SARS-CoV-2 Protein Nanoparticle Vaccines Formed In Situ From Lyophilized Lipids.

The receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) glycoprotein is an appealing immunogen, but associated vaccine approaches must overcome the hapten-like nature of the compact protein and adapt to emerging variants with evolving RBD sequences. Here, a vaccine manufacturing methodology is proposed comprising a sterile-filtered freeze-dried lipid cake formulation that can be reconstituted with liquid proteins to instantaneously form liposome-displayed protein nanoparticles. Mannitol is used as a bulking agent and a small amount of Tween-80 surfactant is required to achieve reconstituted submicron particles that do not precipitate prior to usage. The lipid particles include an E. coli-derived monophosphoryl lipid A (EcML) for immunogenicity, and cobalt porphyrin-phospholipid (CoPoP) for antigen display. Reconstitution of the lipid cake with aqueous protein results in rapid conversion of the RBD into intact liposome-bound format prior to injection. Protein particles can readily be formed with sequent-divergent RBD proteins derived from the ancestral or Omicron strains. Immunization of mice elicits antibodies that neutralize respective viral strains. When K18-hACE2 transgenic mice are immunized and challenged with ancestral SARS-CoV-2 or the Omicron BA.5 variant, both liquid liposomes displaying the RBD and rapid reconstituted particles protect mice from infection, as measured by the viral load in the lungs and nasal turbinates.

Circularized Nanodiscs for Multivalent Mosaic Display of SARS-CoV-2 Spike Protein Antigens.

The emergence of vaccine-evading SARS-CoV-2 variants urges the need for vaccines that elicit broadly neutralizing antibodies (bnAbs). Here, we assess covalently circularized nanodiscs decorated with recombinant SARS-CoV-2 spike glycoproteins from several variants for eliciting bnAbs with vaccination. Cobalt porphyrin-phospholipid (CoPoP) was incorporated into the nanodisc to allow for anchoring and functional orientation of spike trimers on the nanodisc surface through their His-tag. Monophosphoryl-lipid (MPLA) and QS-21 were incorporated as immunostimulatory adjuvants to enhance vaccine responses. Following optimization of nanodisc assembly, spike proteins were effectively displayed on the surface of the nanodiscs and maintained their conformational capacity for binding with human angiotensin-converting enzyme 2 (hACE2) as verified using electron microscopy and slot blot assay, respectively. Six different formulations were prepared where they contained mono antigens; four from the year 2020 (WT, Beta, Lambda, and Delta) and two from the year 2021 (Omicron BA.1 and BA.2). Additionally, we prepared a mosaic nanodisc displaying the four spike proteins from year 2020. Intramuscular vaccination of CD-1 female mice with the mosaic nanodisc induced antibody responses that not only neutralized matched pseudo-typed viruses, but also neutralized mismatched pseudo-typed viruses corresponding to later variants from year 2021 (Omicron BA.1 and BA.2). Interestingly, sera from mosaic-immunized mice did not effectively inhibit Omicron spike binding to human ACE-2, suggesting that some of the elicited antibodies were directed towards conserved neutralizing epitopes outside the receptor binding domain. Our results show that mosaic nanodisc vaccine displaying spike proteins from 2020 can elicit broadly neutralizing antibodies that can neutralize mismatched viruses from a following year, thus decreasing immune evasion of new emerging variants and enhancing healthcare preparedness.

