Polyethylenimine quantity and molecular weight influence its adjuvanting properties in liposomal peptide vaccines.

We recently reported that polyethylenimine (PEI; molecular weight of 600 Da) acted as a vaccine adjuvant for liposomal group A Streptococcus (GAS) vaccines, eliciting immune responses in vivo with IgG antibodies giving opsonic activity against five Australian GAS clinical isolates. However, to date, no investigation comparing the structure-activity relationship between the molecular weight of PEI and its adjuvanting activity in vaccine development has been performed. We hypothesized that the molecular weight and quantity of PEI in a liposomal vaccine will impact its adjuvanting properties. In this study, we successfully formulated liposomes containing different molecular weights of PEI (600, 1800, 10k and 25k Da) and equivalents of PEI (0.5, 1 and 2) of branched PEI. Outbred mice were administrated the vaccine formulations intranasally, and the mice that received a high ratio of PEI 600 reported a stronger immune response than the mice that received a lower ratio of PEI 600. Interestingly, mice that received the same quantity of PEI 600, PEI 10k and PEI 25k showed similar immune responses in vivo and in vitro. This comparative study highlights the ratio of PEI present in the liposome vaccines impacts adjuvanting activity, however, PEI molecular weight did not significantly enhance its adjuvanting properties. We also report that the stability of PEI liposomes is critical for vaccines to elicit the desired immune response.

Peptide-based targeted polymeric nanoparticles for siRNA delivery.

The development of polymer-based nanoparticulate delivery systems for siRNA is important for the clinical success of gene therapy. However, there are some major drawbacks that need to be overcome. Short interfering RNA (siRNA) has been investigated as a potential therapeutic drug to silence disease-associated genes, but its usage is limited due to the lack of effective and safe nanocarriers. In this study, DOPE-PEI, a nanoparticle consisting of the fusogenic lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) conjugated with low-molecular-weight, 600 Da, branched polyethylenimine (PEI) was produced and optimized for siRNA delivery. This delivery system was modified with other components such as 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)2000] (DOPE-PEG2K), DOPE-PEG3.4K-bombesin and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine/1,2-dioleoyl-3-trimethylammonium-propane (DOPE/DOTAP) and tested on PC-3 cells. The conjugation of DOPE to PEI polymer (DOPE-PEI) improved the efficiency of PEI to deliver siRNA into the cytosol and knockdown genes, but demonstrated high toxicity. The addition of DOPE-PEG2K reduced cellular toxicity by masking the surface positive charge of the DOPE-PEI/siRNA complex, with the incorporation of a gastrin-releasing peptide receptor (GRPR) targeting peptide and DOPE/DOTAP components improving the cellular uptake of siRNA into targeted cells and the siRNA knockdown efficiency.

Multiantigenic peptide-polymer conjugates as therapeutic vaccines against cervical cancer.

Immunotherapy is one of the most promising strategies for the treatment of cancer. Human papillomavirus (HPV) is responsible for virtually all cases of cervical cancer. The main purpose of a therapeutic HPV vaccine is to stimulate CD8(+) cytotoxic T lymphocytes (CTLs) that can eradicate HPV infected cells. HPV oncoproteins E6 and E7 are continuously expressed and are essential for maintaining the growth of HPV-associated tumor cells. We designed polymer-based multi-antigenic formulations/constructs that were comprised of the E6 and E7 peptide epitopes. We developed an N-terminus-based epitope conjugation to conjugate two unprotected peptides to poly tert-butyl acrylate. This method allowed for the incorporation of the two antigens into a polymeric dendrimer in a strictly equimolar ratio. The most effective formulations eliminated tumors in up to 50% of treated mice. Tumor recurrence was not observed up to 3months post initial challenge.

Cholesterol as an inbuilt immunoadjuvant for a lipopeptide vaccine against group A Streptococcus infection.

