key: cord-0995788-g05cpzlg
authors: Li, Xinpei; He, Xiaofeng; He, Dongrong; Liu, Yuan; Chen, Kun; Yin, Panchao
title: A polymeric co-assembly of subunit vaccine with polyoxometalates induces enhanced immune responses
date: 2021-12-14
journal: Nano Res
DOI: 10.1007/s12274-021-4004-9
sha: 057e06de87b5271d9854879ea030055ea3ba5974
doc_id: 995788
cord_uid: g05cpzlg

Long-lasting protective immune responses are expected following vaccination. However, most vaccines alone are inability to evoke an efficient protection. The combinatory administration of adjuvants with vaccines is critical for generating the enhanced immune responses. Herein, with biocompatible poly(4-vinylpyridine) (P4VP) as template, 2.5 nm iron/molybdenum oxide cluster, {Mo(72)Fe(30)}, is applied as an adjuvant to co-assemble with antigens of Mycobacterium bovis via hydrogen bonding at molecular scale. Molecular scale integration of the antigens and {Mo(72)Fe(30)} and their full exposure to body fluid media contribute to the augmentation of both humoral and cellular immune responses of the vaccines after inoculation in mice. Anti-inflammatory factor IL-10 gradually increases after 2 weeks followed by a final back to normal level by the 5th week. The balance between proinflammatory cytokines and anti-inflammatory factors suggests that immune system can be activated in the early stage of infection by the antigens carried by the supra-particles and secrete acute inflammatory factors for host defense and antiinflammatory factors for immune protection. [Image: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material (further structural analysis and biological analsyis) is available in the online version of this article at 10.1007/s12274-021-4004-9.

Since the worldwide pneumonia outbreak of coronavirus disease 2019 (COVID- 19) , researchers shoulder the mission of stemming its spread and currently, most of efforts are focused on the development of new detection methods [1] and antiviral drugs, especially, safe and effective vaccines. The COVID-19 pandemic is not a rare exception in human history while the tuberculosis (TB) has afflicted humans since the 18th centuries. Bacillus Calmette Guérin (BCG) vaccine has been proven its protection from COVID-19 and TB since nonspecific immune stimulation is quite useful to organisms to repel most potential pathogens before they cause detectable signs and symptoms of disease [2] [3] [4] . It is wellknown that live-attenuated BCG vaccine (from Mycobacterium bovis (M. bovis)) is effective in protecting young children from severe TB disease; however, it is less efficacious for adults [3] . In essence, systematically administered vaccines like BCG fail to generate adequate protective responses at mucosal surfaces [5] . Nowadays, complex nanoparticles (NPs) carrying antigens are of particular interests for vaccine development because of their adjuvant effects on the modulation of immune response [6] [7] [8] [9] [10] . NPs have been extensively explored to cope with the instability and toxicity frequently interrelated with the administration of soluble molecules [11] . Moreover, the conjunction of antigens to the surfaces of NPs is favorable to B-cell activation since comparatively higher level of antigens can be delivered to antigen presenting cells (APCs) [9, [12] [13] [14] . The delivery of antigens in particulate form of assemblies also shows the potential to induce Tcell activation following vaccination [15] . Therefore, general approaches are urgently needed for the controllable co-assembly of antigens and appropriate NPs to required morphologies for enhanced immune responses.

