.

The pathways that induce macroautophagy (referred to as autophagy hereafter) in response to the stress of starvation are well conserved and essential under nutrient-limiting conditions. However, less is understood about the mechanisms that modulate the autophagy response. Here we present evidence that after induction of autophagy in budding yeast septin filaments rapidly assemble into discrete patches distributed along the cell cortex. These patches gradually mature over 12 h of nutrient deprivation to form extended structures around Atg9 membranes tethered at the cortical endoplasmic reticulum, a class of membranes that are limiting for autophagosome biogenesis. Loss of cortical septin structures alters the kinetics of autophagy activation and most dramatically extends the duration of the autophagy response. In wild-type cells, diffusion of Atg9 membranes at the cell cortex undergoes transient pauses that are dependent on septins, and septins at the bud neck block the diffusion of Atg9 membranes between mother and daughter cells. We conclude that septins reorganize at the cell cortex during autophagy to locally limit access of Atg9 membranes to autophagosome assembly sites, and thus modulate the autophagy response during nutrient deprivation.

Perigraftitis and implant therapy: A case report.

BACKGROUND: A previous case study reported periimplantitis and concomitant perigraftitis of a second implant placed at a site that had alveolar ridge preservation three decades earlier. Infection at the site persisted 4 months after implant removal by flapless implant reversal. A flap was subsequently reflected, the grafted bone was removed, and a second alveolar ridge preservation was performed with a freeze-dried bone allograft. The publication reported infection resolved, and the site healed uneventfully. However, it is unknown if placement of another implant at the site would be successful. The purpose of this paper is to report on the findings at surgical reentry and outcome of the third implant. METHODS: Eleven months after the second alveolar ridge preservation, the site was reentered. The bone graft was found to be partially soft tissue encapsulated. All encapsulated graft materials and soft tissue were removed. An implant was placed, and the alveolar defect was grafted with a demineralized bone allograft. Seventeen months after implant placement, a buccal free gingival graft was performed during which the crestal bone adjacent to the implant was found to be hard and corticated. The implant was deemed to be osseointegrated and restored after soft tissue healing. RESULTS: Twenty-five months after implant placement, the third implant remained functional and asymptomatic with the peri-implant bone exhibiting normal trabeculation. CONCLUSIONS: Implant therapy can be successful following treatment and resolution of perigraftitis. KEY POINTS: Perigraftitis may play a contributing role in the biologic complications of implants that have been placed into grafted bone. Perigraftitis may be successfully resolved by completely removing all grafted bone. Once perigraftitis has been eliminated, an implant may be successfully placed.

Design of universal Ebola virus vaccine candidates via immunofocusing.

Ebola virus causes hemorrhagic fever in humans and poses a significant threat to global public health. Although two viral vector vaccines have been approved to prevent Ebola virus disease, they are distributed in the limited ring vaccination setting and only indicated for prevention of infection from orthoebolavirus zairense (EBOV) - one of three orthoebolavirus species that have caused previous outbreaks. Ebola virus glycoprotein GP mediates viral infection and serves as the primary target of neutralizing antibodies. Here we describe a universal Ebola virus vaccine approach using structure-guided design of candidates with hyperglycosylation that aims to direct antibody responses away from variable regions and toward conserved epitopes of GP. We first determined the hyperglycosylation landscape on Ebola virus GP and used that to generate hyperglycosylated GP variants with two to four additional glycosylation sites to mask the highly variable glycan cap region. We then created vaccine candidates by displaying wild-type or hyperglycosylated GP variants on ferritin nanoparticles (Fer). Immunization with these antigens elicited potent neutralizing antisera against EBOV in mice. Importantly, we observed consistent cross-neutralizing activity against Bundibugyo virus and Sudan virus from hyperglycosylated GP-Fer with two or three additional glycans. In comparison, elicitation of cross-neutralizing antisera was rare in mice immunized with wild-type GP-Fer. These results demonstrate a potential strategy to develop universal Ebola virus vaccines that confer cross-protective immunity against existing and emerging filovirus species.

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.

Decreased efficacy of a COVID-19 vaccine due to mutations present in early SARS-CoV-2 variants of concern.

