A Polysaccharide-RBD-Fc-Conjugated COVID-19 Vaccine, SCTV01A, Showed High Immunogenicity and Low Toxicity in Animal Models.

We previously developed a polysaccharide--RBD-conjugated nanoparticle vaccine which induced protective efficacy against SARS-CoV-2 in a mouse model. Here, we newly developed a vaccine, SCTV01A, by chemically conjugating recombinant SARS-CoV-2 RBD-Fc and PPS14 (Streptococcus pneumoniae serotype type 14 capsular polysaccharide). The immunogenicity and toxicity of SCTV01A were evaluated in animal models. The PPS14 conjugation enhanced the immunogenicity of RBD-Fc in C57BL/6 mice whether formulated with SCT-VA02B or Alum adjuvant. SCTV01A also induced high opsonophagocytic activity (OPA) against S. pneumoniae serotype 14. In addition, SCTV01A stimulated potent neutralizing titers in rhesus macaques and effectively reduced lung inflammation after SARS-CoV-2 infection with neither antibody-dependent enhancement (ADE) nor vaccine-enhanced diseases (VED) phenomenon. Importantly, the long-term toxicity study of SCTV01A in rhesus macaques did not cause any abnormal toxicity and was tolerated at the highest tested dose (120 µg). The existing immunogenicity and toxicological evaluation results have demonstrated the safety and efficacy of SCTV01A, which will be a promising and feasible vaccine to protect against SARS-CoV-2 infection.

Feasibility Studies of Nebulized SARS-CoV-2 Neutralizing Antibody in Mice and Cynomolgus Monkeys.

PURPOSE: Neutralizing antibodies, administrated through intravenous infusion, have shown to be highly efficacious in treating mild and moderate COVID-19 caused by SARS-CoV-2 infection in the lung. However, antibodies do not transport across the plasma-lung barrier efficiently, and up to 100 mg/kg dose was used in human causing significant supply and cost burdens. This study was to explore the feasibility of nebulized antibodies inhalation delivery as an alternative route. METHODS: HB27, a potent RBD-specific humanized monoclonal antibody (Zhu et al. in National Sci Rev. 8:nwaa297, 2020), showed excellent protection against SARS-CoV-2 in animal model and good safety profile in clinical studies. The pharmacokinetics and preliminary safety of HB27 administrated through the respiratory tract were studied in mice and cynomolgus monkeys here. RESULTS: At a single 5 mg/kg dose, the peak HB27 concentration in mice pulmonary epithelial lining fluid (ELF) reached 857.8 µg/mL, 670-fold higher than the PRNT90 value of 1.28 µg/mL, and maintained above PRNT90 over 240 h. In contrast, when administrated by intravenous injection at a 5 mg/kg dose, the antibody concentrations in mice ELF were below PRNT90 value throughout, and were about 50-fold lower than that in the serum. In cynomolgus monkeys administrated with a single dose through inhalation, the antibody concentration in ELF remained high within 3 days. No drug-related safety concerns were observed in the studies. CONCLUSIONS: The study demonstrated that nebulized neutralizing antibody delivery though inhalation could be a more efficient and efficacious alternative approach for treating COVID-19 and other respiratory infectious diseases, and warrants further evaluation in clinical studies.

Immunogenicity correlation in cynomolgus monkeys between Luminex-based total IgG immunoassay and pseudovirion-based neutralization assay for a 14-valent recombinant human papillomavirus vaccine.

