A liposome-displayed hemagglutinin vaccine platform protects mice and ferrets from heterologous influenza virus challenge.

Recombinant influenza virus vaccines based on hemagglutinin (HA) hold the potential to accelerate production timelines and improve efficacy relative to traditional egg-based platforms. Here, we assess a vaccine adjuvant system comprised of immunogenic liposomes that spontaneously convert soluble antigens into a particle format, displayed on the bilayer surface. When trimeric H3 HA was presented on liposomes, antigen delivery to macrophages was improved in vitro, and strong functional antibody responses were induced following intramuscular immunization of mice. Protection was conferred against challenge with a heterologous strain of H3N2 virus, and naive mice were also protected following passive serum transfer. When admixed with the particle-forming liposomes, immunization reduced viral infection severity at vaccine doses as low as 2 ng HA, highlighting dose-sparing potential. In ferrets, immunization induced neutralizing antibodies that reduced the upper respiratory viral load upon challenge with a more modern, heterologous H3N2 viral strain. To demonstrate the flexibility and modular nature of the liposome system, 10 recombinant surface antigens representing distinct influenza virus strains were bound simultaneously to generate a highly multivalent protein particle that with 5 ng individual antigen dosing induced antibodies in mice that specifically recognized the constituent immunogens and conferred protection against heterologous H5N1 influenza virus challenge. Taken together, these results show that stable presentation of recombinant HA on immunogenic liposome surfaces in an arrayed fashion enhances functional immune responses and warrants further attention for the development of broadly protective influenza virus vaccines.

HPV-Associated Tumor Eradication by Vaccination with Synthetic Short Peptides and Particle-Forming Liposomes.

Human papilloma virus (HPV)-16 is associated with cervical cancers and induces expression of the E6 and E7 oncogenes. Using a murine cell line that expresses these, the genes are sequenced, and six predicted major histocompatibility complex (MHC) class I (MHC-I) epitopes are identified. A liposomal vaccine adjuvant based on cobalt-porphyrin-phospholipid (CoPoP) is admixed with synthetic 9-mer epitopes appended with three histidine residues, resulting in rapid formation of peptide-liposome particles. Immunization with multivalent peptides leads to protection from tumor challenge. Of the peptides screened, only the previously identified E749-57 epitope is functional. The peptide-liposome particles that form upon mixing E7HHH49-57 with CoPoP liposomes are stable in serum and are avidly taken up by immune cells in vitro. Immunization results in robust protection from tumor challenge and re-challenge. A 100 ng peptide dose protects mice in a therapeutic tumor challenge when admixed with CoPoP liposomes, whereas 200-fold higher peptide doses are ineffective with the polyinosinic-polycytidylic (poly(I:C)) adjuvant. CoPoP induces a strong infiltrating CD8+ T-cell response within the tumor microenvironment with an improved functional profile. Vaccine monotherapy using nanogram dosing of the E7HHH49-57 peptide admixed with CoPoP reverses the growth of large established tumors, eradicating subcutaneous tumors upwards of 100 mm3 . Immunization also eradicates lung tumors in a metastasis model.

Short Drug-Light Intervals Improve Liposomal Chemophototherapy in Mice Bearing MIA PaCa-2 Xenografts.

Chemophototherapy (CPT) is an emerging tumor treatment that combines phototherapy and chemotherapy. Long-circulating (LC) liposomes can stably incorporate 2 mol % porphyrin-phospholipid (PoP) in the bilayer and load doxorubicin (Dox) to generate LC-Dox-PoP liposomes, for single-agent CPT. Following intravenous administration to mice, LC-Dox-PoP liposomes (2 mg/kg Dox) circulated with similar blood concentration ranges produced by a typical human clinical dose of DOXIL (50 mg/m2 Dox). This dosing approach aims to achieve physiologically relevant Dox and PoP concentrations as well as CPT vascular responses in mice bearing subcutaneous human pancreatic MIA PaCa-2 xenografts. Phototreatment with 2 mg/kg LC-Dox-PoP induced vascular permeabilization, leading to a 12.5-fold increase in Dox tumor influx estimated by a pharmacokinetic model, based on experimental data. Shorter drug-light intervals (0.5-3 h) led to greater tumoral drug deposition and improved treatment outcomes, compared to longer drug-light intervals. At 2 mg/kg Dox, CPT with LC-Dox-PoP liposomes induced tumor regression and growth inhibition, whereas chemotherapy using several other formulations of Dox did not. LC-Dox-PoP liposomes were well tolerated at the 2 mg/kg dose.

