Immunogenicity Of Subunit Vaccine Research Articles

Improved biodistribution and enhanced immune response of subunit vaccine using a nanostructure formed by self-assembly of ascorbyl palmitate

New adjuvant strategies are needed to improve protein-based subunit vaccine immunogenicity. We examined the potential to use nanostructure of 6-O-ascorbyl palmitate to formulate ovalbumin (OVA) protein and an oligodeoxynucleotide (CpG-ODN) (OCC). In mice immunized with a single dose, OCC elicited an OVA-specific immune response superior to OVA/CpG-ODN solution (OC). Rheological studies demonstrated OCC's self-assembling viscoelastic properties. Biodistribution studies indicated that OCC prolonged OVA and CpG-ODN retention at injection site and lymph nodes, reducing systemic spread. Flow-cytometry assays demonstrated that OCC promoted OVA and CpG-ODN co-uptake by Ly6ChiCD11bhiCD11c + monocytes. OCC and OC induced early IFN-γ in lymph nodes, but OCC led to higher concentration. Conversely, mice immunized with OC showed higher serum IFN-γ concentration compared to those immunized with OCC. In mice immunized with OCC, NK1.1+ cells were the IFN-γ major producers, and IFN-γ was essential for OVA-specific IgG2c switching. These findings illustrate how this nanostructure improves vaccine's response.

Utilizing murine dendritic cell line DC2.4 to evaluate the immunogenicity of subunit vaccines in vitro.

Subunit vaccines hold substantial promise in controlling infectious diseases, due to their superior safety profile, specific immunogenicity, simplified manufacturing processes, and well-defined chemical compositions. One of the most important end-targets of vaccines is a subset of lymphocytes originating from the thymus, known as T cells, which possess the ability to mount an antigen-specific immune response. Furthermore, vaccines confer long-term immunity through the generation of memory T cell pools. Dendritic cells are essential for the activation of T cells and the induction of adaptive immunity, making them key for the in vitro evaluation of vaccine efficacy. Upon internalization by dendritic cells, vaccine-bearing antigens are processed, and suitable fragments are presented to T cells by major histocompatibility complex (MHC) molecules. In addition, DCs can secrete various cytokines to crosstalk with T cells to coordinate subsequent immune responses. Here, we generated an in vitro model using the immortalized murine dendritic cell line, DC2.4, to recapitulate the process of antigen uptake and DC maturation, measured as the elevation of CD40, MHC-II, CD80 and CD86 on the cell surface. The levels of key DC cytokines, tumor necrosis alpha (TNF-α) and interleukin-10 (IL-10) were measured to better define DC activation. This information served as a cost-effective and rapid proxy for assessing the antigen presentation efficacy of various vaccine formulations, demonstrating a strong correlation with previously published in vivo study outcomes. Hence, our assay enables the selection of the lead vaccine candidates based on DC activation capacity prior to in vivo animal studies.

Next-generation subunit vaccine delivery systems: Design, applications, and prospects

Subunit vaccine delivery systems have emerged as groundbreaking strategies to enhance immunogenicity and efficacy, overcoming the limitations of traditional vaccine approaches. This review article delves into the vast landscape of subunit vaccine delivery systems, encompassing diverse platforms, such as polymer-based, lipid-based, micelle-based, phage-based, hydrogel-based, inorganic-based, and emulsion-based carriers. This review aimed to comprehensively explore the advancements, challenges, and potential of these delivery systems in revolutionizing vaccine development. Key findings revealed that polymer-based systems offer tunable properties for sustained release, while lipid-based and micelle-based carriers enable efficient encapsulation of hydrophobic antigens. Phage-based platforms leverage host–pathogen interactions, whereas hydrogel-based carriers provide localized delivery and adjuvant effects. Inorganic nanoparticles and emulsions offer targeted delivery and improved immune responses. These findings offer opportunities to enhance the immunogenicity of subunit vaccines, optimize antigen delivery, and tailor responses to specific diseases. This review can guide researchers, clinicians, and policymakers in harnessing the strengths of diverse delivery systems to improve vaccination strategies. By shedding light on their design, applications, and impacts, this review serves as a roadmap for the development of next-generation vaccines with the potential to transform global health-care paradigms.