Antibody induction in mice by liposome-displayed recombinant enterotoxigenic Escherichia coli (ETEC) colonization antigens.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) strains cause infectious diarrhea and colonize host intestine epithelia via surface-expressed colonization factors. Colonization factor antigen I (CFA/I), a prevalent ETEC colonization factor, is a vaccine target since antibodies directed to this fimbria can block ETEC adherence and prevent diarrhea. METHODS: Two recombinant antigens derived from CFA/I were investigated with a vaccine adjuvant system that displays soluble antigens on the surface of immunogenic liposomes. The first antigen, CfaEB, is a chimeric fusion protein comprising the minor (CfaE) and major (CfaB) subunits of CFA/I. The second, CfaEad, is the adhesin domain of CfaE. RESULTS: Owing to their His-tag, recombinant CfaEB and CfaEad, spontaneously bound upon admixture with nanoliposomes containing cobalt-porphyrin phospholipid (CoPoP), as well as a synthetic monophosphoryl lipid A (PHAD) adjuvant. Intramuscular immunization of mice with sub-microgram doses CfaEB or CfaEad admixed with CoPoP/PHAD liposomes elicited serum IgG and intestinal IgA antibodies. The smaller CfaEad antigen benefitted more from liposome display. Serum and intestine antibodies from mice immunized with liposome-displayed CfaEB or CfaEad recognized native CFA/I fimbria as evidenced by immunofluorescence and hemagglutination inhibition assays using the CFA/I-expressing H10407 ETEC strain. CONCLUSION: These data show that colonization factor-derived recombinant ETEC antigens exhibit immunogenicity when delivered in immunogenic particle-based formulations.

Vaccine co-display of CSP and Pfs230 on liposomes targeting two Plasmodium falciparum differentiation stages.

A vaccine targeting multiple stages of the Plasmodium falciparum parasite life cycle is desirable. The sporozoite surface Circumsporozoite Protein (CSP) is the target of leading anti-infective P. falciparum pre-erythrocytic vaccines. Pfs230, a sexual-stage P. falciparum surface protein, is currently in trials as the basis for a transmission-blocking vaccine, which inhibits parasite development in the mosquito vector. Here, recombinant full-length CSP and a Pfs230 fragment (Pfs230D1+) are co-displayed on immunogenic liposomes to induce immunity against both infection and transmission. Liposomes contain cobalt-porphyrin phospholipid (CoPoP), monophosphoryl lipid A and QS-21, and rapidly bind His-tagged CSP and Pfs230D1+ upon admixture to form bivalent particles that maintain reactivity with conformational monoclonal antibodies. Use of multicolor fluorophore-labeled antigens reveals liposome binding upon admixture, stability in serum and enhanced uptake in murine macrophages in vitro. Bivalent liposomes induce humoral and cellular responses against both CSP and Pfs230D1+. Vaccine-induced antibodies reduce parasite numbers in mosquito midguts in a standard membrane feeding assay. Mice immunized with liposome-displayed antigens or that passively receive antibodies from immunized rabbits have reduced parasite liver burden following challenge with transgenic sporozoites expressing P. falciparum CSP.

Vaccination with a structure-based stabilized version of malarial antigen Pfs48/45 elicits ultra-potent transmission-blocking antibody responses.

Malaria transmission-blocking vaccines (TBVs) aim to elicit human antibodies that inhibit sporogonic development of Plasmodium falciparum in mosquitoes, thereby preventing onward transmission. Pfs48/45 is a leading clinical TBV candidate antigen and is recognized by the most potent transmission-blocking monoclonal antibody (mAb) yet described; still, clinical development of Pfs48/45 antigens has been hindered, largely by its poor biochemical characteristics. Here, we used structure-based computational approaches to design Pfs48/45 antigens stabilized in the conformation recognized by the most potently inhibitory mAb, achieving >25°C higher thermostability compared with the wild-type protein. Antibodies elicited in mice immunized with these engineered antigens displayed on liposome-based or protein nanoparticle-based vaccine platforms exhibited 1-2 orders of magnitude superior transmission-reducing activity, compared with immunogens bearing the wild-type antigen, driven by improved antibody quality. Our data provide the founding principles for using molecular stabilization solely from antibody structure-function information to drive improved immune responses against a parasitic vaccine target.

Advanced Materials for SARS-CoV-2 Vaccines.

