Lipid-Based Poly(I:C) Adjuvants Strongly Enhance the Immunogenicity of SARS-CoV-2 Receptor-Binding Domain Vaccine : Infectious Diseases & Immunity

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Original Article

Lipid-Based Poly(I:C) Adjuvants Strongly Enhance the Immunogenicity of SARS-CoV-2 Receptor-Binding Domain Vaccine

Wu, Yixin1; Meng, Liuxian1; Zhang, Huicong2; Hu, Shun3; Li, Fusheng2,4,∗; Yu, Yingjie5,∗

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Infectious Diseases & Immunity 3(1):p 3-12, January 2023. | DOI: 10.1097/ID9.0000000000000074


1. Introduction

The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has greatly threatened public health. Considering the highly contagious nature of SARS-CoV-2, vaccines were the most appropriate choice to combat this pandemic. Subunit and recombinant protein vaccines are considered safer than live attenuated or whole-killed pathogen vaccines because they do not have the risk of reverting to a virulent form. They are also easy to produce and characterize.[1,2] Recent studies have revealed that the spike receptor-binding domain (RBD) protein of SARS-CoV-2 is a potent target for the development of vaccines that interact with the host angiotensin-converting enzyme 2 (ACE2) and initiate membrane fusion mediated by the S2 subunit.[1,3–6] However, adjuvants are usually required to strengthen the immunogenicity of the recombinant antigens.

Synthetic double-stranded (ds) RNA, such as polyriboinosinic acid–polyribocytidylic acid [poly(I:C)], a synthetic analog of viral dsRNA, is a promising immunostimulant candidate for vaccines directed against intracellular pathogens.[7,8] Poly(I:C) is a ligand of the toll-like receptor 3 (TLR3), a transmembrane protein embedded in the membranes of endosomal compartments of most antigen-presenting cells.[9,10] Notably, the length of dsRNA plays a critical role in the differential recognition by RIG-I and MDA5.[11] In this study, poly(I:C) with an average size of 1.5–8 kb was used for MDA-5-dependent poly(I:C) recognition and type I interferon production, which are crucial for the maturation of dendritic cells (DCs), activation of natural killer cells, and induction of CD4+ T-cell immunity.[12] Furthermore, an appropriate delivery vehicle is required to deliver poly(I:C) and avoid the potential pathogenesis of autoimmunity and chronic inflammatory diseases caused by free poly(I:C).[13]

Recent advances in material science and nanotechnology have provided many promising nonviral delivery systems, such as cationic lipids,[14,15] cationic polymers,[16] and cell-penetrating peptides, that protect nucleic acids from serum nucleases.[17,18] In this study, poly-l-lysine (PLL) was used to load poly(I:C), as a poly(I:C)-PLL polyplex, into cationic lipid nanoparticles. We hypothesized that this combination would provide maximal protection for poly (I:C) and exhibit high immunostimulatory effects. Cationic lipid nanoparticles have been widely used as vaccine delivery systems. Over the past few decades, it has become clear that cationic liposomes, such as 1,2-dioleoyl-3-trimethylamonium-propane (DOTAP),[14,19]N-[1-(2,3-dioleyloxy)proply]-n,n,n-trimethylammonium,[20,21] and 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol,[14] are the most effective lipid-based delivery systems for vaccine antigens and adjuvants. Among these, DOTAP is considered an ideal cationic lipid because it can directly interact with cells via electrostatic interactions, and it can be easily taken up by DCs, subsequently allowing DCs to present antigens and enhance cellular immune responses.[19,22]

Adjuvants are widely believed to play an important role in protein-based subunit vaccines. Different types of adjuvants elicit different immune responses.[23] In this study, we compared cationic DOTAP-based adjuvants with different formulations that induce immune responses, using RBD recombinant protein as a model antigen. Cholesterol was added to enhance the stability of liposomes, resulting in a two-component lipid-based adjuvant (group II). In addition to cholesterol, either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), acting as structural lipids, was also added to the liposomes to produce three-component lipid-based adjuvants (group III-DOPE and group III-DSPC, respectively).[24] 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) was further incorporated into the formulation to produce four-component lipid-based adjuvants (group IV-DOPE or IV-DSPC), which could prevent particle aggregation by providing a hydrophilic steric barrier on the liposomal surface.[25]

In this study, we compared the SARS-CoV-2-specific humoral and cellular responses elicited by these adjuvants. Poly(I:C)-PLL loaded into the group IV-DOPE liposomes was the most potent adjuvant, as further demonstrated in the animal challenge study [Figure 1].

