Transformer-Induced Metamorphosis of Polymeric Nanoparticle Shape at Room Temperature.

Controlled polymerizations have enabled the production of nanostructured materials with different shapes, each exhibiting distinct properties. Despite the importance of shape, current morphological transformation strategies are limited in polymer scope, alter the chemical structure, require high temperatures, and are fairly tedious. Herein we present a rapid and versatile morphological transformation strategy that operates at room temperature and does not impair the chemical structure of the constituent polymers. By simply adding a molecular transformer to an aqueous dispersion of polymeric nanoparticles, a rapid evolution to the next higher-order morphology was observed, yielding a range of morphologies from a single starting material. Significantly, this approach can be applied to nanoparticles produced by disparate block copolymers obtained by various synthetic techniques including emulsion polymerization, polymerization-induced self-assembly and traditional solution self-assembly.

Cellular Interactions of Liposomes and PISA Nanoparticles during Human Blood Flow in a Microvascular Network.

A key concept in nanomedicine is encapsulating therapeutic or diagnostic agents inside nanoparticles to prolong blood circulation time and to enhance interactions with targeted cells. During circulation and depending on the selected application (e.g., cancer drug delivery or immune modulators), nanoparticles are required to possess low or high interactions with cells in human blood and blood vessels to minimize side effects or maximize delivery efficiency. However, analysis of cellular interactions in blood vessels is challenging and is not yet realized due to the diverse components of human blood and hemodynamic flow in blood vessels. Here, the first comprehensive method to analyze cellular interactions of both synthetic and commercially available nanoparticles under human blood flow conditions in a microvascular network is developed. Importantly, this method allows to unravel the complex interplay of size, charge, and type of nanoparticles on their cellular associations under the dynamic flow of human blood. This method offers a unique platform to study complex interactions of any type of nanoparticles in human blood flow conditions and serves as a useful guideline for the rational design of liposomes and polymer nanoparticles for diverse applications in nanomedicine.

Human Plasma Protein Corona of Aß Amyloid and Its Impact on Islet Amyloid Polypeptide Cross-Seeding.

Alzheimer's disease (AD) is the most severe form of neurological disorder, characterized by the presence of extracellular amyloid-ß (Aß) plaques and intracellular tau tangles. For decades, therapeutic strategies against the pathological symptoms of AD have often relied on the delivery of monoclonal antibodies to target specifically Aß amyloid or oligomers, largely to no avail. Aß can be traced in the brain as well as in cerebrospinal fluid and the circulation, giving rise to abundant opportunities to interact with their environmental proteins. Using liquid chromatography tandem-mass spectrometry, here we identified for the first time the protein coronae of the two major amyloid forms of Aß-Aß1-42 and Aß1-40-exposed to human blood plasma. Out of the proteins identified in all groups, 58 proteins were unique to the Aß1-42 samples and 31 proteins unique to the Aß1-40 samples. Both fibrillar coronae consisted of proteins significant in complement activation, inflammation, and protein metabolic pathways involved in the pathology of AD. Structure-wise, the coronal proteins often possessed multidomains of high flexibility to maximize their association with the amyloid fibrils. The protein corona hindered recognition of Aß1-42 fibrils by their structurally specific antibodies and accelerated the aggregation but not the ß-cell toxicity of human islet amyloid polypeptide, the peptide associated with type 2 diabetes. This study highlights the importance of understanding the structural, functional, and pathological implications of the amyloid protein corona for the development of therapeutics against AD and a range of amyloid diseases.

Elucidating the Influences of Size, Surface Chemistry, and Dynamic Flow on Cellular Association of Nanoparticles Made by Polymerization-Induced Self-Assembly.

The size and surface chemistry of nanoparticles dictate their interactions with biological systems. However, it remains unclear how these key physicochemical properties affect the cellular association of nanoparticles under dynamic flow conditions encountered in human vascular networks. Here, the facile synthesis of novel fluorescent nanoparticles with tunable sizes and surface chemistries and their association with primary human umbilical vein endothelial cells (HUVECs) is reported. First, a one-pot polymerization-induced self-assembly (PISA) methodology is developed to covalently incorporate a commercially available fluorescent dye into the nanoparticle core and tune nanoparticle size and surface chemistry. To characterize cellular association under flow, HUVECs are cultured onto the surface of a synthetic microvascular network embedded in a microfluidic device (SynVivo, INC). Interestingly, increasing the size of carboxylic acid-functionalized nanoparticles leads to higher cellular association under static conditions but lower cellular association under flow conditions, whereas increasing the size of tertiary amine-decorated nanoparticles results in a higher level of cellular association, under both static and flow conditions. These findings provide new insights into the interactions between polymeric nanomaterials and endothelial cells. Altogether, this work establishes innovative methods for the facile synthesis and biological characterization of polymeric nanomaterials for various potential applications.