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has killed untold millions worldwide and has hurtled vaccines into the spotlight as a go-to approach to mitigate it. Advances in virology, genomics, structural biology, and vaccine technologies have enabled a rapid and unprecedented rollout of COVID-19 vaccines, although much of the developing world remains unvaccinated. Several new vaccine platforms have been developed or deployed against SARS-CoV-2, with most targeting the large viral Spike immunogen. Those that safely induce strong and durable antibody responses at low dosages are advantageous, as well are those that can be rapidly produced at a large scale. Virtually all COVID-19 vaccines and adjuvants possess nanoscale or microscale dimensions and represent diverse and unique biomaterials. Viral vector vaccine platforms, lipid nanoparticle mRNA vaccines and multimeric display technologies for subunit vaccines have received much attention. Nanoscale vaccine adjuvants have also been used in combination with other vaccines. To deal with the ongoing pandemic, and to be ready for potential future ones, advanced vaccine technologies will continue to be developed in the near future. Herein, the recent use of advanced materials used for developing COVID-19 vaccines is summarized.

Lyophilized, thermostable Spike or RBD immunogenic liposomes induce protective immunity against SARS-CoV-2 in mice.

The COVID-19 pandemic has spurred interest in potent and thermostable SARS-CoV-2 vaccines. Here, we assess low-dose immunization with lyophilized nanoparticles decorated with recombinant SARS-CoV-2 antigens. The SARS-CoV-2 Spike glycoprotein or its receptor-binding domain (RBD; mouse vaccine dose, 0.1 µg) was displayed on liposomes incorporating a particle-inducing lipid, cobalt porphyrin-phospholipid (dose, 0.4 µg), along with monophosphoryl lipid A (dose, 0.16 µg) and QS-21 (dose, 0.16 µg). Following optimization of lyophilization conditions, Spike or RBD-decorated liposomes were effectively reconstituted and maintained conformational capacity for binding human angiotensin-converting enzyme 2 (hACE2) for at least a week when stored at 60°C in lyophilized but not liquid format. Prime-boost intramuscular vaccination of hACE2-transgenic mice with the reconstituted vaccine formulations induced effective antibody responses that inhibited RBD binding to hACE2 and neutralized pseudotyped and live SARS-CoV-2. Two days following viral challenge, immunized transgenic mice cleared the virus and were fully protected from lethal disease.

Cross-linked Histone as a Nanocarrier for Gut Delivery of Hydrophobic Cargos.

Delivering hydrophobic molecules through the intestine can be challenging due to limited cargo solubility and the harsh biochemical environment of the stomach. Here, we show that a protein-based nanocarrier system based on the abundant protein histone and the natural cross-linker genipin can deliver hydrophobic cargos, such as dyes and therapeutic molecules, through the gastrointestinal tract. Using hydrophobic near-infrared dyes as model cargos, a panel of potential protein carriers was screened, and histone was identified as the one with the best loading capability. The resulting nanoparticles had a positive ζ potential and were mucoadhesive. Cross-linking of the amine-rich nanocarrier with genipin was particularly effective relative to other proteins and increased the stability of the system during incubation with pepsin. Cross-linking was required for successful delivery of a hydrophobic dye to the colon of mice after oral gavage. To assess the platform for therapeutic delivery, another hydrophobic model compound, curcumin, was delivered using cross-linked histone nanoparticles in a murine colitis model and significantly alleviated the disease. Taken together, these results demonstrate that histone is a cationic, mucoadhesive, and cross-linkable protein nanocarrier that can be considered for oral delivery.

A Potent Cancer Vaccine Adjuvant System for Particleization of Short, Synthetic CD8+ T Cell Epitopes.