Figure 1:
Schematic illustration showing the preparation of lipopolyplex and possible mechanism involved in humoral and cellular immune response. ① Formation of lipopolyplex by microfluidic technology. The lipopolyplex was injected into mice to elicit ②cellular immune and ③ humoral immune response. ④ The lipopolyplex was also verified by animal challenge experiment. B: B cells; DC: dendritic cell; DMG-PEG: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol; DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOTAP:1,2-dioleoyl-3-trimethylamonium-propane; IFN-γ: interferon γ; poly(I:C): polyriboinosinic acid–polyribocytidylic acid; RBD: receptor-binding domain; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; T: T cells.

2. Materials and methods

2.1. Ethical approval

All animal studies were performed at Shanghai Model Organisms Co., Ltd, in strict accordance with the guidelines set by the Chinese Regulations of Laboratory Animals and Laboratory Animal-Requirements of Environment and Housing Facilities. All animal procedures were reviewed and approved by Shanghai Model Organisms Co., Ltd, Institutional Animal Care and Use Committee, China (Assurance Number: 2020-0044).

2.2. Recombinant RBD protein expression and purification

Recombinant RBD protein was chosen to test the adjuvant effects of all the polyplex and lipopolyplex. Codon-optimized genes encoding residues 1–13, followed by 331–524, of SARS-CoV-2 spike protein were fused with Fc of human immunoglobulin G (IgG; TSINGKE Biological Technology, Nanjing, China). The fusion protein with Fc tag was expressed in HEK 293 cell culture supernatant and purified by protein G affinity chromatography (AKTA purifier, GE Healthcare, Piscataway, NJ, USA). The captured protein was incubated with Enterokinase for 21 hours in 1 mM CaCl2 (Sinopharm, Beijing, China) pH 8.0 at room temperature. After cleavage, the resultant protein was captured by protein G affinity chromatography. Protein purity was verified by SDS-PAGE.

2.3. Preparation and characterization of polyplex, poly(I:C)-PLL

TLR 3 agonist poly(I:C) with average size of 1.5–8 kb is chosen for the study. The molecular weight of PLL·HBr (Solarbio, Beijing, China) used to prepare poly(I:C)-PLL is ranging from 30,000 to 70,000 Da. Preparation of polyplex is based on protocol described previously.[26] In brief, poly(I:C)-PLL was prepared in physiological water (InvivoGen, France) by adding the PLL to poly(I:C) (InvivoGen, France) dropwise in a molar ratio of 0.5:1. The process was under magnetic stirring in a sterile environment. After continuous stirring for 24 hours at room temperature, small amount of precipitate can be solubilized. Residual undissolved material, if there any, was removed by centrifugation with 1000 rpm for 3 minutes. The concentration of poly(I:C) was measured by ultraviolet absorption.

2.4. Preparation and characterization of lipopolyplexes

Different groups of lipid-based adjuvant were prepared with the NanoAssemblr Ignite (Precision NanoSystems, Inc, Canada). Lipopolyplexes containing different lipid components as follow: group II: cholesterol and DOTAP with molar ratio of 1:1; group III: DOTAP, cholesterol, and DSPC or DOPE with molar ratio of 5:4:1; and group IV: DOTAP, DSPC or DOPE, cholesterol, and DMG-PEG with molar ratio of 50:10:38.5:1.5. The lipid components were dissolved in ethanol and the poly(I:C)-PLL in physiological water, both of which were injected into the microfluidic mixer at a 1:3 volume with overall flow rate at 10 mL/min (7.5 mL/min aqueous, 2.5 mL/min ethanol). Residual ethanol in the final mixture was then removed by dialysis. Preparation of all lipid-based adjuvants was performed in a sterile environment at room temperature. Dynamic light-scattering (DLS) and transmission electron microscopy (TEM) were used to characterize the lipopolyplexes. All of the lipopolyplex adjuvants were kept at 4°C for 1 week, and the size and polymer dispersity index (PDI) of each group were measured by DLS.