Peptide-based vaccines can trigger highly specific immune responses, although peptides alone are usually unable to confer strong humoral or cellular immunity. Consequently, peptide antigens are administered with immunostimulatory adjuvants, but only a few are safe and effective for human use. To overcome this obstacle, herein a peptide antigen was lipidated to effectively anchor it to liposomes and emulsion. A peptide antigen B cell epitope from Group A Streptococcus M protein was conjugated to a universal T helper epitope, the pan DR-biding epitope (PADRE), alongside a lipidic moiety cholesterol. Compared to a free peptide antigen, the lipidated version (LP1) adopted a helical conformation and self-assembled into small nanoparticles. Surprisingly, LP1 alone induced the same or higher antibody titers than liposomes or emulsion-based formulations. In addition, antibodies produced by mice immunized with LP1 were more opsonic than those induced by administering the antigen with incomplete Freund's adjuvant. No side effects were observed in the immunized mice and no excessive inflammatory immune responses were detected. Overall, this study demonstrated how simple conjugation of cholesterol to a peptide antigen can produce a safe and efficacious vaccine against Group A Streptococcus - the leading cause of superficial infections and the bacteria responsible for deadly post-infection autoimmune disorders.

Self-assembled monovalent lipidated mannose ligand as a standalone nanoadjuvant.

Subunit vaccines require an immunostimulant (adjuvant) and/or delivery system to induce immunity. However, currently, available adjuvants are either too dangerous in terms of side effects for human use (experimental adjuvants) or have limited efficacy and applicability. In this study, we examined the capacity of mannose-lipopeptide ligands to enhance the immunogenicity of a vaccine consisting of polyleucine(L15)-antigen conjugates anchored to liposomes. The clinically tested Group A Streptococcus (GAS) B-cell epitope, J8, combined with universal T helper PADRE (P) was used as the antigen. Six distinct mannose ligands were incorporated into neutral liposomes carrying L15PJ8. While induced antibody titers were relatively low, the ligand carrying mannose, glycine/lysine spacer, and two palmitic acids as liposomal membrane anchoring moieties (ligand 3), induced significantly higher IgG titers than non-mannosylated liposomes. The IgG titers were significantly enhanced when positively charged liposomes were employed. Importantly, the produced antibodies were able to kill GAS bacteria. Unexpectedly, the physical mixture of only ligand 3 and PJ8 produced self-assembled nanorods that induced antibody titers as high as those elicited by the lead liposomal formulation and antigen adjuvanted with the potent, but toxic, complete Freund's adjuvant (CFA). Antibodies produced upon immunization with PJ8 + 3 were even more opsonic than those induced by CFA + PJ8. Importantly, in contrast to CFA, ligand 3 did not induce observable adverse reactions or excessive inflammatory responses. Thus, we demonstrated that a mannose ligand, alone, can serve as an effective vaccine nanoadjuvant.

Self-adjuvanting polymer-peptide conjugates as therapeutic vaccine candidates against cervical cancer.

Dendrimers are structurally well-defined, synthetic polymers with sizes and physicochemical properties often resembling those of biomacromolecules (e.g., proteins). As a result, they are promising candidates for peptide-based vaccine delivery platforms. Herein, we established a synthetic pathway to conjugate a human papillomavirus (HPV) E7 protein-derived peptide antigen to a star-polymer to create a macromolecular vaccine candidate to treat HPV-related cancers. These conjugates were able to reduce tumor growth and eradicate E7-expressing TC-1 tumors in mice after a single immunization, without the help of any external adjuvant.

Activity Relationship of Poly(ethylenimine)-Based Liposomes as Group A Streptococcus Vaccine Delivery Systems.