Progress and challenges in production of recombinant Newcastle disease virus hemagglutinin-neuraminidase subunit vaccine

Recombinant Newcastle disease (ND) subunit vaccines are safe, efficacious and compatible with DIVA strategies. Hence, are promising alternatives to conventional live and inactivated vaccines for ND control and prevention. While production of hemagglutinin-neuraminidase (HN)-based ND subunit vaccines have been extensively evaluated at lab-scale, significant barriers impeding successful translation from high-cost, small bench scale production to low-cost mass production remain. This review details current state and challenges in efficacious HN subunit design, advantages and disadvantages of available production routes using different expression hosts together with gaps and solutions regarding quantitative analytical methods used for formulation in dose-efficiency studies. Critical factors influencing HN subunit vaccine immunogenicity such as protein structure, glycosylation and amino acid variations are discussed. Construct design and available expression hosts are then analyzed from viewpoint of yield and quality measured in terms of efficacy. The current deficiency in available quantitative methods for effective dose-efficacy studies is given emphasis, underscoring the importance of robust analytical methods for determination of optimal vaccine dose. The review outlines current state and way forward for the development of HN ND subunit vaccines and other recombinant subunit vaccines for veterinary applications.

Comparison of effects of multiple adjuvants and immunization routes on the immunogenicity and protection of HSV-2 gD subunit vaccine

Genital herpes caused by herpes simplex virus type 2 (HSV-2) poses a global health issue. HSV-2 infection increases the risk of acquiring HIV infection. Studies have demonstrated that HSV-2 subunit vaccines have potential benefits, but require adjuvants to induce a balanced Th1/Th2 response. To develop a novel, effective vaccine, in this study, a truncated glycoprotein D (aa 1–285) of HSV-2 was formulated with an Al(OH)3 adjuvant, three squalene adjuvants, zMF59, zAS03, and zAS02, or a mucosal adjuvant, bacterium-like particles (BLPs). The immunogenicity of these subunit vaccines was evaluated in mice. After three immunizations, vaccines formulated with Al(OH)3, zMF59, zAS03, and zAS02 (intramuscularly) induced higher titers of neutralizing antibody than that formulated without adjuvant, and in particular, mice immunized with the vaccine plus zAS02 had the highest neutralizing antibody titers and tended to produce a more balanced immune reaction than others. Intranasal gD2-PA-BLPs also induced excellent IgA levels and a more balanced Th1 and Th2 responses than intranasal gD2. After challenge with a lethal dose of HSV-2, all five adjuvants exhibited a positive effect in improving the survival rate. zAS02 and gD2-PA-BLPs enhanced survival by 50% and 25%, respectively, when compared with the vaccine without adjuvant. zAS02 was the only adjuvant that resulted in complete vaginal virus clearance and genital lesion healing within eight days. These results demonstrate the potential of using zAS02 as a subunit vaccine adjuvant, and BLPs as a mucosal vaccine adjuvant.

Developing an efficient MGCR microneedle nanovaccine patch for eliciting Th 1 cellular response against the SARS-CoV-2 infection.

Rationale: Novel vaccine R&D is essential to interrupt the COVID-19 pandemic and other epidemics in the future. Subunit vaccines have received tremendous attention for their low cost and safety. To improve the immunogenicity of subunit vaccines, we developed a novel vaccine adjuvant system. Methods: Here we rationally designed a CpG 1018 and graphene oxide-based bi-adjuvant system to deliver the Receptor-Binding Domain (RBD) of the SARS-CoV-2 spike protein and obtained the graphene oxide-based complex adjuvant nanovaccine (GCR). Furthermore, we developed a microneedle patch vaccine (MGCR) based on the GCR vaccine. Results: GCR nanovaccine displayed superb antigen loading and encapsulation efficiency. Two dosages of vaccination of GCR nanovaccine could elicit adequate RBD-specific binding antibody response with 2.14-fold higher IgG titer than Alum adjuvant vaccine. The peptide pools assay demonstrated the robust RBD-specific Type 1 Cellular response induced by the GCR nanovaccine in CD8+ T cells. Furthermore, we prepared an MGCR microneedle patch, which generated a similar RBD-specific binding antibody response to the GCR vaccine, sustained a high antibody level above 16 weeks, and significantly elevated the Tcm proportion in mouse spleen. The MGCR microneedle patch vaccine also could be stably stored at room temperature for several months and administrated without medical staff, which maximizes the vaccine distribution efficiency. Conclusion: The vaccine system could significantly improve the vaccine distribution rate in low-income areas and offer a potential vaccination approach to fight against the SARS-Cov-2 infection and other pandemics occurred in the future.