Cellular immunity, paired with humoral immunity, is a critical part of a healthy and functioning immune system against infection and reinfection by bacteria or virus [16, 17] . Most NPs are effective in inducing humoral immunity rather than cellular immunity, and necessary functionalization of NPs is required to stimulate comprehensive immune responses. Inorganic nanomaterials have been applied to improve the efficiency of antigen delivery and immunogenicity because of their rigid structures, long shelf life, and the capability to tailor their intrinsic adjuvant-like properties and immunomodulatory functions [18] . Among them, metal oxide clusters (MOCs) are promising candidates since their interaction with biomacromolecules can be facilely designed via regulating their morphologies and surface structures to enhance their beneficial activities on the biological systems [19] [20] [21] . MOCs have been demonstrated to have direct or synergistic antibacterial activities [22, 23] , and the tailor-made MOCs systems, such as virus-like assemblies of MOCs with polymers, excavate a variety of nanocomposites and organic-inorganic hybrids with improved biocompatibility and target activity in anticancer drug delivery systems [21, [24] [25] [26] . Therefore, it would be a beneficial attempt to explore vaccines combined with MOCs to induce qualitatively diverse and longlasting protective immune response [27] . Herein, to enhance BCG trained immunity effects, we develop vaccine adjuvants based on supra-particle assemblies (SPAs) assembled via non-covalent interaction. The enriched hydrogen bonding donor sites (e.g., hydroxy, amine, and amide groups) in antigens and MOCs enable their strong association on the surface of hydrogen bonding acceptor-enriched polymers, e.g., poly(4-vinylpyridine) (P4VP). The co-assembled SPAs of MOCs and antigens of M. bovis with P4VP exhibit high antigen loading and entrapment efficiency. MOCs on the supra-particle surface present the great synergy with P4VP and antigens of M. bovis to enhance the immunogenicity. Our SPA-based vaccine adjuvant can elicit cellular immune responses to promote the activation and multiplication of Tlymphocyte, which is absent in most existing nano-vaccines. No sign of toxicity and mortality was observed in all the tested parameters of SPAs-treated animal models, confirming their great potential in practical medical applications.

To design vaccines that recapitulate the efficacy of NPs and MOCs, biocompatible P4VP is used to template the co-assembly of ultra-small iron/molybdenum oxide clusters 16 (H 2 O) 100 }) with antigens separated from M. bovis. As quasi-spherical metal oxide clusters [28] , {Mo 72 Fe 30 } clusters share the common structural feature with most biomacromolecules whose surfaces are rich in hydrogen bonding donor sites that can bridge their assembly with P4VP via multiple hydrogen bonding into SPAs [29] . Thus, the co-assembly protocol provides potential route to prepare colloidal supraparticles carrying both of the two species (MOCs and whole soluble proteins extracted from M. bovis) to serve as virus-like complex nano-vaccines. By following the co-assembly protocol ( Fig. 1(a) The morphologies of Mb/{Mo 72 Fe 30 }@P4VP and Mb@P4VP virus-like co-assemblies are characterized by light scattering techniques as spherical SPAs, suggested by the angular independent hydrodynamic radius (R h ) ( Fig. 1(b) and Fig. S1 in the Electronic Supplementary Material (ESM)). M. bovis antigens are positively charged and therefore, both Mb/{Mo 72 Fe 30 }@P4VP and Mb@P4VP SPAs, suggested from zeta potential studies, carry positive charges in phosphate buffered saline (PBS) buffers in contrast to the negative charged nature of {Mo 72 Fe 30 }@P4VP SPAs ( Fig. 1(c) ). The charges of SPAs confirm the presence of M. bovis antigens on P4VP (Fig. 1(c) and 

The key steps to trigger immune response are the integration of information about vaccine or microbe by dendritic cells (DCs) and macrophages [30] and the translation of the information to antigen-specific T cells and B cells. DCs and macrophages uptake was combined with anti-mouse Immunoglobulin G-fluorescein isothiocyanate (IgG-FITC) antibody, and the uptake of IgG-FITC was examined by flow cytometry. Murine bone marrow derived dendritic cells (BMDCs) co-incubated with IgG@P4VP or IgG/{Mo 72 Fe 30 }@P4VP uptake 2 times more IgG than that with IgG alone (Fig. S8 in the ESM).