A new virus-like particle based vaccine covering 14 types of high-risk and disease-inducing human papillomavirus (HPV) can offer better coverage against HPV-induced diseases, particularly cervical cancers. However, the assessment of immunogenicity of the vaccine is an important task, representing not only its significant clinical characteristics, but also a major challenge, in terms of both the suitability of methods and the clinical sample testing throughput supporting clinical development. This work covers the development and evaluation of a method based on Luminex technology (a coded-bead and flow-cytometric approach) to assess the HPV-type specific total immunoglobulin G (IgG). This method can evaluate the antibodies in sera post immunization against multiple types of HPV simultaneously (i.e., with multiplexing capability), save time and cost, and improve test throughput with higher sensitivity and precision than the classical, plate-based enzyme-linked immunoassay and competitive Luminex-based immunoassays. Using cynomolgus monkeys as model, we demonstrated the good correlation between the results from the pseudovirion-based neutralization assay (PBNA), and the Luminex-based total IgG assay, supporting that the latter method can be considered as a viable, dependable replacement method for the PBNA supporting immunogenicity evaluation of HPV vaccine in preclinical development and clinical investigation.

Potential Hepatitis B Vaccine Formulation Prepared by Uniform-Sized Lipid Hybrid PLA Microparticles with Adsorbed Hepatitis B Surface Antigen.

For the purpose of strengthening the immunogenicity of the hepatitis B vaccine, which contains hepatitis B surface antigen (HBsAg), the development of biodegradable poly(lactic acid) (PLA) microparticles (MPs) modified with the cationic surfactant didodecyldimethylammonium bromide (DDAB) was attempted. DDAB-PLA MPs with an uniform size of about 1 µm were prepared in a simple and mild way. DDAB-PLA MPs with increased surface charge enhanced antigen adsorption capacity compared to plain PLA MPs. After immunization, DDAB-PLA MPs induced the gene expression of inflammatory cytokines and chemokines, which facilitated the following immune responses. DDAB-PLA MPs augmented the expression of co-stimulatory molecules along with the activation of bone-marrow-derived dendritic cells (BMDCs). DDAB-PLA MP-based vaccine formulations efficiently induced antibody production more than the aluminum-based vaccine and plain PLA MP-based formulation in vivo. Moreover, DDAB-PLA MPs were more likely to generate the polarization of the Th1 response indicating the cytotoxic ability against infectious pathogens. In conclusion, DDAB-PLA MPs could be a potent vaccine formulation to prime robust cellular and humoral immune responses.

Fabrication and characterization of DDAB/PLA-alginate composite microcapsules as single-shot vaccine.

The most effective method to reduce chronic hepatitis B virus infection is the universal implementation of vaccination. The commercial aluminum-based vaccines need multiple-injection protocols for complete protection resulting in poor compliance in developing countries. It is necessary to develop single-shot vaccine formulations. In this study, novel antigen-loaded DDAB/PLA (didodecyldimethylammonium bromide/poly(lactic acid)) nanoparticles (NPs)-alginate composite microcapsules were developed as a single-shot vaccine. The hepatitis B surface antigen (HBsAg)-loaded DDAB/PLA NPs were successfully encapsulated into alginate microcapsules by a modified spray-solidification technique. The response surface method was applied to optimize the preparation parameters employing encapsulation efficiency of HBsAg and particle size of microcapsules as response variables. The antigen-loaded DDAB/PLA NPs-alginate composite microcapsules were prepared under these optimal conditions: the size of composite microcapsules was 24.25 µm, the Span value was 1.627, and the encapsulation efficiency of HBsAg was 68.4%. The obtained microcapsules were spherical gel microparticles with excellent dispersity and narrow size distributions. In vitro release profile indicated a slow release rate of encapsulated HBsAg especially in phosphate buffered saline solution. The microcapsules showed little toxicity in vivo. This vaccine delivery system could induce stronger immune responses by a single shot, which exhibited much higher cytokine secretion levels closely related to cellular immunity and comparable IgG titers to the traditional aluminum-adjuvanted vaccine with three shots.

Adjuvanticity Regulation by Biodegradable Polymeric Nano/microparticle Size.