Display of Streptococcus iniae α-Enolase on the Surface of Virus-Like Particles (VLPs) of Nervous Necrosis Virus (NNV) Using SpyTag/SpyCatcher.

Virus-like particle (VLP)-based vaccines are promising candidates for overcoming the safety problems of live vaccines and weak immunogenicity of subunit vaccines. VLPs can be used as a platform for the development of combined vaccines by expressing foreign antigens, and foreign antigens can be displayed on the surface of VLPs by conjugation. In the present study, to use nervous necrosis virus (NNV) VLPs as a delivery tool for Streptococcus iniae α-enolase by displaying on the VLP's surface, the split-intein (SpyTag/SpyCatcher) conjugation system was used. NNV capsid protein fused to SpyTag (Capsid-SpyTag) and S. iniae α-enolase fused to SpyCatcher (α-enolase-SpyCatcher) were recombinantly produced, then mixed in various ratios. A ratio of Capsid-SpyTag to α-enolase-SpyCatcher of 1 to 1.5 showed the highest coupling efficiency corresponding to 83-92% of coupled capsid protein dimer and 32-52% of coupled capsid protein monomer. In TEM observation, VLP of Capsid-SpyTag had a regular shape and size of about 40 nm, while VLP fused with α-enolase-SpyCatcher showed an irregular shape and size of about 40-50 nm in diameter. In preliminary immunization experiments, olive flounder (Paralichthys olivaceus) and zebrafish (Danio rerio) immunized with VLP fused with α-enolase-SpyCatcher showed the lowest cumulative mortality against S. iniae infection.

Chemically Modified Bacterial Sacculi as a Vaccine Microparticle Scaffold.

Vaccine scaffolds and carrier proteins increase the immunogenicity of subunit vaccines. Here, we developed, characterized, and demonstrated the efficacy of a novel microparticle vaccine scaffold comprised of bacterial peptidoglycan (PGN), isolated as an entire sacculi. The PGN microparticles contain bio-orthogonal chemical handles allowing for site-specific attachment of immunogens. We first evaluated the purification, integrity, and immunogenicity of PGN microparticles derived from a variety of bacterial species. We then optimized PGN microparticle modification conditions; Staphylococcus aureus PGN microparticles containing azido-d-alanine yielded robust conjugation to immunogens. We then demonstrated that this vaccine scaffold elicits comparable immunostimulation to the conventional carrier protein, keyhole limpet hemocyanin (KLH). We further modified the S. aureus PGN microparticle to contain the SARS-CoV-2 receptor-binding domain (RBD)—this conjugate vaccine elicited neutralizing antibody titers comparable to those elicited by the KLH-conjugated RBD. Collectively, these findings suggest that chemically modified bacterial PGN microparticles are a conjugatable and biodegradable microparticle scaffold capable of eliciting a robust immune response toward an antigen of interest.

Fusion of Streptococcus iniae α-enolase to IMX313 enhanced antibody titer and survival rate in olive flounder (Paralichthys olivaceus)

The polymerization of monomeric antigens can be a strategy to overcome the low immunogenicity of subunit vaccines. IMX313 is a hybrid oligomerization domain of chicken C4bp, and has been demonstrated to have potent activity as adjuvants for the fused antigens in mammals. In the present study, we investigated whether the oligomerization of α-enolase of Streptococcus iniae by fusion with IMX313 affected on antibody induction and on protection against S. iniae infection in olive flounder (Paralichthys olivaceus). The oligomerization of S. iniae enolase by fusion with IMX313 (enolase-IMX313) was verified by non-reducing PAGE, and the antibody titer against enolase in olive flounder immunized with enolase-IMX313 was significantly higher than that in fish immunized with enolase alone. Furthermore, although the survival of olive flounder immunized with enolase alone was low, fish immunized with enolase-IMX313 showed much higher survival (RPS 50%) in accordance with higher serum antibody titer, suggesting that fusion of antigens with IMX313 can be an effective way to enhance protective efficacy of subunit vaccines in olive flounder.

