Microparticle Vaccine Research Articles

Buccal Administration of a Zika Virus Vaccine Utilizing 3D-Printed Oral Dissolving Films in a Mouse Model.

Over the years, research regarding the Zika virus has been steadily increasing. Early immunization for ZIKV is a priority for preventing complications such as microencephaly and Guillain-Barré syndrome (GBS). Unlike traditional vaccination approaches, oral dissolving films (ODFs) or mucoadhesive film technology is an emerging, exciting concept that can be used in the field of pharmaceuticals for vaccine design and formulation development. This attractive and novel method can help patients who suffer from dysphagia as a complication of a disease or syndrome. In this study, we investigated a microparticulate Zika vaccine administered via the buccal route with the help of thin films or oral dissolving films (ODFs) with a prime dose and two booster doses two weeks apart. In vitro, the ODFs displayed excellent physiochemical properties, indicating that the films were good carriers for vaccine microparticles and biocompatible with the buccal mucosa. In vivo results revealed robust humoral (IgG, subtypes IgG1 and IgG2a) and T-cell responses (CD4+/CD8+) for ZIKV-specific immunity. Both the Zika MP vaccine and the adjuvanted Zika MP vaccine affected memory (CD45R/CD27) and intracellular cytokine (TNF-α and IL-6) expression. In this study, ZIKV vaccination via the buccal route with the aid of ODFs demonstrated great promise for the development of pain-free vaccines for infectious diseases.

Bioinformatic, Biochemical, and Immunological Mining of MHC Class I Restricted T Cell Epitopes for a Marburg Nucleoprotein Microparticle Vaccine.

The Marburg virus (MARV), the virus responsible for Marburg hemorrhagic fever (MHF), is considered a top-priority pathogen for vaccine development. Recent outbreaks in Equatorial Africa have highlighted the urgency of MARV because of its high fatality rate and historical concerns about potential weaponization. Currently, there are no licensed vaccines for MARV. Existing vaccine candidates rely on attenuated recombinant vesicular stomatitis virus carrying MARV glycoprotein (VSVΔG) or the chimpanzee replication-defective adenovirus 3 vector ChAd3-MARV. Although these platforms provide significant protection in animal models, they face challenges because of their limited thermal stability and the need for cold storage during deployment in resource-poor areas. An alternative approach involves using adjuvanted poly (lactic-co-glycolic acid) (PLGA) microparticles loaded with synthetic peptides representing MHC class I-restricted T cell epitopes. This vaccine platform has demonstrated effectiveness in protecting against SARS-CoV-2 and EBoV disease in animal models and has the advantage of not requiring cold storage and remaining stable at room temperature for over six months. This report outlines the design, manufacturing, and in vivo immunogenicity testing of PLGA microparticle human vaccines designed to prevent Marburg hemorrhagic fever.

Immunogenicity, Efficacy, and Safety of a Novel Synthetic Microparticle Pre-Erythrocytic Malaria Vaccine in Multiple Host Species.

We previously reported a protective antibody response in mice immunized with synthetic microparticle vaccines made using layer-by-layer fabrication (LbL-MP) and containing the conserved T1BT* epitopes from the P. falciparum circumsporozoite protein. To further optimize the vaccine candidate, a benchtop tangential flow filtration method (LbL-by-TFF) was developed and utilized to produce vaccine candidates that differed in the status of base layer crosslinking, inclusion of a TLR2 ligand in the antigenic peptide, and substitution of serine or alanine for an unpaired cysteine residue in the T* epitope. Studies in mice revealed consistent superiority of the Pam3Cys-modified candidates and a modest benefit of base layer crosslinking, as evidenced by higher and more persistent antibody titers (up to 18 months post-immunization), a qualitative improvement of T-cell responses toward a Th1 phenotype, and greater protection from live parasite challenges compared to the unmodified prototype candidate. Immunogenicity was also tested in a non-human primate model, the rhesus macaque. Base layer-crosslinked LbL-MP loaded with T1BT* peptide with or without covalently linked Pam3Cys elicited T1B-specific antibody responses and T1BT*-specific T-cell responses dominated by IFNγ secretion with lower levels of 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. IgG purified from individual macaques immunized with Pam3Cys.T1BT* LbL-MP protected naïve mice from challenges with transgenic P. berghei sporozoites that expressed the full-length PfCS protein, with 50-88% of passively immunized mice parasite-free for ≥15 days. Substitution of serine for an unpaired cysteine in the T* region of the T1BT* subunit did not adversely impact immune potency in the mouse while simplifying the manufacture of the antigenic peptide. In a Good Laboratory Practices compliant rabbit toxicology study, the base layer-crosslinked, Pam3Cys-modified, serine-substituted candidate was shown to be safe and immunogenic, eliciting parasite-neutralizing antibody responses and establishing the dose/route/regimen for a clinical evaluation of this novel synthetic microparticle pre-erythrocytic malaria vaccine candidate.