Short major histocompatibility complex (MHC) class I (MHC-I)-restricted peptides contain the minimal biochemical information to induce antigen (Ag)-specific CD8+ cytotoxic T cell responses but are generally ineffective in doing so. To address this, we developed a cobalt-porphyrin (CoPoP) liposome vaccine adjuvant system that induces rapid particleization of conventional, short synthetic MHC-I epitopes, leading to strong cellular immune responses at nanogram dosing. Along with CoPoP (to induce particle formation of peptides), synthetic monophosphoryl lipid A (PHAD) and QS-21 immunostimulatory molecules were included in the liposome bilayer to generate the "CPQ" adjuvant system. In mice, immunization with a short MHC-I-restricted peptide, derived from glycoprotein 70 (gp70), admixed with CPQ safely generated functional, Ag-specific CD8+ T cells, resulting in the rejection of multiple tumor cell lines, with durable immunity. When cobalt was omitted, the otherwise identical peptide and adjuvant components did not result in peptide binding and were incapable of inducing immune responses, demonstrating the importance of stable particle formation. Immunization with the liposomal vaccine was well-tolerated and could control local and metastatic disease in a therapeutic setting. Mechanistic studies showed that particle-based peptides were better taken up by antigen-presenting cells, where they were putatively released within endosomes and phagosomes for display on MHC-I surfaces. On the basis of the potency of the approach, the platform was demonstrated as a tool for in vivo epitope screening of peptide microlibraries comprising a hundred peptides.

SARS-CoV-2 RBD Neutralizing Antibody Induction is Enhanced by Particulate Vaccination.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a candidate vaccine antigen that binds angiotensin-converting enzyme 2 (ACE2), leading to virus entry. Here, it is shown that rapid conversion of recombinant RBD into particulate form via admixing with liposomes containing cobalt-porphyrin-phospholipid (CoPoP) potently enhances the functional antibody response. Antigen binding via His-tag insertion into the CoPoP bilayer results in a serum-stable and conformationally intact display of the RBD on the liposome surface. Compared to other vaccine formulations, immunization using CoPoP liposomes admixed with recombinant RBD induces multiple orders of magnitude higher levels of antibody titers in mice that neutralize pseudovirus cell entry, block RBD interaction with ACE2, and inhibit live virus replication. Enhanced immunogenicity can be accounted for by greater RBD uptake into antigen-presenting cells in particulate form and improved immune cell infiltration in draining lymph nodes. QS-21 inclusion in the liposomes results in an enhanced antigen-specific polyfunctional T cell response. In mice, high dose immunization results in minimal local reactogenicity, is well-tolerated, and does not elevate serum cobalt levels. Taken together, these results confirm that particulate presentation strategies for the RBD immunogen should be considered for inducing strongly neutralizing antibody responses against SARS-CoV-2.

A surfactant-stripped cabazitaxel micelle formulation optimized with accelerated storage stability.

Pluronic (Poloxomer) micelles can solubilize cabazitaxel (CTX), a second-generation taxane, and then be subjected to low-temperature "surfactant-stripping" to selectively remove loose and free surfactant, thereby increasing the drug-to-surfactant ratio. We previously found that the addition of certain other co-loaded hydrophobic cargo to the micelles can result in stabilized, surfactant-stripped cabazitaxel (sss-CTX) micelles, which resist drug aggregation in aqueous storage, a common challenge for taxanes. Here, we show that elevated temperatures can accelerate the aggregation of sss-CTX micelles, thereby enabling rapid optimization of formulations with respect to the type and ratio of co-loader used for stabilization. A sss-CTX micelle formulation was developed using mifepristone as the co-loader, at a 60% mass ratio to the CTX. Drug release, hemolysis and complement activation were investigated in vitro. Microtubule stabilization and in vitro cytotoxicity were similar for sss-CTX and a conventional Tween-80 micelle formulation. In vivo pharmacokinetics also revealed similar blood circulation of the two formulations. In subcutaneous Lewis lung carcinoma tumors, as well as in an aggressive mouse model of malignant pleural effusion, sss-CTX showed a similar therapeutic effect as the Tween-80 based formulation. Altogether, these data show that sss-CTX can achieve similar efficacy as conventional Tween-80 formulations, albeit with substantially higher drug-to-surfactant ratio and with capability of extended aqueous storage.