Fusion-expressed CtxB-TcpA-C-CPE improves both systemic and mucosal humoral and T-cell responses against cholera in mice

BackgroundDevelopment of an effective oral vaccine against Cholera, a life-threatening dehydrating diarrheal disease, proved to be a challenging task. To improve oral subunit vaccine immunogenicity and to prevent the state of oral tolerance, application of mucosal adjuvants might be a promising approach. In the present study, the CtxB-TcpA-C-CPE fusion was constructed in which CtxB and C-CPE were used as mucosal adjuvants and vaccine delivery system, respectively, to induce mucosal immune responses, and to improve the anti-toxin and anti-colonizing immunity against V. cholerae. Materials & methodsThe fusion construct was synthesized, sub-cloned in pQE30 and expressed in E. coli. The three antigen, making the fusion protein, were also separately expressed in E. coli. The recombinant proteins were purified by affinity chromatography using Ni-NTA agarose. Western blot analysis using anti-His antibody was applied to confirm identity of the purified proteins. BALB/c mice were subcutaneously immunized with CtxB, TcpA, C-CPE and the fusion protein CtxB-TcpA-C-CPE separately. The mice were orally immunized (in 3 boosts) by the same vaccine. Mucosal immune response stimulation was evaluated by measuring the levels of intestinal IgA. Systemic immune response was evaluated by measuring total serum IgG, IgG1, IgG2a, IgG2b subclasses, and also IL-4, IL-5, IL-10 and IFN-γ cytokines in spleen cell culture. ResultsThe recombinant proteins CtxB, TcpA, C-CPE and the fusion protein CtxB-TcpA-C-CPE were expressed in E. coli and highly purified in a single step of chromatography. BALB/c mice immunized with the fusion protein had highest levels of intestinal IgA, serum IgG and IgG subclasses, compared to each of the three proteins making the fusion. Moreover, stimulated splenocytes of mice immunized with the fusion protein displayed significantly higher amounts of IL-5 and IFN-ɣ cytokines. Th2 dominance of the immune response was more evident in mice receiving the fusion protein. ConclusionInclusion of CtxB, as the mucosal adjuvant, and C-CPE, as the vaccine delivery system, in the fusion protein CtxB-TcpA-C-CPE significantly enhanced the elicited mucosal and systemic immune responses, compared to TcpA alone. Of note, significant production of intestinal IgA in mice immunized with the fusion protein is presumably capable of neutralizing TcpA, CtxB and C-CPE antigens, preventing V. cholera colonization, and toxic function of CtxB and C-CPE. Challenge infection of the immunized mice is required to evaluate protective potential of the fusion protein against V. cholera.

AN OVERVIEW OF CLINICAL TRIALS ON VACCINE OF COVID-19 UNDER NOVEL DRUG DELIVERY SYSTEM

The Corona virus disease 2019 (COVID-19) has discovered its underlying foundations from Wuhan (China). Coronavirus is brought about by a novel COVID SARS-CoV2, recently named as 2019-nCoV. Coronavirus has spread across the globe and pronounced as pandemic by World Health Organization (WHO) 0n the 11th March, 2020 as of now, there is no standard medication or vaccine accessible for the treatment, so repurposing of existing medication is the solitary arrangement. vaccine are the arrangements given to patients to summon invulnerable reactions prompting the creation of antibodies (humoral) or cell-interceded reactions that will battle irresistible specialists or non-irresistible conditions, for example, malignancies. Disturbing safety profile of live vaccines, weak immunogenicity of sub-unit vaccines and immunization, failure due to poor patient compliance to booster doses which should potentiate prime doses are rarestoutexplanations, which required the progress of novelgroup of preventive and therapeutic vaccines to encourage effective vaccination. Endeavors are being made to convey immunizations through transporters as they control the spatial and worldly introduction of antigens to invulnerable framework consequently prompting their supported delivery and focusing on. Henceforth, lower dosages of frail immunogens can be successfully coordinated to invigorate safe reactions and kill the requirement for the organization of prime and supporter portions as a section vaccine regimen. Novel Drug Delivery System (NDDS) will be soon for resting of medications. The part of different NDDS in resting of existing medications for treatment of different viral disease, and their importance in COVID-19 has talked about in this paper. It focuses on the currently ongoing research in the implementation and clinical trials on vaccine in the usage of NDDS in COVID-19. In addition, it portrays the part of Liposomes, Virosomes and DNA based vaccine drug delivery advancement for COVID-19

Hemagglutinin Functionalized Liposomal Vaccines Enhance Germinal Center and Follicular Helper T Cell Immunity.

