Microparticle Vaccine Research Articles

Immunization with synthetic LbL microparticle vaccine administered using a microneedle patch elicits humoral and cellular immune responses and protects mice from challenge with respiratory syncytial virus

Abstract Synthetic vaccines made by layer-by-layer fabrication of microparticles (LbL-MP represent a novel method of delivering peptide antigens to elicit protective immune responses in the absence of complex adjuvant and without concomitant inflammation. We incorporated LbL-MP into a microneedle patch that delivers the vaccine directly to the immunologically rich environment of the skin in a minimally-invasive manner. BALB/c mice immunized with microneedle patches loaded with LbL-MP bearing two conserved epitopes of respiratory syncytial virus (RSV) generated humoral and cellular immune responses and were protected from virus challenge. The vaccine contained a conserved CX3C chemokine mimic epitope from the RSV attachment (G) protein that contributes to Th2-skewed inflammatory responses. Patch-immunized mice developed RSV-G-specific antibody titers comparable to the titers induced by direct injection of the vaccine into the footpad. The mice developed T-cell responses, including effector CTL, against a CD8 epitope of the RSV-M2 protein included in the vaccine. While footpad injection elicited both Th2 (IL-5) and Th1 (IFNγ) responses, microneedle patch immunization elicited a dominant Th1 response with very low Th2 response. A similar pattern of Th1>Th2 was seen in C57BL/6 mice immunized with a patch loaded with LbL-MP containing epitopes of the P. falciparum circumsporozoite protein, suggesting that the Th1 dominance was not antigen- or strain-specific, but rather an outcome of LbL-MP microneedle patch immunization. These studies demonstrate that LbL-MP retain their integrity and potency when delivered via microneedle patch, and favor Th1 immune responses that may be more effective against acute infection.

A biomaterial-based microparticle vaccine reverses type 1 diabetes in NOD mice

A biomaterial-based microparticle vaccine reverses type 1 diabetes in NOD mice

Development of a microparticulate prostate cancer vaccine and evaluating the effect of route of administration on its efficacy via the skin

The skin has been identified as a promising target to deliver vaccines. In this study, prostate cancer antigens were delivered in a spray-dried microparticulate carrier to a murine model via the transdermal route and the subcutaneous route. There was a significant increase in the humoral responses as determined by the total serum IgG titres (p < 0.05) and the cellular responses as determined by the T- and B-cells sub-population in spleen samples and delay in tumour growth till 8 weeks post-tumour challenge of both vaccinated groups when compared to the controls. The vaccine microparticles administered via the transdermal route induced a Th2-mediated immune response versus a mixed Th1- and Th2-mediated immune response via the subcutaneous route. Thus, the particulate vaccine delivery system proves to be a promising alternative for generation of a robust immune response against prostate cancer via the skin in a murine model.

A combination dual-sized microparticle system modulates dendritic cells and prevents type 1 diabetes in prediabetic NOD mice

We developed a novel poly(lactic-co-glycolic acid)-based, microparticle (MP) system providing concurrent delivery of multiple encapsulated immuno-suppressive factors and antigen, for in vivo conditioning of dendritic cells (DCs) toward a tolerance promoting pathway. Subcutaneous administration prevents onset of type 1 diabetes (T1D) in NOD mice. Two MP sizes were made: phagocytosable MPs were fabricated encapsulating vitamin D3 or insulin B(9–23) peptide, while unphagocytosable MPs were fabricated encapsulating TGF-β1 or GM-CSF. The combination of Vit D3/TGF-β1 MPs confers an immature and LPS activation-resistant phenotype to DCs, and MP-delivered antigen is efficiently and functionally presented. Notably, two subcutaneous injections into 4week old NOD mice using the combination of MPs encapsulating Vit D3, Ins B, TGF-β1 and GM-CSF protected 40% of mice from T1D development, significant in comparison to the control. This work represents one of the first applications of a biomaterial-based, MP vaccine system to successfully prevent autoimmune diabetes.