Polymeric nano/microparticles as vaccine adjuvants have been researched in experimental and clinical studies. A more profound understanding of how the physicochemical properties regulate specific immune responses has become a vital requirement. Here we prepared poly(d,l-lactic-co-glycolic acid) (PLGA) nano/microparticles with uniform sizes (500 nm, 900 nm, 2.1 µm, and 4.9 µm), and the size effects on particle uptake, activation of macrophages, and antigen internalization were evaluated. Particle uptake kinetic studies demonstrated that 900 nm particles were the easiest to accumulate in cells. Moreover, they could induce macrophages to secrete NO and IL-1ß and facilitate antigen internalization. Furthermore, 900 nm particles, mixed with antigen, could exhibit superior adjuvanticity in both humoral and cellular immune responses in vivo, including offering the highest antibody protection, promoting the maximum secretion levels of IFN-γ and IL-4 than particles with other sizes. Overall, 900 nm might be the optimum choice for PLGA particle-based vaccine adjuvants especially for recombinant antigens. Understanding the effect of particle size on the adjuvanticity based immune responses might have important enlightenments for rational vaccine design and applications.

Facile fabrication of varisized calcium carbonate microspheres as vaccine adjuvants.

Functional calcium carbonate (CaCO3) particles of micron and submicron sizes used in catalysis and biomedicine have attracted considerable attention for decades. In this paper, the process parameters for CaCO3 crystallization were systematically investigated. Our experimental results demonstrated the significance of temperature during fabrication. Under the optimized conditions, various uniform-sized and spherical CaCO3 microparticles (MPs) with average diameters from 0.8 µm to 5 µm were facilely and rapidly fabricated via different mixing strategies including mechanical stirring, homogenization, and ultrasonication. The physicochemical characteristics of the CaCO3 microspheres were evaluated. And, the hepatitis B surface antigen (HBsAg) used as a model antigen was encapsulated into the particles (1 µm and 4 µm) for investigating the immune responses elicited after vaccination. In vitro, dendritic cells (DCs) were significantly activated by the MP-based vaccine formulations with up-regulated co-stimulatory molecules expression of CD40 and CD83. After immunization, CaCO3 MPs loaded with HBsAg induced greater lymphocyte activation, more cytokine secretion, higher antigen-specific IgG titers and more memory T cell generation to protect against reinfection. Therefore, the CaCO3 MPs, especially the 1 µm particles, could induce strong cellular and humoral immune responses, probably because of easier uptake and more efficient antigen-presentation by DCs. With the advantages of good biocompatibility, high loading capacity and easy preparation, they could be potentially useful as vaccine adjuvants. These results might provide further design principles for potent inorganic particulate adjuvant and delivery systems.

Pathogen-Mimicking Polymeric Nanoparticles based on Dopamine Polymerization as Vaccines Adjuvants Induce Robust Humoral and Cellular Immune Responses.

Aiming to enhance the immunogenicity of subunit vaccines, a novel antigen delivery and adjuvant system based on dopamine polymerization on the surface of poly(D,L-lactic-glycolic-acid) nanoparticles (NPs) with multiple mechanisms of immunity enhancement is developed. The mussel-inspired biomimetic polydopamine (pD) not only serves as a coating to NPs but also functionalizes NP surfaces. The method is facile and mild including simple incubation of the preformed NPs in the weak alkaline dopamine solution, and incorporation of hepatitis B surface antigen and TLR9 agonist unmethylated cytosine-guanine (CpG) motif with the pD surface. The as-constructed NPs possess pathogen-mimicking manners owing to their size, shape, and surface molecular immune-activating properties given by CpG. The biocompatibility and biosafety of these pathogen-mimicking NPs are confirmed using bone marrow-derived dendritic cells. Pathogen-mimicking NPs hold great potential as vaccine delivery and adjuvant system due to their ability to: 1) enhance cytokine secretion and immune cell recruitment at the injection site; 2) significantly activate and maturate dendritic cells; 3) induce stronger humoral and cellular immune responses in vivo. Furthermore, this simple and versatile dopamine polymerization method can be applicable to endow NPs with characteristics to mimic pathogen structure and function, and manipulate NPs for the generation of efficacious vaccine adjuvants.

Immunopotentiator-Loaded Polymeric Microparticles as Robust Adjuvant to Improve Vaccine Efficacy.