Untreated group A Streptococcus (GAS) can lead to a range of life-threatening diseases, including rheumatic heart disease. To date, no therapeutic or prophylactic vaccines are commercially available to treat or prevent GAS infection. Development of a peptide-based subunit vaccine offers a promising solution, negating the safety issues of live-attenuated or inactive vaccines. Subunit vaccines administer small peptide fragments (antigens), which are typically poorly immunogenic. Therefore, these peptide antigens require formulation with an immune stimulant and/or vaccine delivery platform to improve their immunogenicity. We investigated polyelectrolyte complexes (PECs) and polymer-coated liposomes as self-adjuvanting delivery vehicles for a GAS B cell peptide epitope conjugated to a universal T-helper epitope and a synthetic toll-like receptor 2-targeting moiety lipid core peptide-1 (LCP-1). A structure-activity relationship of cationic PEC vaccines containing different external PEI-coatings (poly(ethylenimine); 10 kDa PEI, 25 kDa PEI, and a synthetic mannose-functionalized 25 kDa PEI) formed vaccines PEC-1, PEC-2, and PEC-3, respectively. All three PEC vaccines induced J8-specific systemic immunoglobulin G (IgG) antibodies when administered intranasally to female BALB/c mice without the use of additional adjuvants. Interestingly, PEC-3 induced the highest antibody titers among all tested vaccines, with the ability to effectively opsonize two clinically isolated GAS strains. A comparative study of PEC-2 and PEC-3 with liposome-based delivery systems was performed subcutaneously. LCP-1 was incorporated into a liposome formulation (DPPC, DPPG and cholesterol), and the liposomes were externally coated with PEI (25 kDa; Lip-2) or mannosylated PEI (25 kDa; Lip-3). All liposome vaccines induced stronger humoral immune responses compared to their PEC counterparts. Notably, sera of mice immunized with Lip-2 and Lip-3 produced significantly higher opsonic activity against clinically isolated GAS strains compared to the positive control, P25-J8 emulsified with the commercial adjuvant, complete Freund's adjuvant (CFA). This study highlights the capability of a PEI-liposome system to act as a self-adjuvanting vehicle for the delivery of GAS peptide antigens and protection against GAS infection.

Investigation of liposomal self-adjuvanting peptide epitopes derived from conserved blood-stage Plasmodium antigens.

Malaria is a vector born parasitic disease causing millions of deaths every year. Despite the high mortality rate, an effective vaccine against this mosquito-borne infectious disease is yet to be developed. Up to date, RTS,S/AS01 is the only vaccine available for malaria prevention; however, its efficacy is low. Among a variety of malaria antigens, merozoite surface protein-1(MSP-1) and ring-infected erythrocyte surface antigen (RESA) have been proposed as promising candidates for malaria vaccine development. We developed peptide-based Plasmodium falciparum vaccine candidates that incorporated three previously reported conserved epitopes from MSP-1 and RESA into highly effective liposomal polyleucine delivery system. Indeed, MSP-1 and RESA-derived epitopes conjugated to polyleucine and formulated into liposomes induced higher epitope specific antibody titres. However, immunized mice failed to demonstrate protection in a rodent malaria challenge study with Plasmodium yoelii. In addition, we found that the three reported P. falciparum epitopes did not to share conformational properties and high sequence similarity with P. yoelii MSP-1 and RESA proteins, despite the epitopes were reported to protect mice against P. yoelii challenge.

Peptide-Pegylated Lipid Conjugation Via Copper-Catalyzed Alkyne-Azide 1,3-Dipolar Cycloaddition.

Targeted drug delivery is an important strategy in the treatment of many diseases. However, cancer cells are very difficult to target, making this a substantial obstacle in chemotherapy treatment. Bombesin fragment (BBN(6-14)) has been found to target gastrin-releasing peptide receptor (GRPR), which is overexpressed in many cancer cells. In this chapter, BBN peptide was used as a targeting moiety on the surface of polymeric-based nanoparticles to deliver its payload into prostate cancer PC-3 cell lines. Copper-catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) click reaction was utilized to link the BBN peptide with an alkyne derivative of Pegylated lipid.

Peptide-Polymer Conjugation Via Copper-Catalyzed Alkyne-Azide 1,3-Dipolar Cycloaddition.

