DEVELOPMENT OF MICRO-PATTERNED FILMS FOR OPHTHALMIC DRUG DELIVERY

Ocular inflammation is commonly associated with multiple eye disorders such as microbial infections, allergies and post-operative healing. Topical anti-inflammatory agents delivered via eye drops or ointments are commonly used for the management of inflammation. These treatment regimens involve high dosing frequency over prolonged periods depending on the severity of inflammation. Further, only 5% of administered drug may be available for action due to the physiological barriers of the eye and formulation losses. Collectively, this reduces patient compliance and causes irregularity in drug exposure. Therefore, there is a need for prolonged residence time of formulation in the eye to achieve sustained drug delivery. Mucoadhesive polymers and ocular inserts have been used for this purpose. Additionally, topographical features have been explored to enhance adhesion to mucosal surfaces. Taking inspiration from these approaches, we hypothesized that micropatterned polymeric films will prolong residence time by enhancing interactions with ocular mucosal surface and sustain the drug release. We developed micropatterned films containing a hydrophobic drug, hydrocortisone (HCT) or a hydrophilic drug, olopatadine hydrochloride (OLO) using GRAS-compliant cellulose-based polymers and polylactic-co-glycolic acid (PLGA). Films with 100 µm sized square, triangle and circle micropatterns were manufactured and characterized for HCT / OLO release. We demonstrated an in vitro sustained release for up to 72 hours, for both HCT and OLO. The shape of micropatterns did not impact drug release, however, PLGA and hydroxyethylcellulose impacted release of HCT and OLO, respectively. Tensile properties of films were dependent on the film composition and not micropatterns. A novel method to investigate the mucoadhesion potential of micropatterned films was developed. For cellulose and PLGA-based films, micropatterns did not improve adhesion to porcine intestinal tissue. Collectively, our results suggest that sustained release of anti-inflammatory drugs can be achieved by using cellulose and PLGA-based films and the presence of 100 µm sized micropatterns did not improve adhesion to mucosal surface. Further optimization of formulation and micropattern characteristics will be required to maximize sustained release and mucoadhesion in films, which can potentially be used for long-term delivery to the anterior region of the eye

Potently neutralizing and protective anti-human metapneumovirus antibodies target diverse sites on the fusion glycoprotein

Human metapneumovirus (hMPV) is a leading cause of acute lower respiratory tract infections in high-risk populations, yet there are no vaccines or anti-viral therapies approved for the prevention or treatment of hMPV-associated disease. Here, we used a high-throughput single-cell technology to interrogate memory B cell responses to the hMPV fusion (F) glycoprotein in young adult and elderly donors. Across all donors, the neutralizing antibody response was primarily directed to epitopes expressed on both pre- and post-fusion F conformations. However, we identified rare, highly potent broadly neutralizing antibodies that recognize pre-fusion-specific epitopes and structurally characterized an antibody that targets a site of vulnerability at the pre-fusion F trimer apex. Additionally, monotherapy with neutralizing antibodies targeting three distinct antigenic sites provided robust protection against lower respiratory tract infection in a small animal model. This study provides promising monoclonal antibody candidates for passive immunoprophylaxis and informs the rational design of hMPV vaccine immunogens.We acknowledge the Immune Monitoring and Flow Cytometry Resource (IMFCSR) at the Norris Cotton Cancer Center at Dartmouth supported by NCI Cancer Center Support Grant 5P30CA023108-41. This work was funded in part by Welch Foundation grant number F-0003-19620604.S

Broad and potent activity against SARS-like viruses by an engineered human monoclonal antibody

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. We employed a directed evolution approach to engineer three SARS-CoV-2 antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains (RBDs) and neutralizes representative epidemic sarbecoviruses with high potency. Structural and biochemical studies demonstrate that ADG-2 employs a distinct angle of approach to recognize a highly conserved epitope overlapping the receptor binding site. In immunocompetent mouse models of SARS and COVID-19, prophylactic administration of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate against clade 1 sarbecoviruses

Broad neutralization of SARS-related viruses by human monoclonal antibodies

Broadly protective vaccines against known and preemergent human coronaviruses (HCoVs) are urgently needed. To gain a deeper understanding of cross-neutralizing antibody responses, we mined the memory B cell repertoire of a convalescent severe acute respiratory syndrome (SARS) donor and identified 200 SARS coronavirus 2 (SARS-CoV-2) binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the non-neutralizing antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of preexisting memory B cells elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a target for the rational design of pan-sarbecovirus vaccines

Recall of preexisting cross-reactive B cell memory after Omicron BA.1 breakthrough infection

Understanding immune responses after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) breakthrough infection will facilitate the development of next-generation vaccines. Here, we profiled spike (S)-specific B cell responses after Omicron/BA.1 infection in messenger RNA-vaccinated donors. The acute antibody response was characterized by high levels of somatic hypermutation and a bias toward recognition of ancestral SARS-CoV-2 strains, suggesting the early activation of vaccine-induced memory B cells. BA.1 breakthrough infection induced a shift in B cell immunodominance hierarchy from the S2 subunit, which is highly conserved across SARS-CoV-2 variants of concern (VOCs), and toward the antigenically variable receptor binding domain (RBD). A large proportion of RBD-directed neutralizing antibodies isolated from BA.1 breakthrough infection donors displayed convergent sequence features and broadly recognized SARS-CoV-2 VOCs. Together, these findings provide insights into the role of preexisting immunity in shaping the B cell response to heterologous SARS-CoV-2 variant exposure