PURPOSE: Adjuvants are required to ensure the efficacy of subunit vaccines. Incorporating molecular immunopotentiators within particles could overcome drawbacks of molecular adjuvants (such as solubility and toxicity), and improve adjuvanticity of particles, achieving stronger adjuvant activity. Aim of this study is to evaluate the adjuvanticity of immunopotentiator-loaded polymeric particles for subunit vaccine. METHODS: PLGA microparticles (PMPs) and imiquimod (TLR-7 ligand)-loaded PLGA microparticles (IPMPs) were prepared by SPG premix membrane emulsification. In vitro and in vivo studies were performed to their adjuvant activity, using ovalbumin and H5N1 influenza split vaccine as antigens. RESULTS: Incorporating imiquimod into microparticles significantly improved the efficacy of PLGA microparticles in activating BMDCs and pMΦs, and antigen uptake by pMΦs was also promoted. IPMPs showed stronger adjuvanticity to augment OVA-specific immune responses than PMPs. IgG subclass profiles and cytokine secretion levels by splenocytes indicated that IPMPs elicited more Th1-polarized immune response, compared to PMPs. In vivo study using H5N1 influenza split vaccine as antigen also confirmed the effects of IPMPs on antigen-specific cellular immunity. CONCLUSIONS: Considering adjuvanticity and safety profiles (PLGA and IMQ, both approved by FDA), we conclude that IMQ-loaded PLGA microparticles are promising robust adjuvant for subunit vaccines.

pH-Responsive Poly(D,L-lactic-co-glycolic acid) Nanoparticles with Rapid Antigen Release Behavior Promote Immune Response.

In the quest to treat intracellular infectious diseases and virus infection, nanoparticles (NPs) have been considered to be efficient tools for inducing potent immune responses, specifically cellular immunity. Antigen processing and presenting by antigen presenting cells (APCs) could influence immune response, especially the priming of T-cell-mediated cellular immunity. Here, we fabricated pH-responsive poly(D,L-lactic-co-glycolic acid) (PLGA) NPs with rapid antigen intracellular release behavior in APCs. The NPs, which had thin shells and large inner space, contain ammonium bicarbonate (NH4HCO3), which could regulate release in endosomes and lysosomes, acting as an antigen release promoter in dendritic cells (DCs), and were coencapsulated with antigen (ovalbumin, OVA). Hydrogen ions (H(+)) in DC endosomes and lysosomes (pH ∼5.0 and 6.5) could react with NH4HCO3 to generate NH3 and CO2, which broke NPs and released antigens. After uptake by DCs, antigens encapsulated in pH-responsive PLGA NPs could escape from lysosomes into the cytoplasm and be cross-presented. Moreover, the NPs induced up-regulation of co-stimulatory molecules and stimulated cytokine production. Mouse immunization with pH-responsive PLGA NPs induced greater lymphocyte activation, more antigen-specific CD8(+) T cells, stronger cytotoxic capacity (IFN-γ and granzyme B), enhanced antigen-specific IgG antibodies, and higher serum IgG2a/IgG1, indicating cellular immunity. The NPs also improved generation of memory T cells to protect against reinfection. Thus, pH-responsive PLGA NPs, which induced strong cellular immune responses and offered antibody protection, could be potentially useful as effective vaccine delivery and adjuvant systems for the therapy of intracellular infectious diseases and virus infection.

Polycation-decorated PLA microspheres induce robust immune responses via commonly used parenteral administration routes.