To test the stimulating immune response capability of SPAs in mice, female Balb/c mice were subcutaneously vaccinated with Mb/{Mo 72 Fe 30 }@P4VP SPAs diluted with PBS (pH = 7.0). All the animal experiments were approved by the Institutional Animal Care and Use Committee at South China University of Technology (SYXK2017-0178) and were conducted strictly under the Principles of Laboratory Animal Care. The vaccinated mice were kept for 12 weeks while no sign of toxicity, no mortality, and no fever or other symptoms were observed in all the tested parameters of SPAs-treated animal models (Tables S1 and S2 and Figs. S9-S13 in the ESM). {Mo 72 Fe 30 } on the nanocomposite surface presents the synergy with P4VP and M. bovis antigens to enhance the immunogenicity ( Fig. 2(a) ). The mice inoculated with Mb/{Mo 72 Fe 30 }@P4VP keep higher titers of serum TB-specific IgG antibody compared to Mb@P4VP-treated groups for 6 weeks ( Fig.  2(b) ), and maintain comparable titer level with BCG groups for the next 6 weeks (Fig. S14 in the ESM). For the BCG inoculated mice, TB-specific IgG antibody at the 6th week falls more than a half by its level after the first week. While the antibody in Mb/{Mo 72 Fe 30 }@P4VP group at the 6th week remains high at 67.9% of its level at the 1st week.

After uptaken by macrophages, antigens accumulate in the draining lymph nodes (dLNs) and activate the germinal center (GC). GC reactions produce high affinity antibody-secreting plasma cells and memory B-cells necessary for the host defense against invading pathogens [31] . Follicular helper T cells prime B cells to initiate extrafollicular and germinal center antibody responses, which are crucial for affinity maturation and maintenance of humoral memory [32, 33] . Consistently, an obvious increase of B cells and follicular helper T cells in GC was also observed one week after inoculation in the Mb/{Mo 72 Fe 30 }@P4VP treated groups (Fig. 2(c) 

To verify the enhanced immunogenicity of Mb/{Mo 72 Fe 30 }@P4VP SPAs, the production of CD4 + and CD8 + cells was further examined. Local DCs uptake and present antigens, then induce the activation and differentiation of naive T lymphocytes [34, 35] , CD4 + Th cells, and CD8 + cells. It is well-known that B cells mainly regulate the humoral immunity, whereas T cells regulate cellular immunity. Mice inoculated with Mb/{Mo 72 Fe 30 }@P4VP SPAs produce more B cells and T cells than the saline group. Humoral immunity produces an antibody-mediated immune response, whereas cellular immunity produces a cell-mediated immune response. For safety, the endotoxin levels of the formulations are determined (Fig. 3(a) ) and the in vivo safety of Mb/{Mo 72 Fe 30 }@P4VP SPAs is verified (Table S2 in the ESM). All the tested formulations are in a relatively low range of endotoxin level. No significant differences among the groups are observed, indicating the safety of the adjuvants {Mo 72 Fe 30 }@P4VP SPAs. Following the safety evaluation of {Mo 72 Fe 30 }@P4VP SPAs, the production of CD4 + and CD8 + cells in mice is determined. In the antigen-specific immune investigation, Mb/{Mo 72 Fe 30 }@P4VP SPAs maintain high titers of serum anti-TB antibody over time ( Fig. 2(b) ), promoting the humoral immunity. After vaccination, compared with saline group, the production of CD4 + and CD8 + cells in Mb/{Mo 72 Fe 30 }@P4VP-injected mice is distinctly increased stimulates the production of CD4 + cells with little stimulating effect on the production of CD8 + cell. In addition, the increasement of CD8 + cell production exhibits spatiotemporal hysteresis. CD8 + cell production of BCG-injected mice peaks slower than the production of CD4 + cell. Most existing nanovaccines elicit a targeted antibody response, nevertheless, safely activating cellular immunity against many infectious diseases remains a challenging task. As a prerequisite for cellular immunity, the proportion of IFN-γ-secreting CD8 + T cells (Figs. 2(e) and 3(c)) in Mb/{Mo 72 Fe 30 }@P4VP group increases by 42.8% at the end of the first weeks. CD8 + T cells play an important role in mediating the immune response to tuberculosis in spleen, suggesting that CD8 + CTL response is critical for the control of tuberculosis spread in viscera [36] . The increase of CD8 + cells in spleen indicates Mb/{Mo 72 Fe 30 }@P4VP SPAs can control the spread of bacteria in extrapulmonary organs. Also, {Mo 72 Fe 30 } and P4VP components as vaccine adjuvants stimulate both CD4 + and CD8 + cells for antigen presenting and cellular immune response.