Cofibrillization of Pathogenic and Functional Amyloid Proteins with Gold Nanoparticles against Amyloidogenesis.

Biomimetic nanocomposites and scaffolds hold the key to a wide range of biomedical applications. Here we show, for the first time, a facile scheme of cofibrillizing pathogenic and functional amyloid fibrils via gold nanoparticles (AuNPs) and their applications against amyloidogenesis. This scheme was realized by ß-sheet stacking between human islet amyloid polypeptide (IAPP) and the ß-lactoglobulin "corona" of the AuNPs, as revealed by transmission electron microscopy, 3D atomic force microscopy, circular dichroism spectroscopy, and molecular dynamics simulations. The biomimetic AuNPs eliminated IAPP toxicity, enabled X-ray destruction of IAPP amyloids, and allowed dark-field imaging of pathogenic amyloids and their immunogenic response by human T cells. In addition to providing a viable new nanotechnology against amyloidogenesis, this study has implications for understanding the in vivo cross-talk between amyloid proteins of different pathologies.

Star Polymers Reduce Islet Amyloid Polypeptide Toxicity via Accelerated Amyloid Aggregation.

Protein aggregation into amyloid fibrils is a ubiquitous phenomenon across the spectrum of neurodegenerative disorders and type 2 diabetes. A common strategy against amyloidogenesis is to minimize the populations of toxic oligomers and protofibrils by inhibiting protein aggregation with small molecules or nanoparticles. However, melanin synthesis in nature is realized by accelerated protein fibrillation to circumvent accumulation of toxic intermediates. Accordingly, we designed and demonstrated the use of star-shaped poly(2-hydroxyethyl acrylate) (PHEA) nanostructures for promoting aggregation while ameliorating the toxicity of human islet amyloid polypeptide (IAPP), the peptide involved in glycemic control and the pathology of type 2 diabetes. The binding of PHEA elevated the ß-sheet content in IAPP aggregates while rendering a new morphology of "stelliform" amyloids originating from the polymers. Atomistic molecular dynamics simulations revealed that the PHEA arms served as rodlike scaffolds for IAPP binding and subsequently accelerated IAPP aggregation by increased local peptide concentration. The tertiary structure of the star nanoparticles was found to be essential for driving the specific interactions required to impel the accelerated IAPP aggregation. This study sheds new light on the structure-toxicity relationship of IAPP and points to the potential of exploiting star polymers as a new class of therapeutic agents against amyloidogenesis.

Lysophosphatidylcholine modulates the aggregation of human islet amyloid polypeptide.

Amyloid aggregation of human islet amyloid polypeptide (IAPP) is a hallmark of type 2 diabetes (T2D), a metabolic disease and a global epidemic. Although IAPP is synthesized in pancreatic ß-cells, its fibrils and plaques are found in the extracellular space indicating a causative transmembrane process. Numerous biophysical studies have revealed that cell membranes as well as model lipid vesicles promote the aggregation of amyloid-ß (associated with Alzheimer's), α-synuclein (associated with Parkinson's) and IAPP, through electrostatic and hydrophobic interactions between the proteins/peptides and lipid membranes. Using a thioflavin T kinetic assay, transmission electron microscopy, circular dichroism spectroscopy, discrete molecular dynamics simulations as well as free energy calculations here we show that micellar lysophosphatidylcholine (LPC), the most abundant lysophospholipid in the blood, inhibited the amyloid aggregation of IAPP through nonspecific interactions while elevating the α-helical peptide secondary structure. This surprising finding suggests a native protective mechanism against IAPP aggregation in vivo.

Inhibition of hIAPP Amyloid Aggregation and Pancreatic ß-Cell Toxicity by OH-Terminated PAMAM Dendrimer.