Nanoporous polyelectrolyte vaccine microcarriers. A formulation platform for enhancing humoral and cellular immune responses

In this paper we report on the design, characterization and immuno-biological evaluation of nanoporous polyelectrolyte microparticles as vaccine carrier. Relative to soluble antigen, formulation of antigen as a sub-10μm particle can strongly enhance antigen-specific cellular immune responses. The latter is crucial to confer protective immunity against intracellular pathogens and for anti-cancer vaccines. However, a major bottleneck in microparticulate vaccine formulation is the development of generic strategies that afford antigen encapsulation under benign and scalable conditions. Our strategy is based on spray drying of a dilute aqueous solution of antigen, oppositely charged polyelectrolytes and mannitol as a pore-forming component. The obtained solid microparticles can be redispersed in aqueous medium, leading to leaching out of the mannitol, thereby creating a highly porous internal structure. This porous structure enhances enzymatic processing of encapsulated proteins. After optimizing the conditions to process these microparticles we demonstrate that they strongly enhance cross-presentation in vitro by dendritic cells to CD8 T cells. In vivo experiments in mice confirm that this vaccine formulation technology is capable of enhancing cellular immune responses.

Time course study of the antigen-specific immune response to a PLGA microparticle vaccine formulation

Microparticle-based vaccine delivery systems are known to promote enhanced immune responses to protein antigens and can elicit TH1-biased responses when used in combination with Toll-like receptor (TLR) agonists. It is important to understand the kinetics of the immune responses to microparticle-based protein vaccines in order to predict the duration of protective immunity and to optimize prime-boost vaccination regimens. We carried out a 10-week time course study to investigate the magnitude and kinetics of the antibody and cellular immune responses to poly(lactic-co-glycolic acid) (PLGA) microparticles containing 40 μg ovalbumin (OVA) protein and 16 μg CpG-ODN adjuvant (MP/OVA/CpG) in comparison to OVA-containing microparticles, soluble OVA plus CpG, or OVA formulated with Alhydrogel® aluminum adjuvant. Mice vaccinated with MP/OVA/CpG developed the highest TH1-associated IgG2b and IgG2c antibody titers, while also eliciting TH2-associated IgG1 antibody titers on par with Alhydrogel®-formulated OVA, with all IgG subtype titers peaking at day 56. The MP/OVA/CpG vaccine also induced the highest antigen-specific splenocyte IFN-γ responses, with high levels of IFN-γ responses persisting until day 42. Thus the MP/OVA/CpG formulation produced a sustained and heightened humoral and cellular immune response, with an overall TH1 bias, while maintaining high levels of IgG1 antibody equivalent to that seen with Alhydrogel® adjuvant. The time course kinetics study provides a useful baseline for designing vaccination regimens for microparticle-based protein vaccines.

Parasite-neutralizing and protective antibody response in macaques immunized with synthetic LbL microparticle vaccines containing the conserved T1BT* epitope of Plasmodium falciparum (VAC7P.976)

Abstract We have previously shown that synthetic LbL-MP vaccines containing the conserved T1B repeat and T* epitopes from the P. falciparum circumsporozoite (PfCS) protein elicit a protective antibody response in mice. Immunogenicity and efficacy of these LbL-MP were tested in a non-human primate model, the rhesus macaque. LbL-MP were loaded with T1BT* peptide with or without covalently-linked Pam3Cys, a TLR2 ligand. Both constructs elicited T1B-specific antibody responses and T1BT*-specific T-cell responses dominated by IFNγ secretion with little or no IL-5 secretion. The Pam3Cys-modified construct was more potent, generating antibody responses that neutralized wild-type P. falciparum in an in vitro hepatocyte invasion assay. In addition, IgG was purified from the macaque immune sera and passively administered to naïve mice that were then challenged with 5,000 transgenic P. berghei sporozoites that expressed the full-length PfCS protein. IgG from individual macaques immunized with Pam3Cys.T1BT* LbL-MP protected mice from challenge, with 50-88% of mice parasite-free for ≥ 15 days. These results demonstrate that synthetic LbL-MP vaccines bearing the conserved T1BT* epitopes of PfCS can elicit protective antibody responses against P. falciparum in a non-human primate model.