Further, Balb/c mice serums treated with Mb/{Mo 72 Fe 30 }@P4VP were collected to test the cytokine release in eliciting humoral and cellular immune responses. Compared with control groups, the concentrations of Th1 cytokines IFN-γ and IL-2 are increased immediately after inoculation and gradually decreased in the next few days (Figs. 3(d) and 3(e) ), which is conducive to the protective immune response against tuberculosis. Similarly, Th2 cytokines IL-4 and IL-10 are also increased after inoculation (Figs. 3(f) and 3(g) ), and the concentration of IL-10 peaks at the 3rd week. IFN-γ and IL-2 play a major role in establishing cellular immunity, while IL-4 and IL-10 are humoral factors involved in the suppressive function of regulatory T cells [37] . IL-10 cytokines play critical roles in maintaining immune homeostasis [38] . In the later stage, the concentration of IL-10 increases as the antigens are gradually cleared, hence the body secretes anti-inflammatory cytokines to neutralize inflammation in order to prevent the cytokine storm. Through the comparison of cytokine release profiles, {Mo 72 Fe 30 } and P4VP components of SPAs exhibit potent Th-mediated responses. In prophylactic treatment, the activation of B cells by vaccine to produce specific neutralizing antibodies also requires the assistance of Th cells. Cellular immune response determines the vaccine protective cellular immunity and regulates the induction of humoral immunity. Because of their safety profiles, protein-based subunit vaccines are exploited to replace the live attenuated vaccines, such as BCG. To elicit an immune response strong enough for protection from infection by an intracellular pathogen, an adjuvant is required in a subunit vaccine. Our {Mo 72 Fe 30 } and P4VP components of SPAs as vaccine adjuvants not only improve the magnitude and durability of antibody responses but also improve T cell-derived cytokine patterns, leading to the enhanced humoral and cellular immune responses.

A versatile protocol based on polymer-templated assembly strategy is proposed for the facile complexation of vaccines and adjuvants through non-covalent interactions. The sub-nanosized, water-soluble {Mo 72 Fe 30 } clusters with well-defined composition and molecular structure are used as vaccine adjuvants. {Mo 72 Fe 30 } and P4VP are co-assembled with antigenic proteins, and virus-like supra-particle assemblies of Mb/{Mo 72 Fe 30 }@P4VP are formed in buffer solution. Mb/{Mo 72 Fe 30 }@P4VP virus-like SPAs enhance T cell immune response which is highly required in BCG vaccine, since BCG vaccine induces incongruous T cell immunity in immune protection against tuberculosis. Mb/{Mo 72 Fe 30 }@P4VP SPAs can also elicit comparable cellular immune activation which is often hysteretic in BCG immune protection. Compared with pure M. bovis antigenic proteins, {Mo 72 Fe 30 } and P4VP components in Mb/{Mo 72 Fe 30 }@P4VP SPAs can significantly increase the immune protection of vaccines and are potential adjuvant candidates for inclusion in vaccines against tuberculosis. Collectively, the co-assembly method provides the protection against the intractable Mycobacterium infection, opens an avenue of loading antigens for vaccine delivery, and augments the loaded vaccines in low cost and large-scale production.

Preparation of {Mo 72 Fe 30 } was following the Refs. [39, 40] . Considering the ability of {Mo 72 Fe 30 } that can be self-assemble into blackberry-type structure [41] and the stability of the protein antigens, the co-assembly protocol used the freshly prepared {Mo 72 Fe 30 } and was carried out in an ice bath. In addition, constrained by microbiology lab containment, M. bovis bacteria were inactivated and disrupted, and the whole soluble proteins were extracted and used as stimulating antigens to co-assemble the Mb/{Mo 72 Fe 30 }@P4VP SPAs. The detailed co-assembly procedure is as follows: 0.1 mL of bacterial proteins (10 mg/mL) in the vial containing a stirrer was added dropwise with 0.5 mL of P4VP solution (2 mg/mL) along with colloids forming, followed by adding 0.25 mL {Mo 72 Fe 30 } (5 mg/mL) dropwise. The assembled SPAs were characterized by dynamic light scattering (DLS), SEM, and FTIR. FTIR transmittance spectra (Fig. S2 