Adjuvanted-SARS-CoV-2 Spike Protein-Based Microparticulate Vaccine Delivered by Dissolving Microneedles Induces Humoral, Mucosal, and Cellular Immune Responses in Mice.

COVID-19 continues to cause an increase in the number of cases and deaths worldwide. Due to the ever-mutating nature of the virus, frequent vaccination against COVID-19 is anticipated. Most of the approved SARS-CoV-2 vaccines are administered using the conventional intramuscular route, causing vaccine hesitancy. Thus, there is a need for an effective, non-invasive vaccination strategy against COVID-19. This study evaluated the synergistic effects of a subunit microparticulate vaccine delivered using microneedles. The microparticles encapsulated a highly immunogenic subunit protein of the SARS-CoV-2 virus, such as the spike protein's receptor binding domain (RBD). Adjuvants were also incorporated to enhance the spike RBD-specific immune response. Our vaccination study reveals that a microneedle-based vaccine delivering these microparticles induced spike RBD-specific IgM, IgG, IgG1, IgG2a, and IgA antibodies. The vaccine also generated high levels of CD4+ and CD8a+ molecules in the secondary lymphoid organs. Overall, dissolving microneedles delivery spike RBD antigen in microparticulate form induced a robust immune response, paving the way for an alternative self-administrable, non-invasive vaccination strategy against COVID-19.

Gonococcal microparticle vaccine in dissolving microneedles induced immunity and enhanced bacterial clearance in infected mice

There is an alarming rise in the number of gonorrhea cases worldwide. Neisseria gonorrhoeae, the bacteria that causes gonorrhea infection, has gradually developed antimicrobial resistance over the years. To date, there is no licensed vaccine for gonorrhea. This study investigates the in vivo immunogenicity of a whole-cell inactivated gonococci in a microparticle formulation (Gc-MP) along with adjuvant microparticles (Alhydrogel®- Alum MP and AddaVax™ MP) delivered transdermally using dissolving microneedles (MN). The proposed vaccine formulation (Gc-MP + Alum MP + AddaVax™ MP) was assessed for induction of humoral, cellular, and protective immune responses in vivo. Our results show the induction of significant gonococcal-specific serum IgG, IgG1, IgG2a, and vaginal mucosal IgA antibodies in mice immunized with Gc-MP + Alum MP + AddaVax™ MP and Gc-MP when compared to the control groups receiving blank MN or no treatment. The serum bactericidal assay revealed that the antibodies generated in mice after immunization with Gc-MP + Alum MP + AddaVax™ MP were bactericidal towards live Neisseria gonorrhoeae. Gc-MP + Alum MP + AddaVax™ MP and Gc-MP-immunized mice showed enhanced clearance rate of gonococcal bacterial infection post challenge. In contrast, the control groups did not begin to clear the infection until day 10. In addition, the mice which received Gc-MP + Alum MP + AddaVax™ MP showed enhanced expression of cellular immunity markers CD4 and CD8 on the surface of T cells in the spleen and lymph nodes. Taken together, the data shows that microneedle immunization with whole-cell inactivated gonococci MP in mice induced humoral, cellular, and protective immunity against gonococcal infection.