2.5. Animal vaccination and sample collection

Six- to 8-week-old female BALB/c mice were divided into nine groups randomly (n = 6 for each group). Before immunization, the recombinant RBD protein (in 100 μL of physiological water) was mixed with Alum adjuvant, poly(I:C)-PLL, or different groups of lipopolyplexes individually. Then, BALB/c mice were intramuscularly vaccinated with 1 μg/g mouse weight of recombinant RBD protein (in 100 μL of physiological water) in the presence of equal volume of adjuvants from each group in a mass ratio of 90:3:1 (DOTAP:poly(I:C):RBD). Mice were boosted with the same immunogen and respective adjuvants after 2 weeks. Phosphate buffer saline (PBS) group was included as the controls. Serums were collected at different time point as scheduled [Figure 1] to assess SARS-CoV-2 RBD-specific antibody responses. Mice were sacrificed at 14 days after the second immunization. Splenocytes and pneumonocytes were collected to detect SARS-CoV-2 RBD-specific T-cell response.

2.6. Evaluation of vaccine-induced humoral responses

An enzyme-linked immunosorbent assay (ELISA) was used to determine murine antibody response induced by recombinant RBD protein with different adjuvants. Briefly, ELISA plate (Costar Assay Plate Half Area, Corning, USA) was precoated with SARS-CoV-2 recombinant RBD protein overnight at 4°C. After three washes with PBS Tween 20, serial dilution of mouse sera (from 1:100 to 1:204,800) were added to plate and incubated at 37°C for 1 hour. To develop the reaction, the plates were washed 5 times and incubated with horseradish peroxidase-conjugated secondary antibody (antimouse IgG, IgG1, or IgG2a; Invitrogen, USA) for 1 hour at 37°C and washed 5 times. The reaction was visualized by TMB Single-Component Substrate solution (Solarbio, Beijing, China) and stopped with 2 N HCl. Absorbance at 450 nm (A450) was measured on ELISA plate reader (BioTek ELX800, Gene Company Limited, USA).

2.7. In vitro neutralization assay

Vero cells were plated in 96-well plates (2 × 105 cells/well) and incubated overnight. Serial dilutions of serum were incubated with 650 TCID50 (50% tissue culture infective doses) of the pseudovirus of SARS-CoV-2 for 1 hour at room temperature before transfer to Vero cells. After 72 hours of incubation, the supernatant was removed, and luciferase substrate was added. Two minutes later, luciferase activity was measured and neutralization titer was defined as the serum dilution at which the relative light units were reduced by 50% compared with the virus control wells.

2.8. Evaluation of cellular immune responses

An enzyme-linked immune absorbent spot (ELISpot) assay was used to evaluate cellular immune response elicited by recombinant RBD protein with different adjuvants we developed. Briefly, at 14 days after the second immunization, spleens and lungs from immunized mice were harvested, grinded and filtered through 40 μm cell strainers. Splenocytes or pneumonocytes were collected, and then, the lymphocytes were separated by the mouse organ tissue lymphocyte separation kit (Solarbio, Beijing, China). All the lymphocytes were tested by interferon γ (IFN-γ), interleukin 2 (IL-2), and IL-4 ELISpot Kit (MabTech, Sweden). An HIV peptide was used as negative controls.