All the animals were raised in accordance with the National Standard Regulating Welfare of Laboratory Test Animals. The animal study protocol was approved by the Institutional Animal Care and Use Committee at South China University of Technology (SYXK2017-0178). Female Balb/c mice, aged 6-8 week, were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. After one-week acclimation, Balb/c mice were divided into 7 groups. 20 μL of the samples was inoculated subcutaneously in the back of Balb/c mice, separately, and the control mice were injected with saline. Serum and plasma samples were collected every week to test the concentration of IFN-γ, IL-2, IL-4, IL-10, and anti-TB-IgG by using enzyme-linked immunosorbent assay (ELISA) kits following the manufacturer's instruction. In order to measure the concentration of IFN-γ, IL-2, IL-4, IL-10, and anti-TB-IgG in the serum sample, their ELISA kits including a set of calibration standards were applied. The calibration standards were assayed at the same time as the samples and allowed the operator to produce a standard curve of optical density vs. IFN-γ, IL-2, IL-4, IL-10, and anti-TB-IgG concentration. The concentration of IFN-γ, IL-2, IL-4, IL-10, and anti-TB-IgG in the samples was then determined by comparing the OD of the samples to the standard curve. Mouse spleen mononuclear cells were separated by using cell isolation solution kits following the manufacturer's instruction. The CD4 + and CD8 + lymphocytes in mice spleen were analyzed by immunophenotypic method (eBioscience TM ) and counted by flow cytometry.

The serum samples were collected as follows. Before centrifugation for 10 min at approximately 3,000g, the sample in a serum separator tube was allowed to clot for 30 min. Then the serum was removed, and the samples were assayed immediately. The plasma was collected using EDTA or heparin as an anticoagulant. The samples were centrifuged for 30 min at 3,000g at 2-8 °C. The samples were collected and stored at −20 or −80 °C. The samples of cell culture supernates and other biological fluids were centrifuged to remove particulates and assayed immediately. During the operation process, the repeated freezethaw cycles were avoided.

To investigate the antigen uptake, BMDC cells were incubated with anti-mouse IgG-FITC antibody (IgG), IgG@P4VP, and IgG/{Mo 72 Fe 30 }@P4VP for 2 h, respectively. The antibody dilution for flow cytometry staining was performed according to the manufacturer's instructions. Generally, a 1:100 antibody dilution was employed for 10 7 cells. To evaluate the cytotoxicity, BMDC cells were incubated with {Mo 72 Fe 30 }@P4VP, {Mo 72 Fe 30 }, and P4VP for 2 h, respectively. Cell viability was assessed using the CCK-8 kit.

To investigate the germinal center activation and antigen persistence, Balb/c mice were subcutaneously injected with Mb/{Mo 72 Fe 30 }@P4VP, {Mo 72 Fe 30 }@P4VP, Mb@P4VP, and Mb/{Mo 72 Fe 30 } nanocomposites, and {Mo 72 Fe 30 }, P4VP, and BCG, respectively. On the seventh day, mice were euthanized, and their inguinal lymph nodes were stripped out in order to prepare single cell suspensions. Germinal center B cells and follicular helper T cells were labelled with antibodies (FITC rat anti-mouse T-and Bcell activation antigen, Cy5.5 rat anti-mouse CXCR5 from BD, and rabbit anti-CD278/ICOS monoclonal antibody). The antibody dilution for flow cytometry staining was carried out according to the manufacturer's instructions. Each experiment was repeated three times.

Analysis of variance ANOVA was used to compare independent samples from different groups for multiple group comparison. All statistical tests were performed using GraphPad Prism 8.0. All data were presented as mean ± standard deviation (sd). * p < 0.05 was considered statistically significant.

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The work was supported financially by the National Natural Science Foundation of China (Nos. 22101086, 21961142018, and 51873067) and the Natural Science Foundation of Guangdong Province (Nos. 2021A1515012024 and 2021A1515010271).