With the SARS-CoV-2 virus still circulating and evolving, there remains an outstanding question if variant-specific vaccines represent the optimal path forward, or if other strategies might be more efficacious towards providing broad protection against emerging variants. Here, we examine the efficacy of strain-specific variants of our previously reported, pan-sarbecovirus vaccine candidate, DCFHP-alum, a ferritin nanoparticle functionalized with an engineered form of the SARS-CoV-2 spike protein. In non-human primates, DCFHP-alum elicits neutralizing antibodies against all known VOCs that have emerged to date and SARS-CoV-1. During development of the DCFHP antigen, we investigated the incorporation of strain-specific mutations from the major VOCs that had emerged to date: D614G, Epsilon, Alpha, Beta, and Gamma. Here, we report the biochemical and immunological characterizations that led us to choose the ancestral Wuhan-1 sequence as the basis for the final DCFHP antigen design. Specifically, we show by size exclusion chromatography and differential scanning fluorimetry that mutations in the VOCs adversely alter the antigen's structure and stability. More importantly, we determined that DCFHP without strain-specific mutations elicits the most robust, cross-reactive response in both pseudovirus and live virus neutralization assays. Our data suggest potential limitations to the variant-chasing approach in the development of protein nanoparticle vaccines, but also have implications for other approaches including mRNA-based vaccines.

Simplified Subperiosteal Sling Suture for Connective Tissue Graft Stabilization in Root Coverage and Phenotype Modification.

This technical report describes the simplified subperiosteal sling (SPS) suture for connective tissue graft (CTG) stabilization in root coverage and phenotype modification of single and multiple recession defects via the vestibular incisional subperiosteal tunnel access (VISTA). The simplified SPS suture engages the CTG only and stabilizes it to the tooth in the coronal most position inside the subperiosteal tunnel independent of the overlying gingival tissue. The simplified SPS suture differs from the original SPS suture in that it engages the CTG first, and the needle and tail of the suture are knotted before the suture is introduced into the subperiosteal tunnel. This allows the needle to pass through the subperiosteal tunnel only once from the vestibular access to the intended gingival sulcus. When multiple teeth are treated, only one simplified SPS suture traverses the vestibular access at a time as the CTG is incrementally advanced into the tunnel. This prevents suture entanglement and improves the practical application of the technique.

Modified Dual-Zone Therapeutic Concept of Single Immediate Implants with Bone and Soft Tissue Augmentation: Technical Report with Three Cases.

Although connective tissue grafts (CTGs) have been found to increase gingival thickness and reduce facial gingival recession in immediate implant placement and provisionalization (IIPP), they are associated with significant loss of buccal bone thickness. This loss is thought to be related to the preparation of the facial CTG recipient site. This technical report presents a modified dual-zone therapeutic concept in which the bone zone is grafted with bone graft and the tissue zone is grafted with tuberosity CTG without elevation of a facial partial- or full-thickness envelope.

Multiple Subperiosteal Sling Sutures for Connective Tissue Graft Stabilization in the Treatment of Multiple Recession Defects Utilizing Subperiosteal Tunnels via VISTA and Intrasulcular Access.

This report describes the utilization of multiple subperiosteal sling (SPS) sutures to stabilize connective tissue grafts in the treatment of multiple recession defects using subperiosteal tunnels via vestibular and intrasulcular accesses. The SPS sutures engage only the graft and stabilize it against teeth inside the subperiosteal tunnel without engaging the overlying soft tissue, which is neither sutured nor coronally advanced. At sites with deep recessions, the graft is left exposed over the denuded root surfaces and allowed to epithelialize, which results in root coverage and increased attached keratinized tissue. Further controlled studies are required to investigate the predictability of this treatment approach.

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.

Enucleation of recurrent central odontogenic fibroma and bone regeneration of the osseous defect with enamel matrix derivative and bone allograft: Case report with 5-year follow-up.