2.9. Animal challenge experiments

SARS-CoV-2 clinical isolate nCoV-SH01 (GenBank: MT121215.1) was expanded in VeroE6 cells and virus titers were expressed as plaque forming units (PFUs) per milliliter. All the infection experiments were performed in the biosafety level 3 (BSL-3) laboratory of Fudan University. Briefly, male humanized ACE2-IRES-luc transgenic mice (C57BL/6) (Shanghai Model Organisms Center, Inc, China, Cat# NM-HU200218) were divided into two groups randomly (n = 6). The mice in vaccine group were immunized with recombinant RBD protein (1 μg/g mouse weight) plus lipopolyplex IV-DOPE by intramuscular injection preinfection. Selection of this regimen is based on previous experiments. Mice in MOCK group were treated with PBS as control. At 42 days after the first immunization, mice were inoculated via an intranasal route with 4.15 × 104 PFUs of SARS-CoV-2 (nCoV-SH01). Clinical symptoms and body weight were recorded once a day for 5 days.

2.10. Viral RNA determination and histopathological examination

On the fourth-day postinfection, all mice were killed to collect lungs for viral RNA determination and histopathological examination. Specifically, after dissection, the lungs were collected, and the surface blood was washed away in PBS. Then, the tissues were immediately placed in a pathological specimen bottle containing 4% PFA for fixation. The specimen bottle was sealed, sterilized the outer surface with 75% alcohol, and placed it in a 4°C refrigerator for fixation. The remaining lung tissue samples were placed in a tube containing 1 mL of TRIzol lysis solution. The tissue was homogenized into a cell state, placed in −80°C for 24 hours, and then taken out to a BSL-2 laboratory to extract tissue RNA according to the instructions. Real-time quantitative polymerase chain reaction was performed and the primers that target SARS-COV-2 N gene spanning nt 608-706 are as follows: forward primer, 5′-GGGGAACTTCTCCTGCTAGAAT-3′; reverse primer, 5′-CAGACATTTTGCTCTCAAGCTG-3′.

Four percent PFA-fixed tissue was placed in 4°C for 48 hours and then taken out to the BSL-2 laboratory. Mouse lungs were fixed in 4% paraformaldehyde solution (Sigma, Shanghai, China). Tissue paraffin sections (2–4 μm in thickness) were stained with hematoxylin and eosin (H&E). The slices were observed with Olympus microscope.

2.11. Statistical analysis

All statistical analyses were performed using GraphPad Prism 9.0 and SPSS 25.0 statistics software. Statistical significance among different vaccination groups was analyzed by using Mann-Whitney U test, as specified in the figure legends. The values are presented as the means ± SEM unless otherwise noted.

3. Results

3.1. Packaging poly(I:C) with peptides enhanced RBD-induced immune responses

Recombinant RBD protein was obtained through transient transfection of HEK 293 T cells [Figure 2A]. The results showed that the recombinant RBD protein was successfully captured by protein G after cleavage. In this study, PLL was selected to load poly(I:C) as poly(I:C)-PLL for delivery. Dynamic light-scattering results [Figure 2B] suggested that the average size of these polyplexes was 784.22 nm. Next, we evaluated the immunogenicity and efficacy of this polyplex-adjuvanted vaccine. Initially, groups of immunocompetent female BALB/c mice were immunized with RBD protein adjuvanted with poly(I:C)-PLL. Vaccine-induced specific IgG antibody responses were assessed using ELISA. The results showed that the addition of poly(I:C) polyplex improved immune responses to the RBD protein, with antibody titers significantly higher than those of the Alum-adjuvanted group [Figure 2C]. The titers of the different vaccine groups that resulted in neutralization were determined using SARS-CoV-2 pseudovirus and serum collected on day 28 after the initial immunization [Figure 2D]. The results suggested that RBD protein formulated with polyplexes significantly increased the titers of neutralizing antibodies (P < 0.01).

Figure 2:
Characterization of recombinant RBD protein and humoral immune response in RBD-vaccinated mice. (A) SDS-polyacrylamide gel electrophoresis was conducted to demonstrate the recombinant RBD protein. (M: marker; 1: protein captured by protein G, before cleavage; 2: protein captured by protein A, after cleavage; 3: recombinant RBD protein, after purification.) (B) Representative intensity-size graph and zeta potential of poly(I:C)-PLL were measured by dynamic light-scattering method. (C) The enzyme-linked immunosorbent assay was performed to detect the RBD-specific IgG antibody titer. (D) The neutralization ability was determined using SARS-CoV-2 pseudovirus. Neutralizing antibody titers were expressed as the reciprocal of the highest dilutions of specimens that resulted in a complete inhibition of virus-induced CPE in at least 50% of the wells. Data are shown as mean ± SEM of six mice in each group. Significance was calculated using Mann-Whitney U test. *P < 0.05, **P < 0.01. PBS: phosphate buffer saline; PLL: poly-L-lysine; poly(I:C): polyriboinosinic acid–polyribocytidylic acid; RBD: receptor-binding domain.