Despite remarkable successes of immunization in protecting public health, safe and effective vaccines against a number of life‐threatening pathogens such as HIV, ebola, influenza, and SARS‐CoV‐2 remain urgently needed. Subunit vaccines can avoid potential toxicity associated with traditional whole virion‐inactivated and live‐attenuated vaccines; however, the immunogenicity of subunit vaccines is often poor. A facile method is here reported to produce lipid nanoparticle subunit vaccines that exhibit high immunogenicity and elicit protection against influenza virus. Influenza hemagglutinin (HA) immunogens are functionalized on the surface of liposomes via stable metal chelation chemistry, using a scalable advanced microfluidic mixing technology (NanoAssemblr). Immunization of mice with HA‐liposomes elicits increased serum antibody titers and superior protection against highly pathogenic virus challenge compared with free HA protein. HA‐liposomal vaccines display enhanced antigen deposition into germinal centers within the draining lymph nodes, driving increased HA‐specific B cell, and follicular helper T cell responses. This work provides mechanistic insights into highly protective HA‐liposome vaccines and informs the rational design and rapid production of next generation nanoparticle subunit vaccines.

Immunogenicity of Subunit Vaccine of E2 Protein Against Atypical Porcine Pestivirus in Pigs

An atypical porcine pestivirus (APPV) causes congenital tremor (CT) type A-II in piglets and has caused considerable financial losses to the pig industry worldwide. This study aimed to develop an effective vaccine candidate against APPV. In this study, we constructed a prokaryotic expressing APPV E2 protein by Escherichia coli and evaluated the immunogenicity of the APPV E2 subunit vaccine with three different adjuvants in piglets by E2-enzyme-linked immunosorbent assay antibody and the protection against APPV challenge in pigs. E2 protein was successfully expressed by the Escherichia coli prokaryotic expression system firstly, and was confirmed by western blot assay. The E2-enzyme-linked immunosorbent assay antibody detection method was established. Vaccined piglets mounted virus-specific IgG responses. The E2 protein emulsified with ISA 201VG and Gel 02 adjuvant induced significantly higher levels of antibodies than those emulsified with IMS 1313VG adjuvant. The vaccine with ISA 201VG adjuvant had highest antibody levels and can protect against APPV infection. The results demonstrated that Escherichia coli-expressed E2 is mounting an immune response in pigs, E2 subunit vaccine maybe a potential vaccine candidate for the prevention of APPV infection, ISA 201VG and Gel 02 adjuvant can be used as candidate adjuvants for vaccines. This study provides a basis for the further research of APPV vaccine.

Mimicking immune signatures of flavivirus infection with targeted adjuvants improves dengue subunit vaccine immunogenicity

Neutralizing antibodies (nAbs) are a critical component for protection against dengue virus (DENV) infection, but little is known about the immune mechanisms governing their induction and whether such mechanisms can be harnessed for vaccine development. In this study, we profiled the early immune responses to flaviviruses in human peripheral blood mononuclear cells and screened a panel of toll-like receptor (TLR) agonists that stimulate the same immune signatures. Monocyte/macrophage-driven inflammatory responses and interferon responses were characteristics of flavivirus infection and associated with induction of nAbs in humans immunized with the yellow fever vaccine YF-17D. The signatures were best reproduced by the combination of TLR agonists Pam3CSK4 and PolyI:C (PP). Immunization of both mice and macaques with a poorly immunogenic recombinant DENV-2 envelope domain III (EDIII) induced more consistent nAb and CD4+ T-cell responses with PP compared to alum plus monophosphoryl lipid A. Induction of nAbs by PP required interferon-mediated signals in macrophages in mice. However, EDIII + PP vaccination only provided partial protection against viral challenge. These results provide insights into mechanisms underlying nAb induction and a basis for further improving antigen/adjuvant combinations for dengue vaccine development.