Topical Vaccination with Functionalized Particles Targeting Dendritic Cells

Needle-free vaccination, for reasons of safety, economy, and convenience, is a central goal in vaccine development, but it also needs to meet the immunological requirements for efficient induction of prophylactic and therapeutic immune responses. Combining the principles of noninvasive delivery to dendritic cells (DCs) through skin and the immunological principles of cell-mediated immunity, we developed microparticle-based topical vaccines. We show here that the microparticles are efficient carriers for coordinated delivery of the essential vaccine constituents to DCs for cross-presentation of the antigens and stimulation of T-cell responses. When applied to the skin, the microparticles penetrate into hair follicles and target the resident DCs, the immunologically most potent cells and site for induction of efficient immune responses. The microparticle vaccine principle can be applied to different antigen formats such as peptides and proteins, or nucleic acids coding for the antigens.

Novel vaccine formulations against pertussis offer earlier onset of immunity and provide protection in the presence of maternal antibodies

Whooping cough is a respiratory illness most severe in infants and young children. While the introduction of whole-cell (wP) and acellular pertussis (aP) vaccines has greatly reduced the burden of the disease, pertussis remains a problem in neonates and adolescents. New vaccines are needed that can provide early life and long-lasting protection of infants. Vaccination at an early age, however, is problematic due to the interference with maternally derived antibodies (MatAbs) and the bias towards Th2-type responses following vaccination. Here we report the development of a novel vaccine formulation against pertussis that is highly protective in the presence of MatAbs. We co-formulated pertussis toxoid (PTd) and filamentous hemagglutinin (FHA) with cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN), cationic innate defense regulator (IDR) peptide and polyphosphazene (PP) into microparticle and soluble vaccine formulations and tested them in murine and porcine models in the presence and absence of passive immunity. Vaccines composed of the new adjuvant formulations induced an earlier onset of immunity, higher anti-pertussis IgG2a and IgA titers, and a balanced Th1/Th2-type responses when compared to immunization with Quadracel®, one of the commercially available vaccines for pertussis. Most importantly, the vaccines offered protection against challenge infection in the presence of passively transferred MatAbs.

Encapsulation of chimeric protein rSAG1/2 into poly(lactide-co-glycolide) microparticles induces long-term protective immunity against Toxoplasma gondii in mice

In the present study, the poly(lactide-co-glycolide) (PLG) polymer was used as an adjuvant to encapsulate the chimeric protein rSAG1/2 for preparing a microparticle vaccine against Toxoplasma gondii. The resulting PLG-encapsulated rSAG1/2 (PLG-rSAG1/2) microparticles, 1.27–1.65μm in diameter, showed 72–83% entrapment efficiency. The amount of released rSAG1/2 protein from microparticles gradually increased over a long 56-day period and the protein still retained its antigenicity. Intraperitoneal immunization with the microparticles in BALB/c mice elicited significant long-term (10weeks) humoral and cell-mediated immune responses, accompanied by secretion of a large amount of IFN-γ, to achieve strong protection (83%) against a lethal subcutaneous tachyzoite challenge. In conclusion, we have successfully encapsulated rSAG1/2 with the PLG polymer to produce stable microparticles that can effectively induce not only long-term immunity but also high protection against T. gondii. These crucial data would be advantageous for developing long-term Toxoplasma vaccines for future use in humans and animals.