Lyophilized, antigen-bound liposomes with reduced MPLA and enhanced thermostability.

Thermostability and decreased component costs are desirable features for adjuvanted, recombinant vaccines. We previously showed that a model malaria transmission-blocking vaccine candidate antigen, Pfs25, can be rendered more immunogenic when mixed with liposomes containing cobalt porphyrin-phospholipid (CoPoP) and a synthetic monophosphoryl lipid A (MPLA) variant. CoPoP can induce stable particle formation of recombinant antigens based on interaction with their polyhistidine tag. In the present work, different synthetic MPLA variants and concentrations were assessed in CoPoP liposomes. Long-term biophysical stability and immunogenicity were not adversely impacted by a 60% reduction in MPLA content. When admixed with Pfs25, the adjuvant formulations effectively induced functional antibodies in immunized mice and rabbits. Lyophilized, antigen-bound liposomes were formed using sucrose and trehalose cryoprotectants, which improved vaccine reconstitution for a variety of model antigens. Compared to liquid storage, the lyophilized Pfs25 and CoPoP liposomes exhibited thermostability with respect to size, biochemical integrity, binding capacity, protein folding and immunogenicity. Following 6 weeks of storage at 60 °C, the most extended storage period assessed, the lyophilized formulation induced functional antibodies in mice with immunization.

Particle-based, Pfs230 and Pfs25 immunization is effective, but not improved by duplexing at fixed total antigen dose.

BACKGROUND: The Plasmodium falciparum sexual-stage surface proteins Pfs25 and Pfs230 are antigen candidates for a malaria transmission-blocking vaccine (TBV), and have been widely investigated as such. It is not clear whether simultaneously presenting these two antigens in a particulate vaccine would enhance the transmission reducing activity (TRA) of induced antibodies. To assess this, immunization was carried out with liposomes containing synthetic lipid adjuvant monophosphoryl lipid A (MPLA), and cobalt-porphyrin-phospholipid (CoPoP), which rapidly converts recombinant, his-tagged antigens into particles. METHODS: His-tagged, recombinant Pfs25 and Pfs230C1 were mixed with CoPoP liposomes to form a bivalent vaccine. Antigens were fluorescently labelled to infer duplex particleization serum-stability and binding kinetics using fluorescence resonance energy transfer. Mice and rabbits were immunized with individual or duplexed particleized Pfs25 and Pfs230C1, at fixed total antigen doses. The resulting antibody responses were assessed for magnitude and TRA. RESULTS: Pfs230C1 and Pfs25 rapidly bound CoPoP liposomes to form a serum-stable, bivalent particle vaccine. In mice, immunization with 5 ng of total antigen (individual antigen or duplexed) elicited functional antibodies against Pfs25 and Pfs230. Compared to immunization with the individual antigen, Pfs25 antibody production was moderately lower for the bivalent CoPoP vaccine, whereas Pfs230C1 antibody production was not impacted. All antibodies demonstrated at least 92% inhibition in oocyst density at 750 µg/mL purified mouse IgG in the standard membrane feeding assay (SMFA). At lower IgG concentrations, the bivalent vaccine did not improve TRA; antibodies induced by particleized Pfs25 alone showed stronger function in these conditions. In rabbits, immunization with a 20 µg total antigen dose with the duplexed antigens yielded similar antibody production against Pfs25 and Pfs230 compared to immunization with a 20 µg dose of individual antigens. However, no enhanced TRA was observed with duplexing. CONCLUSIONS: Pfs25, Pfs230 or the duplexed combination can readily be prepared as particulate vaccines by mixing CoPoP liposomes with soluble, recombinant antigens. This approach induces potent transmission-reducing antibodies following immunization in mice and rabbits. Immunization with bivalent, particleized, Pfs230 and Pfs25 did not yield antibodies with superior TRA compared to immunization with particleized Pfs25 as a single antigen. Altogether, duplexing antigens is straightforward and effective using CoPoP liposomes, but is likely to be more useful for targeting distinct parasite life stages.