Formulation and Optimization of a New Cationic Lipid-Modified PLGA Nanoparticle as Delivery System for Mycobacterium tuberculosis HspX/EsxS Fusion Protein: An Experimental Design.

Polymeric particles and liposomes are efficient tools to overcome the low immunogenicity of subunit vaccines. The aim of the present study was formulation and optimization of a new cationic lipid-modified PLGA nanoparticles (NPs) as a delivery system for Mycobacterium tuberculosis HspX/EsxS fusion protein. The cationic lipid-modified PLGA NPs containing HspX/EsxS fusion protein were prepared using a modified double emulsion solvent evaporation method. Scanning electron microscopy and dynamic light scattering (DLS) tools were used to determine physical properties of hybrid NPs. A multi-level full factorial design was used to evaluate the influence of two factors of PLGA:DDA weight ratio (w/w) and PVA concentration (%) on size, surface charge, polydispersity index, encapsulation efficiency and yield. Finally, the optimal formulation was achieved based on desired responses. Mathematical models were obtained to indicate the relation between the studied factors and responses. The DDA concentration showed an increasing effect on surface charge and also a decreasing effect on particle size, encapsulation efficiency and yield. Higher amounts of DDA increased surface charge of NPs; however, the size, encapsulation efficiency and yield were decreased. The influence of various concentrations of PVA on different physical characteristics of PLGA:DDA hybrid NPs was variable. The optimal formulation consisted of 0.91 (55:5, w/w) weight ratio of PLGA:DDA and 0.5% PVA. The hybrid NPs showed acceptable particle size distribution, strong positive surface charge, prolonged antigen release and good encapsulation efficiency in comparison to PLGA alone. However, further preclinical and clinical studies are needed.

Gold nanoparticles conjugating recombinant influenza hemagglutinin trimers and flagellin enhanced mucosal cellular immunity

The immunogenicity of subunit vaccines can be augmented by formulating them into nanoparticles. We conjugated recombinant trimetric influenza A/Aichi/2/68(H3N2) hemagglutinin (HA) onto functionalized gold nanoparticle (AuNP) surfaces in a repetitive, oriented configuration. To further improve the immunogenicity, we generated Toll-like receptor 5 (TLR5) agonist flagellin (FliC)-coupled AuNPs as particulate adjuvants. Intranasal immunizations with an AuNP-HA and AuNP-FliC particle mixture elicited strong mucosal and systemic immune responses that protected hosts against lethal influenza challenges. Compared with the AuNP-HA alone group, the addition of AuNP-FliC improved mucosal B cell responses as characterized by elevated influenza specific IgA and IgG levels in nasal, tracheal, and lung washes. AuNP-HA/AuNP-FliC also stimulated antigen-specific interferon-γ (IFN-γ)-secreting CD4+ cell proliferation and induced strong effector CD8+ T cell activation. Our results indicate that intranasal co-delivery of antigen and adjuvant-displaying AuNPs enhanced vaccine efficacy by inducing potent cellular immune responses.

Poloxamer 407-chitosan grafted thermoresponsive hydrogels achieve synchronous and sustained release of antigen and adjuvant from single-shot vaccines.

Sustained-release vaccine delivery systems may enhance the immunogenicity of subunit vaccines and reduce the need for multiple vaccinations. The aim of this study was to develop a thermoresponsive hydrogel using poloxamer 407-chitosan (CP) grafted copolymer as a delivery system for single-shot sustained-release vaccines. The CP copolymer was synthesized using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide chemistry. The CP copolymer was a free flowing solution at ambient temperature and transformed rapidly into a gel at body temperature. The hydrogels were loaded with vaccine antigen and adjuvants or the vaccine components were encapsulated in poly (lactic-co-glycolic acid) nanoparticles in order to ensure synchronous release. The CP hydrogels were stable for up to 18 days in vitro. Release of both nanoparticles and the individual components was complete, with release of the individual components being modulated by incorporation into nanoparticles. In vivo, a single dose of CP hydrogel vaccine induced strong, long lasting, cellular and humoral responses that could protect against the development of tumors in a murine melanoma model.