Human islet amyloid polypeptide (hIAPP, or amylin) forms amyloid deposits in the islets of Langerhans, a phenomenon that is associated with type-2 diabetes impacting millions of people worldwide. Accordingly, strategies against hIAPP aggregation are essential for the prevention and eventual treatment of the disease. Here, it is shown that generation-3 OH-terminated poly(amidoamine) dendrimer, a polymeric nanoparticle, can effectively halt the aggregation of hIAPP and shut down hIAPP toxicity in pancreatic MIN6 and NIT-1 cells as well as in mouse islets. This finding is supported by high-throughput dynamic light scattering experiment and thioflavin T assay, where the rapid evolution of hIAPP nucleation and elongation processes is halted by the addition of the dendrimer up to 8 h. Discrete molecular dynamics simulations further reveal that hIAPP residues bound strongly with the dendrimer near the c-terminal portion of the peptide, where the amyloidogenic sequence (residues 22-29) locates. Furthermore, simulations of hIAPP dimerization reveal that binding with the dendrimer significantly reduces formation of interpeptide contacts and hydrogen bonds, thereby prohibiting peptide self-association and amyloidosis. This study points to a promising nanomedicinal strategy for combating type-2 diabetes and may have broader implications for targeting neurological disorders whose distinct hallmark is also amyloid fibrillation.

Graphene oxide inhibits hIAPP amyloid fibrillation and toxicity in insulin-producing NIT-1 cells.

Human islet amyloid polypeptide (hIAPP or amylin) aggregation is directly associated with pancreatic ß-cell death and subsequent insulin deficiency in type 2 diabetes (T2D). Since no cure is currently available for T2D, it is of great benefit to devise new anti-aggregation molecules, which protect ß-cells against hIAPP aggregation-induced toxicity. Engineered nanoparticles have been recently exploited as anti-aggregation nanomedicines. In this work, we studied graphene oxide (GO) nanosheets for their potential for hIAPP aggregation inhibition by combining computational modeling, biophysical characterization and cell toxicity measurements. Using discrete molecular dynamics (DMD) simulations and in vitro studies, we showed that GO exhibited an inhibitory effect on hIAPP aggregation. DMD simulations indicated that the strong binding of hIAPP to GO nanosheets was driven by hydrogen bonding and aromatic stacking and that the strong peptide-GO binding efficiently inhibited hIAPP self-association and aggregation on the nanosheet surface. Secondary structural changes of hIAPP upon GO binding derived from DMD simulations were consistent with circular dichroism (CD) spectroscopy measurements. Transmission electron microscopy (TEM) images confirmed the reduction of hIAPP aggregation in the presence of GO. Furthermore, we carried out a cell toxicity assay and found that these nanosheets protected insulin-secreting NIT-1 pancreatic ß-cells against hIAPP-induced toxicity. Our multidisciplinary study suggests that GO nanosheets have the potential to be utilized as an anti-aggregation nanomedicine itself in addition to a biosensor or delivery vehicle for the mitigation of T2D progression.

Replacing poly(ethylene glycol) with RAFT lipopolymers in mRNA lipid nanoparticle systems for effective gene delivery.

Lipid nanoparticles (LNPs) have emerged as promising carriers to efficiently transport mRNA into cells for protein translation, as seen with the mRNA vaccines used against COVID-19. However, they contain a widely used polymer - poly(ethylene glycol) (PEG) - which lacks the functionality to be easily modified (which could effectively control the physicochemical properties of the LNPs such as its charge), and is also known to be immunogenic. Thus, it is desirable to explore alternative polymers which can replace the PEG component in mRNA LNP vaccines and therapeutics, while still maintaining their efficacy. Herein, we employed reversible addition-fragmentation chain transfer (RAFT) polymerisation to synthesise five PEG-lipid alternatives that could stabilise LNPs encapsulating mRNA or pDNA molecules. Importantly, the resultant RAFT lipopolymer LNPs exhibit analogous or higher in vivo gene expression and antigen-specific antibody production compared to traditional PEG-based formulations. Our synthesis strategy which allows the introduction of positive charges along the lipopolymer backbone also significantly improved the in vivo gene expression. This work expands the potential of RAFT polymer-conjugated LNPs as promising mRNA carriers and offers an innovative strategy for the development of PEG-free mRNA vaccines and therapeutics.

Patient-Specific Nanoparticle Targeting in Human Leukemia Blood.