Dendrimers are structurally well-defined, artificial polymers with physicochemical characteristics that often imitate biomacromolecules. Consequently, they are encouraging candidates for the delivery of peptide-based vaccines. We developed a synthetic protocol for conjugating a peptide antigen derived from human papillomavirus (HPV) E7 protein to a poly(t-butyl acrylate) dendrimer to construct a vaccine candidate. The synthetic pathway utilized copper-catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) click reaction, and resulted in a 76% substitution ratio of the 8-arm dendrimer. The obtained peptide-polymer construct was self-assembled, dialyzed, and characterized by microanalysis and dynamic light scattering.

Development and Evaluation of a Cryopreserved Whole-Parasite Vaccine in a Rodent Model of Blood-Stage Malaria.

Infection with malaria parasites continues to be a major global public health issue. While current control measures have enabled a significant decrease in morbidity and mortality over the last 20 years, additional tools will be required if we are to progress toward malaria parasite eradication. Malaria vaccine research has focused on the development of subunit vaccines; however, more recently, interest in whole-parasite vaccines has reignited. Whole-parasite vaccines enable the presentation of a broad repertoire of antigens to the immune system, which limits the impact of antigenic polymorphism and genetic restriction of the immune response. We previously reported that whole-parasite vaccines can be prepared using chemically attenuated parasites within intact red blood cells or using killed parasites in liposomes, although liposomes were less immunogenic than attenuated parasites. If they could be frozen or freeze-dried and be made more immunogenic, liposomal vaccines would be ideal for vaccine deployment in areas where malaria is endemic. Here, we develop and evaluate a Plasmodium yoelii liposomal vaccine with enhanced immunogenicity and efficacy due to incorporation of TLR4 agonist, 3D(6-acyl) PHAD, and mannose to target the liposome to antigen-presenting cells. Following vaccination, mice were protected, and strong cellular immune responses were induced, characterized by parasite-specific splenocyte proliferation and a mixed Th1/Th2/Th17 cytokine response. Parasite-specific antibodies were induced, predominantly of the IgG1 subclass. CD4+ T cells and gamma interferon were critical components of the protective immune response. This study represents an important development toward evaluation of this whole-parasite blood-stage vaccine in a phase I clinical trial. IMPORTANCE Malaria is a mosquito-borne infectious disease that is caused by parasites of the genus, Plasmodium. There are seven different Plasmodium spp. that can cause malaria in humans, with P. falciparum causing the majority of the morbidity and mortality. Malaria parasites are endemic in 87 countries and continue to result in >200 million cases of malaria and >400,000 deaths/year, mostly children <5 years of age. Malaria infection initially presents as a flu-like illness but can rapidly progress to severe disease in nonimmune individuals if treatment is not initiated promptly. Existing control strategies for the mosquito vector (insecticides) and parasite (antimalarial drugs) are becoming increasingly less effective due to the development of resistance. While artemisinin combination therapies are frontline treatment for P. falciparum malaria, resistance has been documented in numerous countries. A highly effective malaria vaccine is urgently required to reduce malaria-attributable clinical disease and death and enable progression toward the ultimate goal of eradication.

Pre-clinical evaluation of a whole-parasite vaccine to control human babesiosis.

Babesia spp. are tick-transmitted intra-erythrocytic protozoan parasites that infect humans and animals, causing a flu-like illness and hemolytic anemia. There is currently no human vaccine available. People most at risk of severe disease are the elderly, immunosuppressed, and asplenic individuals. B. microti and B. divergens are the predominant species affecting humans. Here, we present a whole-parasite Babesia vaccine. To establish proof-of-principle, we employed chemically attenuated B. microti parasitized red blood cells from infected mice. To aid clinical translation, we produced liposomes containing killed parasite material. Vaccination significantly reduces peak parasitemia following challenge. B cells and anti-parasite antibodies do not significantly contribute to vaccine efficacy. Protection is abrogated by the removal of CD4+ T cells or macrophages prior to challenge. Importantly, splenectomized mice are protected by vaccination. To further facilitate translation, we prepared a culture-based liposomal vaccine and demonstrate that this performs as a universal vaccine inducing immunity against different human Babesia species.

Mannosylated liposomes formulated with whole parasite P. falciparum blood-stage antigens are highly immunogenic in mice.