Potent anti-viral vaccine adjuvant based on pH-degradable nanogels with covalently linked small molecule imidazoquinoline TLR7/8 agonist

Improving the immunogenicity of subunit vaccines, in particular skewing of the immune response towards Th1 type immunity, is crucial for the development of effective vaccines against intracellular infections and for the development of anti-cancer vaccines. Small molecule TLR7/8 agonist hold high potential for this purpose, but suffer from an undesirable pharmacokinetic profile, resulting in systemic inflammatory responses. An effective solution to this problem is covalent ligation to a larger carrier. Here, a degradable nanogel carrier containing a covalently linked imidazoquinoline (IMDQ) TLR7/8 agonist is explored as adjuvant for vaccination against the respiratory syncytial virus (RSV). In vitro and in vivo experiments in mice provide a solid rational base for preferring nanogels over soluble polymers as IMDQ carrier in terms of cellular uptake and lymph node accumulation.

Cross-Linked Peptide Nanoclusters for Delivery of Oncofetal Antigen as a Cancer Vaccine.

Peptide subunit vaccines are desirable because they increase control over the immune response and safety of the vaccine by reducing the risk of off-target responses to molecules other than the target antigen. The immunogenicity of most peptides, however, is low. Peptide nanoclusters (PNC) are proposed as a subunit peptide vaccine delivery system made completely of cross-linked peptide antigen that could improve the immunogenicity of a peptide vaccine. Proof of concept is demonstrated with oncofetal antigen (OFA), an immature laminin receptor protein expressed by many hematologic cancer cells but not by healthy cells. Peptide epitopes from this protein, called OFA 1, 2, and 3, were synthesized into PNC as a potential cancer peptide vaccine delivery system. PNC were formed by desolvation and stabilized with disulfide bonds using a trithiol cross-linker. Cysteines were added to the C-terminus of each peptide to assist in this cross-linking step, denoted OFA 1C, 2C, and 3C PNC. OFA 2C was found to form the smallest PNC, 148 ± 15 nm in diameter and stable in solution. This size is in the range where particles are readily internalized by dendritic cells (DCs) and may also passively diffuse to regional lymph nodes. OFA 2C PNC and soluble OFA 2C were internalized similarly by DCs in vitro, but only PNC resulted in significant peptide presentation by DCs. This indicates the potential for PNC to improve immune activation against this antigen. Additionally, PNC displayed higher retention at the intradermal injection site in vivo than soluble peptide, allowing more time to interact with DCs in an area of increased DC activity. While offering traditional nanoparticle benefits such as increased DC recognition, slower diffusion, and potential for multivalent cellular interactions, PNC also maximize antigen delivered per particle while minimizing off-target material delivery because the antigens are the main building blocks of the particle. With these properties, PNC are a delivery system with potential to increase peptide subunit vaccine immunogenicity for OFA and other peptide antigens.

Evaluation of lumazine synthase from Bacillus anthracis as a presentation platform for polyvalent antigen display.

Polyvalent antigen display is an effective strategy to enhance the immunogenicity of subunit vaccines by clustering them in an array-like manner on a scaffold system. This strategy results in a higher local density of antigens, increased high avidity interactions with B cells and other antigen presenting cells, and therefore a more effective presentation of vaccine antigens. In this study, we used lumazine synthase (LS), an icosahedral symmetry capsid derived from Bacillus anthracis, as a scaffold to present 60 copies of a linear B cell epitope (PB10) from the ricin toxin fused to the C terminus of LS via four different linkers. We then investigated the effects of linker length, linker rigidity and formaldehyde crosslinking on the protein assembly, conformational integrity, thermal stability, in vitro antibody binding, and immunogenicity in mice. Fusion of the PB10 peptide onto LS, with varying linker lengths, did not affect protein assembly, thermal stability or exposure of the epitope, but had a minor impact on protein conformation. Formaldehyde crosslinking considerably improved protein thermal stability with only minor impact on protein conformation. All LS_PB10 constructs, when administered to mice by injection without adjuvant, elicited measurable anti-ricin serum IgG titers, although the titers were not sufficient to confer protection against a 10× lethal dose ricin challenge. This work sheds light on the biophysical properties, immunogenicity and potential feasibility of LS from B. anthracis as a scaffold system for polyvalent antigen display.