Antibody-directed targeting of chemotherapeutic nanoparticles to primary human cancers holds promise for improving efficacy and reducing off-target toxicity. However, clinical responses to targeted nanomedicines are highly variable. Herein, we prepared and examined a matrix of 9 particles (organic and inorganic particles of three surface chemistries with and without antibody functionalization) and developed an ex vivo model to study the person-specific targeting of nanoparticles in whole blood of 15 patients with chronic lymphocytic leukemia (CLL). Generally, anti-CD20-functionalized poly(ethylene glycol) (PEG) nanoparticles efficiently targeted CLL cells, leading to low off-target phagocytosis by granulocytes and monocytes in the blood. However, there was up to 164-fold patient-to-patient variability in the CLL targeting. This was further exemplified through using clinically relevant PEGylated doxorubicin-encapsulated liposomes, which showed high interpersonal differences in CLL targeting (up to 234-fold differences) and off-target phagocytosis (up to 65- and 112-fold differences in granulocytes and monocytes, respectively). Off-target phagocytosis led to almost all monocytes being killed within 24 h of treatment. Variance of the off-target association of PEGylated liposomes with granulocytes and monocytes significantly correlated to anti-PEG immunoglobulin G levels in the blood of CLL patients. A negative correlation between CLL targeting of PEG particles and anti-PEG immunoglobulin M levels was found in the blood. Taken together, our study identifies anti-PEG antibodies as key proteins in modulating patient-specific targeting of PEGylated nanoparticles in human leukemia blood. Other factors, such as the antigen expression of targeted cells and fouling properties of nanoparticles, also play an important role in patient-specific targeting. The human leukemia blood assay we developed provides an ex vivo model to evaluate interpersonal variances in response to targeted nanomedicines.

Engineered Ferritin Nanoparticle Vaccines Enable Rapid Screening of Antibody Functionalization to Boost Immune Responses.

Employing monoclonal antibodies to target vaccine antigens to different immune cells within lymph nodes where adaptive immunity is initiated can provide a mechanism to fine-tune the magnitude or the quality of immune responses. However, studying the effects of different targeting antibodies head-to-head is challenging due to the lack of a feasible method that allows rapid screening of multiple antibodies for their impact on immunogenicity. Here self-assembling ferritin nanoparticles are prepared that co-display vaccine antigens and the Fc-binding domain of Staphylococcal protein A, allowing rapid attachment of soluble antibodies to the nanoparticle surface. Using this tunable system, ten antibodies targeting different immune cell subsets are screened, with targeting to Clec9a associated with higher serum antibody titers after immunization. Immune cell targeting using ferritin nanoparticles with anti-Clec9a antibodies drives concentrated deposition of antigens within germinal centers, boosting germinal center formation and robust antibody responses. However, the capacity to augment humoral immunity is antigen-dependent, with significant boosting observed for prototypic ovalbumin immunogens but reduced effectiveness with the SARS-CoV-2 RBD. This work provides a rapid platform for screening targeting antibodies, which will accelerate mechanistic insights into optimal delivery strategies for nanoparticle-based vaccines to maximize protective immunity.

In vivo delivery of plasmid DNA by lipid nanoparticles: the influence of ionizable cationic lipids on organ-selective gene expression.

Ionizable cationic lipids play a critical role in developing new gene therapies for various biomedical applications, including COVID-19 vaccines. However, it remains unclear whether the formulation of lipid nanoparticles (LNPs) using DLin-MC3-DMA, an optimized ionizable lipid clinically used for small interfering RNA (siRNA) therapy, also facilitates high liver-selective transfection of other gene therapies such as plasmid DNA (pDNA). Here we report the first investigation into pDNA transfection efficiency in different mouse organs after intramuscular and intravenous administration of lipid nanoparticles (LNPs) where DLin-MC3-DMA, DLin-KC2-DMA or DODAP are used as the ionizable cationic lipid component of the LNP. We discovered that these three benchmark lipids previously developed for siRNA delivery followed an unexpected characteristic rank order in gene expression efficiency when utilized for pDNA. In particular, DLin-KC2-DMA facilitated higher in vivo pDNA transfection than DLin-MC3-DMA and DODAP, possibly due to its head group pKa and lipid tail structure. Interestingly, LNPs formulated with either DLin-KC2-DMA or DLin-MC3-DMA exhibited significantly higher in vivo protein production in the spleen than in the liver. This work sheds light on the importance of the choice of ionizable cationic lipid and nucleic acid cargo for organ-selective gene expression. The study also provides a new design principle towards the formulation of more effective LNPs for biomedical applications of pDNA, such as gene editing, vaccines and immunotherapies.

Anti-PEG Antibodies Boosted in Humans by SARS-CoV-2 Lipid Nanoparticle mRNA Vaccine.

