<em>In Vitro</em> Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

Third Military Medical University of Chinese PLA

The Gram-positive bacterial pathogen methicillin-resistant Staphylococcus aureus (MRSA) can cause infections in the bloodstream, endocardial tissue, respiratory tract, culture-confirmed skin, or soft tissue. There are currently no effective vaccines, and none are expected to become available in the near future. An effective vaccine capable of eliciting both systemic and mucosal immune responses is also urgently needed. Here, we reported a novel oil-in-water nanoemulsion adjuvant vaccine containing an MRSA recombination protein antigen, Cremophor EL-35(®) as a surfactant, and propylene glycol as a co-surfactant. This nanoemulsion vaccine, whose average diameter was 31.34±0.49 nm, demonstrated good protein structure integrity, protein specificity, and good stability at room temperature for 1 year. The intramuscular systemic and nasal mucosal immune responses demonstrated that this nanoemulsion vaccine could improve the specific immune responses of immunoglobulin (Ig)G and related subclasses, such as IgG1, IgG2a, and IgG2b, as well as IgA, in the serum after Balb/c mice intramuscular immunization and C57 mice nasal immunization. Furthermore, this nanoemulsion vaccine also markedly enhanced the interferon-γ and interleukin-17A cytokine cell immune response, improved the survival ratio, and reduced bacterial colonization. Taken together, our results show that this novel nanoemulsion vaccine has great potential and is a robust generator of an effective intramuscular systemic and nasal mucosal immune response without the need for an additional adjuvant. Thus, the present study serves as a sound scientific foundation for future strategies in the development of this novel nanoemulsion adjuvant vaccine to enhance both the intramuscular systemic and nasal mucosal immune responses.

Induction of systemic and mucosal immunity against methicillin-resistant Staphylococcus aureus infection by a novel nanoemulsion adjuvant vaccine.

Multi-drug resistant MRSA (methicillin-resistant Staphylococcus aureus) is a global problem for human health, especially skin burn wound patients. Therefore, we estimated the antibacterial and anti-biofilm activity of a chlorhexidine acetate nanoemulsion (CNE) by previously ourselves designed against skin burn wound MRSA infections. Compared with its water solution (CHX), CNE showed a better and faster action against MRSA both in vitro and in vivo. Importantly, CNE was more effective at inhibiting biofilm formation and clearing the biofilm. We also found that the cell walls and membranes of MRSA were severely disrupted after treatment with CNE. Moreover, the relative electrical conductivity and the leakage of alkaline phosphates, K(+), Mg(2+), DNA and protein obviously increased because the cell wall and membrane were damaged. These data show that novel CNE is a promising potential antimicrobial candidate, especially for skin burn wound MRSA infections.

Enhanced efficacy and anti-biofilm activity of novel nanoemulsions against skin burn wound multi-drug resistant MRSA infections.

As an ingredient of vaccines, adjuvants are indispensable for enhancing and directly inducing robust and extensive adaptive immune responses associated with vaccine antigens. In this study, we initially determined that a new molecular immunopotentiator, ophiopogonin D (OP-D), enhanced the antibody response to antigen. Because OP-D has certain disadvantages, including poor solubility, we next encapsulated OP-D in a nanoemulsion adjuvant (nanoemulsion-encapsulated OP-D, NOD) using low-energy emulsification methods. The NOD thus produced was small, with an average size of 76.45 nm, and exhibited good distribution (PdI value 0.16), significantly increasing the solubility of OP-D. Furthermore, NOD exhibited reduced cellular toxicity and acute toxicity. Our results showed that a fusion antigen of MRSA (HlaHIsdB) formulated with NOD significantly improved humoral and cellular immune responses compared to those observed in the antigen/OP-D and antigen/AlPO groups. Compared with antigen/OP-D, the antigen formulated with NOD more effectively promoted antigen uptake by dendritic cells (DCs) and the activation of antigen-presenting cells (APCs). Moreover, the NOD-formulated antigen had ideal protective efficacy in a MRSA sepsis model by inducing more potent antibody responses and a Th1/Th17-biased CD4 T cell immune response. Therefore, these results suggest that NOD is a promising and robust adjuvant platform for a MRSA vaccine.

An immunopotentiator, ophiopogonin D, encapsulated in a nanoemulsion as a robust adjuvant to improve vaccine efficacy.

Methicillin-resistant  (MRSA) is one of the most predominant and fatal pathogens at wound infection sites. MRSA is difficult to treat because of its antibiotic resistance and ability to form biofilms at the wound site.