Development of a microparticulate HPV vaccine for oral delivery

Current HPV vaccines are effective in preventing infection with the vaccine types. However, the cost of the vaccine, infrastructure for cold chain and trained personnel, is a major barrier to its introduction in low resource settings. An oral microparticulate vaccine could reduce programmatic costs of HPV vaccine implementation. Our aim was to develop a formulation for oral delivery using HPV16 virus like particles (VLPs), and conduct preclinical tests of immunogenicity.Purified VLPs were incorporated into β‐cyclodextrin microparticles using Buchi B191 spray dryer. 3 doses of the vaccine were administered orally to female BALB/c mice at 2 week intervals. Blood samples were collected prior to inoculation and 1 week after each inoculation for up to 7 weeks. Antibody response was determined using direct HPV16 VLP IgG ELISA.Average size of the microparticles was 3.5± 0.6 μm, with encapsulation efficiency of 60% of VLPs. Electron micrographs confirmed the conformational integrity of the VLPs released from the microparticles. Antibody response was observed in 1/6 mice by week 3 which gradually increased to 4/6 mice by week 7. Current formulation shows promise for development of an effective oral vaccine with modifications to boost the positive response observed in this study.“The findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of the funding agency.”

Local and systemic immune responses in mice to intranasal delivery of peptides representing bovine respiratory syncytial virus epitopes encapsulated in poly (dl-lactide-co-glycolide) microparticles

The potential of a microparticulate vaccine delivery system in eliciting a specific mucosal antibody response in the respiratory tract of mice was evaluated. Two vaccine candidate peptides representing epitopes from the G attachment and F fusion antigens from bovine respiratory syncytial virus (BRSV) were encapsulated into poly(dl- lactide co-glycolide) biodegradable microparticles. The encapsulation process did not denature the entrapped peptides as verified by detection of peptide-specific antibodies in mucosal secretions by ELISA using peptide as antigen. Following intranasal immunisation, the encapsulated peptides induced stronger upper and lower respiratory tract specific-IgA responses, respectively, than the soluble peptide forms. Moreover, a strong peptide-specific cell-mediated immune response was measured in splenocytes in vitro from the mice inoculated with the encapsulated peptides compared to their soluble form alone indicating that migration of primed T cells had taken place from the site of mucosal stimulation in the upper respiratory tract to the spleen. These results act as a foundation for vaccine efficacy studies in large animal BRSV challenge models.

Development of a new microparticle vaccine adjuvant with the ability to deliver peptides to dendritic cells.

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Immunogenicity and protection of oral influenza vaccines formulated into microparticles

Influenza is a deadly disease affecting humans and animals. It is recommended that every individual should be vaccinated annually against influenza. Considering the frequency of administration of this vaccine, we have explored the oral route of vaccination with a microparticulate formulation. Microparticles containing inactivated influenza A/PR/34/8 H1N1 virus with Eudragit S and trehalose as a matrix were prepared using the Buchi spray dryer. Particle size distribution of microparticles was measured and the bioactivity of vaccine in a microparticle form was analyzed using a hemagglutination activity test. Furthermore, the efficacy of microparticle vaccines was evaluated in vivo in Balb/c mice. Analysis of serum samples showed that microparticles resulted in enhanced antigen-specific immunoglobulin G (IgG), IgG1, and IgG2a antibodies. Upon challenge with homologous and heterologous influenza viruses, microparticle vaccines showed significantly increased levels of protection. Use of microparticles to deliver vaccines could be a promising tool for the development of an oral influenza vaccine.

Formulation and evaluation of a particulate oral breast cancer vaccine

Breast cancer being the most fatal form of cancer for female population, justifies exploration of immunotherapy as an alternative treatment. Here, we have formulated and evaluated an oral microparticulate breast cancer vaccine to provide a new line of therapy. The whole cell lysate of 4T07 murine breast cancer cells was incorporated in an aqueous polymer matrix and spray dried to formulate an enteric protected vaccine microparticle. These particles were characterized in vitro and then administered orally to female Balb/c mice in successive boosters. Serum antibody titers during the study were analyzed using enzyme‐linked immunosorbent assay. Postvaccination animals were challenged with live 4T07 cells, and tumor growth was monitored. Flow cytometry studies were performed to analyze the role of T cells. Results show that the vaccine microparticles were 1–4 µm in volume diameter and neutral in charge. The particles were protected enterically and had sustained‐release profile. Serum antibody titers of vaccinated animals increased significantly after boosters compared with controls (p < 0.05). Tumor challenge studies revealed that vaccinated animals developed significantly smaller tumors (p < 0.05). Significantly higher numbers of CD4+ cells occurred in vaccinated animals (p < 0.05). Thus, we conclude that the particulate oral breast cancer vaccine was effective in providing protective immune response in the murine model. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:3661–3671, 2012