Controlled release of inactivated influenza virus and α-galactosylceramide from PLGA microparticles results in long-term protection against influenza infection

Abstract The objective of this study was to develop biodegradable microparticles with continuous release that can enhance and extend the long-term protection of single-dose vaccines. Several formulations of Poly (lactic-co-glycolic acid) (PLGA) microparticles were synthesized using a double emulsion solvent evaporation technique to encapsulate inactivated A/PR/8/34 (PR8) H1N1 influenza virus in combination with the invariant natural killer T (NKT) cell agonist α-galactosylceramide (α-GalCer). The most efficient controlled release particle formulations were injected into C57BL/6J mice. Additional control mice were injected with PBS alone, inactivated PR8 dissolved in PBS, or inactivated PR8 and α-GalCer dissolved in PBS. Thirty weeks after vaccination, mice were challenged with a lethal dose of live homologous virus. Mice immunized with the microparticle vaccine survived the infection and had reduced clinical disease and decreased weight loss compared to unvaccinated mice. After vaccination, mice injected microparticles presented a gradual increase production of PR8-specific IgGs that was dominated by IgG1 isotype that is usually produced by T helper (Th) 2 responses. In contrast, mice that received soluble vaccine presented a rapid antibody response that was dominated by antibodies of the Th1-associated IgG2a isotype. Collectively, these results demonstrate the potential of PLGA-based single-dose microparticle vaccines for providing long-term protection against influenza infection and potentially other pathogens. Supported by grants from USAMRAA (W81XWH-19-1-0005)

Monocytes reprogrammed by tumor microparticle vaccine inhibit tumorigenesis and tumor development

Tumor microparticles (T-MPs) are considered as a tumor vaccine candidate. Although some studies have analyzed the mechanism of T-MPs as tumor vaccine, we still lack understanding of how T-MPs stimulate a strong anti-tumor immune response. Here, we show that T-MPs induce macrophages to release a key chemotactic factor CCL2, which attracts monocytes to the vaccine injection site and enhances endocytosis of antigen. Monocytes subsequently enter the draining lymph node, and differentiate into monocyte-derived DCs (moDCs), which present tumor antigens to T lymphocytes and deliver a potent anti-tumor immune response. Mechanically, T-MPs activate the cGAS-STING signaling through DNA fragments, and then induce monocytes to upregulate the expression of IRF4, which is a key factor for monocyte differentiation into moDCs. More importantly, monocytes that have endocytosed T-MPs acquire the ability to treat tumors. Collectively, this work might provide novel vaccination strategy for the development of tumor vaccines and facilitate the application of T-MPs for clinic oncotherapy.

Survivin (BIRC5) Peptide Vaccine in the 4T1 Murine Mammary Tumor Model: A Potential Neoadjuvant T Cell Immunotherapy for Triple Negative Breast Cancer: A Preliminary Study

A triple negative breast cancer model using the murine 4T1 tumor cell line was used to explore the efficacy of an adjuvanted survivin peptide microparticle vaccine using tumor growth as the outcome metric. We first performed tumor cell dose titration studies to determine a tumor cell dose that resulted in sufficient tumor takes but allowed multiple serial measurements of tumor volumes, yet with minimal morbidity/mortality within the study period. Later, in a second cohort of mice, the survivin peptide microparticle vaccine was administered via intraperitoneal injection at the study start with a second dose given 14 days later. An orthotopic injection of 4T1 cells into the mammary tissue was performed on the same day as the administration of the second vaccine dose. The mice were followed for up to 41 days with subcutaneous measurements of tumor volume made every 3-4 days. Vaccination with survivin peptides was associated with a peptide antigen-specific gamma interferon enzyme-linked immunosorbent spot response in the murine splenocyte population but was absent from the control microparticle group. At the end of the study, we found that vaccination with adjuvanted survivin peptide microparticles resulted in statistically significant slower primary tumor growth rates in BALB/c mice challenged with 4T1 cells relative to the control peptideless vaccination group. These studies suggest that T cell immunotherapy specifically targeting survivin might be an applicable neoadjuvant immunotherapy therapy for triple negative breast cancer. More preclinical studies and clinical trials are needed to explore this concept further.

An Adjuvanted Inactivated SARS-CoV-2 Microparticulate Vaccine Delivered Using Microneedles Induces a Robust Immune Response in Vaccinated Mice.