3.2. Characterization of the different lipopolyplex adjuvants

To further optimize the delivery of the immunostimulant and elicit better immune responses, poly(I:C)-PLL was encapsulated in different DOTAP-based lipid nanoparticles using microfluidic mixing technology [Table 1]. The sizes of the five lipopolyplexes were measured by DLS [Figure 3A and Table S1,]. The results showed that except for group II, which had a relatively higher PDI (PDI = 0.463), the other groups of lipopolyplexes had similar PDIs of approximately 0.2, and group IV-DOPE had the smallest size (124.52 nm). Transmission electron microscopy was used to confirm the formation of nanoparticles. The lipopolyplexes were spherical with a diameter of approximately 200 nm, which was consistent with the DLS results [Figure 3A]. All lipopolyplexes were stored at 4°C for 1 week, and their sizes were monitored using DLS. No significant change in the diameter was observed [Figure 3B]. Before immunization, each lipopolyplex formulation was loaded with RBD protein by mixing the nanoparticles with the RBD protein, and the sizes of the particles were measured using DLS. As shown in Figure 3C, the average particle diameter of lipopolyplex IV-DSPC increased by 13.44 nm, from 222.34 to 235.78 nm, after loading with RBD protein. Furthermore, the surface charge decreased from +10.6 to +4.1 mV because of charge neutralization, suggesting that the RBD protein had attached onto the surface of the lipopolyplexes successfully.

Table 1 - Different components of five lipopolyplexes
Groups Cationic lipida Basic lipidb Structural lipid Hydrophilic lipid
Group II DOTAP Cholesterol None None
aAll five groups contain DOTAP.
bAll five groups contain Cholesterol.
DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine; DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOTAP: 1,2-dioleoyl-3-trimethylamonium-propane; DMG-PEG: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol.

Figure 3:
Characterization of lipopolyplexes. (A) Representative intensity-size graph of IV-DSPC measured by dynamic light-scattering method and size characterization of IV-DSPC measured by transmission electron microscopy. (B) Stability of IV-DSPC lipopolyplex (n = 3). (C) Dynamic light-scattering profiles of IV-DSPC and IV-DSPC fused with RBD recombinant protein. DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine; PDI: polymer dispersity index; RBD: receptor-binding domain.

3.3. Lipid-based adjuvants induced higher IgG antibody responses than those of poly(I:C)-PLL alone

The induction of specific IgG antibody responses by the recombinant RBD protein with different lipid-based adjuvants was evaluated. Mice were immunized with recombinant RBD protein with or without adjuvants. Sera were collected at different time points, according to the vaccination schedule shown in Figure 4A. As shown in Figure S1,, without any adjuvants, a low immune response was observed after the first dose of recombinant RBD protein, whereas a high antibody response was elicited by recombinant RBD with IV-DSPC lipopolyplex. After the second immunization, the level of response increased and high antibody titers were sustained for 28 days. The IV-DOPE lipid nanoparticles were also used to package poly(I:C) without PLL, forming poly(I:C)-LNPs. The results demonstrated that the two-step optimization of the delivery system resulted in an improved immune response after the second immunization (P < 0.01).

Figure 4:
Humoral immune response results of different groups of lipopolyplexes in the BALB/c mice. (A) Schematic diagram of immunization, sample collection, and challenge schedule. (B) The SARS-CoV-2-specific IgG antibody titer determined by the enzyme-linked immunosorbent assay. The titers are expressed as the endpoint dilutions that remain positively detectable. PBS was included as the control. (C) The neutralization ability determined using SARS-CoV-2 pseudovirus. Data are shown as mean ± SEM from six mice in each group. Significance was calculated using Mann-Whitney U test. ns: not significant; *P < 0.05, **P < 0.01. DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine; PBS: phosphate buffer saline; PLL: poly-L-lysine; poly(I:C): polyriboinosinic acid–polyribocytidylic acid; RBD: receptor-binding domain.