INTRODUCTION: Odontogenic fibroma is a rare benign neoplasm of mature fibrous connective tissue with variable amounts of inactive-looking odontogenic epithelium. Few recurrences of central odontogenic fibroma (COF) have been reported in the literature. This manuscript reports the enucleation of a recurrent COF and bone regeneration of the osseous defect with enamel matrix derivative and bone allograft. CASE PRESENTATION: A 28-year-old Asian female presented in 2015 with a palatal depression between #10 and 11. The patient previously had an odontogenic fibroma between #10 and 11 removed in 2008. Cone beam computed tomographic evaluation revealed a well-defined, multiloculated radiolucency centered between #10 and 11 that extended from #9-12, and from the alveolar crest to the anterior border and floor of the maxillary sinus. The lesion resulted in splaying of the roots of #10 and 11, external root resorption on #10, loss of crestal and palatal bone cortices, and thinning of labial cortex between #10 and 11. The patient was referred to an oral surgeon for biopsy, and the lesion was diagnosed as odontogenic fibroma. The lesion was enucleated. Enamel matrix derivative was applied to the affected teeth and defect, which was subsequently grafted with bone allograft. At the 5-year follow-up, bone was regenerated to the midroot of #10 and coronal third of #11, with reestablishment of crestal and palatal bone cortices. CONCLUSION(S): Enucleation of COF and regeneration of the osseous defect with enamel matrix derivative and bone allograft appear to be a viable treatment approach that allows for preservation of contiguous teeth. Why is this case new information? This appears to be the first publication to report on bone regeneration following enucleation of a recurrent odontogenic fibroma. What are the keys to successful management of this case? Graft material provided space maintenance and a scaffold for bone regeneration. What are the primary limitations to success in this case? Loss of the labial bone resulted in a through-and-through defect between 10 and 11 that limited the amount of vertical bone regeneration.

Simplified Purification of Glycoprotein-Modified Ferritin Nanoparticles for Vaccine Development.

Ferritin-based, self-assembling protein nanoparticle vaccines are being developed against a range of viral pathogens, including SARS-CoV-2, influenza, HIV-1, and Epstein-Barr virus. However, purification of these nanoparticles is often laborious and requires customization for each potential nanoparticle vaccine. We propose that the simple insertion of a polyhistidine tag into exposed flexible loops on the ferritin surface (His-Fer) can mitigate the need for complex purifications and enable facile metal-chelate-based purification, thereby allowing for optimization of early stage vaccine candidates. Using sequence homology and computational modeling, we identify four sites that can accommodate insertion of a polyhistidine tag and demonstrate purification of both hemagglutinin-modified and SARS-CoV-2 spike-modified ferritins, highlighting the generality of the approach. A site at the 4-fold axis of symmetry enables optimal purification of both protein nanoparticles. We demonstrate improved purification through modulating the polyhistidine length and optimizing both the metal cation and the resin type. Finally, we show that purified His-Fer proteins remain multimeric and elicit robust immune responses similar to those of their wild-type counterparts. Collectively, this work provides a simplified purification scheme for ferritin-based vaccines.

Chemically Modified Bacterial Sacculi as a Vaccine Microparticle Scaffold.

Vaccine scaffolds and carrier proteins increase the immunogenicity of subunit vaccines. Here, we developed, characterized, and demonstrated the efficacy of a novel microparticle vaccine scaffold comprised of bacterial peptidoglycan (PGN), isolated as an entire sacculi. The PGN microparticles contain bio-orthogonal chemical handles allowing for site-specific attachment of immunogens. We first evaluated the purification, integrity, and immunogenicity of PGN microparticles derived from a variety of bacterial species. We then optimized PGN microparticle modification conditions; Staphylococcus aureus PGN microparticles containing azido-d-alanine yielded robust conjugation to immunogens. We then demonstrated that this vaccine scaffold elicits comparable immunostimulation to the conventional carrier protein, keyhole limpet hemocyanin (KLH). We further modified the S. aureus PGN microparticle to contain the SARS-CoV-2 receptor-binding domain (RBD)─this conjugate vaccine elicited neutralizing antibody titers comparable to those elicited by the KLH-conjugated RBD. Collectively, these findings suggest that chemically modified bacterial PGN microparticles are a conjugatable and biodegradable microparticle scaffold capable of eliciting a robust immune response toward an antigen of interest.