SARS-CoV-2, the causal agent of COVID-19, is a contagious respiratory virus that frequently mutates, giving rise to variant strains and leading to reduced vaccine efficacy against the variants. Frequent vaccination against the emerging variants may be necessary; thus, an efficient vaccination system is needed. A microneedle (MN) vaccine delivery system is non-invasive, patient-friendly, and can be self-administered. Here, we tested the immune response produced by an adjuvanted inactivated SARS-CoV-2 microparticulate vaccine administered via the transdermal route using a dissolving MN. The inactivated SARS-CoV-2 vaccine antigen and adjuvants (Alhydrogel® and AddaVax™) were encapsulated in poly(lactic-co-glycolic acid) (PLGA) polymer matrices. The resulting MP were approximately 910 nm in size, with a high percentage yield and percent encapsulation efficiency of 90.4%. In vitro, the vaccine MP was non-cytotoxic and increased the immunostimulatory activity measured as nitric oxide release from dendritic cells. The adjuvant MP potentiated the immune response of the vaccine MP in vitro. In vivo, the adjuvanted SARS-CoV-2 MP vaccine induced high levels of IgM, IgG, IgA, IgG1, and IgG2a antibodies and CD4+ and CD8+ T-cell responses in immunized mice. In conclusion, the adjuvanted inactivated SARS-CoV-2 MP vaccine delivered using MN induced a robust immune response in vaccinated mice.

Zika Vaccine Microparticles (MPs)-Loaded Dissolving Microneedles (MNs) Elicit a Significant Immune Response in a Pre-Clinical Murine Model

Although the global Zika epidemic in 2015-16 fueled vaccine development efforts, there is no approved Zika vaccine or treatment available to date. Current vaccine platforms in clinical trials are administered via either subcutaneous or intramuscular injections, which are painful and decrease compliance. Therefore, in the present study, we explored Zika vaccine microparticles (MPs)-loaded dissolving microneedles (MNs) with adjuvant MPs encapsulating Alhydrogel® and MPL-A® administered via the transdermal route as a pain-free vaccine strategy. We characterized the MNs for needle length, pore formation, and dissolvability when applied to murine skin. Further, we evaluated the in vivo efficacy of vaccine MPs-loaded MNs with or without adjuvants by measuring the immune response after transdermal immunization. The vaccine MPs-loaded dissolving MNs with adjuvants induced significant IgG, IgG1, and IgG2a titers in immunized mice compared to the untreated control group. After the dosing regimen, the animals were challenged with Zika virus, monitored for seven days, and sacrificed to collect spleen and lymph nodes. The lymphocytes and splenocytes from the immunized mice showed significant expressions of helper (CD4) and cytotoxic (CD8a) cell surface markers compared to the control group. Thus, this study puts forth a 'proof-of-concept' for a pain-free transdermal vaccine strategy against Zika.

Boosting In-Vivo Anti-Tumor Immunity with an Oral Microparticulate Breast Cancer Vaccine and Low-Dose Cyclophosphamide.

Tumor cells express antigens that should induce immune-mediated rejection; however, spontaneous rejection of established tumors is rare. Recent evidence suggests that patients suffering from cancer exhibit an elevation in regulatory T cells population, a subset of CD4+ T cells, which suppress tumor recognition and elimination by cytotoxic T cells. This study investigates immunotherapeutic strategies to overcome the immunosuppressive effects exerted by regulatory T cells. A novel immunotherapeutic strategy was developed by simultaneous administration of oral microparticulate breast cancer vaccines and cyclophosphamide, a regulatory T cell inhibitor. Breast cancer vaccine microparticles were prepared by spray drying, and administered orally to female mice inoculated with 4TO7 murine breast cancer cells in combination with a low dose of intraperitoneally administered cyclophosphamide. Mice receiving the combination of vaccine microparticles and cyclophosphamide exhibited maximal tumor regression and the highest survival rate compared with the control groups. This study highlights the importance of cancer vaccination along with regulatory T cell depletion in cancer therapy, and suggests that a low dose of cyclophosphamide that specifically and significantly depletes regulatory T cells may be a highly effective immunotherapeutic strategy for the treatment of cancer.

Self-Boosting Vaccination Based on Pulsatile Antigen Release from Core-Shell Microparticles.