The development of a blood-stage malaria vaccine has largely focused on the subunit approach. However, the limited success of this strategy, mainly due to antigenic polymorphism and the failure to maintain potent parasite-specific immune responses, indicates that other approaches must be considered. Whole parasite (WP) vaccines offer many advantages over sub-units; they represent every antigen on the organism, thus limiting the effects of antigenic polymorphism, and similarly they compensate for individual Immune-Response (Ir) gene-regulated non-responsiveness to any particular antigen. From a development perspective, they negate the need to identify and compare the relative efficacies of individual candidate antigens. WP vaccines induce protective immunity that is largely cell-mediated. However, WP blood-stage vaccines present a number of challenges for the development pathway. Key issues are cryopreservation and storage and the possible induction of antibodies against red blood cell surface antigens, even if the parasites are grown in blood group O, Rh negative blood. Here, we used a novel adaptation of an immunomagnetic method from STEMCELL™ Technologies to remove the red cell membranes from human red blood cells parasitized with P. falciparum. We then used these antigens to construct liposomes which were modified to present mannose on their membrane to target the liposome to antigen presenting cells. We then compared the immunogenicity of freshly prepared and lyophilized liposome vaccines. Following vaccination of mice, liposomes induced significantly lower antibody responses to human red cells but potent strain- and species-transcending cell-mediated immune responses to parasite antigens. These data support transitioning the P. falciparum liposomal vaccine into clinical studies.

Polyethylenimine: An Intranasal Adjuvant for Liposomal Peptide-Based Subunit Vaccine against Group A Streptococcus.

Group A Streptococcus (GAS) and GAS-related infections are a worldwide challenge, with no commercial GAS vaccine available. Polyethylenimine (PEI) attaches to the cells' surface and delivers cargo into endosomal and cytosolic compartments. We hypothesized that this will confer mucosal adjuvant properties for peptide antigens against group A Streptococcus (GAS). In this study, we successfully demonstrated the development of PEI incorporated liposomes for the delivery of a lipopeptide-based vaccine (LCP-1) against GAS. Outbred mice were administrated with the vaccine formulations intranasally, and immunological investigation showed that the PEI liposomes elicited significant mucosal and systemic immunity with the production of IgA and IgG antibodies. Antibodies were shown to effectively opsonize multiple isolates of clinically isolated GAS. This proof-of-concept study showed the capability for PEI liposomes to act as a safe vehicle for the delivery of GAS peptide antigens to elicit immune responses against GAS infection, making PEI a promising addition to liposomal mucosal vaccines.

Poly(amino acids) as a potent self-adjuvanting delivery system for peptide-based nanovaccines.

To be optimally effective, peptide-based vaccines need to be administered with adjuvants. Many currently available adjuvants are toxic, not biodegradable; they invariably invoke adverse reactions, including allergic responses and excessive inflammation. A nontoxic, biodegradable, biocompatible, self-adjuvanting vaccine delivery system is urgently needed. Herein, we report a potent vaccine delivery system fulfilling the above requirements. A peptide antigen was coupled with poly-hydrophobic amino acid sequences serving as self-adjuvanting moieties using solid-phase synthesis, to produce fully defined single molecular entities. Under aqueous conditions, these molecules self-assembled into distinct nanoparticles and chain-like aggregates. Following subcutaneous immunization in mice, these particles successfully induced opsonic epitope-specific antibodies without the need of external adjuvant. Mice immunized with entities bearing 15 leucine residues were able to clear bacterial load from target organs without triggering the release of soluble inflammatory mediators. Thus, we have developed a well-defined and effective self-adjuvanting delivery system for peptide antigens.

Lipid core peptide/poly(lactic-co-glycolic acid) as a highly potent intranasal vaccine delivery system against Group A streptococcus.