Formulation and evaluation of oral microparticulate ovarian cancer vaccines

Ovarian cancer is the fifth most leading cause of cancer related deaths in women in the US. Customized immunotherapeutic strategies may serve as an alternative method to control the recurrence or progression of ovarian cancer and to avoid severe adverse effects of chemotherapy. In this study, a microparticulate vaccine using whole cell lysate of a murine ovarian cancer cell line, ID8 was prepared with the use of a spray dryer. These particles were designed for oral delivery using enteric polymers such as methacrylic copolymer, Eudragit® FS30D and hydroxyl propyl methyl cellulose acetate succinate. These particles were targeted for uptake via microfold cell (M-cell) in Peyer's patches of small intestine using M-cell targeting ligand, Aleuria aurantia lectin. The interleukins (ILs) such as IL-2 and IL-12 were added to the vaccine formulation to further enhance the immune response. The particles obtained were of 1.58±0.62μm size with a charge of 12.48±2.32mV. The vaccine efficacy was evaluated by administering the particles via oral route to C57BL/6 female mice. At the end of vaccination, mice were challenged with live tumor cells. Vaccinated mice showed significant (around six-fold) retardation of tumor volume in comparison to non-vaccinated animals for 3 weeks after the tumor challenge (p<0.001). The serum IgG antibody levels were found to be elevated in case of vaccinated animals in comparison to non-vaccinated group (p<0.05). Analysis of IgG1 titers (indicative of Th2 response) and IgG2a titers (indicative of Th1 response) showed a mixed Th1 and Th2 immune response in case vaccine alone and Th2 response in case of vaccine with interleukins group. Moreover, CD8+ T-cell, CD4+ T-cell and B-cell populations in different lymphatic organs were elevated in case of vaccinated mice. Thus, whole cell lysate vaccine microparticles formulated by spray drying could trigger humoral as well as cellular immune response when administered orally. Such vaccine could potentially be an effective treatment for patients with residual tumor or high tumor-relapse probability.

Formulation of meningococcal capsular polysaccharide vaccine-loaded microparticles with robust innate immune recognition

Neisseria meningitidis is a leading cause of bacterial meningitis and sepsis associated with a high mortality rate. Capsular polysaccharides (CPSs) are a major virulence factor and form the basis for serogroup designation and protective vaccines. The current polysaccharide meningococcal vaccines are available but are very expensive and require chemical conjugation. Here, we report a novel meningococcal vaccine formulation consisting of meningococcal CPS polymers encapsulated in albumin-based biodegradable microparticles that slowly release antigen and induce robust innate immune responses. Vaccines that elicit innate immunity are reported to have enhanced and protective adaptive immune responses. In this study, the meningococcal CPS-loaded microparticles, but not the empty microparticles, induced the release of IL-8, TNF-α and IL-1β, enhanced phagocytic capacity and induced robust autophagy in macrophages. The novel meningococcal vaccine microparticles are robustly taken up by macrophages and elicit strong innate immune responses that enhance antigen presentation which is a prerequisite for inducing adaptive immunity.