Vaccination is among the most effective ways to prevent infectious diseases. Subunit vaccines are safe but usually require multiple booster shots, which may lead to immunity loss and economic consume. In this study, a self-boosting vaccine is developed based on the pulsatile release of antigen from the core-shell microparticle after single-injection immunization. Self-healing technology applied to form an "antigen core" can avoid organic solvents from destroying the spatial structure of the antigen. The "antigen shell" is built-up by self-assemble of the antigen with the opposite charged polypeptide. Primary immunization occurs with the self-assembled film disintegration, and the booster comes with the microparticle degradation. The changing of antigen-specific antibodies after immunization with the core-shell microparticle vaccine is consistent with that caused by the two shots of immunization. The immune effect and safety evaluation results support the translational potential of this self-boosting core-shell microparticle vaccine.

Laser-assisted intradermal delivery of a microparticle vaccine for respiratory syncytial virus induces a robust immune response.

Respiratory syncytial virus (RSV) is an infectious disease that poses a significant public health risk in young children. Vaccine studies conducted in the 1960s using an intramuscular injection of formalin-inactivated respiratory syncytial virus (Fi-RSV) resulted in an enhanced respiratory disease and led to the failure of the vaccine. Thus, the virus-like particles (VLP) of the RSV fusion (F) protein was used as the vaccine antigen in this study. The F-VLP was encapsulated in a microparticle (MP) matrix composed of cross-linked bovine serum albumin (BSA) to enhance the antigen presentation and uptake. Moreover, a painless vaccination method would be desirable for an infectious disease that mainly affects young children. Thus, an ablative laser device, Precise Laser Epidermal System (P.L.E.A.S.E), was utilized to create micropores on the skin for vaccine delivery. We observed enhanced antigen presentation of the vaccine microparticles (F-VLP MP) with and without the adjuvant monophosphoryl lipid A (MPL-A) MP in dendritic cells. Consequently, Swiss Webster mice were immunized with the adjuvanted vaccine microparticles using the P.L.E.A.S.E laser to study the in vivo immunogenicity. The immunized mice had high serum immunoglobulin (IgG, IgG2a) levels, indicating a Th1 response. Subsequent analysis of lung homogenates post- RSV challenge revealed high IgA, indicating generation of a mucosal immune response upon intradermal immunization. Flowcytometry analysis showed high CD8+, and CD4+ expression in the lymph node and spleen of the adjuvanted vaccine microparticle immunized mice. Increased expression of interferon gamma (IFN-γ) in the spleen cells further proved Th1 polarized immune response. Finally, an immune plaque assay indicated significantly low lung viral titer in the mice immunized with intradermal adjuvanted vaccine microparticles. Thus, ablative laser-assisted immunization with the F-VLP based adjuvanted vaccine microparticles could be a promising vaccine candidate for RSV.

Subunit microparticulate vaccine delivery using microneedles trigger significant SARS-spike-specific humoral and cellular responses in a preclinical murine model

The objective of this “proof-of-concept” study was to evaluate the synergistic effect of a subunit microparticulate vaccine and microneedles (MN) assisted vaccine delivery system against a human coronavirus. Here, we formulated PLGA polymeric microparticles (MPs) encapsulating spike glycoprotein (GP) of SARS-CoV as the model antigen. Similarly, we formulated adjuvant MPs encapsulating Alhydrogel® and AddaVax™. The antigen/adjuvant MPs were characterized and tested in vitro for immunogenicity. We found that the antigen/adjuvant MPs were non-cytotoxic in vitro. The spike GP MPs + Alhydrogel® MPs + AddaVax™ MPs showed enhanced immunogenicity in vitro as confirmed through the release of nitrite, autophagy, and antigen presenting molecules with their co-stimulatory molecules. Next, we tested the in vivo efficacy of the spike GP MP vaccine with and without adjuvant MPs in mice vaccinated using MN. The spike GP MPs + Alhydrogel® MPs + AddaVax™ MPs induced heightened spike GP-specific IgG, IgG1 and IgG2a antibodies in mice. Also, spike GP MPs + Alhydrogel® MPs + AddaVax™ MPs enhanced expression of CD4+ and CD8+ T cells in secondary lymphoid organ like spleen. These results indicated spike GP-specific humoral immunity and cellular immunity in vivo. Thus, we employed the benefits of both the subunit vaccine MPs and dissolving MN to form a non-invasive and effective vaccination strategy against human coronaviruses.

