A tale of two fusion proteins: understanding the metastability of human respiratory syncytial virus and metapneumovirus and implications for rational design of uncleaved prefusion-closed trimers.

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) cause human respiratory diseases and are major targets for vaccine development. In this study, we designed uncleaved prefusion-closed (UFC) trimers for the fusion (F) proteins of both viruses by examining mutations critical to F metastability. For RSV, we assessed four previous prefusion F designs, including the first and second generations of DS-Cav1, SC-TM, and 847A. We then identified key mutations that can maintain prefusion F in a native-like, closed trimeric form (up to 76%) without introducing any interprotomer disulfide bond. For hMPV, we developed a stable UFC trimer with a truncated F2-F1 linkage and an interprotomer disulfide bond. Tens of UFC constructs were characterized by negative-stain electron microscopy (nsEM), x-ray crystallography (11 RSV-F and one hMPV-F structures), and antigenic profiling. Using an optimized RSV-F UFC trimer as bait, we identified three potent RSV neutralizing antibodies (NAbs) from a phage-displayed human antibody library, with a public NAb lineage targeting sites Ø and V and two cross-pneumovirus NAbs recognizing site III. In mouse immunization, rationally designed RSV-F and hMPV-F UFC trimers induced robust antibody responses with high neutralizing titers. Our study provides a foundation for future prefusion F-based RSV and hMPV vaccine development.

Adjuvanted-SARS-CoV-2 Spike Protein-Based Microparticulate Vaccine Delivered by Dissolving Microneedles Induces Humoral, Mucosal, and Cellular Immune Responses in Mice.

COVID-19 continues to cause an increase in the number of cases and deaths worldwide. Due to the ever-mutating nature of the virus, frequent vaccination against COVID-19 is anticipated. Most of the approved SARS-CoV-2 vaccines are administered using the conventional intramuscular route, causing vaccine hesitancy. Thus, there is a need for an effective, non-invasive vaccination strategy against COVID-19. This study evaluated the synergistic effects of a subunit microparticulate vaccine delivered using microneedles. The microparticles encapsulated a highly immunogenic subunit protein of the SARS-CoV-2 virus, such as the spike protein's receptor binding domain (RBD). Adjuvants were also incorporated to enhance the spike RBD-specific immune response. Our vaccination study reveals that a microneedle-based vaccine delivering these microparticles induced spike RBD-specific IgM, IgG, IgG1, IgG2a, and IgA antibodies. The vaccine also generated high levels of CD4+ and CD8a+ molecules in the secondary lymphoid organs. Overall, dissolving microneedles delivery spike RBD antigen in microparticulate form induced a robust immune response, paving the way for an alternative self-administrable, non-invasive vaccination strategy against COVID-19.

Enhanced Immunogenicity of an Influenza Ectodomain Matrix-2 Protein Virus-like Particle (M2e VLP) Using Polymeric Microparticles for Vaccine Delivery.

In this study, we demonstrate how encapsulating a conserved influenza ectodomain matrix-2 protein virus-like particle (M2e5x VLP) into a pre-crosslinked bovine serum albumin (BSA) polymeric matrix enhances in vitro antigen immunogenicity and in vivo efficacy. The spray-dried M2e5x VLP-loaded BSA microparticles (MPs) showed enhanced stimulation of antigen presenting cells (APCs), as confirmed through nitrite production and increased antigen-cell interactions seen in real time using live-cell imaging. Next, to further boost the immunogenicity of M2e5x VLP microparticles, M2e5x MPs were combined with Alhydrogel® and monophosphoryl lipid-A (MPL-A®) adjuvant microparticles. M2e5x VLP MPs and the combination VLP M2e5x VLP + Alhydrogel® + MPL-A® MPs elicited a significant increase in the expression of antigen-presenting molecules in dendritic cells compared to M2e5x VLP alone. Lastly, for preliminary evaluation of in vivo efficacy, the vaccine was administered in mice through the skin using an ablative laser. The M2e5x VLP + Alhydrogel® + MPL-A® MPs were shown to induce high levels of M2e-specific IgG antibodies. Further, a challenge with live influenza revealed heightened T-cell stimulation in immune organs of mice immunized with M2e5x VLP + Alhydrogel® + MPL-A® MPs. Hence, we utilized the advantages of both VLP and polymeric delivery platforms to enhance antigen immunogenicity and adaptive immunity in vivo.

Microneedles: A New Generation Vaccine Delivery System.

Transdermal vaccination route using biodegradable microneedles is a rapidly progressing field of research and applications. The fear of painful needles is one of the primary reasons most people avoid getting vaccinated. Therefore, developing an alternative pain-free method of vaccination using microneedles has been a significant research area. Microneedles comprise arrays of micron-sized needles that offer a pain-free method of delivering actives across the skin. Apart from being pain-free, microneedles provide various advantages over conventional vaccination routes such as intramuscular and subcutaneous. Microneedle vaccines induce a robust immune response as the needles ranging from 50 to 900 µm in length can efficiently deliver the vaccine to the epidermis and the dermis region, which contains many Langerhans and dendritic cells. The microneedle array looks like band-aid patches and offers the advantages of avoiding cold-chain storage and self-administration flexibility. The slow release of vaccine antigens is an important advantage of using microneedles. The vaccine antigens in the microneedles can be in solution or suspension form, encapsulated in nano or microparticles, and nucleic acid-based. The use of microneedles to deliver particle-based vaccines is gaining importance because of the combined advantages of particulate vaccine and pain-free immunization. The future of microneedle-based vaccines looks promising however, addressing some limitations such as dosing inadequacy, stability and sterility will lead to successful use of microneedles for vaccine delivery. This review illustrates the recent research in the field of microneedle-based vaccination.

Exploring the Potential of T-Cells for a Universal Influenza Vaccine.

Among the four types of influenza viruses, the influenza A strains and their subtypes have been responsible for causing worldwide pandemics and seasonal epidemics [...].