Humans commonly have low level antibodies to poly(ethylene) glycol (PEG) due to environmental exposure. Lipid nanoparticle (LNP) mRNA vaccines for SARS-CoV-2 contain small amounts of PEG, but it is not known whether PEG antibodies are enhanced by vaccination and what their impact is on particle-immune cell interactions in human blood. We studied plasma from 130 adults receiving either the BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) mRNA vaccines or no SARS-CoV-2 vaccine for PEG-specific antibodies. Anti-PEG IgG was commonly detected prior to vaccination and was significantly boosted a mean of 13.1-fold (range 1.0-70.9) following mRNA-1273 vaccination and a mean of 1.78-fold (range 0.68-16.6) following BNT162b2 vaccination. Anti-PEG IgM increased 68.5-fold (range 0.9-377.1) and 2.64-fold (0.76-12.84) following mRNA-1273 and BNT162b2 vaccination, respectively. The rise in PEG-specific antibodies following mRNA-1273 vaccination was associated with a significant increase in the association of clinically relevant PEGylated LNPs with blood phagocytes ex vivo. PEG antibodies did not impact the SARS-CoV-2 specific neutralizing antibody response to vaccination. However, the elevated levels of vaccine-induced anti-PEG antibodies correlated with increased systemic reactogenicity following two doses of vaccination. We conclude that PEG-specific antibodies can be boosted by LNP mRNA vaccination and that the rise in PEG-specific antibodies is associated with systemic reactogenicity and an increase of PEG particle-leukocyte association in human blood. The longer-term clinical impact of the increase in PEG-specific antibodies induced by lipid nanoparticle mRNA vaccines should be monitored. It may be useful to identify suitable alternatives to PEG for developing next-generation LNP vaccines to overcome PEG immunogenicity in the future.

Stealth nanorods via the aqueous living crystallisation-driven self-assembly of poly(2-oxazoline)s.

The morphology of nanomaterials critically influences their biological interactions. However, there is currently a lack of robust methods for preparing non-spherical particles from biocompatible materials. Here, we combine 'living' crystallisation-driven self-assembly (CDSA), a seeded growth method that enables the preparation of rod-like polymer nanoparticles, with poly(2-oxazoline)s (POx), a polymer class that exhibits 'stealth' behaviour and excellent biocompatibility. For the first time, the 'living' CDSA process was carried out in pure water, resulting in POx nanorods with lengths ranging from ∼60 to 635 nm. In vitro and in vivo study revealed low immune cell association and encouraging blood circulation times, but little difference in the behaviour of POx nanorods of different length. The stealth behaviour observed highlights the promising potential of POx nanorods as a next generation stealth drug delivery platform.

Shape-Controlled Nanoparticles from a Low-Energy Nanoemulsion.

Nanoemulsion technology enables the production of uniform nanoparticles for a wide range of applications. However, existing nanoemulsion strategies are limited to the production of spherical nanoparticles. Here, we describe a low-energy nanoemulsion method to produce nanoparticles with various morphologies. By selecting a macro-RAFT agent (poly(di(ethylene glycol) ethyl ether methacrylate-co-N-(2-hydroxypropyl) methacrylamide) (P(DEGMA-co-HPMA))) that dramatically lowers the interfacial tension between monomer droplets and water, we can easily produce nanoemulsions at room temperature by manual shaking for a few seconds. With the addition of a common ionic surfactant (SDS), these nanoscale droplets are robustly stabilized at both the formation and elevated temperatures. Upon polymerization, we produce well-defined block copolymers forming nanoparticles with a wide range of controlled morphologies, including spheres, worm balls, worms, and vesicles. Our nanoemulsion polymerization is robust and well-controlled even without stirring or external deoxygenation. This method significantly expands the toolbox and availability of nanoemulsions and their tailor-made polymeric nanomaterials.

From influenza to COVID-19: Lipid nanoparticle mRNA vaccines at the frontiers of infectious diseases.