Preclinical evaluation of a microparticle-based transdermal vaccine patch against metastatic breast cancer

Breast cancer is the number one cause of cancer-related deaths among females. Current chemotherapy targets both tumor and normal cells, leading to pronounced side effects. Therefore, therapeutic vaccines acting against specific cancer cells would be the choice of treatment. We prepared microparticles entrapping the antigens obtained from a murine metastatic breast cancer cell line, 4 T1 using the spray drying technology. These microparticles were incorporated into microneedle patches to deliver to the animals for the efficacy study. An antineoplastic drug, cyclophosphamide, in a very low dose has been found to inhibit the immunosuppressive regulatory T cells (Treg) (Le and Jaffee, 2012). In-vivo efficacy of the microparticulate vaccine given along with a low dose of cyclophosphamide was evaluated in a murine breast cancer model. Animals immunized with vaccine microparticles showed considerably slower tumor growth than animals that did not receive the vaccine. The results of the study showed that the Tumor-Associated Antigens (TAAs) within the microparticles were responsible for the delayed tumor growth in vaccinated animals. Vaccinated animals also showed an increase in the population of CD4 and CD8 T cells. Overall, our results demonstrated that immunotherapy with vaccine microparticles encapsulating TAA’s could potentially be an effective treatment for metastatic breast cancer.

Enhanced Immunogenicity of Adjuvanted Microparticulate HPV16 Vaccines Administered via the Transdermal Route.

Human papillomavirus (HPV) causes cervical cancer among women and is associated with other anogenital cancers in men and women. Prophylactic particulate vaccines that are affordable, self-administered and efficacious could improve uptake of HPV vaccines world-wide. The goal of this research is to develop a microparticulate HPV16 vaccine for transdermal administration using AdminPatch® and assess its immunogenicity in a pre-clinical mouse model. HPV16 microparticles were prepared using a biocompatible polymer and characterized in terms of size, zeta potential, encapsulation efficiency and microparticle yield. Scanning and transmission electron microscopy were conducted to confirm particle image and to visualize the conformation of HPV16 vaccine particles released from microparticle formulation. In vivo studies performed to evaluate the potential of the microparticulate vaccine initiated a robust and sustained immune response. HPV16 IgG antibodies were significantly elevated in the microparticle group compared to antigen solutions administered by the transdermal route. Results show significant expansion of CD4+, CD45R, CD27 and CD62L cell populations in the vaccinated mice group, indicating the high efficacy of the microparticulate vaccine when administered via transdermal route. The findings of this study call attention to the use of minimally invasive, pain-free routes to deliver vaccine.

Microneedle Delivery of an Adjuvanted Microparticulate Vaccine Induces High Antibody Levels in Mice Vaccinated against Coronavirus.

This ‘proof-of-concept’ study aimed to test the microparticulate vaccine delivery system and a transdermal vaccine administration strategy using dissolving microneedles (MN). For this purpose, we formulated poly(lactic-co-glycolic) acid (PLGA) microparticles (MP) encapsulating the inactivated canine coronavirus (iCCoV), as a model antigen, along with adjuvant MP encapsulating Alhydrogel® and AddaVax. We characterized the vaccine MP for size, surface charge, morphology, and encapsulation efficiency. Further, we evaluated the in vitro immunogenicity, cytotoxicity, and antigen-presentation of vaccine/adjuvant MP in murine dendritic cells (DCs). Additionally, we tested the in vivo immunogenicity of the MP vaccine in mice through MN administration. We evaluated the serum IgG, IgA, IgG1, and IgG2a responses using an enzyme-linked immunosorbent assay. The results indicate that the particulate form of the vaccine is more immunogenic than the antigen suspension in vitro. We found the vaccine/adjuvant MP to be non-cytotoxic to DCs. The expression of antigen-presenting molecules, MHC I/II, and their costimulatory molecules, CD80/40, increased with the addition of the adjuvants. Moreover, the results suggest that the MP vaccine is cross presented by the DCs. In vivo, the adjuvanted MP vaccine induced increased antibody levels in mice following vaccination and will further be assessed for its cell-mediated responses.