Synthetic LbL microparticle vaccines containing the conserved T1BT* epitope of Plasmodium falciparum elicit protective antibody and cellular immune responses with minimal inflammation (166.11)

Abstract Microparticle vaccines containing the conserved T1B repeat and T* epitopes from the P. falciparum CS protein were synthesized via layer-by-layer (LbL) fabrication on a solid core; microcapsules were prepared by dissolution of the particle core. Mice immunized with microparticles or microcapsules yielded T1B-specific antibody responses that neutralized Plasmodium parasite in vitro. The mice also developed T1BT*-specific cellular responses, including Th1, Th2, and CD4+ cytotoxic effector cells. When challenged with PfPb, a recombinant P. bergheii expressing the T1B repeats from P. falciparum, 70-80% of the mice immunized with microparticle, and 40-50% immunized with microcapsule, showed &amp;gt;90% reduction in liver parasite burden compared to naïve challenged mice. A minority of mice with reduced parasite burden did not have potent PfPb-neutralizing antibody activity. Since depletion of either CD4+ or CD8+ cells prior to challenge did not ablate protection, the vaccine-induced protection appears to be primarily associated with antibody responses but may also involve other mechanisms not yet defined. An examination of cytokine and chemokine gene expression in the liver post-challenge did not reveal any evidence of inflammatory responses following LbL vaccination and PfPb challenge. These results demonstrate that synthetic LbL vaccines bearing the conserved T1BT* epitopes can elicit protective immunity against Plasmodium without triggering unwanted inflammatory responses.

Protective effect of recombinant staphylococcal enterotoxin A entrapped in polylactic-co-glycolic acid microspheres against Staphylococcus aureus infection

Staphylococcus aureus is an important cause of nosocomial and community-acquired infections in humans and animals, as well as the cause of mastitis in dairy cattle. Vaccines aimed at preventing S. aureus infection in bovine mastitis have been studied for many years, but have so far been unsuccessful due to the complexity of the bacteria, and the lack of suitable vaccine delivery vehicles. The current study developed an Escherichia coli protein expression system that produced a recombinant staphylococcal enterotoxin A (rSEA) encapsulated into biodegradable microparticles generated by polylactic-co-glycolic acid (PLGA) dissolved in methylene chloride and stabilized with polyvinyl acetate. Antigen loading and surface properties of the microparticles were investigated to optimize particle preparation protocols. The prepared PLGA-rSEA microspheres had a diameter of approximately 5 μm with a smooth and regular surface. The immunogenicity of the PLGA-rSEA vaccine was assessed using mice as an animal model and showed that the vaccine induced a strong humoral immune response and increased the percent survival of challenged mice and bacterial clearance. Histological analysis showed moderate impairment caused by the pathogen upon challenge afforded by immunization with PLGA-rSEA microspheres. Antibody titer in the sera of mice immunized with PLGA-rSEA microparticles was higher than in vaccinated mice with rSEA. In conclusion, the PLGA-rSEA microparticle vaccine developed here could potentially be used as a vaccine against enterotoxigenic S. aureus.

Oral microparticulate vaccine for melanoma using M-cell targeting

Cancer vaccines are limited in their use, because of their inability to mount a robust anti-tumor immune response. Thus, targeting M-cells in the small intestine, which are responsible for entry of many pathogens, will be an attractive way to elicit a strong immune response toward particulate antigens. Therefore, in the present investigation, we demonstrated that efficient oral vaccination against melanoma antigens could be accomplished by incorporating the antigens in an albumin-based microparticle with a ligand AAL (Aleuria aurantia lectin) targeted specifically to M-cells. The oral microparticulate vaccine effectively protected the mice from subcutaneous challenge with tumor cells in prophylactic settings. The animals were vaccinated with antigen microparticles having a size range of around 1–1.25 µm where one prime and four booster doses were administered every 14 days over 10 weeks of duration, followed by challenge with live tumor cells, which showed complete tumor protection after oral vaccination. With the inclusion of ligand in the microparticles, we observed significantly higher IgG titers (1565 μg/mL) as compared to the microparticle formulations without AAL (872 μg/mL). This data suggests that ligand loaded microparticles may have the potential to target antigens to M-cells for an efficient oral vaccination.