Chitosan-polyvinyl alcohol nanoscale liquid film-forming system facilitates MRSA-infected wound healing by enhancing antibacterial and antibiofilm properties.

This study aimed to improve the solubility, reduce the side effects and enhance the efficacy of gambogic acid against acute myeloid leukemia in vitro and in vivo. This oil-in-water nanoemulsion (average size 17.20 ± 0.11 nm, zeta potential 4.17 ± 0.82 mV) containing Tween-80, glycol, squalene and gambogic acid with improving 4000 times solubility was prepared by pseudoternary phase diagrams. We found that this nanoemulsion successfully encapsulated gambogic acid; it was stable and showed an obvious delayed release effect for the drug in three different phosphate-buffered saline (pH = 2.0, 5.8 and 7.4). The half inhibiting concentration (IC) of this nanoemulsion (480.7 μg/mL and 408 μg/mL) were 1.67 times and 1.98 times higher than those of its water solution (287 μg/mL and 206 μg/mL) after acting on the toxicity standard cell line (L929 line) for 24 h and 48 h, respectively. Importantly, acute injection toxicity indicated that the half lethal dose (LD) of this nanoemulsion (23.25 mg/kg, 95% LD, 21.7-25.16 mg/kg) was 1.26 times higher than that of its water solution (18.59 mg/kg, 95% LD, 16.84-20.53 mg/kg). Compared with its suspension, the bioavailability of this nanoemulsion was 318.2%. Furthermore, this nanoemulsion had a better efficacy against the acute myeloid leukemia in vitro and in vivo by improving the time and percent of survival (MV4-11 engrafts mice) and reducing half inhibiting concentration values in acute myeloid leukemia such as Jurket, HL-60 and MV4-11 cells. Our studies suggested that this nanoemulsion may be a promising therapeutic medicine for acute myeloid leukemia.

Development of a safety and efficacy nanoemulsion delivery system encapsulated gambogic acid for acute myeloid leukemia in vitro and in vivo.

Helicobacter pylori (H. pylori) infection remains a global public health issue, especially in Asia. Due to the emergence of antibiotic-resistant strains and the complexity of H. pylori infection, conventional vaccination is the best way to control the disease. Our previous study found that the N-acetyl-neuroaminyllactose-binding hemagglutinin protein (HpaA) is an effective protective antigen for vaccination against H. pylori infection, and intranasal immunization with the immunodominant HpaA epitope peptide (HpaA 154-171, P22, MEGVLIPAGFIKVTILEP) in conjunction with a CpG adjuvant decreased bacterial colonization in H. pylori-infected mice. However, to confer more robust and effective protection against H. pylori infection, an optimized delivery system is needed to enhance the P22-specific memory T cell response.

Epitope-loaded nanoemulsion delivery system with ability of extending antigen release elicits potent Th1 response for intranasal vaccine against Helicobacter pylori.

Nanoemulsion adjuvants-based vaccines have potent induced immune responses against methicillin-resistant  (MRSA) infection. However, the efficacies and immune responses of different antigen-attaching ways on self-made nanoemulsion adjuvants remain unknown. In this study, we designed three formulations of nanoemulsion adjuvants (encapsulation, mixture, and combination) to explore their immune response-enhancing effects and their underlying mechanism in a systemic infection model of MRSA. Our results showed that the three nanoemulsion-attachment ways formulated with a fusion antigen of MRSA (HlaIsdB) all improved humoral and cellular immune responses. When compared with the mixture and combination formulations, the nanoemulsion-encapsulation group effectively promoted the antigen uptake of dendritic cells (DCs) , the activation of DC in draining lymph nodes and the delayed release of antigen at injection sites . Moreover, the encapsulation group induced a more ideal protective efficacy in a MRSA sepsis model by inducing more potent antibody responses and a Th1/Th17 biased CD4 T cell response when compared with the other two attachment ways. Our findings suggested that the encapsulated formulation of vaccine with nanoemulsion adjuvant is an effective attachment way to provide protective immunity against MRSA infection.

Yanan Tong

Department of Microbiology and Biochemical Pharmacy,
National Engineering Research Centre of Immunological Products,
College of Pharmacy,
Department of Microbiology and Biochemical Pharmacy, National Engineering Research Centre of Immunological Products, College of Pharmacy

Third Military Medical University of Chinese PLA

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D