Novel microparticulate Zika vaccine induces a significant immune response in a preclinical murine model after intramuscular administration

Despite the detrimental effects associated with Zika infection, there are no approved treatments or vaccines available. To address the need for a safe and effective vaccine for Zika, we formulated poly(lactic-co-glycolic) acid (PLGA) polymeric vaccine microparticles (MP) encapsulating the inactivated Zika virus, along with adjuvant MP encapsulating Alhydrogel® and MPL-A®. We characterized the vaccine MP for size, surface charge, morphology, encapsulation efficiency, and antigen integrity. Further, we evaluated immunogenicity and cytotoxicity of vaccine MP in vitro in murine dendritic cells. Vaccine MP with adjuvants induced significantly higher production of nitric oxide, a marker of innate immunity, when compared to the untreated cells. In addition, vaccine MP with or without adjuvants induced increased autophagy in murine dendritic cells when compared to inactivated Zika virus, which is critical in antigen presentation. Next, we evaluated in vivo efficacy of vaccine MP with and without adjuvants in a preclinical murine model by measuring the immune response after intramuscular administration. Vaccine MP with adjuvants induced significant IgG, Ig2a, and IgG1 titers as compared to the control group of untreated mice. Thus, this study provided the ‘proof-of-concept’ for a microparticulate Zika vaccine.

Assessment of In Vitro Immunostimulatory Activity of an Adjuvanted Whole-Cell Inactivated Neisseria gonorrhoeae Microparticle Vaccine Formulation.

The emergence of drug-resistant gonorrhea infections worldwide combined with the lack of a vaccine is alarming. We prepared a novel microparticulate (MP) vaccine formulation using whole-cell inactivated Neisseria gonorrhoeae as the vaccine antigen, with Alum and AddaVax™ as vaccine adjuvants. The adjuvanted vaccine MP formulation was assessed for in vitro immunostimulatory activity, autophagy, and antigen presentation ability. The data shows that the adjuvanted gonococci vaccine MP enhanced autophagy induction in antigen presenting cells (APCs) compared to gonococci vaccine MP without adjuvants, which is important for enhancing antigen presentation. In addition, the adjuvanted vaccine formulation increased the surface expression of antigen presenting molecules MHCI and MHCII as well as co-stimulatory molecules CD40 and CD86 on the surface of dendritic cells. In addition, the gonococci vaccine microparticles at lower doses did not significantly increase the expression of the death receptor CD95 in APCs, which when elevated leads to suboptimal antigen presentation and reduced immune responses. The adjuvanted whole-cell inactivated gonococci microparticle vaccine formulation enhanced antigen uptake, processing, and antigen presentation.

Spray-dried pneumococcal membrane vesicles are promising candidates for pulmonary immunization

Pneumococcal infections represent a global health threat, which requires novel vaccine developments. Extracellular vesicles are secreted from most cells, including prokaryotes, and harbor virulence factors and antigens. Hence, bacterial membrane vesicles (MVs) may induce a protective immune response. For the first time, we formulate spray-dried gram-positive pneumococcal MVs-loaded vaccine microparticles using lactose/leucine as inert carriers to enhance their stability and delivery for pulmonary immunization. The optimized vaccine microparticles showed a mean particle size of 1–2 µm, corrugated surface, and nanocrystalline nature. Their aerodynamic diameter of 2.34 µm, average percentage emitted dose of 88.8%, and fine powder fraction 79.7%, demonstrated optimal flow properties for deep alveolar delivery using a next-generation impactor. Furthermore, confocal microscopy confirmed the successful encapsulation of pneumococcal MVs within the prepared microparticles. Human macrophage-like THP-1 cells displayed excellent viability, negligible cytotoxicity, and a rapid uptake around 60% of fluorescently labeled MVs after incubation with vaccine microparticles. Moreover, vaccine microparticles increased the release of pro-inflammatory cytokines tumor necrosis factor and interleukin-6 from primary human peripheral blood mononuclear cells. Vaccine microparticles exhibited excellent properties as promising vaccine candidates for pulmonary immunization and are optimal for further animal testing, scale-up and clinical translation.