Indocyanine green binds to DOTAP liposomes for enhanced optical properties and tumor photoablation.

Indocyanine green (ICG) is a clinically-approved near infrared (NIR) dye used for optical imaging. The dye is only slightly soluble in water and is prone to aggregation in saline solutions, so that alternative formulations can improve photophysical performance. Numerous nanoscale formulations of ICG have been described in the literature, but we sought to develop an approach that does not require additional purification steps. Pre-formed liposomes incorporating 45 mol% of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) rapidly bind ICG, resulting in enhanced NIR optical properties. ICG binding is dependent on the amount of DOTAP incorporated in the liposomes. A dye-to-lipid mass ratio of [0.5 : 25] is sufficient for full complexation, without additional purification steps following mixing. NIR absorption, fluorescence intensity, and photoacoustic signals are increased for the liposome-bound dye. Not only is the optical character enhanced by simple mixing of ICG with liposomes, but retention in 4T1 mammary tumors is observed following intratumor injection, as assessed by fluorescence and photoacoustic imaging. Subsequent photothermal therapy with 808 nm laser irradiation is effective and results in tumor ablation without regrowth for at least 30 days. Thus, ICG optical properties and photothermal ablation outcomes can be improved by mixing the dye with pre-formed DOTAP liposomes in conditions that result in full dye-binding to the liposomes.

Inhalable lactoferrin-chondroitin nanocomposites for combined delivery of doxorubicin and ellagic acid to lung carcinoma.

AIM: The use of inhalable nanomedicines can overcome the Enhanced permeation and retention effect (EPR)-associated drawbacks in lung cancer therapy via systemic nanomedicines. METHODS: We developed a lactoferrin-chondroitin sulfate nanocomplex for the co-delivery of doxorubicin and ellagic acid nanocrystals to lung cancer cells. Then, the nanocomplex was converted into inhalable nanocomposites via spray drying. RESULTS: The resulting 192.3 nm nanocomplex exhibited a sequential faster release of ellagic acid, followed by doxorubicin. Furthermore, the nanocomplex demonstrated superior cytotoxicity and internalization into A549 lung cancer cells mediated via Tf and CD44 receptors. The inhalable nanocomposites exhibited deep lung deposition (89.58% fine particle fraction [FPF]) with powerful antitumor efficacy in lung cancer bearing mice. CONCLUSION: Overall, inhalable lactoferrin-chondroitin sulfate nanocomposites would be a promising carrier for targeted drug delivery to lung cancer.

Inhalable particulate drug delivery systems for lung cancer therapy: Nanoparticles, microparticles, nanocomposites and nanoaggregates.

There is progressive evolution in the use of inhalable drug delivery systems (DDSs) for lung cancer therapy. The inhalation route offers many advantages, being non-invasive method of drug administration as well as localized delivery of anti-cancer drugs to tumor tissue. This article reviews various inhalable colloidal systems studied for tumor-targeted drug delivery including polymeric, lipid, hybrid and inorganic nanocarriers. The active targeting approaches for enhanced delivery of nanocarriers to lung cancer cells were illustrated. This article also reviews the recent advances of inhalable microparticle-based drug delivery systems for lung cancer therapy including bioresponsive, large porous, solid lipid and drug-complex microparticles. The possible strategies to improve the aerosolization behavior and maintain the critical physicochemical parameters for efficient delivery of drugs deep into lungs were also discussed. Therefore, a strong emphasis is placed on the approaches which combine the merits of both nanocarriers and microparticles including inhalable nanocomposites and nanoaggregates and on the optimization of such formulations using the proper techniques and carriers. Finally, the toxicological behavior and market potential of the inhalable anti-cancer drug delivery systems are discussed.