Recombinant RBD protein alone has low immunogenicity, and the different adjuvants we developed enhanced its immunogenicity in different ways [Figure 4B]. Among the lipopolyplexes, those containing DOPE (III-DOPE and IV-DOPE) elicited RBD-specific IgG antibody responses, and adjuvants with four components elicited better RBD-specific IgG antibody responses than those of adjuvants containing three components (P < 0.05).

Neutralizing antibody titers resulting from the different vaccine adjuvants were also evaluated using the SARS-CoV-2 pseudovirus and sera collected 28 days after the initial immunization [Figure 4C]. The results suggest that the RBD protein formulated with each lipopolyplex could significantly increase the neutralizing titer. The IC50 and IC90 titers shown in Table S2,, indicate that the IV-DOPE lipopolyplex had the best neutralizing ability, with IC50 titers of 1/117,490. These results demonstrated that lipopolyplex adjuvants, especially IV-DOPE, could greatly strengthen specific antibody responses to recombinant proteins.

3.4. Group III and IV lipopolyplex adjuvants induce high T helper 2–biased antibody responses in immunized mice

We evaluated IgG subtypes elicited by the recombinant RBD protein with or without adjuvants using sera collected 14 days after the second immunization. Without any adjuvant, the recombinant RBD protein induced SARS-CoV-2 RBD-specific IgG1 and IgG2a antibodies with a T helper 2 (TH2)–biased (IgG1) response. The addition of any adjuvants regulated IgG subtype responses [Figure 5]. We observed that adjuvants with three or four components significantly strengthened both IgG1 and IgG2a subtypes [Figure 5A and 5B, P < 0.01]. We compared DOPE with DSPC and found that DOPE was more likely to induce a higher IgG1 response than that of DSPC, while DSPC performed better in strengthening the IgG2a subtype antibody than DOPE [Figure 5C, P < 0.01].

Figure 5:
Titers of serum IgG1 and IgG2a antibodies to COVID-19 RBD protein elicited by different group of vaccine-immunized mice. (A) SARS-CoV-2 RBD-specific IgG1 (TH2) titers and (B) IgG2a (TH1) titers of each vaccination group. The antibody titers were expressed as the endpoint dilutions that remain positively detectable. PBS was included as the control. (C) The ratios between specific IgG1 and IgG2a antibody responses. Data are shown as mean ± SEM from six mice in each group. Significance was calculated using Mann-Whitney U test. ns: not significant; *P < 0.05, **P < 0.01, ***P < 0.001. DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine; PBS: phosphate buffer saline; poly(I:C): polyriboinosinic acid–polyribocytidylic acid; RBD: receptor-binding domain; PLL: poly-L-lysine.

3.5. Lipopolyplex adjuvants induce strong SARS-CoV-2–specific cellular immune responses

It has been reported that in patients who have recovered from SARS, the antibodies in sera disappeared after 1 year,[27] while the T cells persisted for 6–11 years,[28,29] indicating that cell-mediated immunity plays an important role in viral immunity. In this study, the secretion of IFN-γ in splenocytes and pneumonocytes was assessed using an ELISpot assay. As shown in Figure 6, the number of IFN-γ–secreting RBD-specific T cells in the lipopolyplex groups was significantly higher than that in the poly(I:C)-PLL and RBD groups. We noticed that in some groups, more IFN-γ–secreting RBD-specific T cells were present in pneumonocytes than in splenocytes (P < 0.01). Because of the ability to induce cellular immune responses in the lungs, the cationic lipopolyplexes developed in this study are promising adjuvants for vaccines against SARS-CoV-2. Interleukin-2– and IL-4–secreting RBD-specific T cells were also evaluated using ELISpot assays. As shown in Figure 6C and 6D, a significantly higher secretion of IL-2 was observed in some lipopolyplex groups than that in the poly (I:C)-PLL group. In contrast, no significant difference was detected in the secretion of IL-4, a TH2 cytokine. These results demonstrated that our two-step optimization of poly(I:C) delivery could evoke high humoral immunity and cellular immunity, suggesting that lipopolyplexes are an excellent adjuvant for RBD proteins.