"Periimplant Mucositis" Secondary to Infection of Residual Bone Allograft Embedded in the Periimplant Sulcus of a Patient With Diabetes: A Case Report.

INTRODUCTION: Currently, inflammation of tissue around implants is diagnosed as either periimplant mucositis or periimplantitis, and the etiology is bacterial biofilm colonization of the implant and its transmucosal component. The purpose of this manuscript is to report a case of "periimplant mucositis" secondary to infection of a residual bone allograft embedded in the periimplant sulcus of a patient with diabetes. CASE PRESENTATION: A #8 implant was placed and provisionalized in a 46-year-old male patient. During implant placement, facial contour augmentation with an allograft (1000-2000 µm), a collagen membrane, and a connective tissue graft was performed. Healing was uneventful. The crown was removed 9 months after implant placement; a graft particulate was observed in the periimplant sulcus and left undisturbed. The subgingival contour of the crown was adjusted, and the crown was reseated. The patient subsequently developed soreness and foul smell at #8. The crown was removed, and the previously observed graft particulate was more exposed and contaminated. It was removed and the crown was reseated. A month later, symptoms persisted. Palpation produced suppuration. A facial flap was reflected, and residual graft particulates embedded in the facial flap were removed. The site was grafted with a connective tissue graft. Subsequently, symptoms resolved, and the implant was restored. CONCLUSION(S): Within the limitation of this case report, it appears residual graft materials can get infected once exposed to the oral cavity and subsequently induce an inflammatory response in the surrounding tissue, especially in a patient with hyperglycemia.

Peri-Implantitis and Concomitant Perigraftitis of an Implant Placed in a Site That Had Alveolar Ridge Preservation Three Decades Earlier: A Case Report With Human Histology.

INTRODUCTION: Although alveolar ridge preservation may minimize alveolar ridge shrinkage following tooth extraction, there is a paucity of data on the effects of alveolar ridge preservation on implant-related outcomes. The purpose of this manuscript is to report on peri-implantitis of an implant placed at a site that had alveolar ridge preservation three decades earlier, and the subsequent dislodgement of an approximately 1-cm3 grafted bone specimen during degranulation 4 months after implant reversal. CASE PRESENTATION: A 58-year-old male had #18 removed and the extraction socket grafted in the 1980s. In 2016, an implant was placed at #18 and restored with a screw-retained restoration. It developed mobility and was removed in 2017; the explantation site was not grafted. In 2018, another implant was placed at #18 and restored in 2019 with a screw-retained restoration. At the 1-year follow-up, the implant had developed peri-implantitis. Consequently, it was reversed without flap elevation. Four months later, infection remained. A buccal mucoperiosteal flap was reflected. Implant threads remained visible in the grafted bone. During degranulation, an approximately 1-cm3 grafted bone specimen dislodged from the mandible. Microscopy of the specimen shows numerous synthetic graft particulates surrounded by vital bone, with a mixed acute and chronic inflammatory infiltrate on its periphery. CONCLUSION(S): Within the limitation of this case report, absence of bone resorption due to impaired/altered bone metabolism of the grafted bone likely allowed bacteria to reach and infect the grafted bone via the implant fixture during development and progression of peri-implantitis.

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.

Bone Regeneration of Maxillary Molar Proximal Class 2 Furcation Defects Treated With Microscope-Assisted Papilla Preservation Technique and DFDBA: A Case Series.

INTRODUCTION: Periodontal regeneration of maxillary molar proximal furcation defects are challenging due to limited access. While combination therapy of open flap debridement with barrier membrane, bone graft, and biologics are reported to be more successful than monotherapeutic approaches, combination therapy can be complicated and costly. CASE PRESENTATION: A total of four teeth in three patients are presented to demonstrate radiographic bone regeneration of deep Class 2 maxillary molar proximal furcation defects (MMPFD) treated with microscope-assisted papilla preservation technique (PPT) and demineralized freeze-dried bone allograft (DFDBA). CONCLUSION(S): Radiographic bone regeneration of deep Class 2 MMPFD is possible with microscope-assisted PPT and DFDBA. Furthermore, treatment outcomes appear to be superior to those from previous clinical trials of Class 2 MMPFD treatment.