Vaccination represents the best line of defense against infectious diseases and is crucial in curtailing pandemic spread of emerging pathogens to which a population has limited immunity. In recent years, mRNA vaccines have been proposed as the new frontier in vaccination, owing to their facile and rapid development while providing a safer alternative to traditional vaccine technologies such as live or attenuated viruses. Recent breakthroughs in mRNA vaccination have been through formulation with lipid nanoparticles (LNPs), which provide both protection and enhanced delivery of mRNA vaccines in vivo. In this review, current paradigms and state-of-the-art in mRNA-LNP vaccine development are explored through first highlighting advantages posed by mRNA vaccines, establishing LNPs as a biocompatible delivery system, and finally exploring the use of mRNA-LNP vaccines in vivo against infectious disease towards translation to the clinic. Furthermore, we highlight the progress of mRNA-LNP vaccine candidates against COVID-19 currently in clinical trials, with the current status and approval timelines, before discussing their future outlook and challenges that need to be overcome towards establishing mRNA-LNPs as next-generation vaccines. STATEMENT OF SIGNIFICANCE: With the recent success of mRNA vaccines developed by Moderna and BioNTech/Pfizer against COVID-19, mRNA technology and lipid nanoparticles (LNP) have never received more attention. This manuscript timely reviews the most advanced mRNA-LNP vaccines that have just been approved for emergency use and are in clinical trials, with a focus on the remarkable development of several COVID-19 vaccines, faster than any other vaccine in history. We aim to give a comprehensive introduction of mRNA and LNP technology to the field of biomaterials science and increase accessibility to readers with a new interest in mRNA-LNP vaccines. We also highlight current limitations and future outlook of the mRNA vaccine technology that need further efforts of biomaterials scientists to address.

Mitigation of Amyloidosis with Nanomaterials.

Amyloidosis is a biophysical phenomenon of protein aggregation with biological and pathogenic implications. Among the various strategies developed to date, nanomaterials and multifunctional nanocomposites possessing certain structural and physicochemical traits are promising candidates for mitigating amyloidosis in vitro and in vivo. The mechanisms underpinning protein aggregation and toxicity are introduced, and opportunities in materials science to drive this interdisciplinary field forward are highlighted. Advancement of this emerging frontier hinges on exploitation of protein self-assembly and interactions of amyloid proteins with nanoparticles, intracellular and extracellular proteins, chaperones, membranes, organelles, and biometals.

The Importance of Poly(ethylene glycol) and Lipid Structure in Targeted Gene Delivery to Lymph Nodes by Lipid Nanoparticles.

Targeted delivery of nucleic acids to lymph nodes is critical for the development of effective vaccines and immunotherapies. However, it remains challenging to achieve selective lymph node delivery. Current gene delivery systems target mainly to the liver and typically exhibit off-target transfection at various tissues. Here we report novel lipid nanoparticles (LNPs) that can deliver plasmid DNA (pDNA) to a draining lymph node, thereby significantly enhancing transfection at this target organ, and substantially reducing gene expression at the intramuscular injection site (muscle). In particular, we discovered that LNPs stabilized by 3% Tween 20, a surfactant with a branched poly(ethylene glycol) (PEG) chain linking to a short lipid tail, achieved highly specific transfection at the lymph node. This was in contrast to conventional LNPs stabilized with a linear PEG chain and two saturated lipid tails (PEG-DSPE) that predominately transfected at the injection site (muscle). Interestingly, replacing Tween 20 with Tween 80, which has a longer unsaturated lipid tail, led to a much lower transfection efficiency. Our work demonstrates the importance of PEGylation in selective organ targeting of nanoparticles, provides new insights into the structure-property relationship of LNPs, and offers a novel, simple, and practical PEGylation technology to prepare the next generation of safe and effective vaccines against viruses or tumours.

Aggregation by peptide conjugation rescues poor immunogenicity of the HA stem.

Influenza virus infection is a global public health threat. Current seasonal influenza vaccines are efficacious only when vaccine strains are matched with circulating strains. There is a critical need for developing "universal" vaccines that protect against all influenza viruses. HA stem is a promising target for developing broad-spectrum influenza vaccines due to its relatively conserved feature. However, HA stem is weakly immunogenic when administered alone in a soluble form. Several approaches have been employed to improve the immunogenicity of HA stem, including conjugation of HA stem with a highly immunogenic carrier protein or displaying HA stem on a nanoparticle scaffold. Converting a weakly immunologic protein into a multimer through aggregation can significantly enhance its immunogenicity, with some multimeric protein aggregates previously shown to be more immunogenic than their soluble counterparts in animal models. Here, we show that a chemically coupling a peptide derived from the head domain of PR8 HA (P35) with the poorly immunogenic HA stem protein results in aggregation of the HA stem which significantly increases stem-specific B cell responses following vaccination. Importantly, vaccination with this conjugate in the absence of adjuvant still induced robust B cell responses against stem in vivo. Improving HA stem immunogenicity by aggregation provides an alternative avenue to conjugation with exotic carrier proteins or nanoparticle formulation.