Figure 6:
Cellular immune response results of different groups of lipopolyplexes. (A) The number of IFN-γ spot counts per 106 splenocytes. (B) The number of IFN-γ spot counts per 106 pneumonocytes. (C) The number of IL-4 spot counts per 106 splenocytes. (D) The number of IL-2 spot counts per 106 splenocytes. Data are shown as mean ± SEM from six mice in each group. Significance was calculated using Mann-Whitney U test. ns: not significant; *P < 0.05, **P < 0.01. DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine; PBS: phosphate buffer saline; PLL: poly-L-lysine; poly(I:C): polyriboinosinic acid–polyribocytidylic acid; RBD: receptor-binding domain.

3.6. Recombinant RBD protein adjuvanted with IV-DOPE lipopolyplex protects mice from COVID-19

Animal challenge experiments were conducted to verify the protective immunity of the IV-DOPE-adjuvanted SARS-CoV-2 recombinant RBD protein vaccine candidate. Male humanized ACE2-IRES-luc mice were inoculated intranasally with 4.15 × 104 PFUs of SARS-CoV-2. The mice in the MOCK group were treated with PBS, and the mice in the vaccine group were immunized with recombinant RBD protein (1 μg/g mouse weight) mixed with IV-DOPE adjuvant, the most effective adjuvant selected from the prior experiments. The clinical symptoms of the mice were observed and recorded daily. During the first 2 days, both groups of mice were vivacious, and no obvious differences were observed. However, on the fourth day, the mice in the MOCK group had difficulty breathing and were all in a moribund and inactive state, resting in curled up positions; their coats were dull, and they had lower body temperatures than those of the vaccinated mice. In contrast, the mice in the vaccine group maintained good conditions before euthanasia. On the fourth day (4 days after infection), the average body weights of the mice were reduced by 18.56% (24.164 ± 0.665 g vs. 19.678 ± 0.455 g) and 0.06% (24.249 ± 0.683 g vs. 24.235 ± 0.681 g) in the MOCK and vaccine groups, respectively [Figure 7A]. On the fourth day, the lungs of the mice were dissected, and the viral loads in the lungs were determined using real-time quantitative polymerase chain reaction. As shown in Figure 7B, the relative expression of viral RNA in the vaccine group was significantly lower than that in the MOCK group (0.46 ± 0.43 vs. 1.06 ± 0.38, P < 0.05). Furthermore, a histopathological assay was performed to evaluate lung damage caused by SARS-CoV-2 after the animal challenge experiment. As shown in Figure 7C, interstitial inflammatory cell infiltration was observed in the MOCK group, while no obvious damage was detected in the vaccine group, indicating that the IV-DOPE-adjuvanted SARS-CoV-2 recombinant RBD protein efficiently protected the mice from SARS-CoV-2. Therefore, IV-DOPE is considered an exceptional adjuvant for SARS-CoV-2 recombinant RBD protein-based vaccines and has the potential to be further developed into a SARS-CoV-2 recombinant RBD protein-based vaccine.

Figure 7:
Protection of receptor-binding domain formulated with IV-DOPE lipopolyplex against SARS-CoV-2 challenge in the humanized ACE2-IRES-luc transgenic mice. (A) Body weight recorded on day 0 and day 4. (B) Virus replication in ACE2-IRES-luc after infection with SARS-CoV-2 determined by quantitative real-time polymerase chain reaction. (C) Hematoxylin-eosin staining of lung tissues after the challenge experiment. Data are shown as mean ± SEM from six mice in each group. Significance was calculated using Mann-Whitney U test (*P < 0.05, ***P < 0.001). DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.