Rheumatic heart disease represents a leading cause of mortality caused by Group A Streptococcus (GAS) infections transmitted through the respiratory route. Although GAS infections can be treated with antibiotics these are often inadequate. An efficacious GAS vaccine holds more promise, with intranasal vaccination especially attractive, as it mimics the natural route of infections and should be able to induce mucosal IgA and systemic IgG immunity. Nanoparticles were prepared by either encapsulating or coating lipopeptide-based vaccine candidate (LCP-1) on the surface of poly(lactic-co-glycolic acid) (PLGA). In vitro study showed that encapsulation of LCP-1 vaccine into nanoparticles improved uptake and maturations of antigen-presenting cells. The immunogenicity of lipopeptide incorporated PLGA-based nanoparticles was compared with peptides co-administered with mucosal adjuvant cholera toxin B in mice upon intranasal administration. Higher levels of J14-specific salivary mucosal IgA and systemic antibody IgG titres were observed for groups immunized with encapsulated LCP-1 compared to LCP-1 coated nanoparticles or free LCP-1. Systemic antibodies obtained from LCP-1 encapsulated PLGA NPs inhibited the growth of bacteria in six different GAS strains. Our results show that PLGA-based lipopeptide delivery is a promising approach for rational design of a simple, effective and patient friendly intranasal GAS vaccine resulting in mucosal IgA response.

Linear and branched polyacrylates as a delivery platform for peptide-based vaccines.

AIM: Peptide-based vaccines are designed to carry the minimum required antigen to trigger the desired immune responses; however, they are usually poorly immunogenic and require appropriate delivery system. RESULTS: Peptides, B-cell epitope (J14) derived from group A streptococcus M-protein and universal T-helper (PADRE) epitope, were conjugated to a variety of linear and branched polyacrylates. All produced conjugates formed submicron-sized particles and induced a high level of IgG titres in mice after subcutaneous immunization. These polymer-peptide conjugates demonstrated high opsonization capacity against group A streptococcus clinical isolates. CONCLUSION: We have successfully demonstrated that submicron-sized polymer-peptide conjugates were capable of inducing strong humoral immune responses after single immunization.

Self-adjuvanting therapeutic peptide-based vaccine induce CD8+ cytotoxic T lymphocyte responses in a murine human papillomavirus tumor model.

Vaccine candidates for the treatment of human papillomavirus (HPV)-associated cancers are aimed to activate T-cells and induce development of cytotoxic anti-tumor specific responses. Peptide epitopes derived from HPV-16 E7 oncogenic protein have been identified as promising antigens for vaccine development. However, peptide-based antigens alone elicit poor cytotoxic T lymphocyte (CTL) responses and need to be formulated with an adjuvant (immunostimulant) to achieve the desired immune responses. We have reported the ability of polyacrylate 4-arm star-polymer (S4) conjugated with HPV-16 E744-57 (8Qmin) epitope to reduce and eradicate TC-1 tumor in the mouse model. Herein, we have studied the mechanism of induction of immune responses by this polymer-peptide conjugate and found prompt uptake of conjugate by antigen presenting cells, stimulating stronger CD8(+) rather than CD4(+) or NK cell responses.

Polyacrylate-based delivery system for self-adjuvanting anticancer peptide vaccine.

Vaccination can provide a safe alternative to chemotherapy by using the body's natural defense mechanisms to create a potent immune response against tumor cells. Peptide-based therapeutic vaccines against human papillomavirus (HPV)-related cancers are usually designed to elicit cytotoxic T cell responses by targeting the HPV-16 E7 oncoprotein. However, peptides alone lack immunogenicity, and an additional adjuvant or external delivery system is required. In this study, we developed new polymer-peptide conjugates to create an efficient self-adjuvanting system for peptide-based therapeutic vaccines. These conjugates reduced tumor growth and eradicated E7-positive TC-1 tumors in mice after a "single shot" immunization, without the help from an external adjuvant. The new conjugates had a significantly higher anticancer efficacy than the antigen formulated with a commercial adjuvant. Furthermore, the polymer-peptide conjugates were promptly taken up by antigen presenting cells, including dendritic cells and macrophages, and efficiently activated CD4(+) T-helper cells and CD8(+) cytotoxic T lymphocyte cells.