Broad anti–SARS-CoV-2 antibody immunity induced by heterologous ChAdOx1/mRNA-1273 vaccination

Heterologous prime-boost immunization strategies have the potential to augment COVID-19 vaccine efficacy We longitudinally profiled severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S)–specific serological and memory B cell (MBC) responses in individuals who received either homologous (ChAdOx1: ChAdOx1) or heterologous (ChAdOx1:mRNA-1273) prime-boost vaccination. Heterologous messenger RNA (mRNA) booster immunization induced higher serum neutralizing antibody and MBC responses against SARS-CoV-2 variants of concern (VOCs) compared with that of homologous ChAdOx1 boosting. Specificity mapping of circulating B cells revealed that mRNA-1273 boost immunofocused ChAdOx1-primed responses onto epitopes expressed on prefusion-stabilized S. Monoclonal antibodies isolated from mRNA-1273–booste participants displayed overall higher binding affinities and increased breadth of reactivity against VOCs relativ to those isolated from ChAdOx1-boosted individuals. Overall, the results provide molecular insight into the enhanced quality of the B cell response induced after heterologous mRNA booster vaccination

Vaccination of SARS-CoV-2-infected individuals expands a broad range of clonally diverse affinity-matured B cell lineages

Vaccination of SARS-CoV-2 convalescent individuals generates broad and potent antibody responses. Here, we isolate 459 spike-specific monoclonal antibodies (mAbs) from two individuals who were infected with the index variant of SARS-CoV-2 and later boosted with mRNA-1273. We characterize mAb genetic features by sequence assignments to the donors' personal immunoglobulin genotypes and assess antibody neutralizing activities against index SARS-CoV-2, Beta, Delta, and Omicron variants. The mAbs used a broad range of immunoglobulin heavy chain (IGH) V genes in the response to all sub-determinants of the spike examined, with similar characteristics observed in both donors. IGH repertoire sequencing and B cell lineage tracing at longitudinal time points reveals extensive evolution of SARS-CoV-2 spike-binding antibodies from acute infection until vaccination five months later. These results demonstrate that highly polyclonal repertoires of affinity-matured memory B cells are efficiently recalled by vaccination, providing a basis for the potent antibody responses observed in convalescent persons following vaccination

Genotype-specific features reduce the susceptibility of South American yellow fever virus strains to vaccine-induced antibodies

The resurgence of yellow fever in South America has prompted vaccination against the etiologic agent, yellow fever virus (YFV). Current vaccines are based on a live-attenuated YF-17D virus derived from a virulent African isolate. The capacity of these vaccines to induce neutralizing antibodies against the vaccine strain is used as a surrogate for protection. However, the sensitivity of genetically distinct South American strains to vaccine-induced antibodies is unknown. We show that antiviral potency of the polyclonal antibody response in vaccinees is attenuated against an emergent Brazilian strain. This reduction was attributable to amino acid changes at two sites in central domain II of the glycoprotein E, including multiple changes at the domain I-domain II hinge, which are unique to and shared among most South American YFV strains. Our findings call for a reevaluation of current approaches to YFV immunological surveillance in South America and suggest approaches for updating vaccines

Human antibody recognizing a quaternary epitope in the Puumala virus glycoprotein provides broad protection against orthohantaviruses

International audienceThe rodent-borne hantavirus Puumala virus (PUUV) and related agents cause hemorrhagic fever with renal syndrome (HFRS) in humans. Other hantaviruses, including Andes virus (ANDV) and Sin Nombre virus, cause a distinct zoonotic disease, hantavirus cardiopulmonary syndrome (HCPS). Although these infections are severe and have substantial case fatality rates, no FDA-approved hantavirus countermeasures are available. Recent work suggests that monoclonal antibodies may have therapeutic utility. We describe here the isolation of human neutralizing antibodies (nAbs) against tetrameric Gn/Gc glycoprotein spikes from PUUV-experienced donors. We define a dominant class of nAbs recognizing the “capping loop” of Gn that masks the hydrophobic fusion loops in Gc. A subset of nAbs in this class, including ADI-42898, bound Gn/Gc complexes but not Gn alone, strongly suggesting that they recognize a quaternary epitope encompassing both Gn and Gc. ADI-42898 blocked the cell entry of seven HCPS- and HFRS-associated hantaviruses, and single doses of this nAb could protect Syrian hamsters and bank voles challenged with the highly virulent HCPS-causing ANDV and HFRS-causing PUUV, respectively. ADI-42898 is a promising candidate for clinical development as a countermeasure for both HCPS and HFRS, and its mode of Gn/Gc recognition informs the development of broadly protective hantavirus vaccines