4. Discussion

Up until May 2022, more than 300 vaccines have been extensively studied worldwide. Recombinant protein vaccines are safer and cheaper than other types of vaccines. However, the immunogenicity of the recombinant proteins is generally weak. Therefore, it is essential to select an excellent adjuvant to achieve optimal immune responses. Here, we applied poly(I:C), a TLR3 agonist, to strengthen the immunogenicity of recombinant proteins, especially for an enhanced TH1 response. To protect poly(I:C) from nucleolytic hydrolysis and elicit better immune responses, we introduced a two-step delivery system. Poly(I:C) was first combined with the cationic polymer PLL, and the resulting poly(I:C)-PLL was then loaded into lipid nanoparticles for further optimization. We immunized mice with the RBD protein, which was encapsulated in either one of the five different formulations of lipopolyplexes that comprised poly(I:C)-PLL, as the polyplex core, and one of the five types of lipid nanoparticles. We compared the resulting RBD-specific antibody titer, neutralization antibody, and T-cell–mediated responses.

Poly-ICLC (Hiltonol, Oncovir, Inc), a combination of poly(I:C) and PLL in carboxymethylcellulose, has been used clinically for its proapoptotic effects. We deduced that a similar delivery strategy for poly(I:C)-PLL may potentially be applied in clinical trials as well. Using the two-step optimization process, the poly(I:C)-PLL polyplexes elicited better immune responses than those of poly(I:C)-LNPs [Figure 4]. The high efficacy could be explained by the fact that the two-step optimization process enabled the lipopolyplex structure to protect poly(I:C) from endosome escape and deliver the RBD to the cytoplasm, consequently activating both TLR3 and MDA-5.

In the second optimization step, we tested the effects of the different liposome components. Our results clearly showed that lipopolyplexes with different components affected RBD-induced immune responses differently. Among the five lipopolyplexes, group II, which contained DOTAP and cholesterol, induced a relatively lower immune response than that of the three- or four-component adjuvants. However, no significant difference was observed between the three- and four-component adjuvants. The adjuvants containing DOPE performed better than those containing DSPC. Furthermore, immunization with the SARS-CoV-2 RBD that was loaded into the group IV-DOPE lipopolyplex led to rapid virus clearance in the mice after SARS-CoV-2 infection.

Some adjuvants may have adverse effects, such as pathological changes at injection sites, significant inflammatory reactions, and experimental colitis due to the activation of innate immunity.[30] In this study, we only observed mild inflammatory reactions at the site of injection on mice vaccinated with the SARS-CoV-2 RBD that was loaded into the group II lipopolyplexes. No obvious adverse effects were observed with other adjuvants. Histological analysis was performed using H&E staining to evaluate pathological changes in important organic tissues. With the exception of group II and group III-DSPC, the different lipopolyplex groups did not cause any obvious inflammatory response or pathological changes in the heart, liver, spleen, lung, and kidney tissues [Figure S2,], indicating the safety and good biocompatibility of the three- and four-component lipid-based adjuvants.

In summary, we have introduced an innovative approach to deliver poly(I:C) as an adjuvant for a recombinant RBD protein vaccine. We constructed five different lipopolyplex groups and selected the IV-DOPE group as the optimal candidate. The use of poly(I:C) delivered by lipid nanoparticles efficiently induced robust humoral and cellular responses. The high neutralizing antibody titers and protection from SARS-CoV-2 infection, which were observed in the animal challenge study, suggest that the IV-DOPE vaccine could be a very promising vaccine candidate. Our lipid-based formulation techniques were similar to those of Moderna’s mRNA-1273 and BioNTech’s BNT162b2. This novel nanovaccine technology has great potential as a future recombinant protein vaccine.

Author Contributions

Yixin Wu conceived the ideas of research, prepared materials, analyzed the data, and wrote the manuscript. Huicong Zhang, Liuxian Meng, and Shun Hu performed the animal surgery. Fusheng Li and Yingjie Yu provided the lab resource and funding, supervised project, revised and edited the manuscript. All authors read and approved the final manuscript.

Conflict of Interest


Data Availability Statement

The data sets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.


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Adjuvants, vaccine; Lipopolyplex; Poly(I:C); RBD protein

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