Recombinant viral subunit-based vaccines have gained increasing attention due to their enhanced safety over the classic live-attenuated or inactivated vaccines. The low immunogenicity of the subunit antigen alone, however, requires the addition of an adjuvant to induce immunity. Particulate-based delivery systems have great potential for developing new vaccine adjuvants, compared to traditional aluminum-based saline adjuvants. The physicochemical properties of particulate vaccines have been extensively investigated; however, few studies have focused on how the administration route of various adjuvant-antigen combinations impacts the efficacy of the immune response. Here, for the first time, the viral Hepatitis B surface antigen (HBsAg) was combined with aluminum-based or cationic-microsphere (MP) based adjuvants to investigate the characteristics of immune responses elicited after immunization via the subcutaneous, intramuscular, or intraperitoneal routes respectively. In vitro, the MP-based vaccine significantly increased dendritic cell (DC) activation with up-regulated CD40 and CD80 expression and IL-12 production compared to alum-based vaccine. After immunization, both MP and alum-based vaccines produced increased IgG titers in mice. The administration route of these vaccines did influenced immune responses. The MP-based vaccine delivered via the intramuscular route yielded the highest levels of the IgG2a isotype. The alum-based vaccine, delivered via the same route, produced an IgG1-dominated humoral immune response. Moreover, subcutaneous and intramuscular immunizations with MP-based vaccine augmented Granzyme B, Th1-type cytokines (IL-2, IL-12, and IFN-γ), and Th2 cytokine IL-4 secretions. These results demonstrate that MP-based vaccines have the capacity to induce higher cellular and humoral immune response especially via an intramuscular administration route than an alum-based vaccine.

Immune responses to vaccines involving a combined antigen-nanoparticle mixture and nanoparticle-encapsulated antigen formulation.

Many physicochemical characteristics significantly influence the adjuvant effect of micro/nanoparticles; one critical factor is the kinetics of antigen exposure to the immune system by particle-adjuvanted vaccines. Here, we investigated how various antigen-nanoparticle formulations impacted antigen exposure to the immune system and the resultant antigen-specific immune responses. We formulated antigen with poly(lactic-co-glycolic acid) (PLGA) nanoparticles by encapsulating antigen within nanoparticles or by simply mixing soluble antigen with the nanoparticles. Our results indicated that the combined formulation (composed of antigen encapsulated in nanoparticles and antigen mixed with nanoparticles) induced more powerful antigen-specific immune responses than each single-component formulation. Mice immunized with the combined vaccine formulation displayed enhanced induction of antigen-specific IgG antibodies with high avidity, increased cytokine secretion by splenocytes, and improved generation of memory T cell. Enhanced immune responses elicited by the combined vaccine formulation might be attributed to the antigen-depot effect at the injection site, effective provision of both adequate initial antigen exposure and long-term antigen persistence, and efficient induction of dendritic cell (DC) activation and follicular helper T cell differentiation in draining lymph nodes. Understanding the effect of antigen-nanoparticle formulations on the resultant immune responses might have significant implications for rational vaccine design.

Oral administration of Clostridium butyricum for modulating gastrointestinal microflora in mice.

This study aimed to evaluate the safety of Clostridium butyricum and to investigate the effect of C. butyricum on mice ecosystem in the intestinal tract by way of examining the population of different microorganisms isolated from caecal contents. We firstly evaluated the safety of C. butyricum using acute toxicity test and Ames test. Then forty male BALB/c mice were divided into the following four treatment groups, each consisting of ten mice: normal group, low-dose group, medium-dose group and high-dose group. Caecal contents were removed aseptically, immediately placed into an anaerobic chamber, and dissolved in sterile pre-reduced PBS. The determination of Enterococcus spp., Enterobacter spp., Lactobacillus spp., Bifidobacterium spp. and Clostridium perfringens was analyzed by the spread plate method, cell morphologies and biochemical profiles. The results showed the oral maximum tolerated dose of C. butyricum was more than 10 g/kg body weight in mice and no mutagenicity judged by negative experimental results of Ames test. And in medium- and high-dose groups, the populations of Bifidobacterium spp. and Lactobacillus spp. increased in caecum, as well as the ratios of Bifidobacterium spp. and Lactobacillus spp. to Clostridium perfringens (P < 0.01) as compared with the normal group. This research showed the intake of C. butyricum significantly improved the ecosystem of the intestinal tract in BALB/c mice by increasing the amount of probiotics and reducing the populations of unwanted bacteria.