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.

Immunogenicity of the Lyme disease antigen OspA, particleized by cobalt porphyrin-phospholipid liposomes.

Outer surface protein A (OspA) is a Borrelia lipoprotein and an established Lyme disease vaccine target. Admixing non-lipidated, recombinant B. burgdorferi OspA with liposomes containing cobalt porphyrin-phospholipid (CoPoP) resulted in rapid, particulate surface display of the conformationally intact antigen. Particleization was serum-stable and led to enhanced antigen uptake in murine macrophages in vitro. Mouse immunization using CoPoP liposomes that also contained a synthetic monophosphoryl lipid A (PHAD) elicited a Th1-biased OspA antibody response with higher IgG production compared to other vaccine adjuvants. Antibodies were reactive with intact B. burgdorferi spirochetes and Borrelia lysates, and induced complement-mediated borreliacidal activity in vitro. One year after initial immunization, mice maintained high levels of circulating borreliacidal antibodies capable of blocking B. burgdorferi transmission from infected ticks to human blood in a feeding chamber.

A malaria vaccine adjuvant based on recombinant antigen binding to liposomes.

Pfs25 is a malaria transmission-blocking vaccine antigen candidate, but its apparently limited immunogenicity in humans has hindered clinical development. Here, we show that recombinant, polyhistidine-tagged (his-tagged) Pfs25 can be mixed at the time of immunization with pre-formed liposomes containing cobalt porphyrin-phospholipid, resulting in spontaneous nanoliposome antigen particleization (SNAP). Antigens are stably presented in uniformly orientated display via his-tag insertion in the cobalt porphyrin-phospholipid bilayer, without covalent modification or disruption of antigen conformation. SNAP immunization of mice and rabbits is well tolerated with minimal local reactogenicity, and results in orders-of-magnitude higher functional antibody generation compared with other 'mix-and-inject' adjuvants. Serum-stable antigen binding during transit to draining lymph nodes leads to enhanced antigen uptake by phagocytic antigen-presenting cells, with subsequent generation of long-lived, antigen-specific plasma cells. Seamless multiplexing with four additional his-tagged Plasmodium falciparum polypeptides induces strong and balanced antibody production, illustrating the simplicity of developing multistage particulate vaccines with SNAP immunization.

RGD peptide-modified multifunctional dendrimer platform for drug encapsulation and targeted inhibition of cancer cells.

Development of multifunctional nanoscale drug-delivery systems for targeted cancer therapy still remains a great challenge. Here, we report the synthesis of cyclic arginine-glycine-aspartic acid (RGD) peptide-conjugated generation 5 (G5) poly(amidoamine) dendrimers for anticancer drug encapsulation and targeted therapy of cancer cells overexpressing αvß3 integrins. In this study, amine-terminated G5 dendrimers were used as a platform to be sequentially modified with fluorescein isothiocyanate (FI) via a thiourea linkage and RGD peptide via a polyethylene glycol (PEG) spacer, followed by acetylation of the remaining dendrimer terminal amines. The developed multifunctional dendrimer platform (G5.NHAc-FI-PEG-RGD) was then used to encapsulate an anticancer drug doxorubicin (DOX). We show that approximately six DOX molecules are able to be encapsulated within each dendrimer platform. The formed complexes are water-soluble, stable, and able to release DOX in a sustained manner. One- and two-dimensional NMR techniques were applied to investigate the interaction between dendrimers and DOX, and the impact of the environmental pH on the release rate of DOX from the dendrimer/DOX complexes was also explored. Furthermore, cell biological studies demonstrate that the encapsulation of DOX within the G5.NHAc-FI-PEG-RGD dendrimers does not compromise the anticancer activity of DOX and that the therapeutic efficacy of the dendrimer/DOX complexes is solely related to the encapsulated DOX drug. Importantly, thanks to the role played by RGD-mediated targeting, the developed dendrimer/drug complexes are able to specifically target αvß3 integrin-overexpressing cancer cells and display specific therapeutic efficacy to the target cells. The developed RGD peptide-targeted multifunctional dendrimers may thus be used as a versatile platform for targeted therapy of different types of αvß3 integrin-overexpressing cancer cells.